Design, synthesis and biological evaluation of novel copper-chelating acetylcholinesterase inhibitors with pyridine N-benzylpiperidine fragments

Article abstract of DOI:10.1016/j.bioorg.2019.103322

Cholinergic depletion is the direct cause of disability and dementia among AD patients. AChE is a classical and key target of cholinergic disorders. Some new inhibitors of AChE combining pyridine, acylhydrazone and N-benzylpiperidine fragments were developed in this work. The hit structure was optimized to yield the compound 21 with an IC₅₀ value of 6.62 nM against AChE, while almost no inhibitory effect against BChE. ADMET predictions and PAMPA permeability evaluation showed good drug-like property. The higher activity with an intermediate alkyl chain substitution indicates a new binding mode of inhibitor with AChE. This finding provides new insights into the binding mechanism and is helpful for discovery of novel high-activity AChE inhibitors.

Full text of DOI:10.1016/j.bioorg.2019.103322

BioorganicChemistry93(2019)103322
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of novel copper-chelating
acetylcholinesterase inhibitors with pyridine N-benzylpiperidine fragments
⁎
Yeheng Zhoua,d,1, Wei Suna,1, Jiale Pengd, Hui Yana, Li Zhangd, Xingyong Liud, Zhili Zuoa,b,c,d,
a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, PR China
b Yunnan Key Laboratory of Natural Medicinal Chemistry, Kunming 650201, PR China
c University of Chinese Academy of Sciences, Beijing 100049, PR China
d School of Chemical Engineering, Sichuan University of Science & Engineering, Zigong 643000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cholinergic depletion is the direct cause of disability and dementia among AD patients. AChE is a classical and
key target of cholinergic disorders. Some new inhibitors of AChE combining pyridine, acylhydrazone and N-
benzylpiperidine fragments were developed in this work. The hit structure was optimized to yield the compound
21 with an IC50 value of 6.62 nM against AChE, while almost no inhibitory effect against BChE. ADMET pre-
dictions and PAMPA permeability evaluation showed good drug-like property. The higher activity with an in-
termediate alkyl chain substitution indicates a new binding mode of inhibitor with AChE. This finding provides
new insights into the binding mechanism and is helpful for discovery of novel high-activity AChE inhibitors.
Acetylcholinesterase
Drug design
N-acylhydrazone
Dementia
Copper-chelating
N-Benzylpiperidine
1. Introduction
AChE is thus a very important target.
In addition, the metal ion hypothesis suggests that the steady-state
Alzheimer's disease (AD) is one of the most popular cause of de-
mentia, accounting for 60–80% of the total number of people with
dementia worldwide [1]. Statistics show that there would be one more
dementia patients every 3 s in the world [2]. The high cost of medicine
and healthcare is a huge burden on the world economy and society.
However, due to the complex pathogenesis of AD, there is no effective
drug to date to cure the disease. Drugs that retard or stop the progress
of AD only are available, such as AChE inhibitors donepezil and pyr-
idostigmine. Therefore, the development of new AD treatment drugs is
still a hot research area.
dysregulation of metal ions is closely related to AD pathology [5–7]. In
particular, steady-state dysregulation of redox-active Cu (I/II) and Fe
(II/III) leads to excess reactive oxygen species (ROS) in the Fenton-like
reaction, which in turn leads to oxidative stress in brain [8,9]. Among
the many metal ions involved in the senile plaques (SPs), many studies
have focused on Cu2+, one of the major cationic elements in the for-
mation of SPs [10,11]. Therefore, removal of Cu2+ may be helpful in
the treatment of AD.
The N-acylhydrazone (NAH) is a kind of chemical structure which
can chelate metal ions, and has both hydrogen bond acceptor and hy-
drogen bond donor. Until 2010, the therapeutic potential of the sub-
family has been concerned by drug developers [12]. In particular, it is
widely applied in drugs for the treatment of chronic pain, inflammatory
diseases and cancer (Fig. 1), [13]. Nitrofurantoin is used as an oral
antibacterial agent for the treatment of genitourinary tract infections
[14]; carbazochrome has been used as a hemostatic agent for capillary
and parenchymal hemorrhage [15]; azumolene sodium salt has good
water solubility and can be used to treat malignant hyperthermia [16];
PAC-1 has been used as a caspase activator and has entered the first
phase of clinical use for anti-cancer [17]. ZT-1, which contains an imine
structure, has been introduced into the clinical phase for the treatment
of AD as a derivative of huperzine-A [18,19]. And it has better
The most important anatomic pathological feature of AD is the
overexpression of β-amyloid (Aβ) aggregated senile plaques and neu-
rofibrillary tangles formed by misfolding of Tau protein after hyper-
phosphorylation [3]. Along with these two organic brain damages, an
important neurotransmitter called acetylcholine (ACh) in the brain cells
of AD patients is less than normal[4]. Because ACh plays an important
role in many human neural activities, such as cognition, memory,
dominating muscle movement, etc., the brain cholinergic system dis-
order in AD patients is the direct cause of disability and dementia. The
cholinergic hypothesis is a classic hypothesis in the field of AD treat-
ment. The most direct pathological factor is the loss of the neuro-
transmitter ACh caused by abnormal brain cholinergic system [3].
⁎ Corresponding authors.
E-mail addresses: sunwei@mail.kib.ac.cn (W. Sun), liuxy@suse.edu.cn (X. Liu), zuozhili@mail.kib.ac.cn (Z. Zuo).
1 Contribute equally to this work.
https://doi.org/10.1016/j.bioorg.2019.103322
Received 23 July 2019; Received in revised form 19 September 2019; Accepted 26 September 2019
Availableonline26September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Fig. 1. Clinical and listed drugs containing hydrazide or imine groups.
selectivity to other marketed medicines for AChE [20]. Its selectivity
may be related to the imine group.
better than the other function of compound 21 was the ability to che-
late metal Cu2+
.
The selectivity of AChE inhibitor over BChE is an important issue in
the drug discovery. Non-selective drug tacrine was withdrawn from the
market because of hepatotoxicity [21]. Pyridostigmine, as an AChE
inhibitor with poor selectivity, was found to cause adverse effects as-
sociated with vegetative nerve system [22]. Binding pocket analysis of
AChE and BChE was shown in Fig. 2(A) and (B) respectively. The
middle part of the AChE active cavity is a narrow channel (PDB code:
4EY7) [23], and the BChE receptor cavity is a tripartite star-shaped
pocket (PDB code: 6QAA) [24]. Therefore, the molecular flexibility may
be the main cause for its selectivity. In addition to the highly conserved
catalytic triad [25], same amino acids are still present in both enzymes,
and several identical amino acids at key sites are shown with green in
Fig. 2.
2. Results and discussion
The three dominant fragments were assembled to give template
compound 1 (Fig. 5). The NAH linker provides both hydrogen bond
acceptor (the imine) and hydrogen bond donor (the amide) to interact
with AChE. We sought to optimize the structure based on the template
of compound 1 by investigation of various type of A ring, B ring and the
linker.
2.1. Chemistry
The structure–activity relationship of A ring was first explored.
Commercially available benzoic acid, nicotinic acid, isonicotinic acid,
and 2-picolinic acid were esterified to give methyl ester 1a-4a, which
were then reacted with hydrazine hydrate to give formylhydrazine in-
termediate 1b-4b. The four formylhydrazides were condensed with
commercially available benzyl-4-piperidone to give the corresponding
acylhydrazones 1–4. Microwave irradiation was used to increase the
reaction rate and yield (see Scheme 1).
Further analysis of the binding pocket of AChE complexed with
donepezil revealed that there is possible a small space available in the
tunnel part, especially around Asp-74. The distance between the me-
thylene group (between the piperidine ring and indanone) of donepezil
to the Asp-74 side chain carboxyl group was 5.8 Å. So, it is possible to
introduce a small substitute at this position to improve the activity.
After a try, this clue brought us a big surprise and implied a new
binding mechanism.
