Molecular-docking-guided design and synthesis of new IAA-tacrine hybrids as multifunctional AChE/BChE inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.10.057

A series of new indole-3-acetic acid (IAA)-tacrine hybrids as dual acetylcholinesterase (AChE)/butyrylcholinesterase (BChE) inhibitors were designed and prepared based on the molecular docking mode of AChE with an IAA derivative (1a), a moderate AChE inhibitor identified by screening our compound library for anti-Alzheimer's disease (AD) drug leads. The enzyme assay results revealed that some hybrids, e.g. 5d and 5e, displayed potent dual in vitro inhibitory activities against AChE/BChE with IC₅₀ values in low nanomolar range. Molecular modeling studies in tandem with kinetic analysis suggest that these hybrids target both catalytic active site and peripheral anionic site of cholinesterase (ChE). Molecular dynamic simulations and Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations indicate that 5e has more potent binding affinity than hit 1a, which may explain the stronger inhibitory effect of 5e on AChE. Furthermore, their predicted pharmacokinetic properties and in vitro influences on mouse brain neural network electrical activity were discussed. Taken together, compound 5e can be highlighted as a lead compound worthy of further optimization for designing new anti-AD drugs.

Full text of DOI:10.1016/j.bioorg.2018.10.057

BioorganicChemistry83(2019)277–288
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Molecular-docking-guided design and synthesis of new IAA-tacrine hybrids
as multifunctional AChE/BChE inhibitors
Zhi-Qiang Chenga,1, Kong-Kai Zhua,b,1, Juan Zhanga,1, Jia-Li Songa,
⁎
⁎
⁎
Luis Alexandre Muehlmannc,d, Cheng-Shi Jianga, , Chang-Liang Liue,f, , Hua Zhanga,
a School of Biological Science and Technology, University of Jinan, Jinan 250022, China
b Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c Institute of Biological Sciences, University of Brasília, Brasilia 70910900, Brazil
d Faculty of Ceilandia, University of Brasília, Brasilia 72220275, Brazil
e Cambrian Discovery Inc., Dover, MA 02115, USA
f Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
A R T I C L E I N F O
A B S T R A C T
A series of new indole-3-acetic acid (IAA)-tacrine hybrids as dual acetylcholinesterase (AChE)/butyr-
Keywords:
Alzheimer’s disease
IAA-tacrine hybrids
Dual AChE/BChE inhibitor
Molecular docking
ylcholinesterase (BChE) inhibitors were designed and prepared based on the molecular docking mode of AChE
with an IAA derivative (1a), a moderate AChE inhibitor identified by screening our compound library for anti-
Alzheimer’s disease (AD) drug leads. The enzyme assay results revealed that some hybrids, e.g. 5d and 5e,
displayed potent dual in vitro inhibitory activities against AChE/BChE with IC50 values in low nanomolar range.
Molecular modeling studies in tandem with kinetic analysis suggest that these hybrids target both catalytic
active site and peripheral anionic site of cholinesterase (ChE). Molecular dynamic simulations and Molecular
Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) calculations indicate that 5e has more potent binding
affinity than hit 1a, which may explain the stronger inhibitory effect of 5e on AChE. Furthermore, their pre-
dicted pharmacokinetic properties and in vitro influences on mouse brain neural network electrical activity were
discussed. Taken together, compound 5e can be highlighted as a lead compound worthy of further optimization
for designing new anti-AD drugs.
Neural network electrical activity
1. Introduction
[4]. ACh in the brain is mainly hydrolyzed by cholinesterase (ChE), and
thus inhibition of ChE has been proved to be an effective way for the
Alzheimer’s disease (AD) is a multifaceted neurodegenerative dis-
order featured by loss of memory, progressive deficits in cognitive
functions and severe behavioral abnormalities [1]. Approximately 46.8
million people worldwide are now believed to suffer from AD, and this
number is expected to triple by 2050 with the aging of global popula-
tion [2].
treatment of AD. There are two types of ChE in the nervous system:
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) [5].
Compared with BChE, AChE is 1013 folds more active and accounts for
nearly 80% ACh hydrolysis in the brain [6]. Four AChE inhibitors in-
cluding tacrine, donepezil, galantamine, and rivastigmine (Fig. 1) have
been approved by FDA [7]. These AChE inhibitors are beneficial to
improving memorial and cognitive functions of AD patients, but tacrine
was withdrawn from clinical in the US in 2013 due to its hepatotoxicity
[8], and others clinically used drugs cannot cure or stop the progress of
AD [9]. Studies indicate that the level and activity of BChE rise with AD
progression while the AChE activity declines [10]. Moreover, the AChE-
knockout mice with normal level and localization of BChE can survive,
which supports the idea that BChE compensates the function of AChE
[8,11]. Additional inhibition on BChE is therefore considered to
The pathogenesis of AD involves several pathways including defi-
ciency in cholinergic neurotransmission, beta-amyloid (Aβ) protein and
tau protein phosphorylation pathways.1 The recently causal treatment
of AD by developing Aβ monoclonal antibodies, Aβ aggregation in-
hibitors or tau aggregation inhibitors have proven to be unsuccessful
[3], while enhancing cholinergic neurotransmission in the brain by
increasing the level of acetylcholine (ACh), as symptomatic drug
therapy, is currently the most efficacious approach for AD treatment
⁎ Corresponding authors at: Cambrian Discovery Inc., Dover, MA 02115, USA (C.L. Liu).
E-mail addresses: jiangchengshi-20@163.com (C.-S. Jiang), Changliang_Liu@hms.harvard.edu (C.-L. Liu), bio_zhangh@ujn.edu.cn (H. Zhang).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2018.10.057
Received 28 August 2018; Received in revised form 26 October 2018; Accepted 27 October 2018
Availableonline29October2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

Z.-Q. Cheng et al.
BioorganicChemistry83(2019)277–288
Scheme 1. Synthetic route of analogues 1a–1p.
reduction of 2-nitrobenzoic acid provided compound 2 which was then
condensed with cyclohexanone in the presence of POCl3 under reflux to
yield compound 3 [22,23]. The reaction of 3 with different diamines in
refluxing pentanol afforded the corresponding intermediates 4a–4g
[24]. Finally, the amidation reaction [17,25] of 4a–4g with IAA yielded
the target compounds 5a–5g (Table 2). The chemical structures of total
twenty-three IAA derivatives were confirmed by 1H, 13C NMR, and MS
data.
Fig. 1. Structures of AChE inhibitors approved for AD treatment and hit 1a.
contribute to ameliorating a cholinergic deficit, and dual inhibition of
AChE/BChE represents a promising therapeutic strategy for AD treat-
ment [12]. Such dual-targeted ChE inhibitors have shown positive
outcomes for the treatment of AD [13,14]. For example, rivastigmine
(Fig. 1), as dual AChE/BChE inhibitor, displays more potent effect than
donepezil [5].
2.2. In vitro inhibition of AChE and BChE, and SAR study
The AChE and BChE inhibitory activities of all the synthesized
compounds were evaluated in vitro, with tacrine as positive control.
From Table 1, all the analogues 1b–1p showed no (IC50 > 100 μM) or
increased ChE inhibitory activity compared with hit 1a. Among them,
only one compound 1n with the same terminal -N(iPr)2 group as 1a
In the course of our research for developing new anti-AD lead
compounds [15,16], an indol-3-acetic acid (IAA) derivative (1a, Fig. 1)
showing moderate to weak anti-ChE activity was identified. Interest-
ingly, this compound did not show obvious cytotoxicity against HepG2
cells even up to 400 μM, indicating that 1a serves as a potential anti-
ChE lead. In the current study, we carried out further structural mod-
ifications of 1a via two steps, which resulted in a panel of IAA-tacrine
hybrids (5a–5g) [17] as a new class of potent multifunctional AChE/
BChE inhibitors. First, molecular-docking-guided design and synthesis
of these new IAA-tacrine AChE/BChE inhibitors, and their inhibitory
activities and kinetic analysis were described. Then the preliminary
structure-activity relationship (SAR) was discussed, and the binding
modes of these hybrids to AChE and BChE were predicted by molecular
docking and molecular dynamics simulation. In addition, the pharma-
cokinetic profiles (solubility, absorption, BBB properties, etc.) and the
effect of 5e on mouse brain neural network electrical activity were
described. (See Fig. 2)
displayed weak AChE inhibition (IC50 = 54.05
3.52 μM). Thus, 1a
was selected as a model compound for further structural modification
using computer-aided drug design (CADD) approach.
Firstly, the binding mode of 1a to AChE was obtained by molecular
docking with bis-tacrine dimer (AA7)-AChE complex (ID: 5EI5).
Analyses of docking result in Fig. 3 revealed that compound 1a bound
well to the active-site gorge of AChE, with partial space superposition
with the tacrine moiety in dimer AA7. Tacrine has been reported to
show excellent inhibition against both AChE and BChE with IC50 values
at nanomolar level, and there has been a great interest in designing
tacrine-based dual binding site ChE inhibitors [25–27]. Based on the
binding mode of 1a in comparison with AA7, the terminal -N(iPr)2
group was then modified by fusion with tacrine pharmacophore to
improve its activity. In addition, the effect of alkyl diamino chain length
was investigated to find the optimal distance between indole and ta-
crine moieties. In the end, a series of hybrids 5a–5g were prepared and
evaluated for their ChE inhibitory activity.
