Synthesis, characterization, monoamine oxidase inhibition, molecular docking and dynamic simulations of novel 2,1-benzothiazine-2,2-dioxide derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.04.012

In this research work, we report the synthesis and biological evaluation of two new series of 1-benzyl-4-(benzylidenehydrazono)-3,4-dihydro-1H-benzo[c] [1,2]thiazine 2,2-dioxides and 1-benzyl-4-((1-phenylethylidene)hydrazono)-3,4-dihydro-1H-benzo[c][1,2]t

Full text of DOI:10.1016/j.bioorg.2018.04.012

Accepted Manuscript
Synthesis, characterization, monoamine oxidase inhibition, molecular docking
and dynamic simulations of novel 2,1-benzothiazine-2,2-dioxide derivatives
Shakeel Ahmad, Sumera Zaib, Saquib Jalil, Muhammad Shafiq, Matloob
Ahmad, Sadia Sultan, Mazhar Iqbal, Sana Aslam, Jamshed Iqbal
PII:
S0045-2068(18)30099-3
DOI:
https://doi.org/10.1016/j.bioorg.2018.04.012
YBIOO 2335
Reference:
Bioorganic Chemistry
To appear in:
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Revised Date:
Accepted Date:
3 February 2018
13 April 2018
17 April 2018
Please cite this article as: S. Ahmad, S. Zaib, S. Jalil, M. Shafiq, M. Ahmad, S. Sultan, M. Iqbal, S. Aslam, J. Iqbal,
Synthesis, characterization, monoamine oxidase inhibition, molecular docking and dynamic simulations of novel
2,1-benzothiazine-2,2-dioxide derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.04.012
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1
Synthesis, characterization, monoamine oxidase inhibition, molecular
docking and dynamic simulations of novel 2,1-benzothiazine-2,2-dioxide
derivatives
Shakeel Ahmada‡, Sumera Zaibb‡, Saquib Jalilb, Muhammad Shafiqa, Matloob Ahmada,*,
Sadia Sultanc,d, Mazhar Iqbale, Sana Aslamf, Jamshed Iqbalb,*
aDepartment of Chemistry, Government College University, Faisalabad-38000, Pakistan.
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan.
cFaculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor Darul Ehsan, Malaysia.
dAtta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi
MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan,
Malaysia.
eDrug Discovery and Structural Biology Group, Health Biotechnology Division, National
Institute for Biotechnology and Genetic Engineering, Faisalabad 38000, Pakistan.
fDepartment of Chemistry, Government College Women University, Faisalabad-38000,
Pakistan.
‡These authors contributed equally.
Correspondence:
Prof. Dr. Jamshed Iqbal, Centre for Advanced Drug Research, COMSATS Institute of
Information Technology, Abbottabad, Postal Code-22060, Pakistan, Tel: +92-992-383591-
96, Fax: +92-992-383441, E-Mail: drjamshed@ciit.net.pk / jamshediqb@googlemail.com
Dr. Matloob Ahmad, Department of Chemistry, Government College University, Faisalabad-
38000, Pakistan, Email: matloob.ahmad@gcuf.edu.pk / matloob_123@yahoo.com
Abstract
In this research work, we report the synthesis and biological evaluation of two new series of
1-benzyl-4-(benzylidenehydrazono)-3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxides and
1-benzyl-4-((1-phenylethylidene)hydrazono)-3,4-dihydro-1H-benzo[c][1,2]thiazine
2,2-
dioxides. The synthetic plan involves the mesylation of methyl anthraniliate with subsequent
N-benzylation of the product. The methyl 2-(N-benzylmethylsulfonamido)benzoate was
subjected to cyclization reaction in the presence of sodium hydride to obtain 1-benzyl-1H-
benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxide which was treated with hydrazine hydrate to get

2
corresponding hydrazone precursor. Finally, the titled compounds were obtained by reaction
of hydrazone with various substituted aldehydes and ketones. The synthesized derivatives
were subjected to carry out their inhibition activities against monoamine oxidases along with
modelling investigations to evaluate their binding interactions and dynamic stability during
the docking studies. The inhibition profile of potent compounds was found as competitive for
both the isozymes. The compounds were more selective inhibitors of MAO-A as compared to
MAO-B. Moreover, drug likeness profile of the derivatives was evaluated to have an
additional insight into the physicochemical properties. The molecular dynamic simulations
predicted the behaviour of amino acids with the active site residues.
Keywords: 2,1-Benzothiazine 2,2-dioxides, Hydrazones, monoamine oxidases, molecular
dynamic simulations, docking studies
1. Introduction
Monoamine oxidase (MAO) is a FAD-dependent oxidoreductase enzyme found in the
mitochondrial outer membrane of neurons, glia and other mammalian tissue cells, such as
kidney, liver, glandular cells and intestinal epithelium [1,2]. Two isoenzymes of MAO have
been identified and distinguished (MAO-A and MAO-B), both have ~ 70% sequence identity,
having non-similar behaviour in their tissue distribution along with substrate and inhibitor
specificity [3,4]. MAO-A is a ubiquitous MAO isoform, that plays an important role in the
deactivation of neurotransmitter, dietary and xenobiotic amines, such as serotonin (5-HT),
norepinephrine, tyramine and dopamine and its selective inhibitor is clorgyline [5,6], while,
MAO-B predominantly exists in brain and helps in the metabolism of dopamine and β-
phenylethylamine, an endogenous amine which promotes dopamine release by inhibiting the
neuronal reuptake, and is inhibited selectively by selegiline [5,6].
Selective MAO-A inhibitors have shown to be effective against anxiety and depression [7],
whereas, selective inhibitors of MAO-B are useful in the treatment of several
neurodegenerative diseases, like Parkinson’s disease (PD) and Alzheimer’s disease (AD) [8].
The increased levels of monoamine oxidases results in elevated production of hydrogen
peroxide, a by-product which is responsible for enhancing the level of reactive oxygen
species (ROS) [1,4]. These ROS are mainly involved in the pathogenesis of
neurodegenerative diseases. Therefore, monoamine inhibitors are feasible and potential drug
target for neurodegenerative diseases as they are involved in the prevention of oxidative

