Design, synthesis, and evaluation of acetylcholinesterase and butyrylcholinesterase dual-target inhibitors against Alzheimer’s diseases

Article abstract of DOI:10.3390/molecules25030489

A series of novel compounds 6a–h, 8i–1, 10s–v, and 16a–d were synthesized and evaluated, together with the known analogs 11a–f, for their inhibitory activities towards acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The inhibitory activities of AChE and BChE were evaluated in vitro by Ellman method. The results show that some compounds have good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Enzyme inhibition kinetics and molecular modeling studies have shown that compound 8i bind simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE. In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential safety as anti-Alzheimer’s disease (anti-AD) agents. In summary, these data suggest that compound 8i is a promising multipotent agent for the treatment of AD.

Full text of DOI:10.3390/molecules25030489

Article
Design, Synthesis, and Evaluation of
Acetylcholinesterase and Butyrylcholinesterase
Dual‐Target Inhibitors against Alzheimer’s Diseases
1
2
1
1
2
2
Yan Guo , Hongyu Yang , Zhongwei Huang , Sen Tian , Qihang Li , Chenxi Du ,
3
2
2,4,
1,
Tingkai Chen , Yang Liu , Haopeng Sun * and Zongliang Liu *
1
School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University),
Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech
Drugs in Universities of Shandong, Yantai University, Yantai 264005, China; 18865672173@163.com (Y.G.);
15809573905@163.com (Z.H.); 18863665626@163.com (S.T.)
2
School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China; yhykjy@sina.com (H.Y.);
liqihangcpu@163.com (Q.L.); 15651670339@163.com (C.D.); lyliuyang1997@163.com (Y.L.)
School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, China;
3
18013972875@163.com
Jiangsu Food and Pharmaceutical Science College, Huaian 223003, China
4
* Correspondences: sunhaopeng@163.com (H.S.); lzl_0_0@126.com (Z.L.); Tel.: +86‐0535‐6706023 (Z.L.)
Received: 24 December 2019; Accepted: 19 January 2020; Published: 23 January 2020
Abstract: A series of novel compounds 6a–h, 8i–1, 10s–v, and 16a–d were synthesized and
evaluated, together with the known analogs 11a–f, for their inhibitory activities towards
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The inhibitory activities of AChE
and BChE were evaluated in vitro by Ellman method. The results show that some compounds have
good inhibitory activity against AChE and BChE. Among them, compound 8i showed the strongest
inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE IC50 = 0.28 μM). Enzyme
inhibition kinetics and molecular modeling studies have shown that compound 8i bind
simultaneously to the peripheral anionic site (PAS) and the catalytic sites (CAS) of AChE and BChE.
In addition, the cytotoxicity of compound 8i is lower than that of Tacrine, indicating its potential
safety as anti‐Alzheimer’s disease (anti‐AD) agents. In summary, these data suggest that compound
8i is a promising multipotent agent for the treatment of AD.
Keywords: Alzheimer’s disease; acetylcholinesterase inhibitor; butyrylcholinesterase inhibitor;
cytotoxicity; molecular docking; structural modification; structure‐activity relationship
1. Introduction
According to the report of the International Alzheimer’s Association in the last three years, there
are more than 36 million AD patients in the world. With the acceleration of the aging process of the
population, the amount of AD incidence is increasing year by year, and the number of AD patients
in the world will exceed 130 million by 2050 [1–3]. The incidence of AD is over 1.9% among people
over 60 years old in China [4]. AD is the third most common disease in the elderly after cardio‐
cerebrovascular disease and cancer. AD can cause dementia, which is one of the six leading causes of
death in the United States [5,6].
AD was initially characterized by memory loss, cognitive dysfunction, inability to take care of
themselves in daily life, and subsequent exacerbations of mental and behavioral abnormalities [7,8].
AD is not only a seriously threaten of human health and life, but also brings heavy mental burden
and economic pressure to the family members and friends of the patients, causing huge fluctuations
Molecules 2020, 25, 489; doi:10.3390/molecules25030489
www.mdpi.com/journal/molecules

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in the social economy. Therefore, it is an important task of medicinal chemists to develop effective
drugs for the treatment of AD [9,10].
AD is a complex neurodegenerative syndrome. Due to the complicated pathogenesis of AD, its
etiology is not completely clear. The therapeutic drugs used in the clinical and research stages can
only delay the course of AD, but there are no drugs that cure or delay the course of AD [11,12]. A
large number of studies have shown that hypotheses of the pathogenesis of AD include the theory of
cholinergic damage, tau protein hyperphosphorylation, amyloid β‐protein (Aβ) cascade hypothesis,
metal ion homeostasis theory, APOE genotype, oxidative stress theory and so on [13–18].
Previous studies have identified three pathological features of AD: decreased levels of AChE in
the neurotransmitter matrix, deposition of Aβ, and hyperphosphorylation of tau protein. Scientists
have been trying to find the etiology and treatment strategy of AD through further study of the above
three pathological characteristics [19,20]. At present, acetylcholinesterase inhibitors (AChEIs) are the
main treatment for AD. Only five drugs have been approved by the Food and Drug Administration
(FDA) to treat AD. Four of them are AChEIs, including Tacrine, Donepezil, Rivastigmine, and
Galantamine. Tacrine is the first generation AChEI, but it is limited in clinical usage because of
hepatotoxicity [11,21,22].
There are two types of ChEs in the central nervous system, namely AChE and BChE. AChE and
BChE are important targets for the development of anti‐AD drugs. The physiological function of
AChE has been preliminarily understood, but the understanding of BChE is less. Studies have shown
that ACh activity in some brain regions of patients with mild to severe AD decreases to 10%–15% of
the normal value, and AChEIs have significant effects on patients with mild to moderate AD and can
repair their cognitive impairment and other symptoms [23].
When AD develops to the middle and late stage, AChE activity decreases, whereas BChE activity
increases, and BChE acts as a metabolic compensation for AChE, partially compensating for the role
of AChE in hydrolyzing ACh [11,23,24]. The regulation of AChE is increasingly dependent on BChE,
so BChE is gradually accepted as a target of anti‐AD drugs. Therefore, the design and development
of dual‐target inhibitors of AChE and BChE may have the following advantages: it can not only
effectively reduce the degradation of AChE and the drug resistance of AChEIs, but also be effective
in patients for moderate to severe symptoms of AD. Some people think that appropriate inhibition of
AChE and BChE is a more ideal treatment for AD [25,26]. Therefore, we are looking for dual‐target
ChEs inhibitors with inhibitory activity on both AChE and BChE.
With the study of AChE crystal structure, it was found that the ligand binding pocket of AChE
in general is a long and narrow channel extending from the surface to the interior [27]. The channel
is dumbbell‐shaped. The opening and bottom of the channel are relatively open, and the middle is
narrow. The active site of AChE contains two important domains: CAS at the bottom of the channel
is the binding site for substrates and inhibitors, consisting of three residues: Ser203, His447, and
Glu334; PAS is situated at the opening of the channel, which is the binding site for the enzyme
inhibitor. It consists of five residues: Tyr72, Tyr124, Trp286, Tyr34 and Asp74 [11,28,29]. It was found
that PAS of AChE could induce the formation of Aβ protein and accelerate its precipitation.
Researchers began to develop dual‐site AChEIs that act on both CAS and PAS sites simultaneously,
which can interfere with the aggregation of Aβ while enhancing the inhibitory activity of AChE, and
play a dual role in the treatment of AD [30].
In our previous studies, we found several new ChEIs through virtual screening based on
pharmacophore. We have found that G801‐0274 AChE (eeAChE IC50 = 2.05 μM), BChE (eqBChE IC50
= 0.03 μM) can inhibit ChEs. In this paper, we used it as the lead compound for structural modification
[31]. It has the property of dual‐site binding and can bind PAS and CAS sites simultaneously.
In this study, we designed and synthesized a series of new derivatives based on G801‐0274 and
evaluated their biological activities, including cytotoxicity and AChEs inhibition. By summarizing
our data, we found a new type of dual‐target inhibitor of AChE and BChE with in vitro activities,
hoping to develop anti‐AD drugs through further efforts (Figure 1).

