Design and synthesis of 2,4-dioxochroman-pyridinium-phenylacetamide derivatives as new anti-Alzheimer agents: in vitro and in silico studies

Article abstract of DOI:10.1002/jccs.202000013

2,4-Dioxochroman-pyridinium-phenylacetamide derivatives 7a–n were synthesized and evaluated for their in vitro cholinesterase (ChE) inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Obtained results demonstrated that, among the synthesized compounds, two compounds, 7j and 7k, were more potent than the standard drug donepezil against BuChE and did not show cytotoxicity and carcinogenicity. Furthermore, through molecular modeling and molecular dynamic studies. we showed that these compounds can be located deep in the gorge cavity of BuChE and that they interacted with catalytic residues, acyl, and cholin-binding pockets of this enzyme. Support information.

Full text of DOI:10.1002/jccs.202000013

Received: 14 January 2020
DOI: 10.1002/jccs.202000013
Revised: 15 April 2020
Accepted: 19 April 2020
A R T I C L E
Design and synthesis of 2,4-dioxochroman-pyridinium-
phenylacetamide derivatives as new anti-Alzheimer
agents: in vitro and in silico studies
1
2
3
Marjan Mollazadeh | Maryam Mohammadi-Khanaposhtani | Homa Azizian
|
1
4
4
5
Afsaneh Zonouzi | Zahra Abdolahi | Hamid Nadri | Bagher Larijani
|
5
5
Mahmood Biglar | Mohammad Mahdavi
1
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
2
3
4
Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran
Department of Medicinal Chemistry, School of Pharmacy-International Campus, Iran University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
5
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences,
Tehran, Iran
Correspondence
Afsaneh Zonouzi, School of Chemistry,
Abstract
College of Science, University of Tehran,
Tehran, Iran.
2,4-Dioxochroman-pyridinium-phenylacetamide derivatives 7a–n were synthe-
sized and evaluated for their in vitro cholinesterase (ChE) inhibitory activities
against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Obtained
results demonstrated that, among the synthesized compounds, two compounds, 7j
and 7k, were more potent than the standard drug donepezil against BuChE and
did not show cytotoxicity and carcinogenicity. Furthermore, through molecular
modeling and molecular dynamic studies. we showed that these compounds can
be located deep in the gorge cavity of BuChE and that they interacted with cata-
lytic residues, acyl, and cholin-binding pockets of this enzyme. Support
information
Email: zonouzi@khayam.ut.ac.ir
Mohammad Mahdavi, Endocrinology and
Metabolism Research Center,
Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran.
Email: momahdavi@tums.ac.ir
K E Y W O R D S
2
,4-Dioxochroman, Alzheimer agents: in vitro, in silico, phenylacetamide, pyridinium
1
| INTRODUCTION
acetylcholine (ACh), abnormal deposits of β-amyloid pep-
tides (Aβ), and presence of neurofibrillary tangles in the
Alzheimer's disease (AD) is a progressive neurodegenera-
tive disorder that is characterized by cognition defects,
hippocampus and cortex areas of the brain that are
related to memory and cognition.
[
3]
[1]
decline in memory, and language deterioration. AD is
the most common cause of dementia that affects elderly
One of the effective treatments for the decrease of the
symptoms of AD is increasing the synaptic levels of ACh
[2]
[4]
people.
At the molecular level, AD was diagnosed
through the inhibition of acetylcholinesterase (AChE).
based on reduced synaptic levels of the neurotransmitter
AChE is responsible for the degradation of ACh in the
©
2020 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
J Chin Chem Soc. 2020;1–19.
http://www.jccs.wiley-vch.de
1

2
MOLLAZADEH ET AL.
FIGURE 1 Design strategy for
new ChE inhibitors 7a–n
O
O
H
N
O
R
N
H
N
N
O
R
B
A
O
O
O
O
H
N
R
N
H
N
R
N
O
H
O
N
O
O
N
H
7
a-k
7l-n
[
5]
synaptic cleft. Today, AChE inhibitors such as donepezil,
rivastigmine, and galantamine are used as the most impor-
tant agents for improvement of Alzheimer's symptoms.
Butyrylcholinesterase (BuChE) is another Ach-degrading
enzyme, which partially acts on the brain. Several studies
revealed that, in the brain of AD patients, AChE activity is
gradually decreased, while BuChE activity is significantly
is shown in Scheme 1. This pathway was a result of
the reaction between 3-(methoxymethylene)-3H-
chromene-2,4-dione 1 and pyridin-3-ylmethanamine
2a or pyridin-4-ylmethanamine 2b in ethanol at reflux
of 4 hr for producing pyridin derivatives 3a–b. On the
other hand, N-phenyl-2-chloroacetamides 6a–l were
obtained from the reaction of anilines 4a–l and
2-chloroacetyl chloride 5 in dimethylformamide (DMF)
at room temperature for 30 min. Finally, the desired
2,4-dioxochroman-pyridinium-phenylacetamides 7a–n
were produced from the reaction between the pyridine
derivatives 3a–b and N-phenyl-2-chloroacetamides 6a–
l in acetonitrile at reflux for 2–3 hr.
[6]
increased. Accordingly, inhibition of BuChE may be use-
ful in the amelioration of AD signs and symptoms.
Over the past year, our research group, using done-
pezil as the template, has introduced numerous struc-
tures with anti-Alzheimer's effects.
[
7,8]
[9,10]
Recently, we
reported 2,4-dioxochroman-benzyl pyridinium deriva-
tives A as potent ChE inhibitors (Figure 1).
[11]
On the
other hand, phenylacetamide derivatives B exhibited
[12]
inhibitory activity against AChE (Figure 1).
Therefore,
2.2 | In vitro ChE inhibitory activity
here, in order to design new ChE inhibitors, we replaced
the pendant phenyl group of the scaffold A with
phenylacetamide moiety of scaffold B; designed
The in vitro ChE inhibitory activity of the synthesized
compounds 7a–n were evaluated against AChE and
BuChE using Ellman's method and compared with stan-
2
,4-dioxochroman-pyridinium-phenylacetamide hybrids
[
13]
7
a-n were synthesized and evaluated for their ChE inhib-
dard inhibitor donepezil.
itory activities against AChE and BuChE (Figure 1).
Docking and toxicity studies of the most potent com-
pounds were also performed.
IC50 values for the inhibition of AChE/BuChE are pres-
ented in Table 1. As can be seen in Table 1, among the syn-
thesized compounds, compounds 7b, 7d, and 7f–k showed
moderate AChE inhibitory activity, with an IC50 ranging
from 5.52 ± 0.71 to 22.25 ± 1.22 μM, in comparison with
donepezil (IC50 = 0.027 ± 0.003 μM), while compounds 7j
and 7k showed more potent BuChE inhibitory activity in
comparison with donepezil (IC50 = 7.79 ± 0.81 μM). More-
over, compounds 7d, 7h, and 7i exhibited good inhibitory
activity against BuChE (IC50 values of 9.90 ± 0.41, 11.0
± 0.19, and 14.77 ± 0.23 μM, respectively).
2
2
| RESULTS AND DISCUSSION
.1 | Chemistry
The synthetic pathway for the synthesis of new
,4-dioxochroman-pyridinium-phenylacetamides 7a-n
2

MOLLAZADEH ET AL.
3
SCHEME 1 Synthesis pathway for produce 2,4-dioxochroman-pyridinium-phenylacetamides 7a–n
TABLE 1
ChE inhibitory activities of the compounds 7a–n
a
a
Compound
R
IC50 AChE (μM)
≥100
IC50 BuChE (μM)
≥100
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
2-CH
3-CH
4-CH
3
3
3
5.52 ± 0.35
≥100
≥100
≥100
2,4-dimethyl
3,4-dimethyl
12.28 ± 0.41
≥100
9.90 ± 0.41
≥100
2-OCH
4-OCH
3-cl
3
3
11.10 ± 0.35
22.25 ± 0.69
5.81 ± 0.27
9.84 ± 0.73
10.30 ± 1.05
14.4 ± 1.23
≥100
65.26 ± 1.31
37.14 ± 1.17
11.0 ± 0.19
14.77 ± 0.23
3.66 ± 0.11
5.60 ± 0.14
≥100
g
h
i
4-cl
j
3,4-Dichloro
2,4-Dichloro
k
l
3-CH
3
m
n
2,5-dimethyl
3,4-Dichloro
—
≥100
≥100
≥100
≥100
Donepezil
0.027 ± 0.003
7.79 ± 0.06
a
Values are the mean ± SD. All experiments were performed at least thrice.
Structurally, the synthesized compounds 7a–n could
group to 4-position of this compound, as in compounds
7a, 7c, and 7e, led to the loss of inhibitory activity.
Although, 2-methyl analog 7a and its regioisomer 7c
with 4-methyl substituent did not show activity against
AChE, the 2,4-dimethyl analog 7d showed moderate
inhibitory activity against this enzyme. Compound 7f
with the 2-methoxy group on the pendant phenyl
group also exhibited moderate inhibitory activity.
Changing the position of the methoxy group from 2- to
4-position, as in compound 7g, reduced activity by half.
In the chloro-substituted compounds 7h–k, the most
be divided into two series: 3-pyridinium derivatives 7a–k
and 4-pyridinium derivatives 7l–m. In each series, the
substituent on the phenyl ring of acetamide moiety was
altered to optimize ChE inhibition.
Among the 3-pyridinium derivatives 7a–k, com-
pound 7b (IC50 = 5.52 ± 0.71 μM), with 3-methyl sub-
stituents on the phenyl ring of acetamide moiety, was
the most active compound against AChE. Movement of
the methyl group of compound 7b from the 3-position
to the 2- or 4-position and or adding another methyl

4
MOLLAZADEH ET AL.
potent compound was 3-chloro analog 7h with
IC50 = 5.81. Changing the position of chloro in this
compound of 3-position to 4-position and/or adding a
chlorin atom to the 4-position of compound 7h did not
improve the anti-AChE potency observed in com-
pounds 7i and 7j. Among the chloro-substituted deriva-
tives, the 2,4-dichloro analog 7k exhibited the lowest
inhibitory activity against AChE. Among the chloro-
substituted derivatives of 3-pyridinium series,
were 3,4 and 2,4-dichloro derivatives 7j and 7k, respec-
tively, with an inhibitory activity that was 2.13- and
1.4-fold greater than donepezil. Other chlorine deriva-
tives (7h with 3-chloro substituent and 7i with 4-chloro
substituent) also showed good inhibitory activity against
BuChE. Another potent BuChE inhibitor in the
3-pyridinium series is 2,4-dimethyl derivative 7d. In this
series, methoxy derivatives 7f and 7g exhibited weak
anti-BuChE activity. The remaining derivatives of
3-pyridinium series had no inhibitory activity against
BuChE.
2
,4-dichloro analog 7k exhibited the lowest inhibitory
activity against AChE.
The observed IC50 values of 3-pyridinium 7a-k
against BuChE revealed that the most active compounds
4-Pyridinium derivatives 7l–n showed no AChE and
BuChE inhibition.
FIGURE 2 Sequence alignment of equine AChE (entry ACE_Equus, F7DYJ5) with human AChE (entry ACE-Human, P22303)
generated by CLUSTALW algorithm. (*) indicates a conserved substitution, (:) indicates a semiconserved substitution, and (.) indicates a
different substitution

