4-Oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide derivatives as new anti-Alzheimer agents: design, synthesis, in vitro evaluation, molecular modeling, and molecular dynamic study

Article abstract of DOI:10.1007/s11224-019-01472-0

A new series of 4-oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide hybrids 8a–p was designed, synthesized, and screened as the potential cholinesterase inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Obtained anti-chol

Full text of DOI:10.1007/s11224-019-01472-0

Structural Chemistry
https://doi.org/10.1007/s11224-019-01472-0
ORIGINAL RESEARCH
4-Oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide derivatives
as new anti-Alzheimer agents: design, synthesis, in vitro evaluation,
molecular modeling, and molecular dynamic study
Fahimeh Hosseini1 & Maryam Mohammadi-Khanaposhtani2 & Homa Azizian3 & Ali Ramazani1
&
Maliheh Barazandeh Tehrani4 & Hamid Nadri5 & Bagher Larijani6 & Mahmoud Biglar6 & Hossein Adibi6
&
Mohammad Mahdavi6
Received: 6 October 2019 /Accepted: 3 December 2019
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A new series of 4-oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide hybrids 8a–p was designed, synthesized, and screened
as the potential cholinesterase inhibitors against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Obtained anti-
cholinesterase activities demonstrated that all the title compounds exhibited excellent inhibition against BuChE and moderate
inhibitory activity toward AChE in comparison to standard cholinesterase inhibitor donepezil. For example, compound 8e
exhibited about 49-fold higher inhibitory activity than donepezil (IC50 = 3.2 0.3 μM) against BuChE. This compound inhibited
BuChE via a mixed-type inhibition mode. This finding demonstrated that compound 8e in addition to catalytic anionic site (CAS)
can also interact with the peripheral anionic site (PAS) of BuChE. Molecular modeling and molecular dynamic studies were also
performed on synthesized compounds.
.
.
.
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Keywords Anti-Alzheimer 4-Oxobenzo[d]1,2,3-triazin Phenylacetamide Molecular dynamic Molecular modeling
Introduction
AD [2]. The exact pathogenic mechanism of this disease re-
mains unclear but researchers suggests that factors such as
decrease neurotransmitter acetylcholine (ACh) level in the
hippocampal and cortical regions of the brain, the ab-
normal deposition of β-amyloid peptides (Aβ), the for-
mation of neurofibrillary tangles, oxidative stress, and
Alzheimer’s disease (AD) is a neurodegenerative disorder
with main clinical manifestations such as progressive memory
loss, language deterioration, and severe cognitive decline [1].
The most common cause of dementia in the elderly people is
Fahimeh Hosseini and Maryam Mohammadi-Khanaposhtani contributed
equally to this work.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-019-01472-0) contains supplementary
material, which is available to authorized users.
3
* Ali Ramazani
Department of Medicinal Chemistry, School of
aliramazani@znu.ac.ir
Pharmacy-International Campus, Iran University of Medical
Sciences, Tehran, Iran
* Mohammad Mahdavi
4
momahdavi@tums.ac.ir
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
1
5
Department of Chemistry, University of Zanjan,
P.O. Box 45195-313, Zanjan, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Shahid Sadoughi
University of Medical Sciences, Yazd, Iran
2
6
Cellular and Molecular Biology Research Center, Health Research
Institute, Babol University of Medical Sciences, Babol, Iran
Endocrinology and Metabolism Research Center, Endocrinology and
Metabolism Clinical Sciences Institute, Tehran University of Medical
Sciences, Tehran, Iran

Struct Chem
biometallic dysregulation can be involved in the crea-
tion and progression of AD [3].
oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide hy-
brids 8a–p was assessed by Ellman’s method [13]. DMSO
(1 mL) was used as the solvent for dissolving the title
compounds and preparing appropriate stock. The stock
of the synthesized compounds was diluted by using phos-
phate buffer to achieve final concentrations. A mixture of
phosphate buffer (0.1 M, pH = 8.0, 50 μL), DTNB
(0.1 M, 125 μL), AChE or BuChE (0.2 U/ml, 25 μL),
and yeast compound (25 μL) was incubated for 5 min at
room temperature. Then, acetylthiocholine iodide or
butyrylthiocholine iodide (0.01 M, 10 μL) was added,
and the change of the absorbance was measured at
405 nm after 15 min. Synergy HTX Multi-Mode Reader
(BioTek) was used for these assays.
Today, the most common treatment for AD is the use of
acetylcholinesterase (AChE) inhibitors in order to in-
crease the synaptic levels of ACh, because AChE is re-
sponsible for the degradation of ACh in the brain [4].
Another enzyme that can partially degrade ACh in the
brain is butyrylcholinesterase (BuChE) [5]. Recently, sev-
eral evaluations revealed that with the progress of AD,
AChE activity in the brain of patients is gradually de-
creased while BuChE activity is significantly increased
[6]. Therefore, inhibition of both of these enzymes may
be useful in the treatment of AD [7, 8].
During the last decade, our research group using the
molecular hybridization approach has introduced numer-
ous structures with anti-Alzheimer’s activities [9, 10].
Recently, we reported 4-oxobenzo[d]1,2,3-triazin-benzyl
pyridinium derivatives A as novel potent AChE inhibitors
[11]. On the other hand, phenylacetamide derivatives B
showed the inhibitory activity against AChE [12].
Therefore, herein, in order to design new cholinesterase
(ChE) inhibitors, we replaced benzyl group of the scaffold
A with phenylacetamide moiety of scaffold B; designed 4-
oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamide hy-
brids 8a–p were synthesized and evaluated for their
in vitro ChE inhibitory activities against AChE and
BuChE. Furthermore, molecular modeling and molecular
dynamic studies were performed to evaluation interactions
of the synthesized compounds with AChE and BuChE.
Kinetic study of BuChE inhibition
The inhibition mode and Ki value of the most BuChE
inhibitor 8e were determined by the modified Ellman’s
method. The related experiments in this step were per-
formed similar to the enzyme inhibition assay. The rate
of enzymatic reaction was measured in the different con-
centrations of the compound 8e (0, 30, 60, and 120 nM)
in order to draw Lineweaver–Burk reciprocal plots to es-
timate the type of inhibition. Finally, slopes of the latter
plots were plotted against the different concentrations of
the test compound 8e in order to determine Ki value.
Enzymatic reactions were performed in triplicate, and data
analysis was performed by Microsoft Excel 2013.
Target and ligand preparation
Experimental details
In order to find out the interaction modes of the synthe-
sized compounds over ChEs, Maestro Molecular
Modeling platform (version 11.5: Schrodinger, LLC:
New York, NY, 2018) was performed [14]. Initially, the
proper crystal structures of AChE (PDB code: 6o4w) and
BuChE (PDB code: 4bds) were retrieved from the Protein
Data Bank (http://www.rcsb.org) [15, 16]. These pdb ids
were selected based on criteria like highest resolution (Å),
being related to Homo sapiens specious, wild type with
no modified residue, and the existence of co-crystalized
ligand. As the prosthetic group and the co-factors are not
directly involved in ChE inhibition, so they totally re-
moved before docking investigation. Except the four
structural waters molecule in the AChE and BuChE active
site, which bridge the receptor important residues by way
of hydrogen bond and conserve among other specious, the
rest of the water molecules were removed from crystallo-
graphic structures of the target enzymes [17]. The 2D
structures of all synthesized compounds were drawn in
Marvin 15.10.12.0 program and converted into pdb
(http://www.chemaxon.com.). The Protein Preparation
Material and methods
Melting points of 4-oxobenzo[d]1,2,3-triazin-pyridinium-
phenylacetamide hybrids 8a–p were measured on a Kofler
hot stage apparatus. The NMR (1H and 13C) and IR spec-
tra of the title compounds were obtained by using a
Bruker FT-500 and Nicolet Magna FTIR 550 spectropho-
tometer (KBr disks), respectively. Elemental analysis was
measured by an Elementar Analysensystem GmbH
VarioEL CHNS mode.
Cholinesterase inhibition assay
The target enzymes (AChE (E.C. 3.1.1.7, type V-S, lyoph-
ilized powder, from electric eel, ≥ 1000 units/mg protein)
and BuChE (E.C. 3.1.1.8, from equine serum)), substrates
(acetylthiocholine iodide and butyrylthiocholine iodide),
indicator (DTNB [5,5′-dithiobis(2-nitrobenzoic acid)]),
and standard drug (donepezil) were purchased from
Sigma-Aldrich. The anti-cholinesterase activity of 4-