3
damage in addition to their role as antidepressant (MAO-A) and neuroprotective (MAO-B)
agents [1,4].
The level of monoamine oxidase increases in the brain of human species during aging and the
oxidative stress which is a potential risk factor for neuronal damage and also causes death in
aging and age related neurodegenerative disorders like AD and PD [9-11]. Selective
inhibitors of monoamine oxidase are therefore considered as a significant therapeutic option
for these disorders so that the level of metabolic degradation of dopamine can be reduced.
Thus, selegiline (deprenyl), rasagiline (Figure 1), two irreversible MAO-B inhibitors, are
commonly used for symptomatic treatment of PD [12]. Another MAOI-B, safinamide (Figure
1), was recently approved in the European Union (EU) for the treatment of PD [13]. In fact,
the interest in selective inhibitors of monoamine oxidase has increased in the last several
years, due to their therapeutic potential for neurodegenerative disorders and other pathologies
in which enzyme is involved [14,15]. The research in the area evolved significant number of
several derivatives based on variety of oxygen, sulphur and nitrogen containing compounds
[16-27].
Figure 1. Structures of some clinically used MAO inhibitor drugs.
One of the main goals of synthetic organic chemistry is the discovery of novel and effective
therapeutic agents. Benzothiazines constitutes a very important class of heterocyclic
compounds, possessing diversified biological activities such as antiviral (reverse transcriptase

4
inhibitors) [28], antimalarial [29] and antibacterial activities [30]. The hydrazone derivatives,
N-benzylidene-N′-(1-methyl-2,2-dioxo-2,3-dihydro-1H-2λ6-benzo[c][1,2]thiazin-4-ylidene)-
hydrazines are reported as antibacterial agents [31]. The benzo[2,1]thiazine-4(3H)-one 2,2-
dioxide core is bioisosteric to the benzo[1,2]thiazine-4(3H)-one which served as a precursor
for oxicams i.e., piroxicam, droxicam and meloxicam etc [32]. 2,1-Benzothiazine derivative,
ethyl
2-(2-(4-hydroxy-1-methyl-2,2-dioxido-1H-benzo[c][1,2]thiazine-3-carboxamido)
thiazol-4-yl)acetate (1) exhibited better analgesic character (Figure 2) as compared to oxicam
drugs, piroxicam (2) and meloxicam (3) [33]. Various other derivatives of 2,1-benzothiazine-
2,2-dioxide displayed their ability as lipoxygenase inhibition [34], treatment for a variety of
heart diseases [34], chiral ligands [35], antipsychotic [36] anti-inflammatory [37] and
anticonvulsant [38]. Moreover, several benzothiazine derivatives have been reported as
anticancer [39], CD73 inhibitors [40], analgesic [41] and neuroprotective agents [42] and also
act as modulator of pro- and anti-inflammatory cytokines in rheumatoid arthritis [43].
Figure 2. Structures of benzothiazine based analgesic agents
In the present study, taking into account of our previous work [22-27], three new series of
novel 2,1-benzothiazine-2,2-dioxide derivatives have been designed, synthesized and
evaluated as monoamine oxidase inhibitors. To investigate the abilities of the synthesized
compounds to access the brain, their permeation across the blood-brain barrier has been
theoretically investigated and predicted in silico. Moreover, the docking studies were carried
out for binding interaction comparison and the dynamic simulation studies were performed to
monitor the behaviour of amino acid with potent compounds inside the active site.
2. Results and Discussion
2.1.
Chemistry

5
The synthetic route for the novel series of hydrazine derivatives of 2,1-benzothiazine-2,2-
dioxide is shown in the Scheme 1. First of all mesylation of methyl anthranilate was carried
out to get sulphonamide (6), which was further N-benzylated (8) and then cyclized to obtain
1-benzyl-1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxide (9). The benzothiazine precursor (9)
was further reacted with hydrazine monohydrate to synthesize corresponding hydrazone
precursor (10), which was then condensed with a series of aromatic aldehydes, acetophenones
and heterocyclic ketones to get the target compounds. The compounds were characterized by
spectroscopic techniques. The SO2CH2 moiety in thiazine ring was observed in the range of 5
to 5.2 (δ, ppm) for the benzylidene derivatives while for phenyl ethylidene derivatives, it
appeared in the shift value of 4-5 ppm. Moreover, PhCH2 protons were observed in the shift
value of 5 to 5.3 ppm. The characteristic singlet for CH=N was observed for benzylidene
derivatives in the range of 8.61 to 8.96 (δ, ppm) while C(Me)=N protons for phenyl
1
ethylidenes were observed in the range of 2.29 to 2.48 (δ, ppm) in H NMR spectra. The
molecular ion peaks in the mass spectrometry experiments were in well agreement to the
assigned structures. Moreover, the phenomenon of tautomerism (Figure 3) was observed in
1
the H NMR of benzylidene derivatives with the appearance of NH peaks in the range of
10.49 to 11.08 (δ, ppm) and corresponding CH peaks in the shift value of 5.19 to 5.22 (δ,
ppm).
Figure 3. Tautomeric structures of benzylidene derivatives