MOLLAZADEH ET AL.
5
FIGURE 3 Sequence alignment of equine BuChE (entry BuCE_Ecuus, P81908) with human BuChE (entry BuCE_Human, P06276)
generated by CLUSTALW algorithm. (*) indicates a conserved substitution, (:) indicates a semiconserved substitution, and (.) indicates a
different substitution
2
.3 | Molecular modeling and dynamic
The experimental cholinesterase process was per-
study
formed on equine serum. As there are no crystal struc-
tures of AChE and BuChE equine in the protein data
bank, human AChE and BuChE were used in in silico
studies. The sequence of equine AChE (entry
ACE_equus, F7DYJ5) and BuChE (entry BuCE_equus,
P81908) derived from the Swiss-Prot database shares 90%
sequence identity with human AChE (entry ACE-human,
P22303) and BuChE (entry P06276, BuCE_human),
respectively (Figures 2 and 3). Consequently, the
2.3.1 | In silico molecular modeling
investigation
A glide docking protocol was performed to predict con-
formation, orientation, and binding energies of the syn-
thesized compounds inside the active sites of AChE (PDB
code: 6o4w) and BuChE (PDB code: 4bds).

6
MOLLAZADEH ET AL.
FIGURE 4 Close-up representation of binding interactions of the superposed docked (cyan) and cocrystalized donepezil and tacrine
green) over AChE (a) and BuChE (b), respectively.
(
investigated equine AChE and BuChE were expected to
exhibit a similar 3D structure with their correlated
human ones, so the result of the atomic level molecular
modeling study on human ChEs can be extrapolated to
equine ChEs as well.
1,2,3,4-tetrahydroacridin moiety and H-bond with the
quaternary amine at a distance of 2.5 Å, respectively
(Figure 4b).
2.3.3 | Docking study
2
.3.2 | Reliability of docking protocol
The performed docking procedure was then applied to
evaluate the interaction between newly effective synthe-
sized compounds 7b, 7d, 7h, 7i, 7j, and 7k over the
AChE and BuChE active sites. The top scoring pose of all
compounds was analyzed inside the binding site of each
ChEs active site.
The applied docking procedure reliability was validated
by redocking donepezil and tacrine over AChE (6o4w)
and BuChE (4bds), respectively, based on glide docking
procedure. The docked conformations corresponding to
the lowest glide energy were selected as the most possible
binding modes. The root mean square deviation (RMSD)
was calculated for each ligand to measure docking pre-
diction accuracy. The pose was counted optimal if its
RMSD was found to be lower than 2 Å. The RMSD of the
redocked conformations of donepezil and tacrine over
In the binding model, all compounds successfully
occupied the active site cavities of AChE and BuChE
(Figure 5). The study by Saxena et al. revealed that the
active site volume on BuChE is larger than that of the
AChE because of aliphatic residue replacement in BuChE
[16]
instead of the bulky aromatic residue of AChE.
6
o4w and 4bds was 0.10 and 0.17 Å, respectively, which
Because of shape enforcements of AChE and BuChE
active sites, compounds were bound to AChE in a narrow
linear-shaped conformation (Figure 5a), while they were
bound to BuChE in a flipped U-shaped conformation
(Figure 5b) in order to occupy the larger BuChE
active site.
In addition, the result of the glide energy of the men-
tioned structures over AChE and BuChE active site var-
ied from −65.94 to −58.46 and −84.06 to −69.937 kcal/
mol, respectively, proposing that the binding interaction
differs as the functional group is varied (Table 2). As can
[14,15]
is considered successfully docked.
validity of docking parameters are reasonable in order to
predict the related cocrystalized structures.
As a result, the
Docking of donepezil over the AChE active site
showed that the ring center of the benzyl and the indene
moiety interacted with the binding pocket by establishing
two π-π stacking with Trp86 and Trp286. In addition, the
quaternary amine of the piperidine ring interacted
through two π–cation interactions with the ring center of
Phe338, and the indene carbonyl group formed a H-bond
interaction with the backbone N-H of Phe295 (2.3 Å)
be seen in Table 2, compound 7b with a 3-CH substitu-
3
(
Figure 4a).
ent on the pendant phenyl ring indicated the highest
glide energy (more negative) of −65.94 kcal/mol over the
AChE active site, while compounds 7j and 7k with
higher bulky dichloro substituents on the pendant phenyl
ring interacted with BuChE through the highest glide
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

MOLLAZADEH ET AL.
7
FIGURE 5 Superimposed binding conformation of compounds 7b, 7d, 7h, 7i, 7j, and 7k over AChE (a) and BuChE (b) active sites. The
active compounds are represented by lines and are colored in black. The acyl-binding pocket, choline-binding pocket, and catalytic residues
at the A-site are in cyan, yellow, and orange, respectively. The P-site residues are located at the top of the gorge are in purple
TABLE 2
AChE and BuChE active site
Glide energy of the most active compounds over
site. In this way, the phenyl amino moiety at the tile part
of the structures acted as the binder for the choline-
binding pocket (yellow residues) of A-site by interacting
with the side chains of Trp86 through π-π stacking inter-
action. Besides, Asp74 and Tyr341 at the P-site interacted
with the pyridinium ring and the quaternary amine
group through π-π stacking and salt–bridge interaction,
respectively.
Moreover, Figure 7a–f show a 3D representation of
ligand–residue interactions over the BuChE active site.
In the case of compound 7b, with a different orienta-
tion relative to other compounds, the small 3-methyl
phenyl group pointed to the choline-binding site (resi-
dues colored in yellow) through π-π stacking interac-
tion with Trp82. The pyridinium quaternary amine
interacted with Asp70 at P-site (colored in purple)
through electrostatic salt–bridge interaction. In addi-
tion, the 2,4-dioxochroman moiety was oriented
toward the acyl-binding pocket (colored in cyan) and
formed π-π stacking interaction with Trp231 and
Phe329 residues.
Obviously, compounds 7d, 7h, 7i, and 7j depicted a
similar orientation in the BuChE active site (Figure 7b–
e). For compound 7j with the highest BuChE inhibition
activity, the pyridinium ring was inserted deeply into the
bottom of the choline-binding pocket of BuChE and
formed strong π-π stacking and π–cation interactions
with the aromatic side chain of Trp82 and Phe329,
respectively (Figure 7e). The 3,4-dichlorophenyl group
also pointed toward the P-site at the mouth of the active
gorge, which is more solvent-accessible through π-π stac-
king interaction with the aromatic side chain of Trp332.
In addition, the 2,4-dioxochroman moiety was oriented
Glide energy (kcal/Mol)
No.
1
Compound
AChE
−65.94
−59.58
−61.88
−62.81
−59.06
−58.46
BuChE
−69.94
−76.19
−74.21
−75.69
−83.94
−84.06
7b
7d
7h
7i
2
3
4
5
7j
6
7k
energy (−83.94 and −84.06 kcal/mol, respectively). These
finding are in accordance with our experimental data,
where compound 7b with an IC50 of 5.52 ± 0.35 μM has
the highest AChE inhibition activity, whereas com-
pounds 7j and 7k demonstrated the most BuChE inhibi-
tion activities (3.66 ± 0.11 and 5.60 ± 0.14 μM,
respectively) among the synthesized compounds. As a
result, it can be proposed that small monosubstitution,
especially at position 3 of the pendant phenyl ring, is
favorable for AChE inhibition activity, while bulkier dis-
ubstitutions at the mentioned part of the compounds
structure is appropriate for BuChE one.
Figure 6a–f shows a 3D representation of ligand–
residue interactions over the AChE active site. For all the
compounds, except compound 7i (Figure 6d),
2
,4-dioxochroman moiety was oriented toward the P-site
and was surrounded by the aromatic residue at the
mouth of the gorge cavity (purple residues), while the
remaining structure was deeply inserted into the active

8
MOLLAZADEH ET AL.
FIGURE 6 Proposed binding mode of compounds 7b (a), 7d (b), 7h (c), 7i (d), 7j (e), and 7k (f) over AChE active site. The active
compound is showed as sticks and colored green. The acyl-binding pocket, choline-binding pocket, and catalytic residues at the A-site are in
cyan, yellow, and orange, respectively. The P-site residues located at the top of the gorge are in purple. The hydrogen bonding, π-π stacking,
π–cation, and electrostatic interactions are represented as yellow, blue, green, and purple dashed lines, respectively
toward the acyl-binding pocket and formed a H-bond
with the catalytic residue Ser198. Considering Figure 7f,
compound 7k (with high BuChE inhibition activity)
interacted with the acyl and choline-binding pockets and
especially formed strong π-π T-shape interactions with
the catalytic residue His438.
2.3.4 | Investigating ligand–ChEs active
site interaction stability based on
molecular dynamic simulation
To study the stability of the ligands in the active site and
to see if the interactions are conserved, it is necessary to

MOLLAZADEH ET AL.
9
FIGURE 7 Proposed binding mode of compounds 7b (a), 7d (b), 7h (c), 7i (d), 7j (e), and 7k (f) over BuChE active site. The active
compound is showed as sticks and colored green. The acyl-binding pocket, choline-binding pocket, and catalytic residues at the A-site are in
cyan, yellow, and orange, respectively. The P-site residues located at the top of the gorge are in purple. The hydrogen bonding, π-π stacking,
π–cation, and electrostatic interactions are represented as yellow, blue, green, and purple dashed lines, respectively
uncover the structural perturbations incurred by the most
active compounds 7b, and 7j over AChE and BuChE
active sites, respectively, and compare them with the ref-
erence compound (donepezil). The appropriate pose for
the molecular dynamics (MD) simulation procedure of
the compounds was achieved using the glide docking
method. The stability of the protein–ligand complex tra-
jectory is defined by the RMSD of the protein's Cα from
its initial to final conformation over an MD simulation
time of 20 ns (Figure 8).

