Graveoline analogs exhibiting selective acetylcholinesterase inhibitory activity as potential lead compounds for the treatment of Alzheimer's disease

Article abstract of DOI:10.3390/molecules21020132

This study designed and synthesized a series of new graveoline analogs on the basis of the structural characteristics of acetylcholinesterase (AChE) dual-site inhibitors. The activity of these analogs was also evaluated. Results showed that the synthesized graveoline analogs displayed stronger inhibitory activity against AChE and higher selectivity than butyrylcholine esterase (BuChE) (Selectivity Index from 45 to 486). When the two sites in the graveoline parent ring substituting phenyl and amino terminal had six chemical bonds (n = 3) and the terminal amino was piperidine, compound 5c showed the best activity. Furthermore, the mechanism of action and binding mode were explored by enzyme kinetic simulation, molecular docking, and thioflavin T-based fluorometric assay. Cytotoxicity assay showed that the low concentration of the analogs did not affect the viability of the neurocyte SH-SY5Y.

Full text of DOI:10.3390/molecules21020132

molecules
Article
Graveoline Analogs Exhibiting Selective
Acetylcholinesterase Inhibitory Activity as Potential
Lead Compounds for the Treatment of
Alzheimer’s Disease
Zeng Li 1,*,†, Chaoyu Mu 1,2,†, Bin Wang 3 and Juan Jin 1
1
College of Pharmacy, Anhui Medical University, Hefei 230032, China; cym19841107@sina.com (C.M.);
aywanganyong@163.com (J.J.)
Department of Clinical Laboratory, Huaibei Miner’s General Hospital, Huaibei 235000, China
School of Pharmaceutical Sciences, Xiangnan University, Chenzhou 423000, China; aymumu@sohu.com
2
3
*
†
Correspondence: lizeng@ahmu.edu.cn; Tel.: +86-551-65161136
These authors contributed equally to this work.
Academic Editors: Michael Decker and Diego Muñoz-Torrero
Received: 11 December 2015 ; Accepted: 20 January 2016 ; Published: 22 January 2016
Abstract: This study designed and synthesized a series of new graveoline analogs on the basis of the
structural characteristics of acetylcholinesterase (AChE) dual-site inhibitors. The activity of these
analogs was also evaluated. Results showed that the synthesized graveoline analogs displayed
stronger inhibitory activity against AChE and higher selectivity than butyrylcholine esterase (BuChE)
(Selectivity Index from 45 to 486). When the two sites in the graveoline parent ring substituting
phenyl and amino terminal had six chemical bonds (n = 3) and the terminal amino was piperidine,
compound 5c showed the best activity. Furthermore, the mechanism of action and binding mode
were explored by enzyme kinetic simulation, molecular docking, and thioflavin T-based fluorometric
assay. Cytotoxicity assay showed that the low concentration of the analogs did not affect the viability
of the neurocyte SH-SY5Y.
Keywords: graveoline analogs; acetylcholinesterase (AChE); butyrylcholine esterase (BuChE);
structure–function relationships (SARs); mechanism
1. Introduction
Alzheimer’s disease (AD) is a chronic progressive degenerative disease of the central nervous
system [
memory, judgment, abstract thinking ability, reasoning ability, and spatial relationship, thus resulting in
the complete loss of a patient’s self-help ability [ ]. This disease is a primary cerebral disease. The onset
1]. The degeneration of the cerebral cortex leads to the loss of its normal function, including
2
of AD occurs slowly and is mostly affected by family history. Furthermore, AD is progressive and is
the most common cause of dementia among the elderly [3].
AD pathogenesis has not yet been fully elucidated, and many hypotheses on AD pathogenesis
have been developed. Among them, the brain cholinergic neuron damage hypothesis and amyloid-
(A ) cascade hypothesis are widely recognized; that is, brain cholinergic neuron damage and A
cascade are speculated to be the dominant causes of AD pathogenesis [
(AChE) inhibitor, based on the cholinergic hypothesis, is the commonly used drug for treating AD
in clinical practice [ ]. However, the therapeutic effect of AChE inhibitor is not ideal. This drug can
β
β
β
4–6]. The acetylcholinesterase
7
improve the patient’s neuropsychiatric symptoms but cannot reverse AD development. The latest
research showed that AChE possibly played multiple roles in AD development because its peripheral
binding site (PAS) could promote the production of A
β
protein and accelerate its deposition.