6
Scheme 1. Synthetic lay out of 1-benzyl-1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxide and
its hydrazone derivatives.
2.2.
Monoamine oxidase inhibition studies
All the synthesized compounds were evaluated for their inhibitory potential against rat MAO-
A and MAO-B enzymes. Clorgyline and deprenyl were used as standard inhibitors of MAO-
A and MAO-B, respectively. The IC50 values and selectivity index (SI for IC50 of MAO-A
over IC50 of MAO-B) for all the evaluated compounds and reference inhibitors were
summarized in Table 1. Selectivity for MAO-A increases as the selectivity index decreases,
while, selectivity towards MAO-B isoform increases as the SI increases. All compounds
exhibited excellent MAO inhibition in the lower micromolar range. By investigating the

7
MAO-A and MAO-B inhibition potencies of the synthesized benzothiazine-2,2-dioxide
derivatives, it was noticed that the unsubstituted benzothiazines (11a) showed significant
inhibition towards MAO-A and MAO-B. Few compounds were found as dual inhibitors of
MAO-A and MAO-B, and were able to inhibit both enzymes efficiently. Most of the
compounds were selective toward MAO-B, while few were selective towards MAO-A.
The most potent inhibitor of monoamine oxidase A was 11i, 1-benzyl-4-((2-
bromobenzylidene)hydrazono)-3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxide, having an
IC50 value of 0.12 ± 0.02 µM, while, the potent inhibitor of monoamine oxidase B was 12d,
4-((1-(2-aminophenyl)ethylidene)hydrazono)-1-benzyl-3,4-dihydro-1H-benzo[c][1,2]thiazine
2,2-dioxide, with an IC50 value of 0.13 ± 0.08 µM. However, the potent inhibitor against
MAO-A, 11i presented ~ 27 fold increased inhibition potential when compared with standard
inhibitor (clorgyline, 0.0045 ± 0.0003 µM) and 12d, MAO-B inhibitor, possess ~ 7 times less
inhibitory profile than standard inhibitor deprenyl (0.0196 ± 0.001 µM). Several other
derivatives exhibited significant inhibitory activities towards monoamine oxidase A, which
included 11b, 11m, 11g and 13b. However, some compounds possessed selective inhibition
for monoamine oxidase B, like 11d, 11k, 12a and 12b. Some of the synthetic compounds
were found dual inhibitors of both the enzymes, 11a, 11c, 11f, 11h and 11j, and were active
against monoamine oxidase A as well as monoamine oxidase B.
The Ki values for MAO-A and MAO-B were reported in nano molar concentration. The
value ranges from 34.2 – 14914.2 nM for MAO-A, whereas, the value 57.5 – 33955.5 nM
was found against MAO-B. However, clorgyline (standard inhibitor, MAO-A) presented Ki
value of 1.28 nM, and deprenyl (standard inhibitor, MAO-B) exhibited inhibition constant of
8.711 nM. The potent inhibitor (11i) of MAO-A showed 34.2 nM, whereas, MAO-B selective
inhibitor (12d) had 57.5 nM. The values were presented in Table 1.

8
Table 1. Structural parameters and in-vitro monoamine oxidase inhibition of synthesized compounds 11(a-m), 12(a-f) and 13(a-c)
Compound
codes
11a
R1
R2
R3
R4
R 5
MAO-A
MAO-B
Ki (nM)
IC50 ± SEMa (µM)
H
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
0.25 ± 0.06
1.87 ± 0.09
0.22 ± 0.04
2.58 ± 0.06
20.6 ± 0.02
0.46 ± 0.03
0.49 ± 0.07
1.33 ± 0.05
0.12 ± 0.02
0.19 ± 0.06
1.26 ± 0.04
52.2 ± 0.18
0.44 ± 0.12
29.9 ± 0.19
40.0 ± 0.14
44.3 ± 0.22
15.5 ± 0.08
1.07 ± 0.02
0.66 ± 0.01
14.1 ± 0.06
0.18 ± 0.03
0.31 ± 0.02
1.61 ± 0.09
0.50 ± 0.08
2.14 ± 0.05
1.02 ± 0.06
3.05 ± 0.01
0.15 ± 0.07
0.14 ± 0.02
68.5 ± 0.27
1.38 ± 0.05
0.64 ± 0.04
0.14 ± 0.05
41.9 ± 0.09
0.13 ± 0.08
76.4 ± 0.11
71.42
293.3
6266.6
80.0
534.3
62.85
11b
11c
H
Cl
OH
H
H
H
OCH3
H
H
737.1
137.7
715.5
222.2
951.1
453.3
1355.5
66.67
62.22
30444.4
613.3
284.4
62.22
18622.2
57.78
33955.5
11d
11e
NO2
H
H
5885.7
131.4
OH
H
H
H
11f
H
OH
NO2
H
H
140.0
11g
H
H
H
380.0
11h
11i
Br
H
H
H
34.28
H
Br
H
H
54.28
11j
OH
H
OCH3
CH3
H
H
360.0
11k
11l
H
H
14914.2
125.7
H
CH3
F
H
11m
12a
H
H
CH3
CH3
CH3
CH3
CH3
8542.8
11428.5
12657.1
4428.51
305.7
Cl
OH
NH2
H
H
Cl
H
12b
12c
H
H
H
12d
12e
H
Cl

9
H
H
H
-
H
-
CH3
-
3.72 ± 0.03
39.9 ± 0.19
59.9 ± 0.13
56.8 ± 0.19
1062.8
26622.2
25244.4
12f
CH3
13a
11400.0
CH3
-
-
-
0.54 ± 0.09
5.62 ± 0.01
49.1 ± 0.07
13b
21822.2
11244.4
154.3
H
-
-
-
-
-
-
25.3 ± 0.05
13c
1605.7
Clorgylineb
-
-
0.0045 ±
0.0003
61.35 ± 1.13
-
27266.6
8.711
1.28
Deprenylb
-
-
-
67.25 ± 1.02
0.0196 ±
0.001
-
19214.2
b
aAll the values are expressed as the mean ± SEM of triplicate determinations; Positive control; cSI = [IC50 MAO-A(μM)]/[IC50 MAO-B (μM)].

