Development of the “hidden” multifunctional agents for Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2019.05.051

Alzheimer's disease (AD)is a chronic, fatal and complex neurodegenerative disorder, which is characterized by cholinergic system dysregulation, metal dyshomeostasis, amyloid-β (Aβ)aggregation, etc. Therefore in most cases, single-target or single-function

Full text of DOI:10.1016/j.ejmech.2019.05.051

Accepted Manuscript
Development of the “hidden” multifunctional agents for Alzheimer's disease
Wenhai Huang, Meihao Liang, Qin Li, Xiaoliang Zheng, Chixiao Zhang, Qiao Wang, Li
Tang, Zhimin Zhang, Beibei Wang, Zhengrong Shen
PII:
S0223-5234(19)30461-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.051
EJMECH 11361
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 March 2019
Revised Date: 26 April 2019
Accepted Date: 18 May 2019
Please cite this article as: W. Huang, M. Liang, Q. Li, X. Zheng, C. Zhang, Q. Wang, L. Tang, Z.
Zhang, B. Wang, Z. Shen, Development of the “hidden” multifunctional agents for Alzheimer's disease,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.05.051.
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Graphical Abstract
Compound 9b could cross the BBB efficiently and release 8a into brain. 9b had a potent AChE
inhibitory activity, while 8a displayed a significant metal ion chelating function, therefore in
combination, both 9b and 8a exhibited a considerable inhibition of Aβ aggregation.

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Development of the “hidden” multifunctional agents for Alzheimer’s disease
Wenhai Huanga,b, Meihao Lianga, Qin Lia, Xiaoliang Zhengc, Chixiao Zhanga, Qiao Wanga, Li
Tangd,e, Zhimin Zhanga, Beibei Wanga, Zhengrong Shena,*.
aKey Laboratory of Neuropsychiatric Drug Research of Zhejiang Province, Institute of Materia Medica,
Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
bState Key Laboratory of Medicinal Chemical Biology, NanKai University, Tianjin, PR China
cCenter for Molecular Medicine, Zhejiang Academy of Medical Sciences, Hangzhou, P. R. China
dDepartment of Pharmacy, School of Medicine, Health Science Center, Shenzhen University, Shenzhen,
P. R. China.
eNational-Regional Key Technology Engineering Laboratory for Synthetic Biology of Medicine,
Shenzhen University, Shenzhen, P. R. China.
*Corresponding author: shenzr@zjams.com.cn, Tianmushan Road. NO.182, Zhejiang Academy of
Medical Sciences, Hangzhou, 310013, P. R. China
Abstract: Alzheimer’s disease (AD) is a chronic, fatal and complex neurodegenerative disorder,
which is characterized by cholinergic system dysregulation, metal dyshomeostasis, amyloid-β (Aβ)
aggregation, etc. Therefore in most cases, single-target or single-functional agents are insufficient
to achieve the desirable effect against AD. Multi-Target-Directed Ligand (MTDL), which is
rationally designed to simultaneously hit multiple targets to improve the pharmacological profiles,
has been developed as a promising approach for drug discovery against AD. To identify the
multifunctional agents for AD, we developed an innovative method to successfully conceal the
metal chelator into acetylcholinesterase (AChE) inhibitor. Briefly, the “hidden” agents first cross
the Blood Brain Barrier (BBB) to inhibit the function of AChE, and the metal chelator will then be
released via the enzymatic hydrolysis by AChE. Therefore, the AChE inhibitor, in this case, is not
only a single-target agent against AD, but also a carrier of the metal chelator. In this study a total
of 14 quinoline derivatives were synthesized and biologically evaluated. Both in vitro and in vivo
results demonstrated that compound 9b could cross the BBB efficiently, then release 8a, the
metabolite of 9b, into brain. In vitro, 9b had a potent AChE inhibitory activity, while 8a displayed
a significant metal ion chelating function, therefore in combination, both 9b and 8a exhibited a
considerable inhibition of Aβ aggregation, one of the observations that plays important roles in the
pathogenesis of AD. The efficacy of 9b against AD was further investigated in both a zebrafish
model and two different mice models.

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Keywords: Alzheimer’s disease; multifunctional agents; Aβ

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1. Introduction
Alzheimer’s disease (AD), the leading cause of dementia in the elderly, is a chronic, fatal, and
complex neurodegenerative disorder.[1,2] In recent decades, only a few drugs were approved
worldwide for treating AD. The failure rate of candidates in clinical trials is about 99.6% from
2002 to 2012, and particularly the failure of Solanezumab at phase III trial cast a shadow on the
AD drug development.[3] Since multiple factors, such as cholinergic system dysregulation, metal
dyshomeostasis, oxidative stress and amyloid-β (Aβ) aggregation, are demonstrated to play
important roles in the development of AD, it is difficult to achieve the desirable therapeutic effects
by single-target strategy. Therefore, Multi-Target-Directed Ligand (MTDL), which is rationally
designed to simultaneously hit multiple targets to improve the pharmacological profiles, has been
developed as a promising approach for drug discovery against AD.[4-6]
To date, most drugs approved for AD are AChE inhibitors, which could improve the level of
ACh, an important neurotransmitter responsible for learning, memory, cognition and maintaining
consciousness.[7-8] Besides the reduced ACh level, dyshomeostasis of biometals are also involved
in the complex interconnected pathogenesis network of AD. The level of metal ions in AD patients
were found to be 3-7 folds higher than healthy individuals,[9] indicating the dyshomeostasis of
biometals (Fe, Cu, Zn) in the brain may contribute to AD pathology.[10] Moreover, reactive oxygen
species (ROS)，which leads to oxidative stress and neuronal cell death, are also related to the
redox-active metal ions, such as Cu(I) and Fe(II).[11-14] The phase II clinical trials suggested that
clioquinol (CQ), a classic metal chelator used as antifungal drug and antiprotozoal drug, could halt
cognitive decline in AD, possibly due to its ability to chelate copper and zinc ions.
Both AChE and metal ions are associated with the aggregation of Amyloid-β (Aβ). AChE forms
a complex with Aβ and changes the conformation of Aβ, therefore promotes the aggregation of
Aβ.[15] Metal ions also bind to the histidine residues[16] of soluble Aβ, thus markedly accelerate Aβ
nucleated aggregation,[17,18] an observation enhanced under mild acidic conditions similar to that
present in aging and AD brains.[19] Therefore, simultaneously inhibiting AChE and decreasing
metal ions, both of which decelerate the Aβ aggregation through different mechanisms, may
render a synergistic effect on the treatment of AD.
Given this, in this study we designed and synthesized a multi-target-directed ligand by