2. Results and discussion
2.1. Chemistry
As shown in Table 1, this class displayed better results with much
stronger ChE inhibitory activity than the 1a series. For example, the
AChE inhibitory activity was increased by 38-fold (5e vs 1a), while the
BChE inhibitory activity was enhanced more significantly (5d and 5e vs
1a). Clearly, this improvement should be related to the insertion of
tacrine moiety. In AChE bioassay, compound 5e with 6-carbon aliphatic
spacer in the linker showed the best activity with an IC50 value of
0.173 μM, similar to that of tacrine. The SAR study revealed that the
activity increased with elongation of the diamine linker (from 5a to 5e)
and then decreased (from 5e to 5g), suggesting that 6-carnon diamino
was the optimal distance between IAA and tacrine. From the results of
BChE inhibitory assay, 5c–5e exhibited the most potent activity with
IC50 values ranging from 0.057 to 0.076 μM, better than tacrine.
A common feature of numerous ChE inhibitors’ structures is the
presence of basic nitrogen atom which can be protonated to form ca-
tion-π interactions with aromatic amino acid residues in the active
pocket of the enzymes [18,19]. Thus, to obtain the preliminary SAR and
improve ChE inhibitory activity, several analogues of 1a were initially
prepared with different amino side chain or structural similar side chain
as the substitution of -N(iPr)2. The synthetic route for 1a analogues was
depicted in Scheme 1, in which analogues 1a–1p (Table 1) were pre-
pared by coupling IAA and corresponding amines in the presence of 1-
ethyl-3-(3-dimethyllaminopropyl)carbodiimide hydrochloride (EDCI)
and 1-hydroxybenzotriazole (HOBT) [20,21].
Next, another batch of IAA-tacrine hybrids 5a–5g was prepared
according to the synthetic route shown in Scheme 2. Briefly, the
Fig. 2. Structural modifications of IAA.
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BioorganicChemistry83(2019)277–288
Table 1
Table 1 (continued)
Structures and ChE inhibitory activities of 1a–1p and 5a–5g.
Compound
IC50, μM
Compound
IC50, μM
AChE
BChE
0.150
AChE
BChE
98.83
5g
0.353
0.003
0.021
0.021
0.037
1a
6.62
0.51
1.78
1b
1c
> 100a
> 100
> 100
> 100
Tacrine
a
0.158
0.268
1d
> 100
> 100
IC50 value above 100 μM was considered to be inactive.
1e
1f
> 100
> 100
> 100
> 100
2.3. Kinetic study of AChE and BChE inhibition
As 5e displayed potent inhibitory activity toward both AChE and
BChE, it was selected as a representative compound for kinetic assay to
obtain information on the mode of action and binding site. The me-
chanisms of AChE and BChE inhibitions were analyzed by recording
substrate concentration–enzyme velocity curves in the presence of dif-
ferent concentrations of 5e.
1g
1h
> 100
> 100
> 100
> 100
1i
1j
> 100
> 100
> 100
> 100
Graphical analyses revealed both decreasing Vmax and increasing
Kmax at rising concentrations of 5e (Figs. 4 and 5). The equilibrium
constants for binding with the free enzyme (KI) and with the enzyme-
substrate complex (KIS) were obtained from the slope and the 1/Vmax
values plotted against inhibitor concentration, respectively [28]. As
shown in Fig. 4, the values of KI and KIS of 5e were 0.065 and 0.198 μM
for AChE and the estimated KI and KIS values for BChE were 0.047 and
0.055 μM, respectively (Fig. 5). These patterns indicate that compound
5e is a linear-mixed type ChE inhibitor (p < 0.05) and suggest that 5e
may not only compete with the substrate for the CAS (catalytic active
site) but also interact with the second binding site, the PAS (peripheral
anionic site). This was in agreement with the subsequent results from
molecular modeling study of 5e (Fig. 6).
1k
1l
> 100
> 100
> 100
> 100
> 100
> 100
1m
1n
1o
54.05
3.52
> 100
> 100
> 100
2.4. Ligand-protein docking study
1p
5a
> 100
1.665
> 100
0.105
Overall, compound 5e showed the best dual inhibition on AChE and
BChE, and was thus selected in the docking simulation. As shown in
Figs. 6 and 7, 5e was able to bind to the active pocket of both AChE and
BChE. Regarding AChE, the tacrine moiety of 5e could spatially overlap
with that of AA7 in the CAS (Fig. 6A), and showed π-π stacking inter-
actions with Trp84 and Phe330 (Fig. 6B). Furthermore, the protonated
nitrogen atom in tacrine ring underwent cation-π interaction with
Phe330 and formed H-bond with the key residue His440. Although the
indole moiety had different orientation at the other end of AA7 in the
PAS (Fig. 6A), it could form π-π stacking interaction with key residue
Trp279 and H-bond interaction with Arg289 (Fig. 6B). It was note-
worthy that the indole moiety of 5e and 1a showed opposite spatial
direction in the proposed binding modes. In addition, hydrophobic in-
teractions between 5e and residues also contributed to the affinity of 5e
to the active site of AChE.
0.142
0.023
0.010
0.008
0.012
0.022
0.022
0.051
0.004
0.008
0.003
0.028
5b
5c
5d
5e
5f
0.998
0.395
0.264
0.173
0.376
0.113
0.076
0.057
0.066
0.246
Protein-ligand interactions between 5e and BChE were shown in
Fig. 7. It deserved to mention that the tacrine moiety of 5e was located
in the PAS of BChE [29]. The protonated nitrogen atom of tacrine ring
could form H-Bond interaction with Pro265 and cation-π interaction
with Tyr 332 of the PAS. The indole moiety could form two types of
interactions with key residue Trp82 of the CAS including π-π stacking
and hydrophobic interactions, which also highly contributed to the
stabilization of the complex. Further, to probe the probable reason why
5e displayed more potent inhibition activity than tacrine, their binding
modes with BChE were compared. As shown in Fig. 8, tacrine over-
lapped with the indole part of 5e in the CAS of BChE, which indicated
that 5e had more interactions with BChE than tacrine. In addition, the
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Scheme 2. The general procedure for the synthesis of 5a–5g. (i): Pd/C (wetted with ca. 55% water), H2, methanol, r.t.; (ii): POCl3, cyclohexanone, 105 °C, 4 h; (iii):
2- to 8-carbon alkyl diamines, pentanol, 140 °C, 12 h; (iv): IAA, HATU, DIEA, CH2Cl2, r.t.
2.5. Molecular dynamics simulation and MM-PBSA calculation
Table 2
Hydrogen bonds existent in the two complexes and their occupancies during the
100 ns MD simulation.
To further probe the molecular mechanism that 5e showed stronger
activity against AChE than 1a, 100 ns molecular dynamics (MD) si-
mulation and Molecular Mechanics/Poisson-Boltzmann Surface Area
(MM-PBSA) calculation were conducted. The time evolution of
weighted root-mean-square deviations (RMSDs) for backbone atoms of
the AChE protein from their initial positions (t = 0) were calculated. As
shown in Fig. 9, RMSDs values of the two complexes were found to be
between 0.75 and 1.75 Å in all simulations. Steady RMSD values for the
heavy atoms of the protein indicated well-equilibrated states of the
systems during MD simulations and showed the reliability of these MD
trajectories, suggesting the suitability for post analysis.
Inhibitors
H-bond Donor
H-bond Acceptor
Occupancy Ratea
1a
Tyr121:OH
1a:N3
1a:O1
92.82%
60.46%
Tyr70:O
5e
Tyr121:OH
5e:O1
41.72%
99.74%
64.32%
5e:N3
Glu199:OE2
Glu278:OE1
5e:N4
a
Only H-bond occupancies > 40% are shown.
H-bonds and hydrophobic interactions in both complexes were
analyzed based on MD trajectories (Tables 2 and 3). In the 1a/AChE
complex, two conserved H-bonds between key residues (Tyr121 and
Tyr70) and 1a in the PAS were revealed by the high H-bond occupancy
rates (92.82% and 60.46%). In the case of 5e/AChE complex, there
were three conserved H-bonds formed between AChE and 5e with a
particularly strong one (99.74%) arising from key residue Gly199 in the
CAS. As shown in Table 3, 5e also had more hydrophobic interactions
with AChE than 1a. Therefore, it was proposed that 5e could have more
potent binding affinity than 1a based on the H-bond and hydrophobic
analyses, which were in good agreement with the experimental results
that 5e displayed better inhibitory activity against AChE than 1a. Then
MM-PBSA calculation was performed to obtain the free binding energy,
and the results were shown in Table 4. Taken together, it was confirmed
that 5e did have more potent binding affinity with AChE than 1a.
Fig. 3. The proposed binding mode of 1a (carbon in blue) to AChE. PDB code
5EI5 was used and shown as cartoon, with ligand AA7 as stick in purple color.
2.6. Prediction of pharmacokinetic properties
docking score for 5e and tacrine is −9.30 and −8.50, respectively,
which further confirmed the tighter binding of 5e. Thus, we proposed
that the potent inhibition of 5e resulted from the more tight binding
with BChE.
From the data in Table 5, the MW (< 500 Da) and PSA-2D value
(< 108 Å2) of each compound fell into the range of marketed CNS drugs
[14,30]. Also, most of them such as 5a–5f were predicted to have high
lipophilicity, acceptable solubility and absorption level, and 5a–5c
Fig. 4. Kinetic assay on AChE inhibition displayed by 5e. (A) Lineweaver-Burk reciprocal plots of initial velocity and increasing substrate concentrations
(0.078–0.625 mM); (B) Secondary plot of slopes versus 5e concentrations; (C) Secondary plot of 1/Vmax versus 5e concentrations.