10
2.2.1. Structure activity relationship
The role of substitution patterns and their nature at the benzylidene ring was investigated in
order to get detail information about the identification and development of selective inhibitors
of monoamine oxidases. Among the three series, 11(a-m) series derivatives showed notable
contribution towards the inhibition of monoamine oxidases (A & B). When substituents were
added to the basic structure, 1-benzyl-4-((E)-(benzylidenehydrazono)-3,4-dihydro-1H-
benzo[c][1,2]thiazine 2,2-dioxide, the significant effect was noted towards monoamine
oxidase A. When series having phenylethylidenehydrazone ring instead of
benzylidenehydrazone ring, was taken into account, the substituent added selective inhibition
profile of compounds towards monoamine oxidase B. Among the third series (13a-c), only
potent compound was 13b, 1-benzyl-4-((E)-(1-(pyridin-2-yl)ethylidene)hydrazono)-3,4-
dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxide, which exhibited selective inhibition for MAO-
A. More specifically, when monoamine oxidase A inhibitors were examined, the structure
activity relationship showed that addition of 2-bromo at benzylidene ring (11i) resulted in the
most potent and selected inhibitor of MAO-A, presenting an IC50 value of 0.12 ± 0.02 µM.
When inhibitory activities of other compounds were compared with the potent inhibitor, it
was noted that 4-hydroxybenzylidene derivative, 11g, showed significant inhibitory values
(0.49 ± 0.07 µM) towards MAO-A. Moreover, 4-methylbenzylidene substituted compound
(11m) was also found as selective inhibitor of MAO-A with an inhibitory concentration of
0.44 ± 0.12 µM. The compounds 12(a-f) did not show significant inhibitions towards
monoamine oxidase A except 12e, which showed an IC50 value of 1.07 ± 0.02 µM, however,
among series 13(a-c), the compound 13b was found as selective inhibitor of MAO-A (0.54 ±
0.09 µM).
When inhibitory profile of monoamine oxidase B inhibitors was investigated, it was noticed
that 12d, 4-((1-(2-aminophenyl)ethylidene)hydrazono)-1-benzyl-3,4-dihydro-1H-benzo[c]

11
[1,2]thiazine 2,2-dioxide, was the most potent and selective inhibitor with an IC50 value of
0.13 ± 0.08 µM. However, other compounds of the series 12a and 12b were also notable and
selective inhibitors of MAO-B with the inhibitory concentrations of 0.64 ± 0.04 and 0.14 ±
0.05 µM, respectively. Among series 13(a-c), none of the compounds exhibited notable
inhibition against monoamine oxidase B. Furthermore, 11d (IC50 ± SEM = 0.31 ± 0.02 µM),
having 4-hydroxy-3-methoxybenzylidene as substituent was the active member among 11(a-
m) series. Moreover, 11k (IC50 ± SEM = 0.14 ± 0.02 µM) was also a significant candidate
when MAO-B inhibitory activity was taken into account.
When the inhibitory activity of derivatives having dual inhibition was taken into account, few
important analysis were made. The compound, 11i, having 2-bromobenzylidene was the
selective inhibitor of MAO-A, whereas, 11j, 4-bromobenzylidene, was found as nonselective
dual inhibitor for MAO-A and MAO-B. Similarly, when change in position of nitro group
was noticed, the compound (11e) having 2-nitrobenzylidene showed less inhibition for MAO-
A, and moderate inhibition for MAO-B, while 4-nitrobenzylidene (11h) was found as dual
inhibitor. The change in position and nature of substituent had profound effect on the
inhibition profile of compounds. Moreover, in case of compound 11g, the substituent,
hydroxyl group, at para-position was selective inhibitor of MAO-A with an inhibitory
concentration of 0.49 ± 0.07 µM, whereas, when the same group was shifted to meta-
position, 11f, was non-selective dual inhibitor of MAO-A and MAO-B with IC50 values of
0.46 ± 0.03 and 0.50 ± 0.08 µM, respectively. The presence of hydroxyl group can be helpful
in the formation of strong hydrogen bonding within the active site, and it can be a reason for
showing dual inhibition of 11f. However, compound 12c, having hydroxyl group at ortho-
position along with chloro group as meta-substituent was weak inhibitor of MAO-A as well
as MAO-B. It suggested that chloro group along with hydroxyl maybe the factor to cause
decrease in inhibition. Furthermore, it was noticed that methoxy group along with hydroxyl
as substituent contributed to enhance inhibitory activity towards MAO-B.

