Design, synthesis, and evaluation of salicyladimine derivatives as multitarget-directed ligands against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2017.08.048

A series of salicyladimine derivatives were designed, synthesized and evaluated as multi-target-directed ligands for the treatment of Alzheimer's disease (AD). Biological activity results demonstrated that some derivatives possessed significant inhibitory activities against amyloid-β (Aβ) aggregation and human monoamine oxidase B (hMAO-B) as well as remarkable antioxidant effects and low cell toxicity. The optimal compound, 5, exhibited excellent potency for inhibition of self-induced Aβ_1–42 aggregation (91.3 ± 2.1%, 25 μM), inhibition of hMAO-B (IC₅₀, 1.73 ± 0.39 μM), antioxidant effects (43.4 ± 2.6 μM of IC₅₀ by DPPH method, 0.67 ± 0.06 trolox equivalent by ABTS method), metal chelation and BBB penetration. Furthermore, compound 5 had neuroprotective effects against ROS generation, H₂O₂-induced apoptosis, 6-OHDA-induced cell injury, and a significant in vitro anti-inflammatory activity. Collectively, these findings highlighted that compound 5 was a potential balanced multifunctional neuroprotective agent for the development of anti-AD drugs.

Full text of DOI:10.1016/j.bmc.2017.08.048

Accepted Manuscript
Design, synthesis, and evaluation of salicyladimine derivatives as multitarget-
directed ligands against Alzheimer’s disease
Hua-Li Yang, Pei Cai, Qiao-Hong Liu, Xue-Lian Yang, Si-Qiang Fang, Yan-
Wei Tang, Cheng Wang, Xiao-Bing Wang, Ling-Yi Kong
PII:
S0968-0896(17)31472-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.048
BMC 13952
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 July 2017
24 August 2017
29 August 2017
Please cite this article as: Yang, H-L., Cai, P., Liu, Q-H., Yang, X-L., Fang, S-Q., Tang, Y-W., Wang, C., Wang,
X-B., Kong, L-Y., Design, synthesis, and evaluation of salicyladimine derivatives as multitarget-directed ligands
against Alzheimer’s disease, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.08.048
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Design, synthesis, and evaluation of salicyladimine derivatives as
multitarget-directed ligands against Alzheimer’s disease
Hua-Li Yangab, Pei Caib, Qiao-Hong Liub, Xue-Lian Yangb, Si-Qiang Fangb,
Yan-Wei Tangb, Cheng Wangb, Xiao-Bing Wangb* and Ling-Yi Kongba*
a
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, Shenyang 110016, China.
b
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China
Pharmaceutical University, Nanjing 210009, China.
* Corresponding author.
Tel/Fax: +86-25-83271405. E-mail: cpu_lykong@126.com; xbwang@cpu.edu.cn

Abstract
A series of salicyladimine derivatives were designed, synthesized and
evaluated as multi-target-directed ligands fo disease
(AD). Biological activity results demonstrated that some derivatives possessed
significant inhibitory activities against amyloid-β (Aβ) aggregation and human
monoamine oxidase B (hMAO-B) as well as remarkable antioxidant effects and
low cell toxicity. The optimal compound, 5, exhibited excellent potency for
inhibition of self-induced Aβ1-42 aggregation (91.3±2.1%, 25 μM), inhibition of
hMAO-B (IC50, 1.73±0.39 μM), antioxidant effects (43.4±2.6 μM of IC50 by DPPH
method, 0.67±0.06 trolox equivalent by ABTS method), metal chelation and BBB
penetration. Furthermore, compound 5 had neuroprotective effects against ROS
generation, H2O2-induced apoptosis, 6-OHDA-induced cell injury, and a significant
in vitro anti-inflammatory activity. Collectively, these findings highlighted that
compound 5 was a potential balanced multifunctional neuroprotective agent for the
development of anti-AD drugs.
Keywords: Alzheimer’s disease, Monoamine oxidases, Aβ1-42 aggregation,
Antioxidant, Metal chelation
1. Introduction
Alzheimer's disease (AD), an age-related progressive neurodegenerative disease
and complicated multi-factorial disorder, is one of the most common forms of
dementia.1 It is characterized by memory loss, decline in language skills and other
cognitive impairments in performing daily activities along with depression.2 The
disorder has shown a surge in the overall mortality rate by over 66%.3 Increased
oxidative stress, dyshomeostasis of biometals, and the formation of toxic Aβ peptide
oligomers are all considered to contribute to the etiology of AD.4,5
One of the main pathological features of AD is the formation of senile plaques
(SP), which caused by amyloid-β (Aβ) deposition.6 The accumulation of toxic Aβ
(especially Aβ1-42) peptide oligomers results in a cascade of biochemical processes
mediating the formation of reactive oxygen species (ROS), calcium dysregulation,

and neuronal cell membrane damage leading to neuronal dysfunction.7 Therefore,
prevention and clearance of Aβ aggregation have been considered as the primary
therapeutic strategy for the neurodegenerative disease.8
Oxidative stress is one of the earliest events in AD pathogenesis.9 As a factor
involved in oxidative stress, monoamine oxidases (MAOs) have drawn much
attention in recent years.10,11 High expression level of MAOs in neuronal tissue relates
to the production of hydrogen peroxide (H2O2), resulting in oxidative damage and
apoptotic signaling events.12,13 MAOs exist as two isoforms, MAO-A and MAO-B,
which exhibit different substrates and inhibitor specificities.14 Human MAO-A
(hMAO-A), preferentially degrading serotonin, adrenaline and noradrenaline, has a
close correlation with depression, whereas human MAO-B (hMAO-B) is specifically
responsible for the neurodegenerative disease such as AD.15 Thus, selective hMAO-B
inhibitors are considered as potential candidates for anti-AD drugs.
In addition, dyshomeostasis of biometals is suggested to play key roles in the
pathogenesis of AD. High levels and miscompartmentalization of metal ions (iron,
copper, zinc, etc.) in the brain can affect the oxidative stress response of mitochondria
and protein misfolding, and ultimately lead to neurodegeneration.16,17 Therefore,
metal chelators have shown promise in the treatment of AD owing to the increased
level of metal ions.
The poor clinical efficacy of single-target drugs raised the possibility that AD
treatments may have to interfere concurrently with more than one neuropathological
mechanism to exert disease modifying benefits. Thus, the multi-target-directed ligand
(MTDL) approach, aiming for this multifaceted disease, has emerged as a new
paradigm in drug discovery.18,19
Resveratrol (trans-3,4,5-trihydroxylstilbene), mainly founded in grapes and red
wine, has shown beneficial effects against neurodegeneration thanks to its antioxidant
and antiinflammatory properties.20 Meanwhile, resveratrol has been extensively
investigated
as
an
inhibitor
of
Aβ1-42
aggregation.21
7-(3-chlorobenzyloxy)-4-formylcoumarin (L1), the cocrystallized ligand in complex
with the human MAO-B active site (PDB: 2V60), has been identified as a new type of

excellent MAO-B inhibitors.22 The benzyloxy group was considered important for
MAO-B inhibitory properties.23,24 The metal-chelating agent, clioquinol (CQ,
5-chloro-7-iodo-8-hydroxyquinoline), demonstrated a significant improvement in
cognition and memory by redistributing metal ions and decreasing plasma Aβ levels
in Phase II clinical trials.25-27
Previously, our group developed several multifunctional frameworks based on
coumarin-benzyloxy and resveratrol-clioquinol scaffolds for the treatment of
neurodegenerative disease.24,28-30 On these bases, we report herein a series of
salicyladimine derivatives as multifunctional anti-AD agents by fusing the structures
of resveratrol, benzyloxy and clioquinol (Fig. 1). These ligands exhibited excellent
biological activities, including the inhibition of self-induced Aβ1-42 aggregation,
inhibition of hMAO-B activitity, metal chelation, antioxidant, neuroprotection and
ability to cross the blood–brain barrier (BBB).
Fig 1. Design strategy for the multifunctional salicyladimine derivatives.
2. Results and discussion
2.1. Chemistry
The synthetic convergent route for accessing the desired compounds 1-18 is
shown in Scheme 1. p-Nitrophenol 1a was used as starting material for the
preparation of the key intermediate 2a, by a O-alkylation reaction with benzyl

