Synthesis, biological activity, molecular docking studies of a novel series of 3-Aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as the acetylcholinesterase inhibitors

Article abstract of DOI:10.1080/07391102.2020.1753576

The acetylcholinesterase inhibitors play a critical role in the drug therapy for Alzheimer’s disease. In this study, twenty-nine novel 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized and assayed for their human acetylcholinesterase (hAChE) inhibitory activities. Inhibitory ratio values of seventeen compounds were above 55% with 4c having the highest value as 77.19%. The compounds with the halogen atoms in the aromatic ring, and N,N-diethylamino or N,N-dimethylamino groups in the side chains at C-3 positions exhibited good inhibitory activity. SAR study was carried out by means of molecular docking technique. According to molecular docking results, the common interacting site for all compounds were found to be peripheral anionic site whereas highly active compounds were interacting with the catalytic active site too. HIGHLIGHTS A novel series of 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized and assayed for their human acetylcholinesterase (hAChE) inhibitory activities. The SAR study of the target 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives was summarized. The active sites in the acetylcholinesterase were analyzed by molecular docking technique. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1753576

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Synthesis, Biological Activity, Molecular
Docking Studies of a Novel Series of 3-Aryl-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-one Derivatives as
the Acetylcholinesterase Inhibitors
Zhe Jin, Chao Zhang, Miao Liu, Simeng Jiao, Jing Zhao, Xiaoping Liu,
Huangquan Lin, David Chi-cheong Wan & Chun Hu
To cite this article: Zhe Jin, Chao Zhang, Miao Liu, Simeng Jiao, Jing Zhao, Xiaoping Liu,
Huangquan Lin, David Chi-cheong Wan & Chun Hu (2020): Synthesis, Biological Activity, Molecular
Docking Studies of a Novel Series of 3-Aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one Derivatives
as the Acetylcholinesterase Inhibitors, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1753576
To link to this article: https://doi.org/10.1080/07391102.2020.1753576
View supplementary material
Accepted author version posted online: 08
Apr 2020.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=tbsd20

Synthesis, Biological Activity, Molecular Docking Studies of a Novel Series of
3-Aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one Derivatives as the Acetylcholinesterase Inhibitors
Zhe Jin1, Chao Zhang1, Miao Liu1, Simeng Jiao1, Jing Zhao1, Xiaoping Liu1,*, Huangquan Lin2,
David Chi-cheong Wan2, Chun Hu1,*
1Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education; Shenyang
Pharmaceutical University, Shenyang 110016, China
2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong
Kong SAR, China
*Correspondences: Xiaoping Liu & Chun Hu, Key Laboratory of Structure-based Drug Design &
Discovery, Ministry of Education; Shenyang Pharmaceutical University, Shenyang 110016, China.
Emails: lxp19730107@163.com (X. Liu) & chunhu@syphu.edu.cn (C. Hu).
ABSTRACT
The acetylcholinesterase inhibitors play a critical role in the drug therapy for Alzheimer’s disease. In
this study, twenty-nine novel 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were
synthesized and assayed for their human acetylcholinesterase (hAChE) inhibitory activities.
Inhibitory ratio values of seventeen compounds were above 55 % with 4c having the highest value as
77.19 %. The compounds with the halogen atoms in the aromatic ring, and N,N-diethylamino or
N,N-dimethylamino groups in the side chains at C-3 positions exhibited good inhibitory activity.
SAR study was carried out by means of molecular docking technique. According to molecular
docking results, the common interacting site for all compounds were found to be peripheral anionic
site whereas highly active compounds were interacting with the catalytic active site too.
KEYWORDS: Heterocycle; Thiazolo[3,2-b]-1,2,4-triazine; Synthesis; Acetylcholinesterase Inhibitor;
SAR; Docking

HIGHLIGHTS:
1. A novel series of 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized and
assayed for their human acetylcholinesterase (hAChE) inhibitory activities.
2. The SAR study of the target 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives was
summarized.
3. The active sites in the acetylcholinesterase were analyzed by molecular docking technique.
1. INTRODUCTION
As Alzheimer’s disease (AD) was first reported in 1907, the number of people with AD has
increased rapidly with the life span of human beings increasing. The pathogenesis of Alzheimer’s
disease (AD) is very complicated, it is generally believed that AD ascribed to multiple factors,
including age, environment and heredity (Eckert et al., 2003; Pascoini, 2019; Rego & Oliveira, 2003).
The acetylcholinesterase (AChE) inhibitors play an important role in drug therapy for AD. So far,
there are only five drugs for AD treatment approved by the FDA (U.S. Food and Drug
Administration) - four are AchE inhibitors, including tacrine, donepezil, rivastigmine, galantamine,
and one N-methyl-D-aspartic acid (NMDA) receptor antagonist memantine (Eslami, Nezafat,
Negahdaripour, & Ghasemi, 2019; Shamsi, et al., 2020; Zheng, Fridkin, & Youdim, 2010). Among
them, tacrine has been discontinued in the US in 2013, due to hepatotoxicity (Atienzar, et al., 2016).
In addition, huperzine A has been approved by China National Medical Products Administration
(CNMPA). On November 2, 2019, CNMPA approved carbohydrate based drug GV-971 for treating
AD. The drug can restore the balance of intestinal flora, reduce the accumulation of peripheral
related metabolites phenylalanine/isoleucine, and improve the cognitive impairment (NMPA, 2019).
Even though AChE inhibitors play an important role in drug therapy for AD, the drugs can only
reduce symptoms and, to some extent, improve quality of life. There are still no effective drugs to
prevent，improve or reverse the occurrence of AD. The development of drug is a time-consuming,
expensive, and interdisciplinary process, which involves medicinal chemistry, pharmacology,
pharmacokinetics and clinical trials (de Almeida, et al., 2019; Taft, V. da Silva, & C. da Silva, 2008) .
The average cost of a newly marketed drug in the USA is $500 million to $800 million, and it takes
11 to 15 years. Several different computer-aided drug design (CADD) techniques have been used to

