Design, synthesis and biological evaluation of tacrine-1,2,3-triazole derivatives as potent cholinesterase inhibitors

Article abstract of DOI:10.1039/c7md00457e

We report herein the design and synthesis of a series of 11 novel tacrine-1,2,3-triazole derivatives via a Cu(i)-catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. The newly synthesized compounds were evaluated for their inhibition activity against Electrophorus electricus acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) as potential drug targets for Alzheimer's disease (AD). Among the designed compounds, compound 8a2 exhibited potent inhibition against AChE and BChE with IC₅₀ values of 4.89 μM and 3.61 μM, respectively. Further structure-activity relationship (SAR) and molecular modeling studies may provide valuable insights into the design of better tacrine-triazole analogues with potential therapeutic applications for AD.

Full text of DOI:10.1039/c7md00457e

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Design, Synthesis and Biological Evaluation of Tacrine 1,2,3-Triazole
Derivatives as Potent Cholinesterase Inhibitors
Gaochan Wua, Yun Gaoa, Dongwei Kanga, Boshi Huanga, Zhipeng Huoa, Huiqing Liuc,
Vasanthanathan Poongavanamb,*, Peng Zhana,*, Xinyong Liua,*
aDepartment of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road,
250012 Ji'nan, Shandong, PR China
bDepartment of Physics, Chemistry, and Pharmacy, University of Southern Denmark, DK-5230
Odense M. Denmark.
cInstitute of Pharmacology, School of Medicine, Shandong University, 44, West Culture Road,
250012 Jinan, Shandong, PR China
*E-mails: nobelvasanth@gmail.com (Poongavanam V.); zhanpeng1982@sdu.edu.cn (Zhan P.);
xinyongl@sdu.edu.cn (Liu X.Y.).
Abstract: We report herein the design and synthesis of a series of 11 novel tacrineꢀ1,2,3ꢀtriazole
derivatives via Cu(I)ꢀcatalyzed, alkyne–azide 1,3ꢀdipolar cycloaddition (CuAAC) reaction. The newly
synthesized compounds were evaluated for their inhibition activity against electrophorus electricus
acetylcholinesterase (AChE) and horse serum butyrylcholinesterase (BChE) as the potential drug
targets for Alzheimer’s disease (AD). Among the designed compounds, compound 8a2 exhibited the
potent inhibition against AChE and BChE with IC50 values of 4.89 ꢀM and 3.61 ꢀM, respectively.
Further structureꢀactivity relationships (SARs) and molecular modeling studies may serve as valuable
insights for the designing of better tacrineꢀtriazole analogues with potential therapeutic applications for
the AD.
Introduction
Alzheimer's disease (AD) characterized by a progressive loss of memory and other cognitive
function impairments is the most common form of senile dementia.1 Environmental, genetic and some
endogenous factors play a role during the development of AD. Recent study estimates over 36 million
people are living with AD worldwide with an estimated global economic impact of approximately $605
billion in 2010,2 however, the figure has changed to $818 billion in 2015,3 an increase of 35% since
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2010. It has been predicted that if an efficient treatment isn’t developed by 2050, the number of
patients with AD will rise to 70 million.4 The impact of this pathology is tremendous for the patients
and their families. According to the Alzheimer’s Association,5 AD is the only one without a means to
prevent, cure, or slow its progression among the top 10 causes of death in the United States. Although
there has been considerable improvement in the management of AD, due to its complex pathogenesis, a
successful AD chemotherapy is still demanding and challenging for medicinal chemist. Given the
background, several hypotheses have been proposed which include the cholinergic hypothesis.6ꢀ8 This
hypothesis suggests that the loss of cholinergic activity is related to the severity of AD.7 Studies have
shown that human acetylcholinesterase (hAChE) or ‘pseudocholinesterase’ (also called as human
butyrylcholinesterase (hBChE) can quickly break down acetylcholine in the body, produce inactive
acetic acid and choline, and thereby terminate nerve impulses,9, 10 which caused the loss of learning
ability and cognition. Thus, demoting acetylcholinesterase (AChE) so as to increase the level of
acetylcholine (ACh) could be beneficial to alleviate AD’s symptoms. Meanwhile, there was evidence
showed that butyrylcholinesterase (BChE), a serine hydrolase related to AChE, catalyzed the
hydrolysis of esters of choline, including ACh, and played important roles in cholinergic
neurotransmission.11 That is to say, the inhibition of butyrylcholinesterase (BChE) can raise ACh levels.
Consequently, dual inhibition of AChE/BChE may be helpful to improve AD symptoms without
remarkable side effects.12 Both hAChE and hBChE structures are very well characterized from Xꢀray
crystallography studies as being small and a narrow gorge structure, mainly composed of the catalytic
active site (CAS) at the bottom of the gorge and the peripheral anionic site (PAS) at the entrance of the
gorge.13 In the hAChE, CAS region is mainly composed of the Ser203, Glu334, Trp86, Pro446, Phe295,
Phe297, Gly120, Gly121, Ala204 and His447 residues.14 Among them, catalytic triad (Ser203, Glu334,
and His447) mainly involved in the hydrolysis of choline, and PAS region is composed of Asp74,
Tyr72, Tyr124, Trp286 and Arg298 residues which stabilize the substrate binding. Although hAChE
and hBChE have high homology (54% identity and 81% similarity), the hAChE catalytic gorge is lined
by fourteen aromatic residues e.g., Tyr72, Tyr124, Trp286, Phe295, Phe297, and Tyr337, whereas six of
these positions were replaced by aliphatic residues (Asn68, Gln119, Ala277, Leu286, Val288, and
Ala328) in hBChE.14 Importantly, πꢀstacking interaction is the main force in the substrate and inhibitor
binding, e.g. tacrine’s aromatic ring.
2

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So far, five cholinergic drugs have been approved for clinical use, namely tacrine, donepezil,
rivastigmine, galantamine, and huperzine A and many others are in various stages of clinical trials.
Among them, tacrine, the first drug approved by FDA that binds at CAS region,15 is a potent
nonselective inhibitor of both hAChE and hBChE and show large modifiable chemical space.
Nonetheless, Tacrine suffers from severe side effects including liver toxicity.16 On the other hand,
17ꢀ20
quinoline derivatives (I-IV)
were reported with specifically binding ability to PAS and displayed
favorable antiꢀAD activity (Fig. 1). The purpose of this study was to investigate the effect of dual
binding site inhibitors on the PAS and CAS of ChE and to find more effective ChE inhibitors.