12
2.2.2. Relationship of novel compounds (benzothiazine-2,2-dioxide derivatives) with
already known MAOI (benzothiazine-3-carbohydrazide 1,1-dioxides)
When relationship of the tested compound was made with already known inhibitors of
monoamine oxidases, it was noted that previously reported derivatives of
benzylidenethiazine-3-carbohydrazide 1,1-dioxide showed notable inhibition towards
monoamine oxidases. However, recent studies were conducted to explore the inhibition effect
of benzylidenethiazine 2,2-dioxide derivatives towards monoamine oxidases. The structures
and their functional groups along with differences in the structures were presented below.
The most interesting feature was the enhanced and selective potential of novel compounds for
monoamine oxidase B. 4-((E)-(1-(2-aminophenyl)ethylidene)hydrazono)-1-benzyl-3,4-
dihydro-1H-benzo[c][1,2] thiazine 2,2-dioxide, exhibited strong inhibition as compared to
already reported inhibitor, methyl 2-benzyl-4-hydroxy-2H-benzo[e][1,2]thiazine-3-
carboxylate 1,1-dioxide for MAO-B.

13
2.2.3. Kinetics Studies
In order to investigate the mode of inhibition, enzyme kinetics studies were carried out for
most active inhibitors of MAO-A (11i) and MAO-B (12d). The results plotted in Figure 3
revealed that the potent compounds inhibit MAO-A and MAO-B in a competitive fashion.
The Lineweaver-Burk plots of compounds 11i and 12d were shown in Figure 3.

14
Figure 3. Lineweaver-Burk plots of MAO-A (left) and MAO-B (right) activity in the absence and
presence of various concentrations of potent inhibitors, 11i for MAO-A and 12d for MAO-B.
2.3.
Molecular docking study
2.3.1. Monoamine oxidase A
In order to justify the in vitro MAO inhibition results, docking study was carried out against
human MAO-A (PDB ID: 2Z5Y) and MAO-B (PDB ID: 2V5Z). There were three functional
domains in the active center of monoamine oxidases, the entrance cavity, the substrate cavity
and the third one was aromatic cage (formed by Tyr398 and Tyr435 and FAD) [44]. For
MAO-A, before docking potent compounds in the active site of the enzyme, cognate ligand
harmine (7-methoxy-1-methyl-9H-β-carboline) was docked. After docking the cognate ligand
successfully an RMSD value of 1.5 Å was obtained.
Amino acid residues found inside the active pocket are Tyr69, Asn181, Phe208, Val210,
Gln215, Cys323, Ile325, Ile335, Leu337, Phe352, Tyr407 and Tyr444 along with cofactor
flavin. For compound 11i 1-Benzyl-4-((2-bromobenzylidene)hydrazono)-3,4-dihydro-1H-
benzo[c][1,2]thiazine 2,2-dioxide, π‒π interactions were observed between 3,4-dihydro-1H-
benzo[c][1,2]thiazine 2,2-dioxide moiety and Tyr407 residue. The same moiety was oriented
towards flavin cofactor, however, no significant interactions were observed. The 2-
bromobenzylidene part was found towards amino acid residues Ile325 and Cys323.
Moreover, non-covalent molecular interactions were noticed with the active site residues
which are consistent to those observed previously [45]. The benzyl-4-((2-bromobenzylidene
moiety was found towards amino acid residues Ile325 and Cys323, whereas, 3,4-dihydro-1H-
benzo[c][1,2]thiazine 2,2-dioxide part was deeply oriented inside the active pocket and may
be the possible reason for the potential inhibition of MAO-A. The possible binding modes of

15
compound 11i and the crystallographic inhibitor harmine were selected after Hyde
assessment and visualization and shown in Figure 4.
Figure 4. Possible binding mode of 11i (brown color) and the crystallographic inhibitor harmine (blue
color) bound to the active site of MAO-A
2.3.2. Monoamine oxidase B
For MAO-B, before docking the potent compounds within the active pocket of the enzyme,
cognate ligand safinamide (S)-(+)-2-[4-(fluorobenzyloxy-benzylamino)propionamide] (SAG)
was docked. After docking the cognate ligand successfully an RMSD value of 2.0 Å was
obtained. MAO-B was a dimer, having globular domain monomers, which were anchored
within the membrane by a C-terminal helix. In the active site of MAO-B, the substrate cavity
was present towards flavin adenine dinucleotide and the entrance cavity located beneath the
protein and the loop having residues 99-112 and aromatic cage were present at the end of
entrance cavity [44,46]. Active site residues included Gln206, Phe103, Trp119, Ile198,
Leu164, Leu167, Tyr188, Phe168, Leu171, Tyr326, Cys172, Ile199, Ile316, Tyr435, Phe343
and Tyr398 and cofactor flavin adenine dinucleotide.

16
The aminophenyl ethylidene ring was oriented towards flavin cofactor in the substrate cavity,
while benzyl-3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxide moiety was located in the
entrance cavity space. Amino acid residues involved in electrostatic interactions were
Phe103, Phe168, Ile198, Gln206, Tyr435, Cys172, Ile199, Tyr326 and Tyr435. Additionally,
π-π interactions were observed between aminophenyl ethylidene ring and residue Tyr398
located in hydrophilic region, as hydrophilic region lies between Tyr398 and Tyr435 [47].
Figure 5 reported possible binding poses of 12d and crystallographic inhibitor safinamide
which were selected after visualization and having lowest binding energies. It was
represented in the Figure 5 that 1-benzyl-3,4-dihydro-1H-benzo[c][1,2]thiazine part of
compound well-occupied the entrance cavity in the same way co-crystallized ligand,
safinamide had. Similarly, benzyl piperazine moiety in the substrate cavity was fitted in the
same orientation as the adenine of cofactor flavin adenine dinucleotide.
Figure 5. Possible binding mode of 12d (black color) and the crystallographic inhibitor SAG (pink
color) within the binding site of MAO-B
Moreover, the dual inhibitors, 11c (1-benzyl-4-((4-chlorobenzylidene)hydrazono)-3,4-
dihydro-1H-benzo[c][1,2]thiazine
2,2-dioxide)
and
11j
(1-benzyl-4-((4-

