Novel 8-hydroxyquinoline derivatives targeting β-amyloid aggregation, metal chelation and oxidative stress against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.04.043

A series of multitargeted 8-hydroxyquinoline derivatives were designed and synthesized for the treatment of Alzheimer's disease (AD). In vitro studies indicated that most of the prepared compounds exhibited significant inhibitory effects against self-induced Aβ_1?42 aggregation and potential antioxidant properties especially compound 5b (IC₅₀ = 5.64 μM for self-induced Aβ aggregation; the oxygen radical absorbance capacity using fluorescein (ORAC-FL) value is 2.63 Trolox equivalents). Notably, 5b can chelate biometals and inhibit Cu²⁺/Zn²⁺-induced Aβ_1?42 aggregation. The cell assays showed that 5b had excellent protective effects against oxidative toxin H₂O₂ and presented low neurotoxicity in PC12 cells. Furthermore, 5b could penetrate the blood–brain barrier (BBB) in vitro and did not show any acute toxicity in mice at doses up to 2000 mg/kg in vivo. Our findings provide a rationale for the potential application of compound 5b as a lead compound in AD therapy.

Full text of DOI:10.1016/j.bmc.2018.04.043

Accepted Manuscript
Novel 8-hydroxyquinoline derivatives targeting β-amyloid aggregation, metal
chelation and oxidative stress against Alzheimer’s disease
Xuelian Yang, Pei Cai, Qiaohong Liu, Jiajia Wu, Yong Yin, Xiaobing Wang,
Lingyi Kong
PII:
S0968-0896(18)30575-3
https://doi.org/10.1016/j.bmc.2018.04.043
BMC 14326
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 March 2018
17 April 2018
20 April 2018
Please cite this article as: Yang, X., Cai, P., Liu, Q., Wu, J., Yin, Y., Wang, X., Kong, L., Novel 8-hydroxyquinoline
derivatives targeting β-amyloid aggregation, metal chelation and oxidative stress against Alzheimer’s disease,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.04.043
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel 8-hydroxyquinoline derivatives targeting β-amyloid aggregation, metal
chelation and oxidative stress against Alzheimer’s disease
Xuelian Yang, Pei Cai, Qiaohong Liu, Jiajia Wu, Yong Yin, Xiaobing Wang* and
Lingyi Kong*
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of
China
*
Corresponding Authors. Tel/Fax: +86-25-83271405; E-mail:cpu_lykong@126.com;
xbwang@cpu.edu.cn

Abstract
A series of multitargeted 8-hydroxyquinoline derivatives were designed and
synthesized for the treatment of Alzheimer’s disease (AD). In vitro studies indicated
that most of the prepared compounds exhibited significant inhibitory effects against
self-induced Aβ1−42 aggregation and potential antioxidant properties especially
compound 5b (IC50 = 5.64 μM for self-induced Aβ aggregation; the oxygen radical
absorbance capacity using fluorescein (ORAC-FL) value is 2.63 Trolox equivalents).
2
+
2+
Notably, 5b can chelate biometals and inhibit Cu /Zn -induced Aβ1−42 aggregation.
The cell assays showed that 5b had excellent protective effects against oxidative toxin
H
2
2
O and presented low neurotoxicity in PC12 cells. Furthermore, 5b could penetrate
the blood−brain barrier (BBB) in vitro and did not show any acute toxicity in mice at
doses up to 2000 mg/kg in vivo. Our findings provide a rationale for the potential
application of compound 5b as a lead compound in AD therapy.
Keywords: 8-Hydroxyquinoline, β-amyloid, neuroprotection, antioxidant, Alzheimer’s
disease.
1
. Introduction
Alzheimer’s disease (AD), which is characterized by cognitive decline, memory loss,
and dementia, is the most common fatal neurodegenerative disorder, and it still lacks
1
a satisfactory therapy or a preventative treatment. Current agents approved for the
treatment of AD include acetylcholinesterase inhibitors (donepezil, rivastigmine, or
galantamine) and an N-methyl-D-aspartic acid (NMDA) receptor antagonist
(
memantine). Unfortunately, these symptomatic treatments do not prevent progressive
neurodegeneration; they only provide a modest improvement in memory and
2
,3
cognitive function. Multiple factors, including β-amyloid (Aβ) deposits, tau protein
phosphorylation, neurofibrillary tangles (NFT), low levels of acetylcholine,
4-9
dyshomeostasis of biometals and oxidative stress, have been proposed to contribute
to the progression of AD, but the etiology of AD remains elusive.

Among the multiple factors that induce AD, Aβ plays a primary role in AD
1
0
pathophysiology with two main isoforms Aβ1−40 and Aβ1−42. Although Aβ1−40 is
enriched in the brain, several lines of evidence actually support Aβ1−42 is more prone
1
1
to aggregation and more neurotoxic. Aβ aggregation forms oligomers, which are
toxic to neuronal and mitochondrial functions and ultimately lead to
1
2
neurodegeneration, cognitive dysfunction, oxidative stress and neuroinflammation.
2
+
2+
2+
3+
Higher levels of metal ions such as Cu , Zn , Fe , and Fe , are found within Aβ
8
,13,14
deposits in AD brain tissue compared to the levels in normal tissue.
These
2
+
2+
metals, especially Cu and Zn , can readily bind to Aβ via His6, His13, His14 and
8
,13
Tyr10 residues, which results in the toxic aggregation of Aβ oligomers.
2
+
3+
Furthermore, redox-active metal ions such as Cu and Fe interact with Aβ to
produce reactive oxygen species (ROS) under physiological conditions by Fenton
1
3,15,16
chemistry,
thus leading to the oxidative stress and ultimately neuronal death.
Oxidative stress appears to be a major determinant of AD pathogenesis and
9
progression. Oxidative damage is marked by nucleic acids oxidation, nitration, lipids
peroxidation, and reactive carbonyls, which could promote the formation of amyloid
9
,17
plaques and neurofibrillary tangles in AD.
Given the multifactorial nature of AD,
the classic drug-discovery approach based on the “one molecule, one target” paradigm
18
might prove inadequate. Accordingly, an appropriate strategy of
1
9
multitarget-directed ligands (MTDLs), that could interact with the multiple targets
that are thought to be responsible for AD pathogenesis was proposed. There has been
2
0-22
growing interest in developing MTDLs for AD networks in recent years.
Clioquinol (CQ Figure 1), an 8-hydroxyquinoline (HQ) with moderate affinity
2
+
2+
for Cu and Zn , can inhibit metal-induced Aβ aggregation and ROS generation in
2
3, 24
vitro.
Long term usage of CQ is, however, limited by the side effect of subacute
2
5
myelo-optic neuropathy. PBT2, a second generation of CQ that shows increased
potency, was demonstrated to cause a significant improvement in cognition and
memory by redistributing metal ions and decreasing plasma Aβ levels in Phase Ⅱ
2
6, 27
clinical trials.
Recently, the HQ-based MTDLs, including M30, HLA20,

WBQ5187, and tacrine-HQ hybrids were developed. These compounds significantly
2
8-31
inhibit Aβ aggregation in vitro and improve cognition in vivo in mouse models.
These results indicated that HQ could be used as a privileged motif in the design of
multitarget-directed drugs in AD therapy. Cinnamic acid has been reported to possess
various pharmacological properties with antioxidant activity being the most important.
3
2
As such, HQ in conjugation with cinnamic acid may enhance the efficacy of AD
therapy through both antioxidation and Aβ aggregation suppression pathways.
Meanwhile, piperazines have been extensively explored due to their proven ability to
3
3, 34
enhance the physicochemical and biological properties of drugs.
In our study,
pharmacophores of HQ were connected to cinnamic acids through a piperazine ring
linker without major structural modifications (Figure 1). Seventeen compounds were
prepared and their biological activities, namely, the inhibition of Aβ1-42 aggregation,
metal-chelating properties, antioxidant activity and blood-brain barrier (BBB)
permeability, were investigated.
Figure 1. Multitarget-directed design strategy by fusing the pharmacophore moieties
of CQ and cinnamic acids through a piperazine linker.
2
. Results and discussion

2
.1. Chemistry
The synthetic route from 8-hydroxyquinoline derivatives is outlined in Scheme 1.
Commercially available 8-hydroxyquinoline was first chloromethylated to give
-chloromethyl-8-hydroxyquinoline (1). Then, 1 was reacted with
5
tertbutylpiperazine-1-carboxylate to give compound 2, which was subsequently Boc
deprotected with trifluoroacetic acid (TFA). Intermediate 3 was further reacted with
the appropriate cinnamic acids or hydrocinnamic acids, using EDCI as a coupling
reagent to give target compounds 4a-4k and 5a-5f, respectively. Structures of all
target compounds were characterized by NMR and MS.
Scheme 1. Synthesis of designed compounds 4a-4k and 5a-5f: (a) 37% formaldehyde,
3
7% hydrochloric acid, hydrogen chloride gas, rt, 8 h; (b) tertbutyl
piperazine-1-carboxylate, K CO , CH CN, rt, overnight; (c) TFA, CH
d) HOBT, EDCI, CH Cl , rt, overnight.
2
3
3
2
Cl , rt, 30 min;
2
(
2
2
2
.2. Pharmacological evaluation