Struct Chem
Wizard and the LigPrep module were used to prepare
protein and ligand structure properly [18].
trajectory-clustering tool, the representative structure with the
largest number of neighbors from the equilibrated trajectory
was evaluated for investigation of each ligand-protein complex.
Docking protocol
Prime MM-GBSA
The appropriate pose for the compounds over AChE and
BuChE was determined by induced fir docking protocol
(IFD) [18]. At the active site of AChE and BuChE, the
co-crystalized ligand (donepezil and tacrine, respectively)
was used to generate the grid for IFD calculation. The
maximum 20 poses with receptor and ligand Van der
Waals radii of 0.7 and 0.5, respectively, were considered.
Residues within 5 Å of the donepezil and tacrine at the
active site were refined followed by side-chain optimiza-
tion. Structures whose prime energy is more than 30 Kcal/
mol eliminated based on extra precious glide docking.
The energetically favorable IFD complexes were obtained
for compounds and applied to MD simulation.
The ligand binding energies (ΔG bind) were calculated
using Molecular Mechanics/Generalized Born Surface
Area (MM-GBSA) modules (Schrödinger LLC 2018)
[18] based on the following equation: ΔG Bind = E
Complex − [E Receptor + E Ligand], where ΔG Bind is
the calculated relative free energy which includes both
ligand and receptor strain energy. E Complex is the
MM-GBSA energy of the minimized complex, and E
Ligand is the MM-GBSA energy of the ligand after re-
moving it from the complex and allowing it to relax. E
Receptor is the MM-GBSA energy of relaxed protein after
separating it from the ligand. The MM-GBSA calculation
performed on the structures with the higher number of
neighbors in cluster was obtained from MD simulation
trajectory.
Molecular dynamics simulation
Molecular simulations of this study were performed using the
Desmond v5.3 using Maestro interface (from Schrödinger
2018–4 suite) [18]. We evaluated the stability of the best drug
candidate from the experimental study, compound 8p and 8e
over AChE and BuChE, respectively, and compared their per-
turbation with the standard inhibitor donepezil.
General procedure for the synthesis of pyridine
derivatives 3a–b
A mixture of isatoic anhydride 1 (1 mmol) and pyridin-3-
ylmethanamine 2a or pyridin-4-ylmethanamine 2b
(1 mmol) in water (15 mL) was stirred at room tempera-
ture for 8 h. Then, the reaction mixture was filtered off
and the precipitates were collected and washed with water
to obtain pure pyridine derivatives 3a–b.
In order to building system for MD simulation, the protein-
ligand complexes were solvated by SPC explicit water molecules
and placed in the center of an orthorhombic box of appropriate
size in the Periodic Boundary Condition (PBC). Sufficient
counter-ions and a 0.15 M solution of NaCl also utilized to
neutralize the system and to simulate the real cellular ionic con-
centrations, respectively. The MD protocol involved minimiza-
tion, pre-production, and finally production of MD simulation
steps. In the minimization procedure, the entire system was
allowed to relax for 2500 steps by the steepest descent approach.
Then, the temperature of the system was raised from 0 to 300 K
by applying a small force constant on the enzyme in order to
restrict any drastic changes. MD simulations were performed via
NPT (constant number of atoms, constant pressure, i.e.,
1.01325 bar and constant temperature, i.e., 300 K) ensemble.
The Nose-Hoover chain method was used as the default thermo-
stat with 1.0 ps interval and Martyna-Tobias-Klein as the default
barostat with 2.0-ps interval by applying isotropic coupling style.
Long-range electrostatic forces were calculated based on particle-
mesh-based Ewald approach with the cut-off radius for columbic
forces set to 9.0 Å. Finally, this system subjected to production
MD simulations for 20 ns for each protein-ligand complex.
During the simulation, every 1000 ps of the actual frame was
stored. The dynamic behavior and structural changes of the sys-
tems were analyzed by the calculation of energy and the root
mean square deviation (RMSD). Subsequently, by using
General procedure for the produce of 3 or 4-(pyridin)
benzo[d][1,2,3]triazin-4(3H)-ones 4a–b
A suspension of the pyridine derivatives 3a–b (1 mmol), sodium
nitrite (1 mmol), and hydrochloride acid 37% (1 mL) in water
(5 mL) was stirred at 0 °C for 10 min. Then, the obtained residue
was cooled to 4 °C and filtered off and the participates were
washed with the mixture of water/ethanol to give pure 3 or
4-(pyridin)benzo[d][1,2,3] triazin-4(3H)-ones 4a–b.
General procedure for the synthesis
of N-phenyl-2-chloroacetamides 7a–h
A mixture of aniline derivatives 5a–h and chloroacetyl chlo-
ride 6 in DMF was stirred at room temperature for 30 min.
Then, the reaction mixture was diluted with water, poured into
crashed ice, and the obtained precipitate were filtered off.
Finally, residue was washed with cold water to obtain pure
N-phenyl-2-chloroacetamides 7a–h.