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Figure 1. Design of a series of derivatives as dual‐target inhibitor of AChE and BChE.
2. Results and Discussion
2.1. Chemistry
The synthesis of the designed compounds 6a–h and 8i–l started from 4‐piperidinecarboxamide
1. At the first step, compound 1 was reacted with appropriate substituted Benzylchloride derivatives
(2a–d) under potassium carbonate (K2CO3) and potassium iodide (KI) conditions, giving relevant N‐
Benzylpiperidin‐4‐carboxamide derivatives (3a–d) with medium yields (67%–81%). In addition, the
obtained compounds 3a–d were transformed into relevant substituted (1‐Benzylpiperidin‐4‐yl)
methanamine derivatives (4a–d) in the reduction reaction, which was carried out in dry
tetrahydrofuran (THF) using lithium aluminum hydride (LiAlH4) under nitrogen atmosphere. The
obtained compounds 4a–d were used in further synthesis without purification. The yields of this step
were between 74% and 82%. Finally, the commercially available nitrogen‐containing heterocyclic
aromatic carboxylic acids (5a–d) or 2‐Oxoindoline‐5‐carboxylic acid 7 were activated with
Carbonyldiimidazole
(CDI)
or
Benzotriazol‐1‐yl‐oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) and reacted with the appropriate amine (4a–d) in dry THF or N,N‐
dimethylformamide (DMF) to give the target compounds 6a–h and 8i–l with moderate to good yield
(35%–80%)(Scheme 1 and Scheme 2). Compounds 10s–v were prepared (73%–88% yield) by reactions
of compounds 4a–d with p‐Toluenesulfonyl chloride (TsCl, 9) under the presence of Triethylamine
(Et3N) in Dichloromethane (DCM) (Scheme 3).
1
13
The structures of the new compounds were confirmed by spectral data ( H‐NMR, C‐NMR, and
HRMS, see Supplementary Material).

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Scheme 1. Synthesis of compounds 6a–h. Reagents and conditions: (a) compounds 2a–d, KI, K2CO3,
acetone, reflux 4 h; (b) LiAlH4, THF, 0 °C, reflux 4 h; (c) 5a–d, CDI, THF, or PyBOP, DIPEA (N,N‐
Diisopropylethylamine), DMF, r.t.
Scheme 2. Synthesis of compounds 8i–l. Reagents and conditions: 2‐Oxoindoline‐5‐carboxylic acid 7,
PyBOP, DIPEA, DMF, r.t. 24 h.
Scheme 3. Synthesis of compounds 10s–v. Reagents and conditions: TsCl 9, Et3N, DCM, 0 °C 4 h.

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The synthetic route of compounds 16a–d has been depicted in Scheme 4. Ethyl 2‐piperidin‐4‐
ylacetate 11 was reacted with appropriate substituted (2‐Bromoethyl) benzene derivatives (12a–b)
under potassium carbonate (K2CO3) and potassium iodide (KI) conditions. Then, the obtained
compounds 13a–b were used in further synthesis without purification. 4 mol/L potassium hydroxide
(KOH) was added to compounds 13a–b in C2H5OH: H2O = 5:1. The reaction mixture was stirred at
room temperature for 7 h to give compounds 14a–d. Finally, compounds 14a–d were activated with
PyBOP and reacted with compounds 15a–b in DMF to give the target compounds 16a–d with
moderate to good yield (30%–80%) (Scheme 4).
Scheme 4. Synthesis of compounds 16a–d. Reagents and conditions: (a) (2‐Bromoethyl) benzene
derivatives 12a–b, K2CO3, catalytic amount of KI, acetone, reflux 4 h; (b) 4 mol/L KOH, C2H5OH: H2O
= 5:1, r.t. 7 h; (c) compounds 15a–b, PyBOP, DIPEA, DMF, r.t. 24 h.
2.2. AChE and BChE Inhibitory Activity of the Target Molecules
Compounds 6a–h, 8i–l, 11a–f, 10s–v, and 16a–d were evaluated for their anti‐ChEs activity.
Tacrine and Donepezil were used as reference drugs. According to the method described by Ellman
[32], the data were expressed by IC50 values. In vitro experiments showed that some of these
compounds could effectively inhibit ChEs in the micromolar range (Table 1).
Table 1. Structures, eeAChE, and eqBChE inhibitory activities of target compounds.
c
AChEa (IC50 , μM or
BChEb (IC50, μM or
Compound
6a
Structure
SIe
IRd, %)
IRd, %)
52.11 ± 30.25
38.08 ± 12.83
15.02 ± 6.17
31.37 ± 15.56
10.75 ± 5.55
9.56 ± 4.52
1.66
11af
6b
3.54
1.57

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0.25
6c
9.06 ± 2.57
48.57 %
36.64 ± 17.54
44.57 %
11bf
11cf
‐‐
5.44 ± 2.22
21.29 ± 5.00
0.26
H
N
N
H
6d
6e
4.80 ± 0.68
2.15 ± 0.63
2.23
N
N
O
48.48 ± 36.19
18.81 %
39.88%
16.51 %
‐‐
‐‐
11df
11ef
7.48 ± 3.10
42.12 %
‐‐
11ff
6f
0.46 ± 0.40
43.07 %
43.07 %
49.63 %
‐‐
‐‐
6g
29.20 ± 16.38
40.20 ± 27.64
49.87 ± 37.29
30.71%
0.58
‐‐
6h
8i
0.39 ± 0.04
no
0.28 ± 0.10
22.26%
1.39
8k
‐‐
8j
8l
14.69%
22.87%
35.20%
‐‐
‐‐
21.54 ± 9.37

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1.03
10s
4.24 ± 2.44
5.50 ± 2.56
4.10 ± 3.74
2.01 ± 0.70
10t
2.74
10u
10v
11.61%
18.31%
‐‐
7.20 ± 10.75
7.14 ± 5.27
1.01
16a
16b
16c
16d
8.61%
8.95%
no
23.49%
31.21%
30.59%
38.04%
‐‐
‐‐
‐‐
‐‐
8.55%
Tacrine
Donepezil
_
_
0.02 ± 0.01
0.008 ± 0.002
0.008 ± 0.004
1.734 ± 0.731
2.50
0.0046
a AChE (EC 3.1.1.7) from electric eel. b BChE (EC 3.1.1.8) from horse serum. c Concentration required
for 50% inhibition of ChEs, data were shown in mean ± SEM of triplicate independent experiments. d
Inhibitory rate of the compounds under 100 μM on ChEs. e Selectivity index (SI) = AChE IC50 / BChE
IC50. f The known analogs 11a–f [33].
First, we synthesized compounds 6a, 6c, 6d, 8i by maintaining the R as H, X as amide and
exploring Y with 1H‐Indole, 1H‐Indazole, 1H‐Benzo[d] imidazole or 2‐Oxoindoline, together with the
known analog 11f.We found that when Y is substituted by a series of different structures, its activity
on AChE are 2‐Oxoindoline (8i) > 1H‐Benzo[d] [1,2,3] triazole (11f) > 1H‐Benzo[d]imidazole (6d) > 1H‐
Indazole (6c) > 1H‐Indole (6a); its activity on BChE are 2‐Oxoindoline (8i) > 1H‐Benzo[d]imidazole
(6d) > 1H‐Indole (6a) > 1H‐Benzo[d] [1,2,3] triazole (11f). Among them, compound 8i (Figure 2)
showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and BChE (eqBChE
IC50 = 0.28 μM), these results indicated that compound 8i was a potent dual inhibitor against AChE
and BChE. It was speculated that 2‐Oxoindoline is the key structure for inhibiting two ChEs and is
not selective for both ChEs. According to the molecular docking results, for eeAChE, 2‐Oxoindoline
of compound 8i bound with Trp286 via π‐π stacking interaction; for huAChE, 2‐Oxoindoline of
compound 8i bound with Trp286 and Tyr341 via π‐π stacking interaction, and for huBChE, 2‐
Oxoindoline of compound 8i bound with Phe329 via π‐π stacking interaction. These interactions
increase the inhibitory activity by enhancing the binding affinity.

