Synthesis, in vitro and in vivo biological evaluation of novel graveolinine derivatives as potential anti-Alzheimer agents

Article abstract of DOI:10.1016/j.bmc.2019.115190

A novel series of graveolinine derivatives were synthesized and evaluated as potential anti-Alzheimer agents. Compound 5f exhibited the best inhibitory activity for acetylcholinesterase (AChE) and had surprisingly potent inhibitory activity for butyrylcho

Full text of DOI:10.1016/j.bmc.2019.115190

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Synthesis, in vitro and in vivo biological evaluation of novel graveolinine de-
rivatives as potential anti-Alzheimer agents
Wen Luo, Jian-Wu Lv, Ting Wang, Zhi-Yang Zhang, Hui-Yan Guo, Zhi-Yi
Song, Chao-Jie Wang, Jing Ma, Yi-ping Chen
PII:
S0968-0896(19)31182-4
https://doi.org/10.1016/j.bmc.2019.115190
BMC 115190
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Bioorganic & Medicinal Chemistry
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Accepted Date:
11 July 2019
28 October 2019
29 October 2019
Please cite this article as: W. Luo, J-W. Lv, T. Wang, Z-Y. Zhang, H-Y. Guo, Z-Y. Song, C-J. Wang, J. Ma, Y-p.
Chen, Synthesis, in vitro and in vivo biological evaluation of novel graveolinine derivatives as potential anti-
Alzheimer agents, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115190
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Synthesis, in vitro and in vivo biological evaluation of novel graveolinine
derivatives as potential anti-Alzheimer agents
Wen Luo §,a, Jian-Wu Lv §,a, Ting Wang a, Zhi-Yang Zhang a, Hui-Yan Guo a, Zhi-Yi Song b, Chao-
Jie Wang a, Jing Ma b,*, Yi-ping Chen c,*
a Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004,
People’s Republic of China
b Institute for Innovative Drug Design and Evaluation, Henan University, Kaifeng 475004, People’s
Republic of China
c
School of Pharmaceutical Sciences, Guangxi University of Chinese Medicine, Nanning 530001,
People’s Republic of China
* Jing Ma: Email majing.1988.ok@163.com, Tel 086 0371-23880680, Fax 086 0371-23880680
* Yi-ping Chen: Email yipchen2009@foxmail.com, Tel 086 0771-4733943, Fax 086 0771-4953042
§ The first two authors contributed equally to this work.
ABSTRACT
A novel series of graveolinine derivatives were synthesized and evaluated as potential anti-Alzheimer
agents. Compound 5f exhibited the best inhibitory activity for acetylcholinesterase (AChE) and had
surprisingly potent inhibitory activity for butyrylcholinesterase (BuChE), with IC50 values of 0.72 M
and 0.16 M, respectively. The results from Lineweaver–Burk plot and molecular modeling study

indicated non-competitive inhibition of AChE by compound 5f. In addition, these derivatives showed
potent self-induced -amyloid (A) aggregation inhibition. Moreover, 5f didn’t show obvious toxicity
against PC12 and HepG2 cells at 50 M. Finally, in vivo studies confirmed that 5f significantly
ameliorates the cognitive performances of scopolamine-treated ICR mice. Therefore, these
graveolinine derivatives should be thoroughly and systematically studied for the treatment of
Alzheimer’s disease.
KEYWORDS
Graveolinine
Acetylcholinesterase
Butyrylcholinesterase
-amyloid
Alzheimer’s disease

1. Introduction
Alzheimer’s disease (AD) is one of the most common diseases in elderly people and is
characterized by irreversible neuronal damage that causes dementia, cognitive impairment, and
memory loss [1]. The observation of a deficiency in cholinergic neurotransmission in AD led to the
cholinergic hypothesis [2]. According to the hypothesis, the current mainstays of AD treatment have
focused on acetylcholinesterase (AChE) inhibitors aimed at increasing acetylcholine (ACh) levels in
the brain [3]. Acetylcholinesterase inhibitors represent a well-established class of drugs for the
symptomatic treatment of AD, which include donepezil, rivastigmine, galantamine and huperzine A.
Among them, galantamine and huperzine A are naturally-occurring alkaloids from the genus Galanthus
(Amaryllidaceae) and the club moss Huperzia serrata (Lycopodiaceae), respectively.
Two major cholinesterases (ChEs), AChE and butyrylcholinesterase (BuChE), are involved in the
hydrolysis and regulation of ACh in vertebrates [4]. Various cholinergic drugs, especially AChE
inhibitors also function as BuChE inhibitors. Moreover, the use of agents with enhanced selectivity for
BuChE, including MF-8622 and cymserine indicated the potential therapeutic benefit of inhibiting
BuChE in AD and related dementias [5]. BuChE specific inhibition is unlikely to be associated with
adverse events and may show efficacy without remarkable side effects [6]. Therefore, BuChE may be
considered as an important target for novel drug development to treat AD. In the future, the
development of specific BuChE inhibitors and the continued use of ChEs inhibitors may lead to
improved clinical outcomes [7].
Graveolinine (Fig. 1) belongs to the 2-phenyl quinolone compounds, alkaloids isolated from Ruta
graveolens L. (Common Rue Herb) in the south of China [8]. It is an isomer of graveoline which is
also an alkaloid from ethanolic extract of Ruta graveolens. Graveolinine can be converted to graveoline

in DMF/MeI at 80 C [9]. Although a few of studies have reported its antibacterial, spasmolytic and
anti-tumor activities [10-12], studies regarding the synthesis and biological activity of graveolinine
derivatives are rare, especially there are almost no reports about the anti-AD activities of graveolinine
and its derivatives. Although in 2016, there was a report by Li et al about the synthesis and AChE
inhibitory activity of graveoline analogs [13], actually, these analogs don’t belong to graveolinine
derivatives.
O
O
O
O
O
O
N
N
Graveolinine
Graveoline
O
O
O
NH2
N
N
O
Cl
O
O
Flavonoid
Tacrine
Berberine
Figure 1. Chemical structures of graveolinine, graveoline, flavonoid, tacrine and berberine.
Our group has focused on the development of ChEs inhibitors as anti-AD agents for many years.
We have previously studied flavonoids and their derivatives as ChEs inhibitors and found that these
derivatives showed moderate inhibitory potency, but lacked good solubility and contained too many
flexible rotation bonds [14-16]. Therefore, we are now focused on the development of more potent
ChEs inhibitors isolated or derived from natural products. Our search indicated graveolinine might be
of interest because its structure is similar to that of flavonoids, two aromatic rings connected by one
rotating bond, and to tacrine (Fig. 1), which contains a quinolone ring. Moreover, the structure of
graveolinine is also similar with berberine, which contains a dioxymethylene group, showed
significant inhibitory activities for both ChEs (IC50 value: 0.37 M for eeAChE, 18.2 M for eqBuChE)