Struct Chem
General procedure for the synthesis of 4-oxobenzo[d]
1,2,3-triazin-pyridinium-phenylacetamides 8a–p
1H, NH), 9.13 (s, 1H), 9.04 (d, J = 5 Hz, 1H), 8.78 (d, J =
7.5 Hz, 1H), 8.19–7.89 (m, 5H), 7.51 (d, J = 7.75 Hz, 2H),
6.86 (d, J = 7.75 Hz, 2H), 5.83 (s, 2H, CH2), 5.65 (s, 2H,
CH2). 13C NMR (CDCl3, 62.9 MHz): δ = 162.8, 155.9,
155.2, 146.2, 145.5, 144.1, 136.4, 135.9, 133.5, 132.4,
128.5, 127.6, 125.0, 124.2, 121.0 (2C), 119.8, 114.4 (2C),
62.5, 55.5, 50.6. Anal. calcd for C22H20ClN5O3 (437.88): C,
60.34; H, 4.60; N, 15.99; found: C, 60.38; H, 4.51; N, 15.78.
The N-phenyl-2-chloroacetamides 7a–h (1 mmol) were
added to the stirred solution of compounds 4a–b
(1 mmol) in dry acetonitrile (10 mL), and the final mix-
ture was heated at reflux for 2–3 h. Upon completion
(checked by TLC), acetonitrile was evaporated and n-
hexane (20 mL) was added to the residue and obtained
precipitates were separated by filtration and washed
with n-hexane to afford the pure title compounds 8a–p.
1-(2-((3,4-Dimethoxyphenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8d)
1-(2-Oxo-2-(phenylamino)ethyl)-3-((4-oxobenzo[d]
[1,2,3]triazin-3(4H)-yl)methyl)pyridin-1-ium chloride
(8a)
Cream solid; yield: 70%; mp: 175–177 °C. IR (KBr): v =
3225, 3172, 3044, 2925, 2849, 1698, 1642, 1596, 1541,
1
1491, 1401, 1248, 1090, 918, 829. H NMR (CDCl3,
Yellow solid; yield: 68%; mp: 198–200 °C. IR (KBr): v =
250 MHz): δ = 10.45 (s, 1H, NH), 9.05–9.01 (m, 2H), 8.78
(d, J = 5 Hz, 1H), 8.20–7.94 (m, 6H), 7.22–7.23 (m, 1H),
7.02–6.86 (m, 2H), 5.78 (s, 2H, CH2), 5.58 (s, 2H, CH2).
13C NMR (CDCl3, 62.9 MHz): δ = 162.0, 155.7, 155.5,
148.7, 146.0, 145.4, 144.0, 136.7, 136.2, 133.8, 131.9,
128.7, 125.0, 119.9, 112.5, 104.5, 62.4, 56.3, 55.9, 50.0.
Anal. calcd for C23H22ClN5O4 (467.9): C, 59.04; H, 4.74;
N, 14.97; found: C, 59.05; H, 4.65; N, 14.73.
3232, 3080, 3042, 2840, 1694, 1682, 1640, 1600, 1546,
1
1465, 1337, 1299, 1250, 1199, 1075, 952, 904. H NMR
(CDCl3, 250 MHz): δ = 10.60 (s, 1H, NH), 9.07–9.02 (m,
2H), 8.75 (s, 1H), 8.21–7.93 (m, 5H), 7.49 (m, 2H), 7.28
(m, 2H), 7.03(s, 1H), 5.80 (s, 2H, CH2), 5.61 (s, 2H,
CH2). 13C NMR (CDCl3, 62.9 MHz): δ = 163.1, 155.5,
146.0 (2C), 145.5, 143.9, 138.2, 136.5, 136.2, 133.8,
129.3 (2C), 128.6, 127.7, 125.0, 124.4, 119.5 (2C),
62.4, 50.0. Anal. calcd for C21H18ClN5O2 (407.85): C,
61.84; H, 4.45; N, 17.17; found: C, 62.87; H, 4.49; N,
17.18.
1-(2-((4-Chlorophenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8e)
1-(2-Oxo-2-(p-tolylamino)ethyl)-3-((4-oxobenzo[d]
[1,2,3]triazin-3(4H)-yl)methyl)pyridin-1-ium chloride
(8b)
Cream solid; yield: 80%; mp: 175–177 °C. IR (KBr): v =
3253, 3187, 3042, 2852, 1697, 1681, 1648, 1601, 1541,
1
1491, 1401, 1290, 1247, 1081, 985, 827. H NMR (CDCl3,
250 MHz): δ = 10.67 (s, 1H, NH), 9.07–9.04 (m, 2H), 8.73 (s,
1H), 8.14–7.93 (m, 5H), 7.49 (d, J = 5 Hz, 2H), 7.30 (d, J =
5 Hz, 2H), 5.75 (s, 2H, CH2), 5.63 (s, 2H, CH2).13C NMR
(CDCl3, 62.9 MHz): δ = 163.4, 156.4, 149.7, 144.0, 141.2,
137.2, 136.5, 136.2, 133.8, 129.7, 129.1 (2C), 128.6, 127.8,
125.0, 121.3 (2C), 120.9, 119.8, 62.6, 50.0. Anal. calcd for
C21H17Cl2N5O2 (442.3): C, 57.03; H, 3.87; N, 15.83; found:
C, 57.07; H, 3.85; N, 15.78.
Cream solid; yield: 70%; mp: 123–125 °C. IR (KBr): v =
3420, 3030, 2923, 1685, 1616, 1577, 1512, 1478, 1428,
1
1332, 1293, 1058, 912. H NMR (CDCl3, 250 MHz): δ =
10.75 (s, 1H, NH), 9.12 (s, 1H), 9.01 (d, J = 5 Hz, 2H),
8.78 (d, J = 7.5 Hz, 1H), 8.17–8.77 (m, 5H), 7.44 (d, J =
5.5 Hz, 2H), 7.08 (d, J = 5.5 Hz, 2H), 5.83 (s, 2H, CH2),
5.59 (s, 2H, CH2), 2.20 (s, 3H, CH3).13C NMR (CDCl3,
62.9 MHz): δ = 164.8, 155.2, 146.1, 145.2, 144.1, 136.2,
135.9, 133.5, 133.2, 132.4, 129.6 (2C), 128.5, 125.0,
124.1, 120.3,119.5 (2C), 62.3, 50.6, 20.8. Anal. calcd
for C22H20ClN5O2 (421.88): C, 62.63; H, 4.78; N,
16.60; found: C, 62.53; H, 4.75; N, 16.67.
1-(2-((2,3-Dichlorophenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)yl)methyl)
pyridin-1-ium chloride (8f)
Cream solid; yield: 68%; mp: 146–148 °C. IR (KBr): v =
1-(2-((4-Methoxyphenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8c)
3455, 3030, 2923, 1686, 1605, 1578, 1478, 1429, 1332,
1293, 1050, 912. H NMR (CDCl3, 250 MHz): δ = 10.53
1
(s, 1H, NH), 9.12 (s, 1H), 9.01 (d, J = 4.6 Hz,1H), 8.76 (d,
J = 7.5 Hz, 1H), 8.24–8.07 (m, 5H), 7.92 (t, J = 7.5 Hz,
1H), 7.69 (t, J = 8 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.37
(d, J = 7.5 Hz, 1H), 5.82 (s, 2H, CH2), 5.69 (s, 2H, CH2).
13C NMR (CDCl3, 62.9 MHz): δ = 163.6, 155.2, 146.3,
Yellow solid; yield: 75%; mp: 144–146 °C. IR (KBr): v =
3425, 3031, 2851, 1685, 1604, 1577, 1510, 1470, 1428,
1294, 1059, 912. 1H NMR (CDCl3, 250 MHz): δ = 10.42 (s,

