Synthesis and α-glucosidase inhibition activity of 2-[3-(Benzoyl/4-bromobenzoyl)-4-hydroxy-1,1-dioxido-2h-benzo[e][1,2]thiazin-2-yl]-n-arylacetamides: An in silico and biochemical approach

Article abstract of DOI:10.3390/molecules26103043

Diabetes mellitus (DM) is a chronic disorder and has affected a large number of people worldwide. Insufficient insulin production causes an increase in blood glucose level that results in DM. To lower the blood glucose level, various drugs are employed th

Full text of DOI:10.3390/molecules26103043

molecules
Article
Synthesis and α-Glucosidase Inhibition Activity of
2-[3-(Benzoyl/4-bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-
benzo[e][1,2]thiazin-2-yl]-N-arylacetamides: An In Silico and
Biochemical Approach
Furqan Ahmad Saddique 1, Sana Aslam 2, Matloob Ahmad 1,
*
, Usman Ali Ashfaq 3,*, Muhammad Muddassar 4,
Sadia Sultan 5,6, Saman Taj 3, Muzammil Hussain 7, Dae Sung Lee 7 and Magdi E. A. Zaki 8,
*
1
Department of Chemistry, Government College University, Faisalabad 38000, Pakistan;
furqanas123@gmail.com
2
3
4
5
6
7
8
Department of Chemistry, Government College Women University, Faisalabad 38000, Pakistan;
Dr.Sana@gcwuf.edu.pk
Department of Bioinformatics and Biotechnology, Government College University,
Faisalabad 38000, Pakistan; saman20220@yahoo.com
Department of Biosciences, COMSATS University Islamabad, Park Road, Islamabad 45500, Pakistan;
mmuddassar@comsats.edu.pk
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300,
Selangor Darul Ehsan, Malaysia; drsadia@uitm.edu.my
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA,
Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor Darul Ehsan, Malaysia
Department of Environmental Engineering, Kyungpook National University, Daegu 41566, Korea;
muzammil@knu.ac.kr (M.H.); daesung@knu.ac.kr (D.S.L.)
Department of Chemistry, Faculty of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU),
Riyadh 11623, Saudi Arabia
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Saddique, F.A.; Aslam, S.;
Ahmad, M.; Ashfaq, U.A.; Muddassar,
M.; Sultan, S.; Taj, S.; Hussain, M.;
Sung Lee, D.; Zaki, M.E.A. Synthesis
and α-Glucosidase Inhibition
*
Correspondence: Matloob.Ahmad@gcuf.edu.pk (M.A.); ashfaqua@gcuf.edu.pk (U.A.A.);
mezaki@imamu.edu.sa (M.E.A.Z.)
Activity of 2-[3-(Benzoyl/4-
Abstract: Diabetes mellitus (DM) is a chronic disorder and has affected a large number of people
worldwide. Insufficient insulin production causes an increase in blood glucose level that results
in DM. To lower the blood glucose level, various drugs are employed that block the activity of
bromobenzoyl)-4-hydroxy-1,1-
dioxido-2H-benzo[e][1,2]thiazin-2-yl]-
N-arylacetamides: An In Silico and
Biochemical Approach. Molecules
2021, 26, 3043. https://doi.org/
10.3390/molecules26103043
the α-glucosidase enzyme, which is considered responsible for the breakdown of polysaccharides
into monosaccharides leading to an increase in the intestinal blood glucose level. We have synthe-
sized novel 2-(3-(benzoyl/4-bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-
N-arylacetamides and have screened them for their in silico and in vitro α-glucosidase inhibition
Received: 3 April 2021
Accepted: 10 May 2021
Published: 20 May 2021
activity. The derivatives 11c, 12a, 12d, 12e, and 12g emerged as potent inhibitors of the α-glucosidase
enzyme. These compounds exhibited good docking scores and excellent binding interactions with
the selected residues (Asp203, Asp542, Asp327, His600, Arg526) during in silico screening. Similarly,
these compounds also showed good in vitro α-glucosidase inhibitions with IC50 values of 30.65,
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
18.25, 20.76, 35.14, and 24.24
µM, respectively, which were better than the standard drug, acarbose
(IC50 = 58.8
of study.
µM). Furthermore, a good agreement was observed between in silico and in vitro modes
Keywords: 1,2-Benzothiazines; synthesis; molecular docking; α-glucosidase inhibition; anti-diabetic
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Diabetes mellitus (DM) is an endocrine system illness and causes metabolic ailment
that leads to hyperglycemia. DM is characterized by increased blood sugar levels either
due to the body’s reduced production of insulin or ineffective utilization of available
insulin by the body. Chronic hyperglycemia results in neuron oxidative damage while
Molecules 2021, 26, 3043. https://doi.org/10.3390/molecules26103043
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3043
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prolonged hyperglycemia causes the production of lower caloric content of sugar in dif-
ferent organs [1,2]. Although genetic predisposition can be a factor in the development
of DM, other major causing factors may include sedentariness, smoking, and intake of
high-saturated fatty meals [
by 58%, for example, overweight people can avoid it by losing weight, a fact explained
experimentally by the Finnish prevention trial [ ] and the US diabetes prevention pro-
gram [ ]. DM also enhances the chances of cardiovascular diseases [ ]. People suffering
with hypertension are already leading a risky life due to high blood pressure [ ] but they
often develop DM and this combination of risks increases cardiovascular transience. The
rate of DM is growing rapidly worldwide especially in developing countries [ ]. The Inter-
3,4]. Change in lifestyle can decrease the risk of type 2 diabetes
5
6
7
8
9
national Diabetes Federation (IDF) has observed that 371 million humans are infected with
T2DM (type 2 diabetes mellitus) while an increase rate of 2% annually has been estimated.
α
-Amylase and
α
-glucosidase are the two enzymes that have a key role in the breakdown
-Glucosidase enzyme, released from mucosal
of polysaccharides to monosaccharides [10].
α
cells, usually converts polysaccharides into monosaccharides causing a gradual increase
in the absorption of glucose and hence increases intestinal blood glucose level [11]. These
enzymes have role in breakdown and absorption of polysaccharides, so by inhibiting the
activity of these enzymes, glucose levels in diabetic patients can be controlled even after
consumption of carbohydrate-rich diets [12].
Different
absorption in the blood, such as acarbose, voglibose, miglitol, etc., but this medication has
many side effects [13 17], such as diarrhea, gastrointestinal distress (flatulence, pain), and
liver disorders (hepatic injury, hepatitis, and renal tumors) [18 22]. Hence, there is a need
α-glucosidase and α-amylase inhibitors are being used for delaying glucose
–
–
in the present era to develop more potent antidiabetic agents with minimum side effects.
Benzothiazines are well-known heterocycles due to their wide spectrum of pharma-
cological activities. Among the various types of benzothiazines, 1,2-benzothiazine 1,1-
dioxides and their derivatives have broadly been studied. Prominently, 1,2-benzothiazine
1,1-dioxide carboxamides have a major footprint in clinical therapy due to their anti-
inflammatory activity. As revealed by the literature, sudoxicam
oxicam having an extended plasma half-life [23], piroxicam (Figure 1), another potent oxi-
cam, is used to cure rheumatoid arthritis, osteoarthritis, and other inflammations [24]. Sim-
ilarly, various 1,2-benzothiazine 1,1-dioxides have been reported as antileukemic [25 27],
11 -HSD1 inhibitors [28], endotheline receptor antagonists [29], antimalarial agents [30],
MAO inhibitors [31], antiallergic agents [32], antithrombotics [33], antiparasitic [34], an-
timicrobials [35 36], antioxidants [37], inhibitors of calpain I [38], and aldose reductase
enzymes [39,40].
1 (Figure 1) is an important
2
–
β
,
Figure 1. Structures of biologically important 1,2-benzothiazine 1,1-dioxides.
Our research group is involved in the search for novel and more effective antidia-
betic agents [41–45], and in this context we have synthesized novel 1,2-benzothiazine-
N-arylacetamides and screened them for their in silico and in vitro α-glucosidase in-
hibitory potential.