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integrating AChE inhibitors and metal chelators, to reduce the hydrolysis of ACh as well as
decrease the metal ions levels in brain, therefore inhibit the aggregation of Aβ. Rivastigmine
(Riva), an AChE inhibitor approved for the treatment of AD, was showed to release metabolite
NAP226-90 AChE in brain.[20] Therefore, this compound could be further modified as a
multi-target ligand by inhibiting the AChE function and chelating the metal ion via the subsequent
hydrolysis products released by AChE. Based on this strategy, quinoline derivatives were designed
by conjugating AChE inhibitor Riva with metal chelator CQ. First, we studied the protein binding
pattern of one of the typical quinoline derivatives 9b (Fig. 1) by a molecular docking model
described previously,[21] and found that 9b had several important interactions with the active-site
of AChE in the same mode as Riva. Next, we synthesized 18 quinoline derivatives, and assessed
their AChE inhibitory activities, AChE enzymatic metabolites, metal chelating effects and Aβ
aggregation inhibitory activities. The efficacies of the candidate compounds were further
evaluated in vivo by multiple models.
HO
N
O
AChE
O
N
N
N
N
NAP226-90
rivastigmine
O
OH
N
OH
OH
N
N
N
O
Fe3+(Zn2+Cu2+
)
N
Design
AChE
Fe3+
N
Cu2+
N
HO
HO
N
N
I
8a
9b
Cl
Clioquinol
N
Fig. 1 The design and functions of quinoline derivative 9b
2. Results and Discussion
2.1 Molecular Docking Study
To understand the molecular interaction between quinoline derivatives and AChE, the
molecular docking studies were performed using the Discovery Studio 4.0/CDOCKER protocol.
[21]
The crystal structure of Riva/AChE complex (PDB ID: 1GQR) was used as the template.
Docking and the subsequent scoring studies were performed using the default parameters. The
results showed that the binding patterns of quinoline derivatives in AChE are similar to Riva (Fig.
2). For example, the result of 9b shows in Fig. 2. Hydrogen bond interactions are observed

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between amide group and Gly119/Ser122 with distance of 2.38Å and 2.96Å, respectively. In
addition, the quinoline ring forms π-π stacking with Trp84. Moreover, same as the binding pattern
of Riva, the carbamate group of 9b is very close to the esteratic site (Ser200, Glu327 and His440),
which is beneficial to hydrolyze the carbamate group and enable a ‘flattening’ of the carbamate
moiety over the esteratic site, contributing to the prolonged inhibition of AChE.
Fig. 2 The molecular docking of rivastigmine (yellow)/9b (gray) with AChE
2.2 Chemistry

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Fig. 3 The synthetic routes of 9a-9n
Reagents and conditions: (a) Br2, acetic acid, 0℃; (b) H2SO4, H2O, nitrobenzene sulfonate,
glycerol, reflux; (c) Pd (OAc)2, DPPP, Et3N, 1-vinyloxy-butane, 145℃; (d) NaBH4, THF, rt; (e)
SOCl2, DCM, rt; (f) R1H, acetonitrile, reflux; (g) 40% HBr, 130℃; (h) NaH, R2COCl, THF, rt.
Quinoline derivatives were prepared according to the procedure depicted in Fig. 3. Reaction of
2-Methoxy-phenylamine with Br2 afforded compound 2, which was refluxed with glycerol in
H2SO4/H2O and nitrobenzene sulfonate by Skraup reaction to structure the quinoline scaffold. The
group Br in compound 3 was substituted by formyl group to yield 4, followed by reduction of
carbonyl group with NaBH4 to get 5. The hydroxyl of compound 5 was substituted by chlorine to
afford 6, and then this key intermediate was followed by reaction with various substituted amine
to yield 7a-7g. While treating with HBr led to the loss of methyl group, 8a-8g were prepared and
subsequently acylated with substituted formyl chloride to obtain target compounds 9a-9n.
In total, 14 quinoline derivatives (i.e., 9a-9n) and 7 intermediates (i.e., 8a-8g), which are the
hydrolytic metabolites of 9a-9n, were designed and synthesized. The chemical structures are
shown in Tables 1, and the details of the synthetic procedures and structural characterization are
described in the experimental section.
2.3 Biological Activity in vitro
2.3.1 AChE Inhibitory Activity
To evaluate the therapeutic potential of the synthesized quinoline derivatives against AD, 9a-9n
were tested for their AChE inhibition by a modified method of Ellman[22] using Riva as control
(Tab. 1). Among the variations of R2 in 9a-9f, methylethylamino- or morpholine- group
demonstrated a significant AChE inhibitory activity. Likewise, the similar SAR was also found in
9g-9i. Specifically, 9b displayed the most potent inhibition (IC50: 1.2±0.5µmol/L, ~7-fold higher
than the positive control Riva). With respect to R1 in 9b and 9j-9n, substitution of
dimethylamino-group by other aliphatic amino- or various types of alicyclic amino-group
remarkably reduced the AChE inhibitory activities. For example, the methylethylamino group at
R1 was 35-fold less potent (9j vs 9b).
Tab. 1 The inhibitions of AChE activity and values of IC50

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O
R2
O
N
R1
Compounds
R1
-
R2
IC50(µmol/L)
9.0±2.9
Compounds
R1
R2
IC50(µmol/L)
48.6±19.6
Rivastigmine
-
9h
N
N
O
O
N
9a
9b
9c
9d
9e
9f
9i
9j
16.3±15.1
1.2±0.5
8.3±3.0
40.7±24.6
41.2±3.9
18.1±3.6
29.0±11.7
54.7±25.8
N
O
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
9k
9l
13.6±3.1
28.6±2.1
20.6±6.0
8.8±0.6
N
N
9m
9n
N
N
N
N
N
O
9g
69.9±34.0
N
O
N
2.3.2 Metal Chelating Effect
The metal chelating effect of compound 8a, a hydrolytic metabolite of 9b, was investigated
using the UV-Vis spectra recorded in methanol at 298K.[23] As shown in Fig. 4, the absorption was
decreased and λmax in the UV-Vis spectra was shifted to red upon the addition of copper ion into
8a, and the similar shift was observed for the iron/zinc ion. These results are similar with the CQ,
which indicated an interaction between 8a and copper/iron/zinc ion, and 8a had a potent metal
chelating activity. As comparison, compound 9b had no significant spectral change in the presence
of FeSO4 or CuCl2, indicating that the formation of metal complex was contributed by the specific
interactions between metal ions and the hydroxyl moiety.

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Fig. 4 The absorption spectra of 8a and CQ (25 µM) chelating with copper, iron and zinc ions
2.3.3 Aβ Aggregation Inhibition
In order to investigate the Aβ aggregation inhibition ability of 9b and 8a, two methods (TEM
and ThT) were used as qualitative and quantitative analyses separately.
2.3.3.1 TEM
The ability of compounds 9b and 8a to inhibit Aβ1 42 self-aggregation was monitored by
−
transmission electron microscopy (TEM) using curcumin as a positive control. After 72h of
incubation at 37℃, Aβ1 42 aggregated into the well-defined amyloid fibrils. In contrast, only a few
−
fibrils could be observed in the presence of curcumin or 9b or 8a under the same conditions.
[24]
Among them, 9b displayed the most potency in inhibition of aggregation of Aβ1 42
.
−
Fig. 5 The transmission electron micrography of Aβ aggregation inhibition
a/b. Aβ; c/d. Aβ+Cur; e/f. Aβ+9b; g/h. Aβ+8a
(a, c, e and g are 10 000×; b, d, f and h are 50 000×)
2.3.3.2 ThT
Thioflavin T (ThT) fluorescence assay was used as quantitative analysis with curcumin as a

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reference compound.[25] As shown in Tab. 2, 9b demonstrated a marked Aβ1 42 self-aggregation
−
inhibitory activity, presenting more potent inhibition (inhibitory=55.88%) than curcumin
(inhibitory=50.19%). Interestingly, the metabolite 8a also displayed a potent aggregation
inhibitory activity. Furthermore, the inhibitory ability of 9b was at time-dependent manner.
Treatment at 24h, 9b could inhibit 31.34% of Aβ1 42 self-aggregation, while 55.88% at 48h and
−
66.09% at 72h.
Tab. 2 The results of the ability to inhibit self-induced Aβ aggregation
Compound
Aβ
Time
48h
Inhibition%
0
Aβ+Cur
48h
50.19
Aβ+9b
Aβ+9b
Aβ+9b
Aβ+8a
24h
48h
72h
48h
31.34
55.88
66.09
44.05
2.4 Pharmacokinetics in vitro and in vivo
2.4.1 AChE Enzymatic Hydrolysis Experiments in vitro
Carbamate AChE inhibitors such as rivastigmine have been shown to be readily hydrolyzed by
AChE with a concomitant release in the brain of the OH metabolite.[26] In order to study the
feasibility of enzymatic removal of the carbamoyl moiety of 9b, we developed an AChE-mediated
hydrolysis model in vitro. As shown in Fig. 6, when 9b was incubated with mouse AChE at 37ºC
for 12h or 48h, small portion of 8a could be identified by HPLC after 12h hydrolysis. At 48h,
more 9b was hydrolyzed by AChE to the OH-metabolite 8a. More interestingly, the
OH-metabolite 8a was the major metabolite of 9b by AChE.