Struct Chem
145.1, 144.1, 138.7, 136.3, 136.0, 133.5, 133.2, 132.4,
128.6, 127.6, 129.6, 128.5 (2C), 125.0, 124.2, 119.8,
62.2, 50.6. Anal. calcd for C21H16Cl3N5O2 (476.74): C,
52.91; H, 3.38; N, 14.69; O, 6.71; found: C, 52.23; H,
3.41; N, 16.79.
1-(2-Oxo-2-(p-tolylamino)ethyl)-4-((4-oxobenzo[d]
[1,2,3]triazin-3(4H)-yl)methyl)pyridin-1-ium chloride
(8j)
Cream solid; yield: 65%; mp: 160–162 °C. IR (KBr): v =
3425, 2924, 2854, 1685, 1604, 1577, 1512, 1459, 1414,
1
1330, 1054, 917. H NMR (CDCl3, 250 MHz): δ = 11.22
1-(2-((4-Bromophenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8g)
(s, 1H, NH), 9.03 (d, J = 5 Hz, 2H), 8.25–7.96 (m, 6H),
7.51(d, J = 7.5 Hz, 2H), 7.10 (d, J = 7.5 Hz, 2H), 5.95 (s,
2H, CH2), 5.70 (s, 2H, CH2), 2.21(s, 3H, CH3). 13C NMR
(CDCl3, 62.9 MHz): δ = 163.3, 156.8, 155.6, 146.6 (2C),
144.3, 136.1, 133.7, 133.2, 129.6 (2C), 128.7(2C), 125.7
(2C), 125.0, 120.07, 119.5 (2C), 62.1, 52.2, 20.8. Anal.
calcd for C22H20ClN5O2 (421.88): C, 62.63; H, 4.78; N,
16.60; found: C, 62.73; H, 4.68; N, 16.61.
Cream solid; yield: 76%; mp: 151–153 °C. IR (KBr): v =
3425, 3030, 2851, 1686, 1606, 1589, 1549, 1478, 1428,
1
1293, 1026, 912. H NMR (CDCl3, 250 MHz): δ = 10.73
(s, 1H, NH), 9.13 (s, 1H), 9.05 (d, J = 4.7 Hz, 1H), 8.78
(d, J = 7.5 Hz, 1H), 8.18–8.01 (m, 5H), 7.57 (d, J =
7.25 Hz, 2H), 7.46 (d, J = 7.25 Hz, 2H), 5.83 (s, 2H,
CH2), 5.69 (s, 2H, CH2). 13C NMR (CDCl3, 62.9 MHz):
δ = 163.7, 155.2, 146.2, 145.5, 144.1, 138.3, 138.1, 136.7,
135.9, 133.5, 132.6, 132.0, 128.5, 127.6, 125.0, 124.3,
1 2 1 . 6 , 11 9 . 8 , 6 2 . 7 , 5 0 . 0 . A n a l . c a l c d f o r
C21H17BrClN5O2 (486.75): C, 51.82; H, 3.52; N, 14.39;
found: C, 51.83; H, 3.56; N, 14.38.
1-(2-((4-Methoxyphenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8k)
Cream solid; yield: 52%; mp: 201–203 °C. IR (KBr): v =
3423, 3030, 2855, 1686, 1604, 1578, 1510, 1471, 1428,
1
1293, 1059, 912. H NMR (CDCl3, 250 MHz): δ = 10.45
(s, 1H, NH), 9.06 (d, J = 5 Hz, 2H), 8.25–7.95 (m, 6H),
7.40 (d, J = 7.5 Hz, 2H), 6.84 (d, J = 7.5 Hz, 2H), 5.90 (s,
2H, CH2), 5.71 (s, 2H, CH2). Anal. calcd for
C22H20ClN5O3 (437.88): C, 60.34; H, 4.60; N, 15.99;
found: C, 60.28; H, 4.68; N, 15.88.
1-(2-((4-Nitrophenyl)amino)-2-oxoethyl)
-3-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8h)
Cream solid; yield: 690%; mp: 158–160 °C. IR (KBr):
v = 3452, 3196, 3044, 2848, 1685, 1642, 1617, 1565,
1508, 1466, 1345, 1254, 1176, 1069, 918. 1H NMR
(CDCl3, 250 MHz): δ = 10.73 (s, 1H, NH), 9.08–9.01
(m, 3H), 8.76 (d, J = 7.7 5 Hz, 1H), 8.36–7.90 (m, 7H),
7.78 (d, J = 8.5 Hz, 2H), 5.78 (s, 2H, CH2), 5.71 (s, 2H,
CH2). Anal. calcd for C21H17ClN6O4 (452.85): C, 55.70;
H, 3.78; N, 18.56; found: C, 55.59; H, 3.77; N, 18.59.
1-(2-((3,4-Dimethoxyphenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8l)
Cream solid; yield: 81%; mp: 190–192 °C. IR (KBr): v =
3224, 3171, 3045, 2922, 2840, 1697, 1643, 1596, 1540,
1491, 1401, 1248, 1090, 918, 829. 1H NMR (CDCl3,
250 MHz): δ = 10.39 (s, 1H, NH), 9.00 (s, 2H), 8.14–
7.93 (m, 6H), 7.18 (s, 1H), 6.88–6.80 (m, 2H), 5.88 (s,
2H, CH2), 5.65 (s, 2H, CH2). 13C NMR (CDCl3,
62.9 MHz): δ = 162.7, 156.6, 155.1, 148.5, 146.3 (2C),
144.0, 136.4, 133.9, 131.7, 128.7, 125.7 (2C), 125.0,
124.4, 119.7, 112.4, 111.6, 104.6, 61.8, 56.6, 56.1, 52.3.
Anal. calcd for C23H22ClN5O4 (467.9): C, 59.04; H, 4.74;
N, 14.97; found: C, 59.13; H, 4.75; N, 14.87.
1-(2-Oxo-2-(phenylamino)ethyl)-4-((4-oxobenzo[d]
[1,2,3]triazin-3(4H)-yl)methyl)pyridin-1-ium chloride
(8i)
Yellow solid; yield: 78%; mp: 170–172 °C. IR (KBr): v =
3186, 3045, 1694, 1640, 1598, 1547, 1466, 1310, 1253,
1
1075, 1016, 908. H NMR (CDCl3, 250 MHz): δ = 10.58
1-(2-((4-Chlorophenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8m)
(s, 1H, NH), 8.99 (s, 2H), 8.15 (s, 2H), 7.54–7.06 (m,
9H), 5.93 (s, 2H), 5.64 (s, 2H). 13C NMR (CDCl3,
62.9 MHz): δ = 163.5, 156.9, 146.6 (2C), 139.5, 138.5,
136.2, 133.8, 129.4, 125.7(2C), 125.0, 124.4, 119.6
(2C), 62.0, 52.2. Anal. calcd for C21H18ClN5O2
(407.85): C, 61.84; H, 4.45; N, 17.17; found: C, 61.86;
H, 4.41; N, 17.08.
Yellow solid; yield: 87%; mp: 140–142 °C. IR (KBr): v =
3225, 3172, 3044, 2925, 2849, 1698, 1642, 1596, 1541,
1491, 1401, 1248, 1090, 918, 829.1H NMR (CDCl3,
250 MHz): δ = 10.73 (s, 1H, NH), 9.02 (d, J = 5.5 Hz,