2
.2.1. Inhibition of self-induced Aβ1−42 aggregation
First, the inhibitory activities of the designed compounds against self-induced
Aβ1−42 aggregation were evaluated using a thioflavin T (ThT) fluorescence assay. The
compounds (10 μM) with inhibition potencies from 32.46 to 73.41%, which are better
than that of CQ (25.72%), were selected for the further complete dose−response
studies. The results presented in Table 1 indicated that compounds 4a, 4g, 4i, and 5b
(
IC50 = 8.86, 5.05, 7.35 and 5.64 μM, respectively) showed more potent Aβ1−42
aggregation inhibition capabilities than that of the reference compound, resveratrol
(
(
IC50 = 12.43 μM). Noticeably, the strongest Aβ1-42 aggregation inhibition potency
IC50 = 5.05 μM) was provided by compound 4g, which features a
2
2
,4-difluorobenzene ring as part of the cinnamic acid. Compounds with
-chlorobenzene rings in their cinnamic acids, such as 4i and 5b, showed good
inhibitory activities against Aβ1−42 aggregation. In addition, from the inhibition values
of compounds 4e, 5f vs. 4g, 4k vs. 4i and 5c vs. 5b, it appeared that fluoro or chloro
substituents at the 2-position of the aromatic ring of the cinnamic acids (compared to
fluoro or chloro substituents at the 4-position ) were beneficial for the Aβ1-42
self-induced aggregation inhibitory activity. According to the inhibition values of
compounds 4b, 4c, 5d and 5e, methoxy substituents were detrimental to the inhibition
activity.
2
.2.2. Antioxidant activity in vitro
The elevated production of ROS has been suggested to promote the progression
9
of AD. Therefore, the antioxidant activities of the compounds were also evaluated. A
fluorescence assay was performed to determine the oxygen radical absorbance
capacity (ORAC) of these compounds, and Trolox, a Vitamin E analogue, was used as
31
the standard. Most of the compounds exhibited much better ORAC values (up to
.74 Trolox equivalents, Table 1) than both CQ and cinnamic acid. These data
3
indicated that we had a rational design that fused the pharmacophores of CQ and
cinnamic acid. Surprisingly, compounds 5a-5f, in which the olefinic bond was

reduced, displayed higher activity (1.86-3.74) than 4a-4k (0.69-1.97). For example, 4i
and its reduced analog 5b, which have the same 2-chloro-substituted benzene ring in
the cinnamic acid, showed significantly different antioxidant activities. Indeed, 5b,
with its olefin reduced, showed an improved antioxidant activity (2.63 Trolox
equivalents). The data in Table 1 showed that analogs 4a, 4e, 4g, 4h, 5b, 5c and 5f
had much better antioxidant activities than Trolox. These results indicated that
electron-withdrawing groups like fluoro, chloro and cyano substituents had substantial
impacts on the antioxidant activities. Among these compounds, 5d and 5e displayed
the antioxidant activities of 3.74 and 2.89 Trolox equivalents, respectively, which
suggested that methoxy group may play a crucial role in determination of antioxidant
activities.
Table 1. Inhibition of self-induced Aβ1-42 aggregation and oxygen radical absorbance
capacity (ORAC, Trolox equivalents) values of compounds 4a-4k and 5a-5f.
Aβ1-42
Inhibition of Aβ 1a -42
b
Compounds
ORAC-FL
aggregation (%)
IC50 (μM)
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
a
b
c
d
e
f
54.98 ± 1.32
12.34 ± 1.27
29.43 ± 1.52
13.18 ± 0.91
49.86 ± 2.31
8.65 ± 0.11
8.86 ± 0.38
1.65 ± 0.07
1.25 ± 0.07
1.08 ± 0.02
0.71 ± 0.09
1.45 ± 0.06
0.93 ± 0.03
1.57 ± 0.07
1.77 ± 0.13
0.71 ± 0.01
1.52 ± 0.03
0.69 ± 0.02
2.60 ± 0.14
2.63 ± 0.12
2.57 ± 0.18
3.74 ± 0.21
2.89 ± 0.13
2.32 ± 0.15
0.53 ± 0.04
3.28 ± 0.17
1.04 ± 0.07
c
n.t.
18.94 ±1.15
c
n.t.
11.44 ± 1.01
c
n.t.
g
h
i
73.41 ± 3.25
27.76 ± 1.16
61.91 ±1.27
26.12 ± 1.04
25.68 ± 0.23
28.15 ± 1.09
65.82 ± 3.30
15.55 ± 0.33
18.31 ± 0.47
34.30 ± 2.75
32.46 ± 1.16
25.72 ± 1.72
51.74 ± 2.33
5.05 ± 0.26
16.81 ± 1.33
7.35 ± 0.37
17.33 ± 1.34
22.55 ± 2.03
19.74 ± 1.43
5.64 ± 0.22
j
k
a
b
c
d
e
f
c
n.t.
c
n.t.
18.28 ± 1.57
20.61 ± 1.86
c
Clioquinol
n.t.
Resveratrol
Cinnamic acid
12.43 ± 2.17
c
c
n.t.
n.t.
c
c
Hydrocinnamic n.t.
n.t.
0
1
.86 ± 0.04
acid
c
c
Trolox
n.t.
n.t.

a
b
All the values were obtained at a compound concentration of 10 µM. The data are
c
expressed as µmol of Trolox equiv/µmol tested compound. n.t. means not tested. The
values are expressed as the mean ± SD of the three replicates.
2
+
2+
2
.2.3. Inhibition of Cu /Zn -induced Aβ1−42 aggregation
2
+
2+
Metal ions such as Cu and Zn can impact the conformation and hence the
8
,14
aggregation properties of Aβ.
Given their overall performance in antioxidant and
self-induced Aβ1−42 aggregation inhibition assays, 4a, 4g and 5b were selected for
further investigation into their ability to inhibit metal-induced Aβ1−42 aggregation. The
Aβ1−42 peptides were first treated with 1 equivalent of CuSO
4
or ZnCl for 2 min at
2
room temperature and then incubated with or without the testing compounds for 24 h
at 37°C. As shown in Figure 2, the fluorescence intensity of Aβ1−42 peptides treated
2
+
2+
with Cu was 173.0% and Zn 168.2% of that of Aβ1−42 alone, which indicated that
2
+
2+
Cu and Zn accelerated Aβ1−42 aggregation. In contrast, the fluorescence of Aβ1−42
2
+
2+
treated with Cu or Zn with the tested compounds dramatically decreased (4a, 69.8%
2
+
2+
inhibition of Cu -induced Aβ1−42 aggregation and 66.5% inhibition of Zn ; 4g, 62.1%
and 52.0% inhibition; 5b, 88.9% and 73.3% inhibition; CQ, 61.4% and 55.3%
inhibition). These results suggested that all selected compounds, especially 5b,
2
+
2+
showed much greater inhibitory activities in Cu - and Zn -induced Aβ1−42
aggregation than those of CQ, a well-known metal chelator.

2
+
2+
Figure 2. The inhibition of Cu -induced (A) and Zn -induced (B) Aβ1-42 aggregation
in the presence of compounds 4a, 4g and 5b. ([Aβ1-42 = 25 μM, [compounds] = 50
2
+
2+
μM, [CQ] = 50 μM, [Cu ] = 25 μM, [Zn ] = 25 μM). Top: Scheme for the inhibition
experiments. Bottom: The fluorescence intensity of the ThT binding assay. The data
**
shown are presented as the mean ± SD of three independent experiments. P < 0.01,
***
###
P < 0.001, P < 0.001, versus Aβ1-42 alone.
2
.2.4. Metal-chelating properties of 5b
2
+
2+
2+
The ability of the HQ derivatives to chelate biometals such as Cu , Zn , Fe ,
3
+
and Fe , was next investigated using UV-vis and fluorescence spectroscopy.
Compound 5b was selected as representative compounds for its balance activities
discussed above. As shown in Figure 3A, after 1 equivalent of either CuSO
FeSO was added to a solution of 5b, the maximum absorption wavelength shifted
from 241 to 261, 260 and 265 nm, respectively, and the absorbance dropped
dramatically. Meanwhile, the absorption band at 311 nm disappeared. These changes
4
, ZnCl or
2
4
2
+
2+
2+
are indicative of the formation of 5b−Cu , 5b−Zn and 5b-Fe complexes, while
3
+
Fe showed very little chelation to 5b. We also collected fluorescence spectra when
different metal ions interacted with 5b (Figure 3B). The specific fluorescence
emission peak of 5b can be observed at 269 nm (λex = 245 nm), and it was the least
2
+
2+
intense emission peak. After Cu and Zn were added to the HEPES solution of 5b,
the fluorescence intensity of the emission peaks dramatically increased. The spectra of
2
+
3+
5
b-Fe and 5b-Fe showed moderate increases in their fluorescence intensity.
Overall, the UV-vis and fluorescence studies both indicated that all test metals,
2
+
2+
especially Zn and Cu , can chelate with compound 5b. Hence, the stoichiometries
2
+
2+
35
of the 5b-Cu and 5b-Zn complexes were determined using Job’s method. The
isosbestic point shown in Figure 3C and 3D at 0.70 and 0.66 implied stoichiometries
2
+
2+
of 2.3:1 for the 5b-Cu complex and 2:1 for the 5b-Zn complex.