and -amyloid (A) aggregation [17-18].
Based on these similar structure features, a series of graveolinine derivatives with 4-methoxyl group
modified on the quinolone ring were designed and synthesized. To explore the structure-activity
relationship, some compounds lacking dioxymethylene were also synthesized. These were then
evaluated for their biological activity, including in vitro ChEs and A aggregation inhibitory activities,
cytotoxicity towards human hepatoma cell line HepG2 and rat pheochromocytoma cell line PC12. The
in vivo pro-cognitive effects of 5f were also evaluated through the application of scopolamine in mice
and exposing to Morris water maze (MWM).
2. Results and discussion
2.1. Chemistry
O
O
O
H
R1
a
b
N
N
H
R1
O
NH2
3a,3b
1
2a,2b
R2
R3
N
Cl
N
c
d
N
R1
R1
5a-5h
6a-6f
R1= 3,4-methylenedioxy
R1= H
4a,4b
Scheme 1. Synthesis of graveolinine derivatives. Reagents and conditions: (a) piperonyloyl chloride or benzoyl
chloride, CH2Cl2, Et3N, r.t. 24 h; (b) (CH3)3COK, (CH3)3COH, 75 C, 16 h; (c) POCl3, reflux, 4 h; (d) amines,
K2CO3, 135 C, 4 h.
The synthetic route for the graveolinine derivatives starting from commercially available 1-(2-
aminophenyl) ethanone (1) was outlined in Scheme 1. Condensation of starting material (1) with
piperonyloyl chloride or benzoyl chloride in the presence of Et3N in CH2Cl2 at room temperature

produced 2a and 2b. The ketone (2a or 2b) was treated with strong base (CH3)3COK to obtain
intermediates (3a or 3b) in satisfactory yield. The chlorination of 3a or 3b with POCl3 provided 4a or
4b in good yields. Finally, the reaction of 4a or 4b with different amines produced the targets
1
compounds 5a-5h and 6a-6f in 51-83% yields. All the compounds were characterized by H NMR,
13C NMR and MS.
2.2. Inhibition studies on AChE and BuChE
The AChE and BuChE inhibition effects of the target compounds were determined using the
spectroscopic method described by Ellman et al [19]. Rivastigmine, a well-known cholinesterase
inhibitor, was used as the positive control. The precursor compound graveolinine was also evaluated
for comparative purpose. The results are listed in Table 1, expressed as IC50 values.
Almost all the tested target compounds showed moderate inhibitory activity for both AChE and
BuChE. The precursor compound graveolinine presented weak ChEs inhibitory potency. Interestingly,
compound 5f possessed a noteworthy BuChE inhibitory activity with IC50 of 0.16 M. The results
showed that introduction of the pyrrolidine group at the 4-position increased the ChEs inhibitory
capacity and improved the selectivity for BuChE over AChE. The selective BuChE inhibitors may
provide therapeutic advantages in patients with advanced stages of AD. Because in AD, the AChE
level in the brain decreases progressively, but BuChE activity remains the same or increases up to 165%
of the normal level. As shown in Table 1, the structure of terminal group of side chain significantly
affected the inhibitory activities for BuChE. The potencies to inhibit BuChE were in the order 5d, 6d
(piperidine substituted) > 5e, 6e (morpholine substituted) > 5a, 6a (hydroxyl substituted). The
screening data also showed that the methylene chain length had significant effects on BuChE inhibitory
activities, the order of potencies is 5c, 6c (6 methylenes) > 5b, 6b (4 methylenes) > 5a, 6a (3

methylenes).
For AChE inhibition, the precursor compound graveolinine did not show potent inhibitory activity,
but introduction of the substituent group (NR2R3) increased the AChE inhibitory capacity. The results
revealed that variation of the substituted group had a smaller influence on their inhibition of AChE
compared with BuChE. Compounds 5f and 6f, containing a pyrrolidine group, exhibited the best
inhibitory activity for AChE, with IC50 values of 0.72 M and 3.71 M, respectively.
Compounds with the same substituted group (such as 5a and 6a), but different mother nucleus were
compared. Compounds containing a graveolinine nucleus (5a-5f) had a greater AChE inhibitory
activity compared with compounds containing a 2-phenyl quinolone nucleus (6a-6f). This indicated
that dioxymethylene is a significant for AChE inhibition activity.
In addition, compounds 5f and 5g attracted our interest, when the pyrrolidine of compound 5f was
replaced with an piperidine group to form compound 5g, the resulting inhibition activity for both ChEs
was decreased about 10-fold compared with 5f. This finding might be explained by an increase in
steric hindrance of the piperidine group, which would prevent compound 5g to interact optimally with
the residues in the binding site of ChEs.
R2
R3
R2
R3
O
N
N
O
O
O
O
N
N
N
Graveolinine
5a-5h
6a-6f
Table 1. Inhibition of ChEs activity and selectivity index of the target compounds
IC50a for
IC50b for
A inhibition
Compds.
5a
SIc
NR2R3
(%) d
AChE (M) BuChE (M)
H
4.23±0.20
4.26±0.12
>10
<0.42
0.44
36.61±1.79
44.13±3.71
N
OH
H
N
5b
9.78±0.28
OH

H
N
5c
5d
5e
5f
4.32±0.07
4.78±0.13
3.22±0.17
0.72±0.06
>10
6.49±0.33
1.45±0.08
8.15±0.39
0.16±0.01
2.09±0.12
7.85±0.12
>10
0.67
3.3
39.37±5.68
65.59±4.29
61.64±4.63
62.52±2.93
48.55±2.38
30.63±2.56
36.80±0.84
40.71±1.61
29.16±3.18
60.31±5.10
55.51±2.44
57.68±3.87
OH
H
N
N
O
H
0.40
4.5
N
N
N
N
5g
5h
6a
6b
6c
6d
6e
6f
>4.8
0.61
-
H
4.80±0.21
>10
N
H
N
OH
H
N
2.3
9.851.35
6.220.37
>10
4.310.16
2.960.11
1.190.09
4.440.21
0.430.05
OH
H
N
2.1
OH
H
N
>8.4
>2.3
8.6
N
O
H
N
>10
N
3.710.24
N
Graveolinine
Rivastigmine
Curcumin
-
1.3
4.2
-
26.92±3.33
-
36.31.40
6.35±1.50
-
28.20.84
1.51±0.09
-
-
-
52.26±3.30
a
b
c
AChE from electric eel; BuChE from equine serum; Selectivity Index = IC50 (AChE) / IC50 (BuChE); d The
thioflavin-T (ThT) fluorescence method was used and the compounds were used at a concentration of 20 μM. Data
are represented as mean ± SD.
2.3 Kinetic characterization of AChE inhibition
We cultured the crystal of 5f with Torpedo californica AChE to elucidate the interaction patterns of
5f with AChE; though, no positive results were obtained. The mechanism of inhibition of AChE was
investigated using the derivative 5f, the most potent AChE inhibitor. Steady state inhibition data of
compound 5f for AChE is shown in Figure 2. Lineweaver-Burk reciprocal plots revealed an increasing