Molecules 2021, 26, 3043
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2. Results and Discussion
2.1. Synthetic Chemistry
The precursors, 3-(benzoyl/4-bromobenzoyl)-1,2-benzothazines 9a
,b, were synthe-
sized via reported methods involving Gabriel–Colman rearrangement [46
3-(benzoyl/4-bromobenzoyl)-1,2-benzothazines 9a were treated with a library of 2-bromo-
N-arylacetamides 10a (synthesized as described previously [48]) in the presence of a
,47]. Later on,
,
b
–
m
potassium carbonate base in a DMF solvent. Reactions were completed in 2–2.5 h (as
indicated by TLC) and then dilution with distilled water followed by acidification with 20%
HCl afforded the targeted 1,2-benzothiazine-N-arylacetamides 11a–m and 12a–m good to
excellent yields (73–92%) (Scheme 1). Increase in reaction time was not helpful regarding
the yields of products. Similarly, use of low boiling solvents like acetonitrile also resulted
in good yields but in a longer reaction time (4 h).
Scheme 1. Synthesis of 1,2-benzothiazine-N-arylacetamides 11a–m and 12a–m.
2.2. Spectroscopic Characterization
All the synthesized derivatives were well characterized with H-NMR, 13C-NMR,
1
and MS spectroscopic techniques. In the 1H-NMR spectra, the methylene (CH2) protons
appeared as a singlet at 3.95–4.20 ppm and the observation was consistent with literature
values of similar compounds [46
appeared as singlets at 1.96–2.17 and 3.64–3.86 ppm, respectively. The NH and enolic–OH
protons were observed as singlets at 9.16–10.54 and 14.92–15.18 ppm, respectively [37 49 50].
,49]. The methyl protons (CH3) and methoxy protons
,
,
Peaks for enolic–OH protons were not observed in some spectra due to the exchangeable
nature of OH protons [49]. All the peak values from 6.50–8.50 ppm were assigned to aro-
matic protons. In MS spectra, the observed molecular ion peaks were found in accordance
with the calculated values.
2.3. Biological Activity
Diabetes mellitus is a severe chronic metabolic syndrome that is illustrated by hy-
perglycemia and escorted by different severe vascular complications. The main therapy
of diabetes is to control the postprandial blood-glucose level, however, limited uptake of

Molecules 2021, 26, 3043
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carbohydrates after meals is also considered as a therapy.
α
-Amylase and
α
-glucosidase
enzymes are responsible for the breakdown of polysaccharides to monosaccharides. Con-
sequently, inhibition of these enzymes leads to treatment postprandial hyperglycemia
by interrupting glucose absorption levels. As described above, the 1,2-benzothiazine
1,1-dioxides are valuable heterocycles and possess various biological activities. In par-
ticular, 1,2-benzothiazine 1,1-dioxide based carboxamides are the most biologically ac-
tive templates. In 2011, Kim and colleagues synthesized different 1,2-benzothiazine-
N-arylacetamides and screened them for their in vitro inhibition toward human 11
HSD1. The derivative (Figure 1) showed the most promising activity with an IC50
value of 0.041 µM 49]. Recently, Shin and coworkers reported novel 1,2-benzothiazine-
N-arylacetamides as SARS-CoV-2 inhibitors and among them compound (Figure 1)
showed robust activity with an IC50 value of 0.88 M [51]. Furthermore, 2-(2-(4-bromo-2-
fluorobenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4(3H)-ylidene)acetic acid was proved
to be a good aldose-reductase inhibitor with an IC50 value of 0.11 M [39]. Similarly, 4-
β-
3
[
4
µ
µ
hydroxy-2-methyl-N’-(1-phenylethylidene)-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-
dioxide and 4-hydroxy-N’-(1-phenylethylidene)-2H-benzo[e[1,2]thiazine-3-carbohydrazide
1,1-dioxide showed excellent inhibitions of
α-glucosidase enzymes with IC50 values of 4.2
and 3.9, respectively [41 45]. Encouraged by these results and to explore further biological
,
potentials, the present study highlighted the antidiabetic activity of novel 1,2-benzothiazine-
N-arylacetamides 11a–m and 12a–m.
2.3.1. Molecular Docking (In Silico Analysis)
In silico screening is an excellent approach for the screening of libraries of compounds
in a short time and thus it minimizes the laborious synthetic work [52–55]. Molecular dock-
ing is a widely used method to predict binding interactions between the 3D conformations
of various ligands and receptor proteins that helps in optimization and leads toward drug
development [56–58].
Regarding the effectiveness of this tool, we executed molecular docking studies of
all the synthesized 1,2-benzothiazine derivatives. Molecular docking was performed via
MOE software to screen out the compounds having the best residue binding interactions
with the receptor protein (α-glucosidase). These compounds were ranked on the basis of
root-mean-square deviation (rmsd) values, bindings with the selected residues (Asp203,
Asp542, Asp327, His600, Arg526) (Figure S1), and docking scores [59].
In the molecular docking experiment, most of the compounds showed good binding
scores and binding interactions with the selected residues (Asp203, Asp542, Asp327, His600,
Arg526) (Table 1, Figure 2). In silico studies of salacinol having a cyclic thiosugar sulfonium
and a sulfate group in its structure and its derivatives found it blocked the α-glucosidase
activity via bindings with the Asp203, Asp327, and Asp542 residues [60]. Recently, in
silico analysis of anthocyanidins and anthocyanins was carried out that showed these
scaffolds to be a good inhibitor of α-glucosidase enzymes. These compounds also showed
their inhibitory action by interacting with the selected residues, Asp203, Asp327, and
Asp542 [61]. Similarly, aglycone of curculigoside A and its derivatives also exhibited
good in silico inhibition of
and Asp542 residues [62]. The ligands 12a
13.47 to 14.23 Kcal/mol were ranked the most effective antidiabetic scaffolds. Moreover,
compounds 11c 11h 11i, and 12m were also considered important due to good binding
energy values in the range of 12.13 to 12.80 Kcal/mol. These above-described ligands
showed binding energy values close to the standard drug, acarbose ( 16.18 Kcal/mol) and
α
-glucosidase enzymes via interactions with Asp203, Asp327,
,
12d 12e, and 12g having docking scores from
,
−
−
,
,
−
−
−
were tightly packed in the selected pocket of the receptor enzyme as shown in 3D maps
(Figure 3). The 2D interaction modes revealed that most of the ligands bind with Asp203,
Asp327, and Asp542 among the selected residues and were supposed to be responsible for
the
α-glucosidase inhibitory activity (Figure 2, Table S1). Acarbose (control) also showed
good interactions with the selected residues, His600, Asp542, Arg526, and Asp327, in the

Molecules 2021, 26, 3043
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re-docking experiment. The docking score and rmsd value for acarbose were found as
−16.18 Kcal/mol and 2.00 Å, respectively.
11c
12d
12g
12a
12e
Acarbose
Figure 2. Two-dimensional interaction modes of the most active compounds (11c, 12a, 12d, 12e, 12g) and the standard drug,
acarbose. Blue color indicates ligand exposure and a green color dotted line indicates the interactions of receptor enzymes
with ligands.
As a whole, 1,2-Benzothiazine-N-arylacetamides 11a
effective compared to derivatives 12a regarding their in silico
potential (Table 1). However, compounds 11c 11h, and 11i showed comparatively better
–
m
were observed to be less
–m
α
-glucosidase inhibitory
,
in silico enzyme inhibitions as revealed from their low binding energies and rmsd values
(Table 1). The compound 11c was observed as the most effective derivative in terms of
binding energy and rmsd value, which were found to be
−
12.80 Kcal/mol and 1.08 Å,