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Fig. 6 The HPLC profiles of 9b and 8a in the hydrolysis with AChE
2.4.2 Stability Test in vitro
Stability of 9b is critical for crossing the BBB (Blood Brain Barrier) to the brain. Therefore,
we assessed the plasma stability, metabolic stability, simulated gastric fluid stability and simulated
intestinal fluid stability of 9b. After incubation with human plasma (from Chempartner), human
liver microsomes (from BD Biosciences Gentest), simulated gastric fluid and simulated intestinal
fluid respectively, 9b was measured at 5min, 15min, 30min, 45min and 60min by HPLC. The
results in Fig. 7 demonstrated that 9b is very stable in all the tests.
Fig. 7 The stability results of 9b. a) Plasma stability; b) Metabolic stability; c) Simulated gastric
fluid stability; d) Simulated intestinal fluid stability
2.4.3 PK and BBB Penetration of 9b in vivo
BBB penetration is an important factor in the development of MTDL against AD.[27] To
investigate the BBB penetration of 9b, plasma and brain pharmacokinetic (PK) studies were

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performed by microdialysis method.[28,29] 9b (25mg/kg) was injected into male Sprague−Dawley
rats by intravenous. As shown in Tab. 3, 9b displayed a high exposure (447.74±149.59 mg/L*min)
and extended MRT (91.75 min) in the plasma. More importantly, the brain/plasma exposure ratio
was 0.524 (calculated with AUC0−∞ exposure measured for brain and plasma dialysis), indicating
that 9b can efficiently cross the BBB (Fig. 8).
Fig. 8 The pharmacokinetics curve of 9b in blood dialysis and brain dialysis
Tab.3 The main pharmacokinetic parameters of 9b in blood dialysis, brain dialysis and plasma
_
of rats. n=6, x±SD.
Blood
Brain
9b
9b
Parameter
Plasma
9b
dialysis
dialysis
AUC0-t(mg/L·min) 444.32±147.815 145.86±50.11 75.69±23.87
AUC0-∞(mg/L·min) 447.74±149.59 147.16±50.97 76.34±23.38
MRT(min)
91.75±17.07
0.062±0.02
69.81±20.65 56.82±13.30
0.19±0.08 0.35±0.10
CLz(L/min/kg)
2.5 Cognitive Improvement in Animal Model of AD
2.5.1 Cognitive Improvement in Mice Model of AD
Since there are no animal models available to investigate the target-directed (AChE and metal
ion) compound simultaneously, we used two mice models separately, the scopolamine-induced
model for targeting AChE, and the Aluminium trichloride-induced model for targeting metal ion,

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in which Rivastigmine (Riva, AChE inhibitor) and Clioquinol (CQ, metal chelating agent) were
used as positive controls.
The effect of compound 9b on cognitive improvement was first assessed in a scopolamine
(Scop)-induced amnesia animal model. Scop can block the cholinergic pathway by antagonizing
the muscarinic receptor, which provides a typical AD model to explore the role of the cholinergic
system in cognition and to evaluate anticholinergic drug candidates. In our preliminary experiment,
CQ had no effect on cognitive improvement in this model, therefore, we used Riva as the positive
control in the following experiment. As shown in Figure 9, the model group exhibited a longer
escape latency (63.2±21.1 sec) with a total swimming distance (785±351cm), as compared to
the normal group (36.1±23.5 sec and 445±301 cm). Compound 9b at 25 mg/kg significantly
rescued the escape latency (43.8±17.0 sec) and reduced the total swimming distance (520±168
cm), compared to the model group (p<0.05). The effect of 9b for the symptom improvement in the
in vivo model indicated the compound 9b can be used as a potential anti-AD lead, especially for
the treatment of cholinergic deficiency in the early stages of the disease.
Fig.9 a) The escape latencies at 5th day of training on the Morris water maze; b) The total
swimming distance at 5th day of training on the Morris water maze
Next, 9b was further assessed in an aluminum trichloride-induced amnesia animal model by a
SMG-2 programmable water maze using Riva (2mg/kg) and CQ (120mg/kg) as positive control
drugs. As shown in Figure 10, the model group exhibited a longer total swimming time (115±19
sec, p<0.05) with more error times (20.7±4.8 sec, p<0.05) than the normal group (66±29 sec and
9.8±5.0 sec). Although the error times for three doses of 9b didn’t show the statistical differences

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as compared to the model group, the numbers were still lower with various degrees after the
treatment with 9b (16.8±6.7 at 8mg/kg, 18.1±4.6 at 2mg/kg, 15.1±6.9 at 0.5mg/kg). In addition,
upon the treatment of 9b with 8 mg/kg and 0.5mg/kg, the total swimming times were significantly
decreased (79±25 sec at 8mg/kg and 73±29 sec at 0.5mg/kg, p<0.05). The efficacy of 9b for
symptom improvement in this AD mice model also demonstrated its potential anti-AD activity. As
comparison, Riva had no effect in this model.
Fig.10 a) The average of error times on the SMG-2 programmable water maze; b) The average
time of total swimming time on the SMG-2 programmable water maze
2.5.2 Cognitive Improvement in a Zebrafish Model of AD
The effect of 9b on cognitive improvement was further assessed in zebrafish model by
aluminum chloride exposure, which was previously developed.[30] We first tested the NOAELs
(No Observed Adverse Effect Levels) of 9b, and then set the high, moderate and low
concentrations of 9b for the efficacy assessment. In the experiment, the zebrafish were first treated
with 150 µM AlCl3 from 3 dpf to 5 dpf, and then treated with 9b at three different doses (1µM,
5µM, 25µM) from 3 dpf to 5 dpf. Each group had 30 zebrafish. As in the Table 5, the results
showed the Dyskinesia Recovery Rate (DRR) measurement was increased in a dose-dependent
manner upon 9b treatment. In addition, the Response Efficiency (RE) measurement was also
increased after adding 9b (Table 4). The effect of 9b was more significant than rivastigmine,
especially in the DRR measurement. The results clearly demonstrated the multi-function of 9b, i.e.
upon penetrating into the brain, 9b first inhibits acetylcholinesterase, and the hydrolysis product
8a then acts as a metal chelator.
Table 4 The RE values of six drugs at low, moderate and high concentrations

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Concentration
Drug
DRR%
p
RE %
p
(µM)
10
50
250
1
53.4
64.0
58.6
70.8
99.3
121
0.00298
0.00012
0.00310
0.09505
0.03251
0.00329
126.3
86.6
0
Rivastigmine
0.00004
0
175.1
112.4
161.4
70.2
0.00228
0.00013
0.02818
9b
5
25
All the values presented are calculated by the average speed of the drug treated fish.
2.6 Toxicity Evaluation
2.6.1 Cyto-inhibition
To investigate the safety of 9b, its cytotoxicities on the cell lines Neuro-2a, MCF-7, A549 were
studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.[25]
The IC50 of compound 9b are > 20µM in all three cell lines, indicating the safety of 9b.
2.6.2 hERG Inhibition
hERG (the human Ether-à-go-go-Related Gene) is a gene that codes for a protein known as the
alpha subunit of a potassium ion channel (sometimes simply denoted as 'hERG'). Since hERG
channel mediates the repolarizing IKrcurrent in the cardiac action potential, it can cause a
potentially fatal disorder called long QT syndrome when this channel's function is inhibited by
application of drugs.[31] A number of clinically successful drugs in the market had the potential of
hERG inhibition, a concomitant risk of sudden death, therefore the hERG inhibition must be
avoided for the drug development.[32] We detected the compound 9b by patch clamp assay using
cisapride as a positive control. The results showed that the IC50 of 9b is > 30 µM, while the
cisapride exhibited 88.99% inhibition at 0.1µM, indicating that 9b has a minimal hERG potassium
channel inhibition.