Figure 3. (A) UV absorbance spectra of 5b (50 μM) alone or in the presence of
CuSO , ZnCl , FeSO , or FeCl (1 equivalent) in buffer (20 mM of HEPES, 150 mM
4
2
4
3
of NaCl, pH 7.4). (B) Fluorescence spectra of 5b (50 μM) with CuSO
or FeCl (1 equivalent) in buffer (20 mM of HEPES, 150 mM of NaCl, pH 7.4). (C)
Determination of the stoichiometry of the 5b-Cu complex by Job’s method. (D)
4
, ZnCl
2
, FeSO ,
4
3
2
+
2
+
Determination of the stoichiometry of the 5b-Zn complex by Job’s method.
2
.2.5. Cytotoxicity and protective effect of 5b against oxidative injuries on PC12 cells
9
As oxidative stress is a key factor in AD onset and progression, successfully
protecting neurons from oxidative damage will potentially prevent the disease. In our
research, we successfully established a rat adrenal pheochromocytoma cell line PC12
36
model with oxidative damage using H
2
O (a radical initiator), rotenone and
2
3
7
oligomycin A (mitochondrial poisons) as stimuli, which mimics arresting
intracellular oxidative stress and neurotoxicity. Compound 5b was first investigated
for cytotoxicity against PC12 cells using the MTT assay. No significant neurotoxicity
was detected against the PC12 cells when they were treated with 5b at a concentration

of 20 μM for 24 h (83% cell viability compared to that of the control). The
neuroprotection effect of 5b was next studied (Figure 4). Intriguingly, compound 5b
showed much stronger antioxidant activities than Trolox against H
2
2
O -induced
oxidative stress in a significantly dose-dependent manner (Figure 4A), whereas 5b
showed relatively poor protective effects against rotenone and oligomycin A-induced
oxidative stress at the same concentrations (1, 2, 5 and 10 μM, Figure 4B). These
results revealed that 5b has interesting protective effects against oxidative stress in
PC12 cells.
Figure 4. (A) The viability of PC12 cells was determined by the MTT assay after 24
h of incubation with 5b at different concentrations (1, 2, 5 and 10 μM) with H
2
2
O (120
μM). Trolox and CQ was used as the reference compounds. Data are expressed as the
percentages of viable cells (compared to the control) and are shown as the mean ± SD
(
n = 3). (B) The PC12 cell viability was determined by the MTT assay after 24 h of
incubation with 5b at different concentrations (1, 2, 5 and 10 μM) with rotenone (10

μM) and oligomycin A (25 μM). Trolox and CQ was used as the reference compounds.
Data are expressed as the percentages of viable cells (compared to the control) and are
shown as the mean ± SD (n = 3). Untreated cells were used as the control. The values
are presented as the mean ± SD of three independent experiments. The data were
*
***
###
analyzed by one-way ANOVA: P < 0.05, P < 0.001, versus stimuli; P < 0.001,
versus control.
2
.2.6. Effects of compound 5b on H
2
2
O -induced intracellular ROS accumulation
Further studies of the protective effects of 5b against oxidative injury of PC12
cells were conducted with a cellular antioxidant assay based on dichlorofluorescein
diacetate (DCFH-DA). When PC12 cells were exposed to 150 μM of H for 24 h,
2
O
2
the intracellular ROS production increased substantially (Figure 5A). However, when
the cells were pretreated with increasing concentration of 5b (2.5, 5, and 10 μM), the
ROS levels decreased in a concentration-dependent manner. This suggested that 5b
inhibited H
2
2
O -induced intracellular ROS accumulation.
2
.2.7. Effects of compound 5b on H
2
2
O -induced apoptosis in PC12 cells
To prove whether compound 5b could protect cells from H
2
2
O -induced apoptosis,
an Annexin V-FITC/PI double staining experiment was performed to analyze the
apoptotic cells detected by flow cytometry. The Annexin V-FITC (-)/PI (-) population
consisted primarily of normal cells, while Annexin V-FITC (+)/PI (-) cells were
considered to be in the early stage of apoptosis, the Annexin V-FITC (+)/PI (+) cells
were considered to be in the late apoptosis stage and the Annexin V-FITC (-)/PI (+)
cells were those considered to be the necrosis. We observed a notable increase in
necrosis and late phase apoptosis in PC12 cells treated with H
B). In contrast, the rates of necrosis and apoptotic cells were significantly reduced
when H -treated PC12 cells were pretreated with compound 5b (2.5, 5, and 10 μM).
2
O
2
for 24 h (Figure
5
2
O
2
These observations suggested that 5b exerts its protective effects through the blocking
of programmed cell death.

Figure 5. (A) Cells were incubated with the indicated concentrations of compound 5b
2.5, 5 and 10 µM) followed by stimulation with H (120 μM). DCFH-DA staining
(
2
O
2
was then performed to determine the intracellular ROS levels by flow cytometry. The
results of the ROS production analysis are presented as the mean fluorescence
intensity (MFI). (B) The PC12 cells were treated as described above. The apoptotic
cells were detected by flow cytometry after Annexin V-FITC and PI double staining.
The values are presented as the mean ± SD of three independent experiments. The
*
***
###
data were analyzed by one-way ANOVA: P < 0.05, P < 0.001, versus H
2
O
2
;
P <
0
.001, versus control.
2
.2.8. In vitro blood−brain barrier permeation assay
Blood−brain barrier (BBB) permeability is essential for successful central
nervous system (CNS) drugs. To measure the in vitro BBB permeability of these
derivatives, a parallel artificial membrane permeation assay of the blood−brain barrier

3
8
(
PAMPA-BBB) as described by Di et al. was performed. First, we compared the
permeability of 8 commercial drugs with their reported values to validate the assay
(
Table 2). A plot of the experimental data versus the reported values showed good
2
linear correlation, Pe (exp) = 0.848Pe (bibl.) + 0.228 (R = 0.983) (Figure S1,).
According to this equation and considering the limit established by Di et al. for BBB
−6
permeation, we concluded that compounds with Pe > 3.62 × 10 cm/s can be
considered to show high BBB permeation (CNS+), compounds with Pe < 1.92 × 10
cm/s can be considered to show low BBB permeation (CNS-), and compounds with
−
6
−
6
−6
3
.62 × 10 cm/s > Pe > 1.92 × 10 cm/s show uncertain BBB permeation (CNS±).
−
6
Based on the measured permeability (Pe = (11.05 ± 0.97) × 10 cm/s), 5b could cross
−
6
the BBB with Pe values higher than 3.62 × 10 cm/s.
-6
-1
Table 2. Permeability results (Pe × 10 cm s ) from the PAMPA-BBB assay for
selected compounds with their predicted penetration into the CNS.
Commercial
drugs
Permeability
a
b
Bibliography
Experiment
Compounds
Prediction
b
(× 10-6 cm s-1)
Testosterone
Verapamil
17
16
12
5.3
15.76 ± 0.89 4c
13.44 ± 1.05 4g
8.82 ± 0.44
18.58 ± 1.12
6.83 ± 0.25
11.05 ± 0.97
9.67 ± 0.51
CNS+
CNS+
CNS+
CNS+
CNS+
β-Estradiol
9.17 ± 0.84
5.00 ± 0.37
4.76 ± 0.43
1.59 ± 0.11
1.97± 0.12
1.80 ± 0.11
4i
Clonidine
5b
5d
Corticosteron
5
1
1
.1
.1
.9
Lomefloxaci
Hydrocortiso
Piroxicam
2.5
a
Taken from Ref.
b
Data are the mean ± SD of three independent experiments.
.2.9. Acute toxicity
2
To investigate the safety profile of the derivatives, the acute toxicity of 5b was
determined in KM mice at doses of 0, 677, 1333, and 2000 mg/kg (n = 5 per group)
by oral administration. At 4 h post-administration, all mice remained alive and