Molecules 2016, 21, 132; doi:10.3390/molecules21020132
www.mdpi.com/journal/molecules

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Therefore, the catalytic activity site (CAS) and PAS dual-binding site inhibitor acting on AChE may
exhibit multiple mechanisms of action for AD treatment. One simple way to design the dual-site
AChE inhibitor is to connect two pharmacophores to a dyad in a certain manner. This dimer can
simultaneously act on the CAS and PAS of AChE, improve the inhibitory activity on AChE, and inhibit
AChE-induced Aβ aggregation [8,9]. This finding has become the new direction in developing new
cholinesterase inhibitors. Butyrylcholinesterase (BuChE) and AChE both exist in the body of mammals.
However, because BuChE mainly exists in the peripheral nervous system, BuChE inhibition will cause
side effects to the peripheral nervous system [10
and salivation (peripheral effect) showed that the highly selective cholinesterase inhibitor donepezil
exerts better therapeutic index inhibition than tacrine, which can inhibit both AChE and BuChE [12 13].
,11]. The dose effect curves of tremor (central effects)
,
Furthermore, recent reports showed that the activity of BuChE decreased in the nerve synapses of AD
patients [14]. These results indicated that AChE inhibitors exhibiting high selectivity and high activity
had excellent development prospects in the research and development of AD drugs.
The discovery of active ingredients from natural products and structural modifications are
important ways of developing new drugs. Graveoline is a 2-phenyl quinolinone compound and
exists in rutaceous plants. Graveoline exhibits extensive pharmacological effects, such as anti-bacteria,
spasmolysis, and anti-tumor [15
graveoline analog 2 was identified as AChE inhibitor with an IC50 value of 3.65
dual-site strategy that was designed according to the structural characteristics of graveoline and benzyl
piperidine AChE inhibitors [18 20], we designed a dual-site AChE inhibitor that acts on both the
–
17]. In our early efforts to search for novel AChE inhibitors, the
µM. On the basis of the
–
AChE central CAS and PAS with 2-phenyl quinolinone as the parent (Figure 1). The overall strategy in
molecular structure design includes the binding of the parent aromatic ring and an appropriate amino
terminal side chain with the central catalytic site of AChE. We aimed to improve AChE inhibitory
1
activity and AChE/BuChE selectivity by substituting the 4 -benzene ring in the 2-phenyl quinolinone
molecule and by introducing an amino terminal side chain. Furthermore, the introduction of amine
side chain could improve the water solubility of the graveoline analogs. This conceptual design was
confirmed by the results of a follow-up study.
Figure 1. Structure of graveoline and the conventional design idea of dual-target AChE inhibitors.
2. Results and Discussion
2.1. Chemistry
Scheme 1 shows the synthetic route of the compound. By reacting 2-aminoacetophenone
(raw material) with benzoyl chloride to obtain amide, we obtained carbostyril midbody
catalyzed by potassium tert-butoxide. To obtain the methylated product , we reacted compound
with the alkylate reagent CH3I under K2CO3. Compound was then mixed 10 times with CCl4
and then reacted with NBS to obtain bromo-product ; this reaction was catalyzed by benzoperoxide.
Product was mixed with various fatty amino to obtain the end-product fatty amino-substituted
analogs 4a–4c, 5a–5c, and 6a–6b by amination reaction.
1 by cyclization
2
1
2
3
3

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Scheme 1. Synthesis of derivatives. Reagent: (i) 4-Rbenzoyl chloride ,THF, rt; (ii) t-BuOH/t-BuOK,
75 ˝C; (iii) CH3I/K2CO3, DMF; (iv) NBS/ benzoyl peroxide, CCl4, reflux; (v) RNH2, CH2Cl2, rt.
2.2. Inhibitory Activity of Graveoline Analogs on AChE and BuChE
The inhibitory activity of the synthesized graveoline analogs on cholinesterase in vitro was studied
using the Ellman method [21]. Tacrine was used as positive control. Table 1 shows the enzymatic
inhibitory activity of the graveoline analogs.
Table 1. In vitro inhibition IC50 and selectivity index of graveoline analogs 4a–4c, 5a–5c and 6a–6b on
Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
AChE Inhibition a,
(IC50) nM
BuChE Inhibition b,
(IC50) nM
Compound
R
n
Selectivity Index c
4a
5a
6a
4b
5b
6b
4c
2
3
4
2
3
4
2
3
78.53 ˘ 1.76
33.36 ˘ 0.78
35.51 ˘ 0.96
10.18 ˘ 1.33
6.47 ˘ 0.15
12.64 ˘ 0.57
9.63 ˘ 0.76
2.36 ˘ 0.24
3646 ˘ 38
3522 ˘ 46
2680 ˘ 32
3674 ˘ 39
2470 ˘85
1886 ˘ 54
2434 ˘ 49
1543 ˘ 29
1147 ˘ 31
9306 ˘ 76
31.26 ˘ 0.34
45
80
103
223
291
193
160
486
3
5c
2
Tacrine
224.7 ˘ 1.24
0.1
a
IC50: 50% inhibitory concentration (means
concentration (means
˘
SEM of three experiments) of AChE; b IC50: 50% inhibitory
˘
SEM of three experiments) of BuChE; c Selectivity Index = IC50 (BuChE)/IC50 (AChE).