speed up the drug development process (Ece, & Sevin, 2010; Ece, 2020; Śledź, & Caflisch, 2018;
Neto, 2017; Taft, et al., 2008; Tahtaci, Karacık, Ece, Er, & Şeker, 2017) . These included molecular
docking, pharmacophore model building, physicochemical and ADME properties calculation, drug
target identification, structure-activity relationships (SAR) explanation. In this study，the structure of
AChE, AChE-ligand interactions and related active sites are analyzed by using CADD technology to
develop new and effective AD lead compounds.
By the end of September 2019, there are 206 AChE crystal structures in Research Collaboratory
for Structural Bioinformatics Protein Data Bank (RCSB PDB). Among them, 87 came from Torpedo
californica (Torpedo californica AChE, TcAChE), 72 came from Mus musculus (mouse AChE,
mAChE), and 39 came from Homo sapiens (human AChE, hAChE). The hAChE-donepezil complex
(PDB code 4EY7, solved at 2.3509 Å) (Cheung, et al., 2012) and the TcAChE-donepezil complex
(PDB code 1EVE, solved at 2.5 Å) (Kryger, Silman, & Sussman, 1999) have been superimposed to
observe the differences, especially the residues of the region near the active site of AChE. There are
significant differences between the binding sites of hAChE and TcAChE, which lead to the binding
of donepezil in different conformations. In this study, the hAChE-ligand complex was selected for
docking (Cheung, et al., 2012).
The most remarkable feature of the structure of AChE is a deep and narrow gorge, about 20 Å
long, the cavity is called ‘active-site gorge’. The catalysis active site (CAS), first discovered, is the
most important site for activity. It is located at the bottom of the active-site gorge and consists of
Ser200, Glu327, and His440 in TcAChE (Dvir, Silman, Harel, Rosenberry, & Sussman, 2010;
Sussman, et al., 1991). The peripheral anionic site (PAS) is located near the entrance of the gorge
and consists of Tyr70, Asp72, Tyr121, Glu278, Trp279 and Tyr334, and some small molecule
inhibitors block the entrance of the active-site gorge to exert the inhibitory effects (Khalid, et al.,
2010; Shiri, Pirhadi, & Ghasemi, 2019; Wan, et al., 2020). In 2005, a series of tacrine-indole
heterodimers were found to display excellent AChE inhibition, with IC50 value of 20 pM on AChE
(Muñoz-Ruiz, et al., 2005). Molecular docking results showed other sites participated in the
interactions. The quaternary ammonium binding site (QABS) consists of Trp84, Tyr130, Glu199,
and Phe330, and is located between CAS and PAS (Harel, Quinn, Nair, Silman, & Sussman, 1996;
Khalid, et al., 2010; Kryger, et al., 1999; Wan, et al., 2020). The amino acid residues of TcAChE

active sites (PDB ID: 1FSS) (Harel, Kleywegt, Ravelli, Silman, & Sussman, 1995), including CAS,
PAS and QABS; and the residues of hAChE active sites (PDB ID: 2X8B) (Carletti, et al., 2010) are
shown in Figure 1. Each color in the Figures was represented one active site: CAS for green, PAS for
blue violet, and QABS for deep pink.
AChE inhibitors, on the market interact with different active sides of the AChE. They are listed in
Table 1.
Table 1 shows that AChE inhibitors interact with CAS, PAS and QABS, and each of them except
rivastigmine, interacts with at least two kinds of active sites. Based on these, twenty-nine
3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed and synthesized, their AChE
inhibitory activities were evaluated, and the active sites of the target compounds were calculated by
molecular modeling.
2. MATERIALS AND METHODS
2.1. Chemistry
The synthetic strategy to prepare the target compounds is illustrated in Schemes 1-2.
All reagents and solvents were purchased from common commercial suppliers and were used
without further purification unless otherwise indicated. Melting points were taken on a Koffler
hot-plate apparatus and were uncorrected. Mass spectra (MS) were obtained using an electronic
impact (EI) ion source at 70 eV in an Agilent spectrometer (with direct insertion probe) or with an
electrospray (ESI) ion source in a Waters spectrometer at 3.5 kV spray voltage, acetonitrile was used
as the solvent. 1H NMR spectra were determined on a Bruker spectrometer operating at 300 MHz or
400 MHz or 600 MHz with TMS as the internal standard, and DMSO-d6 or CDCl3 was used as
solvent. The IR spectra were recorded on a Bruker AFS55 spectrometer.
The styrene derivatives were synthesized according to the previous publications (Marvel, &
Schertz, 1943). The styrene derivatives reacted with KMnO4 alkaline aqueous solution, and
hydrochloric acid was added to acidify the reaction solution to produce keto acids 1. Compounds 2
were prepared by using compound 1 reacted with thiosemicarbazide in sodium hydroxide aqueous
solution. The target compounds 3a-3h were obtained by a condensation reaction of compounds 2
with substituted phenacyl chlorides in acetic acid. The target compounds 4a-4e were prepared by
3f-3h with substituted alkyl chloride by Williamson reaction shown in Scheme 1 (Jin,et al., 2010a;