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BioorganicChemistry83(2019)277–288
Fig. 5. Kinetic assay on BChE inhibition displayed by 5e. (A) Lineweaver-Burk reciprocal plots of initial velocity and increasing substrate concentrations
(0.078–0.625 mM); (B) Secondary plot of slopes versus 5e concentrations; (C) Secondary plot of 1/Vmax versus 5e concentrations.
displayed moderate BBB penetration level. Importantly, all of them may
not bind to PPB and CYP2D6, which possibly indicated fewer side ef-
fects of this series of compounds.
recording continued for another 2 h. 5e caused significant increase in
the frequency (n = 6; p < 0.05; paired t-test), a dramatic decrease in
the duration of each burst, and a moderate decrease in the overall ac-
tivity of the network quantified by the average GCaMP6 intensity
during the recording (Fig. 10B and C). Nevertheless, 5e induced mixed
changes in the peak of the burst firing (Fig. 10C). Similar effects can be
induced using tacrine (n = 6), but with a much higher concentration
(1 µM, Fig. 10D and E). These results indicate that 5e exhibits similar
effects to tacrine in modulating network activity, but most likely with
much stronger efficacy.
2.7. Effect on mouse brain neural network electrical activity study
Synchronized oscillatory activity of the brain represents a core
mechanism for communications within cortical and subcortical net-
works [31], and the progression of AD is associated with abnormal
network rhythmic activities in the brain [32]. Suppression of long-term
fluctuations and enhancement of the frequency of the neural electrical
activity have been shown to increase the ability of neurons to respond
to external information [33]. In addition, cognition-enhancing drugs
elicit similar modulation on network activities [34]. To test the po-
tential effects of 5e on neural network activity, we generated in vitro
mouse brain neural network using primary hippocampal neurons
(Fig. 10A). Co-cultured neuron/glia cells in vitro develop both in-
hibitory and excitatory connections and elicit oscillatory activities
(synchronized periodic bursting) [35]. To monitor the network activity,
we infected the cultures with lentivirus that specifically expressed
GCaMP6 in neurons under a human synapsin I promoter. GCaMP6 is an
ultrasensitive calcium indicator that can get brighter when binding with
calcium [36]. Since the firing activity of a neuron is accompanied by
calcium influx, GCaMP6 can thus be used to report the firing activity of
the neurons. As a result, the cultured neural network will blink under
fluorescent microscope during synchronized network oscillation
(Fig. 10B and D).
3. Conclusion
In summary, a series of novel IAA-tarcine hybrids were prepared
based on the analysis of binding mode of lead 1a with AChE, and their
dual AChE/BChE inhibitory activities were then evaluated. Several
hybrids, e.g., 5d and 5e, displayed potent dual activities against AChE/
BChE with IC50 values at low nanomolar level. The preliminary SAR
analysis indicated that the optimal distance for a diamino linker be-
tween IAA and tacrine should be a 6-carbon chain length in AChE
bioassay. In addition, 5e exhibits much stronger effect in modulating
neural network activity compared to tacrine, indicating better anti-
dementia and nootropic potentials. In the present study, the biological
evaluation together with computational analyses demonstrate that
these IAA-tacrine hybrids may serve as a new template for developing
multifunctional anti-AD drugs. Further structural modifications to im-
prove drug-like properties are in progress and will be reported in due
course.
We first tested the effect of 5e on the network activity. After a stable
baseline recording of 1 h, 50 nM 5e was added into the medium and the
Fig. 6. (A) The proposed orientation of 5e (carbon in blue) in the active of AChE (PDB: 5EI5) with AA7 in purple color; (B) The binding mode of 5e in the active site
of AChE.
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BioorganicChemistry83(2019)277–288
Fig. 7. (A) The proposed orientation of 5e (carbon in blue) in the active of BChE (PDB: 4BDS) active site; (B) The binding mode of 5e in the active site of BChE.
Table 3
Residues involved in hydrophobic interactions (occupancies > 50%) with
the inhibitors during the simulation time and the corresponding occupancy
rates.
Residues
1a
5e
Tyr70
96%
86%
89%
90%
55%
0.0%
0.0%
100%
26%
36%
94%
72%
3%
Asp72
Trp84
100%
90%
83%
64%
67%
52%
50%
95%
93%
Tyr121
Ser122
Gly123
Glu199
Trp279
Phe290
Phe330
Tyr334
Fig. 8. The binding modes of 5e (carbon in blue) and tacrine (carbon in yellow)
generated by molecular docking.
Table 4
The binding free energy of AChE to 1a and 5e.a
4. Experimental section
Inhibitors
ΔGgas
ΔGsolv
ΔG
1a
5e
−222.94
−248.78
195.84
200.58
−27.10
−48.20
4.1. Chemistry
a
4.1.1. General methods
All calculated values obtain by MM-PBSA method were given in kcal/mol.
Melting points were measured by a Melting Point YRY-3 apparatus
(Tianjin Precision Apparatus Factory, China). Commercially available
reagents were used without further purification. Organic solvents were
ΔGgas represents the binding free energy in vacuum; ΔGsolv represents the sol-
vation free energy change; ΔG = ΔGgas + ΔGsolv
.
Fig. 9. Time dependencies of RMSDs for the backbone atoms of the 1a/AChE and 5e/AChE complexes.
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BioorganicChemistry83(2019)277–288
Table 5
Prediction of pharmacokinetic properties of 5a–5g.
No.
MW
PSA-2D
AlogP98
Solubility level
Absorption level
BBB level
PPB
CYP2D6
5a
5b
5c
5d
5e
5f
398
412
426
440
453
468
481
69.237
69.237
69.237
69.237
57.976
69.237
57.976
4.53
2
2
1
1
1
1
1
0
0
0
1
1
2
3
1
1
1
4
4
4
4
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
FALSE
4.592
5.172
5.628
6.8
6.541
7.712
5g
PSA-2D: Polar surface area; AlogP98: Lipophilicity descriptor; Solubility Level: (0, Good; 1, Moderate; 2, Poor; 3, Very poor); Absorption Level: (0, Good; 1,
Moderate; 2, Poor; 3, Very poor); BBB Level: (0, very high blood-brain barrier penetration; 1, high; 2, medium; 3, low; 4, undefined).
evaporated with reduced pressure using a Büchi R-100 evaporator.
Reactions were monitored by TLC using Yantai Jingyou (China) GF254
silica gel plates. Silica gel column chromatography was performed on
silica gel (200–300 mesh) from Qingdao Hailang (China). The purity of
the samples was determined by an analytical Agilent 1260 HPLC with
ZDRBAX SB-C18 column (4.6 mm × 150 mm) using parameters as fol-
lows: H2O/MeOH, 20/80 to 0/100 in 15 min, plus 15 min isocratic
MeOH, flow rate at 1.0 mL/min, λ = 254 nm. NMR spectra were mea-
sured on Bruker Avance III 600 and 400 MHz spectrometers. Chemical
shifts were expressed in δ (ppm) and coupling constants (J) in Hz with
solvent signals as internal standards (CDCl3, δH 7.26 ppm and δC
77.0 ppm; CD3OD, δH 3.31 ppm and δC 49.0 ppm; DMSO‑d6, δH
2.50 ppm and δC 39.5 ppm). ESI-MS analyses were performed on an
Agilent 1260–6460 Triple Quard LC-MS instrument. HR-ESI-MS data
were acquired on an Agilent Q-TOF 6520.
4.1.2. General procedures for the preparation of 1a–1p
The mixtures of IAA (175 mg, 1 mmol), HOBT (153 mg, 1 mmol),
EDCI (170 mg, 1.1 mmol), DIEA (260 μL, 1.5 mmol), and the corre-
sponding amines (1.5 mmol) in 5 mL dichloromethane were stirred at
room temperature for 12 h. The reaction solvent was removed under
reduced pressure, and the obtained residues were purified on silica gel
columns eluted with CH2Cl2-MeOH to yield compounds 1a–1p.
4.1.2.1. N-(2-(Diisopropylamino)ethyl)-2-(1H-indol-3-yl)acetamide
(1a). Yellow solid in 92.2% yield, mp 84.0–85.3 °C. HPLC purity:
97.1% yield, tR = 8.04 min. 1H NMR (600 MHz, CD3OD) δ 7.48 (d,
J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.18 (s, 1H), 7.11 (dd,
J = 8.1, 7.6 Hz, 1H), 7.02 (dd, J = 7.9, 7.6 Hz, 1H), 3.67 (s, 2H), 3.12
(t, J = 6.7 Hz, 2H), 2.85–2.78 (m, 2H), 2.45 (t, J = 6.7 Hz, 2H), 0.80 (d,
J = 7.6 Hz, 12H). 13C NMR (150 MHz, CD3OD) δ 174.9, 138.3, 128.5,
125.3, 122.8, 120.2, 119.2, 112.5, 108.8, 49.9, 44.4, 40.2, 33.9, 20.6.
ESI-MS m/z 302.2 [M+H]+
.
HR-ESIMS: [M+H]+ calcd for
C
18H28N3O+ 302.2227, found 302.2228.