Struct Chem
2H), 8.17–7.91 (m, 6H), 7.52 (d, J = 8.25 Hz, 2H), 7.31
(d, J = 8.25 Hz, 2H), 5.91 (s, 2H, CH2), 5.69 (s, 2H, CH2).
13C NMR (CDCl3, 62.9 MHz): δ = 163.6, 156.8, 155.6,
146.5 (2C), 144.1, 137.3, 136.3, 133.9, 129.3 (2C),
128.7, 127.9, 125.7 (2C), 125.0, 121.3, 119.9 (2C),
61.9, 52.3. Anal. calcd for C21H17Cl2N5O2 (442.3): C,
57.03; H, 3.87; N, 15.83; found: C, 57.05; H, 3.85; N,
15.89.
(2C), 144.6, 144.3, 143.1, 136.1, 133.7, 128.8, 125.8
(2C), 125.6 (2C), 125.0, 120.1, 119.5 (2C), 62.2, 52.3.
Anal. calcd for C21H17ClN6O4 (452.85): C, 55.70; H,
3.78; N, 18.56; found: C, 55.57; H, 3.91; N, 18.50.
Results and discussion
Chemistry
1-(2-((2,3-Dichlorophenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8n)
The synthetic pathway for the preparation of the 4-
oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamides
8a–p is depicted in Scheme 1. It began from the reaction
between isatoic anhydride 1 and pyridin-3-ylmethanamine
2a or pyridin-4-ylmethanamine 2b in H2O at room tem-
perature to give pyridine derivatives 3a–b. Compounds
3a–b in the presence of sodium nitrite and hydrochloride
acid (in H2 O) at 0 °C were converted to 4-
oxobenzo[d]1,2,3-triazin-pyridine derivatives 4a–b. On
the other hand, N-phenyl-2-chloroacetamides 7a–h were
prepared by the reaction between aniline derivatives 5a–h
and chloroacetyl chloride 6 in DMF at room temperature.
Finally, 4-oxobenzo[d]1,2,3-triazin-pyridine derivatives
4a–b reacted with N-phenyl-2-chloroacetamides 7a–h in
acetonitrile at reflux to give the desired compounds 8a–p.
Cream solid; yield: 89%; mp: 117–119 °C. IR (KBr): v =
3425, 2924, 2854, 1682, 1642, 1604, 1577, 1512, 1459,
1
1299, 1054, 979, 917. H NMR (CDCl3, 250 MHz): δ =
10.96 (s, 1H, NH), 9.13 (d, J = 5 Hz, 2H), 8.21–8.02 (m,
4H), 7.93 (t, J = 6.25 Hz, 2H), 7.67 (d, J = 7.5 Hz, 1H),
7.43 (d, J = 6.75 Hz,1H), 7.33 (d, J = 6.25 Hz, 1H), 5.95
(s, 2H, CH2), 5.87 (s, 2H, CH2). 13C NMR (CDCl3,
MHz): δ = 164.7, 156.9, 155.6, 146.7 (2C), 144.2, 136.4,
136.1, 133.7, 132.4, 128.7, 128.4 (2C), 127.7, 125.8 (2C),
125.2, 125.0, 120.0, 62.0, 52.2. Anal. calcd for
C21H16Cl3N5O2 (476.74): C, 52.91; H, 3.38; N, 14.69;
found: C, 52.98; H, 3.31; N, 14.79.
1-(2-((4-Bromophenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8o)
Cholinesterase inhibitory assay
4-Oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamides
8a–p were evaluated for their in vitro AChE/BuChE in-
hibitory activities. The IC50 values of these compounds in
comparison to standard inhibitor donepezil are listed in
Table 1. The ChE inhibitory activities of the compounds
8a–p demonstrated that all these compounds showed
moderate AChE inhibitory activity (IC50 = 2.0 0.1–6.5
Cream solid; yield: 72%; mp: 140–142 °C. IR (KBr): v =
3438, 3171, 2925, 2852, 1694, 1604, 1545, 1488, 1464,
1
1331, 1271, 1007, 920. H NMR (CDCl3, 250 MHz): δ =
11.56 (s, 1H, NH), 9.06 (s, 2H), 8.22–7.94 (m, 6H), 7.61
(d, J = 7.5 Hz, 2H), 7.47 (d, J = 7.5 Hz, 2H), 5.94 (s, 2H,
CH2), 5.74 (s, 2H, CH2). 13C NMR (CDCl3, 62.9 MHz):
δ = 163.9, 156.9, 155.6, 146.7(2C), 144.3, 138.2, 136.1,
133.7, 132.1 (2C), 128.7, 125.8 (2C), 125.0, 121.5, 120.1
(2C), 115.9, 62.2, 52.2. Anal. calcd for C21H17BrClN5O2
(486.75): C, 51.82; H, 3.52; N, 14.39; found: C, 51.70; H,
3.51; N, 14.38.
0.1 μM) in comparison with the donepezil (IC50
=
0.029 0.006 μM) while all the synthesized compounds
showed excellent BuChE inhibitory activity (IC50 = 0.065
0.002–0.279 0.023 μM) in comparison with donepezil
(IC50 = 3.2 0.3 μM).
Structurally, 4-oxobenzo[d]1,2,3-triazin-pyridinium-
phenylacetamides 8a–p could be divided into two se-
ries: 3-pyridinium derivatives 8a–h and 4-pyridinium
derivatives 8i–p.
The inhibitory activity of 3-pyridinium series against
AChE demonstrated that 4-nitro derivative 8h showed
the most potent activity, while 4-methyl and 4-methoxy
derivatives 8b and 8c were less active compounds.
Removal of 4-nitro substituent, and or replacement of this
substituent with bromine atom, led to a significant de-
crease in the inhibitory activity (compound 8h vs. com-
pounds 8a and 8g). Moreover, the introduction of 3,4-
dimethoxy or 2,3-dichloro on pendant phenyl group did
1-(2-((4-Nitrophenyl)amino)-2-oxoethyl)
-4-((4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)methyl)
pyridin-1-ium chloride (8p)
Yellow solid; yield: 73%; mp: 178–180 °C. IR (KBr): v =
3453, 3197, 3044, 2849, 1684, 1643, 1617, 1565, 1507,
1466,1344, 1258, 1179, 1071, 918. 1H NMR (CDCl3,
250 MHz): δ = 11.26 (s, 1H, NH), 8.99 (d, J = 6.25 Hz,
2H), 8.27–8.09 (m, 6H), 7.96 (t, J = 7.75 Hz, 2H), 7.78 (d,
J = 9 Hz, 2H), 5.96 (s, 2H, CH2), 5.69 (s, 2H, CH2). 13C
NMR (CDCl3, 62.9 MHz): δ = 164.8, 157.2, 155.6, 146.7