Jin,et al., 2010b).
The 4-arylmethylideneoxazol-5(4H)-ones 5 were easily converted from the aromatic aldehydes by
cyclization. Then, the hydrolysis of 5 in the acetone aqueous solution resulted in
α-(acetylamino)cinnamic acids 6, subsequently, 6 were converted to aryl pyruvic acids 7 by
treatment with diluted hydrochloric acid. Compounds 7 reacted with thiosemicarbazide to give the
cyclized products 3-thioxo-1,2,4-triazin-5(2H)-ones 8. The target compounds 9a-9d were prepared
by a condensation reaction of compounds 8 with substituted phenacyl chlorides in acetic acid. The
target compounds 10a-10l could be obtained with Williamson reaction shown in Scheme 2 (Liu, et
al., 2009; Liu, Shang, Shi, Wan, & Lin, 2014) .
2.2. hAChE activity assay
The hAChE inhibitory activity values of the tested target compounds were evaluated by means of
Ellman’s test method (Ellman, Courtney, Andres, & Featherstone, 1961; Liu, et al., 2009; Liu, et al.,
2010; Liu, et al., 2013; Liu, et al., 2014; Malinak, et al., 2020; Mosmann, 1983; Xu, et al., 2012).
The hAChE stock solution was prepared by dissolving hAChE 0.5 unit in 100 mM phosphate
buffered saline (PBS) buffer (pH 7.4). The tested target compounds (10 mM) were dissolved in
DMSO. The assay solution consisted of 100 mM PBS buffer (pH 7.4), 10 mM
5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent), 5 mL hAChE, 10 mL drug (the tested
target compounds or positive control huperzine-A), and 12.5 mM acetylthiocholine iodide (ATCh)
water solution. The final assay volume was 900 mL. The tested target compounds were incubated the
reaction at 37 °C for 15 min with continuous gentle shake, added 50mL ATCh and 50 mL DTNB,
incubated at 37 °C for about 20 min with continuous gentle shake, waited until the yellow color
developed. The absorbance values were measured at 412 nm, and the inhibition values were
calculated.
2.3. Computational Methods
The protein structure was obtained from the crystallographic complex of human AChE in complex
with fasciculin-II (RCSB PDB ID: 2X8B), the resolution value of the complex crystal was 2.95 Å
(Carletti, et al., 2010); and the complex of human AChE in complex with donepezil (RCSB PDB ID:
4EY7), the resolution value of the complex crystal was 2.3509 Å (Cheung, et al., 2012).

The complexes were prepared by Schrödinger’s protein preparation wizard, which removed the
hetero atoms and water molecules from the protein, built the missing side chain atoms, corrected the
structure of the protein and added hydrogen atoms before docking (Shivakumar, et al., 2010).
Afterwards, the OPLS_2005 force field was used to optimize protein energy and eliminate steric
hindrance (Itteboina, Ballu, Sivan, & Manga, 2017). The positions of the native ligand was defined
to generate grid for subsequent molecular docking work (Wang, et al., 2018).
The Molegro Virtual Docker (MVD) software version 4.1.0 was used for the molecular docking
process (Han, Lai, & Du, 2014; Khan, Fuskevåg, & Sylte, 2009; Vaqué, et al., 2008), the interactions
between hAChE and the ligands were calculated by using Lamarckian Genetic Algorithm, the grid
resolution was 0.3 Å, the binding site radius was 15 Å, and other parameters were all default values.
All the compounds were docked into the active sites of hAChE (PDB code 2X8B and 4EY7). The
complexes of ligand-receptor were viewed by Discovery Studio ViewerPro ViewerLite50 program
(Kangueane, & Nilofer, 2018; Yang, 2010; Zhu, Wang, Li, Han, & Zhao, 2017).
3. RESULTS AND DISCUSSION
3.1 Chemistry
The structures of all the target compounds are listed in Table 2. The structures of all the newly
1
synthesized compounds were characterized by H-NMR, MS, and IR. The synthetic procedures,
experimental details and spectra data for the target compounds are available as the supplementary
material.
2.2. Biological evaluation
Table 3 lists the hAChE inhibitory activity values of the target compounds, and the positive
control drug was huperzine A (from synthetic, ≥98% (TLC), Sigma), 0.5 unit hAChE (Sigma C-1682)
was used. Each was performed in thrice. The incubation time was 20 min. The assay procedures for
the target compounds are available as the supplementary material.
Some of the target compounds exhibited good hAChE inhibitory activity, the inhibitory activity
values of seventeen compounds were above 55 %, and the highest value of 4c was 77.19 %.