slope and an increasing intercept at higher inhibitor concentrations, and the four lines intersected at
the x-axis. This pattern indicates a non-competitive inhibition which is similar with that of berberine
[20].
Figure 2. Lineweaver-Burk plot and docking model for compound 5f with AChE.
2.4 Molecular modeling
To investigate the possible mode of compound 5f with TcAChE (PDB code: 1ACJ), molecular
modeling was carried out by AUTODOCK 4.0 package shown in Figure 2 [21]. In the 5f-TcAChE
complex, compound 5f occupied the peripheral anionic site (PAS, Asp72, Trp279, Tyr121, Tyr334),
the quinolone moiety establishes a cation- interaction with Trp279, the ring-to-ring distance was 4.0
Å, and the nitrogen atom of pyrrolidine had a hydrogen bond (4.2 Å) interaction with the carbonyl
group of Ser286. The result showed that compound 5f was able to bind PAS of AChE which was in
agreement with the result of kinetic study.
2.5. Inhibition of self-mediated A1-42 aggregation by Thioflavin-T (ThT)
A1-42 and A1-40 are the main forms of A peptides found in amyloid plaques, A1-42 is more
fibrillogenic than A1-40 [22], which inspired us to select A1-42 for evaluating the inhibitory activities
of the derivatives using a ThT assay. The results suggested that these derivatives prevented self-

mediated Aβ1-42 aggregation with percentages of inhibition ranging from 29-66% at 20 M. The most
potent compounds were 5d–f and 6d–f, with percentages of inhibition greater than 50%, which were
better than the reference compound curcumin (52%, Table 1). Compounds 5a-c, 6a-c and 5h coupled
with alkamines or n-butylamine showed moderate or low inhibitory activity. These results implied that
side chain moiety gave a big influence resulting in the inhibitory activity for A1-42 aggregation.
Figure 3. TEM images of Aβ1-42 self-induced aggregation in the presence and absence of test compounds. (A) 50 μM
Aβ1–42 without inhibitors at 0 h, (B) 50 μM Aβ1–42 without inhibitors at 48 h, 37 °C, (C) 50 μM Aβ1–42 and 20 μM 5f,
(D) 50 μM Aβ1–42 and 20 μM curcumin.
Compound 5f showed considerably potent inhibition for both ChEs and Aβ aggregation, therefore,
it was selected as a representative compound and further evaluated by Transmission Electron
Microscopic (TEM) study on Aβ1-42 aggregation potential. Fig 3A represents the Aβ1-42 aggregation at
the start of the incubation at 37 °C without any inhibitor. After 48 h of incubation, the Aβ1-42 had
mostly aggregated into a firm and dense amyloid fibrils (Figure 3B). The inhibitory effect was clear
when treated with the compound 5f at 48 h (20 μM) and lesser numbers of Aβ aggregates were visible
(Figure 3C) compared to the treatment with standard curcumin (Figure 3D). The results of TEM are
consistent with the ThT assay where compound 5f potentially inhibit Aβ self-induced aggregation.
2.6. In vitro cytotoxicity
Tacrine, a well-known AChE inhibitor, was withdrawn from the market for its severe reversible
hepatotoxicity. Our compounds’ structure are similar to tacrine, therefore, to investigate the effect of

the target compounds on cell viability, MTT assay was performed using the human hepatoma cell line
HepG2 and the rat pheochromocytoma cell line PC12. The results showed that most of the compounds
(except for 5d, 6d and 6f) exhibited no obvious toxicity with inhibition rate less than 20% on cell
viability at 10 µM. Among them, the IC50 of compounds 5a, 5c, 5f, 5g, 6a, and 6b were more than 50
µM for both HepG2 and PC12 cell lines, it means they didn’t show obvious toxicity at concentration
of 50 µM (Table 2).
Table 2. Inhibition rate (%)a and IC50 b of HepG2 and PC12 cells
HepG2
PC12
Compds.
NR2R3
µM
µM
10
IC50
µM
10
IC50
µM
Inhibition (%)
Inhibition (%)
(
)
(
)
H
N
5a
5b
5c
5d
5e
5f
<5
>50
>50
8.38 0.12
>50
34.26±2.48
>50
OH
±
H
N
<5
<5
14.68 0.13
±
OH
H
N
>50
<5
OH
H
N
43.70 0.72
9.21 ± 1.98
17.71 ± 2.34
>50
71.47 0.12
7.17 ± 2.35
>50
±
±
N
O
H
<5
<5
<5
<5
<5
<5
N
N
>50
N
N
5g
5h
6a
6b
6c
6d
6e
>50
>50
H
N
15.44 0.82
>50
11.24 0.88
46.24±3.08
>50
±
±
H
<5
<5
>50
<5
<5
N
OH
H
N
>50
>50
OH
H
N
12.03 0.34
44.19±2.87
25.74±2.45
30.06±3.63
<5
>50
±
OH
H
N
24.1 1.33
40.50±2.49
35.23±2.71
±
11.55 ± 0.16
12.56 ± 0.19
N
O
H
N
12.71 0.93
±
N

6f
30.86 ± 1.66
>50
24.22 ± 0.85
<5
24.74±1.83
>50
12.6 ± 0.44
8.76 ± 0.63
N
Graveoli
nine
-
b
aInhibition rate＝(AControl-ASample)/(AControl-ABlank)100%, IC50 values represented the concentration causing 50%
growth inhibition after treatment for 48 h. The values are the mean  SD of three independent experiments.
2.7. ADMET prediction
The in silico prediction of the physicochemical and Absorption, Distribution, Metabolism,
Elimination, and Toxicity (ADMET) properties of compound 5f were calculated using the ACD/Labs
Percepta Platform [23] (License#58830) and the results were compared to those obtained for donepezil
and tacrine. As shown in Table 3, the compound 5f had a similar druglikeness to donepezil, with no
violations of Lipinski's rule of five. The solubility of compounds 5f was calculated as 0.01 mg/mL,
however, it was determined that the solubility of 5f in PBS (0.1 mM, pH=7.4) was 0.26 mg/mL. In
addition, compound 5f was showed to be highly or moderately permeable based on the predicted Log
P, permeability across Caco-2 monolayers (Pe) and human intestinal absorption (HIA) test. The score
of compounds’ Toxicity was predicted as undefined (score > 0.33 and < 0.67), so the data was not
shown.
Table 3. Physico-chemistry properties and ADMET profile of compounds 5f, donepezil and tacrine calculated using
the Program ACD/Percepta.
Compounds
Predicted properties
5f
Donepezil
Tacrine
MW (g/mol)
H-Donors
H-Acceptor
Rot. Bonds
Rings
317.38
379.49
198.26
0
0
2
3
4
2
2
6
0
5
0
4
3
0
Lipinski
0
Solubility
Log P
0.01mg/mL
4.69
6.43 mg/mL
4.23
6 mg/mL
2.86

Pe=23710-6
Pe=19410-6
Pe=6310-6
Caco-2
cm/s
cm/s
cm/s
PPB
CNS
HIA
99%
-2.98
100%
95%
-2.65
100%
78%
-2.59
100%
2.8. Behavioral studies
Cognition-improving potency is utmost importance for anti-AD agents. The in vitro cholinesterase
inhibitory activities must translate into behavioral effects and improve the cognition in vivo. The
ameliorating potential of 5f against scopolamine-induced cognition impairment in ICR mice was
investigated in MWM [24]. The results show that the treatment with 5f (20 μmol/kg body weight)
significantly ameliorated the cognition impairment in mice (Figure 4). In MWM test, mice in control
group exhibited a reduction of mean escape latency from 62.2 to 42.3s (p < 0.01) over the course of 4
training days. Compared to the model group, the 5f-treated and THA-treated group showed
significantly shortened escape latency in the 4th day of training trial sessions (p < 0.05); the compound
5f reduced the mean escape latency (MEL) from 60.2 to 48.1 s while THA decreased the MEL from
59.8 to 48.3 s (p < 0.05) (Figure 4A). Furthermore, in the probe trial on day 15, the administration of
5f significantly improved the overall target quadrant preference (the number to cross the platform, the
percentage of path length in the target quadrant, and the swimming time in the target quadrant)
compared to the model group (p < 0.05) (Figure 4B-D), while no significant differences were observed
between the two groups in the total distance traveled and swimming speed (Supporting Information,
Figure S3). These results indicate that 5f significantly ameliorated the cognition impairment in the
scopolamine treated mice probably by penetrating the blood−brain barrier to affect the central nervous
system.