bromide 1b in the presence of anhydrous K2CO3. The aromatic amine 3a was
obtained by treating 2a with a catalytic amount of 5% Pd/C under hydrogen
atmosphere.31 The target compounds 1-18 were synthesized by the classical method of
imine formation involving condensation between 3a with a variety of salicylaldehyde
derivatives in ethanol under room temperature.29,30,32 All target compounds were
elucidated by spectroscopic measurements (IR, HRMS, 1H NMR and 13C NMR).
Scheme 1. Synthesis of salicyladimine derivatives 1-18. Reagents and conditions: (i)
anhydrous K2CO3, CH3CN, reflux, 24 h; (ii) 5% Pd/C, H2, EtOH, rt, 18 h; (iii)
salicylaldehyde derivatives, EtOH, rt, 1-2 h.
2.2. Biological Assays
2.2.1. Inhibition of self-induced Aβ1-42 aggregation
The inhibitory activities of the target compounds 1-18 on Aβ1-42 self-induced
aggregation were first determined by a thioflavin T (ThT) fluorescence assay using
resveratrol as reference compounds (Table 1).27,33 Most of the target compounds
significantly inhibited self-induced Aβ1-42 aggregation at the tested concentrations. A
structure-activity relationship analysis indicated that the property of substituents in
salicyladimine derivatives had a significant impact on Aβ1-42 inhibitory activity.
Compounds 13, and 17, which have a strong electron-withdrawing group at the R3
position, exhibited much higher inhibitory activities (84.4±2.0%, 70.2±1.6% at 25 μM,
respectively) than resveratrol (65.1±1.3%). The number of phenolic hydroxyl group

was also crucial for proper biological activity. Compounds 5 and 6 (91.3±2.1%,
88.5±1.8% at 25 μM, respectively) with three phenolic hydroxyl groups had more
potent inhibition of self-induced Aβ1-42 aggregation than compounds 2-4 (72.5±1.3%,
59.8±0.8% and 49.2±1.1% at 25 μM, respectively) with two phenolic hydroxyl
groups.
Table 1. Inhibitions of Aβ1-42 self-induced aggregation and human MAOs activities of
the synthesized compounds.
Aβ1-42 aggregation
inhibition (%)
63.3±1.4
IC50 a(μM )
Compd
SI b
＞3.98
1.66
hMAO-A
hMAO-B
25.13±3.39
6.87±1.11
6.78±1.45
1.23±0.42
1.73±0.39
10.14±1.72
15.46±2.18
24.43±3.54
24.62±3.66
6.50±1.82
23.57±2.93
10.71±1.39
15.30±2.05
24.80±2.87
31.74±3.65
10.84±0.91
60.93±5.16
19.15±2.51
0.20±0.04
1
N c
72.5±1.3
11.39±1.04
2
3
N c
＞14.75
15.28
59.8±0.8
49.2±1.1
18.79±1.76
4
5
N c
N c
＞57.80
＞9.86
＞6.47
＞4.09
＞4.06
＞15.38
＞4.24
＞9.34
＞6.54
＞4.03
＞3.15
＞9.23
＞1.64
＞5.22
17.6
91.3±2.1
88.5±1.8
6
7
N c
53.7±1.5
8
N c
60.1±1.4
9
N c
61.9±0.6
10
N c
53.4±0.6
11
N c
69.5±1.8
12
N c
56.6±1.2
13
N c
84.4±2.0
14
15
N c
59.6±1.2
N c
54.3±0.9
16
N c
66.7±2.1
17
N c
70.2±1.6
18
N c
73.1±1.9
d
3.52±0.069
pargyline
resveratrol
-
d
d
d
65.1±1.3
-
-
-

a IC50: 50% inhibitory concentration (means ± SD of three experiments).
b SI: IC50 (hMAO-A)/IC50 (hMAO-B).
c Inactive at 200 μM (highest concentration tested).
d Not tested.
The potent inhibitor 5 was selected with X-ray crystal structure of the protein
Aβ1-42 structure (PDB code 1IYT) for molecular docking studies because of its
remarkable performance regarding self-induced Aβ1-42 aggregation inhibition. As
shown in Fig. 2A and 2B, the benzene ring of compound 5 interacted with the residue
Phe19 via π-π stacking interaction. Hydrogen bond interactions were formed between
phenolic hydroxyl groups of compound 5 and the residues Asp23, Ser26. These
results indicated that the π-π stacking interaction and hydrogen bond interactions
played important roles in the inhibitory activity of compound 5 on Aβ1-42 self-induced
aggregation.
Fig 2. Docking study of compound 5 (colored green) with Aβ1-42 (PDB code 1IYT).
(A) Cartoon representations of compound 5 interacting with Aβ1-42. (B) Association
of 5 (colored green) and the Aβ1-42 obtained from docking calculations. The residues
are highlighted in blue stick representation. The π-π stacking interactions are
depicted with pink dotted lines; H-bonds are represented by green dotted lines.
2.2.2. Inhibition of MAOs in vitro
The hMAO-A and hMAO-B inhibitory activities of all target compounds 1-18
were measured by a previously described fluorescence-based Amplex Red assay using
pargyline as reference compound (Table 1).34 The result showed that compounds 2-5

and 10 exhibit good activities against hMAO-B with IC50 in the micromolar range.
Among them, compound 5 (hMAO-B, IC50=1.73±0.36 μM; SI >57.80) showed the
most potent inhibitory activity and selectivity for hMAO-B. Comparing the IC50
values of 5 and 6 (hMAO-B, IC50=10.14±1.72 μM; SI >9.86), the locality of phenolic
hydroxyl group seemed to be important for the inhibition activities. When the benzene
ring of compound 1 (hMAO-B, IC50=25.13±3.39 μM; SI >3.98) was replaced with
naphthalene, compound 18 (hMAO-B, IC50=19.15±2.51 μM; SI >5.22) also gave
equivalent hMAO-B inhibition activitity.
The molecular modeling study based on MAO-B (PDB code: 2V60) was
performed using docking program, AutoDock 4.2 package with Discovery Studio 2.0.
As shown in Fig. 3, a hydrogen-bonding network was formed between the
phenolic hydroxyl groups of compound
5 and oxygen atoms of Tyr188, Gly434.
In addition, the benzene ring of salicyladimine formed the π-π stacking interaction
with FAD cofactor (FAD 1502). Based on the above discussion, it was evident
that hydrogen-bonding network and π-π stacking interactions were important for
binding patterns of the potential compounds with MAO-B active site.
Fig 3. Docking study of compound 5 (colored green) with MAO-B (PDB code 2V60).
The FAD cofactor (FAD 1502) and residues are highlighted in purple and blue stick
representation, respectively. The π-π stacking interactions are depicted with pink
dotted lines; H-bonds are represented by green dotted lines. The semi-transparent
surface represents the active site cavity.
2.2.3. In vitro antioxidant activity assay

