Imino isatin derivatives; synthesis, in silico molecular dynamic study over monoamine oxidase B, ADME prediction, and in vitro cytotoxicity evaluation

Article abstract of DOI:10.1002/jccs.202000170

The monoamine oxidase B (MAO-B) depicts an attractive drug target for the development of neuro protective agents toward the treatment of neurodegenerative diseases. The current study involved synthesis, in silico and cytotoxic evaluation of N1-alkylated-5-substituted 3-imino isatin derivatives with the proposed MAO-B inhibitory activities. The In silico molecular modeling investigation was performed through the induced fit docking, molecular mechanics-generalized born surface area, and molecular dynamic method in order to uncover the binding mode interaction and their proposed impact on the active site environment and flexibility. The synthesized compounds were characterized by spectroscopic methods. Compound 3-Imino-1-pentyl indolin-2-one (3h) with the highest free binding energy adopts an extended conformation spanning from the flavin ring location to the entrance of the substrate cavity. In this way, Ile199 adopts the “open” conformation so the two separate cavities of MAO-B active site fused and formed a single-space, which abled the compound extending to the MAO-B entrance cavity space. From consideration of the data presented in this paper, we reveal that longer N₁-alkylated-3-imino isatin derivative could be proposed as inhibitor that would occupy both cavities of the MAO-B active site. Furthermore, the mentioned derivatives provided acceptable drug profiling based on in silico ADME calculation and MTT cytotoxicity test evaluation.

Full text of DOI:10.1002/jccs.202000170

Received: 12 June 2020
Revised: 16 January 2021
Accepted: 19 January 2021
DOI: 10.1002/jccs.202000170
A R T I C L E
Imino isatin derivatives; synthesis, in silico molecular
dynamic study over monoamine oxidase B, ADME
prediction, and in vitro cytotoxicity evaluation
Samaneh Ahmadi1 | Homa Azizian2 | Javad Azizian1
1Department of Chemistry, Science and
Abstract
Research Branch, Islamic Azad
University, Tehran, Iran
The monoamine oxidase B (MAO-B) depicts an attractive drug target for the
development of neuro protective agents toward the treatment of neurodegener-
ative diseases. The current study involved synthesis, in silico and cytotoxic
evaluation of N1-alkylated-5-substituted 3-imino isatin derivatives with the
proposed MAO-B inhibitory activities. The In silico molecular modeling inves-
tigation was performed through the induced fit docking, molecular mechanics-
generalized born surface area, and molecular dynamic method in order to
uncover the binding mode interaction and their proposed impact on the active
site environment and flexibility. The synthesized compounds were character-
ized by spectroscopic methods. Compound 3-Imino-1-pentyl indolin-2-one
(3h) with the highest free binding energy adopts an extended conformation
spanning from the flavin ring location to the entrance of the substrate cavity.
In this way, Ile199 adopts the “open” conformation so the two separate cavities
of MAO-B active site fused and formed a single-space, which abled the com-
pound extending to the MAO-B entrance cavity space. From consideration of
the data presented in this paper, we reveal that longer N1-alkylated-3-imino
isatin derivative could be proposed as inhibitor that would occupy both cavities
of the MAO-B active site. Furthermore, the mentioned derivatives provided
acceptable drug profiling based on in silico ADME calculation and MTT cyto-
toxicity test evaluation.
2Department of Medicinal Chemistry,
School of Pharmacy, Iran University of
Medical Sciences, Tehran, Iran
Correspondence
Javad Azizian, Department of Chemistry,
Science and Research Branch, Islamic
Azad University, Tehran, Iran.
Email: azizian@srbiau.ac.ir
K E Y W O R D S
3-Imino isatin, ADME, induced fit docking, MAO-B inhibitor, MM-GBSA, molecular dynamic
1 | INTRODUCTION
target in age-dependent neurodegenerative diseases and
its depression used for the development of neuro protec-
Monoamine oxidase B (MAO-B) is a flavine adenine
dinucleotide (FAD) dependent enzyme responsible for
the oxidative deamination of endogenous neurotransmit-
ters such as dopamine, serotonin, adrenaline, and nor-
adrenaline along with the inactivation of exogenous aryl
alkyl amines. Due to its central role in neurotransmitters
metabolism, this enzyme represents an attractive drug
tive agents.[1–4]
There are many MAO-B inhibitors been developed
and used as anti-Parkinson and anti-Alzheimer agent,
including selegiline and rasagiline, which are known as
irreversible MAO-B inhibitors.[5,6] Propargylamine moi-
ety is a common group among the irreversible inhibitors,
which covalently binds to the FAD cofactor and may
J Chin Chem Soc. 2021;1–14.
http://www.jccs.wiley-vch.de
© 2021 The Chemical Society Located in Taipei & Wiley-VCH GmbH
1