Figure 4. Effect of 5f treatment on spatial memory performance. (A) Escape latencies during acquisition training
trials measured for 90 s for each trial; (B) The number to cross the platform in probe trial without platform; (C) The
percentage of path length in the target quadrant measured for 90 s in probe trial without platform; (D) The swimming
time in the target quadrant measured for 90 s in probe trial without platform. Data are presented as mean ± S.D. (n=8;
*p<0.05, **p<0.01 vs control group, and #p<0.05, ##p<0.01 vs model group).
3. Conclusion
In conclusion, a series of graveolinine derivatives were synthesized and evaluated for anti-AD
agents. Among these compounds 5a-h and 6c-e were novel. The results demonstrated that most of
these compounds exhibited moderate inhibitory activities against AChE and BuChE, with IC50 values
in the sub-micromolar range. In particular, compound 5f exhibited the best inhibitory activity for
AChE and had surprisingly potent inhibitory activity for BuChE, which is remarkably stronger than
Rivastigmine. Lineweaver-Burk plot and molecular modeling studies indicated compound 5f showed
a non-competitive inhibition with AChE. These derivatives also showed significant self-induced A

aggregation inhibition. In addition, 5f had a druglikeness similar to donepezil as calculated by ACD,
with no violations of Lipinski’s rule of five. The derivative 5f didn’t show obvious toxicity towards
HepG2 and PC12 cell lines at 50 μΜ. Furthermore, administration of 5f considerably ameliorated the
cognition impairment in the scopolamine treated ICR mice in MWM test.
Taking together, these graveolinine derivatives especially 5f could be a promising lead compound
for further development of newer anti-AD drugs.
4. Experimental section
4.1 Chemistry
All chemicals (reagent grade) were purchased from J&K and Sigma-Aldrich (China). Graveolinine
was synthesized in our lab (Supporting Information). Flash column chromatography was performed
with silica gel (200 - 300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Melting points
1
13
(mp) were determined using an X-6 hot stage microscope and were not corrected. H and C NMR
spectra data were obtained from a Bruker AV-300 or Bruker AV-400 spectrometer at 300 or 400 MHz
and 75 or 100 MHz, respectively. MS spectra were recorded on a Shimadzu LCMS-2010A instrument
with an ESI mass selective detector. Elemental analyses were performed on a Gmbe VarioEL
Elemental Instrument.
4.2 General procedure for the synthesis of the intermediates (2a, 2b)
To a mixture of 1 (1.35 g, 10.0 mmol) and Et3N (10 mL) in CH2Cl2 (100 mL) was added slowly
piperonyloyl chloride or benzoyl chloride (11.0 mmol) at room temperature. The mixture was stirred
for 24 h and poured into 100 mL ice water. The precipitate was collected and washed with water and
then with methanol, the solid was dried under vacuum and used without further purification.
4.3 General procedure for the synthesis of the intermediates (3a, 3b)

The solid above (2a or 2b) was suspended in 150 mL of tert-butyl alcohol. Potassium tert-butoxide
(15.0 g, 134 mmol) was added, and the mixture was heated at 75 C for 16 h. The mixture was cooled
and poured into 50 mL of ice water. 10% aqueous HCl was added until pH=6. The solid was collected
and washed with water three times. The crude product was recrystallized from CH2Cl2 and petroleum
and afforded the 3a or 3b. Intermediates (3a and 3b) were routinely checked by TLC and 1H NMR,
and subjected to the subsequent synthesis without further purification.
2-(benzo[d][1,3]dioxol-5-yl)quinolin-4(1H)-one (3a)
Dark yellow crystal, yield: 80%. 1H NMR (400 MHz, DMSO-d6) δ 8.24 (d, J = 8.4 Hz, 2H, 2Ar-
H), 7.95 (t, J = 7.8 Hz, 1H, Ar-H), 7.65 (t, J = 7.2 Hz, 1H, Ar-H), 7.60 (d, J = 1.8 Hz, 1H, Ar-H), 7.52
(d, J = 8.0, Hz, 1H, Ar-H), 7.31 – 7.13 (m, 2H, 2Ar-H), 6.20 (s, 2H, OCH2O), 5.51 (s, 1H, NH).
2-phenylquinolin-4(1H)-one (3b)
Light yellow crystal, yield: 82%, 1H NMR (400 MHz, DMSO-d6) δ 8.10 (d, J = 8.0 Hz, 1H, Ar-H),
7.86 (d, J = 3.6 Hz, 1H, Ar-H), 7.84 (d, J = 2.2 Hz, 1H, Ar-H), 7.81 (d, J = 8.4 Hz, 1H, Ar-H), 7.67 (t,
J = 7.6 Hz, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.58 (d, J = 2.6 Hz, 2H, 2Ar-H), 7.34 (t, J = 7.6 Hz, 1H,
Ar-H), 6.36 (s, 1H, Ar-H).
4.4 General procedure for the synthesis of the intermediates (4a, 4b)
Compound 3 (10 mmol) and POCl3 (50 mL) were stirred in ice bath for 30 min, and refluxed for
further 4 h. After cooling, the mixture was poured into 200 mL of ice water. The saturated solution of
NaOH was added until pH=7. The mixture was extracted with CH2Cl2 (3 × 100 mL), and the combined
organic layer was dried with MgSO4 and evaporated. The crude residue was purified by silica gel
column chromatography using CH2Cl2-petroleum (1:5) as an eluent to afford 4a or 4b.
2-(benzo[d][1,3]dioxol-5-yl)-4-chloroquinoline (4a)