The length and flexibility of the linker in the middle were studied
together with the position of nitrogen atom on the benzyl piperidine as
shown in Scheme 2. The intermediates 5b and 6b were obtained by the
Borch reduction reaction and the Dess-Martin oxidation reaction to
change the position of the nitrogen atom on the N-benzyl-4-piperidone,
and to change the length of the linker between the piperidine ring
and the benzene ring. 5b and 6b were condensed with 2-pyr-
idinecarboxhydrazide to obtain the corresponding acylhydrazones 5
and 6. Furthermore, the intermediate 8a was obtained by the Witting
reaction according to the method described in the literature [33]. The
double bond of intermediate 8a was hydrogenated with Pd/C to give
intermediate 9a. 9a was heated to reflux in THF with lithium aluminum
hydride to yield the intermediate 9b. The Dess-Martin oxidation reac-
tion condition was modified to oxidize the intermediate 9b to give the
Donepezil is the most classic AChE inhibitor in the current clinical
treatment of AD. At present, a large number of studies have tried to
design new inhibitors using donepezil as a template [26]. Pyridine al-
kaloids have good biological activity and are widely used in medicines,
such as tipranavir and nexavar (shown in Fig. 3) [27,28]. Currently the
mainstream academia has been trying hard to discover new AChE in-
hibitors with multiple functions, such as antioxidation [29], inhibition
of Aβ aggregation [29–31], inhibition of hyaluronidase [31] and in-
hibition of monoamine oxidases (MAOs) [32]. Some examples with
their IC50 value against electric eel acetyl cholinesterase (eeAChE) are
listed in Fig. 4. In this study, some new structures combined N-ben-
zylpiperidine moiety, a hydrazide group, and a pyridine ring have been
developed. The activity and selectivity of the compound 21 performed
Fig. 2. Binding pocket analysis of AChE (A) and BChE (B).
2

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Fig. 3. The listed drug of Pyridine amide
derivative.
Fig. 4. Examples of multifunctional inhibitors and the IC50 values against eeAChE.
intermediate 9c. Similar reaction route gave the intermediate 8c. 8c
and 9c were condensed with 2-pyridinecarboxhydrazide to obtain the
corresponding acylhydrazones 8 and 9. Commercially available N-
benzylpiperidine-4-carboxaldehyde was directly condensed with 2-
pyridinecarboxhydrazide to obtain the acylhydrazone 7.
amide of NAH according to the method described in the literature [34].
The methyl group was introduced by methyl iodide after the primary
amine of the hydrazide was protected by phthalic anhydride. Depro-
tection gave the key intermediate 20c. 20c was condensed with 2-
pyridinecarboxhydrazide to obtain acylhydrazone 20. The alkyl chain
was introduced to the aldehyde by the Grignard reaction to yield a
secondary alcohol intermediate, which was then oxidized to 19b, 21b-
24b. 19b and 21b-24b were condensed with 2-pyridinecarboxhy-
drazide to obtain the corresponding acylhydrazones 19, 21–24. Trace
of Z-conformation was introduced when compounds 19, 21–24 were
synthesized. The ratio of E/Z conformation was analyzed by the amide
hydrogen with the aid of 2D-NMR (ROESY) (Supporting information).
The percentage of E-conformation of all these compounds was the
majority over 91.9%.
The modification of B ring was shown in Scheme 3. The electron-
withdrawing and electron-pushing groups were introduced to in-
vestigate the effect of electrical properties on activity. Substituted
aromatic aldehyde and 4-piperidinemethanol were subjected to a Borch
reduction to give intermediate 10a-18a. 10a-18a was oxidized by the
Dess-Martin reagent to give intermediates 10b-18b. 10b-18b were di-
rectly condensed with 2-pyridinecarboxhydrazide to obtain the corre-
sponding acylhydrazones 10–18. The E-conformation was determined
by 2D-NMR (COSY and ROESY) with compound 10 as the example.
The introduction of the alkyl chain on the intermediate linker part
was described in Scheme 4. The substituent was introduced to the
2.2. Biological assays
2.2.1. In vitro inhibitory activities against AChE and BChE
The 24 compounds were evaluated against eeAChE and horse serum
butyryl cholinesterase (eqBChE) using the method of Ellman et al.[35].
Donepezil and tacrine were used as the reference drugs.
Initially we looked into the effect of various A ring on compounds
1–4. Only 2-picolinic acid derived compound 1 showed modest activity
with an IC50 value of 135.0 μM, while the other aromatic acids derived
compounds 2–4 gave poor inhibition with 50 μM concentration
(Table 1). So, the optimization with 2-substituted pyridine as the fixed
A ring was further carried out.
The length of the structure and activity of the AChE inhibitors were
closely related [36,37]. We then investigated the spacer between the 2-
substituted pyridine ring and the B ring. Shifting of the nitrogen atom
from the piperidine to the exocyclic position (compound 5) or insertion
extra carbon between the piperidine ring and benzyl group (compound
Fig. 5. Core skeleton design and optimization scheme.
3

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Scheme 1. The study of A ring in the structure. Reagents and conditions: (a) con. H2SO4 (2.5 V%), MeOH, Microwave, 30 min, Yield 87% (b) N2H4·H2O (6 eq), 95 °C,
Microwave, 30 min, Yield 95%. (c) aldehyde or ketone (1.05 eq), MeOH, 100 °C, microwave, 30 min, 80%
6) eliminated the inhibition, which indicated N-benzylpiperidine
moiety from donepezil as the pharmacophore binding to the catalytic
active site of AChE [38]. Elongation the NAH spacer by one carbon
(compound 7) dramatically improved the potency and gave 145-fold
increase in the IC50 value (0.93 μM) compared to compound 1. How-
ever, further elongation with two carbon atom length (compounds 8
and 9) slightly reversed the inhibition increase tendency.
modification.
Synthesized compounds 7, 8 and 9 inhibited BChE activity in a
dose-dependent manner as donepezil did. The data represented the
average of at least three different determinations with the indication of
the standard error. The significance of the data was evaluated by p
value (< 0.005).
Substitution on the benzene ring of the N-benzylpiperidine moiety
had shown influence on the inhibition activity [41]. In this work, ortho-
halogen substitution slightly promoted the potency with the chlorine-
substituted compound 10 as the best example (IC50 = 0.55 µM)
(Table 2). Electron-donating groups, such as methyl group (compound
17) and methoxyl group (compound 14), decreased the inhibition. 2,6-
di-Cl substitution of the benzene ring (compound 18), decreased the
inhibition as well, which might be explained by the spatial limitation at
the binding site.
Selectivity between enzyme subtypes is an important issue in drug
discovery. Compounds with improved selectivity between AChE and
BChE may achieve better safety index[39,40]. So, the inhibition of
BChE with compounds 7, 8, 9 and Donepezil was investigated in this
work (Fig. 6). Dose-dependent effect was observed in all compounds
with the concentration 200 μM, 100 μM, 50 μM and 25 μM. Compound
7 showing the weakest inhibition and Donepezil showing the strongest
inhibition to BChE. Compound 7 was chosen as the template for further
Scheme 2. Modification of the linker, and the position of nitrogen atom on the benzyl piperidine. Reagents and conditions: (d) Na(ACO)3BH (1.6 eq), ACOH (0.9 eq),
DCM, rt, Overnight, yield 90%; (e) Dess-Matin reagent (2 eq), DCM, rt, 2 h; (f) (EtO)2POCH2CO2Et (1 eq), n-BuLi (1 eq), −70 °C, THF; (g) Pd/C (10% on activated
carbon) (5 wt%), H2, MeOH; (h) LiAlH4, THF, reflux, 3 h.
4

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Scheme 3. The modification of B ring. Reagents and conditions: (d), (e) and (c) same as Scheme 2.