2
AHMADI ET AL.
cause a serious side effect like tyramine-induced hyper-
tensive crisis.[7,8] To overcome this problem, new genera-
tion of inhibitors, which perform through reversible
MAO-B inhibition was developed. Today the only FDA
approved reversible MAO-B inhibitor is safinamide used
in Alzheimer disease.[9] Moreover, isatin as an endoge-
nous small molecule (Figure 1) is described to be a
reversible inhibitor of MAO enzyme,[10] which lies in the
substrate cavity close to the FAD co-factor by forming
hydrogen bonding with the conserved water mole-
cules.[11] In this regard, some isatin-based compounds are
shown to possess MAO-B inhibition activity.[12,13] In
addition, the N CH linkage, is well known as a
building block in organic synthesis evaluated for different
kinds of biological activity such as antimicrobial, antifun-
gal, anti-inflammatory, anti-cancer, antitumor, and her-
bicidal.[14,15] Schiff bases of isatin were reported to
energy in order to investigate the active site structures
and environment flexibility based on the best-proposed
compounds interaction over the MAO-B enzyme.
2 | RESULTS AND DISCUSSION
2.1 | Reliability of the IFD protocol
Cross docking is known as an accurate method for the
root mean square deviation (RMSD) validity of docking
protocol.[15] The applied docking procedure reliability
was validated by cross docking of several known crystal-
lographic MAO-B inhibitors over 2v5z. First, all the avail-
able crystallographic structures were aligned over 2v5z
structure. Then the crystallographic ligands were docked
into 2v5z and the docked conformations corresponding
to the lowest IFD Score were selected as the most possi-
ble binding modes. The RMSD was calculated for each
ligand to measure the docking prediction accuracy
(Table 1). The pose was considered optimal if its RMSD
found to be less than 2 Å.[38,39] The RMSD of the re-
docked conformations of safinamide over 2v5z was
obtained to be 0.40 Å, which is considered successfully
docked.
The MAO B active site made of two cavities, the sub-
strate cavity ahead of the flavin as cofactor and the
entrance cavity lied underneath the protein surface,
which blocked by a loop consists of residues 99–112.[40]
Docking of safinamide over the MAO-B active site
showed that it bonded in front of flavin cofactor and
occupied both the substrate and entrance cavity space
(Figure 2). The propanamide group of the syfinamide lied
in the aromatic cage (showed in yellow stick) formed by
Tyr398 and Tyr435 and interacted with Gln206 and
Tyr435 through H-bond and water mediated H-bond,
respectively. In addition, the middle benzyl group stabi-
lized with Phe326 at the substrate cavity through π-π stac-
king interaction (showed in orange stick). Finally, the
end part of safinamide, the fluorophenyl moiety,
extended toward the entrance cavity of the MAO-B
enzyme, which surrounded by Ile199, Ile316, and Phe103
through Van der Waals interactions (showed in purple).
As a result, the validity of docking parameters was rea-
sonable in order to predict the pose of other investigated
structures.
possess
anti-HIV,[16,17]
anticonvulsant,[18]
antibacterial,[19–22] antiprotozoal,[23,24] antifungal,[25–27]
antiviral,[28–30] and antituberculosis[31–33] activity.
Based on these facts, we decided to synthesize
3-imino indolin-2-one derivatives for their biological
evaluation. Previously, the synthesis of 3-imino indolin-
2-one derivatives was carried out using a different kind of
solvents under refluxing conditions.[34–37] In the current
study, the use of solvent has been avoided and the reac-
tions were carried out in the presence of an acid catalyst
process (Scheme 1). While the evolution of biological
activities of the synthesized compounds is currently
ongoing, the cytotoxicity of compounds was assessed in
order to reveal the safety profile of these compounds.
Moreover, the extensive in silico molecular modeling
investigation was performed through the series of compu-
tational methods. At the beginning, the induced fit dock-
ing (IFD) protocol used to uncover the binding mode
interaction of the synthesized compounds over the 5 Å
distance around the MAO-B active site. Thereafter,
molecular mechanics-generalized born surface area
(MM-GBSA) calculation was performed based on the best
posed structure obtained from the IFD complexes to cal-
culate the relative free energy, which reflect the ligand-
binding affinity. Finally, molecular dynamic method was
achieved over the ligands with the highest ligand-binding
2.2 | Investigating IFD interaction of the
synthesized compounds
The performed docking procedure was then applied to
evaluate the interaction between newly synthetized
FIGURE 1 Representation of isatin structure

AHMADI ET AL.
3
SCHEME 1 Preparation of 3-imino indolin-2-one
TABLE 1 The ligands root mean
No.
PDB id
2v5z
Co-crystalized ligand
Safinamide
Rasagiline
Resolution (Å)
RMSD (Å)
0.12
square deviation (RMSD) values of the
1
1.6
1.7
1.7
2.2
2.3
docked and crystalized forms over
2
3
4
5
2c67
0.40
monoamine oxidase B
1oja
Isatin
0.54
2byb
2vz2
Selegiline
0.65
Mofegilide
0.43
FIGURE 2 Close-up
representation of binding
interactions of the superposed
docked (cyan) and co-crystalized
safinamide (green) over
monoamine oxidase
B. Structural waters and their H-
bond interaction render in stick.
Aromatic cage, substrate cavity,
and entrance cavity residues
colored in yellow, orange, and
pink, respectively