4.1.2.2. 2-(1H-Indol-3-yl)acetamide (1b). White solid in 25% yield, mp
158.5–160.6 °C. HPLC purity: 98.7% yield, tR = 9.89 min. 1H NMR
(600 MHz, CD3OD) δ 7.54 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H),
7.18 (s, 1H), 7.10 (dd, J = 8.1, 7.5 Hz, 1H), 7.02 (dd, J = 7.9, 7.5 Hz,
1H), 3.65 (s, 2H). 13C NMR (150 MHz, CD3OD) δ 178.0, 138.1, 128.5,
124.9, 122.5, 119.9, 119.3, 112.2, 109.5, 33.5. ESI-MS m/z 175.4 [M
+H]+. HR-ESIMS: [M+H]+ calcd for C10H11N2O+ 175.0866, found
175.0865.
Fig. 10. Influence of 5e and tacrine on neuronal network activity. (A)
Schematic illustration of the experiment. Hippocampal neurons and glia were
dissected on day 0 and co-cultured in a dish. Cultures were infected on day 5
using lentivirus and images collected on day 15. (B) Representative time-course
plot of the average activity of a neuronal network before and after 50 nM 5e
application. Images of denoted time points are shown on the top of the panel;
(C) Quantification of parameters. Each dot represents an averaged result of a
neural network. (D) and (E) same to (B) and (C) but showing effects of 1 µM
tacrine. **p < 0.01, *p < 0.05, ns non-significant, paired t-test.
4.1.2.3. N-(2-(Dimethylamino)ethyl)-2-(1H-indol-3-yl)acetamide
(1c). Yellow solid in 86.5% yield, mp 98.2–100.4 °C. HPLC purity:
98.1% yield, tR = 5.83 min. 1H NMR (600 MHz, CD3OD) δ 7.54 (d,
J = 8.0 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.20 (s, 1H), 7.12 (dd,
J = 8.1, 7.0 Hz, 1H), 7.03 (dd, J = 8.0, 7.0 Hz, 1H), 3.68 (s, 2H), 3.32
(t, J = 6.7 Hz, 2H), 2.44 (t, J = 6.7 Hz, 2H), 2.24 (s, 6H). 13C NMR
(150 MHz, CD3OD) δ 175.1, 138.2, 128.5, 125.0, 122.6, 120.0, 119.3,
112.3, 109.3, 59.1, 45.3, 38.0, 34.0. ESI-MS m/z 246.2 [M+H]+. HR-
283

Z.-Q. Cheng et al.
BioorganicChemistry83(2019)277–288
4.1.2.10. N-(2,2-Diethoxyethyl)-2-(1H-indol-3-yl)acetamide
ESIMS: [M+H]+ calcd for C14H20N3O+ 246.1601, found 246.1609.
(1j). Yellow oil in 40.4% yield. HPLC purity: 95.7% yield,
tR = 13.97 min. 1H NMR (600 MHz, CDCl3) δ 8.37 (brs, 1H), 7.55 (d,
J = 7.9 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.22 (dd, J = 8.1, 7.4 Hz,
1H), 7.14 (dd, J = 7.9, 7.4 Hz, 1H), 7.12 (s, 1H), 5.91 (brs, 1H, NH),
4.35 (t, J = 5.6 Hz, 1H), 3.74 (s, 2H), 3.58–3.52 (m, 2H), 3.33 (q,
J = 7.0 Hz, 4H), 0.99 (t, J = 7.0 Hz, 6H). 13C NMR (150 MHz, CDCl3) δ
171.7, 136.6, 127.1, 123.8, 122.8, 120.2, 118.8, 111.5, 109.1, 101.0,
63.1, 42.1, 33.5, 15.2. ESI-MS m/z 313.1 [M+Na]+. HR-ESIMS: [M
4.1.2.4. N-(2-(Diethylamino)ethyl)-2-(1H-indol-3-yl)acetamide
(1d). Yellow oil in 68.0% yield. HPLC purity: 98.7% yield,
tR = 7.51 min. 1H NMR (600 MHz, CD3OD) δ 7.51 (d, J = 8.0 Hz,
1H), 7.35 (d, J = 7.5 Hz, 1H), 7.18 (s, 1H), 7.11 (dd, J = 7.5, 7.0 Hz,
1H), 7.02 (dd, J = 8.0, 7.0 Hz, 1H), 3.66 (s, 2H), 3.24 (t, J = 6.7 Hz,
2H), 2.48 (t, J = 6.7 Hz, 2H), 2.44 (q, J = 7.2 Hz, 4H), 0.89 (t,
J = 7.2 Hz 6H). 13C NMR (150 MHz, CD3OD) δ 174.9, 138.2, 128.5,
125.1, 122.6, 120.0, 119.3, 112.4, 109.1, 52.2, 47.9, 37.8, 33.9, 11.6.
+
+Na]+ calcd for C16H22N2NaO3 313.1523, found 313.1527.
ESI-MS m/z 274.7 [M+H]+
.
HR-ESIMS: [M+H]+ calcd for
C16H24N3O+ 274.1914, found 274.1915.
4.1.2.11. 2-(1H-Indol-3-yl)-N-(2-(methylsulfonyl)ethyl)acetamide
(1k). Yellow oil in 54.2% yield. HPLC purity: 95.4% yield,
tR = 10.24 min. 1H NMR (600 MHz, CD3OD) δ 7.53 (d, J = 7.9 Hz,
1H), 7.35 (d, J = 8.2 Hz, 1H), 7.18 (s, 1H), 7.11 (dd, J = 8.2, 7.0 Hz,
1H), 7.02 (dd, J = 7.9, 7.0 Hz, 1H), 3.66 (s, 2H), 3.60 (t, J = 6.6 Hz,
2H), 3.20 (t, J = 6.6 Hz, 2H), 2.79 (s, 3H). 13C NMR (150 MHz, CD3OD)
δ 175.3, 138.1, 128.5, 125.1, 122.7, 120.1, 119.3, 112.4, 109.0, 54.3,
41.1, 34.6, 33.9. ESI-MS m/z 303.0 [M+Na]+. HR-ESIMS: [M+H]+
calcd for C13H17N2O3S+ 281.0954, found 281.0960.
4.1.2.5. N-(2-(Ethylamino)ethyl)-2-(1H-indol-3-yl)acetamide
(1e). Yellow oil in 82.6% yield. HPLC purity: 95.5% yield,
tR = 14.64 min. 1H NMR (600 MHz, CDCl3) δ 8.41 (brs, 1H, NH),
7.30 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 8.1 Hz, 1H), 7.12 (dd, J = 8.1,
5.4 Hz, 1H), 7.10 (dd, J = 8.1, 5.4 Hz, 1H), 6.65 (s, 1H), 6.44 (t,
J = 4.5 Hz, 1H), 3.57 (s, 2H), 3.38 (t, J = 5.3 Hz, 2H), 3.40–3.36 (m,
2H), 3.17 (q, J = 7.1 Hz, 2H), 1.05 (t, J = 7.1 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 172.4, 136.5, 127.2, 124.2, 122.7, 119.7, 118.8,
111.6, 109.1, 53.6, 44.2, 39.2, 33.4, 14.2. ESI-MS m/z 246.1 [M+H]+
.
4.1.2.12. Methyl 5-(2-(1H-indol-3-yl)acetamido)pentanoate (1l). Colorless oil
in 50.8% yield. HPLC purity: 96.2% yield, tR = 13.20 min. 1H NMR
(600 MHz, CDCl3) δ 8.75 (brs, 1H, NH), 7.54 (d, J = 7.9 Hz, 1H), 7.40 (d,
J = 8.2 Hz, 1H), 7.22 (dd, J = 8.2, 7.1 Hz, 1H), 7.13 (dd, J = 7.9, 7.1 Hz,
1H), 7.11 (d, J = 2.3 Hz, 1H), 5.81 (brs, 1H, NH), 3.73 (s, 2H), 3.62 (s, 3H),
3.21–3.14 (m, 2H), 2.24 (t, J = 7.4 Hz, 2H), 1.53–1.47 (m, 2H), 1.43–1,36
(m, 2H). 13C NMR (150 MHz, CDCl3) δ 174.0, 171.8, 136.6, 127.1, 124.0,
122.6, 120.1, 118.7, 111.6, 108.8, 51.6, 39.1, 33.5, 29.0, 22.1. ESI-MS m/z
HR-ESIMS: [M+H]+ calcd for C14H20N3O+ 246.1601, found 246.1611.
4.1.2.6. Tert-butyl
(2-(2-(1H-indol-3-yl)acetamido)ethyl)carbamate
(1f). White solid in 95.2% yield, mp 138.5–141.2 °C. HPLC purity:
96.1% yield, tR = 14.36 min. 1H NMR (600 MHz, CD3OD) δ 7.52 (d,
J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.17 (s, 1H), 7.10 (dd,
J = 8.1, 7.2 Hz, 1H), 7.02 (dd, J = 7.9, 7.2 Hz, 1H), 3.65 (s, 2H), 3.23
(t, J = 6.1 Hz, 2H), 3.10 (t, J = 6.1 Hz, 2H), 1.40 (s, 9H). 13C NMR
(150 MHz, CD3OD) δ 175.3, 158.5, 138.2, 128.5, 125.0, 122.6, 119.8,
119.3, 112.4, 109.2, 80.1, 40.9, 40.6, 34.0, 28.4. ESI-MS m/z 340.1 [M
289.1 [M+H]+. HR-ESIMS: [M+H]+ calcd for C16H21N2O3 289.1547,
+
found 289.1540.