appeared healthy based on criteria related to fur sleekness, water and food
consumption, and body weight (Figure S2). At 14 days after drug administration, all
animals were sacrificed and macroscopically examined, and no damage to the heart,
liver, or kidney was found. The results indicated that the animals treated with
compound 5b did not develop any symptoms of acute toxicity or show mortality
immediately after administration or during the post-treatment period.
3
. Conclusion
In summary, a series of 8-hydroxyquinoline derivatives were designed and
synthesized as multitargeted anti-Alzheimer agents. Among the synthesized
compounds, 5b exerts significant metal-chelating ability, inhibits the formation of Aβ
2
+
2+
fibrils generated by self- and Cu - or Zn -induced Aβ aggregation and shows
antioxidant activity and protective effects with low cytotoxicity in PC12 cells.
Notably, 5b can cross the BBB in vitro and did not exhibit acute toxicity in mice at
doses up to 2000 mg/kg. These properties highlight the potential of compound 5b as a
promising lead compound in the treatment of Alzheimer’s disease.
4
. Experimental section
4
.1. Chemistry
All common reagents and solvents were obtained from commercial suppliers and
used without purification. Reaction progress was monitored using analytical thin layer
chromatography (TLC) on precoated silica gel GF254 plates (Qingdao Haiyang
Chemical Plant, Qing-Dao, China), and the spots were detected under UV light (254
1
nm). Melting points were determined on an XT-4 micromelting point instrument. H
1
3
NMR and C NMR spectra were measured on Bruker AV-600/500 spectrometer (600
1
13
MHz for H NMR and 126 MHz for C NMR, Bruker, Karlsruhe, Germany) and
referenced to TMS. Chemical shifts are reported in ppm (δ) using the residual solvent
line as internal standard. Mass spectra were obtained on a MS Agilent 1100 Series
LC/MSD Trap mass spectrometer (ESI-MS) and an Agilent 6520B Q-TOF

spectrometer (HRESIMS), respectively. HPLC purity analysis was performed using
an Agilent 1200 series instrument, equipped with binary pump, photodiode array
detector (DAD). Agilent Zorbax Exlipse SB-C18 (250×4.6 mm, 5 µm particle size)
analytical column was used.
4
.1.1. General procedure for the preparation of 5-chloromethyl-8-HQ hydrochloride
Into a solution of 8-hydroxyquinoline (HQ, 3.65g) and formaldehyde (4 mL,
7%) in 32% hydrochloric acid (4 mL) was blown a stream of hydrogen chloride gas
3
39
for 6 hours at room temperature. After filtration, the product was crystallized with
ethanol to afford 1 in 80% yield.
4
.1.2. General procedures for the preparation of compound 4a-4k and 5a-5f
Compounds 4a-4k and 5a-5f were prepared as shown in Scheme 1. The common
intermediate 3 was easily synthesized in two steps by acylation of the commercially
available tertbutyl piperazine-1-carboxylate with 8-HQ under basic conditions, which
4
0
was subsequently Boc deprotected with TFA. Then, to solutions of cinnamic acids
1 equivalent) in DCM was added HOBT (1.1 equivalent) and EDCI (1.1 equivalent).
(
After the mixture was stirred at 0 °C for 1 h, the intermediate 3 (1 equivalent) was
added. The reaction mixture was slowly warmed to room temperature and stirred
overnight. The mixture was washed twice with saturated NaHCO , dried over sodium
3
sulfate and concentrated to afford the crude product, which was purified by silica gel
column chromatography (DCM/MeOH as eluent) to provide the target products.
4
.1.2.1. (E)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl)-3-phenylprop-2-en-1-one (4a). The reaction was carried out according
to the general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3)
(
97.32mg, 0.4mmol) and cinnamic acid (59.26mg, 0.4mmol). The title compound was
1
obtained as light green solid (91mg, 61%). m.p. 153-155 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.5 Hz, 1H), 8.68 (dd, J = 8.6, 1.5 Hz,
H), 7.72 – 7.68 (m, 2H), 7.59 (dd, J = 8.5, 4.1 Hz, 1H), 7.48 (d, J = 15.4 Hz, 1H),
.41 – 7.36 (m, 3H), 7.34 (d, J = 7.7 Hz, 1H), 7.24 (d, J = 15.4 Hz, 1H), 7.01 (d, J =
1
7
7
1
3
.7 Hz, 1H), 3.80 (s, 2H), 3.65 (s, 2H), 3.51 (s, 2H), 2.42 (d, J = 24.0 Hz, 4H). C

NMR (126 MHz, DMSO-d
6
) δ 164.4, 153.0, 147.9, 141.6, 138.9, 135.2, 133.9, 129.6,
1
29.1, 128.8, 128.1, 127.9, 123.8, 121.6, 118.2, 110.0, 59.4, 53.3, 52.5, 45.2, 41.8.
+
+
ESI-MS m/z: 374.1 [M + H] ; HRMS: calcd for C23
H
23
N
3
NaO [M + Na] : 396.1682.
2
found: 396.1685. HPLC purity (MeOH / H O / 0.1% FA): 97.6%, 6.6 min.
2
4
.1.2.2. (E)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
3-(4-methoxyphenyl) prop-2-en-1-one (4b). The reaction was carried out according to
the general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3)
-
(
97.32mg, 0.4mmol) and 4-methoxycinnamic acid (71.27mg, 0.4mmol). The title
1
compound was obtained as yellow solid (101mg, 63%). m.p. 171-173 °C; H NMR
(
600 MHz, DMSO-d
.5 Hz, 1H), 7.65 (d, J = 8.8 Hz, 2H), 7.59 (dd, J = 8.6, 4.1 Hz, 1H), 7.44 (d, J = 15.3
Hz, 1H), 7.34 (d, J = 7.7 Hz, 1H), 7.09 (d, J = 15.3 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H),
.95 (d, J = 8.8 Hz, 2H), 3.80 (s, 2H), 3.78 (s, 3H), 3.65 (s, 2H), 3.50 (s, 2H), 2.41 (d,
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.5 Hz, 1H), 8.68 (dd, J = 8.6,
1
6
1
3
J = 22.9 Hz, 4H). C NMR (126 MHz, DMSO-d
6
) δ 164.8, 160.5, 153.0, 148.0, 141.6,
1
5
39.0, 133.9, 129.8, 129.2, 128.0, 127.8, 123.9, 121.7, 115.5, 114.3, 110.1, 59.4, 55.4,
+
3.4, 52.6, 45.2, 41.9. ESI-MS m/z: 404.6 [M + H] ; HRMS: calcd for C24
H
25
N
3
NaO
3
+
[
M + Na] : 427.1788. found : 426.1790. HPLC purity (MeOH / H
2
O / 0.1% FA):
9
9.1%, 6.6 min.
4
.1.2.3. (E)-3-(3,4-dimethoxyphenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl) prop-2-en-1-one (4c) The reaction was carried out according to the
general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg,
0
.4mmol) and 3,4-dimethoxycinnamic acid (83.29mg, 0.4mmol). The title compound
1
was obtained as light green solid (106mg, 61%). m.p. 148-149 °C; H NMR (600
MHz, DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.2, 1.6 Hz, 1H), 8.68 (dd, J = 8.6, 1.6
Hz, 1H), 7.59 (d, J = 8.6, 4.1 Hz, 1H), 7.43 (d, J = 15.2 Hz, 1H), 7.37-7.32 (m, 2H),
7
6
2
1
1
.19 (dd, J = 8.3, 2.0 Hz, 1H), 7.10 (d, J = 15.2 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H),
.95 (d, J = 8.3 Hz, 1H), 3.81(s, 2H) 3.81 (s, 3H), 3.78 (s, 3H), 3.66 (s, 2H), 3.51 (s,
1
3
H), 2.42 (d, J = 31.8 Hz, 4H). C NMR (126 MHz, DMSO-d ) δ 164.6, 153.0, 150.2,
6
48.9, 147.8, 141.9, 138.9, 133.8, 129.1, 128.0, 127.9, 123.8, 122.4, 121.5, 115.5,
11.5, 110.3, 110.0, 59.3, 55.7, 55.5, 53.4, 52.5, 45.0, 41.7. ESI-MS m/z: 434.2 [M +
+
+
H] ; HRMS: calcd for C25
H
28
N
3
O . [M + H] : 434.2074. found: 434.2071. HPLC
4
purity (MeOH / H O / 0.1% FA): 92.4%, 4.8 min.
2