Fig. (1): Tacrine and quinoline derivatives with favorable inhibitory activity, and the newly designed
tacrine derivatives.
Meanwhile, conjugation of functional molecules through 1,2,3ꢀtriazole have received great
attention in drug discovery for the reason that the 1,2,3ꢀtriazole structural motif is a neoclassical
bioisostere of amide, which is a widely employed functional group in approved drugs.21 In general,
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1,2,3ꢀtriazole possessed several advantages in the drug design.22, 23 For instance, the triazole has two
Hꢀbond acceptors which capable of interacting with the biomolecular targets through Hꢀbonding, πꢀπ
stacking, and dipole interaction. Besides, triazole ring exhibits better chemical stability in the
biological environments, and this improves the pharmacokinetic and toxicity properties. All these
advantages made the 1,2,3ꢀtriazole become a remarkable fragment in the drug design. Besides, the
formation of 1,2,3ꢀtriazole compounds via Cu(I)ꢀcatalyzed, alkyneꢀazide 1,3ꢀdipolar cycloaddition
(CuAAC) reaction, often referred to as click chemistry, is widely used for the rapid assembly of
heterocyclic molecules that may be subsequently used as candidate leads in drug development.24
Considering the above benefits and in continuation of our interest in searching for the antiꢀAChE
and antiꢀBChE pharmacological effects of tacrineꢀbased PASꢀCAS dual binders, in the present study,
we describe the synthesis and evaluation of a set of diverse tacrineꢀquinolines (See Figure 1) as
PASꢀCAS dual inhibitors for electric eel AChE and horse serum BChE.
Results and discussions
Chemistry
The synthetic route towards the target tacrineꢀ1,2,3ꢀtriazole derivatives was outlined in Scheme
1-3. Initially, the commercially available starting material indolineꢀ2,3ꢀdione (1) yield intermediate
2ꢀaminobenzoic acid (2a) in the presence of sodium hydroxide and 30% hydrogen peroxide. Under the
presence of POCl3, compound 2a (or 2b) reacted with cyclohexanone to obtain
9ꢀchloroꢀ1,2,3,4ꢀtetrahydroacridine (3a) or 6,9ꢀdichloroꢀ1,2,3,4ꢀtetrahydroacridine (3b), which were
used to yield intermediate 4a (or 4b, or 4c) by nucleophilic substitution reaction. Then 4a (or 4b, or 4c)
reacted with different fragments of alkyne giving the key intermediates 5a1-5a5 and 5b1-5b6.25ꢀ29 In
the meantime, compound 6 was reacted with NaN3 in the presence of NaI to prepare compound 7,
which followed by reaction with 5a1-5a5 and 5b1-5b6 to give the target products 8a and 8b via
CuAAC reaction.30, 31 The newly synthesized compounds were characterized by physicochemical and
spectral means, and the consequence of MS and NMR spectral data were found in agreement with the
assigned molecular structures.
4

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Scheme 1: The synthetic route of intermediates 4a, 4b and 4c. Reagents and Conditions. (i) 30% H2O2,
NaOH, 0 , 3h; (ii) cyclohexanone, POCl3, 100 , 3h; (iii) piperazine, NaI, PhOH, 180 , 3h; (iv)
ethaneꢀ1,2ꢀdiamine, NaI, PhOH, 180 , 3h.
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Scheme 2: The synthetic route of intermediates 5a1-5a5 and 5b1-5b6. Reagents and Conditions. (v)
5a1: 3ꢀbromopropꢀ1ꢀyne, Et3N; 5a2: 5ꢀchloropentꢀ1ꢀyne, K2CO3; 5a3: 6ꢀchlorohexꢀ1ꢀyne, K2CO3; 5a4:
heptꢀ6ꢀynoic acid, EDCI, HOBt, Et3N; 5a5: 1) 4ꢀchlorobutanoyl chloride, Et3N; 2)
Nꢀmethylpropꢀ2ꢀynꢀ1ꢀamine, K2CO3; (vi) 5b1: 3ꢀbromopropꢀ1ꢀyne, Et3N; 5b2: 6ꢀchlorohexꢀ1ꢀyne,
K2CO3; 5b3: heptꢀ6ꢀynoic acid, EDCI, HOBt, Et3N; 5b4: 1) 2ꢀchloroacetyl chloride, Et3N; 2)
Nꢀmethylpropꢀ2ꢀynꢀ1ꢀamine, K2CO3; 5b5: 1): 5ꢀbromopentanoyl chloride, Et3N; 2):
Nꢀmethylpropꢀ2ꢀynꢀ1ꢀamine, K2CO3; (vii): 5b6: 1): ethaneꢀ1,2ꢀdiamine, NaI, PhOH; 2): heptꢀ6ꢀynoic
acid, EDCI, HOBt, Et3N.
Scheme 3: The synthetic route of tacrineꢀ1,2,3ꢀtriazole derivatives. Reagents and Conditions. (viii) NaI,
NaN3, DMF/H2O (v/v = 1:1), 80 , 8h; (ix) Sodium ascorbate, CuSO4, Et3N, nꢀbutanol/H2O (v/v = 1:1).