Scheme 4. The substitution of active hydrogen on the chain. Reagents and conditions: (i) phthalic anhydride, 130℃, 2 h, yield 93%; (j) ICH3, acetone, 50℃, 18 h,
yield 90%; (k) N2H4⋅H2O, MeOH, 50℃, yield 90%. (l) Grignard reagent (2 eq), THF, rt, overnight, yield 70%
Table 1
We sought to modify the NAH spacer on the template of compound
10 to explore possibility to further improve the potency. Methylation
the amide nitrogen yielded compound 20, which lost the inhibition
activity (Table 2). However, introduction of a methyl group to the
imine gave a 6-fold potency increase to yield compound 19 with the
IC50 value of 92.46 nM. Further elongation of the methyl group to ethyl
group and propyl group provided remarkable surprises to yield com-
pound 21 (IC50 = 6.62 nM) and compound 22 (IC50 = 10.25 nM), re-
spectively. A total of 83-fold improvement from compound 10 to
compound 21 indicated that a ligand-induced-fit subpocket might form
in the flexible tunnel of AChE through side-chain fluctuations. It was
obvious that the volume of this ligand-induced-fit subpocket was lim-
ited because compounds 23 and 24 with larger alkyl groups showed a
quick potency decrease. Meanwhile, the selectivity between AChE and
BChE was maintained, which benefited from the structural rigidity of
the NAH spacer. The selectivity index of the most potent compound 21
was over 3000-fold.
Inhibition of eeAChE by compounds 1–9, tacrine and donepezil.
inhibition rate (%)a
IC50
b
Compounds
1
33.5
2.0
0.7
0.1
0.3
0.2
0.1
135.02
–
7.64 µM
2
3
10.3
2.5
–
4
–
5
8.8
–
6
13.6
94.8
90.8
94.3
–
0.5
–
7
0.0
0.3
0.1
0.93
2.38
1.98
20.55
59.73
0.02 µM
8
0.08 µM
0.05 µM
0.16 nM
0.93 nM
9
Donepezil
Tacrine
–
a
Inhibitory rate was measured with a final concentration of 50 μM of each
compound against eeAChE, which was shown in Mean
SD of triplicate wells.
b
Concentration required for 50% inhibition of eeAChE, data were shown in
Mean
SD of triplicate independent experiments.
5

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
hydroxyl group of Tyr-104, but failure to form a hydrogen bond with Phe-
295 may be the main reason for its weaker activity than donepezil.
Comparing the docking results of compounds 1 and 7, it can be seen that
the extension of compound 7 after prolonging one carbon leads to the
closer distance between pyridine ring, carbonyl group and Phe-295, Arg-
296. The position of carbonyl group allows the formation of a key hy-
drogen bond with Phe-295, and the nitrogen on the pyridine ring also
forms hydrogen bond network with Phe-295, Arg-296. Thus, the activity
is greatly increased by about 100 times. Comparing compounds 7 and 10,
it can be seen that after the introduction of the Cl atom at the 2-position of
the benzyl group, Cl and Trp-86 form a weak halogen-Pi interaction, and
thus the activity is slightly enhanced. Comparing compound 10 with
compounds 19, 21, and 22, it can be seen that after the alkyl group is
introduced, the alkyl group is inserted into the hydrophobic pocket
formed by the three aromatic side chains of Tyr-104, Tyr-341 and Trp-
286, and forms Pi-Alkyl interactions. So, the activity is greatly improved.
Since the ethyl group is deeper than the methyl group, the activity is
improved by 14 folds. But the propyl group is too bulky at this site. The
activity test results is thus verified by the molecular docking study.
The binding conformation was further explored by taking the most
active compound 21 as an example for BChE. Although the ligand in the
crystal (PDB code: 6QAA) has a similar molecular length as compound
21, compound 21 has almost no inhibition on BChE. In order to explore
its selective mechanism, we conducted a molecular docking study of
compound 21 with BChE protein. As shown in the Fig. 9, the docking
result implies only a small part of compound 21 enters the binding
pocket. The reason may due to the molecular rigidity of compound 21,
so that it cannot bend like HUN613 and forms a good binding con-
formation with BChE.
Fig. 6. Dose-dependent Inhibition of eqBChE by compounds 7–9, and done-
pezil.
2.2.2. Kinetic characterization of AChE inhibition
The kinetic character describes the interaction patterns of ligands
binding to enzymes. To explore the mechanism of AChE enzyme in-
hibition, the most potent compound 21 was subjected to an enzyme
kinetic study together with donepezil. Lineweaver–Burk reciprocal
plots were generated by plotting the reciprocal of the reaction rates
versus the reciprocal of substrate concentrations using different con-
centrations of the test compounds (Fig. 7). The results suggested that
compound 21 shared a similar non-competitive or mixed type of in-
hibition as donepezil.
2.2.3. Molecular docking study
In order to explore the binding mode of this class of compounds with
AChE, the molecular docking study was carried out (Fig. 8). Compounds 1
and 4 has a similar mode of action as donepezil. The pyridine ring forms a
π-π interaction with Trp-286 and Tyr-341. The piperidine ring simulta-
neously forms a weak Pi-Alkyl interaction with Tyr-341, Phe-338, Tyr-
337. The amide nitrogen forms a hydrogen bond with the phenolic
2.2.4. Prediction of ADME properties
Aqueous solubility, blood brain barrier (BBB) penetration, cyto-
chrome P450 (CYP450) 2D6 inhibition, hepatotoxicity, and human
intestinal absorption can be predicated using the ADMET Descriptors
module in the Discovery Studio 4.0 (DS 4.0) software. A total of 6 key
Table 2
Inhibition of eeAChE and eqBChE by compounds 7, 10–24, tacrine and donepezil.
a
b
c
d
Compounds
AChE inhibition rate (%)
AChE inhibition IC50
BChE inhibition rate (%)
Selectivity Index (SI)
R1
R2
R3
7
H
H
H
H
H
H
H
H
H
H
H
94.8
86.2
49.4
2.8
0.0
0.2
0.7
0.93
0.55
2.54
N/A
N/A
0.02 µM
0.04 µM
0.06 µM
3.2
1.1
0.8
0.4
1.6
0.8
> 21
> 36
> 7.9
N/A
10
11
12
13
o-Cl
m-Cl
p-Cl
16.3
10.8
3.0
0.1
13.5
1.0
−7.0
N/A
14
o-OMe
o-F
H
H
H
H
H
H
H
Me
H
H
H
H
–
14.6
78.0
73.9
54.4
22.8
27.6
N/A
–
0.2
0.3
0.3
0.6
0.9
0.5
N/A
21.0
5.7
0.3
N/A
15
H
0.58
0.03 µM
0.02 µM
0.08 µM
0.4
> 34
> 27
> 10
N/A
16
o-Br
o-Me
2,6-di-Cl
o-Cl
o-Cl
o-Cl
o-Cl
o-Cl
o-Cl
–
H
0.72
20.9
43.6
10.9
13.2
17.4
7.3
1.0
0.2
1.0
0.5
0.5
17
H
1.90
18
H
N/A
19
Me
H
92.46
N/A
2.97 nM
> 216
N/A
20
21
Et
Pr
Bu
iBu
–
6.62
0.38 nM
0.45 nM
1.40 nM
8.43 nM
0.93 nM
0.16 nM
0.7
> 3021
> 1951
N/A
22
–
10.25
35.53
853.00
59.73
20.55
20.1
57.8
31.3
22.99
11.06
0.6
0.1
0.4
23
–
24
–
> 23
0.38
e
e
Tacrine
Donepezil
–
1.15 nM
–
–
–
–
0.55 µM
538
a
b
c
Inhibitory rate was measured with a final concentration of 5 μM of each compound against eeAChE, which was shown in Mean
Concentration required for 50% inhibition of eeAChE, data were shown in Mean SD of triplicate independent experiments.
Inhibitory rate was measured with a final concentration of 20 μM of each compound against eqBChE, and was shown in Mean
SD of triplicate wells.
SD of triplicate wells.
d
Selectivity index (SI) is defined as eqBChE IC50/eeAChE IC50, and the eqBChE IC50 values of compounds 7, 10–22 and 24 were derived to be over 20 μM from the
inhibitory rates measured with the concentration of 20 μM.
e
Concentration required for 50% inhibition of eqBChE, data were shown in Mean
SD of triplicate independent experiments.