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Fig. 11 The hERG inhibition of 9b
3. Conclusions
Because of the complexity of pathogenesis of AD, a chemical entity against multiple targets
might represent a novel therapeutic regimen against AD. In this study we employed the strategy of
MTDLs by integrating the AChE inhibitors with the metal chelators, with the goal of not only
reducing hydrolysis of ACh thus decreasing the levels of metal ions in brain but also slowing
down the aggregation of Aβ.
Due to the high logP values, most metal chelators can’t cross the BBB to the target tissue. We
developed a new strategy to “hide” the metal chelator into the AChE inhibitor, which could further
release the metal chelator after crossing the BBB by metabolism. The AChE inhibitor, in this case,
is not only the anti-AD agent, but also the transporter of the metal chelator. We first showed in
vitro and in vivo that 9b could cross the BBB, and release 8a in brain. Furthermore, in vitro study
found that 9b has the potent AChE inhibitory activity (1.2±0.5µmol/L), which is 7-fold more
important than rivastigmine. After incubation with AChE, 9b releases the OH-metabolite 8a,
therefore chelateing the metal ions. The Aβ aggregation inhibition experiments also suggested that
both 9b and 8a inhibit the Aβ aggregation.
In vivo, two mice models were used to test 9b anti-AD activities. Interestingly, both Riva and
9b are effective on targeting AChE in the scopolamine-induced model, but not CQ. In the
aluminum trichloride-induced model, CQ and 9b are effective on targeting the metal ion, while
Riva not. These results indicated that 9b could reach both targets effectively in vivo. The effect of
9b was further tested in a zebrafish model, showing that 9b has the cognitive improvement.

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In conclusion, our in vitro and in vivo data demonstrated that 9b is a potential candidate for
AD therapy.
4. Experimental
4.1 General
All solvents used were of analytical grade. Melting points were recorded on a Buchi apparatus
1
and uncorrected. H NMR spectra were recorded on a Bruker Avance III 500M instrument
(chemical shifts are expressed as δ values relative to TMS as internal standard). Mass spectra (MS)
were recorded on an Esquire-LC-00075 spectrometer. The database of quinoxaline derivatives was
built by Pipeline Pilot software.
4.2 Chemistry
Compounds 2[33] and 3[34] were synthesized according to the known methods.
4.2.1 1-(8-methoxyquinoline-6-yl) acetone (4)
Compound
3
(2.4g,
10mmol),
Pd(OAc)2
(110mg,
0.5mmol)
and
1,3-Bis(diphenylphosphino)propane (DPPP, 410mg, 1mmol) were dissolved in 20mL alcohol,
triethylamine (25mmol) and 1-vinyloxy-butane (30mmol) were added under nitrogen, and the
mixture was refluxed at 150 for 24h. After cooled to room temperature, 30mL water was added
to the mixture, and extracted with dichloromethane (3×30mL). The organic layer was separated
and dried over Na2SO4. The solvent was evaporated. The residue was purified by silica gel column
chromatography (PE:AcOEt:TEA = 2:1:0.1) to afford 4 as light yellow solid (52%); mp: 64-66℃;
1H-NMR (δ, CDCl3): 9.03 (d, 1H, J = 3.2 Hz, ArH), 8.26 (d, 1H, J = 8.0 Hz, ArH), 8.04 (s, 1H,
ArH), 7.64 (s, 1H, ArH), 7.52 (dd, 1H, J1 = 8.0 Hz, J2 = 3.2 Hz, ArH), 4.16 (s, 3H, -OCH3), 2.75 (s,
3H, -COCH3). MS(ESI): m/z = 202 [M+H]+.
4.2.2 1-(8-methoxyquinine-6-yl) ethanol (5)
Compound 4 (1.02g, 5mmol) was dissolved in 50mL THF, NaBH4 (0.23g, 6mmol) was added
by several times. After the mixture was stirred at room temperature for 3h, 50 mL water was added
to the mixture, and extracted with ethyl acetate (3×50mL). The organic layer was separated and
dried over Na2SO4. The solvent was evaporated and 5 was obtained as light yellow solid (95%);

ACCEPTED MANUSCRIPT
mp: 51-53 ; 1H-NMR (δ, CDCl3): 8.87 (dd, 1H, J1 = 4.4 Hz, J2 = 2.0 Hz, ArH), 8.07 (dd, 1H, J1 =
8.0 Hz, J2 = 2.0 Hz, ArH), 7.40 (dd, 1H, J1 = 8.0 Hz, J2 = 4.4 Hz, ArH), 7.32 (s, 1H, ArH), 7.09 (s,
1H, ArH), 5.04 (q, 1H, J = 6.0 Hz, -CHOHCH3), 4.84 (s, 1H, -CHOHCH3), 4.09 (s, 3H, -OCH3),
1.57 (d, 3H, J = 6.0 Hz, -CHOHCH3). MS(ESI): m/z = 204 [M+H]+.
4.2.3 6-(1-chloroethyl)-8-methoxyquinoline (6)
Compound 5 (1.4g, 5mmol) was dissolved in 10mL dichloromethane, 1mL SOCl2 was added.
After the mixture was stirred at room temperature for 12h, 10mL water was added, the organic
layer was separated and dried over Na2SO4. The solvent was evaporated and 6 was obtained as
light yellow oil (70%); 1H-NMR (δ, CDCl3): 9.03 (dd, 1H, J1 = 4.4 Hz, J2 = 1.6 Hz, ArH), 8.12 (dd,
1H, J1 = 8.4 Hz, J2 = 1.6 Hz, ArH), 7.49 (dd, 1H, J1 = 8.4 Hz, J2 = 4.4 Hz, ArH), 7.38 (s, 1H, ArH),
7.13 (s, 1H, ArH), 4.89 (q, 1H, J = 6.0 Hz, -CHClCH3), 3.98 (s, 3H, -OCH3), 1.64 (d, 3H, J =
6.0 Hz, -CHClCH3). MS(ESI): m/z = 222 [M+H]+.
4.2.4 General procedure for the preparation of compounds 7a-g
Compound 6 (0.22g, 1mmol) was dissolved in 5mL acetonitrile, R1H (5mmol) was added.
After the mixture was refluxed for 3h, 10mL water was added, the organic layer was separated and
dried over Na2SO4. The solvent was evaporated and 7a-g was obtained.
1
4.2.4.1 1-(8-methoxyquinoline-6-yl)-N, N-dimethylamine (7a). Yellow oil (90%); H-NMR (δ,
CDCl3): 8.93 (dd, 1H, J1 = 4.4 Hz, J2 = 2.0 Hz, ArH), 8.07 (dd, 1H, J1 = 8.0 Hz, J2 = 2.0 Hz, ArH),
7.40 (dd, 1H, J1 = 8.0 Hz, J2 = 4.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.16 (s, 1H, ArH), 4.03 (s, 3H,
-OCH3), 3.67 (q, 1H, J = 6.0 Hz, -CHCH3), 2.38 (s, 6H, -N(CH3)2), 1.57 (d, 3H, J = 6.0 Hz,
-CHCH3). MS(ESI): m/z = 231 [M+H]+.
4.2.4.2 N-ethyl-1-(8-methoxyquinolin-6-yl)-N-methylethan-1-amine (7b). Yellow oil (82%);
MS(ESI): m/z = 245 [M+H]+.
4.2.4.3 N, N-diethyl-1-(8-methoxyquinolin-6-yl) ethan-1-amine (7c). Yellow oil (88%) ;
1H-NMR (δ, CDCl3): 8.98 (s, 1H, ArH), 8.18 (d, 1H, J = 8.4 Hz, ArH), 7.52 (dd, 1H, J1 = 8.4 Hz,