Cholinesterase Inhibition Evaluation
Based on the reported protocol,32ꢀ36 all the newly synthesized compounds (8a1-8a5, 8b1-8b6) were
evaluated for their inhibitory activities toward electric eel AChE (SigmaꢀAldrich, USA; EC: 3.1.1.7;
Product Information File Code: GRO, JWM, RBG, MAM 01/08ꢀ1, the download website was shown in
the Supporting Information;) and horse serum BChE (SigmaꢀAldrich, USA; EC: 3.1.1.8; Product
Information File Code: TMG/AJH 10/07, the download website was shown in the Supporting
Information;). The authors declare that all procedures were performed in compliance with the Chinese
relevant laws and institutional guidelines strictly. Tacrine was selected as a reference drug. The initial
in vitro screening was performed using single concentration at 100 ꢀM (see Table 1). Among all the
tested compounds, compound 8a2 exhibited the most potent inhibitory activity against AChE and
BChE with an inhibition ratio of 78.69% and 91.80% at 100 ꢀM. In addition, compounds 8a3, 8a4, 8b2
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and 8b3 showed inhibitory percentage for AChE more than 60% at 100 ꢀM, which comparable to that
of 8a2. Interestingly, compound 8b6 had better selectivity for BChE and displayed inhibitory
percentage of 82.73% against BChE which about two times than that for AChE (49.37%). After the
preliminary screening, compounds 8a2, 8a3, 8a4, 8a5, 8b2, 8b3, and 8b6 were chosen to determine
their IC50 values for cholinesterase. Among them, 8a2 was demonstrated the best antiꢀAChE (IC50
=
4.89 ꢀM) and antiꢀBChE activity (IC50 = 3.61 ꢀM). Besides, the inhibitory efficacy of 8a3, 8a4, 8b3,
and 8b2 demonstrated IC50 values against AChE were 10, 11.07, 18.66 and 19.59 ꢀM, respectively. As
for antiꢀBChE activity, 8b6 was inferior to 8a2, and its IC50 value was 6.06 ꢀM, followd by 8a5(IC50
=
61.13 ꢀM) and 8a3 (IC50 = 66.68 ꢀM). However, all the newly synthesized compounds showed weaker
inhibitory activity compared to the reference drug tacrine.
Preliminary structureꢀactivity relationship (SAR) analysis indicated that the size and length of the
linker between the piperazine and triazole motif is a major determinant for their inhibitory activity. An
alkane chain with three carbons was the most active one (8a2), whether increasing or shortening the
length of the carbon chain would decrease their inhibitory effect for AChE and BChE (Inhibition
activity: 8a2 > 8a3 > 8a1). Compared series a with series b, introducing chlorine atom at the C6
position of tacrine would decrease the inhibitory activity (Inhibition activity: 8a1 > 8b1, 8a3 > 8b2,
8a4 > 8b3). Current SAR will provide additional information to help in the design of inhibitors with
better potency.
Table 1: Inhibition activity of Tacrine derivatives for electrophorus electricus AChE and horse serum
BChE.
Inhibition (%) at 100 ꢀM
IC50 (ꢀM)
Compound
R
Linker
AChE
BChE
AChE
BChE
H
H
39.32
38.56
ꢀ
ꢀ
8a1
8a2
78.69
91.80
4.89
3.61
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H
H
69.31
65.69
47.56
30.38
55.64
46.37
57.90
22.12
10
11.07
>100
ꢀ
66.68
>100
61.13
ꢀ
8a3
8a4
8a5
8b1
H
Cl
Cl
Cl
66.02
64.19
46.68
49.28
19.59
18.66
>100
>100
8b2
8b3
O
N
N
Cl
Cl
41.47
42.31
27.97
39.04
ꢀ
ꢀ
ꢀ
ꢀ
8b4
8b5
Cl
ꢀ
49.37
86.22
82.73
99.63
>100
0.316
6.06
8b6
0.066
Tacrine
ꢀ
Molecular Modeling Analysis
To provide better insight into the binding mode of compound 8a2 to hAChE and hBChE,
molecular docking followed by MMꢀGBSA studies were performed. To this end, compound 8a2 was
docked into the hAChE and hBChE ligandꢀbinding site (see experimental section for detail). The active
site of hAChE has previously been well characterized as having the catalytic site (CAS), which is
composed of the Trp86, Glu334, Tyr336, Trp439, Met443, Pro446 and His447 residues and the
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peripheral site or anionic subsite (PAS) which is composed of Asp74, Tyr72, Tyr124, Trp286 and
Arg298 residues. It is shown in the crystal structure of hAChE complex with tacrine that the acridine
ring involved in the πꢀπ interaction with indole ring of Trp84. In addition to acridine ring surrounded
by the hydrophobic residues such as Phe330, Tyr334, Tyr442 and Trp434.37 The active site of hBChE
is very similar as hAChE as having similar set of residues in both CAS and PAS regions.38 In this study,
the binding mode of compound 8a2 is compared in both hAChE and hBChE active site with Tacrine.
MMꢀGBSA method was employed to rank the bestꢀranked binding pose from the docking solutions,
and the binding pose that has shown the highest binding affinity in the MMꢀGBSA calculation for
hAChE and hBChE is further discussed. It is clear from Fig. 2 that the 8a2 binding mode is similar in
both cases and that the acridine ring involved in πꢀπ interaction with Trp86 (hAChE) and Trp82
(hBChE) as it has previously been shown.37
Moreover, piperazine and triazole rings of 8a2 compound showed the interaction with Tyr124 and
Trp286 in the hAChE. However, compound 8a2 binds slightly better in the hBChE structure, for
instance, quinoline ring strongly binds to Ser287 and Pro285 residues through πꢀπ interactions, and
importantly, electronegative chlorine atom also shows strong interaction with neighboring residue e.g.,
Gln71. Besides, tacrine structure also was prepared in the same manner as compound 8a2 and
reꢀdocked into hAChE and hBChE active site to assess the docking protocol and compared the binding
pose to xꢀray crystal structure. Overall, the docking simulation is able to reproduce the same binding
pose as it was observed in the xꢀray crystallophay with an RMSD of 0.05 A (cf. Figure 2 and
supporting information). In addition to the comparison of 8a2 with tacrine, the binding mode of
compound III and IV (cf. Figure 1) was compared to understand an impact of linker fragment. The
compounds III and IV were also docked same as Tacrine into hAChE and hBChE structures. As clearly
seen from the Fig 2C, the binding mode of 8a2 found to be similar as compound III and IV (see
proteinꢀligand interaction map in the Supporting Information) but showed lesser potency as compared
to tacrine or compound III/IV. The higher IC50 of 8a2 could be due to not only rigid triazole and
piperazine rings, but also having more polar atoms, which may lead to an unfavorable contribution to
the hydrophobicity of active sites, particularly PAS as reported previously.17,18
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Fig. (2): Comparison of the binding mode of compound 8a2 (cyan) at the active site of hAChE (A) and
hBChE (B). Important residues belonging to CAS (green) and PAS (orange) are highlighted. Schematic
representation of binding poses of 8a2 with compounds III, IV and tacrine (for clarity only ligand pose
is shown).