Molecules 2021, 26, 3043
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respectively. This ligand showed interactions with Asp327, Asp474, and Met444 residues.
Asp327 interacted with the benzene ring of the benzothiazine nucleus. Asp474 formed
a hydrogen bond with the chloro and Met444 with the oxygen atom of the SO2 group.
The ligand 11h exhibited a good docking score (
−
12.13 Kcal/mol) and low rmsd value
(
1.21 Å). A hydrogen bonding was observed between the NH group and the Asp542
residue while Thr205 interacted with the oxygen of the CO group. Similarly, a low docking
score (−12.25 Kcal/mol) and rmsd value (1.11 Å) were found for the compound 11i. This
is explained by the strong hydrogen bonding of this compound with the selected residue,
Asp542, via NH functionality.
1,2-Benzothiazine-N-arylacetamides 12a
energies and better interactions with the selected residues compared to 1,2-benzothiazine-
N-arylacetamides 11a (Table 1). Compound 12a was observed as the most potent
compound as it showed a low docking score ( 14.23 Kcal/mol), small rmsd value (1.21 Å),
–m preferentially exhibited good binding
–
m
−
and good interactions with selected residues (Arg526, Asp542, Asp327). This ligand
exhibited a hydrogen bond with Arg526 via the oxygen atom of the OH group. A second
hydrogen interaction was found between the nitrogen atom of the NH group and the
Asp542 residue. Similarly, Asp327 interacted with the benzene ring of the benzothiazine
nucleus of ligand 12a. In addition, the present ligand also formed an interaction with
Trp406 using the oxygen atom of the SO2 group. These interactions and smaller rmsd
value are responsible for the low binding energy value of ligand 12a. The compounds
12d
respectively) due to dipole–dipole interactions with Asp203 and Asp327 via their OH and
Br functionalities, respectively (Figure 2). These ligands (12d 12e, and 12g) also exhibited
,
12e, and 12g also showed good docking scores (
−
13.91,
−
13.47, and
−
13.52 Kcal/mol,
,
low rmsd values of 1.13, 1.27, and 1.01 Å, respectively. In the ligand 12m, the bromo group
showed an interaction with the selected residue, Asp327. Due to this interaction and a low
rmsd value of 1.19 Å, ligand 12m showed a good binding energy value of −12.32 Kcal/mol
The compound 12k was found somewhat less effective as an inhibitor as revealed
from its docking score and rmsd value (Table 1). The compound 12k showed interactions
with Asp327 and Tyr605 via both the bromo groups individually. The other members of
this series showed high rmsd values and no interactions with the selected residues. In
.
addition, the 1,2-benzothiazines 9a,b were also docked into the receptor pocket but these
proved to be noninhibitors of the α-glucosidase enzyme (Table 1 and Table S1).
It is concluded from the above discussion that not only the benzothiazine ring system
but also the presence of the acetamide group was found indispensable for the in silico
inhibition of α-glucosidase enzymes. Furthermore, it was noticed that the presence of
polar as well as nonpolar groups in the structures of scaffolds played a crucial role toward
the inhibition of enzymes by interacting with the selected residue. It is clear from the 2D
diagrams of molecules that the interactions with the selected residues groups, such as CO,
CONH, NH, SO2, Cl, Br, OH, phenyl, and aryl, all participated equally. Compounds with
low rmsd values having good binding energies and interactions with the selected residues
were of our interest.
2.3.2. α-Glucosidase Inhibition (In Vitro Analysis)
In order to assess the in silico results, all the compounds were also subjected to
the in vitro inhibition of
α-glucosidase enzymes, which was investigated using the p-
nitrophenyl- -D-glucopyranoside (PNPG) substrate. Acarbose was employed as the ref-
α
erence drug. In vitro analysis of all the synthesized compounds was performed in order
to justify the validity of in silico outcomes. Results were found in good agreement with
the in silico study. Initially, percentage inhibition values were determined and then the
compounds having 50% inhibition values or above were subjected to the micro dilution ex-
periment (Table 1). As previously stated, 1,2-benzothiazine-N-arylacetamides 12a
witnessed as the most effective antidiabetic agents during the in silico analysis. Herein, dur-
ing in vitro screening these derivatives were also proved effective -glucosidase inhibitors
compared to 1,2-benzothiazine-N-arylacetamides 11a . Among all the compounds, 11c
–m were
α
–m
,

Molecules 2021, 26, 3043
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12a
values of 30.65, 18.25, 20.76, 35.14, and 24.24
have IC50 values even less than the standard drug, acarbose (58.8
compounds showed more potent -glucosidase inhibitions compared to the previously
reported sulfonamides [63], dihydroxy pyrrolidines [64], and thiobarbituric acid enamine
derivatives [65]. The compounds 11h 11i, and 12m having IC50 values 47.13, 40.41, and
58.45 M, respectively, were witnessed as the moderate antidiabetic agents while other
,
12d
,
12e, and 12g showed the most promising
α
-glucosidase inhibition with IC50
µM, respectively (Table 1). These inhibitors
µ
M [42 43]). These
,
α
,
µ
derivatives failed to show any promising inhibitory effects against
and were categorized as weak inhibitors or in-active compounds.
α-glucosidase enzymes
12a
11c
12d
12g
Acarbose
12e
Figure 3. Three-dimensional interaction modes of the most active compounds (11c, 12a, 12d, 12e, 12g) and the standard
drug, acarbose. Color portions show pocket selected and red portions indicate the ligand exposure points.
Derivatives 11a
compared to derivatives 12a
–
m
with 3-benzoyl functionality exhibited low antidiabetic activity
, which was also observed during in silico studies. In this
–m
series, the presence of deactivating groups (like Cl and NO2) in the structure resulted in
better enzyme inhibitions. However, herein, the presence of EWGs at the para-position of
the N-phenyl ring was proved more effective compared to other sites as indicated by the
IC50 values of compounds 11c (30.65
µM), 11h (47.13 µM), and 11i (40.41 µM). Similarly,
for the derivatives 12a , the presence of electron withdrawing groups (weak or strong)
–m
and weak electron donating groups (like CH3) at the ortho-position of the N-phenyl group
resulted in better antidiabetic activity as indicated by low IC50 values of derivatives 12a
(18.25
µM), 12d (20.76
µM), and 12g (24.24
µM). The derivatives 12a and 12d were found
to be roughly three-fold more effective as inhibitors of
α
-glucosidase enzymes compared
to acarbose. The increase in bioactivity with the introduction of EWGs in the benzoyl
and N-phenyl moieties was also witnessed during the evaluation of previously reported
1,2-benzothiazine-N-arylacetamides as SARS-CoV-2 inhibitors [51]. In the series 12a–m,
the variation in the position of nitro, chloro, and bromo substituents failed to enhance the
enzymatic inhibition potential of the compounds.