All the target compounds were substituted in the aromatic rings and the side chains at C-3 and C-6
positions. On the whole, different substituted groups and side chains had great influences on AChE
inhibitory activity. The target compounds of which the halogen atoms in the aromatic rings, and
N,N-diethylamino groups, N,N-dimethylamino groups in the side chains at C-3 positions (compounds
3b, 3c, 3d, 10e, 10f, 10h, 10i, 10k), exhibited good inhibitory activity. The existence of morpholine
rings at C-3 positions decreased the inhibitory activity (compounds 4b, 4d, 10e, 10a, 10g).
2.3. Molecular docking
The model was obtained by docking ligand (the target compounds) to the hAChE (from RCSB
PDB ID: 2X8B) using the methods we have described in section 2.4. Each of the target compounds
was docked to simulate the hydrogen bonds interaction sites of the minimum energy conformation of
the compounds. The detailed interactions for the target compounds are available as the
supplementary material.
The internal validation of molecular docking was carried out. PDB ID of 4EY7 was selected to
check the RMSD value. In the co-crystal complex structure of 4EY7, the ligand was donepezil. The
native ligand in the complex and the docking ligand were overlapped for comparison (Figure 2). The
RMSD value was 0.575 Å, which showed the molecular docking method was reliable.
The target compounds were classified into three groups in accordance to the hAChE inhibitory
activity values of the compounds, highly active compounds (activity values > 55 %), moderately
active compounds (activity values 55 % - 40 %) and inactive compounds (activity values < 40 %).
The hydrogen bond interaction sites of the minimum energy conformation of the compounds are
listed in Tables 4-6.
Table 4-6 show the interaction site in hAChE and the target compounds. For the seventeen group I
compounds, the hydrogen bond interaction sites of the highly active compounds included at least two
active sites: ten interacted with CAS and PAS; five interacted with CAS and QABS; two interacted
with CAS, QABS and PAS. All included CAS active site. In group II, for the six moderately active
compounds. Three compounds interacted with QABS and PAS. Another three only with PAS. In
group III, for the six inactive compounds. Five compounds interacted with PAS, and one with QABS
and PAS.

The active sites of hAChE and compounds 4a, 4d and 10g are drawn in Figure 3.
In Figure 3, the three target compounds 4a, 10g, 4d were embedded in the gorge no matter how
different conformations of the compounds. The hAChE inhibitory activity value of 4a was 58.12 %,
because the compound was at the bottom of the gorge, and there was a hydrogen bond between
Ser203 of CAS and 4a. The inhibitory activity value of 10g was 43.20 %. The compound was at the
middle part of the gorge, and the hydrogen bonds were observed between QABS, PAS and 10g. The
inhibitory activity value of 4d was only 10.65 %, because the compound was located at the upper
part of the gorge, and the hydrogen bonds were available between PAS and 4d.
All the minimum energy conformations of the target compounds are depicted in Figure 4 and
Figure 5. The classified methods of the target compounds were in accordance to Table 2. The target
compounds were divided into 3 groups in accordance with the hAChE inhibitory activity values of
the compounds; above 55 %, below 40 %, and others.
Figure 4 shows that, most of the seventeen target compounds were embedded in the gorge no matter
how different the conformations of the compounds were. All the compounds were at the bottom of
the gorge, and there were hydrogen bonds between the target compounds and Ser203 of CAS.
Figure 5 shows that, most of the six target compounds were inserted into the middle deep of the
gorge at similar conformations. Hydrogen bonds are observed between QABS, PAS and the target
compounds.
Figure 6 shows all the six compounds scattered at the upper part of the gorge, and the hydrogen
bonds interaction are available between PAS and the target compounds.
The hydrogen bond interaction sites of the twenty-nine target compounds are the same as
compounds 4a, 10g, 4d.
2.4 Verification for molecular docking
The molecular docking of the synthesized compounds was also carried out using X-ray crystal
structure of hAChE in complexed with pharmacologically important ligand, Donepezil (PDB ID:
4EY7, 2.35 Å resolution). The docking results used 4EY7 are listed in Tables 7-9. The hydrogen
bonds interaction sites, hydrogen bonds active sites, and other interactions sites are summarized. The
same hydrogen bonds interaction sites and active sites as 2X8B were bold. The details are described
as the supplementary material.

The verification results of hAChE (4EY7) showed that in group I, seventeen highly active
compounds, the hydrogen bond interaction sites of seven compounds were at the catalytic active site
(CAS), one was at CAS and the peripheral anionic site (PAS), three were at CAS and the quaternary
ammonium binding sites (QABS); two were at CAS, QABS, PAS, and the thirteen out of seventeen
included CAS active site.
In comparison, the results of hAChE (2X8B) showed that in group I, the hydrogen bond
interaction sites included at least two active sites: ten with CAS and PAS; five with CAS and QABS;
the other two with CAS, QABS and PAS, and all included CAS active site.
The verification results with hAChE (4EY7) showed that in group II, six moderately active
compounds, the hydrogen bond interaction sites of four compounds were with QABS and PAS, and
another two were with PAS only.
In comparison, the results with hAChE (2X8B) showed that in group II, the hydrogen bond
interaction sites of three compounds were with QABS and PAS, and another three were with PAS
only.
The verification results with hAChE (4EY7) showed that in group III, the six inactive compounds,
the hydrogen bond interaction sites of four compounds were with PAS, and another two were with
QABS and PAS.
In comparison, the results with hAChE (2X8B) showed that in group III, the hydrogen bonds
interaction sites of five compounds were with PAS only, and another one was with QABS and PAS.
Based on above analysis in Tables 4-9, the crucial amino acid residues were identified as Ser203 in
CAS, Trp86 and Tyr133 in QABS, Tyr72, Asp74, Tyr124, Glu202, Tyr337, Tyr341 in PAS.
Interaction of compounds with hAChE were analyzed to verify the involvement of the crucial
amino
acids
found
from
molecular docking,
the
hAChE-ligand
contact
atlas
(https://www.mrc-lmb.cam.ac.uk/rajini/index.html) and the binding interaction of ligands (analyzed
by using Discovery Studio Visualizer v16.1.0.15350) of the three PDB (code 4EY5, ligand is
(-)-huperzine A; code 4EY6, ligand is (-)-galantamine; code 4EY7, ligand is donepezil) were
analyzed. The AChE-ligand contacts are represented by asteroid plot and 3-D binding interaction plot
as shown in Figure 7 to Figure 9.
The asteroid plots of hAChE co-crystal structure and the binding interactions showed the following
amino acid residues in hAChE are important for the interaction with compounds: Tyr72, Asp74,