10
MOLLAZADEH ET AL.
The RMSD of protein and ligand simulation showed
pocket of A-site by hydrophobic interaction with Trp86
and for the side chains of Trp83 and Asp74 through
water-mediated H-bond interaction for 51% and 86% of
MD time, respectively (Figure 9a). In addition, Tyr124,
located at the P-site, has an important role in stabilizing
compound 7b during the entire simulation time. More-
over, the side chain of Tyr341 and Asp74 further stabi-
lized the pyridinium ring through π–cation interaction
and salt–bridge interaction for 45% and 96% of MD simu-
lation time, respectively. Finally, the 2,4-dioxochroman
ring was surrounded by aromatic residue of the P-site
through hydrophobic interaction with Tyr72 and π–
cation interaction with Trp286 for 45% of MD time.
This finding can be used in the interaction mode of
donepezil as a selective AChE inhibitor. Considering
Figure 9b, donepezil stabilized to the P-site of AChE by
interacting with the Asp74, Trp286, and Tyr341 during a
prolonged amount of MD time. In addition, it covered
the choline-binging pocket and acyl-binding pocket
through hydrophobic and water-mediated H-bond inter-
actions with Trp86 and Pro285, respectively.
that all compounds complexed maintained an overall sta-
bility throughout the last 5 ns of MD simulation over
both ChE active sites.
The RMSD of the simulations showed that donepezil
has a mean RMSD value of 1.3 Å over AChE, while it
depicts higher RMSD over the BuChE active site (3.2 Å)
(
Figure 8a,b, orange dash line). In the same manner,
compound 7b has a lower RMSD over AChE (3.2 Å) than
compound 7j over BuChE (5.8 Å) (Figure 8c,d, orange
dash line). In summary, the ligand–protein simulations
showed that ligands have lower RMSD over AChE than
BuChE as result of AChE's narrower-shaped gorge cavity.
Finally, the RMSD value of each protein–ligand com-
plex indicates that the utilized simulation time has been
sufficient to obtain an equilibrium structure. Thus, the
structures at the MD equilibrium state (last 5 ns) were
used to investigate the structural specificity of the ligand–
protein complexes.
The ligand–residue interactions and the fraction
related to each type of interactions during the equili-
brated MD simulation time were used to investigate
ligand–protein complex interactomics (Figure 9).
Compound 7b was bound to AChE, in which the phe-
nyl amide moiety acted as the binder for choline-binding
In the case of compound 7j (Figure 9c), the
3,4-dichloro phenyl ring was surrounded by aromatic side
chains of BuChE P-site pocket. Nitrogen atom of
pyridinium ring and NH unit of amid group formed salt–
FIGURE 8 RMSD of ligand (orange) and protein Cα (green) for donepezil-AChE (a), donepezil-BuChE (b), 7b-AChE (c), and 7j-
BuChE (d) over 20 ns MD simulation time

MOLLAZADEH ET AL.
11
FIGURE 9 2D representation of
ligand–residue interactions that occur
more than 30.0% of the simulation time
at the last 5 ns simulations (equilibratad
simulation phase) for complex 7b-AChE
(
a), donepezil-AChE (b), 7j-BuChE (c),
and donepezil-BuChE (d)
bridge and H-bond interactions with Asp74 during the
majority of the equilibration time. In addition, carbonyl
unit of amide group formed a water-mediated H-bond
interaction with the Pro285 (during 69% of MD time),
which belongs to the acyl-binding pocket of A-site.
Finally, 2,4-dioxochroman moiety formed strong π-π stac-
king interactions with the aromatic side chain of Trp231
and Phe329 of the acyl pocket of BuChE, which lasted for
concentration, the selected compounds were non-
cytotoxic against studied cell lines HDF and MCF-7
(Table 3).
In silico carcinogenicity of the compounds 7j and 7k
was predicted using online server. Obtained results dem-
onstrated that these compounds did not show carcinoge-
nicity in the rat and mice (Table 3).
4
1% and 48% of simulation time, respectively. On the
other hand, we can observe the donepezil-covered P-site
Trp430) and choline-binding pocket (Trp82) and
3 | EXPERIMENTAL
3.1 | General
(
oxonium hole (Gly116) of BuChE in lesser contributed
MD time (during 45%, 55%, and 31%, respectively)
(Figure 9d), which proposed the reason for lower inhibi-
Melting points of the 2,4-dioxochroman-pyridinium-
tion activity of donepezil than compound 7j.
phenylacetamides 7a–n were determined with
a
1
13
In summary, the MD simulation results reveal the
role of 2,4-dioxochroman moiety, pyridinium ring,
and the substituted phenyl moiety in occupying acyl,
choline-binding pockets, and P-site of BuChE, respec-
tively. However, for AChE inhibition, the substituted
phenyl moiety was inserted deep into the acyl-
binding pocket, and the remaining compounds' struc-
ture covered the lining residue at the P-site of the
gorge cavity.
Kofler hot stage apparatus. H and C NMR spectra
of the title compounds were obtained with a Bruker
FT-500 (TMS as an internal standard). IR (infrared)
spectra of these compounds were recorded with a
Nicolet Magna FTIR 550 spectrophotometer (KBr
disks). Elemental analysis was conducted with an
Elementar Analysen system GmbH VarioEL CHN
mode.
3-(Methoxymethylene)-3H-chromene-2,4-dione
1 was prepared according to the procedure described
[
11]
in the literature.
2
.4 | In vitro and in silico toxicity of the
compounds 7j and 7k
3.2 | General procedure for the synthesis
of pyridin derivatives 3a–b
Cytoxicity of the most potent BuChE inhibitors 7j and 7k
was evaluated against normal cell line HDF (human der-
mal fibroblasts ()) and breast cancer cell line MCF-7
A mixture of 3-(methoxymethylene)-3H-chromene-2,-
4-dione 1 (1 mmol) and pyridin-3-ylmethanamine 2a or
pyridin-4-ylmethanamine 2b in ethanol (5 ml) was stirred
at reflux for 4 hr. Then, the reaction was allowed to cool
(Michigan cancer foundation-7) using methyl thiazolyl
[17]
tetrazoliumassay.
Results revealed that, at 100 μM

12
MOLLAZADEH ET AL.
TABLE 3
In vitro and in silico toxicity evaluations of the compounds 7j and 7k
Cytotoxicity (IC50 μM)
Carcinogen
Compound
HDF
≥100
≥100
MCF-7
≥100
Rat
Mouse
7
j
Negative
Negative
Negative
Negative
7
k
≥100
at room temperature and was poured into crushed ices.
Finally, obtained pure pyridin derivatives 3a–b were fil-
tered off. (Yields of compounds 3a and 3b are 91 and
(br. s, 1H, =CNH), 10.44 (br. s, 1H, NH), 9.18 (s, 1H, H2
pyridine), 9.08 (d, 1H, H pyridine, J = 5.0 Hz), 8.74–8.69
6
(m, 2H, =CH, H pyridine), 8.24 (t, 1H, H pyridine,
4
5
8
9%, respectively.)
J = 7.0 Hz), 7.92 (d, 1H, H coumarin, J = 7.5 Hz),
5
7
.69–7.65 (m, 1H, H coumarin), 7.43 (d, 1H, H phenyl,
7
3
J = 7.5 Hz), 7.32–7.29 (m, 2H, H6,8 coumarin), 7.23 (d,
1H, H phenyl, J = 7.0 Hz), 7.18–7.12 (m, 2H, H phe-
3
.3 | General procedure for the synthesis
6
4,5
+
2 2
of N-phenyl-2-chloroacetamides 6a–l
nyl), 5.80 (s, 2H, CH N ), 5.07 (s, 2H, CH N), 2.28 (s, 3H,
13
CH3). CNMR (125 MHz, DMSO-d ) δ: 179.8 (CO), 163.3
6
A mixture of anilines 4a–l and chloroacetyl chloride 5 in
DMF at room temperature was stirred for 30 min. Then,
the reaction mixture was diluted with water, poured into
crushed ice, and the obtained residue was washed with
water to obtain pure N-phenyl-2-chloroacetamides 6a–l.
(CO), 162.8 (CO), 162.6 (CH), 154.2 (C), 145.7 (CH), 145.5
(CH), 144.9 (CH), 137.5 (CH), 137.4 (C), 135.2 (C), 134.6
(CH), 133.0 (C), 129.0 (CH), 126.0 (CH), 125.0 (CH),
124.3 (CH), 124.1 (CH), 120.9 (C), 120.5 (C), 116.0 (CH),
116.2 (CH), 97.2 (C), 62.2 (CH ), 50.2 (CH ), 18.0 (CH ).
NMR data for the Z isomer: H NMR (500 MHz, DMSO-
d6) δ: 10.71 (br. s, 1H, =CNH), 10.44 (br. s, 1H, NH), 9.17
2
2
3
1
(
Yields of compounds 6a–l are 93, 84, 89, 83, 91, 79,
8
6, 88, and 85%).
(
s, 1H, H pyridine), 9.08 (d, 1H, H pyridine, J = 5.0 Hz),
2 6
8
.85 (d, 1H, =CH, J = 15.0 Hz), 8.74–8.69 (m, 1H, H pyr-
4
3
.4 | General procedure for the synthesis
idine), 8.24 (t, 1H, H pyridine, J = 7.0 Hz), 7.96 (d, 1H,
5
of 2,4-dioxochroman-pyridinium-
phenylacetamides 7a–n
H coumarin, J = 7.5 Hz), 7.69–7.65 (m, 1H, H couma-
5
7
rin), 7.43 (d, 1H, H phenyl, J = 7.5 Hz), 7.32–7.29 (m,
3
2
H, H6,8 coumarin), 7.23 (d, 1H, H phenyl, J = 7.0 Hz),
6
+
A solution of the pyridine derivatives 3a–b (1 mmol) and
N-phenyl-2-chloroacetamides 6a–l (1.4 mmol) in dry ace-
tonitrile (10 ml) was stirred under reflux for 2–3 hr. Then,
the formed precipitate was filtered and washed with dry
acetonitrile (3 ml) to produce pure compounds 7a–n. The
latter compounds were obtained as a mixture of E and Z
isomers. The percentage of E and Z isomers was calcu-
lated by NMR.
7.18–7.12 (m, 2H, H4,5 phenyl), 5.80 (s, 2H, CH N ), 5.07
(s, 2H, CH N), 2.28 (s, 3H, CH ). CNMR (125 MHz,
2
13
2
3
DMSO-d ) δ: 177.6 (CO), 164.0 (CO), 162.8 (CO), 162.6
6
(CH), 154.2 (C), 147.7 (CH), 145.5 (CH), 145.0 (CH),
137.5 (CH), 137.4 (C), 135.2 (C), 134.6 (CH), 133.0 (C),
129.0 (CH), 126.0 (CH), 125.0 (CH), 124.3 (CH), 124.1
(CH), 120.9 (C), 120.5 (C), 116.0 (CH), 116.2 (CH), 97.2
(C), 62.2 (CH ), 50.2 (CH ), 18.0 (CH ).
2
2
3
3
.5 | Spectral data
3.5.2 | 3-((([2,4-dioxochroman-3-ylidene]
methyl)amino)methyl)-1-(2-oxo-2-[m-
tolylamino]ethyl)pyridin-1-ium (7b)
3
3
2
.5.1 | 2–3-((([2,4-dioxochroman-
-ylidene]methyl)amino)methyl)-1-(2-oxo-
-[o-tolylamino]ethyl)pyridin-1-ium (7a)
Yield 90% (E isomer: 66% and Z isomer: 34%); white
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3402, 3,196
(NH), 1,707, 1,635, 1,593 (C=O). Anal. Calcd for
C H N O : C, 70.08; H, 5.18; N, 9.81. Found: C,
Yield 92% (E isomer: 62% and Z isomer: 38%); white
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3.426, 3,250
NH), 1,688, 1,631 (C=O). Anal. Calcd for
C H N O : C, 70.08; H, 5.18; N, 9.81. Found: C,
25
22 3 4
1
(
70.21; H, 5.27; N, 9.93. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.89 (s, 1H, =CNH),
25
22
3
4
6
1
7
0.16; H, 5.24; N, 9.67. NMR data for the E isomer: H
10.86 (s, 1H, NH), 9.27 (s, 1H, H pyridine), 9.16 (s, 1H,
2
NMR (500 MHz, DMSO (dimethyl sulfoxide) -d ) δ: 11.88
H pyridine), 8.76–8.73 (m, 2H, =CH and H pyridine),
6
6
4