6

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Fig. 7. Kinetic study of the mechanism of AChE inhibition. Lineweaver − Burk reciprocal plots of the AChE initial velocity at increasing substrate concentrations
(0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM) in the presence of donepezil and compound 21 (20 nM, 40 nM, 80 nM, 160 nM) are shown.
compounds 1, 7, 10, 19, 21 and 22 were selected and analyzed. The
results as shown in Fig. 10, indicating that all compounds have good
ADMET properties.
shown in Table 3. Compounds 1 and 7 showed good solubility, while
compounds 10, 19, 21 and 22 showed low solubility. All six re-
presentative compounds have high blood-brain barrier permeability
and no hepatotoxicity.
Some important parameters from the ADMET predictions were
Fig. 8. The key interaction analysis of representative compounds. A-F illustrate the key interactions between compounds 1, 7, 10, 19, 21, 22 and AChE, respectively.
7

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
Fig. 9. Docking analysis of compound 21 and BChE.
Fig. 11. The metal chelation ability test of compound 21.
Fig. 10. The ADMET predictive analysis of the representative compounds.
2.2.5. Capability to chelate copper ion
2.2.6. PC12 cell toxicity evaluation
The chelation ability of compound 21 against metals such as Cu(II),
Fe(II), Fe(III), Zn(II) and Al(III) was investigated by UV–visible spec-
troscopy (Fig. 11). The wavelength ranges from 190 nm to 600 nm.
When CuCl2 was added, the UV absorption peaks at both 226 nm and
267 nm wavelengths decreased significantly, indicating that 21 may
form a complex with Cu2+. However, there was no significant change
after the addition of FeCl2, FeCl3, AlCl3 and ZnCl2.
In vitro assessment of chemical induced toxicity to cells helps dis-
tinguish non-toxic hit compounds in the very early stage of drug dis-
covery. PC12 cell line was originally isolated from a pheochromocy-
toma developed in the adrenal gland of an irradiated rat. Because PC12
cells treated with nerve growth factor (NGF) resemble sympathetic
neurons, PC12 cells were utilized as a readily manipulable, easily
grown experimental model for sympathetic neurons. Poorly differ-
entiated PC12 cells have been used for cellular toxicity studies of neural
Table 3
PAMPA permeability and theoretical ADMET properties.
Compound
PAMPAa
BBB_Levelb
Solubility Levelc
EXT_Hepatotoxic #Predictiond
Pe (10−6 cm/s)
R (%)
1
3.23
2.91
6.09
6.33
6.72
7.49
6.54
0.24
0.49
0.14
0.32
0.01
0.14
0.57
74.9
7.3
0.8
2
2
2
2
1
1
–
3
3
2
2
2
2
–
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
–
7
1.1
10
4.3
2.2
0.7
19
8.0
21
11.6
9.2
0.6
22
0.8
1.1
Donepezil
8.7
a
Effective permeability coefficients (Pe) and mass retention (R) were calculated as described. Results were given as Mean
Standard Deviation (SD) of quin-
tuplicate wells.
b
Blood-brain barrier (BBB) penetration levels were predicted with Discovery Studio 4.0. There are four prediction levels: 0 (Very high/Brain-Blood ratio > 5:1), 1
(High/Brain-Blood ratio between 1:1 and 5:1), 2 (Medium/Brain-Blood ratio between 0.3:1 and 1:1) and 3 (Low/Brain-Blood ratio less than 0.3:1).
c
Aqueous solubility levels were predicted with Discovery Studio 4.0. There are five prediction levels: 0 (Extremely low), 1 (Very low but possible), 2 (Low), 3
(Good), and 4 (Optimal).
d
Hepatotoxic was predicted with Discovery Studio 4.0 and FALSE means that all properties and OPS components are within expected ranges.
8

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
fit sub-pocket in the tunnel part of AChE may provide new ideas for the
development of novel high active AChE inhibitors.
4. Experimental section
4.1. Chemistry
All commonly used reagents and solvents are available from com-
mercial suppliers and can be used directly. Thin layer chromatography
(TLC) was performed using a GF-254 pre-formed high-performance si-
lica gel plate to detect fluorescent spots at 254 nm UV light. Column
chromatography silica gel was purchased from Qingdao Ocean
Chemical Plant using 200–300 mesh. 1H NMR and 13C NMR spectra
were measured separately at 25 °C using a two-dimensional spectrum
Bruker AV III-400 or 600 spectrometer. The chemical shift is reported in
ppm (d) using the remaining solvent line as an internal standard. Mass
spectrometry and elemental analysis were performed using a Shimadzu
Ultra Performance Liquid Chromatography -Ion Trap Time-of-Flight
(UPLC-IT-TOF) chromatography mass spectrometer.
4.1.1. General procedure for the preparation of 1–24
All compounds were synthesized according to condition c (yield:
45–90%). This general procedure is described by using only Compound
1 as an example. Only the hydrogen, carbon and high-resolution mass
spectra of all final products are listed in the article. See the supporting
information for specific conditions and spectral data for intermediate
synthesis. The reaction conditions were as follows: 0.5 ml of methanol,
22.23 mg (0.16 mmol) of intermediate 1b and 32.2 (0.17 mmol)mg of 4-
benzylpiperidone were added to a 2 ml microwave tube and micro-
waved at 100 °C for 30 min. After the reaction was completed, the
solvent was evaporated under reduced pressure. DCM/MeOH = 40:1
column chromatography gave 49.2 mg, yield 82.0%.
Fig. 12. Cytotoxicity data for compounds 10, 21 and donepezil.
diseases [42]. We turned to utilize this cell line to evaluate the toxicity
of compound 10 and compound 21 as well as the commercially avail-
able drug donepezil. As shown in Fig. 12, at a concentration of 20 µM,
there was apparently no cytotoxicity. This result encourages further
investigation of this series of potent AChE inhibitors.
4.1.1.1. N'-(1-benzylpiperidin-4-ylidene)picolinohydrazide (1). 1H NMR
(400 MHz, CDCl3) δ 2.54–2.74 (8H, m, J = 6.3 Hz), 3.58 (2H, s),
7.23–7.30 (1H, m), 7.30–7.40 (4H, m), 7.45 (1H, dd, J = 4.9, 6.5 Hz),
7.86 (1H, dt, J = 1.3, 7.7 Hz), 8.28 (1H, d, J = 7.8 Hz), 8.53 (1H, d,
J = 4.4 Hz), 10.79 (1H, s). 13C NMR (101 MHz, CDCl3) δ 26.91, 34.77,
52.15, 53.44, 62.35, 122.82, 126.62, 127.31, 128.36, 129.05, 137.61,
137.90, 148.04, 149.45, 159.12, 159.99. HRMS (ESI): m/z 331.1528
[M + Na]+; calcd for C18H20N4ONa, 331.1529. Light yellow oily
liquid, yield 82.0%.
2.2.7. Parallel artificial membrane permeability assay (PAMPA)
The ability to across biological membranes is a fundamental feature
in the absorption and distribution of drugs. And it is the essential re-
quirement for the central nervous system (CNS) drugs to penetrate the
blood-brain barrier (BBB). PAMPA models were developed to evaluate
the passive membrane permeability in vitro. Among the reported
PAMPA models, the Corning Gentest™ Pre-coated PAMPA Plate System
was widely utilized to assess compounds’ passive transport ability
through biological membranes, including numerous reports in CNS field
[43–47]. The Corning Gentest™ Pre-coated PAMPA Plate System was
used and the results were showed in Table 3 with donepezil as the re-
ference compound. Compounds 10, 19, 21 and 22 demonstrated a
significant increase of the passive membrane permeability in compar-
ison with their parent compound 7 with normal mass retention ratio. A
maximal 2.6-fold improvement was achieved. Since these synthesized
compounds shared similar molecule volume and the same pharmaco-
phore warhead with donepezil, the experimental Pe values all fell into
the category (Pe > 1.5 × 10−6 cm/s), which represented a high per-
meability classification according to the literature [48].
4.1.1.2. N'-(1-benzylpiperidin-4-ylidene)isonicotinohydrazide
(2). 1H
NMR (400 MHz, DMSO) δ 2.39–2.59 (8H, m) 3.56 (2H, s), 7.23–7.30
(1H, m), 7.34 (4H, d, J = 4.3 Hz), 7.73 (2H, d, J = 5.6 Hz), 8.72 (2H, d,
J = 5.6 Hz), 10.97 (1H, s). 13C NMR (101 MHz, DMSO) δ 28.76, 34.82,
52.45, 53.52, 61.65, 122.13, 127.47, 128.69, 129.24, 138.69, 141.50,
150.55, 162.26, 165.03. HRMS (ESI): m/z 331.1528 [M + Na]+; calcd
for C18H20N4ONa, 331.1529. Light yellow solid, yield 45.0%.