Conclusion
To summarize, a concise and efficient synthetic approach has been established to readily access two
novel series of tacrineꢀ1,2,3ꢀtriazole derivatives in moderate to high yields. Subsequently, the
synthesized compounds were screened for electric eel AChE and horse serum BChE inhibition. Among
these compounds, compound 8a2 inhibited the AChE and horse serum BChE activity by more than 75%
(IC50: 4.89 ꢀM) and 90% (IC50: 3.61 ꢀM) at a concentration of 100 ꢀM, respectively. Although,
compound 8a2 inhibits cholinesterase slightly lower as compared to reference drug Tacrine, with its
unique binding mode at both at CAS and PAS sites, making it particularly promising lead for the
development of new dual inhibitors of AChE and BChE. Further, molecular docking studies were also
performed to understand its binding mode in the AChE and BChE active, particularly revealing the
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importance of quinoline substituents on the Cꢀ1 position of the 1,2,3ꢀtriazole ring involved in the πꢀπ
interaction with Trp286 (hAChE), Ser287 (hBChE) and Pro285 (hBChE) at PAS region. Meanwhile,
the acridine ring involved in πꢀπ interaction with Trp86 (hAChE) and Trp82 (hBChE) at CAS region
same as the reference compound Tacrine.
Several works have reported on designing of tacrineꢀbased dimers with different linkers (e.g.
disulfide bond, alkane, piperazine, amide etc.), including dual binding site inhibitors on the PAS and
CAS towards AChE and BChE, which is a promising trend deserved to be explored and studied.39 In
this work, we take advantages of CuAAC click chemistry in drug design and 1,2,3ꢀtriazole unique
physiochemical property, thus, adapted a linker including 1,2,3ꢀtriazole group introduced by CuAAC
reaction but maintained its unique binding mode like Tacrineꢀbased dimers. Although a most potent
compound from this series is significantly weaker as compared to Tacrine or previously known
compounds e.g., III and IV, a nonꢀaliphatic linker designing provides further opportunities to design
smarter linker which facilitates the molecules to be able to bind to CAS and PAS sites. With this
understanding, efforts are further expanded in our laboratory for further lead optimization of
Tacrineꢀbased triazole analogues as potent cholinesterase inhibitors with the hope to develop promising
candidates for AD chemotherapy.
Experimental Section
General
All melting points of the compounds were determined on a micro melting point apparatus and
were uncorrected. 1HꢀNMR and 13CꢀNMR spectra were obtained via a Bruker Avanceꢀ400
NMRꢀspectrometer in CDCl3. Chemical shifts were expressed in ꢁ units and TMS as internal reference.
Mass spectra was conducted via a LC Autosampler Device: Standard G1313A instrument. Flash
column chromatography was performed on column packed with Silica Gel 60 (200ꢀ300 mesh).
Meanwhile, TLC was taken on Silica Gel GF254 for TLC and spots were visualized by irradiation with
UV light (ꢂ = 254 nm). Solvents were of reagent grade, which were purified and dried by standard
methods when necessary. Concentration of the reaction solutions was used the rotary evaporator under
reduced pressure condition.
General Procedure for the Synthesis of Compound 7 (4-azido-7-chloroquinoline).
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4,7ꢀDichloroquinoline (6, 3.0 g, 11.4 mmol), NaN3 (2.0 g, 30.0 mmol), and NaI (0.2 g, 1.5 mmol)
were dissolved in the solvent of DMF/H2O (v:v = 1:1, 60 mL). The resulting mixture was stirred at 80
for 8 h. After the reaction was cooled to the room temperature, water (30 mL) was added to the reaction
system. Then the resulting mixture was extracted with ethyl acetate (3 × 40 mL). The combined
organic phase was washed with saturated salt water (3 × 40 mL), dried over anhydrous Na2SO4, filtered,
and concentrated under reduced pressure to give the corresponding crude target product, which was
purified by flash column chromatography with petroleum ether/ethyl acetate to afford compound 7 as
white solid with the yield of 91%.
General Procedure for the Synthesis of 3a (9-chloro-1,2,3,4-tetrahydroacridine) and 3b
(6,9-dichloro-1,2,3,4-tetrahydroacridine).
Indolineꢀ2,3ꢀdione (1, 5.0 g, 34.0 mmol) was dissolved in the solvent of NaOH (67 mL, 1 mol/L).
After hydrogen peroxide (7.3 mL, 30%) was added into the above solution slowly at 0 , the resulting
mixture was stirred at 30ꢀ40 for 3 h. Then the cooling solution was neutralized to pH = 7.5 with
hydrochloric acid (3 mol/L). Subsequently, moderate activated carbon was added and stirred for
another 0.5 h. Then the resulting precipitate was filtered and further the filtrate was acidized until pH =
4ꢀ5 and stirring was continued for another 1 h. The resulting mixture was filtered again and combined
with the filter cake, which was dried in the vacuum drying oven to obtain the crude product 2a. The
crude product 2a was purified by flash column chromatography with dichloromethane/methanol to
afford compound 2a as white hairy solid with the yield of 84%.
2ꢀAminobenzoic acid (2a, 1.0 g, 7.3 mmol) and cyclohexanone (0.9 g, 8.8 mmol) were mixed in
the flask. Then POCl3 (10 mL) was added dropwise into the flask at 0 . The above resulting mixture
was refluxed at 100 for 3 h. Then the mixture was quenched with ice water and neutralized to neutral
with K2CO3 solution (1 mol/L). Subsequently, the neutralized solution was extracted with ethyl acetate
(3 × 15 mL), and the combined organic phase was washed with saturated salt water (3 × 15 mL), dried
over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give the corresponding
crude target product. Finally, crude product was purified by flash column chromatography with
petroleum ether/ethyl acetate to afford product compound 3a as yellow solid. Yield: 88%. To be noted,
synthetic operation of compound 3b (Yellow solid; Yield: 81.5%) was also the same to compound 3a.
General Procedure for the Synthesis of compound 4a (9-(piperazin-1-yl)-1,2,3,4-tetrahydroacridine),
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4b
(6-chloro-9-(piperazin-1-yl)-1,2,3,4-tetrahydroacridine)
and
4c
(N1-(6-chloro-1,2,3,4-tetrahydroacridin-9-yl) ethane-1,2-diamine).