Trp86, Gly120, Gly121, Gly122, Tyr124, Gly126, Tyr133, Glu202, Ser203, Trp286, Ser293, Phe295,
Tyr337, Phe338, Tyr341, His447. Tyr72, Asp74, Trp86, Tyr124, Gly126, Ser203, Ser293, Phe295,
Tyr337, His447 are especially crucial because there were hydrogen bonds between the compounds
and AChE, Trp86, Gly121, Tyr124, Ser203, Tyr337, Phe338, Tyr341, His447 are made weak
interactions with the compounds. The results are basically consistent with molecular docking.
3. CONCLUSION
The goal of this study was to synthesize a series of 3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives, to evaluate their hAChE inhibitory activities, and to study the structure-activity
relationship by using molecular docking method.
Twenty-nine target compounds were synthesized, the structures of all the novel synthesized
compounds were characterized by 1H NMR, MS, and IR. All the target compounds exhibited hAChE
inhibitory activity, the values of seventeen compounds were above 55 %, and the highest value of 4c
was 77.19 %.
The target compounds of which the halogen atoms in the aromatic rings, and N,N-diethylamino
groups, N,N-dimethylamino groups in the side chains at C-3 positions exhibited good inhibitory
activity. The existence of morpholine rings at C-3 positions decreased the inhibitory activity.
Most of the seventeen target compounds of which the hAChE inhibitory activity values were
above 55 % were embedded in the active site gorge disregard the conformations of the compounds,
these conformations were high incidence. Most of the compounds were docked into the bottom of the
gorge, and there were hydrogen bonds between the compounds and Ser203 of CAS. Most of the six
target compounds of which the hAChE inhibitory activity values were between 40 % and 55 % were
inserted into the middle part of the gorge, and hydrogen bond interaction existed between QABS,
PAS and the compounds. All the six compounds with hAChE inhibitory activity values below 40 %
were only inserted into the upper part of the gorge, and hydrogen bond interaction existed between
PAS and the compounds. Ser203 in CAS, Trp86 and Tyr133 in QABS, Tyr72, Asp74, Tyr124,
Glu202, Tyr337, Tyr341 in PAS were crucial amino acid residues. The molecular docking results and
confirmation of crucial amino acid residues have been verified by two hAChE-ligand co-crystal
complexes.

ACKNOWLEDGMENT
This work was supported by the National Science Foundation of China (NSFC) for the grant No.
21072130 & 21342006; the Program for Innovative Research Team of the Ministry of Education of
China (Grant No. IRT_14R36), the Natural Science Foundation of Liaoning Province, China for the
grant No. 201602695 and 2019-ZD-0458; and the Scientific Research Foundation of Department of
Education of Liaoning Province, China for the grant No. L2015517. The authors would like to thank
Molegro ApS for kindly providing a free evaluation copy of their software package, Dassault
Systèmes Biovia Co. for providing Discovery Studio Visualizer v16.1.0.15350, and Accelrys Co. for
allowing the use of ViewerLite 5.0 software.
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
FUNDING
National Science Foundation of China (NSFC) with grant No. 21072130 & 21342006.
REFERENCES
Atienzar, F. A., Blomme, E. A., Chen, M., Hewitt, P., Kenna, J. G., Labbe, G., Moulin, F., Pognan,
F., Roth, A. B., Suter-Dick, L., Ukairo, O., Weaver, R. J., Will, Y., & Dambach, D. M. (2016).
Key challenges and opportunities associated with the use of in vitro models to detect human
DILI: integrated risk assessment and mitigation plans. BioMed Research International, 2016,
9737920. https://doi.org/10.1155/2016/9737920.
Carletti, E., Colletier, J. P., Dupeux, F., Trovaslet, M., Masson, P., & Nachon, F. (2010). Structural
evidence that human acetylcholinesterase inhibited by tabun ages through O-dealkylation.
Journal of Medicinal Chemistry, 53(10), 4002-4008. https://doi.org/10.1021/jm901853b.
Cheung, J., Rudolph, M., Burshteyn, F., Cassidy, M., Gary, E., Love, J., Franklin, M., & Height, J.
(2012). Structures of human acetylcholinesterase in complex with pharmacologically important
ligands.
Journal
of
Medicinal
Chemistry,
55(22),
10282–10286.