4.1.1.3. N'-(1-benzylpiperidin-4-ylidene)nicotinohydrazide (3). 1H NMR
(400 MHz, DMSO) δ 2.27–2.59 (8H, m), 3.54 (2H, s), 7.22–7.29 (1H,
m), 7.33 (4H, d, J = 4.3 Hz), 7.50 (1H, dd, J = 5.1, 7.2 Hz), 8.15 (1H,
3. Conclusion
d, J = 7.7 Hz), 8.71 (1H, d, J = 3.9 Hz), 8.96 (1H, s), 10.89 (1H, s). 13
C
In summary, the study involved the design, synthesis, and biological
evaluation of a series of multifunctional agents against AD by fusing the
N-benzylpiperidine moiety of donepezil and the pharmacophore of
pyridine hydrazide. Most of the compounds have good inhibition of
AChE and show very high selectivity over BChE. ADMET predictions
show that this class of compounds has good drug-like properties. The
most active compound 21 has an IC50 value of 6 nM and the capability
to chelate cupric ion. All these results highlight that compound 21 is a
promising lead compound for the development of anti-AD drugs. In
particular, the introduction of an alkyl group to the NAH linker is
considered to be a highlight of this study. The possible ligand-induced-
NMR (101 MHz, DMSO) δ 28.66, 34.83, 52.47, 53.57, 61.70, 123.87,
127.46, 128.68, 129.23, 130.18, 135.93, 138.73, 149.05, 152.32,
162.42, 163.93. HRMS (ESI): m/z 309.1712 [M + H]+; calcd for
C18H21N4O, 309.1710. Light yellow solid, yield 60.0%.
4.1.1.4. N'-(1-benzylpiperidin-4-ylidene)benzohydrazide (4). 1H NMR
(400 MHz, CDCl3) δ 2.43–2.52 (2H, m), 2.54–2.71 (6H, m), 3.56 (2H,
s), 7.23–7.30 (1H, m,), 7.33 (4H, d, J = 4.2 Hz), 7.42 (2H, t,
J = 7.5 Hz), 7.50 (1H, t, J = 7.2 Hz), 7.78 (2H, d, J = 6.2 Hz), 8.90
(1H, s). 13C NMR (101 MHz, CDCl3) δ 27.11, 34.80, 52.08, 53.45,
62.37, 127.21, 127.28, 128.36, 128.73, 128.99, 131.85, 133.64,
9

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
NMR (101 MHz, CDCl3) δ 29.50, 39.07, 52.93, 59.47, 122.92, 126.62,
126.68, 128.11, 129.40, 130.70, 134.27, 135.98, 137.58, 148.00,
149.14, 155.53, 159.97. HRMS (ESI): m/z 379.1302 [M+Na]+; calcd
for C19H21N4OClNa, 379.1279. Gray-white solid, yield 80%.
138.06, 160.00, 164.25. HRMS (ESI): m/z 330.1576 [M + Na]+; calcd
for C19H21N4ONa, 330.1577. Yellow-green solid, yield 90.0%.
4.1.1.5. N'-(4-(phenylamino)cyclohexylidene)picolinohydrazide (5). 1H
NMR (400 MHz, CDCl3) δ 1.47–1.69 (2H, m), 2.20–2.37 (3H, m),
2.44–2.58 (1H, m), 2.77–2.94 (2H, m), 3.51–3.71 (2H, m), 6.64 (2H,
d, J = 7.8 Hz), 6.72 (1H, t, J = 7.3 Hz), 7.20 (2H, t, J = 7.9 Hz),
7.44–7.50 (1H, m), 7.89 (1H, dt, J = 1.5, 7.7 Hz), 8.30 (1H, d,
J = 7.8 Hz), 8.56 (1H, d, J = 4.3 Hz), 10.82 (1H, s). 13C NMR
4.1.1.11. (E)-N'-((1-(3-chlorobenzyl)piperidin-4-yl)methylene)
picolinohydrazide (11). 1H NMR (400 MHz, CDCl3) δ 1.60–1.74 (2H,
m), 1.85 (2H, d, J = 11.2 Hz), 2.02–2.13 (2H, m), 2.47–2.59 (1H, m,),
2.92 (2H, d, J = 11.5 Hz), 3.49 (2H, s), 7.17–7.30 (3H, m), 7.35 (1H, s),
7.48 (2H, t, J = 6.7 Hz), 7.89 (1H, dt, J = 1.5, 7.7 Hz), 8.30 (1H, d,
J = 7.8 Hz), 8.56 (1H, d, J = 4.4 Hz), 10.69 (1H, s). 13C NMR
(101 MHz, CDCl3) δ 29.41, 39.06, 52.84, 62.67, 122.91, 126.69,
127.18, 127.23, 129.02, 129.49, 134.15, 137.58, 140.53, 148.01,
149.12, 155.45, 159.99. HRMS (ESI): m/z 357.1478 [M+H]+; calcd
for C19H22N4OCl, 357.1477. Yellow-brown solid, yield 60%.
(101 MHz, CDCl3)
δ 29.44, 38.98, 52.61, 55.52, 115.23 (1C, d,
J = 22.2 Hz), 122.90 (1C, s), 123.87 (1C, d, J = 3.5 Hz), 124.67 (1C,
d, J = 14.3 Hz), 126.68, 128.79 (1C, d, J = 8.3 Hz), 131.61 (1C, d,
J = 4.5 Hz), 137.57, 148.00, 149.13, 155.52, 159.97, 161.40 (1C, d,
J = 246.3 Hz). HRMS (ESI): m/z 309.1715 [M + Na]+
;
calcd for
C
18H20N4ONa, 309.1710. White solid, yield 74.6%.
4.1.1.6. N'-(1-phenethylpiperidin-4-ylidene)picolinohydrazide
(6). 1H
4.1.1.12. (E)-N'-((1-(4-chlorobenzyl)piperidin-4-yl)methylene)
picolinohydrazide (12). 1H NMR (400 MHz, CDCl3) δ 1.43–1.57 (2H,
m), 1.69 (2H, d, J = 12.1 Hz), 1.90 (2H, t, J = 10.9 Hz), 2.31–2.43 (1H,
m,), 2.75 (2H, d, J = 11.4 Hz), 3.32 (2H, s), 7.08–7.18 (4H, m), 7.33
(2H, t, J = 5.7 Hz), 7.71–7.78 (1H, m), 8.14 (1H, d, J = 7.8 Hz), 8.40
(1H, d, J = 4.3 Hz), 10.53 (1H, s). 13C NMR (101 MHz, CDCl3) δ 29.41,
39.07, 52.79, 62.51, 122.92, 126.70, 128.37, 130.43, 132.74, 136.77,
137.59, 148.01, 149.11, 155.45, 159.99. HRMS (ESI): m/z 357.1478 [M
+H]+; calcd for C19H22N4OCl, 357.1477. Light yellowish brown solid,
yield 65%.
NMR (400 MHz, CDCl3) δ 2.59–2.66 (2H, m), 2.66–2.74 (6H, m),
2.74–2.79 (2H, m), 2.81–2.89 (2H, m), 7.18–7.26 (3H, m), 7.30 (2H,
t, J = 7.5 Hz), 7.47 (1H, dd, J = 5.0, 6.9 Hz), 7.88 (1H, dt, J = 1.4,
7.7 Hz), 8.30 (1H, d, J = 7.8 Hz), 8.55 (1H, d, J = 4.5 Hz), 10.82 (1H,
s). 13C NMR (101 MHz, CDCl3) δ 26.93, 33.88, 34.73, 52.43, 53.49,
59.74, 122.83, 126.18, 126.63, 128.45, 128.69, 137.62, 140.02,
148.04, 149.45, 158.93, 160.01. HRMS (ESI): m/z 345.1689
[M + Na]+; calcd for C19H22N4ONa, 345.1686. Yellow-green solid,
yield 75%.