17
bromobenzylidene)hydrazono)-3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxide) were
docked inside both the enzymes and their binding modes and interactions with the amino
acids of active site were presented in Figure 6 and 7. Compound 11c contain 4-chloro at
benzylidene)hydrazono ring, whereas, 4-bromo was found at benzylidene)hydrazono in case
of 11j.
Figure 6. Possible binding mode of dual inhibitor 11c in binding site of MAO-A (left) and in binding
site of MAO-B (right) along with the respective crystallographic inhibitors
Figure 7. Possible binding mode of dual inhibitor 11j in binding site of MAO-A (left) and in binding
site of MAO-B (right) along with the respective crystallographic inhibitors

18
2.4.
Dynamic simulation studies
Molecular dynamic simulation method was utilized to find out the transitions and
conformational stability of ligand and protein. Similarly, making and breaking of different
interactions between ligand and the most important amino acids within the active site were
monitored during specific time frame. Dynamic simulation represented the real image of
binding mode of interactions between ligand and protein. The root mean square derivation
(RMSD) of the protein backbone was calculated and data was plotted against time during the
molecular simulations of 5 ns for MAO-A and MAO-B were shown in Figure 8 and 9,
respectively.
2.4.1. Structural stability analysis using Root Mean Square Deviation (RMSD)
In order to understand the structural and dynamic behaviour of best docked pose of the potent
inhibitors and cognate ligands within the active pocket of receptors, MD simulations were
done at 5 ns. In order to distinguish the effect of inhibitor on specific protein, control system
was formed (free of ligand, apo) and simulated by same protocol as for ligand-protein
complex (holo). Stability of the system was achieved for both the receptors and it was shown
in Figure 8 and 9.
2.4.1.1. Monoamine oxidase A
During the course of evaluation, RMSD plot of MAO-A showed that in the start the system
was less stable, but with the passage of time the protein and ligand start stabilizing. It was
also examined that almost no change in the stability of protein-11i complex was found after
0.6 ns till the end of simulation i-e., 5 ns. While the rest of trajectories showed little drift
during the simulation time and can be noted in Figure 8. Conformational changes were
observed throughout the simulation process and flexibilities were noticed in apo and holo
structures. Holo structure of protein complexed with HRM and protein-11i were monitored.
A slight jumps were observed at 2.8 ns and 4.1 ns in the trajectory of protein + 11i, which
may be due to the rotatable bonds in compound 11i. Moreover, system was found stable
when protein-HRM complex was noticed, this decrease in mobility may be due to tight
binding of HRM and protein within the entrance cavity of the active site of protein.

19
Figure 8. Evaluation of RMSD (root-mean-squared-deviation) for protein (MAO-A) backbone during
the production stages of the MD simulation
2.4.1.2. Monoamine oxidase B
When RMSD plot of MAO-B was examined, the system was found in metastable state after
the time frame of 900 ps. This stability started from 0.2 nm and trajectories of whole
simulations, either apo (with protein only) or holo (having FAD, SAG or 12d, the docked
pose of potent inhibitor) were found to converge reasonably between 0.2-0.25 nm. No sudden
jumps in trajectory of protein, protein + SAG and protein + 12d were noticed, but due to the
rotation of FAD, conformational changes were found at 1.1 and 1.6 ns. The trajectories of
protein + SAG and protein + 12d were stable at 0.2 nm throughout the simulation time,
whereas, for protein + FAD, the shift was noticed between 0.25-0.30 nm, as shown in Figure
9. Protein-SAG and protein-12d complex are more stable as compared to protein alone,
which was found to be flexible.

20
Figure 9. Evaluation of RMSD (root-mean-squared-deviation) for protein (MAO-B) backbone during
the production stages of the MD simulation
2.4.2. Residue flexibility analysis using Root Mean Square Fluctuation (RMSF)
2.4.2.1. Monoamine oxidase A
Residues of both the receptors, MAO-A (PDB ID: 2Z5Y) [46] and MAO-B (PDB ID: 2V5Z)
[45] were analyzed in apo state of protein and holo form, i-e., in complex with FAD, cognate
ligand (HRM) and the most potent compounds. The plots are represented in Figures 10 and
11 for MAO-A and MAO-B, respectively. It was illustrated from the RMSF plot of MAO-A,
that residues were found stable for protein, protein with FAD alone and protein with HRM
along with FAD. Whereas, when residues of protein-11i complex were analyzed,
fluctuations at various residues were noticed. The most notable among them were with
residues Ala348, Ile349, Met4350, Gly351 and at second position with residues Tyr491,
Glu492, Arg493, Asn494 and Leu495. Similarly, residues Leu97, Val98, Gln99 and Tyr100
exhibited less flexibility.

21
Figure 10. Root-mean-square fluctuation (RMSF) of values per residue of MAO-A during the MD
simulations
2.4.2.2. Monoamine oxidase B
When root-mean-square fluctuation (RMSF) of MAO-B were examined, it was observed that
residues were found stable in all the 4 simulations, i-e., with protein only, protein with FAD
alone, protein with SAG and protein with 12d in addition to FAD. It was analysed that almost
no noticeable fluctuation was seen except with terminal residues Gly497, Leu498, Thr499,
Thr500 and Ile501 as shown in Figure 11.