Molecules 2021, 26, 3043
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Table 1. Structural parameters, in silico (docking scores, binding residues, and rmsd values) and in vitro (IC50 val-
ues) inhibitory potential of 1,2-benzothiazines 9a , 1,2-benzothiazine-N-arylacetamides 11a , and 12a against
α-glucosidase enzymes.
,b
–m
–m
Docking
Scores (Kcal/mol)
Compound
R2
Binding Residues
Inhibitions (%)
IC50 (µM)
Rmsd Values (Å)
9a
9b
-
−9.12
−10.11
−11.36
−10.33
-
2.54
2.14
2.03
1.50
20
55
39
32
ND
> 100
ND
-
-
11a
11b
2-Cl
3-Cl
Met444
-
ND
Asp327, Asp474,
11c
4-Cl
−12.80
1.08
79
30.65
Met444
11d
11e
11f
2-Me
3-Me
−11.02
−11.42
−10.76
−11.85
−12.13
−12.25
−11.83
−10.23
−11.77
−11.85
Thr205
2.33
1.82
1.72
1.32
1.21
1.11
1.32
1.44
1.39
1.32
10
15
37
58
63
77
50
13
54
69
ND
ND
-
4-Me
-
ND
11g
11h
11i
2-NO2
3-NO2
4-NO2
3-Br
Asp203
88.12
47.13
40.41
> 100
ND
Asp542, Thr205
Asp542
11j
Thr205
11k
11l
4-Br
-
-
-
2-OMe
4-OMe
> 100
88.12
11m
Arg526, Asp542,
Asp327, Trp406
12a
2-Cl
−14.23
1.21
79
18.25
12b
12c
12d
12e
12f
3-Cl
4-Cl
−10.18
−10.95
−13.91
−13.47
−10.86
−13.52
−12.25
−10.82
−11.94
−11.92
−11.60
−12.32
Phe575
1.48
1.81
1.13
1.27
1.09
1.01
1.41
1.27
1.71
1.93
1.23
1.19
47
61
76
71
20
76
58
33
12
48
34
65
> 100
> 100
20.76
35.14
ND
-
2-Me
3-Me
4-Me
2-NO2
3-NO2
4-NO2
3-Br
Asp203, Asp327
Asp203, Asp327
Thr544
12g
12h
12i
Asp203, Asp327
24.24
> 100
ND
-
-
12j
-
ND
12k
12l
4-Br
Asp327, Tyr605
Met444, Phe575
Asp327
96.25
ND
2-OMe
4-OMe
12m
58.45
His600, Asp542,
Arg526, Asp327,
Met444, Lys480
Acarbose
-
−16.18
2.00
45
58.8
ND = not determined; acarbose was used as reference drug; inhibition percentages were determined at 200 µM concentration; reference
(selected) residues are in bold and italic form.
3. Materials and Methods
3.1. General
Different instruments were used for the identification of structural features of syn-
thesized compounds. For the determination of melting points, Stuart apparatus (Open
1
capillary SMP3) was employed. H and 13C-NMR spectra were recorded with the help
of an NMR-spectrophotometer (Bruker/Hazimin) at 600 MHz using a deuterated DMSO

Molecules 2021, 26, 3043
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solvent. Chemical shift values (δ) were given in ppm (parts per million) relative to tetram-
ethylsilane (TMS) as an internal standard. Coupling constants (J) were given in Hertz. For
the progress of chemical reactions, silica-coated TLC-plates (Merk, 0.2 mm 60 HF254) were
used. Spots were visualized under a UV lamp (254 nm).
3.2. General Procedure for the Synthesis of 3-(Benzoyl/4-bromobenzoyl)-1,2-benzothazines (9a
Acetophenones (5a ) (1.0 mmol) and p-toluenesulfonic acid monohydrate (285 mg,
1.5 mmols) were dissolved in acetonitrile (10 mL). Subsequently, N-bromosuccinamide
196 mg, 1.10 mmols) was added in portions and the mixture was refluxed for 2 h. Reaction
,b)
,
b
(
mixture was cooled to room temperature and solvent was evaporated under vacuum. The
residue obtained was dissolved in dichloromethane (DCM) and washed with distilled
water followed by the saturated solution of sodium bicarbonate. After separating the DCM
layer from aqueous layer using a separating funnel, the DCM was evaporated with the
help of a rotary evaporator to get
6a ) (1.0 mmol) were added to a sodium saccharin
made in DMF (5 mL) and the mixture was heated at 110 ◦C for 3 h to get the N-alkylated
saccharin derivatives (8a ). The dried N-alkylated saccharin derivatives (8a ) (1.0 mmol)
α
-bromoacetophenones (6a
,b). α-Bromoacetophenones
(
,
b
7
(226 mg, 1.10 mmol) suspension
,
b
,b
were refluxed with sodium methoxide (270 mg, 5.0 mmols) in dry methanol for 30 min. The
mixture was cooled and treated with 15% cold HCl to get the 3-(benzoyl/4-bromobenzoyl)-
1,2-benzothiazines (9a,b) [46,47] in good isolated yields (given below). The products were
also re-crystallized with methanol.
(4-Hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-3-yl)(phenyl)methanone (9a): Yellow solid; yield:
(232 mg, 77%); m.p. 156–157 ◦C; 1H-NMR (DMSO-d6, 600 MHz)
δ: 7.61 (t, J = 7.2 Hz, 2H,
ArH), 7.68 (t, J =7.2 Hz, 1H, ArH), 7.92–7.96 (m, 3H, ArH), 8.01 (d, J = 7.2 Hz, 2H, ArH),
8.19–8.21 (m, 1H, ArH), 9.91 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 128.2, 128.3,
128.4, 129.3, 130.2, 132.6, 132.8, 133.0, 133.4, 133.5, 134.1, 134.8, 135.2, 148.8, 164.7. MS (ESI+):
m/z calcd. for C15H11NO4NaS [M + Na]+ 324.02; found 324.03.
(4-Bromophenyl)(4-hydroxy-1,1-dioxido-2H-benz◦o[e][1,2]thiazin-3-yl)methanone (9b): Light yel-
low solid; yield: (304 mg, 80%); m.p. 144–146 C; 1H-NMR (DMSO-d6, 600 MHz)
δ: 7.60 (d,
J = 7.9 Hz, 3H, ArH), 7.69 (t, J = 7.6 Hz, 1H, ArH), 7.93 (d, J = 7.5 Hz, 2H, ArH), 8.12–8.17 (m,
1H, ArH), 8.23 (t, J = 7.8 Hz, 1H, ArH), 9.93 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
:
127.4, 127.7, 128.0, 128.3 (2C), 130.2, 130.6 (2C), 131.8, 132.6, 133.2, 134.7, 136.3, 149.2, 165.7.
MS (ESI+): m/z calcd. for C15H10BrNO4NaS [M + Na]+ 401.93; found 401.93.
3.3. General Procedure for the Synthesis of 2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-
benzo[e][1,2]thiazin-2-yl)-N-arylacetamides (11a–m)
3-Benzoyl-1,2-benzothazine (9a) (301 mg, 1.0 mmol), 2-bromo-N-arylacetamides 10a–m
(1.0 mmol), and K2CO3 (207 mg, 1.5 mmols) were added in DMF (5 mL) and heated for
◦
2-2.5 h at 80-100 C. Afterward, the reaction mixture was cooled to room temperature
and diluted with thrice amount of cold distilled water. Acidification with cold dilute
HCl (20%) furnished the colored precipitates of 2-(3-benzoyl-4-hydroxy-1,1-dioxido-2H-
benzo[e][1,2]thiazin-2-yl)-N-arylacetamides (11a–m) (Table 1). Precipitates were filtered,
washed with excess distilled water, and finally dried. All the products were obtained in
good isolated yields (given below) and were also re-crystallized with methanol.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-chlorophenyl)acetamide (11a):
Light yellow solid; yield: (347 mg, 74%); m.p. 141–142 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
: 4.43 (s, 2H, CH2), 7.01–7.13 (m, 2H, ArH), 7.19 (dd, J = 8.2, 2.0 Hz, 2H, ArH), 7.32–7.39
(m, 1H, ArH), 7.63 (t, J = 8.2 Hz, 1H,), 7.90–7.99 (m, 3H, ArH), 8.04 (d, J = 7.6 Hz, 2H,
ArH), 8.23–8.28 (m, 2H, ArH), 10.03 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 53.9,
δ
116.9, 118.6, 119.4, 120.0, 121.7, 122.3, 126.0 (2C), 126.4 (2C), 127.6, 129.0 (2C), 132.6, 133.7,
134.9, 136.2, 138.0, 139.6, 164.9, 167.9, 188.9. MS (ESI+): m/z calcd. for C23H17ClN2O5NaS
[M + Na]+ 491.03; found 491.02.