4.1.1.7. (E)-N'-((1-benzylpiperidin-4-yl)methylene)picolinohydrazide
(7). 1H NMR (400 MHz, CDCl3) δ 1.58–1.73 (2H, m), 1.76–1.88 (2H,
m), 1.99–2.12 (2H, m), 2.45–2.57 (1H, m,), 2.87–2.99 (2H, m), 3.51
(2H, s), 7.20–7.28 (1H, m), 7.32 (4H, d, J = 4.4 Hz), 7.42–7.50 (2H, m),
7.87 (1H, dt, J = 1.5, 7.7 Hz), 8.27 (1H, d, J = 7.8 Hz), 8.53 (1H, d,
J = 4.4 Hz), 10.66 (1H, s). 13C NMR (101 MHz, CDCl3) δ 29.47, 39.16,
52.85, 63.39, 122.93, 126.69, 127.05, 128.23, 129.21, 137.59, 138.20,
148.00, 149.13, 155.64, 159.98. HRMS (ESI): m/z 345.1684
4.1.1.13. (E)-N'-((1-(pyridin-3-ylmethyl)piperidin-4-yl)methylene)
picolinohydrazide (13). 1H NMR (400 MHz, CDCl3) δ 1.56–1.70 (2H,
m), 1.83 (2H, d, J = 11.7 Hz), 2.07 (2H, t, J = 10.7 Hz), 2.44–2.56 (1H,
m), 2.89 (2H, d, J = 11.4 Hz), 3.50 (2H, s), 7.22–7.26 (1H, m),
7.41–7.50 (2H, m), 7.66 (1H, d, J = 7.7 Hz), 7.86 (1H, dt, J = 1.3,
7.7 Hz), 8.27 (1H, d, J = 7.8 Hz), 8.47–8.55 (3H, m), 10.67 (1H, s). 13
C
NMR (101 MHz, CDCl3) δ 29.45, 39.07, 52.86, 60.51, 122.93, 123.33,
126.70, 133.83, 136.71, 137.60, 148.00, 148.56, 149.10, 150.44,
155.39, 159.99. HRMS (ESI): m/z 346.1633 [M+Na]+; calcd for
[M + Na]+
yield 80%.
; calcd for C19H22N4ONa, 345.1686. Gray-white solid,
C18H21N5ONa, 346.1638. Gray-white solid, yield 75%.
4.1.1.8. (E)-N'-(2-(1-benzylpiperidin-4-ylidene)ethylidene)picolinohydrazide
(8). 1H NMR (400 MHz, CDCl3) δ 2.36–2.43 (2H, m), 2.46–2.58 (6H, m),
3.53 (2H, s), 6.22 (1H, d, J = 9.8 Hz), 7.23–7.29 (1H, m), 7.30–7.40 (4H,
m), 7.43–7.49 (1H, m), 7.87 (1H, dt, J = 1.5, 7.7 Hz), 8.19 (1H, d,
J = 9.8 Hz), 8.28 (1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.5 Hz), 10.78
(1H, s). 13C NMR (101 MHz, CDCl3) δ 29.28, 36.49, 53.98, 54.64, 62.61,
120.04, 122.91, 126.66, 127.20, 128.30, 129.12, 137.61, 146.58, 147.99,
149.15, 150.27, 159.91. HRMS (ESI): m/z 335.1868 [M+H]+; calcd for
C20H23N4O, 335.1866. Brown oily liquid, yield 65%.
4.1.1.14. (E)-N'-((1-(2-methoxybenzyl)piperidin-4-yl)methylene)
picolinohydrazide (14). 1H NMR (400 MHz, CDCl3) δ 1.60–1.73 (2H,
m), 1.81 (2H, d, J = 10.9 Hz), 2.11 (2H, t, J = 10.8 Hz), 2.44–2.56 (1H,
m,), 2.97 (2H, d, J = 11.6 Hz), 3.57 (2H, s), 3.82 (3H, s), 6.87 (1H, d,
J = 8.2 Hz), 6.93 (1H, t, J = 7.3 Hz), 7.18–7.25 (1H, m), 7.35 (1H, d,
J = 7.4 Hz), 7.40–7.50 (2H, m), 7.87 (1H, dt, J = 1.4, 7.7 Hz), 8.28
(1H, d, J = 7.8 Hz), 8.53 (1H, d, J = 4.4 Hz), 10.65 (1H, s). 13C NMR
(101 MHz, CDCl3) δ 29.49, 39.12, 52.81, 55.45, 56.28, 110.47, 120.32,
122.90, 126.15, 126.67, 128.07, 130.60, 137.57, 148.00, 149.15,
155.79, 157.82, 159.97. HRMS (ESI): m/z 353.1975 [M+H]+; calcd
for C20H25N4O2, 353.1972. Light yellowish brown solid, yield 77%.
4.1.1.9. (E)-N'-(2-(1-benzylpiperidin-4-yl)ethylidene)picolinohydrazide
(9). 1H NMR (400 MHz, CDCl3) δ 1.34–1.46 (2H, m), 1.56–1.66 (1H,
m), 1.70 (2H, d, J = 13.2 Hz), 1.96 (2H, t, J = 11.1 Hz), 2.40 (2H, t,
J = 6.3 Hz), 2.88 (2H, d, J = 11.4 Hz), 3.49 (2H, s), 7.21–7.26 (1H, m),
7.27–7.34 (4H, m), 7.43–7.49 (1H, m), 7.64 (1H, t, J = 5.9 Hz), 7.87
(1H, dt, J = 1.5, 7.7 Hz), 8.29 (1H, d, J = 7.8 Hz), 8.54 (1H, d,
J = 4.4 Hz), 10.69 (1H, s). 13C NMR (101 MHz, CDCl3) δ 32.07,
34.51, 38.90, 53.58, 63.37, 122.94, 126.68, 126.98, 128.18, 129.22,
137.59, 138.29, 148.00, 149.13, 151.83, 159.91. HRMS (ESI): m/z
337.2024 [M+H]+; calcd for C19H23N4O, 337.2023. White solid, yield
70%.
4.1.1.15. (E)-N'-((1-(2-fluorobenzyl)piperidin-4-yl)methylene)
picolinohydrazide (15). 1H NMR (400 MHz, CDCl3) δ 1.58–1.72 (2H,
m), 1.83 (2H, d, J = 12.4 Hz), 2.11 (2H, t, J = 10.8 Hz), 2.43–2.56 (1H,
m), 2.94 (2H, d, J = 11.5 Hz), 3.58 (2H, s), 7.03 (1H, t, J = 9.1 Hz),
7.10 (1H, t, J = 7.3 Hz), 7.20–7.26 (1H, m), 7.37 (1H, t, J = 6.9 Hz),
7.42–7.51 (2H, m), 7.87 (1H, dt, J = 1.4, 7.7 Hz), 8.28 (1H, d,
J = 7.8 Hz), 8.53 (1H, d, J = 4.4 Hz), 10.65 (1H, s). HRMS (ESI): m/z
341.1770 [M+H]+; calcd for C19H22N4OF, 341.1772. White solid,
yield 70%.