Table 1 shows that all analogs, except for parent compound 2, showed strong inhibitory ability and
selectivity to AChE after the 4’-benzene ring substituted the 2-site and after introducing the side chain.
The selective coefficient ranged from 45 to 486, and the IC50 values were expressed in the nanomole
concentration level; the inhibitory activities of the analogs were better than those of tacrine. On one
hand, we speculated that these side chains were protonated under physiological pH. The protonated
side-chain quaternary ammonium was similar to the structure of the acetylcholine molecule, easily
entered the enzyme activity pocket, and interacted with the amino acid residue in the triplet catalytic
center. Moreover, the protonated side-chain quaternary ammonium can induce the π–π accumulation
of the stabilized ring aromatic structure of the parent compound and the PAS of the aromatic amino
acid residues, thus promoting the combination of parent ring structure and the AChE PAS. On the
other hand, the alkylamino side chain can combine with the active site of the enzyme and guide the
molecules into the narrow active valley of cholinesterase such that the other groups mutually combine
with amino acid residues in the PAS region. BuChE contains no PAS binding site and its active center
is wider than that of AChE. Thus, the introduction of the compound chain exerts a lower effect on
inhibitory activity against BuChE than against AChE.

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The change in the side chains can also significantly affect the inhibitory activity of the graveoline
analogs on cholinesterase. Table 1 shows that if the end groups are the same, the highest inhibitory
activity against AChE is exhibited by compounds 5a
the parent by six chemical bonds (n = 3). The inhibitory activities to shorten (n = 2, 4a
,
5b, and 5c, whose terminal amino is linked with
4b, and 4c) or
,
extend (n = 4, 6a, and 6b) the side chain of the compounds are reduced. A short side chain does not
allow the terminal amino to reach the choline binding site, whereas an excessively long side chain
increases the flexibility and disturbance of the total molecular structure, which was more likely to
cause the side chain to not deeply reach the enzyme active valley, thereby affecting the combination of
the compounds and enzymes.
The analogs bearing N heterocycle as a side-chain terminal structure displayed strong inhibitory
ability (AChE). When the lengths of the side chain were the same, the compounds bearing piperidyl
(
(
4c and 5c) as their terminal group displayed the strongest inhibitory activity, followed by pyrrolidyl
4b 5b, and 6b); the compounds bearing the terminal group N-methyl (4a 5a, and 6a) showed the
,
,
weakest inhibitory activity. Therefore, the size of the terminal groups and the steric hindrance greatly
affected the combination of compounds and enzymes. The relatively large terminal groups increased
the rigidity of the entire molecular structure, thereby reducing the disturbance caused by the side chain
in the enzyme active valley and increasing the binding capacity of the small molecules and enzymes.
These results demonstrated the importance of the length and size of the amide side chain for strong
cholinesterase inhibitory ability.
2.3. Kinetic Characterization of AChE Inhibition
To identify the type of inhibition exerted by the analogs on AChE, we chose compound 5c
,
which exhibits the best activity, to study the inhibition kinetics of AChE. All straight lines in Figure 2
were intersected in the second quadrant of the coordinate axis, which characterizes a typical mixed
inhibition. When the small-molecule ligand was combined with the AChE active center (CAS),
the type of inhibition of the enzyme was classified as competitive inhibition; by contrast, when the
small-molecule ligand was acted on the PAS, the enzymatic inhibition was classified as non-competitive
inhibition. When the small-molecule ligand acted both on CAS and PAS, the enzymatic inhibition
was classified as mixed inhibition [22]. Therefore, we speculated that the synthesized graveoline
analogs interacted with the two AChE functional sites CAS and PAS; thus, the analogs showed a strong
inhibitory effect. This conclusion was verified by subsequent molecular docking experiments.
Figure 2. Lineweaver-Burk plot for the inhibition of AChE by 5c.