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Figure 2. Design of compound 8i for dual‐target inhibitor of AChE and BChE.
Then, we evaluated the effect of R on ChEs activity. We modified Rwith different substituent (2‐
Cl, 4‐OCH3, 4‐CF3), compared with compound 6d, when R is4‐CF3 (6e), 4‐OCH3 (11d), 2‐Cl (11e), the
ChEs inhibitory activity decreased. In particular, compound 11d showed little inhibitory activity on
BChE at 100 μM. In general, with the same Y, when R is H, the compounds have the highest inhibitory
activity against ChEs; when Y is substituted by 2‐Cl or 4‐CF3, the inhibitory activity of the compounds
to ChEs were weakened; and when Y is substituted by 4‐OCH3, the compounds have the worst
inhibitory activity against AChEs. We speculate that enhancing the electron‐withdrawing effect or
the donor effect on the aromatic ring is not conducive to improving the performance of the analog,
and the appropriate space may facilitate the analog to enter the CAS pocket of ChEs.
Next, we investigated the effect of the p‐Toluenesulfonamide moiety on the inhibitory activity
of ChEs. Not only the amide group was replaced by Sulfonamide moiety based on the principles of
bioisosterism, but also the fused nitrogen‐containing bicyclic system (Indole, Indazole, Oxoindoline,
Benzimidazole) in the previous compounds was replaced by Tosyl moiety. We synthesized
compounds 10s–v. Except for compound 10u (eeAChE 11.61% [100 μM], eqBChE 18.31% [100 μM])
has low inhibitory activity against ChEs, the compounds 10s (eeAChE IC50 = 4.24 μM, eqBChE IC50 =
4.10 μM)), 10t (eeAChE IC50 = 5.50 μM, eqBChE IC50 = 2.01 μM), 10v (eeAChE IC50 = 7.20 μM, eqBChE
IC50 = 7.14 μM) all can maintain ChEs inhibitory activity at micromolar levels, indicating that the p‐
Toluenesulfonamide moiety is responsible for maintaining the inhibitory activity of ChEs. On one
hand, we speculate that methyl occupies the pocket of the active site and interacts with amino acid
residues to increase inhibitory activity. On the other hand, Sulfonamide moiety is very important for
maintaining ChEs inhibitory activity, which may be related to the bond angle between Sulfonamide
and molecules [33].
In addition, we tested the number of carbon atoms between benzene and piperidine. When the
number of carbon atoms becomes two (compounds 16a–d), the compounds had low inhibitory
activity against the two kinds of ChEs, in particular, compound 16c has no inhibitory activity against
AChE at 100 μM. We speculate that the decrease in the activity of such compounds may be that the
molecular volume is too large to enter the active pocket of ChEs, indicating that the residue of N‐
Benzylpiperidine in the structure is an essential group for inhibiting both ChEs.
Compounds designed with a Piperazine ring instead of a piperidine ring may also have the same
or higher inhibitory activity on ChEs. Luca P, Tomás Daniel, Asha H, et al. [34] based on the structure
of Donepezil, mainly the conjugation of Benzylpiperidine/ Benzylpiperazine moiety with a
biologically active heterocyclic derivative (Benzimidazole or Benzofuran), which gave the compound
1/4
other relevant properties. It shows good activity (IC 4.0–30.0 μM) for AChE inhibition, and has
50
inhibition of Aβ peptide aggregation, antioxidant activity, and metal chelation.
2.3. Kinetic Studies of AChE and BChE Inhibition
To determine the kinetic types of AChE and BChE inhibition, compounds 8i and 10s were
selected for kinetic studies. In each case, the kinetic types of enzyme inhibition were obtained by the
modified Ellman’s method and the Lineweaver–Burk secondary plots [35]. The Lineweaver–Burk

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plots showed both increasing slope (decreased Vmax) and increasing intercept (higher Km) for higher
inhibitor concentrations, indicating a mixed‐type inhibition, including competitive inhibition and
non‐competitive inhibition, which possibly was because compound 8i could bind to both CAS and
PAS (Figure 3A,B). According to the result of molecular docking study. The same inhibition type
between compound 10s and ChEs was found in graphical analysis (Figure 3C,D).
Figure 3. (A) Lineweaver–Burk plot for the inhibition of eeAChE(A) and eqBChE (B) by compound 8i
at different concentrations of substrate. (B) Lineweaver–Burk plot for the inhibition of eeAChE (C)
and eqBChE (D) by compound 10s at different concentrations of substrate.
2.4. Docking Studies
To further study the binding mode of compound 8i and ChEs, molecular docking was performed
using Discovery Studio software 2016. The predicted binding mode of compound 8i is shown in
Figure 4 and Figure 5. Compound 8i could interact with CAS and PAS of AChE simultaneously. For
AChE (from Electrophorus electricus (electric eeAChE, Sigma‐Aldrich) (Figure 4A), the N‐
Benzylpiperidine moiety interacted with Trp86 in CAS via aromatic π‐π interaction. The Amide
group formed hydrogen bond with Phe295. Moreover, 2‐Oxoindoline of compound 8i bound with
Trp286 via π‐π stacking interaction; for human AChE‐huAChE (Sigma‐Aldrich) (Figure 4B), the N‐
Benzylpiperidine moiety of compound 8i was bound to CAS, displaying a classic aromatic π‐π
interaction with Trp86. Moreover, 2‐Oxoindoline of compound 8i bound with Trp286 and Tyr341 via
π‐π stacking interaction. In addition, the Amide group formed hydrogen bond with Phe295. By
comparison, it was found that the compounds have similar binding patterns to eeAChE and huAChE.
All these facts provide an explanation for the higher inhibitory effects of compound 8i towards AChE.
Molecular docking of compound 8i at the active site of human BuChE‐huBChE (Sigma‐Aldrich,
Munich, Germany) has been shown in Figure 5. The N‐Benzylpiperidine moiety of compound 8i

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interacts with Asp70 via electrostatic interaction. Moreover, the N‐Benzylpiperidine moiety interacts
with Trp82 in CAS by T‐shaped π‐π interaction, and the N‐Benzylpiperidine moiety forms π‐alkyl
interaction with Ala328. 2‐Oxoindoline moiety of compound 8i bound with Phe329 via π‐π stacking
interaction. These interactions increase the inhibitory activity by enhancing the binding affinity.
Figure 4. (A) Binding mode prediction of compound 8i with eeAChE (PDB ID: 1C2B) (B) Binding
mode prediction of compound 8i with huAChE (PDB ID: 4EY7).
Figure 5. Binding mode prediction of compound 8i with huBChE (PDB ID: 4TPK).
From the binding mode prediction of Donepezil with huAChE (Figure 6), we found that the
binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N‐Benzylpiperidine
moiety was bound to CAS, displaying a classic aromatic π‐π interaction with Trp86; (ii) The Oxygen
atom formed hydrogen bond with Phe295; (iii) Aromatic heterocycle moiety bound with Trp286 and
Tyr341 via π‐π stacking interaction. In addition, the Indone moiety of Donepezil interacts with
Trp286 at the center of the PAS; the piperidine moiety of Donepezil interacts with Tyr337, Phe338,
Tyr341.

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Figure 6. (A) Binding mode prediction of Donepezil with eeAChE (PDB ID: 1C2B); (B) Binding mode
prediction of Donepezil with huAChE (PDB ID: 4EY7).
From the binding mode prediction of Donepezil with huBChE (Figure 7), we also found that the
binding pattern of compound 8i is similar to Donepezil in some respects: (i) The N‐Benzylpiperidine
moiety interacts with Asp70 via electrostatic interaction. In addition, the N‐Benzylpiperidine moiety
of Donepezil interacts with Try332 via electrostatic interaction; (ii) the N‐Benzylpiperidine moiety
interacts with Trp82 in CAS by T‐shaped π‐π interaction, and the N‐Benzylpiperidine moiety form
π‐alkyl interaction with Ala328. The difference is that 2‐Oxoindoline of compound 8i bound with
Phe329 via π‐π stacking interaction while the 2‐Oxoindoline moiety of Donepezil interacts with
Gly116 in the center of the PAS.
Figure 7. Binding mode prediction of Donepezil with huBChE (PDB ID: 4TPK).
Compared with Donepezil, the inhibitory activity of compound 8i on BChE is higher than
Donepezil, and has similar inhibitory activity on two ChEs, so that it can exert an anti‐ChEs effect in
a balanced manner. Studies on compound 8i molecular docking have shown that the

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Benzylpiperidine moiety of the compound acts on the CAS of the enzyme, while the 2‐Oxoindoline
moiety binds to the PAS of the enzyme, which is basically consistent with the design idea.
2.5. Cytotoxicity Studies
We focused on the cytotoxicity of the synthetic compounds. The reason for using PC12 is that
our compounds act on the central nervous system, so we need to find out whether the compound has
a toxic effect on normal nerve cells. Compounds 6b, 6c, 6d, 10s, 8i, and 10v were selected as
representative compounds to assess their potential cytotoxic effects. Compounds 6c, 6d, 8i are less
toxic than Tacrine; the toxicity of compounds 6b, 10v are similar to that of Tacrine; compound 10s is
slightly more toxic than Tacrine. Among them, compound 8i has the lowest toxicity (Figure 8).
Figure 8. In vitro cell toxicity of compounds 6b, 6c, 6d, 10s, 8i, and 10v on PC‐12 cell line. Data were
expressed as mean ± SD (n = 3).
3. Materials and Methods
3.1. Chemistry
All reagents were obtained from commercial suppliers and were used without any further
purification unless otherwise stated. Flash column chromatography was performed with silica gel
(200‐300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. Thin layer chromatography was
performed using silica gel 60 F254 precoated plates (purchased from Qingdao Haiyang Inc., Qingdao,
China). Visualization was achieved using Ultraviolet (UV) light (254 nm and 365 nm, Shanghai
Yarong Biochemical Instrument Factory, Shanghai, China). Melting points were determined with a
Mel‐TEMP II melting point apparatus (Beijing Keyi Company, Beijing, China) and was uncorrected.
1
13
H NMR and C NMR spectra were recorded with Bruker AV‐600, AV‐500 or AV‐400 MHz
instruments (Bruker, Ettlingen, Germany) using DMSO‐d6, CD3OD, or CDCl3 as solvent. Chemical
shifts were reported as δ values (ppm) from internal reference tetramethylsilane (TMS). All coupling
constants were reported in hertz (Hz), All chemical shifts are reported in parts per million (ppm),
relative to the internal standard. In addition, proton multiplicities were labeled as br (broad), s
(singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplet). HR‐MS were
performed on a Waters Vion IMS Q‐tof (Waters, MA, USA).