4.1.1.10. (E)-N'-((1-(2-chlorobenzyl)piperidin-4-yl)methylene)
4.1.1.16. (E)-N'-((1-(2-bromobenzyl)piperidin-4-yl)methylene)
picolinohydrazide (10). 1H NMR (400 MHz, CDCl3) δ 1.62–1.76 (2H,
m), 1.83 (2H, d, J = 11.5 Hz), 2.16 (2H, t, J = 10.7 Hz), 2.47–2.59 (1H,
m,), 2.94 (2H, d, J = 11.4 Hz), 3.61 (2H, s), 7.13–7.26 (2H, m),
7.31–7.35 (1H, m), 7.41–7.57 (3H, m), 7.87 (1H, dt, J = 1.5, 7.7 Hz),
8.28 (1H, d, J = 7.8 Hz), 8.53 (1H, d, J = 4.4 Hz), 10.66 (1H, s). 13C
picolinohydrazide (16). 1H NMR (400 MHz, CDCl3) δ 1.60–1.73 (2H,
m), 1.84 (2H, d, J = 11.5 Hz), 2.18 (2H, t, J = 10.7 Hz), 2.47–2.59 (1H,
m), 2.95 (2H, d, J = 11.5 Hz), 3.60 (2H, s), 7.07–7.15 (1H, m), 7.28
(1H, t, J = 7.3 Hz), 7.43–7.51 (3H, m), 7.53 (1H, d, J = 7.9 Hz), 7.88
(1H, dt, J = 1.5, 7.7 Hz), 8.29 (1H, d, J = 7.8 Hz), 8.55 (1H, d,
10

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
J = 4.4 Hz), 10.67 (1H, s). 13C NMR (101 MHz, CDCl3) δ 29.58, 39.13,
52.96, 62.08, 122.94, 124.61, 126.69, 127.21, 128.33, 130.65, 132.70,
137.60, 137.83, 148.01, 149.14, 155.60, 159.98. HRMS (ESI): m/z
401.0971 [M+H]+; calcd for C19H22N4OBr, 401.0971. Yellow-brown
solid, yield 80%.
m), 2.33–2.43 (2H, m), 2.45–2.56 (1H, m), 2.99 (2H, d, J = 11.4 Hz),
3.61 (2H, s), 7.14–7.26 (2H,m), 7.34 (1H, d, J = 8.0 Hz), 7.46 (1H, dd,
J = 4.9, 6.7 Hz), 7.51 (1H, d, J = 7.4 Hz), 7.88 (1H, dt, J = 1.5,
7.7 Hz), 8.30 (1H, d, J = 7.8 Hz), 8.56 (1H, d, J = 4.5 Hz), 10.89 (1H,
s). 13C NMR (101 MHz, CDCl3) δ 14.71, 19.35, 29.57, 29.61, 44.94,
53.64, 59.48, 122.77, 126.47, 126.57, 127.93, 129.33, 130.52, 134.14,
136.36, 137.55, 148.13, 149.74, 159.67, 164.34. HRMS (ESI): m/z
399.1947 [M+H]+; calcd for C22H28N4OCl, 399.1946. Brown solid,
yield 76%.
4.1.1.17. (E)-N'-((1-(2-methylbenzyl)piperidin-4-yl)methylene)
picolinohydrazide (17). 1H NMR (400 MHz, CDCl3) δ 1.55–1.68 (2H,
m), 1.81 (2H, d, J = 11.7 Hz), 2.07 (2H, t, J = 10.7 Hz), 2.46–2.57 (1H,
m), 2.91 (2H, d, J = 11.5 Hz), 3.45 (2H, s), 7.11–7.19 (3H, m),
7.22–7.30 (1H, m), 7.42–7.51 (2H, m), 7.88 (1H, dt, J = 1.5, 7.7 Hz),
8.28 (1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.4 Hz), 10.66 (1H, s). 13C
NMR (101 MHz, CDCl3) δ 19.27, 29.66, 39.34, 53.01, 61.10, 122.94,
125.47, 126.68, 126.94, 129.75, 130.23, 136.69, 137.49, 137.60,
148.00, 149.14, 155.80, 159.98. HRMS (ESI): m/z 337.2025 [M
+H]+; calcd for C20H25N4O, 337.2023. Yellow-brown solid, yield 70%.
4.1.1.23. (E)-N'-(1-(1-(2-chlorobenzyl)piperidin-4-yl)pentylidene)
picolinohydrazide (23). 1H NMR (400 MHz, CDCl3) δ 1.00 (3H, t,
J = 7.1 Hz), 1.43–1.63 (4H, m), 1.71–1.88 (4H, m), 2.07–2.22 (2H,
m), 2.33–2.43 (2H, m), 2.46–2.58 (1H, m), 2.99 (2H, d, J = 11.0 Hz),
3.62 (2H, s), 7.13–7.20 (1H, m), 7.23 (1H, t, J = 7.1 Hz), 7.33 (1H, d,
J = 7.8 Hz), 7.41–7.47 (1H, m), 7.51 (1H, d, J = 7.1 Hz), 7.86 (1H, dt,
J = 1.4, 7.7 Hz), 8.28 (1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.4 Hz),
10.90 (1H, s). 13C NMR (101 MHz, CDCl3) δ 13.68, 23.22, 27.21, 27.72,
29.54, 44.95, 53.59, 59.43, 122.74, 126.48, 126.60, 128.00, 129.34,
130.60, 134.17, 137.55, 148.07, 149.73, 159.66, 164.42. HRMS (ESI):
m/z 413.2105 [M+H]+; calcd for C23H30N4OCl, 413.2103. Brown
solid, yield 74%.
4.1.1.18. (E)-N'-((1-(2,6-dichlorobenzyl)piperidin-4-yl)methylene)
picolinohydrazide (18). 1H NMR (400 MHz, CDCl3) δ 1.52–1.67 (2H,
m), 1.79 (2H, d, J = 11.9 Hz), 2.28 (2H, t, J = 11.0 Hz), 2.46–2.58 (1H,
m), 2.96 (2H, d, J = 11.1 Hz), 3.74 (2H, s), 7.13 (1H, t, J = 8.0 Hz),
7.29 (2H, t, J = 9.1 Hz), 7.40–7.50 (2H, m), 7.87 (1H, dt, J = 1.4,
7.7 Hz), 8.28 (1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.4 Hz), 10.64 (1H,
s). 13C NMR (101 MHz, CDCl3) δ 29.54, 39.11, 52.79, 56.80, 122.92,
126.67, 128.34, 128.74, 134.62, 136.99, 137.58, 147.99, 149.14,
155.73, 159.96. HRMS (ESI): m/z 391.1087 [M+H]+; calcd for
4.1.1.24. (E)-N'-(1-(1-(2-chlorobenzyl)piperidin-4-yl)-3-
methylbutylidene)picolinohydrazide (24). 1H NMR (400 MHz, CDCl3) δ
1.05 (6H, d, J = 6.6 Hz), 1.74–1.94 (4H, m), 2.07–2.23 (3H, m), 2.30
(2H, d, J = 7.5 Hz), 2.36–2.48 (1H, m), 3.00 (2H, d, J = 10.1 Hz), 3.62
(2H, s), 7.17 (1H, t, J = 7.0 Hz), 7.24 (1H, t, J = 7.2 Hz), 7.33 (1H, d,
J = 7.8 Hz), 7.45 (1H, dd, J = 4.9, 6.7 Hz), 7.53 (1H, d, J = 7.3 Hz),
7.87 (1H, dt, J = 1.5, 7.7 Hz), 8.29 (1H, d, J = 7.8 Hz), 8.56 (1H, d,
J = 4.5 Hz), 10.93 (1H, s). 13C NMR (101 MHz, CDCl3) δ 23.19, 26.71,
30.00, 37.02, 44.84, 53.79, 59.34, 122.75, 126.42, 126.62, 127.92,
129.28, 130.54, 134.07, 136.33, 137.54, 148.11, 149.81, 159.55,
163.73. HRMS (ESI): m/z 413.2105 [M+H]+; calcd for C23H30N4OCl,
413.2103. Light yellow solid, yield 51%.
C
19H21N4OCl2, 391.1087. White solid, yield 85%.
4.1.1.19. (E)-N'-(1-(1-(2-chlorobenzyl)piperidin-4-yl)ethylidene)
picolinohydrazide (19). 1H NMR (400 MHz, CDCl3) δ 1.72–1.81 (4H,
m), 2.00 (3H, s), 2.08–2.20 (2H, m), 2.49–2.62 (1H, m), 2.98 (2H, d,
J = 11.2 Hz), 3.60 (2H, s), 7.13–7.20 (1H, m), 7.23 (1H, t, J = 7.4 Hz),
7.33 (1H, d, J = 7.8 Hz), 7.42–7.53 (2H, m), 7.82–7.91 (1H, m), 8.28
(1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.6 Hz), 10.71 (1H, s). 13C NMR
(101 MHz, CDCl3) δ 12.72, 29.30, 45.42, 53.47, 59.50, 122.82, 126.56,
127.96, 129.36, 130.53, 134.19, 136.33, 137.56, 148.04, 149.61,
159.87, 161.15. HRMS (ESI): m/z 371.1634 [M+H]+; calcd for
4.2. Biology
C
20H24N4OCl, 371.1633. Yellow-brown solid, yield 73%.