4
AHMADI ET AL.
compounds (3a–3i) over the MAO-B active site in com-
parison to the reference MAO-B inhibitors. The top scor-
ing pose of all compounds was selected based on IFD
Score and was analyzed for pose investigation and free
binding energy calculation using Prime MM-GBSA
method.
Table 2 shows all of the synthesized isatin analogs
have better glide energy than the mentioned reference
inhibitor except that of safinamide. In addition, it has
been shown that except compounds 3a all the synthesize
compounds have higher free binding energy than isatin
(known as a reversible noncovalent MAO-B inhibitor).[11]
By comparing the values mentioned under the MM-
GBSA column, compounds 3b, 3c, 3g, 3h, and 3i have
higher free binding energy than selegiline. This result
proposed that isatin analogs have good binding affinity
for the active site of MAO-B enzyme.
indicates that the aromatic ring of the compounds adopts
the same orientation and position as isatin except the
N1-alkyl groups, which are extended through the
entrance cavity space (colored in pink), and caused
increasing the free binding energy of the mentioned com-
pounds (Figure 3b). Comparing the free binding energies
of the mentioned compounds reveal that increasing the
length of the N1-alkyl chain from ethyl to butyl (3g and
3h) increase the MM-GBSA value as a result of expan-
ding the contact area over the entrance cavity (Table 2,
highlighted in yellow).
Among the synthesized compounds, the free binding
energy of compound 3h was noticed to be the highest
one with the value of −45.69 kcal/mol, which was higher
than standard inhibitors; isatin, selegiline, and
mofegiline (−28.29, −38.67, and −44.20 kcal/mol,
respectively).
Figure 3a depicts that the polar imino-2-oxoindole
ring of isatin (colored in green) and compounds 3a, 3b,
3c, 3d, and 3e lied in the substrate cavity (colored in
orange) and pointed toward flavin ring at the aromatic
cage (colored in yellow) in a perpendicular manner,
which observed for the most MAO-B-inhibitor com-
plexes[11] and the rest of the structures involved in van
der Waals contacts with hydrophobic residues of the sub-
strate cavity space.
2.3 | Molecular dynamic simulation
In order to understand the criteria for rational designing
of the MAO-B inhibitors, it is necessary to uncover the
structural perturbations incurred by the compound with
the highest free binding energy (compound 3h) over
MAO-B active site (pdb id = 2v5z) and compare the effect
of this compound on the active site environment in com-
parison to selegiline, which is known as the old FDA
approved drug used as anti-Parkinson and anti-
Alzheimer agent.
Furthermore, docking pose of the compounds 3g and
3h with N1-alkyl substitution over MAO-B active site
RMSD of the protein's Cα from its initial to final con-
formation was applied over 20 ns of the MD simulation
in order to study the stability of the protein-ligand com-
plex. The RMSD simulation showed that the MAO-B
complexed with selegiline maintained an overall stability
after 2n of the MD simulation time with higher fluctua-
tion stabilizing at an average of 2.75 Å (Figure 4, blue
line), while the bounded-state of compound 3h displayed
longer equilibration time with obviously maximum fluc-
tuation value around 2 Å (Figure 4, red line). The RMSD
value of each MAO-B ligand complex indicates that the
employed simulation time has been enough to obtain an
equilibrium structure over the simulation time
(Figure 4). Thus, the structures at the MD equilibrium
state used to investigate the structural specificity of the
ligand-protein complexes.
Furthermore, root-mean-square fluctuation (RMSF)
of MAO-B was examined in the bounded-state enzyme
with selegiline and compound 3h (Figure 5). It is
observed that both compounds provided the same struc-
tural flexibility pattern through the MAO-B enzyme
structure in the presence of FAD cofactor except for the
terminal residues. According to the result, the mentioned
TABLE 2 Glide energy and free binding energy (kcal/mol) of
the synthesized compounds over monoamine oxidase B (pdb
id = 2v5z)
Molecular mechanics-
Glide
energy
generalized born surface area
(kcal/mol)
No
3a
−30.378
−30.855
−35.205
−35.100
−34.147
−33.007
−33.450
−42.055
−36.164
−30.300
−30.787
−30.284
−30.423
−20.67
−39.25
−39.30
−37.08
−33.44
−30.56
−40.68
−45.69
−38.84
−28.29
−38.67
−56.47
−44.20
−166.7
3b
3c
3d
3e
3f
3g
3h
3i
Isatin
Selegilin
Rasagilin
Mofegiline
Safinamide −55.362

AHMADI ET AL.
5
FIGURE 3 Close-up representation of synthesized compound over monoamine oxidase B active site, (a) induced fit docking (IFD) pose
of compounds isatin (colored in green), 3a, 3b, 3c, 3d, and 3e (colored in cyan), which do not have N1-alkyl substitution, (b) IFD pose of
compounds 3g, and 3h with N1-alkyl substitution (colored in light green). Aromatic cage, substrate cavity, and entrance cavity residues
colored in yellow, orange, and purple, respectively
FIGURE 4 Root mean square
deviation of the monoamine oxidase B
Cα in complexed with selegiline
(in blue) and compound 3h (in red) for
over 20 ns of the MD simulation time
lower RMSF value occurs upon ligand binding to the
enzyme as a result of non-bonding interaction between
the ligand and the enzyme active site.
aromatic cage, which contribute to the inhibitor
interactions.
Figure 6a shows that selegiline bonded to human
The molecular interactions of selegiline and com-
pound 3h over the binding site of MAO-B involve a num-
ber of hydrophilic and van der Waals contacts with the
active side surrounded residues, which are schematically
shown in Figure 6. There are four ordered water mole-
cules inside the cavity located at the substrate cavity and
MAO-B active site occupying substrate cavity in which
the amino alkyne group formed two water-mediated H-
bond through its amino group with Tyr435 located at
aromatic cage and Gln206 over the second part of MD
simulation with the occupancy time of 44 and 39%,
respectively. In addition, the aromatic ring stabilized