Struct Chem
O
O
O
H2O
N
H
NaNO2/H2O
HCl/0 oC/10 min
+
NH2
N
N
N
H
O
RT/8 h
NH2
3a-b
2a: pyridin-3-ylmethanamine
2b: pyridin-4-ylmethanamine
1
O
H
O
N
Cl
N
H
N
N
N
N
N
Cl
CH3CN
Reflux/2-3h
R
N
N
+
O
O
R
N
7a-h
4a-b
8a-p
DMF RT/30 min
NH2
O
+
Cl
R
Cl
5a-h
6
R = H, 4-CH3, 4-OCH3,
3,4-Dimethoxy,
4-Cl, 2,3-Dichloro,
4-Br, 4-NO2
Scheme 1 Synthesis of 4-oxobenzo[d]1,2,3-triazin-pyridinium-phenylacetamides 8a–p.
not improve inhibitory activity as observed in the com-
pounds 8d and 8f.
pendant phenyl group did not improve anti-BuChE activ-
ity as observed in compounds 8g–h.
The observed IC50 values of 4-pyridinium series
against AChE revealed that the better results were obtain-
ed with 4-nitro, 2,3-dichloro, 4-chloro, 4-methyl substitu-
ents (compounds 8p, 8n, 8m, and 8j). In this series, 4-
methoxy and 3,4-methoxy derivatives 8k and 8l were the
less active compounds. Moreover, un-substituted com-
pound 8i and 4-bromo derivative 8o also showed moder-
ate activity against AChE.
The comparison of IC50 values of 3-pyridinium derivatives
8a–h with their corresponding 4-pyridinium analogs 8i–p
against AChE revealed that 4-pyridinium derivatives were
more potent than their regioisomers.
In the term of BuChE inhibitory activity in 3-
pyridinium series, compounds 8e, 8f, and 8b with 4-
chloro, 2,3-dichloro, and 4-methyl substituents on pen-
dant phenyl group showed highest inhibitory activities.
The fourth potent compound in this series was un-
substituted derivative 8a. The introduction of 4-methoxy
substituent on the pendant phenyl ring of the compound
8a, as in compound 8c, slightly decreased inhibitory ac-
tivity. The addition of the second methoxy group onto 3-
position of the pendant phenyl ring led to a significant
decrease in the inhibitory activity (compound 8c vs. 8d).
Moreover, the introduction of 4-bromo or 4-nitro on the
n the 4-pyridinium series (compounds 8i–p), com-
pounds 8k, 8p, and 8j with 4-methoxy, 4-nitro, and meth-
yl substituents, respectively, were the most active com-
pounds against BuChE. The less active compound in this
series was un-substituted compound 8i. The trend of the
inhibitory activity of the remaining compounds, based on
the substituent on pendant phenyl ring, was 2,3-dichloro
(compound 8n) > 4-Cl (compound 8m) > 4-Br (compound
8o) > 3,4-dimethoxy (compound 8l).
As can be seen in the Table 1, 3-pyridinium deriva-
tives 8a–b and 8d–g were more potent than their
regioisomers of 4-pyridinium series. In contrast, 4-
pyridinium derivatives 8c and 8p were significantly more
active than their corresponding 3-pyridinium regioisomers 8c
and 8h.
The comparison of IC50 values of 4-oxobenzo[d]1,2,3-
triazin-pyridinium-phenylacetamides 8a–p with our previ-
ous reported compounds A against AChE revealed that
compounds A were more active than compounds 8a–p
(Fig. 1) [11]. In contrast, the anti-BuChE activity of the
compounds 8a–p was significantly more than compounds
A. Moreover, AChE and BuChE inhibitory activities of
the compounds 8a–p are more than phenylacetamide de-
rivatives B (Fig. 1) [12].

Struct Chem
Table 1 Inhibitory activities of the synthesized compounds 8a–p against AChE/BuChE
O
O
H
N
N
N
O
R
N
N
N
N+
N
O
N
H
N
R
8a-h
8i-p
Compound
R
AChE IC50 (μM)
BuChE IC50 (μM)
8a
H
5.6 0.2
6.5 0.1
5.9 0.3
4.7 0.2
4.7 0.1
4.1 0.1
5.6 0.5
2.8 0.1
3.1 0.1
2.2 0.1
4.7 0.4
3.9 0.1
2.0 0.1
1.9 0.8
3.5 0.1
1.9 0.2
0.029 0.006
0.097 0.003
0.075 0.004
0.107 0.004
0.193 0.003
0.065 0.002
0.073 0.006
0.147 0.001
0.165 0.003
0.279 0.023
0.107 0.001
0.085 0.003
0.223 0.001
0.190 0.014
0.173 0.16
0.203 0.004
0.094 0.001
3.2 0.302
8b
4-CH3
8c
4-OCH3
3,4-Dimethoxy
4-Cl
8d
8e
8f
2,3-Dichloro
4-Br
8g
8h
4-NO2
8i
H
8j
4-CH3
8k
4-OCH3
3,4-Dimethoxy
4-Cl
8l
8m
8n
2,3-Dichloro
4-Br
8o
8p
4-NO2
Donepezil
–
Kinetic study
compound 8e inhibited BuChE in a mixed-type inhibition
mode. Therefore, this compound can interact with both the
catalytic anionic site (CAS) and peripheral anionic site
(PAS) of BuChE. The Ki value of the compound 8e was cal-
culated by using secondary re-plots of the slope versus the
different concentrations of the compound 8e and was
23.07 nM (Fig. 2b).
The kinetic study of the most potent BuChE inhibitor 8e was
performed in order to determine the inhibition mode of this
compound. As can be seen in Fig. 2a, the graphical analysis of
the Lineweaver–Burk reciprocal plots demonstrated that the
H
N
O
O
N
N
Molecular modeling studies
N
N
N
R
R
Reliability of induced fit docking protocol
B
A
The applied docking procedure reliability was validated
by re-docking of donepezil and tacrine over AChE
(6o4w) and BuChE (4bds), respectively, based on induced
fit docking procedure (IFD). The docked conformations
corresponding to the lowest IFD score were selected as
the most possible binding modes. The RMSD was calcu-
lated for each ligand to measure the docking prediction
accuracy. The pose was counted optimal if its RMSD
found to be less than 2 Å. The RMSD of the re-docked
H
N
O
N
N
R
N
N
O
Designed compounds 8a-p
Fig. 1 Design strategy for new ChE inhibitors 8a–p