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3.2. General Procedure for the Synthesis of Compounds 3a–d
4‐Piperidinecarboxamide (1) (3.00 g, 23.4 mmol) and substituted Benzylchloride derivatives (2a–
d) (28.1 mmol) were dissolved in 20 mL acetone. Then, anhydrous K2CO3 (6.47 g, 46.8 mmol) and
catalytic amount KI were added. The reaction mixture was refluxed for 4 h. After completion of the
reaction, acetone was concentrated, and the residue was dissolved in water (60 mL) and extracted
with ethyl acetate (60 × 3 mL). The combined organic layers were dried over Na2SO4, filtered, and the
solvent was removed under reduced pressure. After concentration, the crude product was purified
by silica gel column chromatograph (DCM: methanol = 60:1–5:1) to give target compounds 3a–d.
3.3. General Procedure for the Synthesis of Compounds 4a–d
Compounds 3a–d (3 g) were dissolved in anhydrous THF (17 mL) and then LiAlH4 (5 equiv.)
was added to the above cooled solution at 0–5 °C in small portions under stirring. The reaction
mixture was further stirred at room temperature for 30 min and finally refluxed for 4 h. After cooling,
water and 10% NaOH solution was added at 0–5 °C. Then, the obtained white precipitate was filtered
off and washed with THF. The filtrate was extracted with ethyl acetate. The combined organic layers
were dried over anhydrous Na2SO4, filtered, and concentrated in vacuum. The obtained compounds
4a–d were used in further synthesis without purification.
3.4. General Procedure for the Synthesis of Compounds 6a–h
CDI (1 equiv.) was added to a solution of the acids (5a–d) ((300 mg, 1 equiv.) in dry THF under
nitrogen atmosphere. After 30 min, the solution of substituted 4‐Amine‐1‐benzylpiperidines (4a–d)
(1.2 equiv.) in THF were added, and the reaction mixture was stirred at room temperature for 24 h.
After the reaction was completed, the solvent was removed under reduced pressure, and then the
reaction mixture was quenched with saturated NaCl solution (25 mL). The aqueous phase was
extracted with DCM (25 × 3 mL). The DCM layer was combined and washed with brine solution (25
× 3 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was removed under
reduced pressure. After concentration, the crude product was purified by silica gel column
chromatograph (DCM: methanol = 60:1–5:1) to give target compounds 6a–h.
N‐((1‐Benzylpiperidin‐4‐yl)methyl)‐1H‐indole‐5‐carboxamide (6a). 1H‐Indole‐5‐carboxylic acid (300 mg,
1.86 mmol), CDI (302 mg, 1.86 mmol), (1‐Benzylpiperidin‐4‐yl)methanamine (457 mg, 2.24 mmol),
1
THF (15 mL) White solid, m.p.: 89–90 °C, yield: 80%, H NMR (500 MHz, DMSO‐d6) δ 11.50 (s, 1H),
8.47 (s, 1H), 8.17 (s, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.41 (d, J = 7.1 Hz, 2H), 7.36–
7.29 (m, 3H), 6.51 (s, 1H), 3.78 (s, 2H), 3.19 (d, J = 5.5 Hz, 2H), 2.99 (d, J = 10.8 Hz, 2H), 2.31 (d, J =
13
11.0 Hz, 2H), 1.72 (d, J = 12.0 Hz, 3H), 1.38 (d, J = 11.4 Hz, 2H). C NMR (126 MHz, DMSO‐d6) δ
168.15, 137.84, 135.07, 130.40, 128.81, 128.39, 127.44, 127.05, 125.94, 120.97, 120.37, 111.37, 102.49,
+
61.15, 52.37, 44.70, 35.20, 28.63. HRMS (ESI): calcd. For C22H25N3O [M + H] 348.2070, found
348.2070.
N‐((1‐(2‐Chlorobenzyl)piperidin‐4‐yl)methyl)‐1H‐Indole‐5‐carboxamide (6b). 1H‐Indole‐5‐carboxylic acid
(300 mg, 1.86 mmol), CDI (302 mg, 1.86 mmol), (1‐(2‐Chlorobenzyl) piperidin‐4‐yl)methanamine
1
(532 mg, 2.24 mmol), THF (15 mL) White solid, m.p.: 88–89 °C, yield: 66%, H NMR (400 MHz,
CD3OD) δ 8.08 (d, J = 1.1 Hz, 1H), 7.58 (dd, J = 8.6, 1.8 Hz, 1H), 7.49 (dd, J = 7.1, 2.2 Hz, 1H), 7.41–
7.35 (m, 2H), 7.30–7.24 (m, 3H), 6.51 (d, J = 3.2 Hz, 1H), 3.78 (s, 2H), 3.05 (d, J = 11.7 Hz, 2H), 2.31 (t, J
13
= 11.9 Hz, 2H), 1.77 (t, J = 14.0 Hz, 3H), 1.45–1.33 (m, 2H). C NMR (101 MHz, CD3OD) δ 170.60,
138.15, 134.64, 131.71, 129.37, 129.10, 127.70, 126.78, 125.91, 125.17, 120.18, 119.95, 110.67, 102.17,
+
58.57, 53.06, 44.79, 35.56, 28.93. HRMS (ESI): calcd. For C22H24ClN3O [M + H] 382.1681, found
382.1703.
N‐((1‐Benzylpiperidin‐4‐yl) methyl)‐1H‐indazole‐5‐carboxamide (6c). 1H‐Indazole‐5‐carboxylic acid (300
mg, 1.86 mmol), CDI (302 mg, 1.86 mmol), (1‐Benzylpiperidin‐4‐yl)methanamine (457 mg, 2.24
1
mmol), THF (15 mL) White solid, m.p.: 111–112 °C, yield: 56%, H NMR (500 MHz, DMSO‐d6) δ
13.30 (s, 1H), 8.48 (s, 1H), 8.35 (s, 1H), 8.20 (s, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H),