6
AHMADI ET AL.
selegiline tertiary amine, except that the mentioned inter-
action persisted for the higher MD simulation time (84%).
This structural finding consistent with the idea that the
aromatic cage plays an important role as a recognition
site for the amine group of the inhibitors.[41]
Furthermore, compound 3h interacted through
hydrophobic interaction with Lue171 and Cys172 located
at the top of the substrate cavity for about the first 12 ns
of MD simulation time, while the mentioned interactions
were disappeared and substituted by hydrophilic water-
mediated H-bonding with Arg197 and His200, which lied
at the separating loop for the rest of the MD simulation
time (with high occupancy time).
FIGURE 5 Root-mean-square fluctuation of the monoamine
oxidase B Cα in complex with selegiline (colored in blue) and
compounds 3h (colored in red) for over 20 ns of the MD
simulation time
Furthermore, we study the active site cavity shape
alongside the interaction mode of the N1-alkylated group
of compound 3h during the MD simulation time. For this
purpose, the distances between Ile199 and Tyr326 located
near the junction of the entrance and substrate cavities in
two opposite side are recorded and analyzed.
with hydrophobic residues include Tyr326, Phe168, and
Ile 171 located at the line of substrate cavity for about the
whole MD simulation time.
On the other way, Figure 6b shows imino group of
compound 3h stabilized through the MAO-B active site
by forming H-bond interaction with Tyr435 the same as
In previous study, it was found that Ile199, located
near the junction of the entrance and substrate cavities,
FIGURE 6 2D (up row) and timeline (below row) representation of ligand-residue interactions that occur during the simulation time,
which include monoamine oxidase B bound-state of selegiline (a) and compound 3h (b), respectively. Aromatic cage and substrate cavity
residues highlighted in yellow and orange, respectively

AHMADI ET AL.
7
engaged in two different conformers relative to the
MAO-B active site cavity; the first one is a “closed confor-
mation” in which its side chain pointed toward the active
site cavity space and the next one is an “open conforma-
tion” merging the two cavities.[11]
Figure 7a displays the distance between Tyr326 (Cα)
and Ile199 (CCD1) over the MAO-B bound-state during
the 20 ns of simulation time. In the case of compound 3h
bound-state, the separation of the mentioned residues at
the beginning of the simulation time was about 9.5 Å and
significantly increased up to 17 Å during the 10 ns
followed by gradually decreased to about 15 Å in the next
4 ns and finally stabilized until the rest of the simulation
time (the red line). On the other hand, in the case of
selegiline bound-state, after a sharp increasing the speci-
fied distance decreased step by step to about below the
11 Å after 14 ns and equilibrated up to the end of simula-
tion time (the blue line).
In order to investigate Ile199 conformers along the
simulation time, three snapshots of the enzyme bound-
states to compound 3h and selegiline provided at the
beginning, middle, and the end of simulation time (the
black dash-lines in Figure 7b,c). Snapshots related to
the enzyme complexed with compound 3h depict that
Ile199 side chain was along the wall of the active site,
which corresponds to the open conformer, while in the
case of selegiline bound-state, the mentioned residue
side chain rotated toward the active site cavity, which
FIGURE 7 MD simulated distance between Tyr326 (Cα)–Ile199 (CCD1) in selegiline (blue) and compound 3h (red) in monoamine
oxidase B bound-state (a). Representative snapshots from MD simulations of compound 3h (b) and selegiline (c) focusing on Ile199
conformations (open/close), distance between Tyr326 (Cα) and Ile199 (CCD1) (showed in black dash-line), and the distance between the tail
of compound and Pro104 (Cα) (showed in red dash-line). Aromatic cage, substrate cavity, and entrance cavity residues colored in yellow,
orange, and purple, respectively