Struct Chem
(a)
(b)
Fig. 2 a Lineweaver–Burk plot for the inhibition of BuChE by compound 8e at different concentrations of ACh. b Steady-state inhibition constant (Ki)
of compound 8e
conformations of donepezil and tacrine over 6o4w and
4bds was 0.40 Å and 0.29 Å, respectively, which is con-
sidered as successfully docked [19, 20]. As a result, the
validity of IFD parameters is reasonable in order to pre-
dict the related co-crystalized structures.
acridine moiety and hydrogen bond with the quaternary
amine at the distance of 2.5 Å, respectively (Fig. 3b).
Investigating BuChE selectivity based on molecular
dynamic simulation
Docking of donepezil over AChE active site showed
that the ring center of the benzyl and the inden moiety
interacted with binding pocket by establishing two π-π
stacking with Trp86 and Trp286, respectively. Also, the
quaternary amine of piperidine ring interacts with the ring
center of Trp86 and Phe338 and carbonyl unit of inden
ring forms a hydrogen bond with NH unit of Phe295 in
the backbone by two π-cation interactions (2.3 Å)
(Fig. 3a).
Furthermore, docking of tacrine over BuChE showed
the prominent role of Trp82 and His438 in stabilizing the
ligand in the binding pocket by constructing π-π stacking
and π-cation interactions with the aromatic part of
In order to study the origin of BuChE selectivity of the
synthesized compounds and represent their stable dynam-
ic interaction over BuChE, it is necessary to uncover the
structural perturbations incurred by the most potent
BuChE inhibitor 8e, the most potent AChE inhibitor 8p,
and the reference compound (donepezil) over ChEs and
the effect of these compounds on the active site environ-
ment. The appropriate pose for MD simulation procedure
of the compounds was achieved by IFD method. The sta-
bility of protein-ligand complex trajectory is defined by
RMSD of the protein’s Cα from its initial to final confor-
mation over 20 ns MD simulation time (Fig. 4). The
Fig. 3 Close-up representation of binding interactions of the superposed docked (green) and co-crystalized donepezil and tacrine (cyan) over AChE (a)
and BuChE (b), respectively, structural waters and their hydrogen interaction render in stick

Struct Chem
Fig. 4 RMSD of ligand (red) and protein Cα (blue) for donepezil-AChE (a), donepezil-BuChE (b), 8e-AChE (c), 8e-BuChE (d), 8p-AChE (e), and 8p-
BuChE (f) over 20 ns MD simulation time
RMSD of protein and ligand simulation showed that the
compounds complexed with AChE maintained an overall
stability throughout the last 5 ns of MD simulation time
with the highest fluctuation stabilizing at an average of
2.2 Å (Fig. 4a, c, e), while the complex of the compounds
with BuChE displayed longer equilibration time (about
the last 15 ns) with lower fluctuations (Fig. 4b, d, f).
The RMSD value of each protein-ligand complex indi-
cates that the employed simulation time has been enough
to obtain an equilibrium structure. Thus, the structures at
the MD equilibrium state were used to investigate the
structural specificity of the ligand-protein complexes.
For compound 8e (Fig. 5a), the 4-oxobenzo[d][1,2,3]triazin-
3(4H)-yl moiety was inserted deeply into the bottom of the
acyl pocket (residues colored in green) of BuChE and
formed strong π-π stacking interactions with the aromatic
side chain of Trp231 and Phe329, which lasts for 32%
and 67% of simulation time, respectively. The protonated
nitrogen atom on the pyridine ring and NH unit of amide
group formed a salt bridge and water-mediated hydrogen
bond with Asp74 and Thr120, respectively, during the
whole time of equilibration. These can provide strong in-
termolecular recognitions between compound 8e and the
PAS region of BuChE, which further improved its binding
affinity. The terminal phenyl group pointed to the choline-
binding pocket of CAS and formed strong π-π stacking inter-
actions for about 62% of equilibrated MD time with the aro-
matic side chain of Trp82 known as secondary door of ChEs
active site [21]. In addition, carbonyl unit of the amide group
formed a water-mediated hydrogen bond with His438 (during
66% of MD time), which belongs to the catalytic triad of CAS.
On the other way, compound 8e became bound to AChE
(Fig. 5b) in which the 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl
moiety acted as the binder for choline-binding pocket of
CAS by interacting with the side chains of Trp84 through
π-π interaction (T-shape) for 79% of MD time. In this way,
Asp74 interacted to pyridinium ring and NH unit of amide
group through a salt bridge and a hydrogen bond, respectively,

Struct Chem
Fig. 5 2D-representation of ligand-residue interactions that occur more than 30.0% of the simulation time at the last 5 ns simulations (equilibrated
simulation phase) for complex 8e-BuChE (a), 8e-AChE (b), 8p-BuChE (c), 8p-AChE (d), donepezil-BuChE (e), and donepezil-AChE (f)
for the completely equilibrated MD time. Moreover, the aro-
matic side chain of Tyr341 further stabilized the pyridinium
ring through a π-cation interaction (during 71% of MD time).
Comparing to the interaction of AChE, compound 8e
interacted to all four parts of BuChE gorge cavity including
PAS, catalytic residue, and choline- and acyl-binding pocket,
while the MD simulation time highlighted PAS and choline-
binding pocket as the main area which was affected by com-
pound 8e over AChE enzyme.
Like compound 8e, compound 8p positioned deeply into the
BuChE active site (Fig. 5c). The ring center of pyridine ring
stabilized into the choline-binding pocket of CAS by interacting
with the aromatic side chains of Trp84 through hydrophobic
interaction. Also, the 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl
moiety (4-oxo) stabilized to the Gly116 located at the
oxonium hole of gorge cavity through water-mediated hy-
drogen bond during 31% of MD time. The amide group
formed a hydrogen bond and a water-mediated hydrogen
bond with Pro285 at the acyl-binding pocket of CAS and
Ser198 as the catalytic residue, respectively.
Considering Fig. 5d, compound 8p became bound to
AChE in which the 4-oxobenzo[d][1,2,3]triazin-3(4H)-yl
moiety interacted to the Phe295 located at the acyl-binding
pocket of CAS through water-mediated hydrogen bond during
79% of MD time. Again, like the 8e-AChE complex, the rest
of the compound 8p located in the PAS in which the
pyridinium ring formed salt bridge and π-π interaction with
Trp286 for more than half of the simulation time.
Furthermore, the nitro group pointed toward the outer part of
PAS and formed salt bridge and water-mediated hydrogen
bond with Glu292 and His287, respectively.
Comparing the interaction of compound 8p over AChE
and BuChE gorge cavity showed that it stabilized BuChE
through interaction with the oxonium hole, catalytic residue,
and acyl- and choline-binding pocket at the same time, while
in the case of AChE, except an indirect interaction to acyl-