4.2.1. In vitro AChE and BChE inhibition Assays.
4.1.1.20. (E)-N'-((1-(2-chlorobenzyl)piperidin-4-yl)methylene)-N-
The enzyme inhibition activity of the test compounds was per-
formed adopting the method of Ellman et al.[35]. Electric eel acetyl
cholinesterase (eeAChE) and horse serum butyryl cholinesterase
(eqBChE) were purchased from Sigma. 5,5′-dithiobis(2-nitrobenzoic
acid) (DTNB-Ellman’s reagent), acetylthiocholine iodide (ATCI), bu-
tyrylthiocholine iodide (BTCI), tacrine and donepezil were purchased
from Aladdin (Shanghai, China). All the experiments were conducted in
Corning Costar 96-well clear flat-bottom plates (9017). The experiment
medium was 50 mM sodium phosphate buffer at pH 8 with a final
concentration of 0.5% DMSO and 0.01% Triton-X100.
methylpicolinohydrazide (20). 1H NMR (400 MHz, CDCl3) δ 1.36–1.50
(2H, m), 1.65 (2H, d, J = 12.3 Hz), 2.07 (2H, t, J = 11.2 Hz), 2.11–2.23
(1H, m), 2.80 (2H, d, J = 10.6 Hz), 3.39 (3H, s), 3.56 (2H, s), 7.03 (1H,
d, J = 3.4 Hz), 7.13–7.25 (2H, m), 7.25–7.36 (2H, m), 7.42 (2H, d,
J = 7.2 Hz), 7.72 (1H, t, J = 7.7 Hz), 8.62 (1H, d, J = 3.9 Hz). 13C NMR
(101 MHz, CDCl3) δ 28.01, 29.26, 38.78, 53.00, 59.43, 122.95, 123.68,
126.55, 128.01, 129.35, 130.60, 134.21, 135.75, 136.13, 146.29,
148.73, 154.86, 169.69. HRMS (ESI): m/z 371.1635 [M+H]+; calcd
for C20H24N4OCl, 371.1633. Yellowish brown oily liquid, yield 80%.
Briefly, 50 μL of AChE or 50 μL of BChE and 100 μL of the compound
were incubated in 96-well plates at room temperature for 10 min.
Further, a mixture of 25 μL of the substrate, ATCI (5 mM) or BTCI
(5 mM), and 25 μL of DTNB (5 mM) were added to each well to start the
reaction. After 30 min of reaction time in the dark, the absorbance was
measured at 415 nm wavelength using microplate reader FlexStation 3
(Molecular Devices, US).
4.1.1.21. (E)-N'-(1-(1-(2-chlorobenzyl)piperidin-4-yl)propylidene)
picolinohydrazide (21). 1H NMR (400 MHz, CDCl3) δ 1.20 (3H, t,
J = 7.7 Hz), 1.71–1.88 (4H, m), 2.07–2.20 (2H, m), 2.42 (2H, q,
J = 7.7 Hz), 2.48–2.60 (1H, m), 2.99 (2H, d, J = 11.3 Hz), 3.61 (2H,
s), 7.13–7.19 (1H, m), 7.19–7.25 (1H, m), 7.29–7.35 (1H, m),
7.41–7.47 (1H, m), 7.50 (1H, d, J = 7.1 Hz), 7.86 (1H, dt, J = 1.5,
7.7 Hz), 8.28 (1H, d, J = 7.8 Hz), 8.54 (1H, d, J = 4.3 Hz), 10.87 (1H,
s). 13C NMR (101 MHz, CDCl3) δ 10.14, 20.34, 29.49, 44.93, 53.59,
59.50, 122.81, 126.49, 126.56, 127.93, 129.35, 130.51, 134.17,
136.38, 137.55, 148.11, 149.72, 159.78, 165.21. HRMS (ESI): m/z
385.1797 [M+H]+; calcd for C21H26N4OCl, 385.1790. Light yellow
oily liquid, yield 70%.
The IC50 value was calculated from the absorbance obtained for
various concentrations of the test and the standard compounds. All the
determinations were performed in triplicate and at least in three in-
dependent runs. The enzyme kinetic study was done to determine the
mechanism of AChE inhibition by donepezil and compound 21 in a
similar manner as described above.
4.1.1.22. (E)-N'-(1-(1-(2-chlorobenzyl)piperidin-4-yl)butylidene)
picolinohydrazide (22). 1H NMR (400 MHz, CDCl3) δ 1.09 (3H, t,
J = 7.3 Hz), 1.60–1.69 (2H, m), 1.74–1.84 (4H, m), 2.09–2.19 (2H,
4.2.2. Metal Chelation [49]
Compound 21 was tested as a metal chelating agent using a differ-
ential UV–visible spectrum with a wavelength range of 190–600 nm
11

Y. Zhou, et al.
BioorganicChemistry93(2019)103322
recorded in phosphate buffer containing 0.05% DMSO in volume in
room temperature. Compound 21 and several metal salts were for-
mulated into 200 μM solutions. The metal salt solution was mixed with
an equal volume of phosphate buffer as the background. A mixture of
the compound 21 solution and an equal volume of phosphate buffer was
used as the control group. The compound 21 solution was mixed with
an equal volume of the metal salt solution and was allowed to stand for
30 min before measuring. The individual metal and solvent spectra
were subtracted from the background to obtain the UV–vis spectrums
that produced a change in absorption peak due to the complexation of
the metal with compound 21.
center points (x, y, z) were (-13.985, −43.975, 27.895) and (19.143,
42.419, 41.008), respectively. The Genetic Optimization of Ligand
Docking (GOLD 5.2.2, CCDC) was used for docking studies. Both radius
of two sphere were set to 10 Å, the template and the scoring function to
the AChE and BChE were set chemscore_kinase and CHEMPLP. Tyr-341,
Tyr-124, Phe-338, Phe-295 were set to be flexible to better predict
possible interaction sites for the alkyl side chain of the compound. The
rotatable torsion in the side chain and other parameters were set as
default parameters. The genetic algorithms (GA) Runs was set to 50
times.
Declaration of Competing Interest
4.2.3. Cytotoxicity Assay
The authors declared that they have no conflicts of interest to this
Compound 10, compound 21 and donepezil were tested for their
cytotoxicity in rat phaeochromocytoma cell line PC12 using the vital
mitochondrial function MTT assay with the Promega CellTiter 96®
AQueous One Solution Reagent (contains MTS (3-(4,5-dimethylthiazol-
2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
and PES (phenazine ethosulfate)). Poorly differentiated PC12 cells were
supplied by the Cell Bank of the Kunming Institute of Zoology, CAS
(Kunming, China). Cells were cultured in Dulbecco's Modified Eagle's
Medium (DMEM, High Glucose) supplemented with 10% FBS, 100
units/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO2
atmosphere. Poorly differentiated PC12 cells (10000 cell/well) were
seeded in 96-well plates and incubated for 24 h. The culture medium
was replaced with fresh medium (with 20 µM of compound 10, com-
pound 21 or donepezil) and the plates were returned to the incubator
for 24 h. After drug treatments, the cells were cultured with fresh
medium (CellTiter 96® AQueous One Solution Reagent/supplemented
DMEM = 1:4 in volume) at 37 °C for 2 h. The absorbance was measured
at 492 nm with a microplate reader. Cell viability was expressed as a
percentage relative to the blank group. Data are expressed as
work.
We declare that we do not have any commercial or associative in-
terest that represents a conflict of interest in connection with the work
submitted.
Acknowledgements
This project was financially supported by the Grant of Sichuan
Science and Technology Program (No: 19ZDYF1034), General Program
of Applied Basic Research of Yunnan Province (No: 2014FB174), and
the State Key Laboratory of Phytochemistry and Plant Resources in
West China (No. P2017-KF07, P2018-KF14).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103322.
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