8
AHMADI ET AL.
correlates to the close rotamer. The conformer of Ile199
affects the structural shape of the active site in the way
that the open conformer merged both of the substrate
and entrance cavity and formed a single space active
site, whereas the close conformer divided the active site
area by separating these two-cavity space.
compound molecular weights, calculated lipophilicity
(log P), number of hydrogen bond acceptors, number of
hydrogen bond donors, number of rotatable bonds, and
the number of violations of Lipinski's rule of five were
used to assess the “drug-likeness” of the synthesized com-
pounds. According to Table 3, it was observed that all
Lipinski rules were in acceptable value and the synthe-
sized compounds showed no Lipinski violations, which
indicates a high probability of finding drug-like potential
within these series.
Furthermore, The blood brain barrier (BBB) absorp-
tion in turn relays on the solubility, oral absorption, and
BBB permeability of the compound. The computed
parameters were used to assess oral absorption, including
the predicted aqueous solubility (log Swat), the predicted
% human oral absorption (%HOA), and the predicted
brain/blood partition coefficient (Table 4). According to
Table 4, it was observed that all of the predicted descrip-
tors are in acceptable value, which indicates the synthe-
sized compounds have adequate BBB bioavailability so
they could emerge good candidate for drug discovery.
Furthermore, analyzing the distance between Pro104
(Cα) located at the active site closing loop and the tail
part of the bound-state compounds depicts the extent of
ligand penetration into the active site from the entrance
space to the substrate cavity pocket. In the case of com-
pound 3h, the mentioned distance decreased from 9.3 Å
at the begin to 4.9 Å at the end of simulation time, while
in the case of selegiline it oscillated within 8.5 Å because
of steric clash of the bottom part of selegiline phenyl ring
with Ile199 side chain (Figure 7b,c, the pink dash-lines).
In summary, according to the compound 3h
bounding-state, Ile199 is in open rotamer conformation
so the two-cavity fused forming a single space, which
abled the N1-pentyl extending to the MAO-B entrance
cavity space. This finding proposed that N1-alkylated-
3-iminoisatin could be considered a new reversible inhib-
itor that would occupy both cavities of the MAO-B
active site.
2.5 | Cytotoxicity assay
Toxicity is one of the most important reason for failure of
new drug entry into the pharmaceutical market. There-
fore, cytotoxicity test is very important filtering stage for
accepting a drug candidate. Figure 8 shows MTT cyto-
toxic activity of compounds 3a–3i against both NIH/3T3
and MCF-7 cells. Our result indicates the IC50 values of
the synthesized compounds were >1,000 μM against both
NIH/3T3 and MCF-7 cells during 24 and 48 hr, which
means the synthesized compounds showed neither
2.4 | In silico ADME properties of
synthesized compounds
The important pharmacokinetic properties of the synthe-
sized compounds, which represent drug- likeness, metab-
olism, BBB transportation, and bioavailability, were
calculated with QikProp module of Schrodinger. Based
on the “Lipinski rule of five” distributions of the
TABLE 3 The Lipinski-rule of five
No.
3a
3b
3c
3d
3e
3f
Molecular weight
146.148
HBDa
HBAb
3.5
3.5
3.5
4.5
3.5
4
Log P o/wc
1.298
Rotord
ROFe
properties of investigated compounds
2
2
2
2
2
1
1
1
1
1
1
1
2
1
1
2
5
2
0
0
0
0
0
0
0
0
0
180.593
1.665
225.044
1.472
191.146
−0.469
1.108
160.175
160.175
0.878
3g
3h
3i
174.202
4
1.032
216.282
4
2.187
174.202
3.5
1.392
Abbreviations: HBA, hydrogen bond acceptors; HBD, hydrogen bond donors; ROF, rule of five.
aNumber of average hydrogen bond donor (recommended value 0.0–6.0).
bNumber of average hydrogen bond acceptor (recommended value 2.0–20.0).
cPredicted octanol/water partition co-efficient (acceptable range from −2 to 6.5).
dNumber of rotatable bond (recommended value 2.0–20.0).
eNumber of violations of Lipinski's rule of five (maximum 4).

AHMADI ET AL.
9
TABLE 4 The calculated ADME properties of synthesized
cytotoxicity toward normal tissue cells nor cancerous
one. Having low toxicity is very important character for
any drug candidate.
compounds
a
No.
3a
3b
3c
3d
3e
3f
Log Swat
−1.391
−1.925
−1.951
−1.507
−1.581
−1.897
−1.861
−2.939
−2.152
PlogBBb
−0.622
−0.479
−1.134
−0.676
−0.344
−0.379
−0.463
−0.396
−0.417
% HOAc
81.400
83.549
82.416
54.491
80.291
85.323
87.139
93.933
89.490
Metabd
RO3e
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
3 | EXPERIMENTAL
3.1 | Chemistry
All reagents were purchased from Sigma Aldrich Com-
pany (Tehran, Iran). All chemicals were reagent grade
and were without further purification. Melting points
were detected with an Electrothermal 9300 apparatus
3g
3h
3i
1
(Ontario, Canada). H-NMR and 13C-NMR spectra were
obtained by a Brucker AVANCE 500 Ultra shield Spec-
trometer (Brucker, Rheinstetten, Germany). Tetra-
methylsilane used as an internal standard. The IR spectra
were obtained using Jasco FT-IR-410 spectrophotometer
(KBr disks). Mass spectra were obtained using a Finnigan
Abbreviation: HOA, human oral absorption.
aPredicted aqueous solubility in mol/dm3 (−6.5 to 0.5) (QPlogS >−5.7).
bPredicted brain/blood partition coefficient (acceptable range: −3.0 to 1.2).
cPercentage human oral absorption (<25% is poor and >80% is high).
dNumber of likely metabolic reactions (primary metabolites <7).
eNumber of violations of Jorgensen's rule of three. Compounds with fewer
(preferably no) violations of these rules are more likely to be orally available.
FIGURE 8 Viability test of the synthesized compounds on NIH3T3 and MCF-7 cells after 24 and 48 hr by MTT assay. Percentage of live
cells was calculated relative to control group. Values are presented as mean SD of five independent experiments