4
.1.2.4. (E)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl)-3-(4-prop-2-en-nitrophenyl)1-one (4d). The reaction was carried out
according to the general procedure, starting from 5-(piperazin-1-ylmethyl)
quinolin-8-ol (3) (97.32mg, 0.4mmol) and 4-Nitrocinnamic acid (77.26mg, 0.4mmol).
The title compound was obtained as pale yellow solid (100mg, 60%). m.p.
1
1
1
2
89-190 °C; H NMR (600 MHz, DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.6 Hz,
H), 8.68 (dd, J = 8.6, 1.6 Hz, 1H), 8.23 (d, J = 8.8 Hz, 2H), 8.00 (d, J = 8.8 Hz,
H),7.61 – 7.59 (m, 1H), 7.57 (d, J = 15.5 Hz, 1H), 7.49 (d, J = 15.5 Hz, 1H), 7.35 (d,
J = 7.7 Hz, 1H), 7.01 (d, J = 7.7 Hz, 1H), 3.81 (s, 2H), 3.68 (s, 2H), 3.53 (s, 2H), 2.46
13
(
t, J = 5.1 Hz, 2H), 2.41 (t, J = 5.1 Hz, 2H). C NMR (126 MHz, DMSO-d
6
) δ 163.6,
1
1
53.0, 147.8, 147.5, 141.8, 139.0, 138.9, 133.8, 129.1, 129.0, 127.9, 123.9, 123.7,
+
22.8, 121.5, 110.0, 59.3, 53.3, 52.3, 45.2, 41.8. ESI-MS m/z: 419.2 [M + H] ; HRMS:
+
calcd for C23
H
23
N
4
O
4
. [M +H] : 419.1714. found: 419.1715. HPLC purity (MeOH /
2
H O / 0.1% FA): 90.2%, 4.9 min.
4
.1.2.5. (E)-3-(4-fluorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
prop-2-en-1-one (4e). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
4
-fluorocinnamic acid (66.46mg, 0.4mmol). The title compound was obtained as light
1
green solid (105mg, 67%). m.p. 154-156 °C; H NMR (600 MHz, DMSO-d
6
) δ 9.75
(
s, 1H), 8.86 (dd, J = 4.1, 1.5 Hz, 1H), 8.68 (dd, J = 8.6, 1.5 Hz, 1H), 7.78 (m, 2H),
7
7
2
1
1
.59 (dd, J = 8.6, 4.1 Hz, 1H), 7.48 (d, J = 15.4 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H),
.24 (m, 2H), 7.21 (d, J = 15.4 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 3.80 (s, 2H), 3.65 (s,
1
3
H), 3.51 (s, 2H), 2.41 (d, J = 26.6 Hz, 4H). C NMR (126 MHz, DMSO-d ) δ 164.3,
6
62.0, 153.0, 147.9, 140.4, 138.9, 133.9, 131.8, 130.3, 129.1, 127.9, 123.8, 121.6,
+
18.1, 115.7, 110.0, 59.3, 53.3, 52.4, 45.1, 41.8. ESI-MS m/z: 392.1 [M + H] ;
+
HRMS: calcd for C23
purity (MeOH / H
H
22FN
3
NaO
2
. [M + Na] : 414.1588. found: 414.1587. HPLC
2
O / 0.1% FA): 100%, 4.2 min.
4
.1.2.6. (E)-3-(4-bromophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
prop-2-en-1-one (4f). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
4
-bromocinnamic acid (90.82mg, 0.4mmol). The title compound was obtained as pale

1
yellow solid (121mg, 67%). m.p. 189-190 °C; H NMR (600 MHz, DMSO-d
6
) δ 9.75
(
s, 1H), 8.86 (dd, J = 4.1, 1.5 Hz, 1H), 8.68 (dd, J = 8.6, 1.5 Hz, 1H), 7.70 (d, J =
8
7
2
1
1
.5 Hz, 2H), 7.63 – 7.58 (m, 3H), 7.46 (d, J = 15.4 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H),
.31 (d, J = 15.4 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 3.82 (s, 2H), 3.67 (s, 2H), 3.53 (s,
1
3
H), 2.44 (d, J = 20.0 Hz, 4H). C NMR (126 MHz, DMSO-d ) δ 164.2, 153.0, 147.9,
6
40.3, 138.9, 134.5, 133.8, 131.7, 130.1, 129.1, 127.9, 123.8, 122.8, 121.5, 119.2,
+
10.0, 59.3, 53.3, 52.4, 45.1, 41.8. ESI-MS m/z: 452.1 [M + H] ; HRMS: calcd for
+
C
23
H22BrN
3
NaO
2
. [M +Na] : 474.0788. found: 474.0787. HPLC purity (MeOH / H
2
O
/
0.1% FA): 93.2%, 5.1 min.
4
.1.2.7 (E)-3-(2,4-difluorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
prop-2-piperazin-1-yl)en-1-one (4g). The reaction was carried out according to the
general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg,
0
.4mmol) and 2,4-difluorocinnamic acid (73.66mg, 0.4mmol). The title compound
1
was obtained as light brown solid (111mg, 68%). m.p. 186-187 °C; H NMR (600
MHz, DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.5Hz, 1H), 8.68 (dd, J = 8.6,
.5Hz, 1H), 7.59 (dd, J = 8.6, 4.1 Hz, 1H), 7.54 (d, J = 15.5 Hz, 1H), 7.36-7.30 (m,
H), 7.28 (d, J = 15.5 Hz, 1H), 7.17 m, 1H), 7.00 (d, J = 7.7 Hz, 1H), 3.80 (s, 2H),
1
2
3
1
3
.64 (s, 2H), 3.52 (s, 2H), 2.42 (d, J = 22.6 Hz, 4H). C NMR (126 MHz, DMSO-d )
6
δ164.0, 153.0, 147.9, 138.9, 133.9, 132.0, 130.1, 129.2, 128.0, 123.8, 121.6, 120.4,
1
4
4
19.6, 112.4, 112.2, 110.7, 110.1, 104.5, 59.3, 53.3, 52.4, 45.2, 41.9. ESI-MS m/z:
+
+
10.1 [M + H] ; HRMS: calcd for C23
32.1496. HPLC purity (MeOH / H
H
21
F
2
N
3
NaO
2
[M +Na] : 432.1494. found:
2
O / 0.1% FA): 92.3%, 3.4 min.
4
.1.2.8. (E)-4-(3-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl)-3-oxoprop-1-en-1-yl) benzonitrile (4h). The reaction was carried out
according to the general procedure, starting from 5-(piperazin-1-ylmethyl)
quinolin-8-ol (3) (97.32mg, 0.4mmol) and 4-cyanocinnamic acid (69.27mg, 0.4mmol).
The title compound was obtained as light brown solid (100mg, 63%). m.p.
1
1
1
7
7
07-108 °C; H NMR (600 MHz, DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.5Hz,
H), 8.68 (dd, J = 8.6, 1.5Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H),
.59 (dd, J = 8.6, 4.1 Hz, 1H), 7.52 (d, J = 15.4 Hz, 1H), 7.43 (d, J = 15.4 Hz, 1H),
.35 (d, J = 7.8 Hz,, 1H), 7.00 (d, J = 7.8 Hz, 1H), 3.81 (s, 2H), 3.67 (s, 2H), 3.52 (s,