https://doi.org/10.1021/jm300871x.
de Almeida, J. S. F. D., Cavalcante, S. F. A., Dolezal, R., Kuca, K., Musilek, K., Jun, D., & França, T.
C. C. (2019). Molecular modeling studies on the interactions of aflatoxin B1 and its metabolites
with the peripheral anionic site of human acetylcholinesterase. Journal of Biomolecular
Structure and Dynamics, 37(8), 2041-2048. https://doi.org/10.1080/07391102.2018.1475259.
Dvir, H., Silman, I., Harel, M., Rosenberry, T., & Sussman, J. (2010). Acetylcholinesterase: From 3D
structure
to
function.
Chemico-Biological
Interactions,
187(1-3),
10-22.
https://doi.org/10.1016/j.cbi.2010.01.042.
Ece, A. (2020). Towards more effective acetylcholinesterase inhibitors: a comprehensive modelling
study based on human acetylcholinesterase protein-drug complex. Journal of Biomolecular
Structure and Dynamics, 38(2), 565-572. https://doi.org/10.1080/07391102.2019.1583606.
Ece, A., & Sevin, F. (2010). Exploring QSAR on 4-cyclohexylmethoxypyrimidines as antitumor
agents for their inhibitory activity of CDK2. Letters in Drug Design & Discovery, 7(9), 625-631.
https://doi.org/10.2174/157018010792929612.
Eckert, A., Keil, U., Marques, C., Bonert, A., Frey, C., Schüssel, K., & Müller, W. (2003).
Mitochondrial dysfunction, apoptotic cell death, and Alzheimer's disease. Biochemical
Pharmacology, 66(8), 1627-1634. https://doi.org/10.1016/S0006-2952(03)00534-3.
Ellman, G., Courtney, K., Andres, V., & Featherstone, R. (1961). A new and rapid colorimetric
determination of acetylcholinesterase activity. Biochemical Pharmacology, 7(2), 88-95.
https://doi.org/10.1016/0006-2952(61)90145-9.
Eslami, M., Nezafat, N., Negahdaripour, M., & Ghasemi, Y. (2019). Computational approach to
suggest a new multi-target-directed ligand as a potential medication for Alzheimer’s disease.
Journal
of
Biomolecular
Structure
and
Dynamics,
37(18),
4825-4839.
https://doi.org/10.1080/07391102.2018.1564701.
Han, L., Lai, P., & Du, J.-R. (2014). Deciphering molecular mechanism underlying hypolipidemic
activity of echinocystic acid. Evidence-Based Complementary and Alternative Medicine, 2014,
1-7. https://doi.org/10.1155/2014/823154.
Harel, M., Kleywegt, G. J., Ravelli, R. B., Silman, I., & Sussman, J.L. (1995). Crystal structure of an
acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom
with its target. Structure, 3(12),1355-1366. https://doi.org/10.1016/s0969-2126(01)00273-8.

Harel, M., Quinn, D., Nair, H., Silman, I., & Sussman, J. (1996). The X-ray structure of a transition
state analog complex reveals the molecular origins of the catalytic power and substrate
specificity of acetylcholinesterase. Journal of the American Chemical Society, 118(10),
2340-2346. https://doi.org/10.1021/ja952232h.
Itteboina, R., Ballu, S., Sivan, S. & Manga, V. (2017). Molecular modeling-driven approach for
identification of Janus kinase 1 inhibitors through 3D-QSAR, docking and molecular dynamics
simulations. Journal of Receptors and Signal Transduction, 37(5), 453-469.
https://doi.org/10.1080/10799893.2017.1328442.
Jin, Z., Yang, L., Liu, S.-J., Wang, J., Li, S., Lin, H.-Q., Wan, D., & Hu, C. (2010a). Synthesis and
biological evaluation of 3,6-diaryl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives as
acetylcholinesterase inhibitors. Archives of Pharmacal Research, 33(10), 1641–1649.
https://doi.org/10.1007/s12272-010-1013-8.
Jin, Z., Yang, L., Xu, X. N., Huang, E. R., Wan, D. C. Li, S., Huang, H. Q., & Hu, C. (2010b).
Synthesis and biological activity of 3,6-diaryl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one derivatives
as novel acetylcholinesterase inhibitors. Science China Chemistry, 53(11), 2297-2303.
https://doi.org/10.1007/s11426-010-4094-9.
Kangueane, P., & Nilofer, C. (2018). Patented Protein Structural Complexes in Discovery Platform.
In:
Protein-Protein
and
Domain-Domain
Interactions.
Springer,
Singapore.
https://doi.org/10.1007/978-981-10-7347-2_17.
Khalid, A., Zaheer-ul-Haq, Anjum, S., Khan, M., Atta-ur-Rahman, & Choudhary, M. (2004).
Kinetics and structure–activity relationship studies on pregnane-type steroidal alkaloids that
inhibit cholinesterases. Bioorganic
&
Medicinal Chemistry, 12(9), 1995-2003.
https://doi.org/10.1016/j.bmc.2004.03.002.
Khan, M., Fuskevåg, O.-M., & Sylte, I. (2009). Discovery of potent thermolysin inhibitors using
structure based virtual screening and binding assays. Journal of Medicinal Chemistry, 52(1),
48-61. https://doi.org/10.1021/jm8008019.
Kryger, G., Silman, I., & Sussman, J. (1999). Structure of acetylcholinesterase complexed with
E2020 (Aricept): implications for the design of new anti-Alzheimer drugs. Structure, 7(3),
297-307. https://doi.org/10.1016/S0969-2126(99)80040-9.
Liu, S., Shang, R., Shi, L., Wan, D., & Lin, H. (2014). Synthesis and biological evaluation of