MOLLAZADEH ET AL.
13
8
.27 (br. s, 1H, H pyridine), 7.91 (d, 1H, H coumarin,
J = 14.5 Hz), 8.72 (d, 1H, H pyridine, J = 7.5 Hz), 8.24
5
5
4
J = 5.0 Hz), 7.70–7.24 (m, 6H, H6 coumarin, H phenyl),
(br. s, 1H, H5 pyridine), 7.96 (d, 1H, H5 coumarin,
,8
+
5
.91 (s, 2H, CH N ), 5.11 (s, 2H, CH N), 2.09 (s, 3H,
J = 7.5 Hz), 7.67–7.65 (m, 1H, H coumarin), 7.51 (d, 2H,
2
2
7
13
CH3). CNMR (125 MHz, DMSO-d ) δ: 179.4 (CO), 163.3
H phenyl, J = 8.0 Hz), 7.32–7.28 (m, 2H, H coumarin),
6
α
6,8
+
(CO), 162.6(CO), 161.9 (CH), 154.2 (C), 146.5 (CH), 145.8
7.12 (d, 2H, H phenyl, J = 8.0 Hz), 5.72 (s, 2H, CH N ),
5.07 (s, 2H, CH N), 2.25 (s, 3H, CH ) CNMR (125 MHz,
β
2
13
(CH), 145.5 (CH), 137.6 (CH), 137.4 (C), 134.5 (CH),
2
3
1
34.0 (C), 129.7 (C),129.5 (CH), 127.5 (CH), 125.7
DMSO-d ) δ: 177.3 (CO), 163.4 (CO), 163.0 (CO), 162.6
6
(
(
CH),125.2 (CH), 124.0 (CH), 123.8 (CH), 120.1 (C), 116.8
CH), 96.7 (C), 62.1 (CH ), 50.2 (CH ), 30.6 (CH ).
(CH), 154.2 (C), 147.0 (CH), 145.5 (CH), 145.0 (CH),
137.6 (C), 137.6 (CH), 135.7(C), 134.5 (CH), 132.9 (C),
129.2 (CH), 127.2 (CH), 124.1 (CH), 120.1 (C), 119.2
2
2
3
1
NMR data for the Z isomer: H NMR (500 MHz,
DMSO-d ) δ: 10.86 (s, 1H, NH), 10.74 (s, 1H, =CNH),
(CH), 119.0 (CH), 116.8 (CH), 96.5 (C), 62.2 (CH ), 50.2
6
2
9
.27 (s, 1H, H2 pyridine), 9.16 (s, 1H, H6 pyridine),
(CH ), 20.4 (CH ).
2
3
8
.86 (d, 1H, J = 14.5 Hz, =CH), 8.74 (d, 1H, H pyri-
4
dine, J = 7.0 Hz), 8.27 (br. s, 1H, H pyridine), 7.96
5
(
d, 1H, H coumarin, J = 5.0 Hz), 7.70–7.24 (m, 6H,
3.5.4 | 1-(2-([2,4-dimethylphenyl]amino)-
2-oxoethyl)-3-((([2,4-dioxochroman-
3-ylidene]methyl)amino)methyl)pyridin-
1-ium (7d)
5
+
H6,8 coumarin, H phenyl), 5.91 (s, 2H, CH N ), 5.11
(
DMSO-d ) δ: 177.4 (CO), 163.8 (CO), 163.3(CO), 162.9
(
1
(
1
(
2
13
s, 2H, CH N), 2.09 (s, 3H, CH ). CNMR (125 MHz,
2 3
6
CH), 154.2 (C), 147.0 (CH), 146.0 (CH), 145.8 (CH),
37.6 (CH), 137.4 (C), 134.5 (CH), 134.0 (C), 129.7
C),129.5 (CH), 127.5 (CH), 125.7 (CH),125.2 (CH),
24.0 (CH), 123.8 (CH), 120.1 (C), 116.8 (CH), 96.5
C), 62.1 (CH ), 50.2 (CH ), 30.6 (CH ).
Yield 95% (E isomer: 65% and Z isomer: 35%); white solid;
ꢀ
−1
mp > 250 C. IR (KBr, cm ) ѵmax: 3,433, 3,281 (NH), 1,670,
1,632 (C=O). Anal. Calcd for C H N O : C, 70.57; H,
26 24 3 4
5.47; N, 9.50. Found: C, 70.41; H, 5.33; N, 9.61. NMR data
2
2
3
1
for the E isomer: H NMR (500 MHz, DMSO-d ) δ: 11.95
6
(
s, 1H, =CNH), 10.38 (s, 1H, NH), 9.20 (s, 1H, H pyridine),
2
3
.5.3 | 3-((([2,4-dioxochroman-3-ylidene]
9.09 (s, 1H, H pyridine), 8.74–8.72 (m, 2H, =CH and H
6
4
methyl)amino)methyl)-1-(2-oxo-2-[p-
pyridine), 8.25 (br. s, 1H, H pyridine), 7.92 (d, 1H, H cou-
5 5
tolylamino]ethyl)pyridin-1-ium (7c)
marin, J = 7.5 Hz), 7.68–7.65 (m, 1H, H7 coumarin),
.32–7.29 (m, 2H, H6,8 coumarin), 7.26 (s, 1H, H phenyl),
7
3
Yield 90% (E isomer: 65% and Z isomer: 35%); white
7.10 (d, 1H, H phenyl, J = 7.5 Hz), 6.92 (d, 1H, H phenyl,
5 6
+
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,448, 3,271
NH), 1,692, 1,633 (C=O). Anal. Calcd for
C H N O : C, 70.08; H, 5.18; N, 9.81. Found: C,
J = 7.5 Hz), 5.81 (s, 2H, CH N ), 5.09 (s, 2H, CH N), 2.23
2 2
13
(
(s, 6H, 2CH3). CNMR (125 MHz, DMSO-d ) δ: 179.4
6
(CO), 163.2 (CO), 162.9 (CO), 162.6 (CH), 154.2 (C), 145.7
(CH), 145.5 (CH), 145.0 (CH), 137.4 (CH), 135.0 (CH),134.9
(C), 134.5 (C), 130.2 (C), 128.4 (CH), 127.1 (CH), 126.4
(CH), 126.2 (CH), 125.2 (CH), 123.9 (CH), 116.8 (CH), 96.7
(C), 62.0 (CH ), 50.2 (CH ), 20.5 (CH ), 17.5 (CH ). NMR
25
22 3 4
1
6
9.94; H, 5.07; N, 9.97. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.84 (br. s, 1H, =CNH),
6
1
1
1.09 (br. s, 1H, NH), 9.16 (s, 1H, H pyridine), 9.05 (br. s,
H, H6 pyridine, J = 6 Hz), 8.72 (d, 1H, =CH,
2
2
2
3
3
1
J = 14.5 Hz), 8.71 (d, 1H, H pyridine, J = 7.5 Hz), 8.24
data for the Z isomer: H NMR (500 MHz, DMSO-d ) δ:
4
6
(br. s, 1H, H5 pyridine), 7.91 (d, 1H, H5 coumarin,
10.72 (s, 1H, =CNH), 10.38 (s, 1H, NH), 9.18 (s, 1H, H pyr-
2
J = 7.5 Hz), 7.67–7.65 (m, 1H, H coumarin), 7.51 (d, 2H,
idine), 9.09 (s, 1H, H6 pyridine), 8.85 (d, 1H, =CH,
7
H phenyl, J = 8.0 Hz), 7.32–7.28 (m, 2H, H coumarin),
J = 15.0 Hz), 8.73 (d, 1H, H pyridine, J = 7.0 Hz), 8.25
α
6,8
4
+
7
5
.12 (d, 2H, H phenyl, J = 8.0 Hz), 5.72 (s, 2H, CH N ),
.07 (s, 2H, CH N), 2.25 (s, 3H, CH ) CNMR (125 MHz,
(br. s, 1H, H5 pyridine), 7.97 (d, 1H, H5 coumarin,
β
2
13
J = 7.5 Hz), 7.68–7.65 (m, 1H, H coumarin), 7.32–7.29(m,
2
3
7
DMSO-d ) δ: 179.4 (CO), 162.9 (CO), 162.6 (CO), 162.0
2H, H6,8 coumarin), 7.26 (s, 1H, H phenyl), 7.10 (d, 1H, H
6
3
5
(
1
1
CH), 154.2 (C), 145.7 (CH), 145.5 (CH), 144.8 (CH),
37.6 (C), 137.4 (CH), 135.7(C), 134.5 (CH), 132.9 (C),
29.2 (CH), 127.2 (CH), 125.3 (CH), 120.1 (C), 119.2
phenyl, J = 7.5 Hz), 6.92 (d, 1H, H phenyl, J = 7.5 Hz),
6
+
5.81 (s, 2H, CH N ), 5.09 (s, 2H, CH N), 2.23 (s, 6H, 2CH ).
2
2
3
13
CNMR (125 MHz, DMSO-d ) δ: 177.3 (CO), 165.0 (CO),
6
(
(
(
(
CH), 119.0 (CH), 116.8 (CH), 96.7 (C), 62.2 (CH ), 50.2
162.9 (CO), 162.6 (CH), 154.2 (C), 147.0 (CH), 145.5 (CH),
145.0 (CH), 137.4 (CH), 135.0 (CH),134.9 (C), 134.5 (C),
130.2 (C), 128.4 (CH), 127.1 (CH), 126.4 (CH), 126.2 (CH),
125.2 (CH),123.9 (CH), 116.8 (CH), 96.7 (C), 62.0 (CH2),
50.2 (CH ), 20.5 (CH ), 17.5 (CH ).
2
1
CH ), 20.4 (CH ). NMR data for the Z isomer: H NMR
2
3
500 MHz, DMSO-d ) δ: 11.09 (br. s, 1H, NH), 10.70
6
br. s, 1H, =CNH), 9.14 (s, 1H, H pyridine), 9.05 (br. s,
2
1
H, H6 pyridine, J = 6.0 Hz), 8.84 (d, 1H, =CH,
2
3
3