Free radicals-induced oxidative stress plays an important role on AD
pathogenesis. Drugs, which remove the free radicals in the brain would benefit the
AD treatment. The antioxidant activities of our derivatives were evaluated by the
DPPH (diphenyl-1-picrylhydrazyl) radical scavenging method and ABTS (2,
2’-azino-bis(3-ethylbenzthiazoline-6-sulfonicacid)) radical scavenging method. For
comparison purpose, resveratrol was used as the reference compound. The test results
were summarized in Table 2.
Table 2. DPPH and ABTS scavenging activities of the synthesized compounds.
Compd
IC50 a (μM )
ABTS assay b
DPPH scavenging activities
c
1
＞200
13.3±0.8
＞200
-
0.60±0.12
2
c
3
-
21.3±1.8
43.4±2.6
23.9±0.7
＞200
0.97±0.11
0.67±0.06
0.69±0.08
4
5
6
c
7
-
c
8
＞200
-
c
9
＞200
-
c
10
＞200
-
c
11
＞200
-
c
12
＞200
-
c
13
＞200
-
c
14
＞200
-
c
15
＞200
-
c
16
17
＞200
-
c
＞200
-
c
18
＞200
-
82.7±2.1
1.57±0.19
resveratrol

a IC50: 50% inhibitory concentration (means ± SD of three experiments).
b The means ± SD of three independent experiments. Data are expressed as μmol of
trolox equivalent/μmol of tested compound.
c Not tested.
2.2.3.1. DPPH radical scavenging assay
DPPH radicals can be utilized in preliminary screening of compounds capable of
scavenging reactive oxygen species (ROS), since these nitrogen radicals are much
more stable and easier to handle than oxygen free radicals.35,36 From Table 2, it could
be seen that compounds 2,4-6 had much better scavenging activities compared with
resveratrol (DPPH, IC50=82.7±2.1 μM), while others had a negligible effect on
DPPH-scavenging. From the IC50 values of compounds 2-4, it appeared that R2-OH
group on the benzene ring had less positive effect than R1- or R3-OH. Replacement of
the R1- or R3-OH group on the benzene ring by other group was found to reduce the
ability of scavenging the DPPH free radical. This observation indicated that other
group was negative for the activity compared with hydroxyl group.
2.2.3.2. ABTS radical scavenging assay
Some selected compounds tested for their antioxidant activities (DPPH) were
also tested for their ABTS radical scavenging activities.37 Trolox, a water-soluble
vitamin E analog, was used as a reference standard. Their antioxidant activities were
provided as a trolox equivalent, with their relative potency at 25 μM compared with
trolox. As shown in Table 2, compounds 2, 4-5 demonstrated moderate antioxidant
activities ranging from 0.60- to 0.97-fold of the trolox value.
2.2.4. Metal-chelating properties of compound 5
An UV-vis spectroscopy assay was performed to evaluate the ability of the
compound 5 to chelate biometals such as Cu2+, Zn2+, Fe2+, and Fe3+.27,38,39 As shown
in Fig. 4A, new optical bands were detected at 370, 386 and 379 nm after the
addition of CuSO4, FeSO4 or FeCl3 to the solution of compound 5, which
demonstrated the production of the corresponding complex via metal chelation.
To determine the stoichiometry of the complex, the Job’s method was used by
preparing solutions of compound 5 and ion so that the sum of concentrations of both

species was constant in all samples, but the proportions of both components varied
between 0 and 100%.27,38,40 Taking complex 5-Cu2+ as an example, the UV spectra
were recorded and the absorbance change at 370 nm was plotted versus the mole
fraction of compound 5, giving two straight lines whose equations were calculated
(Fig. 4B). The two straight lines intersected at a mole fraction of 0.49, revealing
about a 1:1 stoichiometry for complex 5-Cu2+. Similar Job plots analysis for
complexes 5-Fe2+ and 5-Fe3+ were shown in the Supporting Information (Figs.
S2-S3), suggesting the formation of both 1:1 complexes. A higher value of the
stability constant (log K) indicates a higher affinity of metal ion and compound. The
log K values of Cu2+, Fe2+, Fe3+ with compound 5 for 1:1 ratio were 8.01, 6.30, 6.19,
respectively. The affinity order of different metals and compound 5 was Cu2+
>
Fe2+ > Fe3+.
Fig 4. (A) UV absosobance spectrum of compound 5 (50 μM) alone or in the presence
of CuSO4 (50 μM), ZnCl2 (50 μM), FeSO4 (50 μM), or FeCl3 (50 μM) in buffer (20
mM HEPES, 150 mM NaCl, pH 7.4). (B) Determination of the stoichiometry of
complex 5-Cu2+ by Job’s method
2.2.5. Cell viability and neuroprotective effect against H2O2-induced toxicity
The safety is extraordinary important for the CNS drugs, so the potential toxicity
effect of compounds 1-18 was investigated on compound-treated PC12 cells. The
cells were exposed to compound for 24 h and the cell viability was determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay.41,42 As shown in
Fig. 5, the results revealed that most of the compounds at a concentration of 25 μM
did not show significant neurotoxicity.

Fig 5. Effect of compounds on cell viability in PC12 cells. The results were expressed
as a percentage of control cells. Values are reported as the mean ± SD of three
independent experiments.
Compounds 2, 4, 5 and 6 with remarkable radical scavenging activities, were
tested for their capacities to protect PC12 cells against oxidative stress-associated
death induced by H2O2.41,43 Resveratrol was used as the reference compound. As
shown in Fig. 6, compounds 2, 4, 5 showed strong protective capabilities almost the
same as resveratrol at the concentration of 25 μM except compound 6. Compound 5
could significantly inhibit cell death at concentrations ranging from 6.25 to 25 μM.
These observations further indicated that compound 5 had the potential to be an
efficient multifunctional agent, including antioxidant activity, for the treatment of AD.

Fig 6. Neuroprotection against H2O2 toxicity. Compounds 2, 4, 5 and 6 were tested
for neuroprotective activity against H2O2 toxicity in PC12 cells. Resveratrol 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) < 0.001.
p
2.2.6. Compound 5 attenuated H2O2-induced ROS production in PC12 cells
The oxidative stress response to ROS has been proposed to be the key initiator
for H2O2-induced cell oxidative damage.44 Therefore, the effects of compound 5 on
ROS production in PC12 cells exposed to H2O2 at the concentration of 180 μM was
evaluated. While treated with compound 5 at the concentrations of 10, 15, 20 and 25
μM, the intracellular ROS levels were effectively suppressed in
a
concentration-independent manner, which was significantly lower than that in the
H2O2 group (Fig. 7). The result suggested that H2O2 could induce ROS production in
PC12 cells and compound 5 effectively reduced H2O2-induced ROS accumulation.
Fig 7. The effect of compound 5 on H2O2-induced intracellular ROS production in
PC12 cells. The percentage of cells for ROS (H2O2) was stained with DCFH-DA and
the stained cells were immediately determined by flow cytometry. ***p (###p) < 0.001.
2.2.7. Compound 5 prevented H2O2-induced apoptosis in PC12 cells
AnnexinV/PI double staining method was used to evaluate the effect of different
concentrations of compound 5 on cell apoptosis. As shown in Fig. 8, the percentage of
apoptotic cells was increased from 3.7% (normal group) to 52.3% (H2O2 group). In
contrast, rates of apoptotic cells reduced to 38.6%, 29.9%, 11.2%, 6.1% when PC12
cells were pretreated with 6.25, 12.5, 25, 50 μM of compound 5. This result indicated

that compound 5 prevented H2O2-induced apoptosis in PC12 cells.
Fig 8. The effect of compound 5 on H2O2-induced apoptosis in PC12 cells. Apoptotic
cells detected by flow cytometry after AnnexinV and PI double staining. ***p (###p) <
0.001, **p < 0.01.
2.2.8. Neuroprotective effects of compound 5 against 6-OHDA-induced toxicity in
PC12 cells
6-hydroxydopamine (6-OHDA) is one of the widely used neurotoxins for
neuronal degeneration by simulating oxidative stress in vitro as well as in vivo
experiments. Like dopamine, 6-OHDA is also a substrate for the oxidation by MAO-B.
6-OHDA crosses the cell membrane through dopamine transporters/receptors and
inhibits mitochondrial respiration. It has been shown to induce toxicity by
autooxidation and H2O2 generation.45,46 Therefore, compound 5 with remarkable
MAO-B inhibitory activitity, was tested for their capacity to protect PC12 cells
against oxidative stress-associated death induced by 6-OHDA.46 PC12 cells were
treated with compound 5 at different concentrations in presence/absence of 6-OHDA
at 50 μM concentration. As the results were summarized in Fig. 9, compound 5
exhibited significant protective effect (65.9-75.4%) at the concentrations of 10, 15, 20
and 25 μM in 6-OHDA-treated PC12 cell. This result suggested that compound 5 had
the potential to be an antioxidant agent.