7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as dual binding site acetylcholinesterase
inhibitors. European Journal of Medicinal Chemistry, 81, 237-244.
https://doi.org/10.1016/j.ejmech.2014.05.020.
Liu, S.-J., Cui, L.-B., Xu, H.-L., Wang, T.-Y., Hu, C., Li, S., Lin, H.-Q., & Wan, D. (2013). Design,
synthesis, and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as
dual binding site acetylcholinesterase inhibitors. Heterocycles, 87(12), 2607-2614.
https://doi.org/10.3987/COM-13-12820.
Liu, S.-J., Yang, L., Jin, Z., Huang, E.-F., Wan, D., Lin, H.-Q., & Chun H. (2009). Design, synthesis,
and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as novel
acetylcholinesterase
inhibitors.
ARKIVOC,
(x),
333-348.
https://doi.org/10.3998/ark.5550190.0010.a30.
Liu, S.-J., Yang, L., Liu, X.-G., Luo, Y., Cao, Z.-J., Wan, D., Lin, H.-Q., & Hu, C. (2010). Design,
synthesis, and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as
acetylcholinesterase inhibitors. Letters in Drug Design & Discovery, 7(1), 5-8.
https://doi.org/10.2174/157018010789869343.
Malinak, D., Dolezal, R., Hepnarova, V., Hozova, M., Andrys, R., Bzonek, P., Racakova, V.,
Korabecny, J., Gorecki, L., Mezeiova, E., Psotka, M., Jun, D., Kuca, K., & Musilek, K. (2020).
Synthesis, in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as
acetylcholinesterase and butyrylcholinesterase reactivators. Journal of Enzyme Inhibition and
Medicinal Chemistry, 35(1), 478-488. https://doi.org/10.1080/14756366.2019.1710501.
Marvel, C., & Schertz, G. (1943). Copolymers of p-chlorostyrene and methyl methacrylate. Journal
of the American Chemical Society, 65(11), 2054–2058. https://doi.org/10.1021/ja01251a006.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. Journal of Immunological Methods, 65(1-2), 55-63.
https://doi.org/10.1016/0022-1759(83)90303-4.
Muñoz-Ruiz, P., Rubio, L., García-Palomero, E., Dorronsoro, I., del Monte-Millán, M., Valenzuela,
R., Usán, P., de Austria, C., Bartolini, M., Andrisano, V., Bidon-Chanal, A., Orozco, M., Luque,
F., Medina, M., & Martínez, A. (2005). Design, synthesis, and biological evaluation of dual
binding site acetylcholinesterase inhibitors: new disease-modifying agents for Alzheimer's
disease.
Journal
of
Medicinal
Chemistry,
48(23),
7223-7233.

https://doi.org/10.1021/jm0503289.
National Medical Products Administration. The National Medical Products Administration
conditional approved GV-971 as the mild to moderate Alzheimer's disease drug. [2019-11-09].
http://www.nmpa.gov.cn/WS04/CL2056/359779.html.
Neto, D., Lima, J., de Almeida, J., França, T., Nascimento, C., & Villar, J. (2017). New
semicarbazones as gorge-spanning ligands of acetylcholinesterase and potential new drugs
against Alzheimer’s disease: Synthesis, molecular modeling, NMR, and biological evaluation.
Journal
of
Biomolecular
Structure
and
Dynamics,
1-15.
https://doi.org/10.1080/07391102.2017.1407676.
Pascoini, A. L., Federico, L. B., Arêas, A. L. F., Verde, B. A., Freitas P. G., & Camps, I. (2019). In
silico development of new acetylcholinesterase inhibitors. Journal of Biomolecular Structure
and Dynamics, 37(4), 1007-1021. https://doi.org/10.1080/07391102.2018.1447513.
Polinsky, R. (1998). Clinical pharmacology of rivastigmine: a new-generation acetylcholinesterase
inhibitor for the treatment of alzheimer's disease. Clinical Therapeutics, 20(4), 634–647.
https://doi.org/10.1016/S0149-2918(98)80127-6.
Raves, M., Harel, M., Pang, Y.-P., Silman, I., Kozikowski, A., & Sussman, J. (1997). Structure of
acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A. Nature Structural
& Molecular Biology, 4(1), 57-63. https://doi.org/10.1038/nsb0197-57.
Rego, A., & Oliveira, C. (2003). Mitochondrial disfunction and reactive oxygen species in
excitotoxicity and apoptosis: implications for the pathogenesis of neurodegenerative diseases.
Neurochemical Research, 28(10), 1563-1574. https://doi.org/10.1023/A:1025682611389.
Shamsi, A., Al Shahwan, M., Ahamad, S., Hassan, M. I., Ahmad, F., & Islam, A. (2020).
Spectroscopic, calorimetric and molecular docking insight into the interaction of Alzheimer’s
drug donepezil with human transferrin: implications of Alzheimer’s drug. Journal of
Biomolecular
Structure
and
Dynamics,
38(4),
1094-1102.
https://doi.org/10.1080/07391102.2019.1595728.
Shiri, F.,Pirhadi, S., & Ghasemi, J. B. (2019). Dynamic structure based pharmacophore modeling
of the Acetylcholinesterase reveals several potential inhibitors. Journal of Biomolecular
Structure and Dynamics, 37(7), 1800-1812. https://doi.org/10.1080/07391102.2018.1468281.
Shivakumar, D., Williams, J., Wu, Y., Damm, W., Shelley, J. & Sherman, W. (2010). Prediction of