Compound 3a (or 3b, 13.8 mmol), piperazine (5.9 g, 69.0 mmol), NaI (0.2 g, 13.8 mmol) and
phenol (2.6 g, 27.6 mmol) were mixed and stirred at 180 for 3 h in the flask. Then the reaction
system was cooled to 80 and subsequently quenched with NaOH solution (30 mL, 1 mol/L). The
quenched mixture was extracted with dichloromethane (3 × 15 mL), and the combined organic phase
was washed with saturated salt water (3 × 15 mL), dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure to give the corresponding crude target product, which was
purified by flash column chromatography with dichloromethane/methanol to afford compound 4a (4b)
as yellow solid (yellow oil) with the yield of 81% (42%). The same procedure was used to obtain
product 4c with yield of 82%, only with the difference that the piperazine was changed to
ethaneꢀ1,2ꢀdiamine.
General Procedure for the Synthesis of compound 5a1-3 and 5b1-2.
Compound 4a (4b, 3.7 mmol), 3ꢀbromopropꢀ1ꢀyne (0.49 g, 4.1 mmol), and triethylamine (0.6 g,
5.6 mmol) were stirred at 50 for 3 h in the solvent of dichloromethane. Then the reaction mixture was
cooled to the room temperature and concentrated under the reduced pressure to obtain the crude
product, which was purified by flash column chromatography with dichloromethane/methanol to get
compound 5a1 (5b1) as yellow solid (yellow solid). Yield: 70% (82%). If 3ꢀbromopropꢀ1ꢀyne was
changed to 5ꢀchloropentꢀ1ꢀyne or 6ꢀchlorohexꢀ1ꢀyne, 5a2, 5a3, and 5b2 would be obtained with yield
of 81%, 69% and 49%, respectively.
General Procedure for the Synthesis of compound 5a4, 5b3 and 5b6.
Heptꢀ6ꢀynoic acid (0.5 g, 4.0 mmol), EDCI (1.0 g, 5.0 mmol), and HOBt (0.7 g，5.0 mmol) were
stirred at room temperature for 0.5 h in dichloromethane. Then compound 4a (4b or 4c, 3.3 mmol,) and
triethylamine (0.7 g, 6.6 mmol) were added, and the reaction was continued at the room temperature
for another 5 h. Once the reaction was finished (monitored by TLC), the reaction mixture was
quenched with saturated salt water (3 × 30 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure to give the corresponding crude target product, which was
purified by flash column chromatography with dichloromethane/methanol to get compound 5a4 (5b3or
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5b6) as yellow solid. Yield: 50% (70% or 86%).
General Procedure for the Synthesis of compound 5a5, 5b4, 5b5, and 5b6.
4ꢀchlorobutanoyl chloride (0.8 g, 5.6 mmol) was added dropwise into the solvent of
dichloromethane (25 mL) at 0 , which previously stirred with triethylamine (0.7 g, 7.4 mmol) and
compound 4a (1.0 g, 3.7 mmol). Then the reaction was continued at 0 under magnetic stirring for 0.5
h. Subsequently, stirring was continued for another 2 h at room temperature. Once the reaction was
completed (monitored by TLC), the resulting mixture was washed with saturated salt water (3 × 25
mL), dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure to give the
corresponding crude target product, which was purified by flash column chromatography with
petroleum ether/ethyl acetate to obtain the precursor of compound 5a5. Yield: 82%. And then
compound 5a5 reacted with Nꢀmethylpropꢀ2ꢀynꢀ1ꢀamine as the synthetic operation of compound 4a,
4b and 4c, and was purified by flash column chromatography. Yellow solid. When the same procedure,
only with the difference that procedure 4ꢀchlorobutanoyl chloride was changed to 2ꢀchloroacetyl
chloride or 5ꢀbromopentanoyl chloride, product 5b4 and 5b5 were obtained with yield of 83% and 89%,
respectively.
General Procedure for the Synthesis of compound 8a1-8a5 and 8b1-8b6.
Intermediate 5a1-5a5 (1.0 eq, or 5b1-5b6), 4ꢀazidoꢀ7ꢀchloroquinoline (compound 7, 1.0 eq),
ascorbic acid sodium (0.6 eq), CuSO4▪5H2O (0.3 eq) and triethylamine (1.2 eq) were dissolved in the
solution of nꢀbutyl alcohol/water (v:v = 1:1). The resulting mixture was stirred at room temperature for
5 h. Then the reaction mixture was extracted with ethyl acetate (3 × 10 mL), and the combined organic
phase was washed with saturated salt water (3 × 10 mL), dried over anhydrous MgSO4, filtered, and
concentrated under reduced pressure to give the corresponding crude target product, which was
purified by flash column chromatography to afford product 8a1-8a5 (or 8b1-8b6). Yield: 37%ꢀ92%.
9-(4-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1,2,3,4-tetrahydroacridi
1
ne (8a1). Yield: 74%; White solid; mp:105ꢀ107°C. H NMR (400 MHz, CDCl3, ppm) δ: 9.08 (d, J =
4.6 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 8.07ꢀ8.05 (m, 2H), 7.97 (d, J = 8.3 Hz,
1H), 7.64ꢀ7.57 (m, 2H), 7.54 (d, J = 4.6 Hz, 1H), 7.42 (t, J = 7.2 Hz, 1H), 3.99 (s, 2H), 3.41 (s, 4H),
3.13 (t, J = 6.6 Hz, 2H), 2.96ꢀ2.87 (m, 6H), 1.97ꢀ1.82 (m, 4H); 13C NMR (100 MHz, CDCl3, ppm) δ:
14

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160.3, 152. 8, 151.4, 150.3, 147. 7, 145.6, 141.1, 137.0, 129.5, 129.1, 128.7, 128.4, 127.7, 125.8, 125.1,
124.7, 124.5, 123.9, 120.6, 115.8, 54.4, 53.7, 50.4, 34.0, 26.9, 23.0, 22.8. ESIꢀMS: C29H28ClN7, Exact
Mass: 509.21, m/z 511.5 (M + 2)+.