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7.33–7.27 (m, 3H), 7.24 (d, J = 6.8 Hz, 1H), 3.45 (s, 2H), 3.18 (d, J = 6.0 Hz, 2H), 2.81 (d, J = 11.1 Hz,
13
2H), 1.92 (t, J = 11.1 Hz, 2H), 1.67 (d, J = 12.4 Hz, 2H), 1.59 (s, 1H), 1.22 (d, J = 9.4 Hz, 2H). C NMR
(126 MHz, DMSO‐d6) δ 167.07, 141.33, 138.83, 135.11, 129.24, 128.56, 127.67, 127.62, 127.30, 125.79,
+
122.79, 120.90, 110.16, 62.78, 53.38, 45.31, 36.17, 30.23. HRMS (ESI): calcd. For C21H24N4O [M + H]
349.2023, found 349.2019.
N‐((1‐Benzylpiperidin‐4‐yl)methyl)‐1H‐benzo[d]imidazole‐5‐carboxamide (6d). 1H‐Benzo[d]imidazole‐5‐
carboxylic acid (300 mg, 1.85 mmol), CDI (300 mg, 1.85 mmol), (1‐Benzylpiperidin‐4‐
1
yl)methanamine (453 mg, 2.22 mmol), THF (15 mL) Yellow oil, yield: 72%, H NMR (500 MHz,
DMSO‐d6) δ 8.53 (s, 1H), 8.27 (d, J = 68.8 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.70–7.61 (m, 1H), 7.28 (d, J
= 6.9 Hz, 3H), 7.21 (d, J = 5.6 Hz, 1H), 7.04 (s, 1H), 3.39 (s, 2H), 3.19 (s, 2H), 2.76 (s, 2H), 1.85 (d, J =
13
11.8 Hz, 2H), 1.65 (d, J = 11.8 Hz, 3H), 1.20 (d, J = 10.9 Hz, 2H). C NMR (126 MHz, DMSO‐d6) δ
167.40, 144.24, 139.08, 135.64, 129.16, 128.51, 127.18, 121.88, 115.66, 114.85, 62.93, 53.43, 53.35, 45.39,
+
36.25, 30.32. HRMS (ESI): calcd. For C21H24N4O [M + H] 349.2023, found 349.2023.
N‐((1‐(4‐(Trifluoromethyl)benzyl)piperidin‐4‐yl)methyl)‐1H‐benzo[d]imidazole‐5‐carboxamide (6e). 1H‐
Benzo[d]imidazole‐5‐carboxylic acid (300 mg, 1.85 mmol), CDI (300 mg, 1.85 mmol), (1‐(4‐
(Trifluoromethyl)benzyl)piperidin‐4‐yl)methanamine (604 mg, 2.22 mmol), THF (15 mL) White
1
solid, m.p.: 92–93 °C, yield: 77%, H NMR (400 MHz, CD3OD) δ 8.29–8.27 (m, 1H), 8.12 (d, J = 1.0 Hz,
1H), 7.87–7.77 (m, 2H), 7.62 (d, J = 7.8 Hz, 1H), 7.56 (t, J = 8.9 Hz, 2H), 7.39 (p, J = 5.5 Hz, 2H), 3.66 (d,
J = 6.7 Hz, 2H), 3.30 (s, 1H), 3.27 (s, 1H), 2.90 (d, J = 11.4 Hz, 2H), 2.15–2.06 (m, 2H), 1.79–1.63 (m,
13
3H), 1.44–1.24 (m, 3H). C NMR (126 MHz, CD3OD) δ 169.23, 141.32, 134.81, 131.92, 130.75, 130.34,
128.00, 127.37, 127.30, 125.41, 122.55, 120.67, 114.13, 113.58, 109.69, 58.94, 57.96, 53.34, 53.01, 44.93,
+
44.83, 35.64, 35.52, 29.26, 29.06. HRMS (ESI): calcd. For C22H23F3N4O [M + H] 417.1897, found
417.1895.
N‐((1‐(4‐Methoxybenzyl)piperidin‐4‐yl)methyl)‐1H‐benzo[d][1,2,3]triazole‐5‐carboxamide (6f). 1H‐
Benzo[d][1,2,3]triazole‐5‐carboxylic acid (300 mg, 1.84 mmol), CDI (298 mg, 1.84 mmol), (1‐(4‐
Methoxybenzyl)piperidin‐4‐yl)methanamine (517 mg, 2.21 mmol), THF (15 mL) Yellow oil, yield:
1
63%, H NMR (500 MHz, DMSO‐d6) δ 8.53 (s, 1H), 8.32 (s, 1H), 8.16 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H),
7.61 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.3 Hz, 2H), 6.91 (d, J = 8.3 Hz, 2H), 3.74 (s, 3H), 3.69 (s, 2H), 3.18
(d, J = 5.9 Hz, 2H), 2.97 (d, J = 11.1 Hz, 2H), 2.26 (s, 2H), 1.73 (d, J = 13.0 Hz, 2H), 1.68 (s, 1H), 1.34 (d,
13
J = 11.2 Hz, 2H). C NMR (126 MHz, DMSO‐d6) δ 167.31, 159.33, 144.22, 131.57, 128.92, 121.94,
+
114.16, 60.78, 55.52, 52.34, 44.84, 35.34, 28.86. HRMS (ESI): calcd. For C21H25N5O2 [M + H] 380.2081,
found 380.2077.
N‐((1‐(2‐Chlorobenzyl)piperidin‐4‐yl)methyl)‐1H‐benzo[d][1,2,3]triazole‐5‐carboxamide (6g). 1H‐
Benzo[d][1,2,3]triazole‐5‐carboxylic acid (300 mg, 1.84 mmol), CDI (298 mg, 1.84 mmol), (1‐(2‐
Chlorobenzyl)piperidin‐4‐yl)methanamine (526 mg, 2.21 mmol), THF (15 mL) White solid, m.p.: 77–
1
79 °C, yield: 57%, H NMR (500 MHz, DMSO‐d6) δ 8.63 (s, 1H), 8.44 (s, 1H), 7.87 (d, J = 2.7 Hz, 2H),
7.47 (d, J = 7.1 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 6.9 Hz, 1H), 7.27–7.25 (m, 1H), 7.03 (s, 1H),
3.52 (s, 2H), 3.21 (t, J = 6.1 Hz, 2H), 2.82 (d, J = 11.3 Hz, 2H), 2.00 (t, J = 10.4 Hz, 2H), 1.68 (d, J = 12.0
13
Hz, 2H), 1.61 (s, 1H), 1.23 (d, J = 10.5 Hz, 2H). C NMR (126 MHz, DMSO‐d6) δ 166.50, 140.30,
139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08, 30.16.
+
HRMS (ESI): calcd. For C20H22ClN5O [M + H] 384.1586, found 384.1584.
N‐((1‐(4‐(Trifluoromethyl)benzyl)piperidin‐4‐yl)methyl)‐1H‐benzo[d][1,2,3]triazole‐5‐carboxamide (6h).
1H‐Benzo[d][1,2,3]triazole‐5‐carboxylic acid (300 mg, 1.84 mmol), CDI (298 mg, 1.84 mmol), (1‐(4‐
(Trifluoromethyl)benzyl)piperidin‐4‐yl)methanamine (601 mg, 2.21 mmol), THF (15 mL) White
1
solid, m.p.: 90–91 °C, yield: 73%, H NMR (400 MHz, CD3OD) δ 8.38 (td, J = 1.5, 1.0 Hz, 1H), 7.92–
7.90 (m, 1H), 7.88 (t, J = 1.0 Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 7.8 Hz, 1H), 7.61 (dt, J = 7.7,
4.1 Hz, 1H), 7.51–7.42 (m, 2H), 3.89 (d, J = 22.6 Hz, 2H), 3.33 (dd, J = 6.5, 3.7 Hz, 2H), 3.13–3.01 (m,
13
2H), 2.47–2.31 (m, 2H), 1.90–1.68 (m, 3H), 1.52–1.32 (m, 2H). C NMR (126 MHz, DMSO‐d6) δ 166.50,
140.30, 139.38, 138.70, 131.96, 129.27, 128.56, 127.32, 125.46, 115.61, 114.32, 62.73, 53.33, 45.41, 36.08,
+
30.16. HRMS (ESI): calcd. For C21H22F3N5O [M + H] 418.1849, found 418.1850.