ACCEPTED MANUSCRIPT
J2 = 5.6 Hz, ArH), 7.40 (s, 1H, ArH), 7.22 (s, 1H, J = 5.6 Hz, ArH), 4.22 (s, 3H, -OCH3), 4.03 (q,
1H, J = 6.0 Hz, -CHCH3), 2.72 (m, 4H, -N(CH2CH3)2), 1.53 (d, 3H, J = 6.0 Hz, -CHCH3), 1.08 (m,
6H, -N(CH2CH3)2). MS(ESI): m/z = 259 [M+H]+.
4.2.4.4 8-methoxy-6-(1-(pyrrolidin-1-yl) ethyl) quinoline (7d). Yellow oil (85%); MS(ESI): m/z
= 257 [M+H]+.
4.2.4.5 8-methoxy-6-(1-(piperidin-1-yl) ethyl) quinoline (7e). Yellow oil (90%); MS(ESI): m/z
= 271 [M+H]+.
4.2.4.6 4-(1-(8-methoxyquinin-6-yl) ethyl) morpholine (7f). Yellow oil (92%);1H-NMR (δ,
CDCl3): 8.83 (dd, 1H, J1 = 4.0 Hz, J2 = 2.0 Hz, ArH), 8.00 (dd, 1H, J1 = 8.0 Hz, J2 = 2.0 Hz, ArH),
7.34 (dd, 1H, J1 = 8.0 Hz, J2 = 4.0 Hz, ArH), 7.23 (s, 1H, ArH), 7.11 (s, 1H, ArH), 4.04 (s, 3H,
-OCH3), 3.61 (m, 5H, morpholine, -CHCH3), 2.32 (m, 4H, morpholine), 1.57 (d, 3H, J = 6.4 Hz,
-CHCH3). MS(ESI): m/z = 273 [M+H]+.
4.2.4.7 8-methoxy-6-(1-(4-methylpiperazin-1-yl) ethyl) quinoline (7g). Yellow oil (90%);
MS(ESI): m/z = 286 [M+H]+.
4.2.5 General procedure for the preparation of compounds 8a-g
Compound 7 (1mmol) was dissolved in 5mL HBr, after the mixture was stirred at 100 for
24h, the pH of the mixture was adjusted to 8 by NaHCO3, and extracted with ethyl acetate (3×
10mL). The organic layer was separated and dried over Na2SO4. The solvent was evaporated and
the residue was purified by silica gel column chromatography (PE:AcOEt:TEA = 2:1:0.1) to
afford 8.
1
4.2.5.1 6-(1-(dimethylamino) ethyl) quinolin-8-ol (8a). Yellow oil (46%); H-NMR (δ, CDCl3):
8.75 (s, 1H, ArH), 8.13 (d, 1H, J = 8.4 Hz, ArH), 7.41 (dd, 1H, J1 = 8.4 Hz, J2 = 4.0 Hz, ArH),
7.23 (s, 1H, ArH), 7.13 (d, 1H, J = 4.0 Hz, ArH), 3.71 (q, 1H, J = 6.8 Hz, -CHCH3), 2.28 (s, 6H,
-N(CH3)2), 1.46 (d, 3H, J = 6.8 Hz, -CHCH3). MS(ESI): m/z = 217 [M+H]+.

ACCEPTED MANUSCRIPT
1
4.2.5.2 6-(1-(ethyl(methyl) amido) ethyl) quinolin-8-ol (8b). Brown oil (44%); H-NMR (δ,
CDCl3): 8.73 (d, 1H, J = 2.8, ArH), 8.10 (d, 1H, J = 8.4 Hz, ArH), 7.40 (dd, 1H, J1 = 8.4 Hz, J2 =
2.8 Hz, ArH), 7.27 (s, 1H, ArH), 7.25 (s, 1H, ArH), 3.68 (q, 1H, J = 6.4 Hz, -CHCH3), 2.42 (q, 2H,
J = 2.8, -N(CH3)CH2CH3), 2.16 (s, 3H, -N(CH3)CH2CH3), 1.43 (d, 3H, J = 6.4 Hz, -CHCH3), 1.04
(q, 3H, J = 2.8, -N(CH3)CH2CH3). MS(ESI): m/z = 231 [M+H]+.
4.2.5.3 6-(1-(diethylamino) ethyl) quinolin-8-ol (8c). Yellow solid (46%); mp: 65-67 ; 1H-NMR
(δ, CDCl3): 8.72 (s, 1H, ArH), 8.11 (d, 1H, J = 8.4 Hz, ArH), 7.39 (dd, 1H, J1 = 8.4 Hz, J2 = 5.2
Hz, ArH), 7.32 (s, 1H, ArH), 7.20 (d, 1H, J = 5.2 Hz, ArH), 3.91 (q, 1H, J = 6.0 Hz, -CHCH3),
2.57 (m, 4H, -N(CH2CH3)2), 1.42 (d, 3H, J = 6.0 Hz, -CHCH3), 1.01 (m, 6H, -N(CH2CH3)2).
MS(ESI): m/z = 245 [M+H]+.
1
4.2.5.4 6-(1-(pyrrolidin-1-yl) ethyl) quinolin-8-ol (8d). Yellow oil (45%); H-NMR (δ, CDCl3):
8.73 (d, 1H, J = 4.0, ArH), 8.08 (d, 1H, J = 8.0 Hz, ArH), 7.39 (dd, 1H, J1 = 8.0 Hz, J2 = 4.0 Hz,
ArH), 7.30 (s, 1H, ArH), 7.17 (s, 1H, ArH), 3.30 (q, 1H, J = 6.0 Hz, -CHCH3), 2.42 (m, 4H,
pyrrolidine), 1.77 (m, 4H, pyrrolidine), 1.46 (d, 3H, J = 6.0 Hz, -CHCH3). MS(ESI): m/z = 243
[M+H]+.
4.2.5.5 6-(1-(piperidin-1-yl) ethyl) quinolin-8-ol (8e). Light yellow solid (52%); mp: 104-106 ;
1H-NMR (δ, CDCl3): 8.74 (d, 1H, J = 4.4, ArH), 8.12 (d, 1H, J = 8.4 Hz, ArH), 7.41 (dd, 1H, J1 =
8.4 Hz, J2 = 4.4 Hz, ArH), 7.28 (s, 1H, ArH), 7.25 (s, 1H, ArH), 3.49 (q, 1H, J = 3.6 Hz, -CHCH3),
2.39 (m, 4H, piperidine), 1.55 (m, 4H, piperidine), 1.41 (m, 5H, -CHCH3, piperidine). MS(ESI):
m/z = 257 [M+H]+.
4.2.5.6 6-(1-morpholinoethyl) quinolin-8-ol (8f). Brown oil (45%); 1H-NMR (δ, CDCl3): 8.75 (s,
1H, ArH), 8.12 (d, 1H, J = 8.4 Hz, ArH), 7.34 (dd, 1H, J1 = 8.4 Hz, J2 = 5.2 Hz, ArH), 7.28 (s, 1H,
ArH), 7.26 (d, 1H, J = 5.2 Hz, ArH), 3.72 (m, 4H, morpholine), 3.41 (q, 1H, J = 6.4 Hz, -CHCH3),
2.36 (m, 4H, morpholine), 1.41 (d, 3H, J = 6.4 Hz, -CHCH3). MS(ESI): m/z = 259 [M+H]+.
4.2.5.7 6-(1-(4-methylpiperazin-1-yl) ethyl) quinolin-8-ol (8g). Yellow solid (45%); mp:
1
117-119 ; H-NMR (δ, CDCl3): 8.72 (d, 1H, J = 4.0, ArH), 8.09 (d, 1H, J = 8.0 Hz, ArH), 7.40
(dd, 1H, J1 = 8.0 Hz, J2 = 4.0 Hz, ArH), 7.27 (s, 1H, ArH), 7.24 (s, 1H, ArH), 3.44 (q, 1H, J = 6.4
Hz, -CHCH3), 2.47 (m, 8H, 1-methylpiperazine), 2.29 (s, 3H, -NCH3), 1.40 (d, 3H, J = 6.4 Hz,
-CHCH3). MS(ESI): m/z = 272 [M+H]+.
4.2.6 General procedure for the preparation of compounds 9a-n
Compound 8 (0.5mmol) was dissolved in 10mL THF, NaH (0.6mmol) was added. After the