Fig 9. Neuroprotective effects of compound 5 against 6-OHDA-induced toxicity in
PC12 cells. Results are expressed as percent viability compared to cells not treated
with neurotoxins. ***p (###p) < 0.001, **p < 0.01.
2.2.9. Neuroprotective effects of the compound 7d against LPS-stimulated
inflammation in BV2 Microglial Cells
Neuroinflammation plays a vital role in Alzheimer’s disease (AD) and other
neurodegenerative conditions.47,48 The activation of brain microglial cells in the CNS
and the subsequent excess production of inflammatory mediators, such as nitric oxide
(NO), may result in uncontrolled neuroinflammation in neurodegenerative diseases.27
Therefore, inhibition of inflammatory mediators NO production in microglial cells is
considered to be a promising strategy in the onset and progression of
neurodegenerative diseases. The antineuroinflammatory properties of target
compound 5 were determined by the exhibited significantly higher inhibition of NO
production in the LPS-stimulated BV2 cells than resveratrol (Fig. 10). This result
further demonstrated that the unique structure of compound 5 resulted in improved
anti-neuroinflammatory activities compared to the leading compound resveratrol.

Fig 10. NO production inhibiting effect of resveratrol (Res) and compound 5.
2.2.10. In vitro blood-brain barrier permeation assay
The blood-brain barrier (BBB) protects the brain from harmful chemicals, and
makes it difficult for drugs to reach the target cells. Thus, it is important to cross the
blood-brain barrier (BBB) for the drugs targeting central nervous system (CNS). On
this basis, the parallel artificial membrane permeation assay (PAMPA)-BBB method
was used to evaluate the brain penetration ability of compound 5.49 Table 3 showed
the permeability values of eight reference drugs with known CNS penetration and
compound 5. The experimental and reported permeability values of eight reference
drugs provided a good linear correlation: Pe (exp) = 0.6701 Pe (Bibl) + 0.7106 (R2
=0.9898) (Fig 11). From this equation and considering the limits established by Di et
al. for BBB permeation, we established that compound with Pe value above 3.39×10-6
cm s-1could penetrate into the CNS. Compound 5 showed a Pe value of 5.75×10-6 cm
s-1, indicating that it could easily cross the BBB.
Table 3. Permeability (Pe × 10-6 cm s-1) in the PAMPA-BBB assay for 8 commercial
drugs (used in the experiment validation) and compound 5.
Commercial drugs
Testosterone
Verapamil
Bibliography a
Experiment b
11.757 ± 2.014
11.439 ± 1.538
9.173 ± 1.0695
5.003 ± 0.835
17
16
12
5.3
β-Estradiol
Clonidine

Corticosterone
Piroxicam
5.1
2.5
1.9
1.1
-
3.763 ± 0.571
1.801 ± 0.291
1.968 ± 0.355
1.592 ± 0.194
5.751 ± 0.917
Hydrocortisone
Lomefloxacin
5
a Taken from Ref.49
b Data are the mean ± SD of three independent experiments.
Fig 11. Linear correlation between experimental and reported permeability of
commercial drugs using the PAMPA-BBB assay.
3. Conclusion
In summary, a series of salicyladimine derivatives were designed and
synthesized as effective MTDL against AD by fusing the pharmacophore of
resveratrol with a similar pattern with clioquinol and L1. Among the synthesized
compounds, compound 5 exhibited significant inhibition of self-induced Aβ
aggregation/hMAO-B, antioxidant activity, metal-chelating ability and low cell
toxicity as well as remarkable BBB penetration. Furthermore, compound 5 had
neuroprotective effects against ROS generation, H2O2-induced apoptosis and
6-OHDA-induced cell injury, and a significant in vitro anti-inflammatory activity.

These properties highlighted that the compound 5 could be considered as a potential
multifunctional neuroprotective agent for the development of anti-AD drugs.
4. Experimental section
4.1. Chemistry
All chemicals (reagent grade) used were purchased from Sino pharm Chemical
Reagent Co., Ltd. (China). Reaction progress was monitored using analytical thin
layer chromatography (TLC) on precoated silica gel GF254 (Qingdao Haiyang
Chemical Plant, Qing-Dao, China) plates and the spots were detected under UV light
(254 nm). Column chromatography was performed on silica gel (90-150 μm; Qingdao
1
13
Marine Chemical Inc.). H NMR and C NMR spectra were measured on a Bruker
ACF-500 spectrometer at 25°C and referenced to TMS. Chemical shifts are reported
in ppm (δ) using the residual solvent line as internal standard. Splitting patterns are
designed as s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectra were obtained
on a MS Agilent 1100 Series LC/MSD Trap mass spectrometer (ESI-MS) and a
Mariner ESI-TOF spectrometer (HRESIMS), respectively.
4.1.1. Synthesis of 1-(benzyloxy)-4-nitrobenzene 2a
To a solution of p-nitrophenol 1a (15 mmol) and powdered K2CO3 (30 mmol) in
acetonitrile (30 mL), benzyl bromide 1b (18 mmol) was added. The reaction mixture
was refluxed for 24 h. Upon completion, K2CO3 was removed by filtration and the
solvent was concentrated under vacuum, the residue was purified on silica gel
chromatography with petroleum ether/ethyl acetate (10:1, v/v) as elution solvent to
give the desired product 2a. White solid, 85% yield;1H NMR (500 MHz, DMSO-d6) δ
8.22 (d, J = 9.3 Hz, 2H, Ar-H), 7.48 (d, J = 7.3 Hz, 2H, Ar-H), 7.41 (t, J = 7.5 Hz, 2H,
Ar-H), 7.36 (t, J = 7.3 Hz, 1H, Ar-H), 7.23 (d, J = 9.2 Hz, 2H, Ar-H), 5.27 (s, 2H,
OCH2)..
4.1.2. Synthesis of 4-(benzyloxy)aniline 3a
The compound 2a (10 mmol) was dissolved in ethanol and a catalytic amount of
5% Pd/C (2 mmol) was added to the mixture. The solution was stirred at room
temperature under H2 atmosphere for 18 h. After the completion of the reaction, the