10
AHMADI ET AL.
1
Mat TSQ-70 spectrometer at 70 eV (Finnigan Mat, Bre-
men, Germany).
(CO, CN), 1,428.7 (C C). HNMR (δ, DMSO-d6): 11.13
(1H, s, NH), 6.69 (1H, d, J = 7.8 Hz, Ar), 7.17 (1H, t,
J = 7.4 Hz, Ar), 6.46 (1H, d, J = 8.1 Hz, Ar), 7.52 (1H, t,
J = 8.1 Hz, Ar), 7.04 (1H, s, NH). 13CNMR (δ, DMSO_d6):
197.1, 169.1, 150.7, 133.6, 133.0, 114.3, 113.0, 112.9. MS
(ESI, 70 eV) m/z (%): 146.05 (100), 147.05 (8.7). Elemental
Analysis for C8H6N2O: C, 65; H, 4.1; N, 20.5; O, 10.9.
3.2 | Preparation of 3-imino indolin-
2-one derivatives
The syntheses of N1-alkylated-5-substituted isatin have
been done according to the previously published arti-
cle.[42]
A
mixture of N1-alkyl-5-substituted isatins
3.2.2 | 5-Chloro-3-imino indolin-
2-one (3b)
(0.1 mmol) and malononitrile (0.1 mmol) stirred at room
temperature for 15 min. Then after, AlCl3 (0.01 mol)
added to the reaction mixture, and heated up to 100ꢀC.
In continue, ammonium bromide (0.1 mol) added,
stirred, and heated at 100ꢀC for about 2 hr (for synthesiz-
ing compound 3L, ethylamine was used). The process
followed by cooling the mixture, adding NaOH 10%, and
diluting with DCM. The organic layer washed with water,
dried on Na2SO4, and evaporated. Finally, the compounds
crystallized from ethanol and the purity of the com-
pounds was monitored by thin layer chromatography.
The melting points and yields reported in Table 5. The
structures of the products were confirmed by elemental
and spectral analysis as follows and on supplementary
material.
Yield 92%; IR (KBr) cm−1: 3,428.47, 3,428.47 (NH), 641.94
(CO), 1,517.89 (CN), 1,457.92 (C C), 657.80 (C Cl).
1HNMR (δ, DMSO-d6): 11.13 (1H, s, NH), 8.23 (1H, s,
NH), 7.95 (1H, dd, Ar), 7.62 (1H, d, Ar), 7.55 (1H, s, Ar).
13CNMR (δ, DMSO-d6): 169.4, 144.4, 132.0, 124.6, 124.0,
115.0, 113.9. MS (ESI, 70 eV) m/z (%): 180.01 (100),
182.01 (32), 181.01 (8.7), 183.01 (2.8). Elemental Analysis
for C8H5ClN2O: C, 53.33; H, 2.77; Cl, 19.72;N,
16.66; O, 8.88.
3.2.3 | 5-Bromo-3-imino indolin-
2-one (3c)
Yield 93%. IR (KBr) cm−1: 3,444.7, 3,444.7 (NH), 1,619.90
1
3.2.1 | 3-imino indolin-2-one (3a)
(CO, CN), 1,428.15 (C C), 882.32 (C Br). HNMR (δ,
DMSO-d6): 11.22 (1H, s, NH), 8.36 (1H, s, NH), 7.72 (1H,
dd, Ar), 7.76 (1H, d, Ar), 7.74 (1H, s, Ar). 13CNMR (δ,
DMSO-d6): 116.8, 122.2, 125.8, 129.6, 133.0, 145.1, 166.8,
Brown solid. Yield 90%, MP: 142–143ꢀC. IR (KBr) cm−1
3,473.09 (NH), 3,348.77 (NH), 3,028.49 (Ar), 1,625.35
:
TABLE 5 Physico-chemical properties of the synthesized compounds (3a–3i)
Entry
3a
Molecular weight
146.05
R1
R2
H
R3
H
m.p. (ꢀC)
142
Yield (%)
Molecular formula
C8H6N2O
H
90
92
93
95
90
75
73
65
80
3b
3c
180.01
H
Cl
H
225
C8H5ClN2O
C8H5BrN2O
C8H6N3O3
223.96
H
Br
NO2
CH3
H
H
253
3d
3e
191.03
H
H
281
160.06
H
H
210
C9H8N2O
3f
160.06
CH3
C2H5
C4H11
H
H
186
C9H8N2O
3g
174.08
H
H
208
C10H10N2O
C13H16N2O
C10H10N2O
3h
3i
216.03
H
H
277
174.08
H
C2H5
229