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2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(3-chlorophenyl)acetamide (11b):
Light yellow solid; yield: (366 mg, 78%); m.p. 182–184 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ: 4.02 (s, 2H, CH2), 7.03 (dd, J = 7.9, 2.1 Hz, 1H, ArH), 7.12 (dd, J = 8.2, 2.0 Hz, 1H, ArH),
7.22 (t, J = 8.0 Hz, 1H, ArH), 7.36–7.37 (m, 1H, ArH), 7.63 (t, J = 7.5, 2H, ArH), 7.89–7.97
(m, 4H, ArH), 8.06 (d, J = 7.7 Hz, 2H, ArH), 8.20–8.22 (m, 1H, ArH), 10.08 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ: 53.7, 117.0, 118.3, 118.8, 119.0, 119.8, 121.9, 123.8, 127.8,
128.7 (3C), 131.6 (2C), 134.2 (2C), 134.9, 135.2, 139.0, 139.9, 165.1, 168.1, 189.1. MS (ESI+):
m/z calcd. for C23H17ClN2O5NaS [M + Na]+ 491.03; found 491.03.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-chlorophenyl)acetamide (11c):
◦
1
Yellow solid; yield: (347 mg, 73%); m.p. 146-148 C. H-NMR (DMSO-d6, 600 MHz) δ:
4.00 (s, 2H, CH2), 7.20–7.26 (m, 4H, ArH), 7.63 (t, 2H, J = 7.6 Hz, ArH), 7.70 (t, J = 7.3 Hz,
1H, ArH), 7.87–7.90 (m, 1H, ArH), 7.90–7.94 (m, 2H, ArH), 8.05 (d, J = 7.6 Hz, 2H, ArH),
8.18–8.19 (m, 1H, ArH), 10.01 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 53.8, 116.2,
120.3, 121.8, 126.9, 127.0, 128.4, 128.5 (3C), 128.6 (2C), 128.7 (2C), 132.7, 132.9, 133.8, 135.2,
137.0, 138.5, 164.9, 168.0, 189.1. MS (ESI+): m/z calcd. for C23H17ClN2O5NaS [M + Na]+
491.0397; found 491.0295.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(o-tolyl)acetamide (11d): Light
Brown powder; yield: (359 mg, 80%); m.p. 165-167 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ:
2.20 (s, 3H, CH3), 3.94 (s, 2H, CH2), 6.96 (t, J = 7.8 Hz, 2H, ArH), 7.05 (dd, J = 7.4, 2.4 Hz, 2H
ArH), 7.65 (d, J = 7.8 Hz, 3H, ArH), 7.70–7.76 (m, 2H, ArH), 7.87 (d, 1H, ArH), 7.94–8.01 (m,
1H, ArH), 8.04 (t, J = 7.9 Hz, 1H, ArH), 8.16–8.21 (m, 1H, ArH), 9.73 (s, 1H, NH). 13C-NMR
(DMSO-d6, 150 MHz) δ: 21.2, 54.0, 115.1, 116.6, 119.3, 121.4, 124.2 (2C), 127.6, 128.4 (2C),
128.6 (2C), 128.7, 128.8, 132.2, 132.8, 133.9, 134.4, 137.2, 139.0, 164.4, 167.0, 187.9. MS (ESI+):
m/z calcd. for C24H20N2O5NaS [M + Na]+ 471.09; found 471.09.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(m-tolyl)acetamide (11e): Light
orange powder; yield: (350 mg, 78%); m.p. 177–178 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ:
2.16 (s, 3H, CH3), 4.00 (s, 2H, CH2), 6.78 (d, J = 7.2 Hz, 1H, ArH), 7.02 (d, J = 8.3 Hz, 2H,
ArH), 7.05 (t, J = 7.7 Hz, 2H, ArH), 7.63 (t, J = 7.7 Hz, 2H, ArH), 7.70 (t, J = 7.4 Hz, 1H, ArH),
7.88–7.93 (m, 2H, ArH), 8.07 (d, J = 7.6 Hz, 2H, ArH), 8.19–8.21 (m, 1H, ArH), 9.78 (s, 1H,
NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 21.0, 53.7, 115.2, 116.2, 119.4, 121.8, 124.1, 127.1
(2C), 128.4, 128.5, 128.6 (2C), 128.7 (2C), 132.6, 132.9, 133.8, 134.1, 137.0, 138.9, 164.6, 166.3,
187.8. MS (ESI+): m/z calcd. for C24H20N2O5NaS [M + Na]+ 471.09; found 471.08.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(p-tolyl)acetamide (11f): Yel-
◦
1
low powder; yield: (341 mg, 76%); m.p. 136-137 C. H-NMR (DMSO-d6, 600 MHz) δ:
2.16 (s, 3H, CH3), 3.97 (s, 2H, CH2), 6.97 (d, J = 8.2 Hz, 2H, ArH), 7.09 (d, J = 7.9 Hz, 2H,
ArH), 7.63 (t, J = 7.2 Hz, 2H, ArH), 7.70 (t, J = 7.2 Hz, 1H, ArH), 7.86–7.89 (m, 1H, ArH),
7.90–7.93 (m, 2H, ArH), 8.05 (d, J = 7.2 Hz, 2H, ArH), 8.17–8.19 (m, 1H, ArH), 9.77 (s, 1H,
NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 20.3, 53.7, 116.2, 118.8, 121.8, 127.0, 128.5, 128.5
(2C), 128.6 (2C), 128.7 (2C), 128.9, 132.3, 132.6, 132.9, 133.8, 135.2, 135.6, 138.6, 164.5, 168.2,
188.9. MS (ESI+): m/z calcd. for C24H20N2O5NaS [M + Na]+ 471.09; found 471.09.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-nitrophenyl)acetamide (11g):
◦
Light brown solid; yield: (398 mg, 83%); m.p. 157-158 C. 1H-NMR (DMSO-d6, 600 MHz)
δ:
4.06 (s, 2H, CH2), 7.23–7.34 (m, 2H, ArH), 7.37 (d, J = 8.2 Hz, 2H, ArH), 7.55 (d, J = 8.2 Hz,
2H, ArH), 7.53–7.58 (m, 1H, ArH), 7.62 (d, J = 7.8 Hz, 1H, ArH), 7.84–7.87 (m, 2H, ArH),
7.88–7.91 (m, 2H, ArH), 8.14–8.12 (m, 1H, ArH), 9.87 (s, 1H, NH). 13C-NMR (DMSO-d6,
150 MHz) δ: 53.8, 113.1, 116.1, 118.0, 121.8, 124.8, 127.2 (2C), 128.3 (2C), 128.7, 128.8, 130.1,
132.6, 132.7, 133.9, 135.3, 138.3, 139.3, 146.9, 165.5, 168.2, 188.9. MS (ESI+): m/z calcd. for
C23H17N3O7NaS [M + Na]+ 502.06; found 502.06.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(3-nitrophenyl)acetamide (11h):
◦
Light yellow powder; yield: (403 mg, 84%); m.p. 170-172 C. 1H-NMR (DMSO-d6, 600 MHz
)
δ: 4.04 (s, 2H, CH2), 7.49 (t, J = 8.1 Hz, 1H, ArH), 7.56 (d, J = 8.1 Hz, 1H, ArH), 7.63 (t,
J = 7.5 Hz, 2H, ArH), 7.69 (t, J = 7.3 Hz, 1H, ArH), 7.84 (dd, J = 8.3, 4.2 Hz, 1H, ArH),