mixture was filtered to eliminate the catalyst. The obtained crude product was then
purified by recrystallization to give the desired intermediate 3a as a brown solid with
72% yield; 1H NMR (500 MHz, DMSO-d6) δ 7.40 (d, J = 7.1 Hz, 2H, Ar-H), 7.36 (t, J
= 7.5 Hz, 2H, Ar-H), 7.30 (t, J = 7.1 Hz, 1H, Ar-H), 6.73 (d, J = 8.7 Hz, 2H, Ar-H),
6.54 (d, J = 8.7 Hz, 2H, Ar-H), 4.94 (s, 2H, OCH2).
4.1.3. General procedures for the preparation of compound 1-18.
The respective salicylaldehyde derivatives (5.0 mmol) were added to a solution
of compound 3a (5.0 mmol) in ethanol (10 mL). The mixture was stirred under room
temperature for 2-5 h then followed by filtration and recrystallization in EtOAc or
MeOH to obtain the pure compounds.
4.1.3.1. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)phenol (1)
Yellow solid, 82% yield; m.p. 119-120 °C; IR (KBr) ν 3033, 2914, 1617, 1496,
1452, 1255, 1037, 836, 728, 693, 530 cm-1;1H NMR (500 MHz, DMSO-d6) δ 13.28 (s,
1H, OH), 8.94 (s, 1H, CH=N), 7.61 (dd, J = 7.7, 1.7 Hz, 1H, Ar-H), 7.47 (d, J = 7.3
Hz, 2H, Ar-H), 7.45-7.37 (m, 5H, Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.11 (d, J =
8.9 Hz, 2H, Ar-H), 7.00-6.92 (m, 2H, Ar-H), 5.15 (s, 2H, OCH2).13C NMR (125 MHz,
DMSO-d6) δ 161.9, 160.6, 158.1, 141.4, 137.4, 133.3, 132.8, 128.9, 128.4, 128.2,
123.1, 119.9, 119.5, 117.0, 116.1, 70.0. HRMS: calcd for C20H17NO2 [M + H]+
304.1332, found 304.1332.
4.1.3.2. (E)-3-(((4-(benzyloxy)phenyl)imino)methyl)benzene-1,2-diol (2)
Orange solid, 79% yield; m.p. 174-175 °C; IR (KBr) ν 3033, 2897, 1623, 1509,
1456, 1249, 1038, 832, 742, 696, 564 cm-1;1H NMR (500 MHz, DMSO-d6) δ 13.41 (s,
1H, OH), 9.15 (s, 1H, OH), 8.90 (s, 1H, CH=N), 7.47 (d, J = 7.3 Hz, 2H, Ar-H),
7.45-7.37 (m, 4H, Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.11 (d, J = 8.9 Hz, 2H,
Ar-H), 7.06 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 6.92 (dd, J = 7.8, 1.6 Hz, 1H, Ar-H), 6.78
(t, J = 7.8 Hz, 1H, Ar-H), 5.15 (s, 2H, OCH2).13C NMR (125 MHz, DMSO-d6) δ
162.3, 158.0, 149.6, 146.0, 141.3, 137.4, 128.9, 128.4, 128.2, 123.0, 119.9, 119.1,
119.1, 116.1, 70.0. HRMS: calcd for C20H17NO3 [M + Na]+ 342.1101, found
342.1103.
4.1.3.3. (E)-4-(((4-(benzyloxy)phenyl)imino)methyl)benzene-1,3-diol (3)

Yellow solid, 85% yield; m.p. 177-178 °C; IR (KBr) ν 3032, 2859,1630, 1513,
1
1459, 1243, 1132, 834, 743, 697, 599 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.68
(s, 1H, OH), 10.20 (s, 1H, OH), 8.76 (s, 1H, CH=N), 7.46 (d, J = 7.3 Hz, 2H, Ar-H),
7.41-7.38 (m, 3H, Ar-H), 7.35-7.32 (m, 3H, Ar-H), 7.07 (d, J = 8.9 Hz, 2H, Ar-H),
6.40 (d, J = 8.6 Hz, 1H, Ar-H), 6.29 (s, 1H, Ar-H), 5.13 (s, 2H, OCH2).13C NMR (125
MHz, DMSO-d6) δ 163.3, 162.5, 161.3, 157.4, 141.7, 137.5, 134.6, 128.9, 128.3,
128.2, 122.6, 116.0, 112.6, 108.2, 102.9, 69.9. HRMS: calcd for C20H17NO3 [M + H]+
320.1281, found 320.1280.
4.1.3.4. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)benzene-1,4-diol (4)
Yellow solid, 89% yield; m.p. 181-182 °C; IR (KBr) ν 3037, 2878, 1601, 1501,
1
1458, 1242, 1003, 834, 742, 700, 548 cm-1; H NMR (500 MHz, DMSO-d6) δ 12.44
(s, 1H, OH), 9.07 (s, 1H, OH), 8.82 (s, 1H, CH=N), 7.47 (d, J = 7.3 Hz, 2H, Ar-H),
7.44-7.37 (m, 4H, Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.09 (d, J = 8.9 Hz, 2H,
Ar-H), 7.00 (d, J = 2.9 Hz, 1H, Ar-H), 6.83 (dd, J = 8.8, 2.9 Hz, 1H, Ar-H), 6.77 (d, J
= 8.8 Hz, 1H, Ar-H), 5.15 (s, 2H, OCH2).13C NMR (125 MHz, DMSO-d6) δ 161.6,
157.9, 153.4, 150.0, 141.8, 137.4, 128.9, 128.3, 128.2, 123.1, 121.0, 119.8, 117.5,
117.3, 116.0, 69.9. HRMS: calcd for C20H17NO3 [M + H]+ 320.1281, found 320.1282.
4.1.3.5. (E)-4-(((4-(benzyloxy)phenyl)imino)methyl)benzene-1,2,3-triol (5)
Orange solid, 75% yield; m.p. 197-198 °C; IR (KBr) ν 3033, 2884, 1631, 1511,
1
1458, 1248, 1044, 824, 738, 695, 563 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.77
(s, 1H, OH), 9.64 (s, 1H, OH), 8.74 (s, 1H, CH=N), 8.44 (s, 1H, OH), 7.46 (d, J = 7.3
Hz, 2H, Ar-H), 7.40 (t, J = 7.3 Hz, 2H, Ar-H), 7.37-7.32 (m, 3H, Ar-H), 7.08 (d, J =
8.9 Hz, 2H, Ar-H), 6.91 (d, J = 8.4 Hz, 1H, Ar-H), 6.40 (d, J = 8.4 Hz, 1H, Ar-H),
13
5.14 (s, 2H, OCH2). C NMR (125 MHz, DMSO-d6) δ 161.8, 157.4, 151.7, 150.4,
141.3, 137.5, 132.8, 128.9, 128.3, 128.2, 124.2, 112.8, 122.6, 116.0, 108.1, 69.9.
HRMS: calcd for C20H17NO4 [M + H]+ 336.1230, found 336.1233.
4.1.3.6. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)benzene-1,3,5-triol (6)
Red brown solid, 77% yield; m.p. 198-199 °C; IR (KBr) ν 3036, 2884, 1623,
1
1511, 1450, 1247, 1077, 824, 738, 696, 566 cm-1; H NMR (500 MHz, DMSO-d6) δ
12.26 (s, 2H, OH), 10.15 (s, 1H, OH), 8.87 (s, 1H, CH=N), 7.46 (d, J = 7.3 Hz, 2H,

Ar-H), 7.40 (t, J = 7.3 Hz, 2H, Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.29 (d, J = 8.9
Hz, 2H, Ar-H), 7.06 (d, J = 8.9 Hz, 2H, Ar-H), 5.84 (s, 2H, Ar-H), 5.13 (s, 2H,
OCH2).13C NMR (125 MHz, DMSO-d6) δ 163.9, 162.7, 157.2, 156.5, 141.9, 137.5,
128.9, 128.3, 128.1, 122.3, 116.1, 101.9, 94.6, 70.0. HRMS: calcd for C20H17NO4 [M
+ H]+ 336.1230, found 336.1231.
4.1.3.7. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-6-methylphenol (7)
Red brown solid, 85% yield; m.p. 95-96 °C; IR (KBr) ν 3031, 2893, 1616, 1506,
1
1455, 1250, 1040, 838, 735, 695, 591 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.74
(s, 1H, OH), 8.93 (s, 1H, CH=N), 7.47 (d, J = 7.3 Hz, 2H, Ar-H), 7.46-7.38 (m, 5H,
Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.28 (d, J = 7.5 Hz, 1H, Ar-H), 7.11 (d, J = 8.9
Hz, 2H), 6.88 (t, J = 7.5 Hz, 1H, Ar-H), 5.15 (s, 2H, OCH2), 2.22 (s, 3H, CH3).13C
NMR (125 MHz, DMSO-d6) δ 162.3, 159.0, 158.0, 141.1, 137.4, 134.1, 130.8, 128.9,
128.3, 128.2, 125.4, 123.1, 119.0, 118.9, 116.1, 69.9, 15.7. HRMS: calcd for
C21H19NO2 [M + H]+ 318.1489, found 318.1490.
4.1.3.8. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-5-methylphenol (8)
Yellow solid, 81% yield; m.p. 178-179 °C; IR (KBr) ν 3040, 2916, 1619, 1504,
1
1453, 1249, 1018, 858, 742, 698, 536 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.28
(s, 1H, OH), 8.88 (s, 1H, CH=N), 7.61-7.22 (m, 8H, Ar-H), 7.09 (s, 2H, Ar-H), 6.78
(s, 2H, Ar-H), 5.14 (s, 2H, OCH2), 2.31 (s, 3H, CH3).13C NMR (125 MHz, DMSO-d6)
δ 161.7, 160.7, 157.9, 143.9, 141.5, 137.4, 132.7, 128.9, 128.3, 1282, 123.0, 120.6,
117.5, 117.3, 116.0, 69.9, 21.9. HRMS: calcd for C21H19NO2 [M + H]+ 318.1489,
found 318.1491.
4.1.3.9. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-methylphenol (9)
Yellow solid, 78% yield; m.p. 144-145 °C; IR (KBr) ν 3035, 2912, 1624, 1505,
1
1489, 1244, 1007, 822, 734, 696, 549 cm-1; H NMR (500 MHz, DMSO-d6) δ 12.98
(s, 1H, OH), 8.87 (s, 1H, CH=N), 7.47 (d, J = 7.3 Hz, 2H, Ar-H), 7.43-.37 (m, 5H),
7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.20 (dd, J = 8.3, 2.2 Hz, 1H, Ar-H), 7.10 (d, J = 8.9
Hz, 2H, Ar-H), 6.85 (d, J = 8.3 Hz, 1H, Ar-H), 5.15 (s, 2H, OCH2), 2.27 (s, 3H,
CH3).13C NMR (125 MHz, DMSO-d6) δ 161.8, 158.4, 158.0, 141.6, 137.4, 134.0,