1
3
2
1
1
H), 2.43 (d, J = 28.3 Hz, 4H). C NMR (126 MHz, DMSO-d ) δ163.9, 153.0,
6
47.9, 139.8, 139.6, 138.9, 133.9, 132.7, 129.1, 128.8, 128.0, 123.8, 122.0, 121.6,
+
18.8, 111.5, 110.0, 59.3, 53.3, 52.4, 45.2, 41.9. ESI-MS m/z: 399.1 [M+H] ; HRMS:
+
calcd for C24
H
23
N
4
O
2
[M + H] : 399.1816. found: 399.1814. HPLC purity (MeOH /
H
2
O / 0.1% FA): 97.8%, 4.9 min.
4
.1.2.9. (E)-3-(2-chlorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
prop-2-en-1-one (4i). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
2
-chlorocinnamic acid (73.04mg, 0.4mmol). The title compound was obtained as light
1
brown solid (98mg, 60%). m.p. 187-188 °C; H NMR (600 MHz, DMSO-d
6
) δ 9.74 (s,
1
7
7
1
H), 8.86 (dd, J = 4.1, 1.7 Hz, 1H), 8.68 (dd, J = 8.5, 1.7 Hz, 1H), 8.02 – 7.96 (m, 1H),
.80 (d, J = 15.4 Hz, 1H), 7.59 (dd, J = 8.5, 4.1 Hz, 1H), 7.53 – 7.48 (m, 1H), 7.40 –
.37 (m, 2H), 7.34 (d, J = 7.7 Hz, 1H), 7.31 (d, J = 15.4 Hz, 1H), 7.00 (d, J = 7.7 Hz,
1
3
H), 3.81 (s, 2H), 3.65 (s, 2H), 3.52 (s, 2H), 2.42 (d, J = 19.0 Hz, 4H). C NMR (126
) δ 163.8, 153.0, 147.9, 138.9, 136.5, 133.8, 133.4, 132.8, 131.0,
29.8, 129.1, 128.2, 127.9, 127.5, 123.7, 121.5, 121.3, 110.0, 59.3, 53.2, 52.4, 45.1,
MHz, DMSO-d
6
1
4
4
+
+
1.8. ESI-MS m/z: 408.1 [M + H] ; HRMS: calcd for C23
H23ClN
3
O
2
. [M + H] :
08.1473. found: 408.1471. HPLC purity (MeOH / H O / 0.1% FA): 98.4%, 5.1 min.
2
4
.1.2.10. (E)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)-3-(o-tolyl)
prop-2-en-1-one (4j). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
2
-methylcinnamic acid (64.88mg, 0.4mmol). The title compound was obtained as
1
light brown solid (94mg, 61%). m.p. 145-147 °C; H NMR (600 MHz, DMSO-d
6
) δ
9
.75 (s, 1H), 8.86 (dd, J = 4.2, 1.6 Hz, 1H), 8.68 (dd, J = 8.6, 1.6 Hz, 1H), 7.78 – 7.74
(
m, 1H), 7.72 (d, J = 15.2 Hz, 1H), 7.59 (dd, J = 8.5, 4.2 Hz, 1H), 7.34 (d, J = 7.7 Hz,
1
2
H), 7.28 – 7.18 (m, 3H), 7.11 (d, J = 15.2 Hz, 1H), 7.00 (d, J = 7.7 Hz, 1H), 3.81 (s,
1
3
H), 3.64 (s, 2H), 3.52 (s, 2H), 2.42 (d, J = 14.1 Hz, 4H), 2.36 (s, 3H). C NMR (126
) δ 164.3, 153.0, 147.8, 138.9, 138.7, 136.7, 133.9, 133.8, 130.6,
29.3, 129.1, 128.0, 126.5, 126.2, 123.8, 121.5, 119.2, 110.0. 59.3, 53.2, 52.4, 45.1,
MHz, DMSO-d
6
1

+
+
4
3
1.7, 19.4. ESI-MS m/z: 388.2 [M+H] ; HRMS: calcd for C24
H
26
N
3
O
2
[M + H] :
88.2020. found: 388.2022. HPLC purity (MeOH / H O / 0.1% FA): 91.5%, 4.8 min.
2
4
.1.2.11. (E)-3-(4-chlorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl) prop-2-en-1-one (4k). The reaction was carried out according to the
general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg,
0
.4mmol) and 4-chlorocinnamic acid (73.04mg, 0.4mmol). The title compound was
1
obtained as white solid (119mg, 73%). m.p. 156-158 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.75 (s, 1H), 8.86 (dd, J = 4.1, 1.6 Hz, 1H), 8.68 (dd, J = 8.6, 1.6 Hz,
H), 7.75 (d, J = 8.6, 2H), 7.59 (dd, J = 8.6, 4.1 Hz, 1H), 7.46 (d, J = 15.4 Hz, 1H),
.45 (d, J = 8.6, 2H),7.34 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 15.4 Hz, 1H), 7.00 (d, J =
1
7
7
1
3
.7 Hz, 1H), 3.81 (s, 2H), 3.65 (s, 2H), 3.51 (s, 2H), 2.42 (d, J = 27.9 Hz, 4H). C
) δ 164.1, 153.0, 147.8, 140.1, 138.9, 134.1, 133.9, 133.8,
29.8, 129.1, 128.8, 127.9, 123.7, 121.5, 119.1, 110.0. 59.3, 53.3, 52.4, 45.1, 41.8.
NMR (126 MHz, DMSO-d
6
1
+
+
ESI-MS m/z: 408.1 [M + H] ; HRMS: calcd for C23
H23ClN
3
O . [M + H] : 408.1473.
2
found: 408.1472. HPLC purity (MeOH / H O / 0.1% FA): 97.8%, 5.3 min.
2
4
.1.2.12. 1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl)-3-phenylpropan-1-one (5a). The reaction was carried out according to
the general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3)
(
97.32mg, 0.4mmol) and 3-phenylpropionic acid (60.07mg, 0.4mmol). The title
1
compound was obtained as light green solid (93mg, 62%). m.p. 98-99 °C; H NMR
(
600 MHz, DMSO-d
6
) δ 9.73 (s, 1H), 8.85 (dd, J = 4.1, 1.5 Hz, 1H), 8.64 (dd, J = 8.5,
1
2
3
.5 Hz, 1H), 7.58 (dd, J = 8.5, 4.1 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.28 – 7.24 (m,
H), 7.22 (m, 2H), 7.16 (m, 1H), 6.99 (d, J = 7.8 Hz, 1H), 3.75 (s, 2H), 3.39 (s, 2H),
.34 (s, 2H), 2.79 (t, J = 7.7 Hz, 2H), 2.58 (t, J = 7.7 Hz, 2H), 2.34 – 2.27 (m, 4H).
1
3
C NMR (126 MHz, DMSO-d
6
) δ 169.8, 152.9, 147.8, 141.4, 138.9, 133.8, 129.0,
1
28.4, 128.3, 127.9, 125.9, 123.8, 121.5, 109.9, 59.4, 52.7, 52.4, 45.0, 41.2, 33.9, 30.8.
+
+
ESI-MS m/z: 376.1 [M+H] ; HRMS: calcd for C23
H
25
N
3
NaO . [M+Na] : 398.1839.
2
found: 398.1837. HPLC purity (MeOH / H O / 0.1% FA): 98.3%, 5.6 min.
2

4
.1.2.13. 3-(2-chlorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
propan-1-one (5b). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
3
-(2-chlorophenyl) propionic acid (73.85mg, 0.4mmol). The title compound was
1
obtained as light brown solid (98mg, 60%). m.p. 72-73 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.73 (s, 1H), 8.85 (dd, J = 4.1, 1.6 Hz, 1H), 8.65 (dd, J = 8.5, 1.6 Hz,
1
1
3
2
1
1
H), 7.58 (dd, J = 8.5, 4.1 Hz, 1H), 7.40 (dd, J = 7.4, 1.4 Hz, 1H), 7.36 (dd, J = 7.4,
.4 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.28 – 7.20 (m, 2H), 6.99 (d, J = 7.8 Hz, 1H),
.76 (s, 2H), 3.40 (s, 2H), 3.36 (s, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.58 (t, J = 7.7 Hz,
1
3
H), 2.34 – 2.29 (m, 4H). C NMR (126 MHz, DMSO-d ) δ 169.4, 152.8, 147.8,
6
38.8, 138.6, 133.7, 132.9, 130.8, 129.1, 128.9, 127.9, 127.8, 127.2, 123.7, 121.4,
+
09.9, 59.3, 52.7, 52.3, 44.9, 41.2, 32.1, 28.6. ESI-MS m/z: 410.1 [M + H] ; HRMS:
+
calcd for C23
H25ClN
3
O
2
. [M + H] : 410.1630. found: 410.1633. HPLC purity (MeOH
/
H
2
O / 0.1% FA): 98.8%, 4.9 min.
4
.1.2.14. 3-(4-chlorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
propan-1-one (5c). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
3
-(4-chlorophenyl) propionic acid (73.85mg, 0.4mmol). The title compound was
1
obtained as white solid (108mg, 66%). m.p. 145-146 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.74 (s, 1H), 8.85 (dd, J = 4.1, 1.6 Hz, 1H), 8.64 (dd, J = 8.6, 1.6 Hz,
H), 7.58 (dd, J = 8.6, 4.1 Hz, 1H), 7.31 (m, 3H), 7.25 (m, 2H), 6.99 (d, J = 7.7 Hz,
H), 3.75 (s, 2H), 3.38 (s, 2H), 3.34 (s, 2H), 2.78 (t, J = 7.6 Hz, 2H), 2.58 (t, J = 7.6
1
1
13
Hz, 2H), 2.30 (d, J = 17.9 Hz, 4H). C NMR (126 MHz, DMSO-d
6
) δ 169.6, 152.9,
1
5
47.8, 140.4, 138.8, 133.7, 130.5, 130.4, 128.9, 128.1, 127.8, 123.7, 121.5, 109.9,
+
9.4, 52.7, 52.4, 44.9, 41.1, 33.6, 30.0. ESI-MS m/z: 410.1 [M + H] ; HRMS: calcd
+
for C23
H24ClN
3
NaO
2
. [M +Na] : 432.1449. found: 432.1448. HPLC purity (MeOH /
H
2
O / 0.1% FA): 98.8%, 5.1 min.
4
.1.2.15. 3-(3,4-dimethoxyphenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl)
piperazin-1-yl) propan-1-one (5d). The reaction was carried out according to the
general procedure, starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg,
0
.4mmol) and 3-(3,4-dimethoxyphenyl) propanoic acid (84.09mg, 0.4mmol). The title
1
compound was obtained as light brown solid (109mg, 63%). m.p. 96-97 °C; H NMR