+Na]+. HR-ESIMS: [M+Na]+ calcd for C17H23N3NaO3 340.1632,
4.1.2.13. Tert-butyl
(5-(2-(1H-indol-3-yl)acetamido)pentyl)carbamate
+
found 340.1633.
(1m). Yellow oil in 69.6% yield. HPLC purity: 97.4%,
tR = 15.37 min. 1H NMR (600 MHz, CDCl3) δ 9.00 (s, 1H), 7.52 (d,
J = 7.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.21 (dd, J = 8.0, 7.4 Hz,
1H), 7.12 (dd, J = 7.8, 7.4 Hz, 1H), 7.10 (d, J = 2.3 Hz, 1H), 5.80 (t,
J = 5.7 Hz, 1H, NH), 4.57 (brs, 1H, NH), 3.72 (s, 2H), 3.17–3.11 (m,
2H), 3.03–2.92 (m, 2H), 1.44 (s, 9H), 1.37–1.30 (m, 4H), 1.16–1.09 (m,
2H). 13C NMR (150 MHz, CDCl3) δ 171.8, 156.2, 136.6, 127.1, 124.1,
122.5, 119.9, 118.7, 111.7, 108.8, 79.2, 40.4, 39.3, 33.5, 29.6, 29.1,
28.5, 23.9. ESI-MS m/z 394.1 [M + Cl]−. HR-ESIMS: [M+Na]+ calcd
4.1.2.7. 2-(1H-indol-3-yl)-N-(2-(pyrrolidin-1-yl)ethyl)acetamide
(1g). White solid in 86.2% yield, mp 106.5–107.4 °C. HPLC purity:
98.6% yield, tR = 8.66 min. 1H NMR (600 MHz, CDCl3) δ 8.83 (brs, 1H,
NH), 7.55 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.20 (dd,
J = 8.1, 7.2 Hz, 1H), 7.12 (dd, J = 7.9, 7.1 Hz, 1H), 6.97 (d, J = 2.1 Hz,
1H), 6.28 (s, 1H), 3.70 (s, 2H), 3.34–3.29 (m, 2H), 2.49 (t, J = 6.2 Hz,
2H), 2.37–2.32 (m, 4H), 1.62–1.55 (m, 4H). 13C NMR (150 MHz, CDCl3)
δ 171.9, 136.6, 127.2, 123.9, 122.4, 119.8, 118.8, 111.5, 109.0, 54.4,
53.8, 38.3, 33.6, 23.5. ESI-MS m/z 272.2 [M+H]+. HR-ESIMS: [M
+H]+ calcd for C16H22N3O+ 272.1757, found 272.1755.
+
for C20H29N3NaO3 382.2101, found 382.2100.
4.1.2.14. 5-(2-(1H-indol-3-yl)acetamido)-N-(2-(diisopropylamino)ethyl)
pentanamide (1n). Colorless oil in 71.4% yield. HPLC purity: 99.0%,
tR = 8.25 min. 1H NMR (600 MHz, CDCl3) δ 9.19 (brs, 1H, NH), 7.51 (d,
J = 7.7 Hz, 1H), 7.37 (d, J = 8.3 Hz, 1H), 7.18 (dd, J = 7.7, 7.3 Hz,
1H), 7.09 (dd, J = 8.3, 7.3 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 6.24 (brs,
1H, NH), 5.94 (t, J = 5.7 Hz, 1H, NH), 3.69 (s, 2H), 3.20–3.16 (m, 2H),
3.14 (q, J = 6.7 Hz, 2H), 3.02–2.96(m, 2H), 2.54 (t, J = 6.1 Hz, 2H),
2.09 (t, J = 7.5 Hz, 2H), 1.54–1.47 (m, 2H), 1.41–1.34 (m, 2H), 0.99 (d,
J = 6.7 Hz, 12H). 13C NMR (150 MHz, CDCl3) δ 172.7, 171.9, 136.6,
127.1, 124.1, 122.3, 119.8, 118.6, 111.6, 108.6, 57.0, 45.6, 39.1, 38.2,
36.0, 33.5, 29.0, 22.8, 20.7. ESI-MS m/z 401.3 [M+H]+. HR-ESIMS:
4.1.2.8. 1. 2-(1H-Indol-3-yl)-N-(2-morpholinoethyl)acetamide (1h). Yellow
solid in 83.0% yield, mp 108.5–110.4 °C. HPLC purity: 97.1% yield,
tR = 6.56 min. 1H NMR (600 MHz, CD3OD) δ 7.51 (d, J = 7.9 Hz, 1H),
7.37 (d, J = 8.2 Hz, 1H), 7.21 (s, 1H), 7.12 (dd, J = 8.2, 7.4 Hz, 1H), 7.02
(dd, J = 7.9, 7.4 Hz, 1H), 3.67 (s, 2H), 3.43–3.37 (m, 4H), 3.26 (t,
J = 6.3 Hz, 2H), 2.35 (t, J = 6.3 Hz, 2H), 2.28–2.22 (m, 4H). 13C NMR
(150 MHz, CD3OD) δ 174.8, 138.3, 128.5, 125.3, 122.7, 120.1, 119.3,
112.5, 109.2, 67.7, 57.8, 54.2, 37.0, 34.0. ESI-MS m/z 288.2 [M+H]+
.
+
HR-ESIMS: [M+H]+ calcd for C16H22N3O2 288.1707, found 288.1704.
[M+H]+ calcd for C23H37N4O2 401.2911, found 401.2914.
+
4.1.2.9. N-(2,2-Dimethoxyethyl)-2-(1H-indol-3-yl)acetamide (1i). White
solid in 59.8% yield, mp 58.5–60.0 °C. HPLC purity: 97.2% yield,
tR = 12.13 min. 1H NMR (600 MHz, CDCl3) δ 8.39 (brs, 1H, NH), 7.55
(d, J = 7.9 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.22 (dd, J = 8.2, 7.0 Hz,
1H), 7.14 (dd, J = 7.9, 7.0 Hz, 1H), 7.12 (s, 1H), 5.88 (brs, 1H, NH),
4.26 (t, J = 5.5 Hz, 1H), 3.74 (s, 2H), 3.33 (dd, J = 5.8, 5.5 Hz, 2H),
3.24 (s, 6H). 13C NMR (150 MHz, CDCl3) δ 171.8, 136.5, 127.0, 123.9,
122.7, 120.1, 118.9, 111.5, 109.0, 102.9, 54.6, 41.2, 33.6. ESI-MS m/z
4.1.2.15. 5-(2-(1H-indol-3-yl)acetamido)-N-(2,2-dimethoxyethyl)
pentanamide (1o). Yellow oil in 92.4% yield. HPLC purity: 98.2%,
tR = 12.09 min. 1H NMR (600 MHz, CDCl3) δ 9.02 (brs, 1H, NH), 7.52
(d, J = 7.9 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.19 (dd, J = 8.2, 7.1 Hz,
1H), 7.11 (dd, J = 7.9, 7.1 Hz, 1H), 7.08 (d, J = 2.3 Hz, 1H), 5.92 (t,
J = 5.6 Hz, 1H, NH), 5.87 (t, J = 5.6 Hz, 1H, NH), 4.34 (t, J = 5.2 Hz,
1H,), 3.70 (s, 2H), 3.37–3.33 (m, 8H), 3.17–3.12 (m, 2H), 2.09 (t,
J = 7.5 Hz, 2H), 1.52–1.46 (m, 2H), 1.40–1.34 (m, 2H). 13C NMR
(150 MHz, CDCl3) δ 173.1, 171.9, 136.6, 127.1, 124.1, 122.5, 119.9,
285.1 [M+Na]+. HR-ESIMS: [M+Na]+ calcd for C14H18N2NaO3
+
285.1210, found 285.1216.
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Z.-Q. Cheng et al.
BioorganicChemistry83(2019)277–288
118.7, 111.6, 108.7, 102.6, 54.4, 40.9, 38.9, 35.8, 33.5, 28.9, 22.7. ESI-
4.1.5.3. N1-(1,2,3,4-Tetrahydroacridin-9-yl)butane-1,4-diamine
MS m/z 396.1 [M + Cl]−
.
HR-ESIMS: [M+Na]+ calcd for
(4c). Brown oil in 21.6% yield. 1H NMR (600 MHz, CDCl3) δ 7.98 (d,
J = 8.5 Hz, 2H), 7.96 (d, J = 8.5 Hz, 1H), 7.58 (dd, J = 8.5, 7.0 Hz,
1H), 7.37 (dd, J = 8.5, 7.0 Hz, 1H), 3.62 (t, J = 7.1 Hz, 2H), 3.05 (brs,
2H), 2.77 (t, J = 7.0 Hz, 2H), 2.67 (brs, 2H), 1.93–1.87 (m, 4H),
1.77–1.71 (m, 2H), 1.63–1.56 (m, 2H). 13C NMR (150 MHz, CDCl3) δ
157.2, 152.2, 145.5, 129.6, 126.4, 124.2, 123.2, 119.2, 114.9, 49.2,
+
C
19H27N3NaO4 384.1894, found 384.1883.