14
MOLLAZADEH ET AL.
3
2
3
1
.5.5 | 1-(2-([2,4-dimethylphenyl]amino)-
-oxoethyl)-3-((([3,4-dioxochroman-
-ylidene]methyl)amino)methyl)pyridin-
-ium (7e)
134.5 (CH), 127.2 (C), 126.2 (C), 125.8 (CH), 125.2
(CH), 124.1 (CH), 121.8 (CH), 120.1 (CH), 117.0 (CH),
111.4 (CH), 96.6 (C), 62.4 (CH ), 55.6 (OCH ), 50.2
2
3
1
(CH2). NMR data for the
Z
isomer:
H NMR
(
500 MHz, DMSO-d ) δ: 10.80 (br. s, 1H, =CNH), 10.20
6
Yield 93% (E isomer: 66% and Z isomer: 34%); white
(br. s, 1H, NH), 9.16 (s, 1H, H pyridine), 9.06 (d, 1H,
2
H6 pyridine, J = 5.0 Hz), 8.84 (d, 1H, =CH,
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,420, 3,270
NH), 1,685, 1,630 (C=O). Anal. Calcd for
C H N O : C, 70.57; H, 5.47; N, 9.50. Found: C,
(
J = 15.0 Hz), 8.72 (d, 1H, H pyridine, J = 7.0 Hz),
4
8.24 (t, 1H, H pyridine, J = 7.0 Hz), 7.96 (d, 1H, H
5
2
6
24
3
4
5
1
7
0.62; H, 5.54; N, 9.39. NMR data for the E isomer: H
coumarin, J = 7.5 Hz), 7.92 (d, 1H, H3 phenyl,
7.5 Hz), 7.67–7.65 (m, 1H, H7 coumarin),
NMR (500 MHz, DMSO-d ) δ: 10.75 (s, 1H, NH), 9.12 (s,
J
=
6
1
H, H pyridine), 9.01 (d, 1H, H pyridine, J = 5.7 Hz),
7.34–7.29 (m, 2H, H6,8 coumarin), 7.14–7.09 (m, 2H,
H , phenyl), 6.91 (t, 1H, H phenyl, J = 7.0 Hz), 5.87
2
6
8
.72 (d, 1H, =CH, J = 15 Hz), 8.71 (br. s, 1H, H pyri-
4
4 6
5
+
dine), 8.23 (t, 1H, H pyridine, J = 7.5 Hz), 7.91 (d, 1H,
(s, 2H, CH N ), 5.08 (s, 2H, CH N), 3.88 (s, 3H,
2 2
OCH3). CNMR (125 MHz, DMSO-d ) δ: 177.0 (CO),
5
13
H coumarin, J = 8.15 Hz), 7.68–7.67 (m, 1H, H couma-
5
7
6
rin), 7.38 (s, 1H, H phenyl), 7.34–7.29 (m, 3H, H cou-
165.0 (CO), 162.6 (CO), 162.1 (CH), 154.2 (C), 149.7
(C), 145.7 (CH), 145.5 (CH), 145.4 (CH), 137.4 (CH),
134.5 (CH), 127.2 (C), 126.2 (C), 125.8 (CH), 125.2
(CH), 124.1 (CH), 121.8 (CH), 120.1 (CH), 117.0 (CH),
3
6.8
marin, H phenyl), 7.08 (d, 1H, H phenyl, J = 8.1), 5.66
5
6
+
2 2 3
(
2
s, 2H, CH N ), 5.05 (s, 2H, CH N), 2.17 (s, 3H, OCH ),
.16 (s, 3H, CH ). NMR data for the Z isomer: H NMR
3
1
(
500 MHz, DMSO-d ) δ: 10.75 (s, 1H, NH), 9.10 (s, 1H, H2
111.4 (CH), 96.6 (C), 62.4 (CH ),55.6 (OCH3),
6
2
pyridine), 9.01 (d, 1H, H pyridine, J = 5.7 Hz), 8.85 (d,
50.2 (CH2).
6
1
H, =CH, J = 15 Hz), 8.71 (br. s, 1H, H pyridine), 8.23
4
(
t, 1H, H pyridine, J = 7.5 Hz), 7.95 (d, 1H, H coumarin,
5
5
J = 8.15 Hz), 7.68–7.67 (m, 1H, H coumarin), 7.38 (s,
3.5.7 | 3-((([2,4-dioxochroman-3-ylidene]
methyl)amino)methyl)-
1-(2-([4-methoxyphenyl]amino)-2-oxoethyl)
pyridin-1-ium (7g)
7
1
H, H phenyl), 7.34–7.29 (m, 3H, H coumarin, H phe-
3
6.8
5
nyl), 7.08 (d, 1H, H6 phenyl, J = 8.1), 5.66 (s, 2H,
+
CH N ), 5.05 (s, 2H, CH N), 2.17 (s, 3H, OCH ), 2.16 (s,
2
2
3
3
H, CH3).
Yield 90% (E isomer: 66% and Z isomer: 34%); white
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,428, 3,270
(NH), 1,685, 1,630, 1,600 (C=O). Anal. Calcd for
C H N O : C, 67.56; H, 4.99; N, 9.45. Found: C,
3
.5.6 | 3-((([2,4-dioxochroman-3-ylidene]
methyl)amino)methyl)-
-(2-([2-methoxyphenyl]amino)-2-oxoethyl)
25
22 3 5
1
1
67.45; H, 4.84; N, 9.31. NMR data for the E isomer: H
pyridin-1-ium (7f)
NMR (500 MHz, DMSO-d ) δ: 11.84 (s, 1H, =CNH),
6
1
1.04 (s, 1H, NH), 9.17 (s, 1H, H pyridine), 9.06 (br. s,
2
Yield 87% (E isomer: 65% and Z isomer: 35%); white
1H, H pyridine), 8.81–8.73 (m, 2H, =CH and H pyri-
6
4
ꢀ
−1
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,426, 3,250
NH), 1,712, 1,693, 1,626 (C=O). Anal. Calcd for
C H N O : C, 67.56; H, 4.99; N, 9.45. Found: C,
dine), 8.25 (br. s, 1H, H pyridine), 7.94 (br. s, 1H, H
5
5
(
coumarin), 7.67 (br. s, 1H, H coumarin), 7.55 (br. s, 2H,
7
H phenyl), 7.31 (br. s, 2H, H phenyl), 6.92–6.90 (m, 2H,
2
5
22
3
5
α
β
1
+
2 2
6
7.68; H, 5.12; N, 9.64. NMR data for the E isomer: H
H6,8 coumarin), 5.72 (s, 2H, CH N ), 5.08 (s, 2H, CH N),
3.72 (s, 3H, OCH3). CNMR (125 MHz, DMSO-d ) δ:
13
NMR (500 MHz, DMSO-d ) δ: 11.90 (br. s, 1H, =CNH),
6
6
1
1
0.20 (br. s, 1H, NH), 9.18 (s, 1H, H pyridine), 9.06 (d,
179.5 (CO), 164.0 (CO), 162.7 (CO), 162.3 (CH), 155.6 (C),
154.1 (C), 145.7 (CH), 145.5 (CH), 144.9 (CH), 137.5
(CH), 134.5 (CH), 131.3 (C), 127.1 (C), 125.4 (CH),124.1
(CH), 120.7 (CH), 120.6 (CH), 120.2 (C),116.8 (CH), 114.1
(CH), 113.8 (CH), 96.6 (C), 62.1 (CH ), 55.2 (OCH ), 50.3
2
H, H pyridine, J = 5.0 Hz), 8.74–8.71 (m, 2H, =CH and
6
H4 pyridine), 8.24 (t, 1H, H5 pyridine, J = 7.0 Hz),
7
.93–7.89 (m, 2H, H coumarin and H phenyl), 7.67–7.65
5
3
(
7
m, 1H, H coumarin), 7.34–7.29 (m, 2H, H coumarin),
7
6,8
2
1
3
.14–7.09 (m, 2H, H4,6 phenyl), 6.91 (t, 1H, H phenyl,
(CH ). NMR data for the Z isomer: H NMR (500 MHz,
5
2
+
J = 7.0 Hz), 5.77 (s, 2H, CH N ), 5.07 (s, 2H, CH N), 3.88
DMSO-d ) δ: 11.04 (s, 1H, NH), 10.80 (s, 1H, =CNH),
2
2
6
13
(
(
(
s, 3H, OCH3). CNMR (125 MHz, DMSO-d ) δ: 179.4
CO), 163.2 (CO), 162.6 (CO), 162.1 (CH), 154.2 (C), 149.7
C), 145.7 (CH), 145.5 (CH), 145.4 (CH), 137.4 (CH),
9.16 (s, 1H, H pyridine), 9.06 (br. s, 1H, H pyridine),
6
2
6
8.81–8.73 (m, 2H, =CH and H pyridine), 8.25 (br. s, 1H,
4
H pyridine), 7.94 (br. s, 1H, H coumarin), 7.67 (br. s,
5
5