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7.89–7.97 (m, 3H, ArH), 8.05 (d, J = 7.6 Hz, 2H, ArH), 8.09 (s, 1H, ArH), 8.15–8.22 (m,
1H, ArH), 10.38 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 53.9, 113.0, 116.2, 117.9,
121.9, 124.9, 127.1, 128.4, 128.6 (2C), 128.7 (2C), 130.1, 132.8, 132.9, 133.9, 135.1, 138.4, 139.1,
147.7, 165.5, 167.9, 189.1. MS (ESI+): m/z calcd. for C23H17N3O7NaS [M + Na]+ 502.0697;
found 502.0531.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-nitrophenyl)acetamide (11i):
Light brown solid; yield: (369 mg, 77%); m.p. 194–195 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
: 4.08 (s, 2H, CH2), 7.44–7.47 (m, 2H, ArH), 7.63 (td, J = 7.2, 1.6 Hz, 2H, ArH), 7.68–7.71 (m,
1H, ArH), 7.89–7.95 (m, 3H, ArH), 8.05 (d, J = 7.2 Hz, 2H, ArH), 8.09–8.11 (m, 2H, ArH),
8.20–8.21 (m, 1H, ArH), 10.48 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 53.9, 116.1,
δ
118.6 (2C), 121.8, 124.8 (2C), 127.1, 128.3, 128.6 (2C), 128.7 (2C), 132.8, 132.9, 133.9, 135.2,
138.5, 142.2, 144.2, 165.9, 167.7, 189.4. MS (ESI+): m/z calcd. for C23H17N3O7NaS [M + Na]+
502.0697; found 502.0675.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(3-bromophenyl)acetamide (11j):
◦
Yellow solid; yield: (416 mg, 81%); m.p. 163-166 C. 1H-NMR (DMSO-d6, 600 MHz)
δ: 4.04
(s, 2H, CH2), 7.04 (d, J = 8.5 Hz, 2H, ArH), 7.21 (t, J = 7.6 Hz, 2H, ArH), 7.56–7.59 (m, 3H,
ArH), 7.68 (t, J = 7.5 Hz, 1H, ArH), 7.88–7.90 (m, 3H, ArH), 7.98 (d, J = 7.2 Hz, 1H, ArH),
8.12 (s, 1H, ArH), 9.46 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ: 53.9, 116.4, 119.3,
119.7, 122.0, 126.4, 127.3 (2C), 128.3 (3C), 128.5, 130.6, 132.0, 134.5, 136.6 (2C), 138.6, 139.2,
140.5, 165.2, 167.2, 189.2. MS (ESI+): m/z calcd. for C23H17BrN2O5NaS [M + Na]+ 534.98;
found 534.98.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-bromophenyl)acetamide (11k):
◦
Light yellow solid; yield: (421 mg, 82%); m.p. 150-153 C. 1H-NMR (DMSO-d6, 600 MHz)
δ:
4.09 (s, 2H, CH2), 7.05 (d, J = 7.9 Hz, 2H, ArH), 7.22–7.35 (m, 2H, ArH), 7.56 (d, J = 7.9 Hz
,
3H, ArH), 7.69–7.74 (m, 2H, ArH), 7.95 (t, J = 7.9 Hz, 2H, ArH), 8.09–8.11 (m, 1H, ArH),
8.12–8.14(m, 1H, ArH), 9.48 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
: 54.0, 116.9,
119.4, 119.8, 120.2, 126.4, 126.9 (2C), 127.2 (2C), 128.3 (2C), 128.6, 130.5, 132.1, 134.4, 136.4
(2C), 138.5, 139.9, 140.2, 165.3, 167.4, 188.9. MS (ESI+): m/z calcd. for C23H17BrN2O5NaS
[M + Na]+ 534.98; found 534.97.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-methoxyphenyl)acetamide
◦
1
(
11l): Light brown solid; yield: (372 mg, 80%); m.p. 199-200 C. H-NMR (DMSO-d6,
600 MHz : 3.72 (s, 3H, CH3), 4.09 (s, 2H, CH2), 6.74 (t, J = 7.6 Hz, 1H, ArH), 6.93 (d,
)
δ
J = 8.0 Hz, 1H, ArH), 6.98 (t, J = 8.1 Hz, 1H, ArH), 7.49 (d, J = 7.9 Hz, 1H, ArH), 7.63
(t, J = 7.6 Hz, 2H, ArH), 7.70 (t, J = 7.4 Hz, 1H, ArH), 7.86–7.91 (m, 3H, ArH), 8.06 (d,
J = 7.5 Hz, 2H, ArH), 8.16–8.18 (m, 1H, ArH), 9.17 (s, 1H, NH). 13C-NMR (DMSO-d6, 150
MHz) δ: 53.8, 55.5, 111.0, 116.4, 120.0, 121.4, 121.8, 124.4, 126.3, 127.0, 128.5, 128.6 (2C), 128.9
(2C), 132.6, 132.9, 133.7, 135.1, 138.4, 149.2, 165.0, 168.2, 188.8. MS (ESI+): m/z calcd. for
C24H20N2O6NaS [M + Na]+ 487.08; found 487.07.
2-(3-Benzoyl-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-methoxyphenyl)acetamide
◦
11m): Yellow solid; yield: (367 mg, 79%); m.p. 164-165 C. 1H-NMR (DMSO-d6, 600 MHz)
(
δ: 3.64 (s, 3H, OCH3), 3.95 (s, 2H, CH2), 6.75 (d, J = 7.8 Hz, 2H, ArH), 7.11 (d, J = 7.8 Hz,
2H, ArH), 7.63 (t, J = 7.2 Hz, 2H, ArH), 7.70 (t, J = 7.2 Hz, 1H, ArH), 7.86–7.92 (m, 3H, ArH),
8.04 (d, J = 7.2 Hz, 2H, ArH), 8.17 (d, J = 7.8 Hz, 1H, ArH), 9.71 (s, 1H, NH). 13C-NMR
(DMSO-d6, 150 MHz) δ: 53.7, 55.0, 113.7 (3C), 116.2, 120.4 (3C), 121.8, 127.0, 128.6, 128.7
(2C), 131.2, 132.6, 132.9, 133.7, 135.1, 138.6, 155.2, 164.1, 168.3, 188.8. MS (ESI+): m/z calcd.
for C24H20N2O6NaS [M + Na]+ 487.08; found 487.07.
3.4. General Procedure for the Synthesis of 2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-
benzo[e][1,2]thiazin-2-yl)-N-arylacetamides (12a–m)
3-(4-Bromobenzoyl)-1,2-benzothazine (9b) (380 mg, 1.0 mmol), 2-bromo-N-arylacetamides
10a
–m (1.0 mmol), and K2CO3 (207 mg, 1.5 mmols) were added in DMF (5 mL) and heated
for 2-2.5 h at 80–100 ◦C. Afterward, the reaction mixture was cooled to room temperature