ACCEPTED MANUSCRIPT
mixture was stirred at room temperature for 4h, R2COCl (0.6mmol)was added drop by drop, and
the mixture was continue to stirred at room temperature for 12h. After the solvent was evaporated,
20mL water was added to the mixture, and extracted with ethyl acetate (3×20mL). The organic
layer was separated and dried over Na2SO4. The solvent was evaporated and the residue was
purified by silica gel column chromatography (PE:AcOEt:TEA = 2:1:0.1) to afford 9.
4.2.6.1 6-(1-(dimethylamine) ethyl) quinolin-8-yl-dimethylcarbamate (9a).Yellow oil (48%);
1H-NMR (δ, CDCl3): 8.90 (d, 1H, J = 2.8 Hz, ArH), 8.12 (d, 1H, J = 8.0 Hz, ArH), 7.60 (s, 1H,
ArH), 7.51 (d, 1H, J = 8 Hz, ArH), 7.39 (d, 1H, J = 4 Hz, ArH), 3.50(q, 1H, J = 6.8 Hz, -CHCH3),
3.28 (s, 3H, -CON(CH3)2), 3.07 (s, 3H, -CON(CH3)2), 2.28 (s, 6H, -CHN(CH3)2), 1.46 (d, 3H, J =
6.8 Hz, -CHCH3). 13C NMR (125 MHz, DMSO-d6) δ 154.1, 149.2, 147.1, 140.5, 134.9, 128.2,
122.4, 120.6, 64.6, 41.9, 35.9, 28.7, 18.5. HRMS (ESI) m/z Calcd C16H22N3O2 (M+H) 288.1712,
found 288.1715.
4.2.6.2 6-(1-(dimethylamino) ethyl) quinolin-8-yl ethyl(methyl)carbamate (9b). Brown oil
1
(51%); H-NMR (δ, CDCl3): 8.91-8.90 (d, 1H, J = 4.0 Hz, ArH), 8.15-8.12 (d, 1H, J = 12.0 Hz,
ArH), 7.60 (s, 1H, ArH), 7.52-7.53 (d, 1H, J = 4.0 Hz, ArH), 7.41-7.38 (dd, 1H, J1 = 8.0 Hz, J2 =
4.0 Hz, ArH), 3.71-3.66 (m, 1H, -NCH2CH3), 3.46-3.52 (m, 2H, -NCH2CH3), 3.27 (s, 1.5H,
-NCH3), 3.07 (s, 1.5H, -NCH3), 2.28 (s, 6H, -NCH3), 1.48 (d, 3H, J = 4.0 Hz, -CHCH3), 1.38 (m,
1.5H, -NCH2CH3), 1.25 (m, 1.5H, -NCH2CH3). 13C NMR (125 MHz, DMSO-d6) δ 154.7, 145.0,
148.2, 143.1, 141.4, 135.9, 129.1, 123.2, 121.5, 65.5, 44.4, 43.2, 34.2, 19.9, 13.1. HRMS (ESI)
m/z Calcd C17H24N3O2 (M+H) 302.1869, found 302.1880.
4.2.6.3 6-(1-(dimethylamine) ethyl) quinolin-8-yl diethylcarbamate (9c). Yellow oil (58%);
1H-NMR (δ, DMSO-d6): 8.87(d, 1H, J= 4.0 Hz, ArH), 8.11 (d, 1H, J= 8.4 Hz，ArH), 7.57 (dd, 1H,
J1= 8.4 Hz, J2 = 4.0 Hz, ArH), 7.52 (s, 1H, ArH), 7.36 (d, 1H, J= 4.0 Hz, ArH), 3.66 (q, 2H, J =
7.2 Hz, -NCH2CH3), 3.40 (m, 3H, -CHN(CH3)2, -NCH2CH3), 2.26 (s, 6H, -CHN(CH3)2), 1.46 (d,
3H, J = 6.4 Hz, -CHCH3), 1.38 (t, 2H, J = 7.2 Hz, -CHCH3). 1.26 (t, 4H, J = 7.2 Hz, -CHCH3).
13C NMR (125 MHz, DMSO-d6) δ 154.6, 149.8, 148.2, 142.7, 141.6, 135.6, 129.4, 123.0, 121.5,
65.5, 43.2, 42.3, 29.7, 20.0, 14.5, 13.4. HRMS (ESI) m/z Calcd C18H26N3O2 (M+H) 316.2025,
found 316.2031.
4.2.6.4 6-(1-(dimethylamino) ethyl) quinolin-8-yl pyrrolidine-1-carboxylate (9d). Brown oil
(41%); 1H-NMR (δ, CDCl3): 8.91 (d, 1H, J= 4.0 Hz, ArH), 8.14 (d, 1H, J= 8.0 Hz, ArH), 7.61 (s,
1H, ArH), 7.54 (s, 1H, ArH), 7.38 (d, 1H, J= 8.0 Hz, ArH), 3.81 (t, 1H, J = 6.0 Hz, -CHN(CH3)2),
3.52-3.56 (m, 4H, pyrrolidine), 2.27 (s, 6H, -CHN(CH3)2), 1.42 (t, 3H, J = 6.0 Hz, -CHCH3), 1.23
(m, 4H, pyrrolidine). 13C NMR (125 MHz, DMSO-d6) δ 155.5, 149.8, 143.0, 135.5, 129.0, 121.6,
111.4, 101.0, 66.4, 36.2, 47.9, 43.3, 25.7, 20.1. HRMS (ESI) m/z Calcd C18H24N3O2 (M+H)
314.1869, found 314.1870.
4.2.6.5 6-(1-(dimethylamino) ethyl) quinolin-8-yl piperidine-1-carboxylate (9e). Brown oil
(46%); 1H-NMR (δ, CDCl3): 8.89 (d, 1H, J = 6.8 Hz, ArH), 8.12 (d, 1H, J = 8.4 Hz, ArH), 7.57(s,
1H, ArH), 7.51 (s, 1H, ArH), 7.40(m, 1H, ArH),3.81-3.56 (m, 4H, pyridine), 3.45-3.40 (t, 1H, J =