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2.4. Study on the Interaction between 5c and Cholinesterase by Molecular Docking Method
AUTODOCK docking and PyMOL observation were used in the molecular docking between 5c
,
which exhibited the strongest activity, and cholinesterase. Figure 3 shows that the optimal structural
conformation of the ligand molecule 5c was based on the docking energy value during docking
between 5c and cholinesterase. The docking result showed that the ligand molecule interacted
with the functional groups of the amino acid residues found along the active valley of the AChE
(Figure 3A). The parent structure of 5c was bound to the PAS region and displayed a classic π–π
stacking interaction between Tyr334, the 2-phenyl of 5c, and Phe331, with a ring-to-ring distance of
3.6 and 3.4 Å. The carbonyl group of the quinolinone moiety established a hydrogen bond (3.7 Å) with
the nitrogen atom of the main chain of Gly335. Furthermore, the conformation of the amino alkyl
amine side chain matched the enzyme active valley well. The N atoms in the terminal piperidine
ring were also positively charged after being solvated and formed positive ion-π interaction with
the tryptophan Trp84 residue in the catalytic activity region in the bottom valley. The distance
was 3.2 Å. These results showed that compound 5c could react with both the PAS and the catalytic
activity region of the enzyme, thus resulting in the strong inhibitory effect against AChE. At the same
time, different interaction modes were found in 5c in the complex with human butyrylcholinesterase
(HuBuChE, PDB code: 1POI). The quinolinone moiety of 5c reached the valley bottom with a classic
π´π stacking interaction with the Trp82 residue of catalytic activity, and the ring-to-ring distance
was 3.6 Å (Figure 3B). Furthermore, no obvious interaction between the ligand and the enzyme was
observed. Thus, the inhibitory activity against BuChE was weak and the analogs had high selectivity.
(A)
(B)
Figure 3. Docking models of the 5c–enzyme complex. Representations of compound 5c interacting
with residues in the binding site of Torpedo californica acetylcholinesterase (TcAChE) ( ) and human
butyrylcholinesterase (HuBuChE) ( ). The compounds are rendered in green stick models, and the
A
B
amino acid residues are rendered in golden sticks.
2.5. AChE-Induced Aβ Aggregation: Inhibition Studies
To explore the combining effect of graveoline analogs on the AChE functional site PAS,
compound 5c was chosen to study the inhibitory activity of AChE-induced A aggregation using
thioflavin T-based fluorometric assay [ ]. As shown in Figure 4, compound 5c can significantly
inhibit AChE-induced Aβ42 aggregation. The inhibitory effect of 5c was related to the compound
concentration. The combinations of the inhibitory activity of 5c on AChE and AChE-induced A
β
8,9
β
aggregation indicated that the graveoline analog was a dual-site AChE inhibitor that acts on both the
AChE central CAS and PAS.

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Figure 4. Determination of 5c inhibition potency on AChE-induced amyloid-β (Aβ)42 aggregation.
2.6. Effects of Graveoline Analogs on Neurocyte Viability
As potential anti-AD drugs, the graveoline analogs must exhibit low toxicity. Thus, we used
the methyl thiazolyl tetrazolium (MTT) method [23] to perform a neurocyte (SH-SY5Y) toxicity test
in vitro. The preliminary results (Figure 5) showed that the incubation of the SH-SY5Y cell strain with
0.1 and 1.0
the cell viability of the strain. Furthermore, the neurocyte inhibition rate was below 15%. However,
cell viability was greatly influenced at 20 and 40 M concentrations and the inhibition rates were
more than 90%. By contrast, the inhibition rate of the reference substance curcumin (40 M) on the
µM concentrations of compounds 4a–4c, 5a–5c, and 6a–6b for 48 h did not greatly influence
µ
µ
neurocytes was 57.3%. This result showed that the synthesized compounds still exerted a certain level
of cytotoxicity. However, compared with the cholinesterase activity at the nanomole level, this level of
cytotoxicity is acceptable. As a drug, the blood drug concentration in vivo did not reach the micromole
level. Cholinesterase could also be inhibited under 100 nmol or lower concentrations to prevent severe
toxicity to the body.
Figure 5. Inhibition for SH-SY5Y of compounds 4a–4c, 5a–5c, 6a–6b and curcumin at different concentrations.
3. Materials and Methods
3.1. Chemistry
1H-NMR spectra was measured in DMSO-d6 or CDCl3 on a Bruker 400 NMR (Bruker, Karlsruhe,
Germany) by using tetramethylsilane as internal standard. The mass spectrometer (MS) were recorded
using Shimadzu LC-MS-2010A spectrometer (Shimadzu, Kyoto, Japan) with an ESI or ACPI mass
selective detector. Elemental analysis was measured by the Elementar Vario EL CHNS Elemental
Analyzer (Elementar, Hanau, Germany). The melting points (m.p.) were measured using the X-6

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microscope melting point instrument (Beijing Tech Instrument Co. Ltd., Beijing, China), and data were
not corrected.