9-(4-(3-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)propyl)piperazin-1-yl)-1,2,3,4-tetrahydroacri
dine (8a2). Yield：47%; White solid; mp: 98ꢀ100°C. 1H NMR (400 MHz, CDCl3, ppm) δ: 9.03 (d, J =
4.6 Hz, 1H), 8.22 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.95 (d, J = 8.3
Hz, 1H), 7.86 (s, 1H), 7.59ꢀ7.54 (m, 2H), 7.48 (d, J = 4.6 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 3.37 (s, 4H),
3.11 (t, J = 6.6 Hz, 2H), 3.01ꢀ2.92(m, 4H), 2.72ꢀ2.63 (m, 6H), 2.14 ꢀ2.06 (m, 2H), 1.96 ꢀ1.80 (m, 4H)；
13C NMR (100 MHz, CDCl3, ppm) δ: 160.3, 152.8, 151.4, 150.2, 148.7, 147.8, 141.1, 136.6, 129.2,
128.9, 128.7, 128.3, 127.6, 125.9, 124.9, 124. 8, 123.9, 122.6, 120.5, 115.8, 58.2, 54.3, 50.5, 34.1, 26.9,
26.5, 23.5, 23.0, 22.8. ESIꢀMS: C31H32ClN7, Exact Mass: 537.24, m/z 539.2 (M + 2)+.
9-(4-(4-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)butyl)piperazin-1-yl)-1,2,3,4-tetrahydroacridi
ne (8a3). Yield: 43%; White solid; mp:112ꢀ115 °C. 1H NMR (400 MHz, CDCl3, ppm) δ: 9.05 (d, J =
4.6 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.15 (d, J = 8.3 Hz, 1H), 8.06ꢀ8.00 (m, 2H), 7.83 (s, 1H),
7.61ꢀ7.56 (m, 2H), 7.50 (d, J = 4.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 3.38 (s, 4H), 3.15 (t, J = 6.6 Hz,
2H), 2.98ꢀ2.92 (m, 4H), 2.72 (s, 4H), 2.61ꢀ2.57 (m, 2H),1.97ꢀ1.72 (m, 8H); 13C NMR (100 MHz,
CDCl3, ppm) δ: 160.3, 153.1, 151. 6, 150.3, 149.0, 147.5, 141.2, 136.8, 129.3, 129.0, 128.6, 128.4,
127.6, 125.9, 125.0, 124.8, 124.0, 122. 6, 120.7, 115.8, 58.7, 54.4, 50.4, 34.1, 27.3, 26.9, 26.5, 25.5,
23.0, 22.8. ESIꢀMS: C32H34ClN7, Exact Mass: 551.26, m/z 553.2 (M + 2)+.
5-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)-1-(4-(1,2,3,4-tetrahydroacridin-9-yl)piperazin-1-y
1
l)pentan-1-one (8a4). Yield: 64%; White solid; mp: 98ꢀ101°C. H NMR (400 MHz, CDCl3, ppm) δ
9.03 (d, J = 4.6 Hz, 1H), 8.21 (d, J = 2.0 Hz, 1H), 8.09ꢀ8.03 (m, 2H), 7.97ꢀ7.91 (m, 2H), 7.61ꢀ7.55 (m,
2H), 7.49 (d, J = 4.6 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 4.08ꢀ3.71 (m, 4H), 3.33ꢀ3.28 (m, 4H), 3.12 (t, J
= 6.5 Hz, 2H), 2.98ꢀ2.89 (m, 4H), 2.52 (t, J = 7.0 Hz, 2H), 1.97ꢀ1.82 (m, 8H); 13C NMR (100 MHz,
CDCl3, ppm) δ: 171.4, 160.6, 152.0, 151.4, 150.2, 148.8, 147.8, 141.1, 136.7, 129.2, 129.0, 128. 9, 128.
5, 127.9, 125.8, 125.3, 124.8, 123.4, 122.7, 120.6, 115.8, 50.6, 50.2, 46.8, 42.8, 34.1, 32.9, 28.9, 26.8,
25.4, 24.7, 22.9, 22.7. ESIꢀMS: C33H34ClN7O, Exact Mass: 579.25, m/z 581.5 (M + 2)+.
4-(((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)methyl)(methyl)amino)-1-(4-(1,2,3,4-tetrahydroa
1
cridin-9-yl)piperazin-1-yl)butan-1-one (8a5). Yield: 43%; White solid; mp:110ꢀ112°C. H NMR (400
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MHz, CDCl3, ppm) δ: 9.05 (d, J = 4.3 Hz, 1H), 8.23 (d, J = 1.7 Hz, 1H), 8.08ꢀ8.04 (m, 3H), 7.96 (d, J
= 8.3 Hz, 1H), 7.61ꢀ7.57 (m, 2H), 7.54 (d, J = 4.6 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 3.91ꢀ3.70 (m, 6H),
3.31ꢀ3.25 (m, 4H), 3.12 (t, J = 6.5 Hz, 2H), 2.89 (t, J = 6.2 Hz, 2H), 2.62ꢀ2.41 (m, 7H), 2.03ꢀ1.83 (m,
6H); 13C NMR (100 MHz, CDCl3, ppm) δ: 171.4, 160.5, 152.0, 151.4, 150.2, 147.9, 145.8, 141.1,
137.0, 129.4, 129.0, 128.5, 127. 9, 125.8, 125.3, 124. 8, 124.5, 123.4, 120.6, 116.0, 56.3, 52.0, 50.7,
50.2, 46.8, 42.8, 42.6, 34.1, 30.7, 26.8, 22.93, 22.7. ESIꢀMS: C34H37ClN8, Exact Mass: 608.28, m/z
609.4 (M + 1)+.
6-Chloro-9-(4-((1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4yl)methyl)piperazin-1-yl)-1,2,3,4-tetrahy
1
droacridine (8b1). Yield: 92%; Yellow solid; mp:115ꢀ117°C. H NMR (400 MHz, CDCl3, ppm) δ:
9.08 (d, J = 4.6 Hz, 1H), 8.26 (d, J = 1.9 Hz, 1H), 8.10ꢀ8.04 (m, 3H), 7.96 (d, J = 1.6 Hz, 1H), 7.63 (dd,
J = 9.1, 2.0 Hz, 1H), 7.53 (d, J = 4.6 Hz, 1H), 7.36 (dd, J = 9.0, 2.0 Hz, 1H), 3.99 (s, 2H), 3.39 (s, 4H),
3.11 (t, J = 6.6 Hz, 2H), 2.94ꢀ2.88 (m, 6H), 2.88 (s, 4H), 1.97ꢀ1.82 (m, 4H); 13C NMR (100 MHz,
CDCl3, ppm) δ: 161.8, 152.8, 151.4, 150.3, 148.3, 145.6, 141.0, 137.0, 134.1, 129.4, 129.1, 127.9,
127.7, 125.9, 125.4, 124.7, 124.5, 124.3, 120.6, 115.9, 54.3, 53.6, 50.4, 34.1, 26.9, 22.9, 22.7. ESIꢀMS:
C29H27Cl2N7, Exact Mass: 543.17, m/z 545.2 (M + 2)+.