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and diluted with thrice amount of cold distilled water. Acidification with cold dilute
HCl (20%) furnished the colored precipitates of 2-(3
dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-aryl acetamides (12a
-
(4-bromobenzoyl)-4-hydroxy-1,1-
) (Table 1). Precipitates
–
m
were filtered, washed with excess distilled water and then dried. The products were
re-crystallized from methanol and were dried.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-chlorophenyl)
◦
acetamide (12a): Light yellow solid; yield: (438 mg, 80%); m.p. 180-181 C. 1H-NMR (DMSO-
d6, 600 MHz) δ: 4.12 (s, 2H, CH2), 7.12 (t, J = 7.2 Hz, 1H, ArH), 7.19 (t, J = 7.5 Hz, 1H, ArH),
7.34 (d, J = 7.8 Hz, 1H, ArH), 7.39 (d, J = 7.9 Hz, 1H, ArH), 7.85–7.90 (m, 5H, ArH), 7.96
(d, J = 8.1 Hz, 2H, ArH), 8.13–8.15 (m, 1H, ArH), 9.53 (s, 1H, NH), 14.99 (s, 1H, OHenolic).
13C-NMR (DMSO-d6, 150 MHz)
δ: 53.5, 116.3, 122.0, 125.4, 126.0, 126.3, 126.9, 127.1, 127.3,
128.5, 129.4, 130.7 (2C), 131.8 (2C), 132.7, 133.8, 133.9, 134.3, 138.3, 165.4, 168.1, 187.8. MS
(ESI+): m/z calcd. for C23H16BrClN2O5NaS [M(81Br) + Na]+ 570.9497; found 570.9286.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-◦2-yl)-N-(3-chlorophenyl)
acetamide (12b): Yellow powder; yield: (460 mg, 84%); m.p. 148-150 C. 1H-NMR (DMSO-d6,
600 MHz)
7.59–7.60 (m, 2H, ArH), 7.74 (d, J = 7.6 Hz, 2H, ArH), 7.88–7.95 (m, 3H, ArH), 8.17–8.23
(m, 1H, ArH), 9.55 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 53.8, 116.3, 121.9, 126.0,
δ: 4.00 (s, 2H, CH2), 7.12–7.17 (m, 3H, ArH), 7.27 (dd, J = 7.8, 2.7 Hz, 1H, ArH),
δ
126.2, 126.2, 126.9, 127.1, 127.4 (2C), 128.3 (2C), 129.8, 130.6 (2C), 132.8, 133.6, 133.9, 134.3,
138.4, 165.1, 168.7, 188.0. MS (ESI+): m/z calcd. for C23H16BrClN2O5NaS [M + Na]+ 568.94;
found 568.94.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-chlorophenyl)
acetamide (12c): Yellow solid; yield: (477 mg, 87%); m.p. 181-184 ◦C. 1H-NMR (DMSO-d6,
600 MHz)
2H, ArH), 7.74 (d, J = 7.6 Hz, 1H, ArH), 7.88–7.95 (m, 3H, ArH), 8.08–8.13 (m, 2H, ArH),
8.19–8.21 (m, 1H, ArH), 9.59 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 54.0, 116.8,
δ: 4.08 (s, 2H, CH2), 7.24–7.30 (m, 1H, ArH), 7.44–7.48 (m, 2H, ArH), 7.54–7.57 (m,
δ
116.1, 118.6 (2C), 124.9 (2C), 126.3 (2C), 126.8, 127.1 (2C), 128.2 (2C), 129.8, 132.8, 133.7, 133.9,
136.4, 138.5, 165.0, 167.4, 188.3. MS (ESI+): m/z calcd. for C23H16BrClN2O5NaS [M + Na]+
568.94; found 568.93.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(o-tolyl)acetamide
(
12d): Light orange powder; yield: (464 mg, 88%); m.p. 193-194 ◦C. 1H-NMR (DMSO-d6,
600 MHz)
1H, ArH), 7.86–7.92 (m, 5H, ArH), 7.98 (d, J = 8.2 Hz, 2H, ArH), 8.13–8.15 (m, 1H, ArH),
9.29 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 17.4, 53.5, 116.3, 121.9, 124.5, 125.3,
δ: 1.96 (s, 3H, CH3), 4.07 (s, 2H, CH2), 6.98–7.02 (m, 3H, ArH), 7.10 (d, J = 6.9 Hz,
δ
125.8, 127.0, 127.5, 128.5, 130.2, 130.7 (2C), 131.5, 131.8 (2C), 132.7, 133.8, 134.2, 135.2, 138.4,
164.9, 168.3, 187.2. MS (ESI+): m/z calcd. for C24H19BrN2O5NaS [M + Na]+ 551.0097;
found 550.9858.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(m-tolyl)acetamide
◦
1
(
12e): Light yellow solid; yield: (427 mg, 81%); m.p. 143-144 C. H-NMR (DMSO-d6,
600 MHz) : 2.17 (s, 3H, CH3), 3.99 (s, 2H, CH2), 6.79 (d, J = 7.3 Hz, 1H, ArH), 7.01 (d,
J = 8.9 Hz, 2H, ArH), 7.06 (t, J = 7.6 Hz, 1H, ArH), 7.83–7.95 (m, 7H, ArH), 8.16–8.19 (m, 1H,
ArH), 9.37 (s, 1H, NH), 14.92 (s, 1H, OHenolic). 13C-NMR (DMSO-d6, 150 MHz)
: 21.0, 53.9,
δ
δ
116.1, 116.3, 119.3, 122.0, 124.1, 126.9, 127.1, 128.4 (2C), 130.7 (2C), 131.7 (2C), 132.8, 133.9,
134.3, 137.8, 138.0, 138.4, 164.6, 168.0, 187.6. MS (ESI+): m/z calcd. for C24H19BrN2O5NaS
[M + Na]+ 549.0097; found 550.0000.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(p-tolyl)acetamide
◦
1
(
12f): Light brown solid; yield: (454 mg, 86%); m.p. 190-192 C. H-NMR (DMSO-d6,
600 MHz) : 2.43 (s, 3H, CH3), 3.98 (s, 2H, CH2), 6.98 (d, J = 8.3 Hz, 2H, ArH), 7.09 (d,
δ
J = 7.9 Hz, 1H, ArH), 7.70 (t, J = 7.2 Hz, 1H, ArH), 7.74 (t, J = 7.2 Hz, 2H, ArH), 7.87–7.90
(m, 1H, ArH), 7.91–7.94 (m, 2H, ArH), 8.05 (d, J = 7.2 Hz, 2H, ArH), 8.17–8.19 (m, 1H,
ArH), 9.48 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
: 21.0, 53.8, 116.2, 118.8, 121.8,
127.0, 128.3 (2C), 128.5, 128.6 (2C), 128.7 (2C), 128.9, 132.3, 132.6, 132.9, 133.8, 135.2, 135.6,