2-p-Tolylquinolin-4(1H)-one (1). A solution of the p-Toluoyl chloride (10 mmol) in THF (10 mL)
was added to a well-stirred m˝ixture of 2-Aminoacetophenone (10 mmol) and Et3N (10 mL) in
anhydrous THF (150 mL) at 0 C. After this addition, the solution was stirred for 12 h at room
temperature. The precipitated solid was collected through filtration, washed with ethanol, and then
dried. To mechanically stir the suspension of the dried solid in 100 mL tert-butyl alcohol, potassium
tert-butoxide was added (15.0 g, 130 mmol) under N2 atmosphere. The reaction was heated to 75 ˝C
for 16 h, allowed to cool to room temperature, and then washed with ice water. The precipitate
was removed and recrystallized by using CH2Cl2 and methanol to give compound
1
a yellow solid
˝
1
in 76% yield. m.p. 169–172 C. H-NMR (400 MHz, DMSO-d6)
δ (ppm): 8.10 (d, 1H, J = 8.1 Hz),
7.84 (d, 1H, J = 8.5 Hz), 7.74 (d, 2H, J = 8.1 Hz), 7.65 (t, 1H, J = 8.0 Hz), 7.39 (d, 2H, J = 8.0 Hz),
7.33 (t, 1H, J = 8.0 Hz), 6.33 (s, 1H), 2.41 (s, 3H). APCI-MS m/z: 236.1 [M + H]+.
1-Methyl-2-p-tolylquinolin-4(1H)-one (2). Methyl iodide (3 mL) was added to a well-stirred mixture of
the intermediate
1
(10 mmol) and K2CO3 (10 mmol) in anhydrous DMF (60 mL) at 0 ˝C under N2
atmosphere. The reaction was then allowed to warm up to 50 ˝C for 5 h, washed with ice water, and
then eluted with CH2Cl2. The organic solvent was removed in vacuo, and the residue was purified
using column˝chromatography (eluent CHCl3/MeOH = 40:1) to give compound 2 (0.71 g, 14%).
1
m.p. 171–175 C. H-NMR (400 MHz, CDCl3):
δ 8.50 (d, J = 8.0 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H),
7.55 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.30 (s, 4H), 6.30 (s, 1H), 3.61 (s, 3H), 2.44 (s,3H).
APCI-MS m/z: 250.1 [M + H]+.
General Procedure for the Synthesis of Graveoline Analogs (4a–4c, 5a–5c, and 6a–6b)
A mixture of intermediate
8.55 mmol) in anhydrous CCl4 (50 mL) were stirred at 50 ˝C for 5 h. After the reaction was complete,
the mixture was filtered through a pad of celite and then concentrated in vacuo. The residue (
2 (1.8 g, 7.2 mmol), benzoyl peroxide (0.08 g), and NBS (1.56 g,
3
)
was suspended in CH2Cl2 (5 mL), and amine (2.0 mL) was added dropwise in CH2Cl2 (5 mL).
The reaction was stirred overnight and then washed with 30 mL water. Finally, the solution was
extracted with CH2Cl2 (3
Na2SO4. The solvent was then removed under vacuum. The residue was chromatographed on silica
ˆ
100 mL), and the combined organic layers were dried over anhydrous
gel eluting with CHCl3/MeOH (30:1) to obtain 4a–4c, 5a–5c, and 6a–6b yields of 54%–73%.
2-(4-((2-(Dimethylamino)ethylamino)methyl)phenyl)-1-methylquinolin-4(1H)-one (4a). A light-yellow solid.
˝
1
m.p. 197–199 C; H-NMR (400 MHz, CDCl3):
δ 7.96 (dd, J = 7.5, 1.4 Hz, 1H), 7.68–7.55 (m, 3H),
7.32 (d, J = 7.4 Hz, 2H), 7.08 (td, J = 7.6, 1.6 Hz, 1H), 6.90 (dd, J = 7.6, 1.4 Hz, 1H), 6.85 (s, 1H),
6.45 (s, 1H), 3.92 (s, 2H), 3.52 (s, 3H), 2.63 (t, 2H), 2.54 (t, 2H), 2.32 (s, 6H); APCI-MS m/z: 336.2 [M + H]+;
Anal. Calcd. for C21H25N3O. H2O: C, 71.36; H, 7.70; N, 11.89. Found: C, 71.28; H, 7.69; N, 11.95.
1-Methyl-2-(4-((2-(pyrrolidin-1-yl)ethylamino)methyl)phenyl)quinolin-4(1H)-one (4b). A light-yellow solid.