(
600 MHz, DMSO-d
.6 Hz, 1H), 7.58 (dd, J = 8.6, 4.1 Hz, 1H), 7.31 (d, J = 7.7 Hz, 1H), 6.99 (d, J = 7.7
Hz, 1H), 6.85 – 6.79 (m, 2H), 6.71 (dd, J = 8.2, 2.0 Hz, 1H), 3.75 (s, 2H), 3.72 (s, 3H),
6
) δ 9.73 (s, 1H), 8.85 (dd, J = 4.1, 1.6 Hz, 1H), 8.64 (dd, J = 8.6,
1
3
2
1
1
.70 (s, 3H), 3.39 (s, 2H), 3.34 (s, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.55 (t, J = 7.6 Hz,
1
3
H), 2.29 (d, J = 25.8 Hz, 4H). C NMR (126 MHz, DMSO-d ) δ 169.9, 152.9, 148.6,
6
47.8, 147.1, 138.9, 133.8, 133.8, 129.0, 127.9, 123.8, 121.5, 120.2, 112.5, 111.9,
10.0, 59.4, 55.6, 55.4, 52.8, 52.5, 45.0, 41.1, 34.3, 30.6. ESI-MS m/z: 436.1 [M +
+
+
H] ; HRMS: calcd for C25
H
29
N
3
NaO . [M +Na] : 458.2050. found: 458.2053. HPLC
4
purity (MeOH / H O / 0.1% FA): 98.3%, 3.3 min.
2
4
.1.2.16. 1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)-3-(4-methoxyphenyl)
propan-1-one (5e). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
3
-(4-methoxyphenyl) propanoic acid (72.08mg, 0.4mmol). The title compound was
1
obtained as light brown solid (108mg, 67%). m.p. 106-108 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.73 (s, 1H), 8.85 (dd, J = 4.1, 1.6 Hz, 1H), 8.64 (dd, J = 8.6, 1.6 Hz,
1
6
2
H), 7.57 (dd, J = 8.6, 4.1 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.13 (d, J = 8.6 Hz,, 2H),
.99 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 3.75 (s, 2H), 3.71 (s, 3H), 3.38 (s,
H), 3.32 (s, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.54 (t, J = 7.6 Hz, 2H), 2.30 (d, J = 24.8
13
Hz, 4H). C NMR (126 MHz, DMSO-d
6
) δ 169.8, 157.5, 152.8, 147.7, 138.8, 133.7,
1
4
33.2, 129.3, 128.9, 127.8, 123.7, 121.4, 113.6, 109.9, 59.3, 55.0, 52.7, 52.3, 44.9,
+
1.1, 34.2, 29.9. ESI-MS m/z: 406.1 [M + H] ; HRMS: calcd for C24
H
27
N
3
NaO [M
3
+
+
Na] : 428.1945. found: 428.1944. HPLC purity (MeOH / H
2
O / 0.1% FA): 99.3%,
3
.9 min.
4
.1.2.17. 3-(4-fluorophenyl)-1-(4-((8-hydroxyquinolin-5-yl) methyl) piperazin-1-yl)
propan-1-one (5f). The reaction was carried out according to the general procedure,
starting from 5-(piperazin-1-ylmethyl) quinolin-8-ol (3) (97.32mg, 0.4mmol) and
3
-(4-fluorophenyl) propionic acid (67.27mg, 0.4mmol). The title compound was
1
obtained as light brown solid (108mg, 69%). m.p. 118-120 °C; H NMR (600 MHz,
DMSO-d
6
) δ 9.73 (s, 1H), 8.85 (dd, J = 4.1, 1.2 Hz, 1H), 8.64 (dd, J = 8.5, 1.2 Hz,
1
H), 7.57 (dd, J = 8.5, 4.1 Hz, 1H), 7.31 (d, J = 7.8 Hz, 1H), 7.25 (m, 2H), 7.08 (m,

2
H), 6.99 (d, J = 7.8 Hz, 1H), 3.75 (s, 2H), 3.38 (s, 2H), 3.32 (s, 2H), 2.78 (t, J = 7.6
1
3
Hz, 2H), 2.57 (t, J = 7.6 Hz, 2H), 2.35 – 2.25 (m, 4H). C NMR (126 MHz,
DMSO-d ) δ170.1, 162.1, 160.2, 153.4, 148.3, 139.3, 138.0, 134.2, 130.6, 129.4,
28.3, 121.9, 115.3, 110.4, 59.8, 53.2, 52.8, 45.4, 41.6, 34.4, 30.4. ESI-MS m/z: 394.1
6
1
+
+
[
M + H] ; HRMS: calcd for C23
H25FN
3
O [M + H] : 394.1925. found: 394.1924.
2
HPLC purity (MeOH / H O / 0.1% FA): 99.8%, 4.3 min.
2
4
.2. Pharmacology
4
.2.1. Aβ aggregation assay
Inhibition of Aβ aggregation was measured using a Thioflavon T (ThT)-binding
2
2,31
assay.
Aβ1−42 (Sigma) was pretreated with 1 mL Hexafluoroisopropanol (HFIP) to
give a stock solution, which was aliquot into small samples. These samples were
incubated at room temperature to evaporate the solvent, then stored at –80 °C. For the
inhibition of self-mediated Aβ1−42 aggregation experiment, the Aβ stock solution was
diluted with 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,
different concentrations) was incubated at 37 °C for 48 h to induce aggregation.
Blanks using 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 (excitation, 450 nm; emission, 485 nm). The percent
inhibition of aggregation was calculated by the expression (1 − IF
i c
/IF ) × 100, in
which IF and IF are the fluorescence intensities obtained for Aβ in the presence and
i
c
absence of inhibitors after subtracting the background, respectively.
For the inhibition of copper-mediated Aβ1−42 aggregation experiment, the Aβ1−42
stock solution was diluted in 20 µM HEPES (pH 6.6) with 150 µM NaCl. The
mixture of the peptide (10 µL, 25 µM, final concentration) with or without copper (10
µL, 25 µM, final concentration) and the tested compound (10 µL, 50 µM, final
concentration) was incubated at 37 °C for 24 h. Then 20 µL of 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). The detection method was the same as that of self-mediated
Aβ1−42 aggregation experiment.
4
.2.2. Oxygen radical absorbance capacity (ORAC-FL) assay
The tested compounds and fluorescein (FL) stock solution were diluted with
phosphate buffer (pH 7.4) to 10 and 0.117 µM, respectively. The solution of
±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox, Sigma-Aldrich)
was diluted with the same buffer to 5, 2, 1, 0.5, 0.2 and 0.1 µM. The solution of
,2′-azobis-(amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich) was prepared
before the experiment by dissolving 21.69 mg of AAPH in 2 mL of phosphate buffer
pH 7.4) to a final concentration of 40 mM. The mixture of the tested compound (20
(
2
(
µL) and fluorescein (FL, Sigma-Aldrich) (120 µL, 70 nM, final concentration) in a
black 96-well plate was preincubated for 15 min at 37 °C, and then 60 µL of the
AAPH solution was added. The fluorescence was recorded every minute for 120 min
(
(
excitation, 485 nm; emission, 538 nm) using Varioskan Flash Multimode Reader
Thermo Scientific). A blank using phosphate buffer instead of the tested compounds
were also carried out. All reaction mixtures were prepared in triplicate. The
antioxidant curves (fluorescence versus time) were normalized to the curve of the
blank. The area under the fluorescence decay curve (AUC) was calculated using
following equation:
ꢈꢌꢍ
ꢀ
ꢁꢂꢃꢄ ꢅꢆꢇꢉꢇꢋ
ꢈ ꢊ
ꢈꢌꢍꢎꢊ
where f
at time i. The net AUC was calculated by the expression AUCsample − AUCblank
Regression equations between net AUC and Trolox concentrations were calculated.
0
is the initial fluorescence reading at 0 min and f is the fluorescence reading
i
.
2
6,31
ORAC-FL
value for each sample was calculated by using the standard curve,
which means the ORAC-FL value of tested compound expressed as Trolox
equivalents.
4
.2.3. Metal-chelating study