MOLLAZADEH ET AL.
15
1
2
H, H coumarin), 7.55 (br. s, 2H, H phenyl), 7.31 (br. s,
3.5.9 | 1-(2-([4-chlorophenyl]amino)-
2-oxoethyl)-3-((([2,4-dioxochroman-
3-ylidene]methyl)amino)methyl)pyridin-
1-ium (7i)
7
α
H, H phenyl), 6.92–6.90 (m, 2H, H coumarin), 5.72
β
6,8
+
2 2 3
(
s, 2H, CH N ), 5.08 (s, 2H, CH N), 3.72 (s, 3H, OCH ).
1
3
CNMR (125 MHz, DMSO-d ) δ: 177.5 (CO), 165.0 (CO),
6
1
1
62.7 (CO), 162.3 (CH), 155.6 (C), 154.1 (C), 147.0 (CH),
45.5 (CH), 145.9 (CH), 137.5 (CH), 134.5 (CH), 131.3
Yield 89% (E isomer: 66% and Z isomer: 34%); white
ꢀ
−1
(
(
(
C), 127.1 (C), 125.4 (CH),124.1 (CH), 120.7 (CH), 120.6
CH), 120.2 (C),116.8 (CH), 114.1 (CH), 113.8 (CH), 96.6
C), 62.1 (CH ), 55.2 (OCH ), 50.3 (CH ).
solid; mp > 250 C; IR (KBr, cm ) ѵmax: 3,426, 3,259
(NH), 1,688, 1,631, 1,595 (C=O); Anal. Calcd for
C H ClN O : C, 64.22; H, 4.27; N, 9.36. Found: C,
2
3
2
24 19
3 4
1
64.14; H, 4.35; N, 9.45. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.84 (br. s, 1H, =CNH),
6
3.5.8 | 1-(2-([3-chlorophenyl]amino)-
2-oxoethyl)-3-((([2,4-dioxochroman-
3-ylidene]methyl)amino)methyl)pyridin-
1-ium (7h)
11.50 (br. s, 1H, NH), 9.16 (s, 1H, H pyridine), 9.06 (d,
2
1H, H pyridine, J = 5.5 Hz), 8.71–8.73 (m, 2H, =CH, H
6
4
pyridine), 8.24 (t, 1H, H pyridine, J = 6.0 Hz), 7.92 (br. s,
5
1H, H coumarin), 7.69–7.65 (m, 1H, H coumarin), 7.68
5
7
(
d, 2H, H phenyl, J = 8.0 Hz), 7.38 (d, 2H, H phenyl,
α
β
Yield 83% (E isomer: 68% and Z isomer: 32%); white
J = 8.0 Hz), 7.32–7.28 (m, 2H, H6 coumarin), 5.77 (s,
,8
13
ꢀ
−1
+
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,420, 3,250
NH), 1,710, 1,693 (C=O). Anal. Calcd for
C H ClN O : C, 64.22; H, 4.27; N, 9.36. Found: C,
2H, CH N ), 5.07 (s, 2H, CH N). CNMR (125 MHz,
2
2
(
DMSO-d ) δ: 179.5 (CO), 163.1 (CO), 162.5 (CO), 162.0
6
(CH), 154.1 (C), 145.7 (CH), 145.6 (CH), 145.3 (CH),
137.5 (C), 137.2 (CH), 134.5 (C), 132.9 (C), 128.9 (C),128.7
(CH), 127.5 (CH),127.2 (CH), 125.3 (CH), 124.1 (CH),
2
4
19
3 4
1
6
4.37; H, 4.13; N, 9.45. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.90 (br. s, 1H, =CNH),
6
1
1.50 (s, 1H, NH), 9.17 (s, 1H, H pyridine), 9.07 (d, 1H,
123.9 (CH), 120.7 (C), 116.8 (CH), 96.7 (C), 62.2 (CH ),
2
2
1
H6 pyridine, J = 4.5 Hz), 8.74 (d, 1H, H4 pyridine,
J = 6.5 Hz), 8.72 (d, 1H, =CH, J = 15.0 Hz), 8.26 (br. s,
50.2 (CH ). NMR data for the Z isomer: H NMR
2
(500 MHz, DMSO-d ) δ: 11.50 (br. s, 1H, NH), 10.70
6
1
7
7
H, H pyridine), 7.92 (d, 1H, H coumarin, J = 7.0 Hz),
(br. s, 1H, =CNH), 9.16 (s, 1H, H pyridine), 9.06 (d, 1H,
5
5
2
.83 (s, 1H, H phenyl), 7.68–7.65 (m, 1H, H coumarin),
H pyridine, J = 5.5 Hz), 8.71–8.73 (m, 2H, =CH, H pyri-
2
7
6
4
.55 (d, 1H, H phenyl, J = 8.0 Hz), 7.38 (t, 1H, H phe-
dine), 8.24 (t, 1H, H pyridine, J = 6.0 Hz), 7.92 (br. s, 1H,
4
5
5
nyl, J = 8.0 Hz), 7.34–7.29 (m, 2H, H6,8 coumarin), 7.16
H coumarin), 7.69–7.65 (m, 1H, H coumarin), 7.68 (d,
5
7
+
(
(
(
(
(
d, 1H, H phenyl, J = 7.5 Hz), 5.77 (s, 2H, CH N ), 5.08
2H, Hα phenyl, J = 8.0 Hz), 7.38 (d, 2H, H phenyl,
6
2
β
13
s, 2H, CH N). CNMR (125 MHz, DMSO-d ) δ: 179.4
J = 8.0 Hz), 7.32–7.28 (m, 2H, H coumarin), 5.77 (s, 2H,
2
6
6,8
+
2 2
13
CO), 163.4 (CO), 162.6 (CO), 162.5 (CH), 154.2 (C), 145.7
CH), 145.5 (CH), 145.3 (CH), 139.6 (C), 137.6 (C), 137.4
CH), 134.5 (CH), 133.1 (C), 130.7 (CH), 127.1 (CH),
CH N ), 5.07 (s, 2H, CH N). CNMR (125 MHz, DMSO-
d6) δ: 177.5 (CO), 164.0 (CO), 162.5 (CH), 162.0 (CO), 154.1
(C), 147.0 (CH), 145.7 (CH), 145.5 (CH), 137.5 (C), 137.2
(CH), 134.5 (C), 132.9 (C), 128.9 (C),128.7 (CH), 127.5
(CH),127.2 (CH), 127.1 (CH), 124.1 (CH), 123.9 (CH), 120.7
(C), 117.0 (CH), 96.7 (C), 62.2 (CH ), 50.2 (CH ).
1
25.8 (CH), 125.2 (CH), 123.6 (CH), 120.1 (C), 118.6
(
CH), 117.0 (CH), 96.6 (C), 62.6 (CH ), 50.2 (CH ). NMR
2
2
1
data for the Z isomer: H NMR (500 MHz, DMSO-d ) δ:
6
2
2
1
1.50 (s, 1H, NH), 10.73 (br. s, 1H, =CNH), 9.16 (s, 1H,
H pyridine), 9.07 (d, 1H, H pyridine, J = 4.5 Hz), 8.85
2
6
(
d, 1H, =CH, J = 15.0 Hz), 8.74 (d, 1H, H pyridine,
3.5.10 | 1-(2-([3,4-dichlorophenyl]
amino)-2-oxoethyl)-3-((([2,4-dioxochroman-
3-ylidene]methyl)amino)methyl)pyridin-
1-ium (7j)
4
J = 6.5 Hz), 8.26 (br. s, 1H, H pyridine), 7.97 (d, 1H, H5
coumarin, J = 7.0 Hz), 7.83 (s, 1H, H phenyl), 7.68–7.65
(
5
2
m, 1H, H7 coumarin), 7.55 (d, 1H, H4 phenyl,
J = 8.0 Hz), 7.38 (t, 1H, H phenyl, J = 8.0 Hz), 7.34–7.29
5
(
m, 2H, H6,8 coumarin), 7.16 (d, 1H, H phenyl,
Yield 81% (E isomer: 66% and Z isomer: 34%); white
6
+
ꢀ
−1
J = 7.5 Hz), 5.77 (s, 2H, CH N ), 5.08 (s, 2H, CH N).
CNMR (125 MHz, DMSO-d ) δ: 176.3 (CO), 164.0 (CO),
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,454, 3,245
(NH), 1,687, 1,630, 1,590 (C=O). Anal. Calcd for
C H Cl N O : C, 59.64; H, 3.75; N, 8.69. Found: C,
2
2
13
6
1
62.6 (CO), 162.5 (CH), 154.2 (C), 147.0 (CH), 145.5
2
4
18
2 3 4
1
(
(
CH), 145.3 (CH), 139.6 (C), 137.6 (C), 137.4 (CH), 134.5
CH), 133.1 (C), 130.7 (CH), 127.1 (CH), 125.8 (CH),
59.57; H, 3.68; N, 8.52. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.90 (br. s, 1H, =CNH),
6
1
25.2 (CH), 123.6 (CH), 120.1 (C), 118.6 (CH), 117.0
11.85 (s, 1H, NH), 9.17 (s, 1H, H pyridine), 9.07 (d, 1H,
2
(
CH), 96.6 (C), 62.6 (CH ), 50.2 (CH ).
H pyridine, J = 5.0 Hz), 8.74–8.71 (m, 2H, H pyridine
2
2
6
4