absolute solvation free energies using molecular dynamics free energy perturbation and the
OPLS force field. Journal of Chemical Theory and Computation, 6(5), 1509-1519.
https://doi.org/10.1021/ct900587b.
Śledź, P., & Caflisch, A. (2018). Protein structure-based drug design: from docking to molecular
dynamics.
Current
Opinion
in
Structural
Biology,
48,
93-102.
https://doi.org/10.1016/j.sbi.2017.10.010.
Sussman, J., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., & Silman, I. (1991). Atomic
structure of acetyl-cholinesterase from Torpedo californica: a prototypic acetylcholine-binding
protein. Science, 253(5022), 872-879. https://doi.org/10.1126/science.1678899.
Taft, C., da Silva, V., & da Silva, C. (2008). Current topics in computer‐aided drug design. Journal of
Pharmaceutical Sciences, 97(3), 1089-1098. https://doi.org/10.1002/jps.21293.
Tahtaci, H., Karacık, H., Ece, A., Er, M., & Şeker, M. (2017). Design, synthesis, SAR and molecular
modeling studies of novel imidazo[2,1-b][1,3,4]thiadiazole derivatives as highly potent
antimicrobial
agents.
Molecular
Informatics,
37(3),
1700083.
https://doi.org/10.1002/minf.201700083.
Vaqué, M., Ardévol, A., Bladé, C., Salvadó, M., Blay, M., Fernández-Larrea J., Arola, L., & Pujadas,
G. (2008). Protein-ligand docking: a review of recent advances and future perspectives. Current
Pharmaceutical Analysis, 4(1), 1-19. https://doi.org/10.2174/157341208783497597.
Wan, Y.-F., Guan, S.-S., Qian, M.-D., Huang, H.-H., Han, F., Wang, S., & Zhang, H.
(2020). Structural basis of fullerene derivatives as novel potent inhibitors of protein
acetylcholinesterase without catalytic active site interaction: insight into the inhibitory
mechanism through molecular modeling studies. Journal of Biomolecular Structure and
Dynamics, 38(2), 410-425. https://doi.org/10.1080/07391102.2019.1576543.
Wang, M., Li, W., Wang, Y., Song, Y., Wang, J., & Cheng, M. (2018). In silico insight into
voltage-gated sodium channel 1.7 inhibition for anti-pain drug discovery. Journal of Molecular
Graphics and Modelling, 84, 18-28. https://doi.org/10.1016/j.jmgm.2018.05.006.
Xu, H., Jin, Z., Liu, S., Liu, H., Li, S., Lin, H., Wan, D., & Hu, C. (2012). Design, synthesis
characterization
3-aryl-6-(bromoarylmethyl)-7H-thiazolo[3,2-b]-1, 2, 4-triazin-7-one derivatives as the novel
acetylcholinesterase inhibitors. Chinese Chemical Letters, 23(7), 765-768.
and
in
vitro
biological
activity
of
a
series
of

https://doi.org/10.1016/j.cclet.2012.04.022.
Yang, S. Y. (2010). Pharmacophore modeling and applications in drug discovery: challenges and
recent
advances.
Drug
Discovery
Today,
2010,
15(11-12):444-450.
https://doi.org/10.1016/j.drudis.2010.03.013.
Zheng, H., Fridkin, M., & Youdim, M. B. (2010). Site-activated chelators derived from
anti-Parkinson drug rasagiline as a potential safer and more effective approach to the treatment
of
Alzheimer's
disease.
Neurochemical
Research,
35(12),
2117-23.
https://doi.org/10.1007/s11064-010-0293-1.
Zhu, J., Wang, Y., Li, X., Han, W., & Zhao, L. (2017). Understanding the interactions of different
substrates with wild-type and mutant acylaminoacyl peptidase using molecular dynamics
simulations. Journal of biomolecular Structure
&
Dynamics, 36(16), 1-43.
https://doi.org/10.1080/07391102.2017.1414634.

Figure 1. The amino acid residues of TcAChE active site (a. left, 1FSS), hAChE active site (b. right,
2X8B). CAS for green, PAS for blue violet, and QABS for deep pink.

Figure 2. The overlapping conformations of the native ligand and the docking ligand of 4EY7.

Figure 3. Compounds 4a, 10g, 4d in the hAChE active sites (PDB code: 2X8B) (a. left, viewed along
the “gorge”; b. right, viewed clearly different active sites). CAS for green, PAS for blue violet,
QABS for deep pink. Compound 4a for red, 4d for blue, 10g for dark orange.

Figure 4. The highly active compounds in the hAChE active sites (PDB code: 2X8B)

Figure 5. The moderately active compounds in the hAChE active sites (PDB code: 2X8B)

Figure 6. The inactive compounds in the hAChE active sites (PDB code: 2X8B)

Figure 7. The asteroid plot (left) and binding interaction (right) of (-)-huperzine A at the active sites
of hAChE (PDB code: 4EY5).

Figure 8. The asteroid plot (left) and binding interaction (right) of (-)-galantamine at the active sites
of hAChE (PDB code: 4EY6).

Figure 9. The asteroid plot (left) and binding interaction (right) of donepezil at the active sites of
hAChE (PDB code: 4EY7).

Scheme 1. The synthetic route of 3a-3h and 4a-4e

Scheme 2. The synthetic route of 9a-9d and 10a-10l