˝
1
m.p. 187–190 C; H-NMR (400 MHz, CDCl3):
δ 8.13 (dd, J = 7.5, 1.4 Hz, 1H), 7.71–7.60 (m, 3H),
7.31 (d, J = 7.4 Hz, 2H), 7.12 (td, J = 7.6, 1.6 Hz, 1H), 6.93 (dd, J = 7.6, 1.4 Hz, 1H), 6.86 (s, 1H),
6.45 (s, 1H), 3.92 (s, 2H), 3.52 (s, 3H), 3.03 (t, 2H), 2.64 (t, 2H), 2.56–2.43 (t, 4H), 1.66 (m, 4H). APCI-MS
m/z: 362.1 [M + H]+. Anal. Calcd. for C23H27N3O. H2O: C, 72.79; H, 7.70; N, 11.07. Found: C, 72.83;
H, 7.53; N, 11.02.
1-Methyl-2-(4-((2-(piperidin-1-yl)ethylamino)methyl)phenyl)quinolin-4(1H)-one (4c). A light-yellow solid.
˝
1
m.p. 185–188 C; H-NMR (400 MHz, CDCl3):
δ 8.04 (dd, J = 6.8, 1.4 Hz, 1H), 7.70–7.62 (m, 3H),
7.35 (d, J = 7.4 Hz, 2H), 7.14 (td, J = 7.4, 1.6 Hz, 1H), 6.98 (dd, J = 7.4, 1.4 Hz, 1H), 6.83 (s, 1H),
6.45 (s, 1H), 3.92 (s, 2H), 3.55 (s, 3H), 2.69 (t, J = 5.4 Hz, 2H), 2.66 (t, 2H), 2.36 (t, J = 5.4 Hz, 4H),
1.64 (m, 4H), 1.52 (m, 2H); APCI-MS m/z: 376.2 [M + H]+. Anal. Calcd. for C24H29N3O. 2H2O:
C, 70.04; H, 8.08; N, 10.21. Found: C, 69.98; H, 8.15; N, 10.11.

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2-(4-((3-(Dimethylamino)propylamino)methyl)phenyl)-1-methylquinolin-4(1H)-one (5a). A light-yellow solid.
˝
1
m.p. 182–185 C; H-NMR (400 MHz, CDCl3):
δ 8.17 (dd, J = 7.6, 1.4 Hz, 1H), 7.95–7.64 (m, 3H),
7.46 (d, J = 7.6 Hz, 2H), 7.22 (td, J = 7.6, 1.6 Hz, 1H), 7.08 (dd, J = 7.6, 1.4 Hz, 1H), 7.01 (s, 1H),
6.50 (s, 1H), 3.92 (s, 2H), 3.54 (s, 3H), 2.61 (t, J = 7.6 Hz, 2H), 2.50 (t, J = 7.6 Hz, 2H), 2.27 (s, 6H),
1.42 (m, 2H); APCI-MS m/z: 350.2 [M + H]+. Calcd. for C22H27N3O. H2O: C, 71.90; H, 7.95; N, 11.43.
Found: C, 72.03; H, 7.94; N, 11.37.
1-Methyl-2-(4-((3-(pyrrolidin-1-yl)propylamino)methyl)phenyl)quinolin-4(1H)-one (5b). A light-yellow solid.
˝
1
m.p. 176–180 C; H-NMR (400 MHz, CDCl3):
δ 8.31 (dd, J = 7.6, 1.4 Hz, 1H), 7.89–7.64 (m, 3H),
7.48 (d, J = 7.6 Hz, 2H), 7.32 (td, J = 7.6, 1.6 Hz, 1H), 6.99 (dd, J = 7.6, 1.4 Hz, 1H), 6.88 (s, 1H),
6.45 (s, 1H), 3.94 (s, 2H), 3.52 (s, 3H), 3.07 (t, 2H), 2.62 (t, J = 7.6 Hz, 2H), 2.48 (t, 2H), 2.32 (m, 4H),
1.66 (m, 4H); APCI-MS m/z: 376.2 [M + H]+. Calcd. for C24H29N3O. 2H2O: C, 70.04; H, 8.08; N, 10.21.
Found: C, 70.09; H, 7.99; N, 10.34.
1-Methyl-2-(4-((3-(piperidin-1-yl)propylamino)methyl)phenyl)quinolin-4(1H)-one (5c). A light-yellow solid.
˝
1
m.p. 177–179 C; H-NMR (400 MHz, CDCl3):
δ 8.26 (dd, J = 7.4, 1.4 Hz, 1H), 7.65 (ddd, J = 10.8,
8.2, 4.4 Hz, 3H), 7.32 (d, J = 7.5 Hz, 2H), 7.28 (td, J = 7.6, 1.6 Hz, 1H), 7.16 (dd, J = 7.6, 1.4 Hz, 1H),
6.88 (s, 1H), 6.45 (s, 1H), 3.97 (s, 2H), 3.53 (s, 3H), 3.14 (t, J = 5.4 Hz, 2H), 2.60 (dd, J = 16.0, 7.8 Hz, 2H),
2.37 (dt, J = 10.7, 6.5 Hz, 4H), 1.67–1.60 (m, 6H), 1.52 (m, 2H); APCI-MS m/z: 390.2 [M + H]+. Calcd. for
C25H31N3O. H2O: C, 73.68; H, 8.16; N, 10.31. Found: C, 73.54; H, 8.27; N, 10.26.