22
Figure 11. Root-mean-square fluctuation (RMSF) values per residue of MAO-B during the MD
simulations
2.5.
HYDE assessment of novel 2,1-benzothiazine-2,2-dioxide derivatives
The HYDE affinity assessment was done for the first 30 top ranking docked conformations
within the active sites of monoamine oxidase A and monoamine oxidase B and it helped in
the selection of correct binding mode. The binding free energy ΔG, FlexX docking score and
the most favourable poses for all the synthetic derivatives 11 (a-m), 12 (a-f) and 13(a‒c)
were given in Table 2 (for MAO-A) & 3 (for MAO-B). Most of the compounds bind to the
receptor with a very high binding affinity and give favourable contributions. Safinamide
(SAG) is the co-crystallized ligand for MAO-B and by Hyde scoring showed binding free
energy of -26 kJ mol‒1, whereas all the derivatives showed more than the binding affinity of
reference ligand. Similarly, in case of MAO-A, most of the compounds bind to the receptor
with a very high binding affinity as compared to the reference ligand harmine (HRM). The
potent and selective inhibitors (11i and 12d) presented the lowest energy scores and the
binding free energies ΔG also predicted their high binding affinity inside the active pocket of
respective receptors.
Table 2. Docking and Hyde scores and their corresponding ranks by Hyde affinity
assessment (MAO-A)
Codes FlexX score of the top ranking
pose
Binding free energy
Poser rank
ΔG (kJ mol‒1)

23
‒12.66
‒11.98
‒14.92
‒15.95
‒16.06
‒11.52
‒17.39
‒13.23
‒19.88
‒18.58
‒10.81
‒12.14
‒11.93
‒16.43
‒10.16
‒13.90
‒18.10
‒18.51
‒14.38
‒10.64
‒16.10
‒10.07
‒16.29
1
3
5
6
27
2
1
3
4
1
2
7
6
3
12
7
8
5
9
‒21
‒18
‒29
‒18
‒22
‒25
‒26
‒23
‒37
‒20
‒17
‒11
‒34
‒26
‒23
‒21
‒18
‒16
‒15
‒24
‒21
‒19
‒25
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
12a
12b
12c
12d
12e
12f
13a
13b
13c
HRM
10
4
1
3
Table 3. Docking and Hyde scores and their corresponding ranks by Hyde affinity
assessment (MAO-B)
Codes FlexX score of the top ranking
pose
Binding free energy
Poser rank
ΔG (kJ mol‒1)
‒14.95
‒11.05
‒15.14
‒16.82
‒17.93
‒18.45
‒14.56
‒19.01
‒10.19
‒18.57
‒19.63
‒16.41
‒15.32
‒23.85
‒21.98
‒19.76
5
4
1
3
6
1
2
9
3
2
4
8
1
7
5
3
‒21
‒19
‒18
‒26
‒19
‒23
‒20
‒28
‒17
‒26
‒20
‒14
‒21
‒25
‒29
‒22
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
12a
12b
12c

24
‒25.92
‒16.-3
‒15.63
‒17.43
‒18.19
‒15.33
‒26.13
4
6
23
11
17
3
‒32
‒28
‒19
‒14
‒17
‒15
‒36
12d
12e
12f
13a
13b
13c
2
SAG
2.6. In silico physicochemical properties
Drug-likeness and in silico physicochemical properties for all the tested compounds were
predicted using MedChem Designer3.0 [48]. Compounds having properties by which they
distinguish from chemicals are identified as critical properties. These parameters work out to
Lipinski’s rule of five, following which, molecular weight should be less than 500 Da,
calculated octanol/water partition coefficient less than five (lipophilicity), number of H-bond
donors less than five and number of H-bond acceptors less than 10 [49]. Studies showed that
CNS drugs possess low level of lipophilicities and less molecular weight. In addition, drugs
are also characterized by low H-bond capacity and less number of rotatable bonds [50].
Herein, we determined the octanol-water distribution and partition coefficients (S + log D),
octanol-water partition coefficient (S + log P), no. of H-bond donors (HBD), H-bond acceptor
(MNO) and topological polar surface area (TPSA), an important descriptor in drug
absorption. A drug having less than 60-140 Å2 polar surface area, is expected to be
sufficiently bio-available [51]. The partition coefficient is generally representative of
distribution of drug in the body. Octanol-water partition is helpful in the estimation of
hydrophobicity and hydrophilicity of any drug. Most of the synthesized compounds exhibited
a narrow range of lipophilicity with some deviations. Majority of the compounds showed less
than 100 Å2, while only few have values within the range of 100-130 Å2. Therefore, in the
form of drugs, all the derivatives can easily be penetrated. Typically, compounds having
molecular weight < 500, M log P value < 5 are thought to be orally bioavailable with
favorable ADME profile [52-53]. All the compounds possessed molecular weight less than
500. All the compounds have equal to or less than 10 rotatable bonds. The calculated ADME
properties of 11(a-m), 12(a-f) and 13(a-c) derivatives are given in Table 4.
Table 4. Calculated ADME properties of 11(a-m), 12(a-f) and 13(a-c) derivatives.