Light white feathery crystal, Yield: 41%, m.p. 63-65 C. 1H NMR (300 MHz, Chloroform-d) δ 8.17
(t, J = 7.5 Hz, 2H, 2Ar-H), 7.86 (s, 1H, Ar-H), 7.75 (t, J = 7.8 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H),
7.65 – 7.57 (m, 2H, Ar-H), 6.93 (d, J = 8.1 Hz, 1H, Ar-H), 6.04 (s, 2H, OCH2O).
4-chloro-2-phenylquinoline (4b)
Pale solid, Yield: 56%, m.p. 122-124 C. 1H NMR (300 MHz, Chloroform-d) δ 8.36 (d, J = 8.4 Hz,
1H, Ar-H), 8.25 (d, J = 8.4 Hz, 1H, Ar-H), 8.17 (d, J = 7.2 Hz, 2H, 2Ar-H), 7.99 (s, 1H, Ar-H), 7.82
(t, J = 7.5 Hz, 1H, Ar-H), 7.70 – 7.62 (m, 1H, Ar-H), 7.58 - 7.48 (m, 3H, 3Ar-H).
4.5 General procedure for the synthesis of the target compounds (5a-5h, 6a-6f)
A mixture of intermediates (4a or 4b, 1 mmol), K2CO3 (2 mmol) and amine (2 mmol) were stirred
at 135 C under for 4 h. After cooling, 10 mL of distillated water was added, the precipitate was filtered
and dried, then purified by silica gel column chromatography using CH2Cl2/MeOH (20:1) as an eluent.
4.5.1 3-((2-(benzo[d][1,3]dioxol-5-yl)quinolin-4-yl)amino)propan-1-ol (5a)
Sandybrown solid, yield: 51%,m.p. 126-128 C. 1H NMR (300 MHz, Methanol-d4) δ 8.00 (d, J =
8.4 Hz, 1H, Ar-H), 7.87 (d, J = 8.4 Hz, 1H, Ar-H), 7.61 (t, J = 7.8 Hz, 1H, Ar-H), 7.46 – 7.35 (m, 3H,
3Ar-H), 6.90 (d, J = 8.7 Hz, 1H, Ar-H), 6.75 (s, 1H, Ar-H), 5.99 (s, 2H, OCH2O), 3.75 (t, J = 6.0 Hz,
2H, CH2), 3.49 (t, J = 7.0 Hz, 2H, CH2), 1.98 (p, J = 6.4 Hz, 2H, CH2). 13C NMR (75 MHz, Methanol-d4)
δ 157.9, 151.8, 148.7, 148.1, 147.1, 134.3, 129.5, 127.0, 124.0, 121.7, 120.5, 117.8, 107.8, 107. 7,
101.4, 95.8, 59.5, 40.0, 30.9. ESI-MS m/z: 323.2 [M+H]+. Elemental Anal. Calcd for C19H18N2O3: C,
70.79; H, 5.63; N, 8.69. Found: C, 71.06; H, 5.83; N, 8.41.
4.5.2 4-((2-(benzo[d][1,3]dioxol-5-yl)quinolin-4-yl)amino)butan-1-ol (5b)
Sandybrown solid, yield: 62.2%, m.p. 118-120 C. 1H NMR (300 MHz, Methanol-d4) δ 8.07 (d, J
= 8.4 Hz, 1H, Ar-H), 7.89 (d, J = 8.1 Hz, 1H, Ar-H), 7.62 (t, J = 7.8 Hz, 1H, Ar-H), 7.48 – 7.44 (m,

2H, 2Ar-H), 7.40 (t, J = 7.5 Hz, 1H, Ar-H), 6.93 (d, J = 8.7 Hz, 1H, Ar-H), 6.74 (s, 1H, Ar-H), 6.02
(s, 2H, OCH2O), 3.65 (t, J = 6.3 Hz, 2H, CH2), 3.43 (t, J = 6.9 Hz, 2H, CH2), 1.91 – 1.81 (m, 2H, CH2),
1.76 – 1.67 (m, 2H, CH2). 13C NMR (75 MHz, Methanol-d4) δ 158.3, 151.6, 148.7, 148.1, 147.6, 134.8,
129.3, 127.4, 123.9, 121.6, 120.6, 117.9, 107.8, 107.7, 101.4, 95.9, 61.2, 42.5, 29.8, 24.7. ESI-MS m/z:
337.2 [M+H]+. Elemental Anal. Calcd for C20H20N2O3: C, 71.41; H, 5.99; N, 8.33. Found: C, 71.61;
H, 5.65; N, 8.18.
4.5.3 6-((2-(benzo[d][1,3]dioxol-5-yl)quinolin-4-yl)amino)hexan-1-ol (5c)
Sandybrown oil, yield: 59.8%. 1H NMR (300 MHz, Acetone-d6) δ 8.13 (d, J = 8.1 Hz, 1H, Ar-H),
7.90 (d, J = 8.4 Hz, 1H, Ar-H), 7.84 – 7.76 (m, 2H, 2Ar-H), 7.60 (t, J = 7.5 Hz, 1H, Ar-H), 7.36 (t, J
= 7.5 Hz, 1H, Ar-H), 7.01 (s, 1H, Ar-H), 6.95 (d, J = 8.1 Hz, 1H, Ar-H), 6.49 (t, J = 4.5 Hz, 1H, NH),
6.07 (s, 2H, OCH2O), 3.55 – 3.48 (m, 4H, 2CH2), 2.97 (brs, 1H), 1.86 – 1.78 (m, 2H, CH2), 1.55 –
13
1.46 (m, 6H, 3CH2). C NMR (75 MHz, Acetone-d6) δ 156.5, 150.9, 148.7, 148.4, 148.1, 135.2,
129.7, 128.8, 123.6, 121.2, 120.6, 118.3, 107.9, 107.5, 101.4, 94.9, 61.5, 42.9, 32.85, 28.5, 27.0, 25.7.
ESI-MS m/z: 365.2 [M+H]+. Elemental Anal. Calcd for C22H24N2O3: C, 72.51; H, 6.64; N, 7.69. Found:
C, 72.23; H, 6.67; N, 7.35.
4.5.4 2-(benzo[d][1,3]dioxol-5-yl)-N-(3-(piperidin-1-yl)propyl)quinolin-4-amine (5d)
Sandybrown solid, yield: 79%, m.p. 109-111 C. 1H NMR (300 MHz, Methanol-d4)  8.16 (d, J =
8.1 Hz, 1H, Ar-H), 7.91 (d, J = 8.1 Hz, 1H, Ar-H), 7.71 (t, J = 7.8 Hz, 1H, Ar-H), 7.53 – 7.45 (m, 3H,
3Ar-H), 6.98 (d, J = 7.9 Hz, 1H, Ar-H), 6.81 (s, 1H, Ar-H), 6.06 (s, 2H, OCH2O), 3.56 (t, J = 6.9 Hz,
2H, CH2), 2.80 – 2.72 (m, 6H, 3CH2), 2.12 – 2.03 (m, 2H, CH2), 1.75 – 1.68 (m, 4H, 2CH2), 1.60 –
1.54 (m, 2H, CH2). 13C NMR (75 MHz, Methanol-d4) δ 157.1, 152.4, 149.2, 148.2, 145.6, 132.8, 130.3,
125.8, 124.5, 122.0, 121.0, 117.5, 108.0, 107.7, 101.6, 96.0, 56.3, 53.9, 41.1, 24.7, 24.1, 23.2. ESI-MS