ACCEPTED MANUSCRIPT
6.0 Hz, -CHN(CH3)2), 2.26(s, 6H, -CHN(CH3)2), 1.69(s, 6H, pyridine), 1.44 (d, 3H, J = 6.8 Hz,
-CHCH3). 13C NMR (125 MHz, DMSO-d6) δ 154.0, 149.8, 148.4, 143.0, 141.4, 135.8, 129.2,
122.9, 121.5, 65.6, 46.0, 45.3, 43.2, 29.6, 25.9. HRMS (ESI) m/z Calcd C19H26N3O2 (M+H)
328.2025, found 328.2028.
4.2.6.6 6-(1-(dimethylamino) ethyl) quinolin-8-yl morpholine-4-carboxylate (9f). Yellow oil
(59%); 1H-NMR (δ, CDCl3): 8.91 (d, 1H, J = 4.4 Hz, ArH), 8.12 (d, 1H, J = 8.4 Hz, ArH), 7.60(dd,
1H, J1= 8.4 Hz, J2 = 4.4 Hz, ArH), 7.55 (d, 1H, J = 1.6 Hz, ArH), 7.41 (d, 1H, J = 1.6 Hz, ArH),
3.73 (s, 4H, morphine), 3.50 (m, 1H, J = 4.8 Hz, -CHN(CH3)2),3.21(s, 3H, -CHN(CH3)2),3.09 (s,
3H, -CHN(CH3)2), 2.57-2.44(m, 4H, morphine) 1.44 (d, 3H, J = 6.4 Hz, -CHCH3). 13C NMR (125
MHz, DMSO-d6) δ 154.5, 147.8, 142.6, 138.8, 134.8, 128.1, 120.7, 116.4, 106.1, 65.6, 55.0, 46.2,
42.7, 28.7, 19.4, 16.6. HRMS (ESI) m/z Calcd C18H24N3O3 (M+H) 330.1818, found 330.1823.
4.2.6.7 6-(1-morpholinoethyl) quinolin-8-yl dimethylcarbamate (9g). Orange oil (42%);
1H-NMR (δ, CDCl3): 8.90(d, 1H, J = 4.4 Hz, ArH), 8.12(d, 1H, J = 8.0 Hz, ArH), 7.60 (s, 1H,
ArH), 7.55 (d, 1H, J = 8.0 Hz, ArH), 7.40 (d, 1H, J2 = 4.0 Hz, ArH), 3.72 (m, 4H, morpholine),
3.50 (q, 1H, J = 5.6 Hz, -CHCH3), 3.31 (s, 3H, -NCH3), 3.09(s, 3H, -NCH3), 2.44-2.57 (m, 4H,
morpholine), 1.45 (d, 3H, J = 5.6 Hz, -CHCH3). 13C NMR (125 MHz, DMSO-d6) δ 155.1, 150.0,
148.0, 142.7, 141.4, 135.8, 129.2, 123.3, 121.6, 121.5, 67.0, 65.0, 51.2, 36.8, 29.7, 19.4. HRMS
(ESI) m/z Calcd C18H24N3O3 (M+H) 330.1818, found 330.1829.
4.2.6.8 6-(1-morpholinoethyl) quinolin-8-yl pyrrolidine-1-carboxylate (9h). Brown oil (43%);
1H-NMR (δ, CDCl3): 8.92 (d, 1H, J = 2.4 Hz, ArH), 8.15 (d, 1H, J = 8.4 Hz, ArH), 7.61 (s, 1H,
ArH), 7.43 (dd, 1H, J1 = 8.4 Hz, J2 = 2.4 Hz, ArH), 7.30 (s, 1H, ArH), 4.14 (m, 2H, pyrrolidine),
3.82 (m, 1H, pyrrolidine), 3.74 (m, 4H, morpholine), 3.56 (m, 1H, pyrrolidine), 3.57(m, 1H,
-CHCH3), 2.57-2.45 (m, 4H, morpholine), 2.00 (m, 4H, pyrrolidine), 1.45 (m, 3H, -CHCH3). 13C
NMR (125 MHz, DMSO-d6) δ 154.58, 149.0, 146.9, 140.4, 134.7, 128.2, 122.1, 120.8, 116.7,
109.5, 66.1, 64.0, 55.1, 50.5, 45.6, 28.7, 24.8, 24.0, 13.2. HRMS (ESI) m/z Calcd C20H26N3O3
(M+H)356.1974, found 356.1987.
4.2.6.9 6-(1-morpholinoethyl) quinolin-8-yl morpholine-4-carboxylate (9i). Yellow oil (61%);
1H-NMR (δ, CDCl3): 8.88 (d, 1H, J = 4 Hz, ArH), 8.11 (d, 1H, J = 8.4 Hz, ArH), 7.60 (s, 1H, ArH),
7.55 (s, 1H, ArH), 7.40 (m, 1H, ArH), 3.88-3.83 (m, 6H, morpholine), 3.71-3.64 (m, 6H,
morpholine), 3.50 (q, 1H, J = 6.4 Hz, -CHCH3), 2.55-2.44 (m, 4H, morpholine), 1.41 (d, 3H, J =
6.4 Hz, -CHCH3). 13C NMR (125 MHz, DMSO-d6) δ 154.1, 150.0, 148.0, 141.2, 135.8, 129.2,
123.5, 121.7, 121.3, 67.1, 66.6, 65.0, 51.3, 45.5, 44.5, 29.7, 19.5. HRMS (ESI) m/z Calcd
C20H26N3O4 (M+H) 372.1923, found 372.1930.
4.2.6.10 6-(1-(ethyl (methyl) amino) ethyl) quinolin-8-yl ethyl (methyl) carbamate (9j).
1
Yellow oil (55%); H-NMR (δ, CDCl3): 8.91 (d, 1H, J = 3.6 Hz, ArH), 8.10 (d, 1H, J = 8.4 Hz,
ArH), 7.43 (dd, 1H, J1 = 8.4 Hz, J2 = 3.6 Hz, ArH), 7.30 (s, 1H, ArH), 7.24 (s, 1H, ArH), 4.14 (m,
3.5H, -CONCH3, -CONCH2CH3), 3.71 (m, 1.5H, -CONCH3), 3.50 (m, 1H, -CHCH3), 2.58 (m, 2H,