4.1.2.16. 5-(2-(1H-indol-3-yl)acetamido)-N-(2,2-diethoxyethyl)
pentanamide (1q). Colorless oil in 62.3% yield. HPLC purity: 99.4%,
tR = 13.71 min. 1H NMR (600 MHz, CDCl3) δ 9.08 (brs, 1H, NH), 7.51
(d, J = 8.3 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.19 (dd, J = 8.1, 7.2 Hz,
1H), 7.10 (dd, J = 8.3, 7.1 Hz, 1H), 7.07 (d, J = 2.3 Hz, 1H), 5.93 (t,
J = 5.8 Hz, 1H, NH), 5.87 (t, J = 5.5 Hz, 1H, NH), 4.47 (t, J = 5.2 Hz,
1H), 3.71–3.65 (m, 4H), 3.54–3.48 (m, 2H), 3.34 (t, J = 5.5 Hz, 2H),
3.14 (q, J = 7.1 Hz,2H), 2.08 (t, J = 7.5 Hz, 2H), 1.52–1.45 (m, 2H),
1.40–1.33 (m, 2H), 1.19 (t, J = 7.1 Hz, 6H). 13C NMR (150 MHz,
CDCl3) δ 173.1, 172.0, 136.6, 127.1, 124.1, 122.4, 119.9, 118.6,
111.6, 108.7, 100.8, 62.9, 41.9, 38.9, 35.8, 33.5, 28.9, 22.7, 15.4.
41.4, 32.3, 30.2, 29.1, 24.7, 22.8, 22.3. ESI-MS m/z 270.2 [M+H]+
.
4.1.5.4. N1-(1,2,3,4-Tetrahydroacridin-9-yl)pentane-1,5-diamine
(4d). Brown oil in 76.3% yield. 1H NMR (600 MHz, CDCl3) δ 7.94 (d,
J = 8.4 Hz, 1H), 7.90 (d, J = 8.5 Hz, 1H), 7.54 (dd, J = 8.5, 7.0 Hz,
1H), 7.34 (dd, J = 8.4, 7.0 Hz, 1H), 3.49 (t, J = 7.2 Hz, 2H), 3.06 (brs,
2H), 2.70 (brs, 2H), 2.69 (t, J = 7.7, 2H), 1.93–1.89 (m, 4H), 1.70–1.64
(m, 2H), 1.51–1.45 (m, 2H), 1.45–1.40 (m, 2H). 13C NMR (150 MHz,
CDCl3) δ 158.5, 150.9, 147.5, 128.8, 128.5, 123.8, 122.9, 120.3, 116.0,
49.6, 42.1, 34.1, 31.8, 24.9, 24.4, 23.2, 23.2, 22.9. ESI-MS m/z 284.2
ESI-MS m/z 390.1 [M+H]+
.
HR-ESIMS: [M+Na]+ calcd for
+
C
21H31N3NaO4 412.2207, found 412.2210.
[M+H]+
.
4.1.3. 2-Aminobenzoic acid (2)
4.1.5.5. N1-(1,2,3,4-Tetrahydroacridin-9-yl)hexane-1,6-diamine
2-Nitrobenzoic acid (3.006 g, 18 mmol) and Pd/C (954 mg, 9 mmol)
were added into 20 mL methanol under hydrogen, and the mixture was
stirred at room temperature for 12 h. The solution was filtered to re-
move the catalyst, and then the filtrate was concentrated under reduced
pressure to give 2-aminobenzoic acid 2. This compound was used in
next step without further purification.
(4e). Brown oil in 76.4% yield. 1H NMR (600 MHz, CDCl3) δ 7.95 (d,
J = 8.4 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.55 (dd, J = 8.5, 7.9 Hz,
1H), 7.34 (dd, J = 8.4, 7.9 Hz, 1H), 3.49 (t, J = 7.2 Hz, 2H), 3.06 (brs,
2H), 2.70 (brs, 2H), 2.68 (t, J = 7.0 Hz, 2H), 1.95–1.88 (m, 4H),
1.71–1.60 (m, 2H), 1.48–1.43 (m, 2H), 1.43–1.38 (m, 2H), 1.38–1.33
(m, 2H). 13C NMR (150 MHz, CDCl3) δ 158.4, 151.0, 147.3, 128.6,
128.5, 123.8, 123.0, 120.2, 115.9, 49.6, 42.1, 34.0, 33.4, 31.9, 26.9,
4.1.4. 9-Chloro-1,2,3,4-tetrahydroacridine(3)
26.8, 24.9, 23.2, 22.9. ESI-MS m/z 298.2 [M+H]+
.
Cyclohexanone (1.88 g, 19.2 mmol) was added to a solution of
compound 2 (2.20 g, 16 mmol) in 15 mL phosphorus oxychloride. The
reaction mixture was stirred at 100 °C for 4 h, and then diluted in ethyl
acetate, washed with potassium carbonate solution. The organic phase
was dried over sodium sulphate. The product 9-chloroacridine (3) was
purified by flash column chromatography using mixtures of petroleum
ether-acetone. Yellow solid in 61.0% yield, mp 121.8–122.6 °C. 1H
NMR (600 MHz, CDCl3) δ 8.16 (d, J = 8.3 Hz, 1H), 7.97 (d, J = 8.4 Hz,
1H), 7.66 (dd, J = 8.3, 7.4 Hz, 1H), 7.53 (dd, J = 8.4, 7.4 Hz, 1H), 3.13
(t, J = 5.6 Hz, 2H), 3.02 (t, J = 6.4 Hz, 2H), 1.98–1.91 (m, 4H). 13C
NMR (150 MHz, CDCl3) δ 159.7, 146.8, 141.6, 129.4, 129.0, 128.8,
126.6, 125.5, 123.8, 34.4, 27.7, 22.8, 22.8. ESI-MS m/z 218.2 [M
4.1.5.6. N1-(1,2,3,4-Tetrahydroacridin-9-yl)heptane-1,7-diamine
(4f). Brown oil in 69.1% yield. 1H NMR (600 MHz, CDCl3) δ 7.94 (d,
J = 8.5 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.53 (dd, J = 8.4, 7.3 Hz,
1H), 7.33 (dd, J = 8.5, 7.3 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 3.05 (brs,
2H), 2.69 (brs, 2H), 2.65 (t, J = 7.0 Hz, 2H), 1.92–1.88 (m, 4H),
1.67–1.61 (m, 2H), 1.44–1.40 (m, 2H), 1.39–1.35 (m, 2H), 1.33–1.28
(m, 4H). 13C NMR (150 MHz, CDCl3) δ 158.5, 151.0, 147.5, 128.7,
128.4, 123.7, 123.0, 120.1, 115.9, 49.6, 42.1, 34.0, 33.5, 31.8, 29.3,
27.0, 26.9, 24.9, 23.1, 22.9. ESI-MS m/z 312.2 [M+H]+
.
4.1.5.7. N1-(1,2,3,4-Tetrahydroacridin-9-yl)octane-1,8-diamine
+H]+
.
(4g). Brown oil in 49.1% yield. 1H NMR (600 MHz, CDCl3) δ 7.95 (d,
J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 8.4, 7.7 Hz,
1H), 7.33 (dd, J = 8.4, 7.7 Hz, 1H), 3.47 (t, J = 7.3 Hz, 2H), 3.05 (brs,
2H), 2.70 (brs, 2H), 2.65 (t, J = 7.1 Hz, 2H), 1.93–1.87 (m, 4H),
1.68–1.61 (m, 2H), 1.46–1.4 (m, 4H), 1.32–1.26 (m, 6H). 13C NMR
(150 MHz, CDCl3) δ 158.5, 151.0, 147.5, 128.7, 128.4, 123.7, 123.0,
120.3, 115.9, 49.6, 42.2, 34.1, 33.7, 31.9, 29.5, 29.4, 27.0, 26.9, 24.9,
4.1.5. General procedures for the preparation of 4a–4g
To a solutions of 3 (235 mg, 2 mmol) in 5 mL pentanol were added
the corresponding diamines (6 mmol). The reaction mixtures were
stirred at 140 °C overnight. After cooling to room temperature, the re-
sidues were purified on silica gel columns using CH2Cl2-CH3OH with
0.5% Et3N to give the corresponding intermediates 4a–4g.
23.2, 22.9. ESI-MS m/z 326.2 [M+H]+
.
4.1.5.1. N1-(1,2,3,4-Tetrahydroacridin-9-yl)ethane-1,2-diamine
4.1.6. General procedures for the preparation of 5a–5g
(4a). Brown oil in 33% yield. 1H NMR (600 MHz, CDCl3) δ 8.03 (d,
J = 9.1 Hz, 2H), 8.01 (d, J = 9.8 Hz, 2H), 7.56 (dd, J = 9.8, 7.0 Hz,
1H), 7.35 (dd, J = 9.1, 7.0 Hz, 1H), 3.57 (t, J = 5.8 Hz, 2H), 3.10 (brs,
2H), 2.98 (t, J = 5.8 Hz,2H), 2.74 (brs, 2H), 1.95–1.86 (m, 4H). 13C
NMR (150 MHz, CDCl3) δ 157.4, 151.8, 146.2, 129.1, 127.5, 123.9,
123.2, 119.7, 115.7, 50.6, 42.3, 33.2, 24.7, 23.0, 22.6. ESI-MS m/z
To the mixtures of corresponding diamines 4a–4g (1 mmol) in 5 mL
dichloromethane, IAA (175 mg, 1 mmol), HATU (304 mg, 1.1 mmol),
and DIEA (139 μL, 1.5 mmol) were added. The reaction mixtures were
stirred at room temperature for 12 h and then concentrated under re-
duced pressure. The obtained residues were purified on silica gel col-
umns eluted with CH2Cl2-CH3OH with 0.5% Et3N to give target com-
pounds 5a–5g.