2-(4-((4-(Dimethylamino)butylamino)methyl)phenyl)-1-methylquinolin-4(1H)-one (6a). A light-yellow
˝
1
solid. m.p. 173–176 C; H-NMR (400 MHz, CDCl3):
δ 8.25 (dd, J = 7.6, 1.4 Hz, 1H), 7.68–7.55
(m, 3H), 7.32 (d, J = 7.6 Hz, 2H), 7.17 (td, J = 7.6, 1.4 Hz, 1H), 6.99 (dd, J = 7.4, 1.5 Hz, 1H),
6.85 (s, 1H), 6.50 (s, 1H), 3.95 (s, 2H), 3.54 (s, 3H), 2.73 (t, 2H), 2.62 (t, 2H), 2.39 (s, 6H), 1.76 (m, 2H),
1.63 (m, 2H); APCI-MS m/z: 364.2 [M + H]+. Calcd. for C23H29N3O. 2H2O: C, 69.14; H, 8.33; N, 10.52.
Found: C, 69.34; H, 8.39; N, 10.47.
1-Methyl-2-(4-((4-(pyrrolidin-1-yl)butylamino)methyl)phenyl)quinolin-4(1H)-one (6b). A light-yellow solid.
˝
1
m.p. 169–172 C; H-NMR (400 MHz, CDCl3):
δ 8.21 (dd, J = 7.6, 1.4 Hz, 1H), 7.68–7.55 (m, 3H),
7.44 (d, J = 7.6 Hz, 2H), 7.23 (td, J = 7.6, 1.6 Hz, 1H), 7.14 (dd, J = 7.6, 1.4 Hz, 1H), 7.02 (s, 1H),
6.45 (s, 1H), 3.98 (s, 2H), 3.50 (s, 3H), 3.13 (t, 2H), 2.68 (t, 2H), 2.40 (t, 2H), 2.14 (m, 4H), 1.98–1.31
(m, 6H); APCI-MS m/z: 390.3 [M + H]+. Calcd. for C25H31N3O. 2H2O: C, 70.56; H, 8.29; N, 9.87. Found:
C, 70.44; H, 8.31; N, 9.93.
3.2. Materials
Stock solutions of all graveoline analogs (10 mM) were prepared using DMSO and stored at
˝
´
80 C. Further dilutions to working concentrations were made with double-distilled deionized
water. AChE (E.C. 3.1.1.7, obtained from electric eel), BuChE (E.C. 3.1.1.8, obtained from equine
serum), 5,51-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent, DTNB), acetylthiocholine chloride
(ATC), butylthiocholine chloride, thioflavin T, and tarcine hydrochloride were purchased from
Sigma-Aldrich (St. Louis, MO, USA). Aβ42 was purchased from Millipore (Billerica, MA, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased formal Aladdin
(Shanghai, China). The SH-SY5Y cell line was obtained from the Animal experimental center of
Sun Yat-sen University.
3.3. In Vitro Inhibition of AChE and BuChE
In vitro AChE assay: all methods were performed under 0.1 M KH2PO4/K2HPO4 buffer at pH
8.0 by using a Shimadzu 2450 spectrophotometer. The solutions of the enzyme were prepared to
achieve 2.0 units/mL in 2 mL aliquots. The experiment system contained phosphate buffer, pH 8.0
(1 mL), 50
µL of 0.01 M substrate (ATC), 50 µL of 0.01 M DTNB, and 10 µL of enzyme. The substrate
was added into the experiment system containing the DTNB, buffer, and enzyme with inhibitors

Molecules 2016, 21, 132
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after 15 min of incubation. The activity of the enzyme was determined by recording the increase in
˝
absorbance at 412 nm in 1 min intervals at 37 C. Calculations were conducted according to the assay
of Ellman et al. [18]. The in vitro BuChE assay was performed using the method described above.
The kinetic characterization of AChE was performed using the reported method [19].
Three concentrations (2, 4, and 8 nM) of the substrate were mixed in 1 mL 0.1 M KH2PO4/K2HPO4
buffer (pH 8.0) containing 10 µL AChE, 50 µL DTNB, and 50 µL substrate. The test compound was
added into the assay solution and was pre-incubated with the AChE at 37 ˝C for 15 min followed by
addition of the substrate. The kinetic characterization of the AChE-catalyzed ATC hydrolysis was
performed spectrometrically at 412 nm. A parallel control with no inhibitor in the mixture allowed
adjusting activities to be measured at various times.