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3.5. General Procedure for the Synthesis of 8i–l
Intermediates (7) (140 mg, 1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were added to
DMF and stirred at room temperature for 20 min. Then, intermediates (4a–d) (1.0 equiv.) was added
and stirred at room temperature for 4 h. After completion of the reaction, the reaction mixture was
quenched with saturated NaCl solution (25 mL). The aqueous phase was extracted with DCM (25 ×
3 mL). The DCM layer was combined and washed with brine solution (25 × 3 mL). The organic layer
was dried over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After
concentration, the crude product was purified by silica gel column chromatograph using a methanol
in DCM gradient (DCM: methanol= 60:1–5:1) yielded compounds 8i–l.
N‐((1‐Benzylpiperidin‐4‐yl)methyl)‐2‐Oxoindoline‐5‐carboxamide (8i). 2‐Oxoindoline‐5‐carboxylic acid
(140 mg, 0.79 mmol), (1‐Benzylpiperidin‐4‐yl)methanamine (135 mg, 0.66 mmol), PyBOP (412 mg,
1
0.79 mmol), DIPEA(128 mg, 0.99 mmol), DMF (6 mL). White solid, m.p.:155–156 °C, yield: 56%, H
NMR (400 MHz, CD3OD) δ 7.73–7.68 (m, 2H), 7.53–7.42 (m, 5H), 6.91 (d, J = 8.1 Hz, 1H), 4.26 (s, 2H),
3.44 (d, J = 12.5 Hz, 2H), 3.31 (s, 1H), 2.98 (t, J = 12.9 Hz, 2H), 1.97 (d, J = 13.6 Hz, 3H), 1.54 (q, J = 13.1,
13
12.1 Hz, 2H). C NMR (101 MHz, CD3OD) δ 178.52, 168.90, 146.69, 130.99, 129.80, 129.40, 128.99,
127.92, 127.60, 125.95, 123.42, 123.40, 108.97, 60.21, 51.86, 43.82, 42.11, 34.01, 26.82. HRMS (ESI):
+
calcd. For C22H25N3O2 [M + H] 364.2020, found 364.2032.
N‐((1‐(2‐Chlorobenzyl)piperidin‐4‐yl)methyl)‐2‐Oxoindoline‐5‐carboxamide (8j). 2‐Oxoindoline‐5‐
carboxylic acid (140 mg, 0.79 mmol), (1‐(2‐Chlorobenzyl)piperidin‐4‐yl)methanamine (157 mg, 0.66
mmol), PyBOP (412 mg, 0.79 mmol), DIPEA(128 mg, 0.99 mmol), DMF (6 mL). White solid, m.p.:
1
151–152 °C, yield: 35%, H NMR (600 MHz, CD3OD) δ 7.74–7.69 (m, 2H), 7.50 (d, J = 7.2 Hz, 1H), 7.39
(d, J = 7.6 Hz, 1H), 7.28 (dt, J = 20.1, 7.1 Hz, 2H), 6.93 (d, J = 8.1 Hz, 1H), 3.71 (s, 2H), 3.27 (d, J = 6.8
13
Hz, 2H), 3.00 (s, 2H), 2.21 (s, 2H), 1.76 (d, J = 12.7 Hz, 2H), 1.69 (s, 1H), 1.37 (q, J = 11.5 Hz, 2H).
C
NMR (101 MHz, CD3OD) δ 168.83, 146.63, 135.02, 132.40, 130.20, 129.68, 128.07, 127.58, 127.18,
125.93, 123.41, 108.96, 57.94, 52.76, 44.36, 34.83, 28.01. HRMS (ESI): calcd. For C22H24ClN3O2 [M + H]
398.1630, found 398.1652.
+
N‐((1‐(4‐Methoxybenzyl)piperidin‐4‐yl)methyl)‐2‐Oxoindoline‐5‐carboxamide (8k). 2‐Oxoindoline‐5‐
carboxylic acid (140 mg, 0.79 mmol), (1‐(4‐Methoxybenzyl)piperidin‐4‐yl)methanamine (154 mg,
0.66 mmol), PyBOP (412 mg, 0.79 mmol), DIPEA(128 mg, 0.99 mmol), DMF (6 mL). White solid,
1
m.p.: 158–159 °C, yield: 43%, H NMR (400 MHz, CD3OD) δ 7.74–7.68 (m, 2H), 7.40 (d, J = 8.7 Hz,
2H), 6.98 (d, J = 8.7 Hz, 2H), 6.91 (d, J = 8.0 Hz, 1H), 4.19 (s, 2H), 3.79 (s, 3H), 3.43 (d, J = 12.0 Hz, 2H),
13
3.31 (s, 1H), 2.95 (t, J = 11.7 Hz, 2H), 1.97 (d, J = 13.9 Hz, 3H), 1.60–1.45 (m, 2H), 1.26 (s, 1H).
C
NMR (101 MHz, CD3OD) δ 178.54, 168.87, 161.10, 146.66, 132.51, 127.95, 127.62, 125.93, 123.46,
121.09, 114.21, 108.98, 54.55, 51.51, 43.78, 34.03, 27.22, 26.85. HRMS (ESI): calcd. For C23H27N3O3 [M +
+
H] 394.2125, found 394.2131.
2‐Oxo‐N‐((1‐(4‐(trifluoromethyl)benzyl)piperidin‐4‐yl)methyl)indoline‐5‐carboxamide (8l). 2‐Oxoindoline‐
5‐carboxylic acid (140 mg, 0.79 mmol), (1‐(4‐(Trifluoromethyl)benzyl)piperidin‐4‐yl)methanamine
(179 mg, 0.65 mmol), PyBOP (412 mg, 0.79 mmol), DIPEA(128 mg, 0.99 mmol), DMF (6 mL). White
1
solid, m.p.: 113–115 °C, yield: 40%, H NMR (400 MHz, CD3OD) δ 7.79 (d, J = 7.8 Hz, 1H), 7.73–7.69
(m, 2H), 7.62 (d, J = 8.2 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.44–7.32 (m, 2H), 6.95–6.90 (m, 1H), 3.60 (d, J
= 17.7 Hz, 2H), 3.24 (dd, J = 9.1, 6.8 Hz, 2H), 2.91–2.77 (m, 2H), 2.09–1.93 (m, 2H), 1.78–1.61 (m, 3H),
13
1.40–1.21 (m, 3H). C NMR (126 MHz, CD3OD)) δ 168.70, 146.44, 131.73, 130.35, 130.30, 128.23,
127.47, 127.30, 126.79, 125.81, 125.26, 123.34, 108.90, 59.79, 59.79, 58.24, 53.43, 53.14, 45.07, 36.04,
+
36.04, 36.00, 36.00, 29.82, 29.76. HRMS (ESI): calcd. For C23H24F3N3O2 [M + H] 432.1893, found
432.1886.
3.6. General Procedure for the Synthesis of 10s–v
To a solution of (1‐Benzylpiperidin‐4‐yl) methanamine derivatives (4a–d) (150 mg, 1 equiv.) in
DCM (5 mL) was added Et3N (1.8 equiv.). The mixture was cooled to 0 °C, TsCl (1.3 equiv.) in DCM
(5 mL) was added dropwise, and the reaction mixture was stirred for an additional 1 h at 0 °C. After

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completion of the reaction, the reaction mixture was dissolved in 15 mL saturated sodium bicarbonate
(NaHCO₃) and extracted with DCM (25 × 3 mL). Organic phases were combined and washed with
saturated NaCl solution (25 × 3 mL). The organic layer was dried over anhydrous Na2SO4 and the
solvent was removed under reduced pressure. After concentration, the crude product was purified
by silica gel column chromatograph using
a
methanol in dichloromethane gradient
(dichloromethane: methanol = 60:1–5:1) yielded compounds 10s–v.
N‐((1‐Benzylpiperidin‐4‐yl)methyl)‐4‐methylbenzenesulfonamide (10s). (1‐Benzylpiperidin‐4‐yl)
methanamine (150mg,0.73mmol), TsCl (181mg, 0.95 mmol), Et3N (133 mg, 1.31 mmol), DCM (9 mL)
1
White solid, m.p.:78–79 °C, yield: 80%, H NMR (400 MHz, CD3OD) δ 7.68 (d, J = 8.3 Hz, 2H), 7.36–
7.24 (m, 7H), 3.60 (s, 2H), 2.93 (d, J = 12.0 Hz, 2H), 2.67 (d, J = 6.8 Hz, 2H), 2.39 (s, 3H), 2.09 (t, J = 12.6
13
Hz, 2H), 1.68 (d, J = 13.8 Hz, 2H), 1.44 (s, 1H), 1.28–1.13 (m, 2H). C NMR (101 MHz, CD3OD) δ
143.22, 137.71, 136.09, 129.69, 129.36, 128.05, 127.39, 126.67, 62.59, 52.72, 35.66, 28.79, 20.11. HRMS
+
(ESI): calcd. For C20H26N2O2S [M + H] 359.1788, found 359.1802.
N‐((1‐(2‐Chlorobenzyl)piperidin‐4‐yl)methyl)‐4‐methylbenzenesulfonamide (10t). (1‐(4‐
Chlorobenzyl)piperidin‐4‐yl)methanamine (150 mg,0.63 mmol), TsCl (156 mg, 0.82 mmol), Et3N
1
(114 mg, 1.13 mmol), DCM (9 mL) White solid, m.p.: 111–112 °C, yield: 73%, H NMR (400 MHz,
CD3OD) δ 7.69 (d, J = 8.3 Hz, 2H), 7.44 (dd, J = 7.4, 2.0 Hz, 1H), 7.37–7.31 (m, 3H), 7.23 (td, J = 7.1, 1.9
Hz, 2H), 3.62 (s, 2H), 2.89 (d, J = 11.8 Hz, 2H), 2.67 (d, J = 6.8 Hz, 2H), 2.39 (s, 3H), 2.06 (t, J = 11.7 Hz,
13
2H), 1.65 (d, J = 13.1 Hz, 2H), 1.37 (s, 1H), 1.20 (dd, J = 12.3, 3.6 Hz, 1H), 1.15 (s, 1H). C NMR (101
MHz, CD3OD) δ 143.42, 137.49, 135.59, 133.53, 131.79, 130.10, 129.48, 127.74, 127.35, 126.68, 126.68,
+
20.13. HRMS (ESI): calcd. For C20H25ClN2O2S [M + H] 393.1398, found 393.1397.
N‐((1‐(4‐Methoxybenzyl)piperidin‐4‐yl)methyl)‐4‐methylbenzenesulfonamide (10u). (1‐(4‐
Methoxybenzyl)piperidin‐4‐yl)methanamine (150 mg, 0.64 mmol), TsCl (158 mg, 0.83 mmol), Et3N
1
(117 mg, 1.15 mmol), DCM (9 mL) White solid, m.p.: 76–77 °C, yield: 87%, H NMR (600 MHz,
CD3OD) δ 7.61 (d, J = 8.3 Hz, 2H), 7.25 (dd, J = 27.4, 8.3 Hz, 4H), 6.85 (d, J = 8.6 Hz, 2H), 3.80 (s, 2H),
3.70 (s, 3H), 3.10 (d, J = 10.7 Hz, 2H), 2.63 (d, J = 6.7 Hz, 2H), 2.44 (s, 2H), 2.32 (s, 3H), 2.05 (s, 1H),
13
1.73 (s, 2H), 1.52 (s, 1H), 1.27–1.19 (m, 2H). C NMR (101 MHz, CD3OD) δ 160.41, 143.33, 137.60,
131.82, 129.42, 126.66, 113.87, 60.67, 54.47, 51.88, 34.72, 27.47, 20.10. HRMS (ESI): calcd. For
+
C21H28N2O3S [M + H] 389.1893, found 389.1894.
4‐Methyl‐N‐((1‐(4‐(trifluoromethyl)benzyl)piperidin‐4‐yl)methyl)benzenesulfonamide (10v). (1‐(4‐
(Trifluoromethyl)benzyl)piperidin‐4‐yl)methanamine (150 mg, 0.55 mmol), TsCl (136 mg, 0.72
1
mmol), Et3N (100 mg, 0.99 mmol), DCM (9 mL) White solid, m.p.: 87–88 °C, yield: 88%, H NMR
(400 MHz, CD3OD) δ 7.76 (d, J = 7.8 Hz, 1H), 7.71–7.66 (m, 2H), 7.61 (d, J = 7.9 Hz, 1H), 7.57–7.51 (m,
1H), 7.40–7.31 (m, 4H), 3.58 (d, J = 14.0 Hz, 2H), 2.79 (t, J = 12.2 Hz, 2H), 2.67 (t, J = 6.9 Hz, 2H), 2.40
(s, 3H), 1.97 (t, J = 11.6 Hz, 2H), 1.69–1.59 (m, 2H), 1.40 (dtt, J = 14.5, 7.3, 3.2 Hz, 1H), 1.15 (dqd, J =
13
24.7, 12.2, 3.7 Hz, 2H). C NMR (126 MHz, CD3OD) δ 143.14, 137.67, 133.70, 131.73, 130.36, 130.33,
130.03, 129.28, 127.36, 126.84, 126.60, 125.27, 113.19, 59.65, 58.14, 53.26, 52.97, 48.19, 48.15, 48.12,
48.06, 47.95, 47.89, 47.78, 47.72, 47.61, 47.44, 47.32, 47.27, 47.10, 35.94, 35.86, 29.45, 20.03. HRMS (ESI):
+
calcd. For C21H25F3N2O2S [M + H] 427.1662, found 427.1660.
3.7. General Procedure for the Synthesis of 16a–d
Ethyl 2‐piperidin‐4‐ylacetate 11 (1 g, 5.84 mmol, 1.0 equiv.) and substituted (2‐
romoethyl)benzene derivatives (12a–b) (7.01 mmol, 1.2 equiv.) were dissolved in 20 mL acetone.
Then, anhydrous K2CO3 (11.68 mmol, 2 equiv.) and catalytic amount KI were added. The reaction
mixture was refluxed for 4 h. After completion of the reaction, acetone was concentrated, and the
residue was dissolved in water (60 mL) and extracted with ethyl acetate (60 × 3 mL). The combined
organic layers were dried over Na2SO4, filtered, and concentrated in vacuum. The obtained oil was
used in further synthesis without purification yielded compounds 13a–b (Yields were 67% and 72%).
Then, 4 mol/L KOH (2.5 equiv.) was added to the solution of compounds 13a–b in C2H5OH: H2O =
5:1(6 mL). The reaction mixture was stirred at room temperature for 7 h. After completion of the