242.6 [M+H]+
.
4.1.5.2. N1-(1,2,3,4-Tetrahydroacridin-9-yl)propane-1,3-diamine
4.1.6.1. 2-(1H-Indol-3-yl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)
ethyl)acetamide (5a). White solid in 43.3% yield, mp 202.1–203.5 °C.
HPLC purity: 98.8%, tR = 12.20 min. 1H NMR (400 MHz, DMSO‑d6) δ
10.96 (brs, 1H, NH), 8.58 (t, J = 5.7 Hz, 1H, NH), 8.41 (d, J = 8.7 Hz,
1H), 7.97 (d, J = 8.1 Hz, 1H), 7.81 (dd, J = 8.1, 7.4 Hz, 1H), 7.49 (dd,
J = 8.7, 7.4 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H),
7.16 (brs, 1H), 6.98 (dd, J = 8.1, 7.3 Hz, 1H), 6.72 (dd, J = 7.9, 7.3 Hz,
1H), 3.99–3.91 (m, 2H), 3.52 (s, 2H), 3.50–3.44 (m, 2H), 2.96–2.88 (m,
(4b). Brown oil in 68.2% yield. 1H NMR (600 MHz, CD3OD) δ 8.07 (d,
J = 8.6 Hz, 1H), 7.71 (t, J = 6.9 Hz, 1H), 7.68 (dd, J = 8.3, 6.9 Hz, 1H),
7.40 (dd, J = 8.6, 8.3 Hz, 1H), 3.93 (t, J = 6.0 Hz, 2H), 2.75 (brs, 2H),
2.41 (brs, 2H), 1.97 (t, J = 6.0 Hz, 2H), 1.81–1.71 (m, 6H). 13C NMR
(150 MHz, CD3OD) δ 156.7, 153.2, 141.7, 132.9, 134.2, 126.1, 125.6,
120.2, 117.4, 52.8, 45.9, 32.6, 30.3, 25.0, 22.9, 22.1. ESI-MS m/z 256.2
[M+H]+
.
285

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BioorganicChemistry83(2019)277–288
2H), 2.46–2.37 (m, 2H), 1.73–1.62 (m, 4H). 13C NMR (100 MHz,
DMSO‑d6) δ 172.6, 155.6, 150.3, 138.1, 136.1, 132.3, 127.0, 125.3,
124.8, 123.9, 120.8, 119.2, 118.4, 118.0, 115.3, 111.3, 111.1, 108.4,
C29H35N4O+ 455.2805, found 455.2803.
4.1.6.6. 2-(1H-Indol-3-yl)-N-(7-((1,2,3,4-tetrahydroacridin-9-yl)amino)
heptyl)acetamide (5f). Yellow oil in 42% yield. mp 74.3–75.4 °C. HPLC
purity: 96.0%, tR = 16.01 min. 1H NMR (600 MHz, DMSO‑d6) δ 10.87
(brs, 1H, NH), 8.12 (d, J = 8.5 Hz, 1H), 7.82 (t, J = 5.5 Hz, 1H, NH),
7.71 (d, J = 8.4 Hz, 1H), 7.53 (dd, J = 8.4, 7.8 Hz, 1H), 7.52 (d,
J = 7.5H, 1H), 7.34 (dd, J = 8.5, 7.8 Hz, 2H), 7.16 (brs, 1H), 7.04
(dd, J = 8.0, 7.3 Hz, 1H), 6.94 (dd, J = 7.5, 7.3 Hz, 1H), 3.47 (s, 2H),
3.42–3.37 (m, 2H), 3.02–2.97 (m, 2H), 2.90 (t, J = 6.1 Hz, 2H), 2.69 (t,
J = 6.2 Hz, 2H), 1.85–1.75 (m, 4H), 1.56–1.49 (m, 2H), 1.37–1.29 (m,
2H), 1.26–1.17 (m, 6H). 13C NMR (150 MHz, DMSO‑d6) δ 170.5, 157.4,
150.7, 146.3, 136.1, 128.2, 127.7, 127.2, 123.7, 123.3, 123.2, 120.9,
119.9, 118.7, 118.2, 115.5, 111.3, 109.0, 47.9, 38.5, 33.2, 32.8, 30.5,
48.6, 39.1, 32.6, 27.9, 23.4, 21.3, 20.2. ESI-MS m/z 399.2 [M+H]+
.
HR-ESIMS: [M+H]+ calcd for C25H27N4O+ 399.2179, found 399.2179.
4.1.6.2. 2-(1H-Indol-3-yl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)
propyl)acetamide (5b). Yellow oil in 62.9% yield. mp 84.2–85.6 °C.
HPLC purity: 96.5%, tR = 13.61 min. 1H NMR (600 MHz, DMSO‑d6) δ
10.88 (brs, 1H, NH), 8.12 (d, J = 8.5 Hz, 1H), 7.98 (t, J = 5.7 Hz, 1H,
NH), 7.72 (d, J = 8.4 Hz, 1H), 7.54 (dd, J = 8.4, 7.0 Hz, 1H), 7.52 (d,
J = 7.6 Hz, 1H), 7.34 (dd, J = 8.5, 7.0 Hz, 1H), 7.33 (d, J = 8.1 Hz,
1H), 7.18 (brs, 1H), 7.04 (dd, J = 8.1, 7.2 Hz, 1H), 6.92 (dd, J = 7.6,
7.2 Hz, 1H), 3.50 (s, 2H), 3.41–3.37 (m, 2H), 3.14–3.10 (m, 2H), 2.90
(t, J = 6.3 Hz, 2H), 2.66 (t, J = 6.3 Hz, 2H), 1.83–1.78 (m, 2H),
1.78–1.73 (m, 2H), 1.70–1.64 (m, 2H). 13C NMR (150 MHz,
DMSO‑d6) δ 171.1, 157.4, 150.6, 146.2, 136.1, 128.3, 127.6, 127.2,
123.8, 123.5, 123.0, 120.9, 119.9, 118.6, 118.3, 115.7, 111.3, 108.8,
45.0, 36.1, 33.1, 32.7, 30.7, 25.0, 22.7, 22.3. ESI-MS m/z 413.2 [M
+H]+. HR-ESIMS: [M+H]+ calcd for C26H29N4O+ 413.2336, found
413.2330.
29.1, 28.5, 26.3, 26.3, 25.0, 22.7, 22.3. ESI-MS m/z 469.2 [M+H]+
.
HR-ESIMS: [M+H]+ calcd for C30H37N4O+ 469.2962, found 469.2965.
4.1.6.7. 2-(1H-Indol-3-yl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)
octyl)acetamide (5g). Yellow solid in 53.2% yield, mp 70.9–71.6 °C.
HPLC purity: 95.4%, tR = 17.16 min. 1H NMR (400 MHz, DMSO‑d6) δ
10.92 (brs, 1H, NH), 8.31 (d, J = 8.6 Hz, 1H), 7.92 (t, J = 5.4 Hz, 1H,
NH), 7.89 (d, J = 8.6 Hz, 1H), 7.74 (dd, J = 8.6, 7.4 Hz, 1H), 7.52 (d,
J = 7.9 Hz, 1H), 7.49 (dd, J = 8.6, 7.4 Hz, 1H), 7.32 (d, J = 8.1 Hz,
1H), 7.16 (brs, 1H), 7.02 (dd, J = 8.1, 7.2 Hz, 1H), 6.92 (dd, J = 7.9,
7.2 Hz, 1H), 3.73–3.65 (m, 2H), 3.47 (s, 2H), 3.04–2.98 (m, 2H),
2.98–2.92 (m, 2H), 2.69–2.60 (m, 2H), 1.86–1.76 (m, 4H), 1.68–1.58
4.1.6.3. 2-(1H-Indol-3-yl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)
butyl)acetamide (5c). Yellow oil in 62.3% yield. mp 98.4–100.2 °C.
HPLC purity: 96.4%, tR = 13.90 min. 1H NMR (600 MHz, DMSO‑d6) δ
10.89 (brs, 1H, NH), 8.29 (d, J = 8.6 Hz, 1H), 7.96 (t, J = 5.6 Hz, 1H,
NH), 7.87 (d, J = 8.3 Hz, 1H), 7.73 (dd, J = 8.3, 7.4 Hz, 1H), 7.51 (d,
J = 7.9 Hz, 1H), 7.47 (dd, J = 8.6, 7.4 Hz, 1H), 7.31 (d, J = 8.1 Hz,
1H), 7.15 (brs, 1H), 7.02 (dd, J = 8.1, 7.2 Hz, 1H), 6.89 (dd, J = 7.9,
7.2 Hz, 1H), 3.71–3.65 (m, 2H), 3.46 (s, 2H), 3.08–3.02 (m, 2H),
2.97–2.93 (m, 2H), 2.67–2.62 (m, 2H), 1.84–1.76 (m, 4H), 1.68–1.61
(m, 2H), 1.48–1.40 (m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ 170.6,
153.9, 152.9, 140.7, 136.1, 131.1, 127.2, 124.5, 124.4, 123.7, 122.0,
120.9, 118.6, 118.2, 117.0, 112.6, 111.3, 108.9, 47.1, 38.2, 32.8, 29.7,
27.6, 26.4, 24.3, 21.9, 21.0. ESI-MS m/z 427.2 [M+H]+. HR-ESIMS:
[M+H]+ calcd for C27H31N4O+ 427.2492, found 427.2491.