m/z: 390.2 [M+H]+. Elemental Anal. Calcd for C24H27N3O2: C, 74.01; H, 6.99; N, 10.79. Found: C,
73.93; H, 6.79; N, 11.07.
4.5.5 2-(benzo[d][1,3]dioxol-5-yl)-N-(3-morpholinopropyl)quinolin-4-amine (5e)
1
Sandybrown solid，yield: 82.8%，m.p. 142-144 C. H NMR (300 MHz, Chloroform-d) δ 8.19
(d, J = 8.1 Hz, 1H, Ar-H), 7.97 (d, J = 8.1 Hz, 1H, Ar-H), 7.60 (d, J = 8.1 Hz, 2H, 2Ar-H), 7.55 (s,
1H, Ar-H), 7.40 (t, J = 6.9 Hz, 1H, Ar-H), 6.86 (d, J = 7.8 Hz, 1H, Ar-H), 6.64 (s, 1H, Ar-H), 5.98 (s,
2H, OCH2O), 3.83 (s, 4H, CH2), 3.52 (s, 2H, CH2), 2.71 – 2.47 (m, 6H, 3CH2), 1.99 (s, 2H, CH2).
13C NMR (75 MHz, Chloroform-d) δ 156.5, 152.0, 149.0, 148.1, 145.9, 132.8, 130.1, 127.7, 124.5,
122.3, 120.6, 117. 6, 108.4, 108.2, 101.5, 95.9, 66.9, 58.7, 54.0, 44.1, 30.0, 29.7, 23.5. ESI-MS m/z:
392.2 [M+H]+. Elemental Anal. Calcd for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found: C, 70.65;
H, 6.35; N, 10.81.
4.5.6 2-(benzo[d][1,3]dioxol-5-yl)-4-(pyrrolidin-1-yl)quinoline (5f)
1
Sandybrown solid，yield: 88.0%，m.p. 165-167 C. H NMR (300 MHz, Chloroform-d) δ 8.15
(d, J = 8.7 Hz, 1H, Ar-H), 8.08 (d, J = 8.4 Hz, 1H, Ar-H), 7.61 – 7.54 (m, 3H, 3Ar-H), 7.31 – 7.25
(m, 1H, Ar-H), 6.90 (d, J = 8.1 Hz, 1H, Ar-H), 6.76 (s, 1H, Ar-H), 5.99 (s, 2H, OCH2O), 3.73 – 3.64
13
(m, 4H, 2CH2), 2.07 – 1.97 (m, 4H, 2CH2). C NMR (75 MHz, Chloroform-d) δ 152.2, 149.6,
144.6, 144.1, 125.2, 125.0, 121.0, 119.1, 117.9, 116.0, 104.4, 104.2, 97.4, 96.3, 48.4, 22.0. ESI-MS
m/z:319.1 [M+H]+. Elemental Anal. Calcd for C20H18N2O2: C, 75.45; H, 5.70; N, 8.80. Found: C, 75.62;
H, 5.36; N, 8.80.
4.5.7 2-(benzo[d][1,3]dioxol-5-yl)-4-(piperidin-1-yl)quinoline (5g)
Sandybrown solid，yield:62.6%，m.p. 143-146 C. 1H NMR (300 MHz, Chloroform-d) δ 8.10 (d, J
= 8.4 Hz, 1H, Ar-H), 7.99 (d, J = 8.4 Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.63 (t, J = 8.4 Hz, 2H, 2Ar-

H), 7.42 (t, J = 7.5 Hz, 1H, Ar-H), 7.19 (s, 1H, Ar-H), 6.93 (d, J = 8.1 Hz, 1H, Ar-H), 6.01 (s, 2H,
OCH2O), 3.25 – 3.14 (m, 4H, 2CH2), 1.85 (p, J = 6.0 Hz, 4H, 2CH2), 1.73 – 1.64 (m, 2H, CH2). 13C
NMR (75 MHz, Chloroform-d) δ 158.6, 157.4, 149.5, 148.6, 148.2, 134.9, 130.0, 129.1, 124.7, 123.7,
122.7, 121.7, 108.4, 108.0, 106.3, 101.3, 53.7, 26.2, 24.5. ESI-MS m/z: 333.2 [M+H]+. Elemental Anal.
Calcd for C21H20N2O2: C, 75.88; H, 6.06; N, 8.43. Found: C, 75.71; H, 5.91; N, 8.32.
4.5.8 2-(benzo[d][1,3]dioxol-5-yl)-N-butylquinolin-4-amine (5h)
Sandybrown solid，yield: 55.24%，m.p. 86-88 C. 1H NMR (300 MHz, Chloroform-d) δ 8.04 (d, J
= 8.4 Hz, 1H, Ar-H), 7.70 (d, J = 8.1 Hz, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.60 (d, J = 8.1 Hz, 2H, 2Ar-
H), 7.37 (t, J = 7.5 Hz, 1H, Ar-H), 6.92 (d, J = 8.1 Hz, 1H, Ar-H), 6.76 (s, 1H, Ar-H), 6.01 (s, 2H,
OCH2O), 3.39 – 3.32 (m, 2H, CH2), 1.76 (p, J = 7.2 Hz, 2H, CH2), 1.58 – 1.45 (m, 2H, CH2), 1.01 (t,
J = 7.2 Hz, 3H, CH3). 13C NMR (75 MHz, Chloroform-d) δ 157.7, 150.2, 148.4, 148.4, 148.1, 135.3,
130.0, 129.2, 124.2, 121.6, 119.1, 117.7, 108.3, 108.1, 101.3, 96.2, 43.0, 31.1, 20.4, 13.9. ESI-MS m/z:
321.2 [M+H]+. Elemental Anal. Calcd for C20H20N2O2: C, 74.98; H, 6.29; N, 8.74. Found: C, 74.63;
H, 6.55; N, 8.41.
4.5.9 3-((2-phenylquinolin-4-yl)amino)propan-1-ol (6a)
Sandybrown solid, Yield: 51%, m.p. 134-137 C. 1H NMR (300 MHz, Acetone-d6)  8.28 (d, J =
6.9 Hz, 2H, 2Ar-H), 8.11 (d, J = 7.8 Hz, 1H, Ar-H), 8.00 (d, J = 8.4 Hz, 1H, Ar-H), 7.66 (t, J = 7.5
Hz, 1H, Ar-H), 7.52 (t, J = 7.2 Hz, 2H, 2Ar-H), 7.49 – 7.39 (m, 2H, 2Ar-H), 7.12 (s, 1H, Ar-H),
6.81 (s, 1H, NH), 3.83 (t, J = 5.7 Hz, 2H, CH2), 3.67 – 3.61 (m, 2H, CH2), 3.51 (brs, 1H, OH), 2.09 –
2.05 (m, 2H). 13C NMR (75 MHz, Acetone-d6) δ 157.3, 151.1, 148.7, 140.8, 129.7, 129.0, 128.8, 128.3,
127.3, 124.0, 120.6, 118.4, 95.4, 60.0, 40.7, 31.3. ESI-MS m/z: 279.1 [M+H]+. Elemental Anal. Calcd
for C18H18N2O: C, 77.67; H, 6.52; N, 10.06. Found: C, 77.55; H, 6.70; N, 10.35.