25
Codes M log P S + log P S + log D Mol. Wt. MNO
TPSA
HBDH
3.486
3.969
3.969
2.948
3.572
2.982
2.982
3.572
4.075
4.075
2.948
3.701
3.701
4.075
4.387
3.919
3.708
4.180
3.701
2.565
2.714
2.99
3.829
4.338
4.398
3.569
3.836
3.647
3.517
4.006
4.422
4.445
3.777
4.179
4.317
4.371
5.131
4.669
3.575
4.603
4.046
3.122
3.067
3.137
3.829
4.338
4.398
3.554
3.836
3.642
3.507
4.006
4.422
4.445
3.716
4.179
4.317
4.371
5.131
4.669
3.575
4.603
4.046
3.122
3.067
3.137
389.47
423.924
423.924
435.504
434.476
405.478
405.478
434.476
468.38
5
5
5
7
8
6
6
8
5
5
7
5
5
5
5
6
6
5
5
6
6
5
62.10
62.10
62.10
91.56
107.92
82.33
82.33
107.92
62.10
62.10
91.56
62.10
62.10
62.10
62.10
82.33
88.12
62.10
62.10
75.24
74.99
62.10
0
0
0
1
0
1
1
0
0
0
1
0
0
0
0
1
2
0
0
0
0
0
11a
11b
11c
11d
11e
11f
11g
11h
11i
468.38
11j
11k
11l
435.504
403.506
403.506
421.496
472.396
453.95
418.52
437.95
403.506
393.467
404.493
367.472
11m
12a
12b
12c
12d
12e
12f
13a
13b
13c
2.7. Cytotoxic Potential by MTT Assay
In vitro evaluation of anticancer activity of all the compounds 11(a-m), 12(a-f) and 13(a-c)
was carried out using the 3–(4,5–dimethylthiazol– 2–yl)–2,5–diphenyltetrazolium bromide
(MTT) assay. The cell viability was determined by measurement of purple formazan that was
formed after metabolization of yellow colored tetrazolium bromide (MTT). This conversion
of yellow tetrazolium bromide to purple formazan is brought about by oxidoreductase
enzymes that are typically present in the cytosol of living cells, thereby providing an estimate
of number of viable (healthy (control) and cancerous) cells present after treatment with test
compounds. The screening of all the derivatives was carried out using cervical cancer cells
(HeLa). The viability of cells was carried out after 24 h treatment with 100 µM concentration
of the synthetic derivatives. The cytotoxic potential was measured at the final concentration
of 100 μM of tested compounds. The obtained results are expressed as percentage of viability
stated in untreated cells and are presented in Table 5. Carboplatin was used as a standard
drug.

26
The most potent inhibitor was 11l, with an inhibition potential of 83.8% inhibition against
cervical cancer cell lines, which was followed by 13b having 83.2%. All the synthetic
derivatives presented more than 50% inhibition against cervical cancer (HeLa) which
indicated that the compounds had significant inhibitory effects against cancer cell lines.
Moreover, it was worth noting that all the derivatives exhibited maximum inhibition towards
HeLa cells with the percentage inhibition of 60-85%.
Table 5. Cytotoxic potential of synthetic derivatives 11(a-m), 12(a-f) and 13(a-c) and
standard carboplatin against cervical cancer cells (HeLa)
Compound codes
HeLa
%age inhibition ± SEM
72.1 ± 1.99
76.3 ± 1.34
65.2 ± 3.05
69.7 ± 2.49
75.4 ± 1.44
82.6 ± 2.28
79.5 ± 1.73
64.8 ± 2.12
82.6 ± 3.18
71.1 ± 2.25
69.7 ± 3.17
83.8 ± 4.36
70.9 ± 2.02
78.5 ± 1.74
69.4 ± 1.92
82.2 ± 2.34
76.4 ± 2.18
69.9 ± 3.01
75.8 ± 1.88
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
12a
12b
12c
12d
12e
12f

27
13a
13b
70.1 ± 2.05
83.2 ± 2.57
79.3 ± 1.12
84.9 ± 1.97
13c
Carboplatin
3. Conclusion
In summary, the present study reported an efficient synthetic plan that involved the
mesylation of methyl anthraniliate with subsequent N-benzylation of the product. The
structural diversity of the synthesized derivatives was ensured by the addition of a diverse
range of substituents including electron-donating and electron-withdrawing groups on the
aromatic ring. The synthesized conjugates 11 (a-m), 12 (a-f) and 13 (a-c) were assayed for
their monoamine oxidase inhibition potential, resulting in some highly active and selective
inhibitors towards monoamine oxidase A. However, few compounds were found potent
inhibitors of monoamine oxidase B. The detailed binding mode analysis with docking
simulation at molecular level provided an insight into the biological properties of the
analyzed compounds. Both kinetic analysis of MAO inhibition and molecular modelling
study suggested that potent compound bound simultaneously to the catalytic active site of
MAO-A and MOA-B. Observation of the docked poses revealed many interactions with
already reported residues that effect the inhibition of MAO enzymes. These interactions were
further investigated and validated through molecular simulation studies of most potent
compounds. These results suggested that the inhibitors could be stabilized in the active site
through the formation of multiple interactions with catalytic residues and the establishment of
hydrophobic interactions in an interactive fashion. Finally, based on the activity findings and
docking analysis, these compounds could potentially be developed as a novel family of
structurally diverse, potent, and highly selective monoamine oxidase inhibitors. The studies
revealed an interesting multi-targeted active molecules, which offer an attractive starting
point for further lead optimization in the drug-discovery process against Parkinson’s disease.
4. Material and methods
Please see supporting information file for material and method section.

28
Acknowledgements
The author, Matloob Ahmad acknowledges the financial support from 'Higher Education
Commission of Pakistan' through project No. 20-3715/NRPU/R&D/HEC/14/162 and
Government College University, Faisalabad, Pakistan. Sadia Sultan would like to
acknowledge Universiti Teknologi MARA for the financial support under the reference
number UiTM 600- IRMI/FRGS 5/3 (0119/2016) and LESTARI grant 600-RMI/DANA5/3
LESTARI (92/2015). Jamshed Iqbal is thankful to the Organization for the Prohibition of
Chemical Weapons (OPCW), The Hague, The Netherlands and Higher Education
Commission of Pakistan for the financial support through Project No. 20-
3733/NRPU/R&D/14/520.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
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