Molecules 2020, 25, 489
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reaction, the reaction mixture was evaporated to dryness after neutralization with dilute hydrochloric
acid solution. Poured into ethyl acetate to deposit the solid, after cooling off, the mixture was filtered
and washed with cold ethyl acetate to give compounds 14a–d.
Finally, intermediates (14a–b) (1.2 equiv.), PyBOP (1.2 equiv.) and DIPEA (1.5 equiv.) were
added to 6 mL DMF and stirred at room temperature for 20 min. Then, intermediates (15a–b) (1.0
equiv.) was added and stirred at room temperature for 4 h. After completion of the reaction, the
reaction mixture was quenched with saturated NaCl solution. The aqueous phase was extracted with
DCM. The DCM layer was combined and washed with brine solution. The organic layer was dried
over anhydrous Na2SO4 and the solvent was removed under reduced pressure. After concentration,
the crude product was purified by silica gel column chromatograph using a methanol in
dichloromethane gradient (DCM:methanol = 60:1–5:1) yielded compounds 16a–d.
N‐(1H‐Indol‐5‐yl)‐2‐(1‐phenethylpiperidin‐4‐yl)acetamide (16a). 2‐(1‐Phenethylpiperidin‐4‐yl) acetic
acid (140 mg, 0.57 mmol), 1H‐Indol‐5‐amine (63 mg, 0.48 mmol), PyBOP (295 mg, 0.57 mmol),
1
DIPEA (93 mg, 0.72 mmol), DMF (6 mL). Yellow solid, m.p.: 165–167 °C, yield: 80.70%, H NMR
(400 MHz, CD3OD) δ 7.74 (d, J = 2.0 Hz, 1H), 7.28 (tt, J = 13.1, 6.8 Hz, 6H), 7.20 (d, J = 3.1 Hz, 1H),
7.15 (dd, J = 8.6, 2.0 Hz, 1H), 6.37 (d, J = 3.1 Hz, 1H), 3.60 (d, J = 14.9 Hz, 2H), 3.26 (d, J = 5.0 Hz, 1H),
3.08–3.01 (m, 3H), 2.39 (d, J = 7.0 Hz, 2H), 2.21–2.13 (m, 1H), 2.04 (d, J = 15.4 Hz, 2H), 1.74–1.60 (m,
13
2H), 1.27 (s, 2H). C NMR (151 MHz, CD3OD) δ 170.66, 136.38, 133.81, 129.79, 128.59, 128.42, 127.97,
126.86, 125.29, 115.83, 115.76, 112.47, 110.75, 101.07, 72.24, 70.09, 60.80, 57.69, 53.42, 52.37, 41.90,
+
31.68, 31.23, 29.38, 28.89, 22.34, 13.05. HRMS (ESI): calcd. For C23H27N3O [M + H] 362.2227, found
362.2242.
2‐(1‐(2‐Chlorophenethyl)piperidin‐4‐yl)‐N‐(1H‐indol‐5‐yl)acetamide (16b). 2‐(1‐(2‐
Chlorophenethyl)piperidin‐4‐yl)acetic acid (140 mg, 0.50 mmol), 1H‐Indol‐5‐amine (55 mg, 0.42
mmol), PyBOP (260 mg, 0.50 mmol), DIPEA(80 mg, 0.62 mmol), DMF (6 mL). Yellow solid, m.p.:
1
172–173 °C, yield: 30.41%, H NMR (400 MHz, CD3OD) δ 7.75 (d, J = 1.9 Hz, 1H), 7.39–7.29 (m, 3H),
7.28–7.20 (m, 2H), 7.19 (d, J = 3.1 Hz, 1H), 7.16 (dd, J = 8.7, 2.0 Hz, 1H), 6.37 (d, J = 4.0 Hz, 1H), 3.45
(d, J = 12.3 Hz, 2H), 3.15–3.09 (m, 2H), 3.08–3.01 (m, 2H), 2.79 (t, J = 13.0 Hz, 2H), 2.36 (d, J = 7.1 Hz,
13
2H), 2.10 (s, 1H), 2.00–1.92 (m, 2H), 1.60 (q, J = 11.5 Hz, 2H). C NMR (151 MHz, CD3OD) δ 171.22,
135.56, 133.78, 133.57, 130.84, 129.85, 129.30, 128.28, 127.96, 127.17, 125.21, 115.80, 112.48, 110.73,
101.06, 56.91, 52.68, 42.55, 32.24, 29.95, 29.32, 29.01. HRMS (ESI): calcd. For C23H26ClN3O [M + H]
+
396.1837, found 396.1838.
N‐(1H‐Benzo[d]imidazol‐5‐yl)‐2‐(1‐phenethylpiperidin‐4‐yl)acetamide (16c). 2‐(1‐phenethylpiperidin‐4‐
yl)acetic acid (140 mg, 0.57 mmol), 1H‐Benzo[d]imidazol‐5‐amine (64 mg, 0.48 mmol), PyBOP (295
mg, 0.57 mmol), DIPEA(93 mg, 0.72 mmol), DMF (6 mL). Red solid, m.p.: 167–168 °C, yield: 45.92%,
1
H NMR (400 MHz, CD3OD) δ 8.09 (s, 1H), 8.03 (s, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.29–7.12 (m, 6H),
3.07 (d, J = 12.0 Hz, 2H), 2.84–2.78 (m, 2H), 2.64–2.58 (m, 2H), 2.32 (d, J = 7.2 Hz, 2H), 2.17 (t, J = 11.8
13
Hz, 2H), 1.93 (s, 1H), 1.81 (d, J = 12.9 Hz, 2H), 1.42 (q, J = 12.1, 10.5 Hz, 2H). C NMR (101 MHz,
CD3OD) δ 171.84, 141.67, 139.67, 133.93, 128.33, 128.21, 128.20, 125.93, 116.32, 60.33, 53.19, 47.89,
+
43.30, 33.27, 32.51, 31.15. HRMS (ESI): calcd. For C22H26N4O [M + H] 363.2179, found 363.2197.
N‐(1H‐Benzo[d]imidazol‐5‐yl)‐2‐(1‐(2‐chlorophenethyl)piperidin‐4‐yl)acetamide (16d). 2‐(1‐(2‐
Chlorophenethyl)piperidin‐4‐yl)acetic acid (140 mg, 0.50 mmol), 1H‐Benzo[d]imidazol‐5‐amine (55
mg, 0.42 mmol), PyBOP (259 mg, 0.50 mmol), DIPEA(80 mg, 0.62 mmol), DMF (6 mL). Red solid,
m.p.: 176–177 °C, yield: 40.33%, 1H NMR (600 MHz, CDCl3) δ 7.95 (s, 1H), 7.70 (s, 1H), 7.66 (d, J =
8.1 Hz, 1H), 7.48 (d, J = 7.4 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.19 (t, J = 7.3 Hz,
1H), 6.90 (d, J = 8.1 Hz, 1H), 3.62 (s, 2H), 3.57 (d, J = 11.7 Hz, 1H), 3.37 (d, J = 6.5 Hz, 2H), 2.95 (d, J =
11.2 Hz, 2H), 2.10 (t, J = 11.3 Hz, 2H), 1.75 (d, J = 12.4 Hz, 2H), 1.66 (s, 1H), 1.60 (s, 1H), 1.39 (q, J =
13
11.5 Hz, 2H). C NMR (151 MHz, CD3OD) δ 171.90, 141.65, 137.34, 134.98, 133.95, 133.58, 130.73,
129.20, 127.73, 126.97, 121.27, 116.28, 58.31, 58.27, 53.17, 53.06, 50.62, 43.39, 40.15, 33.39, 32.59, 31.29,
+
31.15, 30.24. HRMS (ESI): calcd. For C22H25ClN4O [M + H] 397.1790, found 397.1797.
3.8. AChE and BChE Inhibition Assay