6-Chloro-9-(4-(4-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-triazol-4-yl)butyl)piperazin-1-yl)-1,2,3,4-tetrah
ydroacridine (8b2). Yield: 52.6%; White solid; mp:128ꢀ130°C. 1H NMR (400 MHz, CDCl3, ppm) δ:
8.87 (d, J = 4.6 Hz, 1H), 8.05 (d, J = 1.8 Hz, 1H), 7.95ꢀ7.9 (m, 2H), 7.76 (s, 2H), 7.42 (dd, J = 9.1, 2.0
Hz, 1H), 7.34 (d, J = 4.6 Hz, 1H), 7.17 (dd, J = 9.0, 1.9 Hz, 1H), 3.20 (s, 4H), 2.93 (t, J = 6.5 Hz, 2H),
2.83 (t, J = 7.6 Hz, 2H), 2.77 (t, J = 6.2 Hz, 2H), 2.56ꢀ2.42 (m, 4H), 1.83ꢀ1.59 (m, 8H); 13C NMR (100
MHz, CDCl3, ppm) δ: 161.6, 152.8, 151.3, 150.1, 148.8, 148.0, 141.0, 136.6, 133. 8, 129.1, 128.8,
127.8, 127.5, 125.6, 125.5, 124.8, 124.2, 122.5, 120.5, 115. 7, 58.6, 54.2, 50.4, 34.0, 27.3, 26. 8, 26.5,
25.5, 22.8, 22.6. ESIꢀMS: C32H33Cl2N7, Exact Mass: 585.22, m/z 586.9 (M + 1)+.
1-(4-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)piperazin-1-yl)-5-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-t
riazol-4-yl)pentan-1-one (8b3). Yield: 80%; Yellow solid; mp:108ꢀ110°C. 1H NMR (400 MHz, CDCl3,
ppm) δ: 9.06 (d, J = 4.6 Hz, 1H), 8.24 (d, J = 1.9 Hz, 1H), 8.06ꢀ8.0 (m, 3H), 7.87 (s, 1H), 7.60 (dd, J =
9.1, 2.0 Hz, 1H), 7.50 (d, J = 4.6 Hz, 1H), 7.38 (dd, J = 9.0, 2.0 Hz, 1H), 3.72 (s, 4H), 3.32ꢀ3.28 (m,
4H), 3.13 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 7.0 Hz, 2H), 2.89 (t, J = 6.3 Hz, 2H), 2.52 (t, J = 7.0 Hz, 2H),
1.97ꢀ1.84 (m, 8H); 13C NMR (100 MHz, CDCl3, ppm) δ: 171.4, 161.7, 152.3, 151.3, 150.3, 148.6,
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148.0, 141.1, 136.8, 134.4, 129.3, 129.0, 128.1, 127. 8, 126.3, 125.0, 124.2, 122.7, 115.8, 50.7, 50.3,
46.8, 42.7, 34.0, 32.9, 28.9, 26.8, 25.3, 24.6, 22.8, 22.6. ESIꢀMS: C33H33Cl2N7O, Exact Mass: 613.21,
m/z 615.4 (M + 2)+.
1-(4-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)piperazin-1-yl)-2-(((1-(7-chloroquinolin-4-yl)-1H-1,2,3
1
-triazol-4-yl)methyl)(methyl)amino)ethan-1-one (8b4). Yield: 70%; White solid; mp:113ꢀ116 °C. H
NMR (400 MHz, CDCl3, ppm) δ: 9.06 (d, J = 4.6 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.14 (s, 1H), 8.09ꢀ
8.02 (m, 2H), 7.94 (d, J = 2.0 Hz, 1H), 7.59 (dd, J = 9.1, 2.1 Hz, 1H), 7.53 (d, J = 4.6 Hz, 1H), 7.35 (dd,
J = 9.0, 2.1 Hz, 1H), 4.01 (s, 2H), 3.97ꢀ3.49 (m, 4H), 3.44 (s, 2H), 3.30 (d, J = 29.1 Hz, 4H), 3.09 (t, J
= 6.6 Hz, 2H), 2.90 (t, J = 6.2 Hz, 2H), 2.51 (s, 3H), 2.03ꢀ1.77 (m, 4H); 13C NMR (100 MHz, CDCl3,
ppm) δ: 168.6, 161.9, 152.1, 151.4, 150.2, 148.3, 144.5, 141.0, 136. 9, 134.2, 129.4, 129.0, 128.1,
127.8, 126.2, 125.2, 125.0, 124.62, 124.2, 120.5, 115.9, 59.4, 51.4, 50.8, 50.3, 46.9, 43.0, 34.1, 26.8,
22.8, 22.6. ESIꢀMS: C32H32Cl2N8O, Exact Mass: 614.21, m/z 616.4 (M + 2)+.
1-(4-(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)piperazin-1-yl)-5-(((1-(7-chloroquinolin-4-yl)-1H-1,2,3
-triazol-4-yl)methyl)(methyl)amino)pentan-1-one (8b5). Yield: 37%; White solid; mp:127ꢀ130°C. 1H
NMR (400 MHz, CDCl3, ppm) δ: 9.05 (d, J = 4.6 Hz, 1H), 8.23 (d, J = 2.0 Hz, 1H), 8.09 (s, 1H),
8.05ꢀ8.00 (m, 2H), 7.94 (d, J = 2.1 Hz, 1H), 7.59 (dd, J = 9.1, 2.1 Hz, 1H), 7.53 (d, J = 4.6 Hz, 1H),
7.35 (dd, J = 9.0, 2.1 Hz, 1H), 4.09ꢀ3.64 (m, 6H), 3.09 (t, J = 6.6 Hz, 2H), 2.87 (t, J = 6.2 Hz, 2H), 2.59
(t, J = 7.0 Hz, 2H), 2.48ꢀ2.40 (m, 5H), 1.96ꢀ1.66 (m, 8H); 13C NMR (100 MHz, CDCl3, ppm) δ: 171.5,
161.9, 152.1, 151.4, 150.2, 148.3, 145.9, 141.0, 136.8, 134.2, 129.4, 129.0, 128.1, 127.8, 126.1, 125.0,
124.7, 124.60, 124.2, 120.5, 115.9, 56.8, 52.2, 50.7, 50.3, 46.8, 42.7, 42.3, 34.1, 33.1, 27.0, 26.8, 23.0,
22.8, 22.6. ESIꢀMS: C35H38Cl2N8O, Exact Mass: 656.25, m/z 657.2 (M + 1)+.