16
MOLLAZADEH ET AL.
and = CH), 8.26 (t, 1H, H pyridine, J = 7.5 Hz), 8.03 (s,
J = 4.5 Hz), 8.74 (d, 1H, H pyridine, J = 6.5 Hz), 8.72 (d,
5
4
1
7
H, H phenyl), 7.93 (d, 1H, H coumarin, J = 7.5 Hz),
1H, =CH, J = 15.0 Hz), 8.26 (br. s, 1H, H pyridine,
3
5
5
.70–7.57 (m, 3H, H coumarin, H phenyl), 7.33–7.29
J = 7.0 Hz), 7.92 (d, 1H, H coumarin, J = 7.5 Hz), 7.88
7
5,6
5
+
(m, 2H, H coumarin), 5.79 (s, 2H, CH N ), 5.08 (s, 2H,
(s, 1H, H phenyl), 7.67 (t, 1H, H coumarin, J = 7.5 Hz),
6,8
13
2
3
7
CH N). CNMR (125 MHz, DMSO-d ) δ: 179.4 (CO),
7.59 (d, 1H, H phenyl, J = 7.0 Hz), 7.33–7.29 (m, 3H,
2
6
5
+
1
63.7 (CO), 163.5 (CO), 162.6 (CH), 154.2 (C), 145.6 (CH),
H6,8 coumarin, H phenyl), 5.83 (s, 2H, CH N ), 5.08 (s,
2H, CH N). CNMR (125 MHz, DMSO-d ) δ: 177.5 (CO),
6
2
13
1
45.5 (CH), 144.8 (CH), 138.4 (CH), 137.5 (C), 134.5
2
6
(
(
1
CH), 131.0 (C), 127.2 (C), 127.1 (C), 125.4 (CH), 125.2
CH), 120.4 (CH), 120.3 (CH), 120.1 (CH), 119.3 (C),
166.0 (CO), 163.4 (CO), 162.6 (CH), 154.2 (C), 147.6 (CH),
145.5 (CH), 145.3 (CH), 137.5 (CH), 135.2 (C), 134.5
(CH), 131.5 (C), 131.1 (C), 131.0 (C), 127.1 (CH), 126.4
(CH),125.7 (CH), 125.2 (CH), 124.8 (CH), 124.0 (C), 117.0
(CH), 97.2 (C), 62.2 (CH ), 50.2 (CH ).
17.0 (CH), 96.7 (C), 62.2 (CH ), 50.2 (CH ). NMR data
2 2
1
for the Z isomer: H NMR (500 MHz, DMSO-d ) δ: 11.90
(
6
br. s, 1H, =CNH), 11.85 (s, 1H, NH), 9.16 (s, 1H, H pyri-
2
2
2
dine), 9.07 (d, 1H, H pyridine, J = 5.0 Hz), 8.50(d, 1H,
6
=
CH, J = 15 Hz), 8.74–8.71 (m, 1H, H pyridine), 8.26 (t,
4
1
H, H pyridine, J = 7.5 Hz), 8.03 (s, 1H, H phenyl), 7.97
3.5.12 | 4-((([2,4-dioxochroman-
3-ylidene]methyl)amino)methyl)-1-(2-oxo-
2-[m-tolylamino]ethyl)pyridin-1-ium (7l)
5
3
(
d, 1H, H coumarin, J = 7.5 Hz), 7.70–7.57 (m, 3H, H7
5
coumarin, H5,6 phenyl), 7.33–7.29 (m, 2H, H couma-
rin), 5.79 (s, 2H, CH N ), 5.08 (s, 2H, CH N). CNMR
6,8
13
+
2
2
(
(
125 MHz, DMSO-d ) δ: 177.3 (CO), 165.0 (CO), 163.5
CO), 162.6 (CH), 154.2 (C), 147.0 (CH), 145.5 (CH),
Yield 70% (E isomer: 67% and Z isomer: 33%); white
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,037 (NH),
6
ꢀ
−1
1
1
1
6
45.0 (CH), 138.4 (CH), 137.5 (C), 134.5 (CH), 131.0 (C),
27.2 (C), 127.1 (C), 125.4 (CH), 125.2 (CH), 120.4 (CH),
20.3 (CH), 120.1 (CH), 119.3 (C), 117.0 (CH), 96.7 (C),
2.2 (CH ), 50.2 (CH ).
1,701, 1,641 (C=O). Anal. Calcd for C H N O : C,
25
22 3 4
70.08; H, 5.18; N, 9.81. Found: C, 70.01; H, 5.04; N, 9.95.
1
NMR data for the E isomer: H NMR (500 MHz, DMSO-
d6) δ: 11.90 (br. s, 1H, =CNH), 11.20 (s, 1H, NH), 9.05 (d,
2
2
2H, Hα pyridine, J = 6.5 Hz), 8.72 (d, 1H, =CH,
J = 14.5 Hz), 8.12 (d, 2H, H pyridine, J = 6.5 Hz), 7.95
β
3
.5.11 | 1-(2-([2,4-dichlorophenyl]
(d, 1H, H coumarin, J = 7.6 Hz), 7.69–7.66 (m, 1H, H
5
7
amino)-2-oxoethyl)-3-((([2,4-dioxochroman-
coumarin), 7.47 (s, 1H, H phenyl), 7.43 (d, 1H, H phe-
2 4
3
1
-ylidene]methyl)amino)methyl)pyridin-
-ium (7k)
nyl, J = 7.5 Hz), 7.35–7.30 (m, 2H, H6,8 coumarin), 7.21
(t, 1H, H phenyl, J = 7.5 Hz), 6.90 (d, 1H, H phenyl,
5
6
+
J = 7.5 Hz), 5.72 (s, 2H, CH N ), 5.18 (s, 2H, CH N), 2.26
2
2
13
Yield 80% (E isomer: 66% and Z isomer: 34%); white
(s, 1H, CH3). CNMR (125 MHz, DMSO-d ) δ: 179.0
6
ꢀ
−1
solid; mp > 250 C; IR (KBr, cm ) ѵmax: 3,315, 3,153
NH), 1,697, 1,636, 1,592 (C=O). Anal. Calcd for
C H Cl N O : C, 59.64; H, 3.75; N, 8.69. Found: C,
(CO), 163.2 (CO), 162.5 (CO), 162.0 (CH), 157.7 (C),
154.3 (C), 146.1 (CH), 145.5 (CH), 138.3 (CH), 138.1
(C), 128.7 (CH), 125.4 (CH), 124.6 (CH), 120.2 (C),
119.7 (CH), 117.1 (CH), 117.0 (CH), 116.3 (CH), 96.9
(C), 61.7 (CH ), 50.2 (CH ), 21.2 (CH ). NMR data
(
24
18
2 3 4
1
5
9.73; H, 3.61; N, 8.74. NMR data for the E isomer: H
NMR (500 MHz, DMSO-d ) δ: 11.90 (br. s, 1H, =CNH),
6
2
2
3
1
1
1.50 (s, 1H, NH), 9.18 (s, 1H, H pyridine), 9.07 (d, 1H,
for the E isomer: H NMR (500 MHz, DMSO-d ) δ:
2
6
H6 pyridine, J = 4.5 Hz), 8.74 (d, 1H, H4 pyridine,
J = 6.5 Hz), 8.72 (d, 1H, =CH, J = 15.0 Hz), 8.26 (t, 1H,
11.20 (s, 1H, NH), 10.70 (br. s, 1H, =CNH), 9.05 (d,
2H, Hα pyridine, J = 6.5 Hz), 8.85 (d, 1H, =CH,
H pyridine, J = 7.0 Hz), 7.92 (d, 1H, H coumarin,
J = 14.5 Hz), 8.12 (d, 2H, H pyridine, J = 6.5 Hz),
5
5
β
J = 7.5 Hz), 7.88 (s, 1H, H phenyl), 7.67 (t, 1H, H cou-
7.96 (d, 1H, H5 coumarin, J = 7.6 Hz), 7.69–7.66
(m, 1H, H7 coumarin), 7.47 (s, 1H, H2 phenyl), 7.43
(d, 1H, H4 phenyl, J = 7.5 Hz), 7.35–7.30 (m, 2H,
3
7
marin, J = 7.5 Hz), 7.59 (d, 1H, H phenyl, J = 7.0 Hz),
5
7
.33–7.29 (m, 3H, H coumarin, H phenyl), 5.83 (s, 2H,
6
,8
6
+ 13
CH N ), 5.08 (s, 2H, CH N). CNMR (125 MHz, DMSO-
H6,8 coumarin), 7.21 (t, 1H, H phenyl, J = 7.5 Hz),
2
2
5
d6) δ: 179.5 (CO), 164.3 (CO), 163.4 (CO), 162.6 (CH),
54.2 (C), 145.6 (CH), 145.5 (CH), 144.9 (CH), 137.5
6.90 (d, 1H, H6 phenyl, J = 7.5 Hz), 5.72 (s, 2H,
+
1
CH N ), 5.18 (s, 2H, CH N), 2.26 (s, 1H, CH ).
2
2
3
13
(
(
(
(
CH), 135.2 (C), 134.5 (CH), 131.5 (C), 131.1 (C), 131.0
C), 127.1 (CH), 126.4 (CH),125.7 (CH), 125.2 (CH), 124.8
CNMR (500 MHz, DMSO-d ) δ: 177.0 (CO), 164.0
6
(CO), 162.5 (CO), 162.0 (CH), 157.7 (C), 154.3 (C),
146.1 (CH), 145.5 (CH), 138.3 (CH), 138.1 (C), 128.7
(CH), 125.4 (CH), 124.6 (CH), 120.2 (C), 119.7 (CH),
117.1 (CH), 117.0 (CH), 116.3 (CH), 96.9 (C), 61.7
(CH ), 50.2 (CH ), 21.2 (CH ).
CH), 124.0 (C), 117.0 (CH), 97.2 (C), 62.2 (CH ), 50.2
2
1
CH ). NMR data for the Z isomer: H NMR (500 MHz,
2
DMSO-d ) δ: 11.50 (s, 1H, NH), 10.75 (br. s, 1H, =CNH),
6
9
.17 (s, 1H, H2 pyridine), 9.07 (d, 1H, H6 pyridine,
2
2
3