The chelating studies were performed with an UV−vis spectrophotometer. The
absorption spectra of each compound (50 µM, final concentration) alone or in the
presence of CuSO
4
, FeSO
4
, FeCl
3
, or ZnCl
2
(50 µM, final concentration) for 30 min
in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded at room temperature.
2
+
Fluorescence spectra of 5b (50 μM) to Cu (1 equiv.) and other metal ions (1 equiv.)
in buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were recorded after the incubation
for 20 min at 25 °C (λex = 245 nm).
2
+
2+
The stoichiometry of the 5b−Cu and 5b−Zn complexes were determined by
3
5,41
Job’s method.
Separate solutions of 5b and CuSO
4
/ZnCl were used to prepare
2
-5
solutions in which the sum of the concentrations of both species (2.5 × 10 M) was
constant in all samples, 21 solutions were obtained with the condition but the
proportions of either component varied between 0% and 100%. Blanks using buffer
2
+
2+
(
20 mM HEPES, 150 mM NaCl, pH 7.4) instead of Cu /Zn were also carried out.
The UV spectra were normalized to the respective blanks. The difference UV−vis
spectra were examined to investigate the ratio of 5b/metals in the complexes.
4
.2.4. Cell viability and neuroprotection activity assay
The rat adrenal pheochromocytoma cell line PC12 was obtained from the Cell
Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained
in RPMI-1640 medium supplemented with 10% horse serum (GIBCO) and 5% fetal
bovine serum (FBS, Gemini), 100 IU/mL penicillin, and 100 µg/mL streptomycin.
Cultures were seeded into flasks containing supplemented medium and maintained at
3
7 °C in a humidified atmosphere of 5% CO
2
and 95% air. For assays, PC12 cells
3
were sub-cultured in 96-well plates at a seeding density of 5×10 cells per well. When
the PC12 cells reached 80% confluence, the medium was replaced with fresh medium
containing compounds. All compounds were dissolved in pure DMSO, but the final
DMSO concentration was 0.05% in each well. Cell viability was measured by
quantitative colorimetric assay with
3
-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma
4
2
Aldrich, Madrid, Spain), as described previously. Briefly, 10 μL of the MTT
labeling reagent, at a final concentration of 0.5 mg/mL, was added to each well at the
end of the incubation period and the plate was placed in a humidified incubator at
3
7 °C with 5% CO and 95% air (v/v) for an additional 4 h period. Then, the insoluble
2

formazan was dissolved with dimethylsulfoxide (DMSO); colorimetric determination
of MTT reduction was measured at 570 nm using a microplate reader (SpectraMax
plus 384, Molecular Devices, Sunnyvale, CA, 176 USA). Control cells treated with
RPMI-1640 were taken as 100% viability.
For antioxidant assay, PC12 cells were sub-cultured in 96-well plates at a
4
seeding density of 1.2×10 cells per well. The cells were then incubated with H
2
O
2
(
150 µM), or 10 µM Rotenone or 25 µM Oligomycin-A, with or without test
compound at various concentrations for 24 h before assayed with MTT. Trolox was
used as the control with the same concentrations. Control cells treated with
RPMI-1640 were taken as 100% viability.
4
.2.5. Evaluation of ROS
The level of intracellular ROS was measured by using the ROS-sensitive dye,
2
',7'-dichloro-fluorescein diacetate (DCFH-DA (Beyotime, Nantong, China)), as a
5
probe. In brief, PC12 cells were seeded in six-well plates at 3×10 cells/well, the
medium was replaced with fresh medium containing various concentrations of tested
samples after 24 h. A H
2
O
2
model group was carried out with fresh medium instead of
(150 μM) 4h later and incubated at
7 °C for 24 h and then washed three times and incubated with final concentration of
0 mM DCFH-DA for 30 min at 37 °C in the dark. After incubation, cells were
tested samples. The cells were exposed to H
2
O
2
3
1
washed three times and harvested in free-serum medium. The fluorescence of
4
3,44
2
',7'-dichlorofluorescein (DCF) was detected by flow cytometry
excitation and 525 nm emission filters) using BD Accuri C6 flow cytometer (Becton
Dickinson Company, Miami, FL, USA). Data were processed by using cell quest
software (Becton & Dickinson Company, Miami, FL, USA).
(488 nm
&
4
.2.6. Apoptosis detection
Annexin V-FITC and propidium iodide were used to evaluate apoptotic cells by
5
flow cytometry. PC12 cells were seeded in six-well plates at 3×10 cells/well, and
were treated with various concentrations of tested samples 24 h later. Then the cells
were exposed to H
2
2
O (150 μM) 4h later and incubated at 37 °C for 24 h. Each well
cells were harvested and 200 µL binding buffer was added. Then the cells were
stained with 5 µL °C Annexin V-FITC and 5 µL propidium iodide at room

temperature for 20 min. Cells were then analyzed by flow cytometry. Cells
undergoing apoptosis are both Annexin V positive and PI negative.
4
.2.7. In vitro blood−brain barrier permeation assay
In order to predict passive blood-brain penetration of novel compounds, the
parallel artificial membrane permeation assay (PAMPA) described by Di et al has
3
8
been used. Porcine brain lipid (PBL) was obtained from Avanti Polar Lipids. The
donor microplate (PVDF membrane, pore size 0.45 mm) and acceptor microplate
were both from Millipore. The 96-well UV plate (COSTAR) was from Corning
Incorporated. The acceptor 96-well microplate was filled with 300 μL of PBS/EtOH
(
7:3), and the filter membrane was impregnated with 4 μL of PBL in dodecane (20
mg/mL). Compounds were dissolved in DMSO at 5 mg/mL and diluted 50-fold in
PBS/EtOH (7:3) to a final concentration of 100 μg/mL. Then 200 μL of the solution
was added to the donor wells. The acceptor filter plate was carefully placed on the
donor plate to form a sandwich, which was left undisturbed for 10 h at 25 °C. After
incubation, the donor plate was carefully removed, and the concentration of
compounds in the acceptor wells was determined using the UV plate reader
(
SpectraMax plus 384, Molecular Devices, Sunnyvale, CA, 176 USA). Every sample
was analyzed at five wavelengths in four wells and in at least three independent runs.
Pe was calculated using the following expression: Pe = {−V /[(V + V )At]}ln(1 −
is the volume of donor well, V is the volume in the
d
V
a
d
a
drugacceptor/drugequilibrium) where V
d
a
acceptor well, A is the filter area, t is the permeation time, drugacceptor is the
absorbance obtained in the acceptor well, and drugequilibrium is the theoretical
equilibrium absorbance. The results are given as the mean ± standard deviation. In the
experiment, 8 quality control standards of known BBB permeability were included to
validate the analysis set. A plot of the experimental data versus literature values gave
2
a strong linear correlation, Pe(exp) = 0.848Pe(bibl.) +0.228 (R = 0.983) (Figure S2).
−
6
From this equation and the limit established by Di et al. (Pe(lit.) = 4.0 × 10 cm/s)
for blood−brain barrier permeation, we concluded that compounds with Pe > 3.62 ×
−
6
1
×
×
0 cm/s for high blood-brain barrier permeation (CNS+), compounds with Pe < 1.92
−
6
10 cm/s for low blood-brain barrier permeation (CNS-), and compounds with 3.62
−
6
−6
10 cm/s > Pe > 1.92 × 10 cm/s for uncertain blood-brain barrier permeation
(
CNS±).

4
.2.8. Acute toxicity
A total of 20 KM mice (male, 22 days, 18−20 g) purchased from Comparative
Medicine Center of Yangzhou University (Yangzhou, China) were used to evaluate
the acute toxicity of compound 5b. Mice were maintained with a 12 h light/dark cycle
at 25 °C with a relative humidity of 60−70%. Animals were reared and handled
strictly according to the obligations of the Animal Ethics Committee of China
Pharmaceutical University and the guidelines for the care and use of laboratory
animal from the National Institute of Health. Sterile food and water were provided
according to institutional guidelines. The animals were acclimated for 5 days prior to
operation. Compound 5b was suspended in 0.5% carboxymethylcellulose sodium
(
CMC-Na) salt solution (677, 1333, and 2000 mg/kg) and given via oral
22
administration according to the divided experimental groups. After the
administration of the compound, the mice were observed continuously for the first 4 h
for any abnormal behavior and mortality changes, intermittently for the next 24 h, and
occasionally thereafter for 14 days for the onset of any delayed effects. All animals
were sacrificed on the 14th day after drug administration and were macroscopically
examined for possible damage to the heart, liver, and kidneys.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgment
We gratefully acknowledge the financial support of the National Natural Science
Foundation of China (81573313 and 81602985), the Qing Lan Project of Jiangsu
Province in China, the Innovative Research Team in University (IRT_15R63) and the
Priority Academic Program Development of Jiangsu Higher Education Institutions
(
PAPD).
References
1
. Finder VH. Alzheimer's disease: A general introduction and pathomechanism. J
Alzheimers Dis. 2010; 22:S5-S19.