3.4. Molecular Modeling
The crystal structure of AChE (code ID: 1EVE) and of human BuChE (code ID: 1POI) were obtained
from the Protein Data Bank after eliminating the inhibitor and water molecules. The 3D structure of 5c
was built and geometry optimization was performed using molecular mechanics. Docking studies
were performed using the AUTODOCK 4.0 program (The Scripps Research Institute, San Diego, CA.,
USA). By using the AUTODOCK 4.0 package, we adopted the hybrid Lamarckian genetic algorithm as
the searching algorithm to provide the compound with full flexibility. AUTODOCK Tools was used
to assign Solvation parameters and Kollman charges for all atoms in AChE and BuChE. The grid for
energy evaluation was placed at the bottom of the active site gorge (AChE 2.781 64.383 67.971; BuChE
112.0 20.0 40.0) with grid points in the x-, y-, and z-axes set to 50 Å
ˆ
50 Å
ˆ
50 Å and separated by
0.375 Å. The flexible ligand docking of the compounds was also performed. The docking calculations
were performed using the Lamarckian genetic algorithm, and all parameters were the same for each
docking. We initially used a population of random individuals (population size: 150), with a maximum
number of 2,500,000 energy evaluations. At the end of a docking procedure (150 docking runs),
the resulting positions were clustered according to a root mean square criterion of 1.5 Å.
3.5. Thioflavin T-Based Fluorometric Assay
The self-mediated Aβ42 aggregation was evaluated through the thioflavin-T fluorescence assay [
A mixture of 230 M Aβ42 peptide, 2.30 M AChE (Aβ42/AChE molar ratio 100:1), and the indicated
concentrations of compound 5c (5, 10, 20 ,40 and 80 M) were added to the phosphate buffer (pH 7.40,
0.01 M). After co-incubated in 37 C for 48 h, thioflavin-T (5 M in 50 mM glycine-NaOH buffer,
pH 8.00) was added. Fluorescence was monitored with excitation at 450 nm ( ex) and 485 nm ( em) by
9].
µ
µ
µ
˝
µ
λ
λ
the Spectrofluorimeter LS55. The fluorescence intensity values reached at the plateau (around 300 s)
were recorded after subtracting the background fluorescence. The solutions containing Aβ42, and
Aβ42 plus HuAChE was checked as control. Each experiment was run in triplicate.
3.6. Cellular Toxicity
The SH-SY5Y cell line was seeded on 96-well plates at a density of 1.0
After overnight incubation, the cells were treated with various concentrations (0.1, 1, 20, and 40
graveoline derivatives for 48 h. Subsequently, 10 L of 5 mg/mL MTT solution was added to each well
and further incubated for 4 h at 37 C. The solution was carefully removed, and each well was treated
with dimethyl sulfoxide (DMSO) (200 L for each well). The optical density of the wells was then read
ˆ
104 cells/well.
M) of
µ
µ
˝
µ
on the microplate reader at 490 nm. The parallel tests of all drug doses were performed in triplicate.
4. Conclusions
This study designed and synthesized a series of new graveoline analogs on the basis of the
structural characteristics of AChE dual-site inhibitors. The activity, mechanism of action, binding
mode, and neurotoxicity were explored. The results showed that all analogs displayed inhibitory
ability and inhibitory selectivity against AChE after 41-benzene ring substituted the 2-site and after

Molecules 2016, 21, 132
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introducing the side chain. Compared with that of the parent compounds, the selectivity of the analogs
was reversed. The structure analysis showed the best activity (5c) when the two sites in the graveoline
parent ring substituting phenyl and amino terminal had six chemical bonds (n = 3), and the terminal
amino among these graveoline analogs was piperidine. The enzyme kinetics, molecular docking
studies, and thioflavin T-based fluorometric assay showed that these compounds could combine both
with the AChE central catalytic site and PAS. Furthermore, the cytotoxicity study showed that the
neurocyte (SH-SY5Y) viability was unaffected at low compound concentrations. The results indicated
that these compounds offer potential applications in AD treatment.
Acknowledgments: We thank the National Nature Science Foundation of China (81302701), the Grants for
Scientific Research of BSKY (XJ201226) from Anhui Medical University and the Natural Science Foundation of
Anhui Province (1308085QH140) for financially supporting this study.
Author Contributions: Zeng Li gave the concepts of work, interpreted the results and prepared the manuscript;
Chaoyu Mu and Bin Wang performed the experiments and cooperated in the preparation of the manuscript;
Juan Jin analyzed the data and cooperated in the preparation of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).