4.5.10 4-((2-phenylquinolin-4-yl)amino)butan-1-ol (6b)
Sandybrown solid，yield:75.0%，m.p. 132-134 C. 1H NMR (300 MHz, Acetone-d6) δ 8.26 (d, J =
6.9 Hz, 2H, 2Ar-H), 8.15 (d, J = 8.4 Hz, 1H, Ar-H), 7.96 (d, J = 8.4 Hz, 1H, Ar-H), 7.62 (t, J = 7.5
Hz, 1H, Ar-H), 7.49 (t, J = 7.2 Hz, 2H, 2Ar-H), 7.43 (d, J = 6.9 Hz, 1H, Ar-H), 7.38 (t, J = 7.5 Hz,
1H, Ar-H), 7.07 (s, 1H, Ar-H), 6.66 (s, 1H, NH), 3.65 (t, J = 6.3 Hz, 2H, CH2), 3.55 – 3.48 (m, 2H,
13
CH2), 1.91 (p, J = 6.9 Hz, 2H, CH2), 1.77 – 1.66 (m, 2H, CH2). C NMR (75 MHz, Acetone-d6) δ
157.2, 151.1, 148.8, 140.8, 129.8, 128.9, 128.7, 128.3, 127.3, 123.9, 120.7, 118.4, 95.4, 61.3, 42.9,
30.3, 25.1. ESI-MS m/z: 293.1 [M+H]+. Elemental Anal. Calcd for C19H20N2O: C, 78.05; H, 6.90; N,
9.58. Found: C, 78.00; H, 6.57; N, 9.49.
4.5.11 6-((2-phenylquinolin-4-yl)amino)hexan-1-ol (6c)
Sandybrown solid，yield: 63.2%，m.p. 116-118 C. 1H NMR (300 MHz, Acetone-d6) δ 8.25 (d, J =
6.9 Hz, 2H, 2Ar-H), 8.16 (d, J = 8.4 Hz, 1H, Ar-H), 7.97 (d, J = 8.4 Hz, 1H, Ar-H), 7.65 – 7.58 (m,
1H, Ar-H), 7.49 (t, J = 7.2 Hz, 2H, 2Ar-H), 7.45 – 7.34 (m, 2H, 2Ar-H), 7.06 (s, 1H, Ar-H), 6.56
(s, 1H, NH), 3.54 (t, J = 6.3 Hz, 2H, CH2), 3.49 – 3.44 (m, 2H, CH2), 1.85 – 1.76 (m, 2H, CH2), 1.58
– 1.39 (m, 6H, 3CH2). 13C NMR (75 MHz, Acetone-d6) δ 157.3, 151.1, 148.8, 140.8, 129.7, 128.9,
128.7, 128.3, 127.3, 123.9, 120.7, 118.4, 95.4, 61.5, 43.0, 32.9, 27.0, 25.7. ESI-MS m/z: 321.2 [M+H]+.
Elemental Anal. Calcd for C21H24N2O: C, 78.71; H, 7.55; N, 8.74. Found: C, 78.82; H, 7.68; N, 8.98.
4.5.12 2-phenyl-N-(3-(piperidin-1-yl)propyl)quinolin-4-amine (6d)
Sandybrown solid，yield: 80.2%，m.p. 147-149 C. 1H NMR (300 MHz, Chloroform-d) δ 8.11 –
8.06 (m, 3H, 3Ar-H), 7.95 (d, J = 8.4 Hz, 1H, Ar-H), 7.69 – 7.61 (m, 2H, 2Ar-H), 7.50 (t, J = 7.2
Hz, 2H, 2Ar-H), 7.44 (d, J = 7.2 Hz, 1H, Ar-H), 7.38 (d, J = 7.2 Hz, 1H, Ar-H), 6.77 (s, 1H, NH),
3.46 – 3.39 (m, 2H, CH2), 2.57 – 2.53 (m, 2H, CH2), 1.98 – 1.90 (m, 2H, CH2), 1.73 (q, J = 5.4 Hz,

13
4H, 2CH2), 1.60 – 1.54 (m, 2H, CH2). C NMR (75 MHz, Chloroform-d) δ 158.6, 151.2, 148.5,
141.2, 129.9, 129.0, 128.7, 128.5, 127.6, 127.5, 123.7, 120.7, 118.3, 96.0, 59.4, 55.0, 44.5, 26.0, 24.4,
23.6. ESI-MS m/z: 346.3 [M+H]+. Elemental Anal. Calcd for C23H27N3: C, 79.96; H, 7.88; N, 12.16.
Found: C, 79.98; H, 7.83; N, 12.28.
4.5.13 N-(3-morpholinopropyl)-2-phenylquinolin-4-amine (6e)
1
Sandybrown solid，yield: 79.5%，m.p. 152-154 C. H NMR (300 MHz, Chloroform-d) δ 7.97
(d, J = 7.8 Hz, 3H, 3Ar-H), 7.75 (d, J = 8.4 Hz, 1H, Ar-H), 7.52 (t, J = 7.5 Hz, 1H, Ar-H), 7.37 (t, J
= 7.2 Hz, 2H, 2Ar-H), 7.29 (q, J = 7.5, 7.2 Hz, 2H, 2Ar-H), 6.90 (t, J = 4.5 Hz, 1H, Ar-H), 6.65 (s,
1H, NH), 3.70 (t, J = 4.5 Hz, 4H, 2CH2), 3.27 (q, J = 5.4 Hz, 2H, CH2), 2.45 - 2.37 (m, 6H, 3CH2),
1.77 (p, J = 6.0 Hz, 2H, CH2). 13C NMR (75 MHz, Chloroform-d) δ 158.5, 151.0, 148.6, 141.1, 130.1,
129.2, 128.8, 128.6, 127.6, 124.0, 120.2, 118.2, 96.2, 67.0, 58.8, 54.0, 44.0, 23.6. ESI-MS m/z: 348.2
[M+H]+. Elemental Anal. Calcd for C22H25N3O: C, 76.05; H, 7.25; N, 12.09. Found: C, 76.39; H, 7.47;
N, 12.16.
4.5.14 2-phenyl-4-(pyrrolidin-1-yl)quinoline (6f)
1
Sandybrown solid，yield: 78.9%，m.p. 166-169 C. H NMR (300 MHz, Chloroform-d) δ 8.45
(d, J = 8.1 Hz, 1H, Ar-H), 8.21 (d, J = 8.4 Hz, 1H, Ar-H), 8.09 (d, J = 1.8 Hz, 1H, Ar-H), 8.06 (d, J =
1.5 Hz, 1H, Ar-H), 7.62 (t, J = 7.8 Hz, 1H, Ar-H), 7.51 – 7.41 (m, 3H, 3Ar-H), 7.34 (t, J = 7.8 Hz,
1H, Ar-H), 6.78 (s, 1H, Ar-H), 3.86 – 3.76 (m, 4H, 2CH2), 2.13 – 2.05 (m, 4H, 2CH2). 13C NMR
(75 MHz, Chloroform-d) δ 155.7, 153.9, 147.4, 138.1, 129.8, 129.6, 128.7, 127.9, 127.7, 125.0, 123.6,
119.4, 100.6, 52.6, 25.9. ESI-MS m/z: 275.1 [M+H]+. Elemental Anal. Calcd for C19H18N2: C, 83.18;
H, 6.61; N, 10.21. Found: C, 83.43; H, 6.34; N, 10.02.