AHMADI ET AL.
11
171.2. MS (ESI, 70 eV) m/z (%): 223.96 (100), 225.96 (97),
224.96 (8.7), 226.96 (8.4). Elemental Analysis for
C8H5BrN2O: C, 42.86; H, 2.23; Br, 35.27; N,
13.39; O, 7.14.
3.2.8 | 3-Imino-1-pentyl indolin-2-
one (3h)
Yield 65%. IR (KBr) cm−1: 3,430.20 (CH), 3,443.5 (NH),
1
1,642.61 (CO, CN), 1,387.78 (C C). 1,428.77. HNMR (δ,
DMSO-d6). 7.04 (1H, s, NH), 6.69 (1H, d, J = 8.0 Hz, Ar),
7.17 (1H, t, Ar), 6.46 (1H, t, Ar), 7.5198 (1H, d, J = 8.0 Hz,
Ar), 2.89–0.9 (m, 8H, CH2), 0.29 (3H, CH3). 13CNMR (δ,
DMSO-d6): 11.5, 18.4, 25.7, 28.1, 41.3, 41.3, 111.5, 118.4,
130.9, 131.8, 135.4, 138.4, 159.2, 163.0. Elemental Analy-
sis for C13H16N2O: C, 72.21; H, 7.40; N, 13.88; O, 7.4.
3.2.4 | 3-Imino-5-nitro indolin-2-one (3d)
Yield 95%. IR (KBr) cm−1: 3,435.09, 3,554.09 (NH),
1,642.73 (CO), 1,445.90 (C C), 1,313.20, 1,569.84 (NO2).
1HNMR (δ, DMSO-d6): 11.79 (1H, s, NH), 8.45 (1H, s,
NH), 8.37 (1H, dd, Ar), 8.17 (1H, d, Ar), 8.21 (1H, s, Ar).
13CNMR (δ, DMSO-d6): 174.5, 149.8, 148.9, 141.1, 138.1,
135.7, 118.4, 111.5. MS (ESI, 70 eV) m/z (%): 191.03 (100),
192.04 (8.7). Elemental Analysis for C8H6N3O3: C,
50.25; H, 3.14; N, 23.55; O, 25.12.
3.2.9 | 3-(ethyl imino) indolin-2-one (3i)
Brown solid. Yield 80%, m.p. 142–143ꢀC; IR (KBr) cm−1
:
3,349.51 (NH), 3,472.90 (NH), 1,638.51 (CO, CN),
1,382.23 (C C). 1HNMR (δ, DMSO-d6): 7.01 (1H, s, NH),
6.92 (1H, t, Ar), 6.33 (1H, t, Ar), 7.74 (1H, d, Ar), 7.01
(1H, d, NH), 2.49 (2H, q, CH2), 1.0305 (3H, t, CH3).
13CNMR (δ, DMSO-d6):11.5, 59.2, 111.5, 118.4, 130.9,
131.8, 135.5, 138.4, 159.2, 163.0. MS (ESI, 70 eV) m/z (%):
188.09 (100), 189.10 (11.9). Elemental Analysis for
C11H12N2O: C, 68.93; H, 5.71; N, 17.23; O, 9.19.
3.2.5 | 3-Imino-5-methyl indolin-2-
one (3e)
Yield 90%. IR (KBr) cm−1: 3,473.09, 3,340.77 (NH), 3,020.49
(Ar), 2,970.12 (CH), 1,620.35 (CO,CN), 1,428.7 (C C):
1HNMR (δ, DMSO-d6): 11.23 (1H, s, NH), 7.41 (1H, s,
NH), 7.33 (1H, dd, Ar), 7.14 (1H, d, Ar), 6.82 (1H, s, Ar),
2.23 (3H, s, CH3). 13CNMR (δ, DMSO-d6): 187.9, 157.0,
131.8, 130.9, 125.7, 118.4, 111.5, 14.3. MS (ESI, 70 eV) m/z
(%): 160.06 (100), 161.07 (9.7). Elemental Analysis for
C9H8N2O: C, 67.47; H, 4.99; N, 18.74; O, 9.99.
3.3 | Target and ligand preparation
In order to find out the interactions mode of designed
molecules over MAO-B enzyme, Maestro Molecular
Modeling platform (version 11.5) by Schrödinger, LLC
was used.[43] Initially, the crystal structures of the five
human MAO-B enzyme were retrieved from the Pro-
tein Data Bank (PDB: 1oja, 2v5z, 2byb, 2vz2, and 2c67
(http://www.rcsb.org).[11,40,44–46] 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, which related to the FDA approved
drugs. As the prosthetic group and the co-factors are
not directly involved in MAO-B inhibition, they totally
removed before docking investigation. Except several
ordered water molecules in the MAO-B active site,
which bridge the receptor important residues by the
way of H-bonds, and conserve among other
specious,[47] the rest of the water molecules removed
from the enzymes crystallographic structures. The 2D
structures of all the synthesized compounds were
drawn in Marvin 15.10.12.0 program (http://www.
chemaxon.com)[48] and converted into pdb file. The
Protein Preparation Wizard[49] and the LigPrep[50]
module were used to prepare protein and ligands
structure properly.
3.2.6 | 3-Imino-1-methyl indolin-2-
one (3f)
Yield 75%. IR (KBr) cm−1: 2,921.89 (CH), 3,442.50 (NH),
1
1,637.72 (CO, CN), 1,455.19 (C C). HNMR (δ, DMSO-
d6): 8.23 (1H, s, NH), 7.95 (1H, dd, Ar), 7.62 (1H, dd, Ar),
7.60 (1H, d, Ar), 7.55 (1H, d, Ar), 3.55 (3H, s, CH3).
13CNMR (δ, DMSO-d6): 166.8, 159.8, 151.2, 138.8, 124.9,
123.0, 118.3, 113.8, 55.8. Elemental Analysis for
C9H8N2O: C, 67.47; H, 4.99; N, 18.74; O, 9.99.
3.2.7 | 1-Ethyl-3-imino indolin-2-one (3g)
Yield 73%. IR (KBr) cm-1: 2,970.63, 2,872.96 (CH),
3,471.92 (NH), 1,613.40 (CO, CN), 1,444.41 (C C).
1HNMR (δ, DMSO-d6): 8.00 (1H, s, NH), 7.68 (1H, d,
J = 8.0 Hz, Ar), 7.64 (1H, t, Ar), 7.14 (1H, d, J = 8.0 Hz,
Ar), 7.10 (1H, t, H), 2.89 (2H, q, CH2), 0.89 (3H, t, CH3).
13CNMR (δ, DMSO-d6): 25.7, 41.3, 111.5, 118.4, 130.9,
131.8, 135.4, 138.4, 159.2, 163.0. Elemental Analysis for
C10H10N2O: C, 68.93; H, 5.71; N, 17.23; O, 9.19.