Struct Chem
Fig. 6 Proposed binding modes for compound 8e (a, b), 8p (c, d), and
donepezil (e, f) over BuChE and AChE, respectively. The active
compound is showed as sticks and colored cyan. The acyl-binding pock-
et, choline-binding pocket, and catalytic residues at the CAS are in green,
yellow, and red, respectively. The PAS residues located at the top of the
gorge are in purple. The hydrogen bonding, π-π stacking, and electrostat-
ic interactions are represented as yellow, blue, and purple dashed lines,
respectively
binding pocket, the main affecting region was the PAS at the
top of the gorge cavity. Therefore, it can propose that
interacting with the part of CAS cavity (catalytic residue,
acyl- and choline-binding pocket) which is located deeper to
the gorge cavity is essential for ChE selectivity. This finding
can be addressed to the interaction mode of donepezil, as a
selective AChE inhibitor. Considering Fig. 5e and f, donepezil
stabilized to the PAS of AChE through interacting with the
Asp74, Trp286, and Tyr341 during prolonged amount of MD
time. In addition, it covered choline-binding pocket and acyl-
binding pocket through hydrophobic and water-mediated hy-
drogen bond interaction with Trp86 and Pro285, respectively.
On the other hand, we can observe donepezil covered
PAS, choline-binding pocket, and oxonium hole of
BuChE with lower contribution MD time (during 45%,
55%, and 31%, respectively). Our molecular dynamics
study suggests that the more interaction of ligand with
the PAS, catalytic residues, and acyl- and choline-
binding pockets at the same time has a significant con-
tribution for selective inhibition of ChEs.

Struct Chem
Table 2 The ligand
interaction energy over
ChEs based on MM-
GBSA method
gorge cavity of BuChE, developing bulky substitution at this
position creates high steric clash inside the AChE narrower
gorge, while it causes improved coverage into the larger gorge
cavity of BuChE, so improving BuCuE selectivity.
In addition to the interaction analysis, the Prime/MM-
GBSA module was used to estimate the strengths of interac-
tions between the ligand-protein complexes which were gen-
erated by the clustering method used for energy calculation
(Table 2).
Compound
MM-GBSA (kcal/mol)
BuChE
AChE
8e
− 84.83
− 78.49
− 74.96
− 86.85
− 91.86
− 104.70
8p
Donepezil
The ligand-residue interactions and the fraction related to
each type of interactions during the equilibrated MD simula-
tion time were used to investigate ligand-protein complex in-
teratomic (Fig. 5).
ΔG bind of 8e-BuChE complex and 8p-BhChE complex
was estimated to be − 84.83 and − 78.49 kcal/mol, respective-
ly, revealing stronger binding interaction of compound 8e than
8p which also supported by experimental assay.
Comparing Fig. 5a and c, compound 8e generated
narrower-mouth and longer length U-shape conformation than
compound 8p into the BuChE active site, which caused more
coverage of PAS site and CAS through its bottom and wall of
the U-shape conformation.
In silico conformational study of the most active
compounds over ChEs gorge cavity
The representative structures based on hierarchical clustering
method were used in uncovering the conformational differ-
ence between compounds 8e and 8p over the ChEs active site
(Fig. 6). As it observed in Fig. 6a, c, e, compounds 8e, 8p, and
donepezil became bound to BuChE in a U-shaped conforma-
tion in order to occupy the larger active site, while they be-
came bound to AChE in a linear-shaped conformation
(Fig. 6b, d, f).
Compared to the conformation in AChE, compound 8e not
only exhibited a more contracted pose in BuChE gorge cavity,
but also showed flipped orientation in which 4-chloro phenyl
moiety deeply inserted to the choline-binding pocket of
BuChE while in the case of AChE, it pointed toward the most
outer part of PAS which is close to the solvent accessible area
(Fig. 6a, b). These flipping and changing of the orientation
take place because of the bulky phenomena of chlorine group
that is not tolerated by AChE gorge cavity. In view of larger
The reason for difference in compactness of compound 8e
and 8p might be pointed to the difference in the position of
benzo[1,2,3]triazin-3(4H) aromatic ring substitution over the
pyridine ring which could affect the flexibility of the whole
structure. The mentioned group existed at meta-position in
compound 8e while in case of compound 8p it is located at
para-position with less ability for bending the whole molecule.
The hypothesis is consistent to some extent with our experi-
mental enzymatic assay, in which the potent inhibition of
BuChE are acquired through compounds which are related
to the 3-benzo [1,2,3]triazin-3(4H) substitution over the pyri-
dine ring rather than 4-substituted one (compounds 8e, 8f, 8b,
8a, 8c). Therefore, besides the impact of substitutions on ChE
selectivity, the core structural flexibility of compounds has
outstanding role in the ChE inhibition potency. Finally,
Fig. 7 summarized the two-design strategy for developing
more selective and potent compound for the designed
scaffold.
Conclusion
In conclusion, 16 new 4-oxobenzo[d]1,2,3-triazin-
pyridinium-phenylacetamide hybrids 8a–p were synthesized
and evaluated as potent cholinesterase inhibitors for treatment
of AD. The in vitro inhibition assay of these compounds re-
vealed that all the synthesized compounds had significant in-
hibitory activity against BuChE. Among them, compound 8e
displayed the highest anti-BChE activity. Furthermore, kinetic
study revealed that this compound inhibited BChE via a
mixed-type inhibition mode. Furthermore, compound 8e as
the most potent inhibitors against BuChE was inserted well
into the gorge cavity of BuChE by simultaneously interacting
with CAS and PAS.
Fig. 7 Detailed modification strategy of the synthesized scaffold to
improve the BuChE selectivity

Struct Chem
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Soyer Z (2016) Synthesis and cholinesterase inhibitory activity of
some phenylacetamide derivatives bearing 1H-pyrazole and 1H-1,
2, 4-triazole. Marmara Pharm J 20:21–27
Conflict of interest The authors declare that they have no conflict of
interest.
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