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The inhibitory activity of the target compound against AChE (from Electrophorus electricus
(eeAChE), Sigma‐Aldrich, Munich, Germany) and horse serum BChE (eqBChE, Sigma‐Aldrich,
Munich, Germany) were measured by Ellman’s method [32]. In 96‐well plates, a mixture of phosphate
buffer (0.1 M, pH 8.0, 2 mL), 5,5′‐dithiobis‐2‐nitrobenzoic acid (DTNB, 60 μL), acetylcholinesterase or
butyrylcholinesterase (20 μL, 5 IU/mL) and different concentrations of the compounds solution (30
μL) was pre‐incubated for 5 min and then substrates (acetylthiocholine iodide or butyrylthiocholine
iodide, 20 μL) were added.
Changes in absorbance were measured at 412 nm by using microplate reader (Thermo,
Varioskan Flash 3001, Thermo Fisher Scientific, Agawam, MA, USA). The measurement of each
concentration for each compound was detected in triplicate. GraphPad Prism 6.0 (GraphPad
Software, Inc., La Jolla, CA, USA) was used for data processing. The inhibition curve was fitted by
plotting the logarithm of the concentration of the tested compounds with the percentage of enzyme
activity (reference to 100%). The (IC50) value was calculated according to the inhibition curve and the
data were shown in the layout of mean ± SEM by GraphPad Prism 6.0.
3.9. Kinetics of AChE and BChE Inhibition
Kinetic studies were performed in the same manner as the determination of ChEs inhibition,
with substrate (ATC/BTC) concentrations of 90, 150, 226, 452 and 904 μM. The concentration of
compound 8i was set to 0, 0.25, 0.5, 0.75, 1 μM, and the concentration of compound 10s was set to 1,
2.5, 5, 7.5, 10 μM. The enzymatic reaction was extended to 7 min for eeAChE and eqBChE before the
determination of the absorption. The Vmax and Km values of Michaelis‐Menten kinetics were
calculated by nonlinear regression from the substrate‐velocity curves using GraphPad Prism 6.0.
Linear regression was used to fit the Lineweaver–Burk plot.
3.10. Molecular Docking Study
The crystal structures of eeAChE (PDB:1C2B) [36], huAChE (PDB ID: 4EY7) [32] and huBChE
(PDB ID: 4TPK) [37] were obtained from the Research Collaboratory for Structural Bioinformatics
Protein Data Bank (RCSB PDB). The Discovery Studio software 2016 (DS 2016, BIOVIA, San Diego,
CA, USA) was used to study the docking of compound 8i. The three protein structures are pretreated
(i.e., protonated, removed water, added Miss sidechains, etc.) by the “prepare protein” module in DS
to provide the structures suitable for docking. The “prepare ligand” module in DS is used to test the
structural preparation of the compound. The native ligand in the crystal structure was used to define
the binding site. The binding site was defined as the site sphere ((in 10 Å radius) around the original
ligand in the co‐crystal structures. The docking program CDOCKER encoded in DS 2016 was applied
to identify the potential binding of compound 8i to eeAChE, huAChE, and huBChE. Other
CDOCKER parameters were set to default values. Compound 8i was chosen for molecular modeling
as the most active compounds in the series (Table 1). Compound 8i produced 10 poses to eeAChE,
huAChE, and huBChE. These postures were visually examined, and the most appropriate docking
pose was selected according to the scores and interactions with key residues of the eeAChE, huAChE,
and huBChE active sites.
3.11. Cell Studies in Vitro
Pheochromocytoma‐derived cell line (PC12 cells) were grown in Dulbeccoʹs Modified Eagle
Medium (DMEM) supplemented with 10% FBS at 37 °C in a humidified atmosphere containing 5%
3
CO2. To carry out the experiment, cells (6 × 10 cells/well) were seeded in 96‐well plate in complete
medium. After 24 h, the culture medium was removed and the cells were exposed to increasing
concentrations of compounds 6b, 6c, 6d, 8i, 10s, 10v or Tacrine (10, 20, 30 and 50 μM) in DMEM for
further 24 h. Cell survival was measured by 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium
Bromide (MTT) assay [37].
4. Conclusions

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In this paper, a series of novel compounds 6a–h, 8i–1, 10s–v, and 16a–d were synthesized and
evaluated, together with the known analogs 11a–f, for their inhibitory activities towards AChE and
BChE. The results show that most of the compounds have AChE and/or BChE inhibitory activity.
Compound 8i showed the strongest inhibitory effect on both AChE (eeAChE IC50 = 0.39 μM) and
BChE (eqBChE IC50 = 0.28 μM). Compared with compound G801‐0274, compound 8i has comparable
inhibitory activity against two ChEs, so that it can exert an anti‐ChEs effect in a balanced manner.
Kinetic studies indicated a mixed‐type inhibition of compound 8i, including competitive inhibition
and non‐competitive inhibition. Subsequently, molecular docking was performed to evaluate the
interaction mechanism between compound 8i and enzymes. Enzyme inhibition kinetics and
molecular modeling studies have shown that compound 8i bind simultaneously to the PAS and the
CAS of AChE and BChE. Therefore, compounds 8i may be promising scaffold for treatment, and
further modifications have been made to obtain novel AChE and BChE dual‐target inhibitors.
Supplementary Materials: Copies of the 1H‐NMR and 13C‐NMR spectra of the compounds are available online.
Author Contributions: Conceptualization, H.S. and Z.L.; Data curation, Y.G., H. Y. and Q.L.; Formal analysis,
Y.G., H.Y. and Q.L.; Methodology, Y.G., H.Y., Z.H., S.T., Q.L., C.D., T.C. and Y.L.; Project administration, H.S.
and Z.L.; Validation, Y.G., H.Y., Z. H., S.T., Q.L., C.D., T.C. and Y.L.; Writing–original draft, Y.G.; Writing–review
and editing, H.S. and Z.L. All authors have read and agreed to the published version of the manuscript.
Funding: The authors acknowledge the financial support of the National Natural Science Foundation of China
(No. 81502983) and the Taishan Scholar Project.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: All samples of the compounds are available from the authors.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).