12
AHMADI ET AL.
3.4 | IFD protocol
water molecules and placed in the center of an ortho-
rhombic box of appropriate size in the Periodic Boundary
Condition. Sufficient counter-ions and a 0.15 M solution
of NaCl were also utilized to neutralize the system and to
simulate the real cellular ionic concentrations, respec-
tively. The MD protocol involved minimization, pre-pro-
duction, 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 with a small force constant on the
enzyme in order to restrict any drastic changes. MD sim-
ulations were performed via NPT (constant number of
atoms, constant pressure, that is, 1.01325 bar and con-
stant temperature, that is, 300 K) 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 electrostatic forces were calcu-
lated based on Particle-mesh-based Ewald approach with
the cut-off radius for columbic forces set to 9.0 Å. Finally,
this system was subjected to the production of MD simu-
lations 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, the energy-minimized
structure calculated from the equilibrated trajectory sys-
tem was evaluated for investigation of each ligand-
protein complex interaction.
The IFD (flexible docking) was carried out using Glide
software (Schrödinger LLC 2018) to predict accurate con-
comitant structural movements, including side-chain (χ
angles) or backbone (Cα) conformational changes or both
during ligand binding at the active site of the MAO-B
enzyme.[51] The energy minimized human MAO-B/
safinamide complex was subjected for IFD studies as it has
the highest resolution among the other pdb structures. At
the active site of MAO-B the safinamide binding 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 Å, respectively, considered. Residues within
5 Å of the safinamide at the active site were refined
followed by side-chain optimization. Structures whose
Prime energy is more than 30 kcal/mol are eliminated
based on extra precious Glide docking. The energetically
favorable IFD complexes were obtained for all compounds
and the best poses based on IFD Score were selected for
MM-GBSA and analysis.
3.5 | Prime MM-GBSA
The ligand binding energies (ΔGBind) were calculated for each
synthesized and MAO-B inhibitors using molecular mechan-
ics/generalized born surface area (MM-GBSA) modules
(Schrödinger LLC 2018)[52] based on the following equation;
Â
Ã
ΔGBind = EComplex – EReceptor + ELigand
3.7 | In silico ADME properties of
where ΔGBind is the calculated relative free energy, which
includes both ligand and receptor strain energy. EComplex
is the MM-GBSA energy of the minimized complex, and
ELigand is the MM-GBSA energy of the ligand after removing
it from the complex and allowing it to relax. EReceptor is the
MM-GBSA energy of relaxed protein after separating it from
the ligand. The MM-GBSA calculation was performed based
on the best pose structure obtained from IFD complexes.
synthesized compounds
The important pharmacokinetic properties of the synthe-
sized compounds, which represent drug- likeness, metab-
olism, cell permeation, and bioavailability, were
calculated with QikProp module of Schrodinger.[54]
3.8 | Cell viability assay
3.6 | Molecular dynamic (MD)
simulation
As toxicity is one of the filtering stage for drug candidate,
in preclinical phases, cytotoxicity assays performed on
new drug candidate to select more safe and appropriate
drugs.[55] Based on ISO recommendation (10993-5, 2009),
NIH/3T3 is a good choice for evaluation of cytotoxic
potential of candidate drugs.[56] In addition to NIH/3T3
(murine embryonic fibroblast) as a normal cell line, we
also examined the effects of the synthesized substances
on breast cancer cells (MCF-7). After culturing in RPMI
1640 medium, cells were seeded in 96-well plates
Molecular simulations of this study were performed using
the Desmond v5.3 using Maestro interface (from
Schrödinger 2018-4 suite) [53]. We evaluated the stability
of the best MM-GBSA compound and compared their
perturbation with the standard inhibitor.
In order to build the system for MD simulation, the
protein-ligand complexes were solvated with SPC explicit

AHMADI ET AL.
13
(1 × 104 cells per well). After 24 hr, fresh medium con-
taining different concentrations of synthesized com-
pounds were added at concentrations between 0.1 and
50 μM. At the end of exposure time (24 and 48 hr), super-
natant removed and 20 μl of MTT solution (5 mg/ml)
was added to each well and incubated. After 4 hr,
formazan crystals were dissolved in 100 μl DMSO and
optical density was measured with plate reader at
570 and 690 nm. The cell viability was presented as a per-
centage of the control group.
ORCID
Javad Azizian https://orcid.org/0000-0002-1392-0274
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How to cite this article: Ahmadi S, Azizian H,
Azizian J. Imino isatin derivatives; synthesis, in
silico molecular dynamic study over monoamine
oxidase B, ADME prediction, and in vitro
cytotoxicity evaluation. J Chin Chem Soc. 2021;
1–14. https://doi.org/10.1002/jccs.202000170