N-(2-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-5-(1-(7-chloroquinolin-4-yl)-1H-1,2,3-t
riazol-4-yl)pentanamide (8b6). Yield: 65%; White solid; mp:112ꢀ114 °C. 1H NMR (400 MHz, CDCl3,
ppm) δ: 8.98 (d, J = 3.9 Hz, 1H), 8.17 (s, 1H), 7.94ꢀ7.73 (m, 4H), 7.51ꢀ7.31 (m, 3H), 7.11 (d, J = 8.5
Hz, 1H), 5.19 (s, 1H), 3.61 (d, J = 23.7 Hz, 4H), 3.92ꢀ3.33 (m, 8H), 1.80 (s, 8H); 13C NMR (100 MHz,
CDCl3, ppm) δ: 174.7, 159.1, 151.3, 150.9, 150.0, 148.3, 147.4, 140.9, 136.7, 134.0, 129.2, 128.8,
126.6, 124.6, 124.6, 124.0, 122.8, 120.4, 117.8, 115.8, 115.4, 50.2, 40.5, 36.0, 33.8, 28.6, 25.1, 25.0,
22.9, 22.5. ESIꢀMS: C31H31Cl2N7O, Exact Mass: 587.20, m/z 589.0 (M + 2)+.
Acetylcholinesterase and Butyrylcholinesterase Inhibition Assay.
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The inhibition ability of tacrineꢀderivatives 8a1-8a5 and 8b1-8b6 for electric eel acetylcholinesterase
(eeAChE; SigmaꢀAldrich, USA; Enzyme Information Download Website:
https://www.sigmaaldrich.com/content/dam/sigmaꢀaldrich/docs/Sigma/Product_Information_Sheet/c33
89pis.pdf) and horse serum butyrylcholinesterase (hsBChE; SigmaꢀAldrich, USA; Enzyme Information
Download
Website:
https://www.sigmaaldrich.com/content/dam/sigmaꢀaldrich/docs/Sigma/Datasheet/c1057dat.pdf) were
tested using the 5,5’ꢀdithiobisꢀ(2ꢀnitrobenzoic acid) (DTNB) method with the related assay kit (Keygen,
China). DTNB would generate 5ꢀmercaptoꢀ2ꢀnitrobenzoic acidꢀa detectable chromophore at the
405ꢀ412 nm range. The final concentrations (100 ꢀM, 20 ꢀM, 4 ꢀM, 0.8 ꢀM, 0.16 ꢀM, 0.032 ꢀM) of
screening compounds were prepared using DMSO. In 96ꢀwell plates, 20 ꢀL eeAChE (or hsBChE, 0.5
u/mL diluted using saline) was incubated with above different concentrations test compounds at room
temperature for 15 min followed by the addition of relative agents according to the kit instruction.
Finally, the chromophore absorbance was examined at the wavelength of 405 nm via microplate reader
(Thermo MK3). Percentage of inhibition was calculated by the comparison of compoundꢀtreated to
various control incubations.Molecular simulations
Preparation of hAChE/hBChE structures and ligands
The atomic coordinates of the hAChE and hBChE were obtained from the Protein Data Bank
(PDB ID: hAChE: 1B41 (resolution 2.76 Å)40 and hBChE: 1P0I (resolution 2.0 Å).41 Protein structures
42
were imported into the Maestro module available in the Schrödinger Suite (Schrödinger, LLC) and
subsequently, both structures were optimized separately by using the Protein Preparation Wizard.43
This protein structure optimization includes adding hydrogen atoms, assigning bond orders and
building disulfide bonds. The protonation states of the ionisable residues (pH = 7) were predicted by
the PROPKA tool provided in the Protein Preparation Wizard.41 An optimized structure was finally
found by energy minimization (i.e., position of the hydrogen atoms) with the OPLS_2005 force field.
The 3D structures of 8a2 and Tacrine were built with Maestro and preprocessed using the LigPrep
module (v2.5)44 of the Schrödinger modeling software. Preprocessing includes Energy minimization
using the OPLS_2005 force field and determination of ionization states at pH = 7 (+/ꢀ2).45
Molecular docking simulation
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The receptor grid generation module of Glide 46 was used to define both peripheral site (PAS) and
catalytic sites (CAS) for the docking experiments in hAChE and hBChE. A set of PAS and CAS
residues was as the centroid of the grid box (of size 20 Å).40,41 Water molecules at the active site were
deleted. Glide uses an inꢀbuild docking scoring function resulting in a Glidescore (Standard Precision).
The docking and scoring function parameters and settings used in this study are described in detail
elsewhere.47 The best 25 docking solutions for each protein structure were selected for investigation,
followed by MMꢀGBSA calculation.48ꢀ50 The Prime MMꢀGBSA method calculates the binding affinity
of the ligand (ꢁGbind) by energy minimization procedures. The binding energy of the ligand was
extracted from an energyꢀoptimized proteinꢀligand complex and Prime MMꢀGBSA by using the VSGB
2.0 solvation (implicit) model.51,50 During binding affinity calculation, the program offers the option to
treat the ligand and protein as flexible; in this study, a 5Å region of the protein around the ligand was
treated as flexible.
Conflict of Interest
The authors declare no conflict of interests.
Ethical Statement
All procedures were conducted in strict accordance with the Chinese relevant laws and institutional
guidelines (School of Pharmaceutical Sciences, Shandong University).This study was performed in
strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication
No. 85ꢀ23 Rev. 1985) and was approved by the Institutional Animal Care and Use Committee of
Shandong University (Shandong, China)".
Acknowledgements
Financial support from the Key Research and Development plan of Shandong Province (No.
2017CXGC1401), Major Project of Science and Technology of Shandong Province (No.
2015ZDJS04001) is gratefully acknowledged.
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the
final version of the manuscript.
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