132.6, 128.9, 128.3, 128.2, 128.0, 123.0, 119.5, 116.8, 116.0, 69.9, 20.4. HRMS:
calcd for C21H19NO2 [M + H]+ 318.1489, found 318.1490.
4.1.3.10. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-6-methoxyphenol (10)
Red brown solid, 83% yield; m.p. 116-117 °C; IR (KBr) ν 3033, 2932, 1619,
1
1508, 1463, 1248, 1017, 828, 731, 699, 511 cm-1; H NMR (500 MHz, DMSO-d6) δ
13.47 (s, 1H, OH), 8.92 (s, 1H, CH=N), 7.46 (d, J = 7.3 Hz, 2H, Ar-H), 7.42-7.38 (m,
4H), 7.33 (t, J = 7.3 Hz, 1H, Ar-H), 7.20 (d, J = 7.7 Hz, 1H, Ar-H), 7.13-7.09 (m, 3H,
Ar-H), 6.89 (t, J = 7.7 Hz, 1H, Ar-H), 5.14 (s, 2H, OCH2), 3.81 (s, 3H, OCH3).13C
NMR (125 MHz, DMSO-d6) δ 162.0, 158.0, 150.9, 148.3, 141.2, 137.4, 128.9, 128.34,
128.2, 124.2, 123.1, 119.7, 119.0, 116.0, 115.7, 69.9, 56.3. HRMS: calcd for
C21H19NO3 [M + H]+ 334.1438, found 334.1437.
4.1.3.11. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-5-methoxyphenol (11)
Yellow solid, 74% yield; m.p. 158-159 °C; IR (KBr) ν 3033, 2937, 1624, 1516,
1
1467, 1241, 1004, 836, 750, 700, 540 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.81
(s, 1H, OH), 8.83 (s, 1H, CH=N), 7.49 (d, J = 8.6 Hz, 1H, Ar-H), 7.46 (d, J = 7.3 Hz,
2H, Ar-H), 7.40 (t, J = 7.3 Hz, 2H, Ar-H), 7.37-7.32 (m, 3H, Ar-H), 7.08 (d, J = 8.9
Hz, 2H, Ar-H), 6.55 (dd, J = 8.6, 2.5 Hz, 1H, Ar-H), 6.48 (d, J = 2.5 Hz, 1H, Ar-H),
5.14 (s, 2H, OCH2), 3.80 (s, 3H, OCH3).13C NMR (125 MHz, DMSO-d6) δ 163.7,
163.4, 161.2, 157.6, 141.3, 137.4, 134.3, 128.9, 128.3, 128.2, 122.7, 116.0, 113.5,
107.1, 101.3, 69.9, 55.9. HRMS: calcd for C21H19NO3 [M + H]+ 334.1438, found
334.1440.
4.1.3.12. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-methoxyphenol (12)
Yellow solid, 78% yield; m.p. 126-127 °C; IR (KBr) ν 3038, 2937, 1599, 1490,
1
1467, 1241, 1155, 823, 738, 700, 528 cm-1; H NMR (500 MHz, DMSO-d6) δ 12.63
(s, 1H, OH), 8.91 (s, 1H, CH=N), 7.47 (d, J = 7.3 Hz, 2H, Ar-H), 7.42-7.36 (m, 4H,
Ar-H), 7.34 (t, J = 7.3 Hz, 1H, Ar-H), 7.21 (d, J = 3.1 Hz, 1H, Ar-H), 7.10 (d, J = 8.8
Hz, 2H, Ar-H), 7.01 (dd, J = 8.9, 3.1 Hz, 1H, Ar-H), 6.89 (d, J = 8.9 Hz, 1H, Ar-H),
5.15 (s, 2H, OCH2), 3.75 (s, 3H, OCH3).13C NMR (125 MHz, DMSO-d6) δ 161.5,
158.0, 154.6, 152.3, 141.6, 137.4, 128.9, 128.3, 128.2, 123.1, 120.4, 119.7, 117.8,

116.0, 115.6, 69.9, 56.0. HRMS: calcd for C21H19NO3 [M + H]+ 334.1438, found
334.1436.
4.1.3.13. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-fluorophenol (13)
Yellow solid, 77% yield; m.p. 135-136 °C; IR (KBr) ν 3068, 2915, 1599, 1493,
1
1451, 1274, 1037, 826, 733, 694, 529 cm-1; H NMR (500 MHz, DMSO-d6) δ 12.93
(s, 1H, OH), 8.91 (s, 1H, CH=N), 7.48-7.46 (m, 3H), 7.44-7.37 (m, 4H), 7.34 (t, J =
7.3 Hz, 1H, Ar-H), 7.25 (td, J = 8.7, 3.2 Hz, 1H, Ar-H), 7.11 (d, J = 8.9 Hz, 2H,
Ar-H), 6.97 (dd, J = 9.1, 4.5 Hz, 1H, Ar-H), 5.15 (s, 2H, OCH2).13C NMR (125 MHz,
DMSO-d6) δ 160.5, 158.3, 156.9, 156.3, 154.5, 141.3, 137.4, 128.9, 128.4, 128.2,
123.2, 120.2, 118.4, 117.4, 116.1, 70.0. HRMS: calcd for C20H16FNO2 [M + H]+
322.1238, found 322.1237.
4.1.3.14. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-chlorophenol (14)
Yellow solid, 79% yield; m.p. 159-160 °C; IR (KBr) ν 3035, 2861, 1620, 1505,
1
1478, 1249, 1016, 824, 738, 694, 549 cm-1; H NMR (500 MHz, DMSO-d6) δ 13.25
(s, 1H, OH), 8.92 (s, 1H, CH=N), 7.70 (d, J = 2.6 Hz, 1H, Ar-H), 7.47 (d, J = 7.2 Hz,
2H, Ar-H), 7.45-7.37 (m, 5H, Ar-H), 7.34 (t, J = 7.2 Hz, 1H, Ar-H), 7.12 (d, J = 8.9
Hz, 2H, Ar-H), 6.98 (d, J = 8.8 Hz, 1H, Ar-H), 5.16 (s, 2H, OCH2).13C NMR (125
MHz, DMSO-d6) δ 160.4, 159.3, 158.3, 141.2, 137.4, 132.7, 131.4, 128.9, 128.4,
128.2, 123.2, 122.9, 121.2, 119.0, 116.1, 70.0. HRMS: calcd for C20H16ClNO2 [M +
H]+ 338.0942, found 338.0944.
4.1.3.15. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-bromophenol (15)
Yellow solid, 88% yield; m.p. 168-169 °C; IR (KBr) ν 3037, 2870, 1618, 1505,
1474, 1246, 998, 821, 747, 700, 546 cm-1; 1H NMR (500 MHz, DMSO-d6) δ 13.28 (s,
1H, OH), 8.92 (s, 1H, CH=N), 7.83 (d, J = 2.6 Hz, 1H, Ar-H), 7.52 (dd, J = 8.8, 2.6
Hz, 1H, Ar-H), 7.47 (d, J = 7.3 Hz, 2H, Ar-H), 7.45-7.38 (m, 4H), 7.34 (t, J = 7.3 Hz,
1H, Ar-H), 7.11 (d, J = 8.9 Hz, 2H, Ar-H), 6.93 (d, J = 8.8 Hz, 1H, Ar-H), 5.16 (s, 2H,
OCH2).13C NMR (125 MHz, DMSO-d6) δ 160.3, 159.7, 158.3, 141.1, 137.4, 135.5,
134.3, 128.9, 128.4, 128.2, 123.3, 121.8, 119.5, 116.1, 110.3, 70.0. HRMS: calcd for
C20H16BrNO2 [M + H]+ 382.0437, found 382.0439.
4.1.3.16. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4,6-dibromophenol (16)