(m, 2H), 1.38–1.30 (m, 2H), 1.28–1.23 (m, 2H), 1.22–1.14 (m, 6H). 13
C
NMR (100 MHz, DMSO‑d6) δ 170.5, 154.1, 152.5, 140.3, 136.1, 131.2,
127.2, 124.5, 124.4, 123.7, 121.7, 120.8, 118.7, 118.1, 116.8, 112.4,
111.3, 109.0, 47.4, 38.5, 32.8, 32.2, 30.0, 29.5, 29.1, 28.6, 26.2, 26.0,
24.3, 21.8, 20.9. ESI-MS m/z 483.2 [M+H]+. HR-ESIMS: [M+H]+
calcd for C31H39N4O+ 483.3118, found 483.3116.
4.2. AChE/BChE bioassay
AChE and BChE inhibitory activities of compounds were determined
by using modified Ellman’s method [37,38]. Electric eel AChE, equine
serum BChE, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), phosphate
buffer solution (PBS, pH 8.0), acetylthiocholine (ATC) iodide, and bu-
tyrylthiocholine (BUC) iodide were purchased from Sigma-Aldrich
(Steinheim, Germany). Tacrine was used as positive control. Enzyme
solutions were prepared at 2.0 U/mL in 2 mL aliquots. The assay
medium consisted of 10 μL of enzyme, 40 μL of PBS, 20 μL of 0.01 M
DTNB and 10 μL of tested compound. Assayed solutions of tested
compounds were pre-incubated with corresponding ChE for 5 min. The
reaction was initiated by addition of 20 μL of 0.01 M substrate (ATC or
BUC). The activity was determined by measuring the increase in ab-
sorbance at 410 nm at 37 °C in 2 min intervals using Tecan Spark
multimode microplate reader (Mannedorf, Switzerland). The percen-
tage of inhibition (I) was calculated from the measured data as follows:
I = (Ac − Ai)/Ac × 100%, where Ai and Ac represent the change in the
absorbance in the presence of inhibitor and without inhibitor, respec-
tively.
4.1.6.4. 2-(1H-Indol-3-yl)-N-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)
pentyl)acetamide (5d). Yellow solid in 72.2% yield, mp 65.4–67.6 °C.
HPLC purity: 95.1%, tR = 14.67 min. 1H NMR (600 MHz, DMSO‑d6) δ
10.92 (brs, 1H, NH), 8.37 (d, J = 8.5 Hz, 1H), 8.00–7.94 (m, 2H), 7.82
(dd, J = 8.4, 7.9 Hz, 1H), 7.53 (dd, J = 8.5, 7.9 Hz, 1H), 7.52 (d,
J = 7.7 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.16 (brs, 1H), 7.01(dd,
J = 8.1, 7.1 Hz, 1H), 6.91 (dd, J = 7.7, 7.1 Hz, 1H), 3.80–3.72 (m, 2H),
3.47 (s, 2H), 3.07–3.03 (m, 2H), 3.01–2.97 (m, 2H), 2.67–2.60 (m, 2H),
1.84–1.76 (m, 4H), 1.74–1.66 (m, 2H), 1.44–1.37 (m,2H), 1.32–1.27
(m, 2H). 13C NMR (150 MHz, DMSO‑d6) δ 170.6, 155.4, 150.8, 138.3,
136.1, 132.3, 127.2, 125.0, 124.9, 123.7, 120.8, 119.5, 118.7, 118.1,
115.7, 111.3, 111.2, 109.0, 47.2, 38.3, 32.8, 29.5, 28.7, 28.1, 24.0,
23.4, 21.5, 20.3. ESI-MS m/z 441.2 [M+H]+. HR-ESIMS: [M+H]+
calcd for C28H33N4O+ 441.2649, found 441.2653.
4.1.6.5. 2-(1H-Indol-3-yl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)
hexyl)acetamide (5e). Yellow solid in 62.3% yield, mp 70.5–72.3 °C.
HPLC purity:95.5%, tR = 15.18 min. 1H NMR (600 MHz, DMSO‑d6) δ
10.88 (brs, 1H, NH), 8.16 (d, J = 8.5 Hz, 1H), 7.85 (t, J = 5.5 Hz, 1H,
NH), 7.74 (d, J = 8.4 Hz, 1H), 7.58 (dd, J = 8.4, 7.7 Hz, 1H), 7.52 (d,
J = 7.9 Hz, 1H), 7.37 (dd, J = 8.5, 7.7 Hz, 1H), 7.32 (d, J = 8.1 Hz,
1H), 7.16 (brs, 1H), 7.02 (dd, J = 8.1, 7.6 Hz, 1H), 6.91 (dd, J = 7.9,
7.6 Hz, 1H), 3.47 (s, 2H), 3.46–3.42 (m, 2H), 3.03–2.98 (m, 2H), 2.92
(t, J = 5.8 Hz, 2H), 2.68 (t, J = 5.4 Hz, 2H), 1.85–1.78 (m, 4H),
1.57–1.50 (m, 2H), 1.37–1.31 (m, 2H), 1.28–1.18 (m, 4H). 13C NMR
(150 MHz, DMSO‑d6) δ 170.5, 156.4, 151.4, 145.0, 136.1, 128.8, 127.2,
126.4, 123.7, 123.6, 123.5, 120.9, 119.3, 118.7, 118.2, 114.8, 111.3,
109.0, 47.7, 38.5, 32.8, 32.4, 30.4, 29.1, 26.1, 26.0, 24.9, 22.5, 22.0.
4.3. Kinetic assay
Kinetic studies of inhibition on AChE and BChE were performed by
using Ellman’s method as described above. The concentrations of used
substrates were 0.07813, 0.1563, 0.3125, and 0.625 μM. Linear re-
gression was used for calculation of Lineweaver–Burk plots, and all the
calculations were performed using GraphPad Prism 5.0 software.
4.4. Molecular docking
GLIDE 5.5 program [39,40] was employed to perform molecular
docking studies. The LigPrep [41] panel was used to produce multiple
ESI-MS m/z 455.2 [M+H]+
.
HR-ESIMS: [M+H]+ calcd for
286

Z.-Q. Cheng et al.
BioorganicChemistry83(2019)277–288
output structures of inhibitors by generating different protonation
states, stereochemistry, tautomers, and ring conformations for mole-
cular docking. The Protein Preparation Wizard Workflow was used to
process and minimize the protein structures. Residues located within
20 Å around AA7 on AChE (PDB: 5EI5) [17,42] and tacrine on BChE
(4BDS) [43] were defined as binding sites in which the docking grids
were created. The default settings were used. The inhibitors were
docked into the defined binding site using extra precision (XP) mode
without any constraint and were ranked by Glide-score.
mm2) was generated using a 470 nm LED (Thorlabs) and images were
taken every 2.5 s for 2 h before and after adding the drug. A self-written
Matlab program was produced to analyze the images. Briefly, in-
dividual neurons were first detected using Otsu’s algorithm and by a
size filter of 16–200 µm2. The time courses of each cell were then
generated and averaged, and physiological parameters were derived
from the time courses.
Conflicts of interest
4.5. Molecular dynamics simulation
There are no conflicts to declare.
Acknowledgements
Molecular dynamics (MD) simulations were performed on 1a/AChE
and 5e/AChE complexes obtained from molecular docking. Before si-
mulation, the protonation states of ionizable residues of AChE were
determined using the H++ program [44]. Each complex model was
surrounded by a periodic box of transferable intermolecular potential
3P [45] water molecules that extend 10 Å from the protein atoms.
Counter-ions (Na+ × 2) were added to neutralize each simulation
system. The parm99SB version of the all-atom assisted model building
and energy refinement (AMBER) force field was used to represent the
protein system. Atom types and charges of 1a and 5e were generated by
antechamber package [46,47].
This research work was financially supported by the Natural Science
Foundation of China [Nos. 21672082, 81803438], Shandong Key
Development Project [No. 2016GSF201209], the Young Taishan
Scholars Program [No. tsqn20161037], Natural Science Foundation of
Shandong Province [No. ZR2017BH038, JQ201721, ZR2018ZC0944],
and Shandong Talents Team Cultivation Plan of University
Preponderant Discipline [No. 10027]. Authors are grateful to Jin-Tong
Song (School of Biological Science and Technology, University of Jinan)
for NMR analytical support.
Firstly, energy minimization (8000 steps for the water molecules
followed by 6000 steps for the whole system) for each solvated system
was done to remove poor contacts between the solute and the solvent.
All MD simulations were conducted using the SANDER program in-
cluded in the AMBER package (version 10.0) [48] at constant tem-
perature and pressure and periodic boundary conditions with a time
step of 10 fs. The SHAKE algorithm [49] was used to constrain all hy-
drogen atom bonds. Electrostatic interactions were calculated using the
particle-mesh Ewald method [50]. The non-bonded cutoff was set to
10.0 Å, and the non-bonded pairs were updated every 25 steps. The
temperature (300 K) and pressure (1 atm = 101.3 kPa) of the system
were controlled during the MD simulations by applying the algorithm
developed by Berendsen et al. [51] The temperature and pressure
coupling parameters were set to 1 ps.
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4.6. Binding free energy calculation by MM-PBSA method
Based on the equilibrated dynamic trajectory, the binding free en-
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