MOLLAZADEH ET AL.
17
3
.5.13 | 1-(2-([2,5-dimethylphenyl]
(m, 1H, H coumarin), 7.69–7.60 (m, 4H, H , phenyl, H7
5
4 5,6
amino)-2-oxoethyl)-4-((([2,4-dioxochroman-
coumarin), 7.35–7.30 (m, 2H, H6 coumarin), 5.68 (s, 2H,
,8
+
3
1
-ylidene]methyl)amino)methyl)pyridin-
CH N ), 5.17 (s, 2H, CH N). NMR data for the E isomer:
H NMR (500 MHz, DMSO-d ) δ: 11.80 (s, 1H, =CNH),
2
2
1
-ium (7m)
6
1
0.70 (s, 1H, NH), 9.05 (d, 2H, H pyridine, J = 6.5 Hz),
α
Yield 80% (E isomer: 67% and Z isomer: 33%); white
solid; mp > 250 C. IR (KBr, cm ) ѵmax: 3,037 (NH),
8.85 (d, 1H, =CH, J = 14.5 Hz), 8.13 (d, 2H, H pyridine,
β
ꢀ
−1
J = 6.0 Hz), 8.02 (s, 1H, H phenyl), 7.97–7.94 (m, 1H, H
3
5
1
7
,701, 1,641 (C=O). Anal. Calcd for C H N O : C,
coumarin), 7.69–7.60 (m, 4H, H4,5,6 phenyl, H coumarin),
26
24
3
4
7
+
0.57; H, 5.47; N, 9.50. Found: C, 70.67; H, 5.58; N, 9.39.
7.35–7.30 (m, 2H, H6,8 coumarin), 5.68 (s, 2H, CH N ),
2
1
NMR data for the E isomer: H NMR (500 MHz, DMSO-
5.17 (s, 2H, CH N).
2
d6) δ: 11.90 (br. s, 1H, =CNH), 10.39 (s, 1H, NH), 9.05 (d,
2
H, Hα pyridine, J = 6.6 Hz), 8.70 (d, 1H, =CH,
J = 14.5 Hz), 8.12 (d, 2H, H pyridine, J = 6.6 Hz), 7.94
3.6 | ChE inhibition assay
β
(
d, 1H, H coumarin, J = 8.0 Hz), 7.69–7.66 (m, 1H, H7
5
coumarin), 7.35–7.30 (m, 2H, H6,8 coumarin), 7.22 (s, 1H,
H phenyl), 7.10 (d, 1H, H phenyl, J = 7.6 Hz), 6.92 (d,
All the synthesized 2,4-dioxochroman-pyridinium-
phenylacetamides 7a–n were screened for ChE inhibitory
activities against AChE (E.C. 3.1.1.7, typeV-S, lyophilized
powder, from electric eel, 1,000 unit) and BuChE
(E.C.3.1.1.8, from equine serum) using Ellman's
3
5
+
1
2
H, H phenyl, J = 7.6 Hz), 5.75 (s, 2H, CH N ), 5.18 (s,
6 2
13
H, CH N), 2.22 (s, 6H, 2CH ). CNMR (125 MHz,
2
3
DMSO-d ) δ: 179.8 (CO), 163.5 (CO), 163.4 (CO), 162.0
6
[
13]
(
(
(
CH), 158.0 (C), 154.3 (C), 145.5 (CH), 137.0 (C), 135.2
C), 135.1 (CH), 135.0 (CH), 130.3 (C),128.6 (CH), 126.3
CH), 125.4 (CH), 125.3 (CH), 124.0 (CH), 120.0 (C),
method.
These enzymes, acetylthiocholine iodide
(ATCI), and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)
were purchased from Sigma-Aldrich. Potassium phos-
phate buffer components, including potassium hydrox-
ide, potassium dihydrogen phosphate, dipotassium
hydrogen phosphate, and sodium hydrogen carbonate,
were purchased from Fluka. The title compounds 7a–n
were dissolved in a mixture of 5 ml of DMSO and 5 ml of
methanol and diluted in potassium phosphate buffer
(pH 8.0, 0.1 M). Then, 50 μl of potassium phosphate
buffer, 25 μl of prepared sample (as described above), and
25 μl of target enzyme (AChE or BuChE, final concentra-
tion of 0.22 U/ml) were added to each well of a 96-well
plate and were preincubated for 15 min at room tempera-
ture. After that, 125 μl of DTNB (3 mM in the buffer) was
added to each well, and the change of absorbance was
determined at 412 nm for 15 min. The IC50 values were
calculated graphically from the inhibition curves (log
inhibitor concentration vs. percentage of inhibition).
1
2
19.7 (CH), 117.0 (CH), 96.9 (C), 62.0 (CH ), 52.3 (CH ),
2 2
1
0.5 (CH ), 17.5 (CH ). NMR data for the Z isomer: H
3
3
NMR (500 MHz, DMSO-d ) δ: 10.80 (br. s, 1H, =CNH),
6
1
0.39 (s, 1H, NH), 9.05 (d, 2H, H pyridine, J = 6.6 Hz),
α
8
.85 (d, 1H, =CH, J = 14.5 Hz), 8.12 (d, 2H, H pyridine,
β
J = 6.6 Hz), 7.96 (d, 1H, H coumarin, J = 8.0 Hz),
5
7
.69–7.66 (m, 1H, H coumarin), 7.35–7.30 (m, 2H, H
7
6,8
coumarin), 7.22 (s, 1H, H phenyl), 7.10 (d, 1H, H phe-
3
5
nyl, J = 7.6 Hz), 6.92 (d, 1H, H phenyl, J = 7.6 Hz), 5.75
6
+
2 2 3
(
s, 2H, CH N ), 5.18 (s, 2H, CH N), 2.22 (s, 6H, 2CH ).
1
3
CNMR (125 MHz, DMSO-d ) δ: 177.0 (CO), 164.0 (CO),
6
1
1
63.4 (CO), 162.0 (CH), 158.0 (C), 154.3 (C), 145.5 (CH),
37.0 (C), 135.2 (C), 135.1 (CH), 135.0 (CH), 130.3
(
(
(
C),128.6 (CH), 126.3 (CH), 125.4 (CH), 125.3 (CH), 124.0
CH), 120.0 (C), 119.7 (CH), 117.0 (CH), 96.9 (C), 62.0
CH ), 52.3 (CH ), 20.5 (CH ), 17.5 (CH ).
2
2
3
3
3
.5.14 | 1-(2-([3,4-dichlorophenyl]
3.7 | Molecular modeling and dynamic
study
amino)-2-oxoethyl)-4-((([2,4-dioxochroman-
3
1
-ylidene]methyl)amino)methyl)pyridin-
-ium (7n)
3.7.1 | Target and ligand preparation
Yield 73% (E isomer: 67% and Z isomer: 33%); white solid;
mp > 250 C. IR (KBr, cm ) ѵmax: 3,037 (NH), 1,701, 1,641
In order to find out interaction modes of the synthesized
molecules over ChE enzymes, the Maestro Molecular
Modeling platform (version 11.4) by Schrödinger, LLC
ꢀ
−1
(
C=O). Anal. Calcd for C H Cl N O : C, 59.64; H,
24 18 2 3 4
[
18]
3
.75; N, 8.69. Found: C, 59.71; H, 3.82; N, 8.47. NMR data
was used.
Initially, the crystal structures of AChE and
1
for the E isomer: H NMR (500 MHz, DMSO-d ) δ: 11.96
BuChE enzymes were retrieved from the Protein Data
Bank (PDB: 6o4w and 4bds, respectively) (http://www.
6
(
s, 1H, NH), 11.80 (s, 1H, =CNH), 9.05 (d, 2H, H pyridine,
α
[
19,20]
J = 6.5 Hz), 8.70 (d, 1H, =CH, J = 14.5 Hz), 8.11 (d, 2H,
H pyridine, J = 6.0 Hz), 8.02 (s, 1H, H phenyl), 7.97–7.94
rcsb.org).
These PDB IDs were selected based on the
following criteria: highest resolution (Å), being related to
β
3

18
MOLLAZADEH ET AL.
Homo sapiens species, wild type with no modified resi-
due, and the existence of a cocrystalized ligand. As the
prosthetic group and the cofactors are not directly
involved in ChE inhibition, they were completely
removed before the docking investigation. Except the
four structural water molecules in the AChE and BuChE
active site, which bridge the receptor's important residues
through of H-bonds and conservation among other
respectively—and compared their perturbation with the
standard inhibitor donepezil.
In order to build a system for MD simulation, the
protein–ligand complexes were solvated by simple point
charge explicit water molecules and placed in the center
of an orthorhombic box of appropriate size in the Peri-
odic Boundary Condition (PBC). Sufficient counterions
and a 0.15 M solution of NaCl were also utilized to neu-
tralize the system and to simulate real cellular ionic con-
centrations, respectively. The MD protocol involved
minimization, preproduction, and finally production MD
simulation steps. In the minimization procedure, the
entire system was allowed to relax for 2,500 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 an
NPT (the number of particles, pressure, and temperature)
ensemble. The Nose-Hoover chain method was used as
the default thermostat 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 electro-
static forces were calculated based on particle mesh-
based Ewald approach, with the cut-off radius for colum-
bic forces set to 9.0 Å. Finally, this system was subjected
to production MD simulations for 20 ns for each protein–
ligand complex. During the simulation, every 1,000 ps of
the actual frame was stored. The dynamic behavior and
structural changes of the systems were analyzed by the
calculation of energy and the RMSD. Subsequently, by
using a trajectory-clustering tool, the representative struc-
ture with the largest number of neighbors from the equil-
ibrated trajectory was evaluated for investigation of each
ligand–protein complex.
[21]
species,
the remaining water molecules were removed
from the enzymes' crystallographic structures. The 3D
structures of all synthesized compounds were drawn in
Marvin 15.10.12.0 program (http://www.chemaxon.
[
22]
com)
Wizard
and converted to PDB. The Protein Preparation
[23]
[24]
and the LigPrep
module were used to prop-
erly prepare protein and ligand structures.
The glide receptor grids were constructed based on
the cocrystalized ligand-binding sites in the Glide appli-
cation (Glide, version) of Maestro. The center of each grid
was arranged at the centroid of the crystalized ligand-
binding site, which was set with inner (acceptable space
for the ligand center) and outer (search space surrounded
all the ligand atoms) box sizes of 10 and 20 Å, respec-
tively. In the case of AChE (PDB ID: 6o4w), the hydroxyl
group of active site residues of Thr72, Thr75, Tyr124,
Tyr133, Tyr151, Ser203, Ser293, Tyr337, Tyr341, Ser438,
Tyr449, and GLH202 are treated as flexible, and for
BuChE (PDB ID: 4bds), Ser72, Ser79, Thr120, Tyr128,
Ser198, Ser224, Thr327, Tyr332, Tyr440, and GLH197
were set to rotate during the docking experiment.
3.7.2 | Molecular docking protocol
The glide docking protocol implemented in Maestro was
[25]
used to dock and score the compounds.
The applied
docking consisted of Standard Precision (SP) and Extra
Precision (XP) in order to increase the computational
complexity. full Flexibility Of molecular docking was
assumed for the ligands. During the SP step, up to five
poses were generated, and energetically favorable com-
plexes were obtained for compounds and applied to XP
docking. In this step, up to three poses were generated
for each compound state, and only the best glide energy
states were retained.
3.8 | In vitro and in silico toxicity assays
In vitro evaluation of cytotoxic effects of the compounds
7j and 7k was performed based on our previous
[
17]
reports.
In silico carcinogenicity assay of these com-
pounds was determined by using an online server (http://
preadmet.bmdrc.org/).
4
| CONCLUSIONS
3.7.3 | Molecular dynamic procedure
A
new series of 2,4-Dioxochroman-pyridinium-
Molecular simulations of this study were performed using
the Desmond v5.3 using Maestro interface (from
phenylacetamide derivatives 7a–n was synthesized and
evaluated as ChE inhibitors against AChE and BuChE.
The two compounds 7j and 7k showed greater inhibitory
activities against BuChE compared to donepezil as the
standard drug. Moreover, docking and dynamic studies
[26]
Schrödinger 2018-4 suite).
We evaluated the stability
of the best drug candidates from the experimental
study—compounds 7b and 7j over AChE and BuChE,

MOLLAZADEH ET AL.
19
confirmed the ability of compounds 7j and 7k to be
located deep in the gorge cavity of BuChE and interacted
with important residues in the active site of this enzyme.
Furthermore, this studies permitted us to rationalize the
observed structure–activity relationship in the synthe-
sized compounds against BuChE. in vitro and in silico
toxicity of the compounds 7j and 7k demonstrated that
these compounds are nontoxic
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How to cite this article: Mollazadeh M,
Mohammadi-Khanaposhtani M, Azizian H, et al.
Design and synthesis of 2,4-dioxochroman-
pyridinium-phenylacetamide derivatives as new
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