ACCEPTED MANUSCRIPT
-NCH2CH3), 2.33 (s, 3H, -NCH3), 1.50 (d, 3H, J = 6 Hz, -CHCH3), 1.26 (m, 3H, -CONCH2CH3),
1.10 (m, 3H, -NCH2CH3). 13C NMR (125 MHz, DMSO-d6) δ 153.9, 148.8, 146.9, 142.7, 140.3,
134.7, 128.1, 121.8, 121.1, 120.4, 59.4, 43.4, 41.9, 20.1, 13.1, 11.1.HRMS (ESI) m/z Calcd
C18H26N3O2 (M+H) 316.2025, found 316.2031.
4.2.6.11 6-(1-(diethylamino) ethyl) quinolin-8-yl ethyl (methyl) carbamate (9k). Yellow oil
(54%); 1H-NMR (δ, CDCl3): 8.89 (d, 1H, J = 4.0 Hz, ArH), 8.12 (d, 1H, J = 8.0 Hz, ArH), 7.43 (s,
1H, ArH ), 7.30 (d, 1H, J = 6.8 Hz, ArH), 7.20(dd, 1H, J1 = 8.0 Hz, J1 = 6.8 Hz, ArH), 4.15(d, 4 H,
J = 6.0 Hz, -CONCH3, -CONCH2CH3), 3.37（m, 2H, -CHCH3, -CONCH2CH3）2.55 (s, 2H,
-NCH2CH3), 2.45(s, 2H, -NCH2CH3), 2.05(s, 2H, -CHNCH2CH3), 1.82(s, 4H, -CHNCH2CH3),
1.52 (d, 3H, J = 6.0 Hz, -CHCH3), 1.28(m, 3H, -CONCH2CH3). 13C NMR (125 MHz, DMSO-d6)
δ 154.9, 154.8, 149.6, 147.9, 144.1, 141.3, 135.6, 129.1, 122.6, 122.0, 121.3, 58.7, 45.0, 42.9, 35.6,
34.2, 17.1, 13.1, 12.3.HRMS (ESI) m/z Calcd C19H28N3O2 (M+H) 330.2182, found 330.2187.
4.2.6.12 6-(1-(pyrrolidin-1-yl) ethyl) quinolin-8-yl ethyl (methyl) carbamate (9l). Yellow oil
1
(52%); H-NMR (δ, CDCl3): 8.89 (d, 1H, J = 4.0 Hz, ArH), 8.10 (d, 1H, J = 8.8 Hz, ArH), 7.63
(dd, 1H, J1 = 8.8 Hz, J2 = 4.0 Hz, ArH), 7.30 (s, 1H, ArH), 7.20 (s, 1H, ArH), 4.15(s, 5H, -NCH3,
-NCH2CH3), 3.37 (q, 1H, J = 5.6 Hz, -CHCH3), 2.65-2.47 (m, 4H, pyrrolidine), 1.82 (m, 4H,
pyrrolidine), 1.52 (d, 3H, J = 5.6 Hz, -CHCH3), 1.28 (t, 3H, J = 6.8 Hz, -NCH3). MS(ESI): m/z =
328 [M+H]+. 13C NMR (125 MHz, DMSO-d6) δ 151.4, 149.1, 147.1, 141.6, 140.4, 134.8, 128.4,
121.3, 120.6, 120.3, 64.9, 59.3, 52.1, 43.5, 37.7, 33.15,22.4,12.21, 11.53. HRMS (ESI) m/z Calcd
C19H26N3O2 (M+H) 328.2025, found 328.2027.
4.2.6.13 6-(1-(piperidin-1-yl) ethyl) quinolin-8-yl ethyl (methyl) carbamate (9m). Yellow oil
(57%); 1H-NMR (δ, CDCl3): 8.87 (d, 1H, J = 3.8 Hz, ArH), 8.10 (d, 1H, J = 8.3Hz, ArH), 7.57 (s,
1H, ArH), 7.53 (d, 1H, J = 6.8 Hz, ArH), 7.37 (dd, 1H, J1= 8.2 Hz, J2 = 4.1 Hz, ArH), 3.68 (q, 1H,
J = 6.8 Hz, -NCHCH3), 3.56 (q, 1H, J = 6.8 Hz, -NCH2CH3), 3.48 (dd, 1H, J1= 13.9 Hz, J2 = 6.8
Hz, -NCH2CH3), 3.26 (s, 1.5H, -NCH3), 3.06 (s, 1.5H, -NCH3), 2.41 (m, 4H, piperidine), 1.57 (m,
4H, piperidine), 1.44 (d, 3H, J = 6.8 Hz, -CHCH3), 1.39 (m, 3.5H, piperidine，-CH2CH3), 1.25 (t,
1.5H, J = 6.8 Hz, -CH2CH3). 13C NMR (125 MHz, DMSO-d6) δ 154.8, 149.9, 147.9, 142.6, 141.3,
135.8, 129.2, 123.2, 121.9, 121.5, 64.9, 51.6, 44.5, 34.3, 29.7, 26.1, 24.5, 18.9, 13.2, 12.5. HRMS
(ESI) m/z Calcd C20H28N3O2 (M+H) 342.2182, found 342.2188.
4.2.6.14 6-(1-(4-methylpiperazin-1-yl) ethyl) quinolin-8-yl ethyl (methyl) carbamate (9n).
Yellow oil (40%); 1H-NMR (δ, CDCl3): 8.88 (d, 1H, J = 3.2 Hz, ArH), 8.12 (d, 1H, J = 8 Hz, ArH),
7.58 (s, 1H, ArH), 7.53 (d, 1H, J = 8 Hz, ArH), 7.39 (dd, 1H, J1 = 8.0 Hz, J2 = 3.2 Hz, ArH), 4.13
(q, 1H, J = 6.8 Hz, -CHCH3), 3.67 (q, 1H, J = 7.2 Hz, -NCH2CH3), 3.27 (m, 2.5H, -CONCH3,
-NCH2CH3), 3.06 (s, 1.5H, -CONCH3), 2.51 (m, 8H, 1-Methyl-piperazine), 2.32 (s, 3H, -NCH3),

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1.43 (d, 3H, J = 6.8 Hz, -CHCH3), 1.41 (t, 1.5H, J = 7.2 Hz, -NCH2CH3), 1.12 (t, 1.5H, J = 7.2 Hz,
-NCH2CH3). 13C NMR (125 MHz, DMSO-d6) δ 154.7, 149.9, 148.0, 142.7, 141.44, 135.7, 129.2,
123.2, 121.4, 64.4, 55.4, 50.2, 45.5, 44.5, 44.0, 34.3, 19.6, 13.2, 12.4. HRMS (ESI) m/z Calcd
C20H29N4O2 (M+H) 357.2291, found 357.2300.
Acknowledgments
This work was supported by Natural Science Foundation of Zhejiang Province (LY17H310008
and LQ17H300003), Health Commission of Zhejiang Province (XKQ-010-001, WJK-ZJ-1918
and 2019KY365), and the State Key Laboratory of Medicinal Chemical Biology.
Authors' contributions
Wenhai Huang designed the study, and drafted the manuscript; Meihao Liang, Chixiao Zhang,
Beibei Wang and Zhimin Zhang performed chemistry; Li Tang performed Aβ Aggregation
Inhibition; Qiao Wang performed PK; Qin Li and Xiaoliang Zheng performed animal models;
Zhengrong Shen revised the manuscript; All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
List of abbreviations:
AChE:acetylcholinesterase
Aβ:amyloid-β
BBB: Blood Brain Barrier
AD:Alzheimer’s disease
ACh:acetylcholine
MTDL:Multi-Target-Directed Ligand
Riva:Rivastigmine
TEM:transmission electron microscopy
ThT:Thioflavin T
PK:pharmacokinetic
MRT:Mean Retention Time
AUC:Area Under Curve
CQ:Clioquinol
Scop:scopolamine
No Observed Adverse Effect Levels:NOAELs
DRR:Dyskinesia Recovery Rate

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RE:Response Efficiency
hERG:the human Ether-à-go-go-Related Gene
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A total of 18 quinoline derivatives were synthesized. Both in vitro and in vivo results demonstrated
that compound 9b could cross BBB, then release 8a into brain. 9b could inhibit AChE, while 8a
displayed metal ion chelating function, therefore in combination, both 9b and 8a exhibited a
considerable inhibition of Aβ aggregation, one of the observations that plays important roles in the
pathogenesis of AD. The efficacy of 9b against AD was further investigated in both a zebrafish
model and two different mice models.