5. Biological activity
5.1. In vitro inhibition studies on AChE and BChE
AChE (E.C.3.1.1.7, Type VI−S, from electric eel) and BuChE (E.C.3.1.1.8, from equine serum),
5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride (ATC), butylthiocholine
chloride (BTC) were purchased from Sigma-Aldrich. The compounds were dissolved in a minimum
volume of DMSO (1%) and diluted in phosphate-buffered solution (0.1 M, pH 8.0) to provide a final
concentration range. All the assays were under the phosphate-buffered solution, using a Shimadzu UV-
2450 Spectrophotometer. Enzyme solutions were prepared to give 2.0 units/mL in 2 mL aliquots. The
assay medium contained 10 L of enzyme, 50 L of DTNB (0.01 M) and 50 L of substrate (ATC,
0.01 M). The substrate was added to the assay medium containing enzyme, buffer, and DTNB with
inhibitor after 15 min of incubation time at 37 C. The activity was determined by measuring the
increase in absorbance at 412 nm at 1 min intervals at 37 C. Calculations were performed according
to the method of the equation in Ellman et al [19]. In vitro BuChE assay use the similar method
described above. Each concentration was assayed in triplicate.
5.2. Kinetic characterization of AChE inhibition
Three different concentrations of substrate were mixed in the 1.0 mL phosphate buffer (0.1 M, pH
8.0), containing 50 L of DTNB, 10 L AChE, and 50 L substrate. Test compound was added into
the assay solution and pre-incubated with the enzyme at 37C for 15 min, followed by the addition of
substrate. Kinetic characterization of the hydrolysis of ATC catalyzed by AChE was done
spectrometrically at 412 nm. A parallel control with no inhibitor in the mixture, allowed adjusting
activities to be measured at various times.
5.3. Molecular modeling

The crystal structure of the torpedo AChE (code ID: 1ACJ) were obtained in the Protein Data Bank
after eliminating the inhibitor and water molecules. The 3D structure of 5f was built and performed
geometry optimization by molecular mechanics. Further preparation of substrates included addition of
Gasteiger charges, removal of hydrogen atoms and addition of their atomic charges to skeleton atoms,
and finally, assignment of proper atomic types. Autotors was then used to define the rotatable bonds
in the ligand.
Docking studies were carried out using the AutoDock 4.2 program (The Scripps Research Institute,
San Diego, CA, USA), polar hydrogen atoms were added and Gasteiger charges were assigned to the
enzyme. The resulting enzyme structure was used as an input for the AUTOGRID program.
AUTOGRID performed a precalculated atomic affinity grid maps for each atom type in the ligand plus
an electrostatics map and a separate desolvation map present in the substrate molecule. All maps were
calculated with 0.375 Å spacing between grid points. The center of the grid box was placed at the
bottom of the active site gorge (AChE [2.781 64.383 67.971]. The dimensions of the active site box
were set at 50  46  46 Å. Flexible ligand docking was performed for the compounds. Docking
calculations were carried out using the Lamarckian genetic algorithm (LGA) and all parameters were
the same for each docking. To ensure the reliability of the results, the docking procedures were
repeated 10 independent times for the compounds and the obtained orientations were analyzed.
5.4. Inhibition of self-mediated Aβ(1-42) aggregation [25]
Aβ1-42 peptide (Chinapeptides) was dissolved in ammonium hydroxide (1% v/v), and diluted with
phosphate buffer (pH 7.40, 20 mM) to 200 μM before use. Compounds were firstly dissolved in DMSO
to obtain a 10 mM solution. The peptide was incubated in phosphate buffer (pH 7.40, 20 mM) in 37 °C
for 48 h (final Aβ concentration of 50 μM) with or without the tested compounds at 20 μM. After

incubation, thioflavin-T (5 μM in 50 mM glycine-NaOH buffer, pH 8.00) was added. Then a scan of
fluorescence intensity was performed (λex = 450 nm; λem = 485 nm). The fluorescence intensities were
recorded, and the percentage of inhibition on aggregation was calculated by the following expression:
(1-IFi/IFc)*100% in which IFi and IFc were the fluorescence intensities obtained for absorbance in the
presence and absence of inhibitors, respectively, after subtracting the fluorescence of respective blanks.
Each assay was performed in triplicate.
5.5. MTT assay of HepG2 and PC12 cell viability
MTT assay of PC12 and HepG2 was conducted according to the previous report [16].
5.6. Behavioral Studies
All experiments were performed in accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. The procedures were approved by the Animal Care and Use
Committee of the Henan University. Kunming Mice, (male, 20 ± 2g) were purchased form
Experimental Animal Center of Zhengzhou University (Zhengzhou, China). The mice were housed in
SPF grade animal room, and kept in plastic cages in the Experimental Animal Center of Henan
University (Kaifeng, China). The animals were provided with free access to food and water.
Behavioral studies were performed with adult male ICR mice (8−10 weeks old, weight 20−25 g)
from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) by MWM. The
mice were divided into four groups: control (vehicle), model (scopolamine), THA (scopolamine plus
THA), and 5f (scopolamine plus compound 5f). THA and 5f at the dose of 20 μmol/kg body weight
per day were orally administered to the mice at 30 min before the intraperitoneal (ip) administration
of scopolamine (1 mg/kg). Memory impairment was induced by administering scopolamine and the
maze task was performed after 30 min of scopolamine administration. The schematic diagram (Figure

S1) depicts the experimental procedure.
MWM is a circular pool (100 cm in diameter and 50 cm in height) with visual cues. The circular
pool was filled at a depth of 30 cm with water (25 °C). A platform (6 cm in diameter and 29 cm in
height) was centered in one of four quadrants of the pool. Mice were trained during trial sessions of
four trials each day for four consecutive days. During each trial, the mouse’s escape latency was
recorded. Once the mouse located the platform, it was permitted to remain on it for 10s. If the mouse
did not locate the platform within 90 s, it was placed on the platform for 10s. On the fifth day after
training, mice were given a probe trial session in which the platform was moved from the pool and
allowing the mice to swim for 90s. The time and the distance taken in the target quadrant and the
number of times the animals crossed the platform location were recorded.
Acknowledgements
We thank the Natural Science Foundation of China (No. U1704176, 81460528, 21762010), the
Foundation for University Key Teacher by Educational Commission of Henan Province (No.
2016GGJS-030), the Natural Science Foundation of Guangxi Province of China
(2018GXNSFAA281135）
Conflict of Interest
We declare that we have no conflict of interest.
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Synthesis, in vitro and in vivo biological evaluation of novel graveolinine
derivatives as potential anti-Alzheimer agents
Wen Luo §,a, Jian-Wu Lv §,a, Ting Wang a, Zhi-Yang Zhang a, Hui-Yan Guo a, Zhi-Yi Song b, Chao-Jie Wang a , Jing
Ma b,*, Yi-ping Chen c,*
> A novel series of graveolinine derivatives were synthesized and evaluated as potential anti-Alzheimer’s agents.
> Compound 5f showed moderate inhibitory activities for AChE, BuChE and A self-aggregation.
> Compound 5f significantly ameliorates the cognitive performances of scopolamine-treated ICR mice.