Orange solid, 82% yield; m.p. 155-156 °C; IR (KBr) ν 3034, 2944, 1613, 1505,
1
1442, 1245, 1167, 1002, 837, 751, 700, 542 cm-1; H NMR (500 MHz, DMSO-d6) δ
14.85 (s, 1H, OH), 8.97 (s, 1H, CH=N), 7.90 (d, J = 2.4 Hz, 1H, Ar-H), 7.82 (d, J =
2.4 Hz, 1H, Ar-H), 7.52-7.44 (m, 4H, Ar-H), 7.41 (t, J = 7.3 Hz, 2H, Ar-H), 7.34 (t, J
= 7.3 Hz, 1H, Ar-H), 7.14 (d, J = 8.9 Hz, 2H, Ar-H), 5.17 (s, 2H, OCH2).13C NMR
(125 MHz, DMSO-d6) δ 160.1, 158.7, 157.8, 139.3, 137.4, 137.3, 134.4, 129.0, 128.4,
128.2, 123.4, 121.5, 116.2, 111.9, 109.7, 70.0. HRMS: calcd for C20H15Br2NO2 [M +
Na]+ 481.9362, found 481.9359.
4.1.3.17. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-nitrophenol (17)
Orange solid, 71% yield; m.p. 188-189 °C; IR (KBr) ν 3032, 2942, 1624, 1481,
1
1353, 1295, 1172, 1011, 822, 734, 690, 512 cm-1; H NMR (500 MHz, DMSO-d6) δ
14.74 (s, 1H, OH), 9.17 (s, 1H, CH=N), 8.63 (s, 1H, Ar-H), 8.23 (s, 1H, Ar-H),
13
7.79-7.25 (m, 7H), 7.15-7.08 (m, 3H), 5.17 (s, 2H, OCH2). C NMR (125 MHz,
DMSO-d6) δ 167.7, 160.0 , 158.7, 139.4, 137.3, 129.0, 128.9, 128.6 , 128.4, 128.2,
123.3, 118.9, 118.9, 116.2, 70.0. HRMS: calcd for C20H16N2O4 [M + Na]+ 371.1002,
found 371.1000.
4.1.3.18. (E)-2-(((4-(benzyloxy)phenyl)imino)methyl)-4-nitrophenol (18)
Yellow solid, 84% yield; m.p. 146-147 °C; IR (KBr) ν 3034, 2895, 1606, 1503,
1
1384, 1246, 1175, 1042, 825, 743, 698, 516 cm-1; H NMR (500 MHz, DMSO-d6) δ
15.98 (d, J = 4.5 Hz, 1H, OH), 9.64 (d, J = 4.5 Hz, 1H, CH=N), 8.50 (d, J = 8.5 Hz,
1H, Ar-H), 7.91 (d, J = 9.1 Hz, 1H, Ar-H), 7.80 (d, J = 8.0 Hz, 1H, Ar-H), 7.62 (d, J =
8.9 Hz, 2H, Ar-H), 7.54 (t, J = 7.3 Hz, 1H, Ar-H), 7.48 (d, J = 7.3 Hz, 2H, Ar-H),
7.41 (t, J = 7.3 Hz, 2H, Ar-H), 7.35 (t, J = 7.4 Hz, 2H, Ar-H), 7.14 (d, J = 8.9 Hz, 2H,
Ar-H), 7.03 (d, J = 9.1 Hz, 1H, Ar-H), 5.17 (s, 2H, OCH2).13C NMR (125 MHz,
DMSO-d6) δ 169.4, 157.7, 155.2, 138.0, 137.4, 136.5, 133.5, 129.4, 128.9, 128.4,
128.4, 128.2, 127.2, 123.8, 122.5, 122.3, 120.8, 116.2, 109.1, 70.0. HRMS: calcd for
C24H19NO2 [M + H]+ 354.1489, found 354.1488.
4.2. Pharmacology
4.2.1. Inhibition of self-induced Aβ1-42 aggregation

Inhibition of Aβ1-42 aggregation was measured using a thioflavin T
(ThT)-binding assay.27,33 Resveratrol were used as reference compounds. Aβ1-42
(Millipore, counterion: NaOH) was dissolved in ammonium hydroxide (1% v/v) to
give a stock solution (2000 μM), which was aliquoted into small samples and stored at
−80 °C. For the experiment of self-mediated Aβ1-42 aggregation inhibition, the Aβ
stock solution was diluted with 50 mM phosphate buffer (pH 7.4) to 50 μM before use.
A mixture of the peptide (10 μL, 25 μM, final concentration) with or without the
tested compound (10 μL) was incubated at 37 °C for 48 h. Blanks using 50 mM
phosphate buffer (pH 7.4) instead of Aβ with or without inhibitors were also carried
out. The sample was diluted to a final volume of 200 μL with 50 mM glycine-NaOH
buffer (pH 8.0) containing thioflavin T (5 μM). Then the fluorescence intensities were
recorded 5 min later (excitation, 446 nm; emission, 490 nm). The percent inhibition of
aggregation was calculated by the expression (1 − IFi/IFc) × 100%, in which IFi and
IFc are the fluorescence intensities obtained for Aβ in the presence and absence of
inhibitors after subtracting the background, respectively.
4.2.2. MAO inhibition assay
Human MAO-A and MAO-B were purchased from Sigma-Aldrich. The capacity
of the test compounds to inhibit hMAO-A and hMAO-B activities was assessed by
Amplex Red MAO assay.34 Firstly, MAOs activity were adjusted to obtain in our
experimental conditions the same reaction velocity in the presence of both isoforms
(i.e., to oxidize (in the control group) the same concentration of substrate: 165 pmol
of p-tyramine/min(hMAO-A: 1.1µg protein; specific activity: 150 nmol of p-tyramine
oxidized to p-hydroxyphenylacetaldehyde/min/mg protein; hMAO-B: 7.5µg protein;
specific activity: 22 nmol of p-tyramine transformed/min/mg protein). Then
compounds were dissolved in DMSO (10 mM) and diluted in 0.05 M
KH2PO4/K2HPO4 buffer (pH = 7.4) to the desired final concentration. All the
compounds are soluble at the tested concentration. Test drugs (20 μL) and MAO (80
μL) were incubated at 37 °C for 15 min in a flat-black-bottom 96-well microtest plate
in dark. The reaction was started by adding 200 μM Amplex Red reagent, 2 U/mL
horseradish peroxidase, and 2 mM p-tyramine for hMAO at 37 °C for 20 min. The

reaction was quantified in a multidetection microplate fluorescence reader based on
the fluorescence generated (excitation, 545 nm; emission, 590 nm). The specific
fluorescence emission was calculated after subtraction of the background activity,
which was determined from vials containing all components except the hMAO
isoforms, which were replaced by a sodium phosphate buffer solution. The percent
inhibition was calculated by the following expression: (1 − IFi/IFc) × 100% in which
IFi and IFc are the fluorescence intensities obtained for hMAO in the presence and
absence of inhibitors after subtracting the respective background.
4.2.3. Docking study
Molecular docking studies were performed using the Discovery Studio 2.0
software program (DS 2.0).50 The X-ray crystallographic structures of Aβ1-42 (PDB
code1IYT) and hMAO-B (PDB code 2V60) were obtained from the PDB (the Protein
Data Bank). First, Water molecules were removed. Second, hydrogen atoms were
added. Third, side chain amides and side chains bumps were fixed. The compound 5
was imported to DS 2.0 and docked into the active site to investigate the binding
modes.
4.2.4. In vitro antioxidant assays
4.2.4.1. DPPH free radical-scavenging assay
Diphenyl-1-picrylhydrazyl stabile free radical assay (DPPH), one of the few
stable and commercially available organic nitrogen radicals, has a UV-vis absorption
maximum at 517 nm and was used to assess free radical-scavenging activity.35,36 The
assay was carried out in methanol. DPPH solution was prepared at 0-200 μM
concentration, and compounds 1-18 were adjusted with methanol solution to final
concentrations of 0-200 μM. Serial dilutions of the test sample (20 mL) were
combined with the DPPH (180 mL, 400 μM) solution in a 96-well microtitre plate.
MeOH was used as a negative control and resveratrol was used as a positive control.
The antioxidant activity was determined after 30 min of incubation by measuring the
increase in absorbance at 517 nm at laboratory temperature. Mean values were
obtained from triplicate experiments. The IC50 values of test compounds were

determined using the Graph Pad Prism 4.03 software (San Diego, CA, USA).
4.2.4.2. ABTS radical cation scavenging activity assay
2, 2’-azino-bis-2-ethybenz-thiazoline-6-sulfonic acid (ABTS) was dissolved in
＋
purified water to a 7 mM concentration. ABTS radical cation (ABTS• ) was produced
by reacting ABTS stock solution with 2.45 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at room temperature for
at least 18 h before use.37 The stock solution of ABTS was serially diluted with
sodium phosphate buffer (50 mM, pH 7.4) to 100 μM. Trolox and tested compounds
at different concentrations (total volume of 50 μL) were added to 150 μL of 100 μM
ABTS solution, respectively. After the addition of either trolox or another antioxidant
to the ABTS solution, complete mixing of reactants was achieved by bubbling three to
four times using plastic pipettes. The optical absorbance of ABTS at 415 nm was
measured at 6 min after addition and equilibrated at 30°C. Each individual treatment
was repeated for three times and the results of the experiments were compared.
4.2.5. Metal-chelating study
The chelating studies were performed in methanol at 298 K using UV−vis
spectrophotometer (SHIMADZU UV-2450PC).27 The absorption spectra of each
compound (50 μM, final concentration) alone or in the presence of CuSO4, FeSO4,
FeCl3 and ZnCl2 (50 μM, final concentration) for 30 min in 20% (v/v) ethanol/buffer
(20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at room temperature. The
stoichiometry of the 5-Cu2+ complex was determined by employing Job’s method.
The 21 solutions were obtained with the condition that the sum of concentrations of
both species was a constant in all samples but the proportions of both components
varied between 0 and 100%. The difference UV−vis spectra were examined to
investigate the ratio of ligand/metal in the complex.
4.2.6. Cell culture and MTT assay for cell viability
PC12 cells was obtained from the Cell Bank of the Chinese Academy of
Sciences (Shanghai, China) and grown in RPMI-1640 medium containing 10% (v/v)
foetal bovine serum, 100 U penicillin/mL and 100 mg streptomycin/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 6,000 cells per well and allowed
to adhere and grow. When cells reached the required confluence, they were placed
into serum-free medium and treated with compounds. The survival of cells was
determined by MTT assay after 24h.51 Briefly, after incubation with 20 μL of MTT at
37 °C for 4 h, living cells containing MTT formazan crystals were solubilized in 200
μL 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.
4.2.7. Neuroprotective effect against H2O2-induced toxicity
PC12 cells were seeded at 1.2×104 cells/well in 96-well plates. After 24 h, the
medium was removed and replaced with the tested compounds (6.25, 12.5, 25 μM) at
37 °C and incubated for another 24 h. Then, the cells were exposed to H2O2 (120 μM)
and incubated at 37 °C for 24 h before assayed with MTT.41,51 Results are expressed
as percent viability compared to untreated cells. PC12 cells were cultured without test
compound or H2O2 as control group and the results were expressed by percentage of
control. Results are expressed as the mean ± SD of three independent experiments.
4.2.8. Evaluation of ROS
The level of intracellular ROS was measured by using the ROS-sensitive dye,
2’,7’-dichloro-fluorescein diacetate (DCFH-DA), as a probe.52 In brief, PC12 were
seeded in six-well plates at 30×104 cells/well, culturing in the presence or absence of
various concentrations of tested samples for additional 48 h, and then washed three
times and incubated with final concentration of 10 μM DCFH-DA for 30 min at 37 °C
in the dark. After incubation, cells were washed three times and harvested in
free-serum medium. The fluorescence of 2’,7’-dichlorofluorescein (DCF) was
detected by flow cytometry (488 nm excitation and 525 nm emission filters) using BD
Accuri C6 flow cytometer (Becton & Dickinson Company, Franklin Lakes, NJ, USA).
Data were processed by using cell quest software (Becton & Dickinson Company,
Franklin Lakes, NJ).
4.2.9. Apoptosis detection

Annexin V-FITC and propidium iodide were used to evaluate apoptotic cells by
flow cytometry.52 PC12 cells were treated with various concentrations of tested
sample for 24 h. Then the cells were washed twice with phosphate-buffered saline
(PBS) (centrifugation at 2000 rpm, 5 min). The collected cells were then resuspended
in 500 μL of binding buffer. After stained with 5 μL AnnexinV-FITC and 5 μL
propidium iodide at room temperature for 15 min. Cells were then analyzed by flow
cytometer (FACSCalibur; Becton–Dickinson, USA). Cells undergoing apoptosis are
both Annexin V positive and PI negative.
4.2.10. Neuroprotective effect against 6-OHDA-induced toxicity
PC12 cells were seeded at 1.2×104 cells/well in 96-well plates. After 24 h, the
medium was removed and replaced with the tested compounds (10, 15, 20, 25 μM) at
37 °C and incubated for another 24 h. Resveratrol was used as the control with
concentrations of 25 μM. Then, the cells were exposed to 6-OHDA (50 μM) and
incubated at 37 °C for 24 h before assayed with MTT.24,46 Results are expressed as
percent viability compared to untreated cells. PC12 cells were cultured without test
compound or 6-OHDA as control group and the results were expressed by percentage
of control. Results are expressed as the mean ± SD of three independent experiments.
4.2.11. Inhibiting NO Production of LPS-Stimulated BV2 Microglial Cells
The BV2 microglial cells were maintained in high-glucose DMEM
supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL
streptomycin in a humidified atmosphere of 5% CO2 at 37 °C. The BV2 microglial
cells were plated in 96-well microplates at a density of 3 × 104 cells/well. After the
cells had attached, the medium was removed, and the BV2 microglial cells were
incubated with the compounds and LPS (purchased from Sigma, diluted with medium
to a final concentration 1μg/ml) for 24 h.53 The same volume of Griess assay agent
was added into the 96-well plates and mixed with the culture medium. The mixture
solution was incubated at room temperature for 10 min, and the absorbance at 540nm
was measured in a multifunctional microplate reader. The percent of inhibition of NO
production was calculated by the formula: (FL − FC)/(FL − F0) × 100, where FC =