Molecules 2021, 26, 3043
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138.6, 164.4, 168.1, 188.0. MS (ESI+): m/z calcd. for C24H19BrN2O5NaS [M + Na]+ 549.00;
found 549.01.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-nitrophenyl)
acetamide (12g): Light brown powder; yield: (497 mg, 89%); m.p. 177–178 ◦C. 1H-NMR
(DMSO-d6, 600 MHz)
1H, ArH), 7.58 (q, J = 7.2 Hz, 1H, ArH), 7.83–7.90 (m, 6H, ArH), 7.98 (t, J = 8.4 Hz, 2H,
ArH), 8.11–8.15 (m, 1H, ArH), 10.17 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 53.7,
δ: 4.08 (s, 2H, CH2), 7.29 (q, J = 7.2, 1H, ArH), 7.40 (t, J = 8.4,
δ
116.2, 122.0, 124.8, 125.1, 125.4, 126.9, 127.2 (2C), 128.4, 130.2, 130.6 (2C), 131.8 (2C), 132.8,
133.9, 134.0, 134.5, 138.2, 165.5, 168.3, 187.7. MS (ESI+): m/z calcd. for C23H16BrN3O7NaS
[M + Na]+ 579.97; found 579.98.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(3-nitrophenyl)
◦
acetamide (12h): Light yellow solid; yield: (469 mg, 84%); m.p. 208-209 C. 1H-NMR (DMSO-
d6, 600 MHz)
ArH), 7.82–7.87 (m, 3H, ArH), 7.91–8.07 (m, 5H, ArH), 8.16–8.17 (m, 1H, ArH), 8.19–8.21
(m, 1H, ArH), 10.39 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
: 54.1, 112.9, 116.3, 118.0,
δ: 4.05 (s, 2H, CH2), 7.50 (t, J = 7.8 Hz, 1H, ArH), 7.58 (dd, J = 6.0, 2.0 Hz, 1H,
δ
126.2, 126.5, 127.4 (2C), 127.9 (2C), 128.4 (2C), 130.0, 131.7, 132.9, 134.0, 134.2, 138.2, 139.1,
147.7, 165.5, 167.9, 188.1. MS (ESI+): m/z calcd. for C23H16BrN3O7NaS [M + Na]+ 579.97;
found 579.98.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-◦2-yl)-N-(4-nitrophenyl)
acetamide (12i): Yellow powder; yield: (491 mg, 88%); m.p. 210-212 C. 1H-NMR (DMSO-d6,
600 MHz)
7.88–7.95 (m, 5H, ArH), 8.11 (d, J = 7.8 Hz, 3H, ArH), 8.19 (d, J = 7.0 Hz, 1H, ArH), 10.04
(s, 1H, NH), 15.18 (s, 1H, OHenolic). 13C-NMR (DMSO-d6, 150 MHz),
: 53.8, 114.1, 116.2,
δ: 4.08 (s, 2H, CH2), 7.47 (d, J = 7.8 Hz, 1H, ArH), 7.84 (d, J = 7.8 Hz, 2H, ArH),
δ
118.5, 122.0, 124.9, 127.2 (2C), 130.0 (2C), 130.2, 131.5, 131.7, 132.9, 133.9, 136.9, 138.4, 142.2,
144.2, 148.2, 165.9, 167.9, 188.7. MS (ESI+): m/z calcd. for C23H16BrN3O7NaS [M + Na]+
579.97; found 579.97.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(3-bromophenyl)
◦
acetamide (12j): Light orange solid; yield: (527 mg, 89%); m.p. 145–147 C. 1H-NMR (DMSO-
d6, 600 MHz)
3H, ArH), 7.84 (d, J = 7.8 Hz, 2H, ArH), 7.88–7.94 (m, 2H, ArH), 8.17–8.19 (m, 1H, ArH),
10.05 (s, 1H, NH), 14.97 (s, 1H, OHenolic). 13C-NMR (DMSO-d6, 150 MHz)
: 54.0, 116.3,
δ: 4.00 (s, 2H, CH2), 7.13–7.17 (m, 3H, ArH), 7.48 (s, 1H, ArH), 7.55–7.59 (m,
δ
117.6, 121.1, 121.3 (2C), 122.0, 126.0, 127.2 (2C), 130.7 (2C), 130.7 (3C), 131.7 (2C), 132.8, 133.9,
138.3, 139.6, 165.4, 168.7, 186.9. MS (ESI+): m/z calcd. for C23H16Br2N2O5NaS [M + Na]+
612.89; found 612.89.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-bromophenyl)
acetamide (12k): Yellow solid; yield: (545 mg, 92%); m.p. 200-203 ◦C. 1H-NMR (DMSO-d6,
600 MHz)
δ: 4.00 (s, 2H, CH2), 7.19 (d, J = 8.0 Hz, 3H, ArH), 7.37 (dt, J = 9.0, 3.0 Hz, 2H,
ArH), 7.83–7.94 (m, 3H, ArH), 8.16–8.18 (m, 3H, ArH), 10.12 (s, 1H, NH). 13C-NMR (DMSO-
d6, 150 MHz) δ: 54.0, 114.9, 116.2, 120.7 (3C), 121.1, 127.9, 130.7 (3C), 131.4 (3C), 131.7 (2C),
132.8, 133.9, 137.4, 138.0, 165.2, 167.9, 187.0. MS (ESI+): m/z calcd. for C23H16Br2N2O5NaS
[M + Na]+ 612.89; found 612.88.
2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(2-methoxyphenyl)
acetamide (12l): Yellow solid; yield: (484 mg, 89%); m.p. 186–187 ◦C. 1H-NMR (DMSO-d6,
600 MHz) δ: 3.73 (s, 3H, OCH3), 4.11 (s, 2H, CH2), 6.75 (t, J = 7.8 Hz, 1H, ArH), 6.94 (dd,
J = 7.8, 1.4 Hz, 1H, ArH), 6.98 (t, J = 7.8 Hz, 1H, ArH), 7.51 (d, J = 7.8 Hz, 1H, ArH), 7.85 (d,
J = 7.8 Hz, 1H, ArH), 7.86–7.91 (m, 4H, ArH), 7.95 (d, J = 7.8 Hz, 2H, ArH), 8.15–8.18 (m, 1H,
ArH), 9.17 (s, 1H, NH), 14.92 (s, 1H, OHenolic). 13C-NMR (DMSO-d6, 150 MHz)
δ: 53.7, 55.1,
111.7, 116.2, 120.4 (2C), 121.3, 121.8, 124.6, 126.5, 127.0, 127.6, 128.7 (2C), 131.2, 132.6, 132.9,
133.7, 135.1, 138.6, 149.2, 164.9, 167.6, 187.8. MS (ESI+): m/z calcd. for C24H19BrN2O6NaS
[M + Na]+ 564.99; found 564.98.

Molecules 2021, 26, 3043
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2-(3-(4-Bromobenzoyl)-4-hydroxy-1,1-dioxido-2H-benzo[e][1,2]thiazin-2-yl)-N-(4-methoxyphenyl)
acetamide (12m): Yellow powder; yield: (489 mg, 90%); m.p. 198-199 ◦C. 1H NMR (DMSO-
d6, 600 MHz) δ: 3.73 (s, 3H, OCH3), 4.01 (s, 2H, CH2), 6.75 (t, J = 7.8 Hz, 1H, ArH), 6.93 (dd,
J = 7.8, 1.4 Hz, 1H, ArH), 6.98 (t, J = 7.8 Hz, 1H, ArH), 7.50 (d, J = 7.8 Hz, 1H, ArH), 7.84
(d, J = 7.8 Hz, 1H, ArH), 7.86–7.91 (m, 4H, ArH), 7.96 (d, J = 7.8 Hz, 2H, ArH), 8.15–8.18
(m, 1H, ArH), 9.17 (s, 1H, NH), 14.92 (s, 1H, OHenolic). 13C-NMR (DMSO-d6, 150 MHz)
δ
: 54.0, 55.5, 111.0, 116.4, 120.0, 121.2, 121.9, 124.4, 126.4, 126.9, 127.1, 128.4, 130.7 (2C),
131.7 (2C), 132.7, 133.8, 134.3, 138.4, 149.1, 165.0, 168.0, 187.8. MS (ESI+): m/z calcd. for
C24H19BrN2O6NaS [M + Na]+ 566.99; found 564.98.
3.5. In Silico α-Glucosidase Inhibition
The Molecular Operating Environment (MOE) Ver.2014.0901 software [66–68] was
utilized for the in silico analysis of all the 1,2-benzothiazine derivatives along with the
reference acarbose as follows.
3.5.1. Ligand Preparation
Structures of all these compounds were designed with the help of ChemDraw Ultra
12.02. In order to display these files in the MOE, structures of these compounds were
saved in an MDL file (“.sdf”). The 2D structure of the reference acarbose in the SDF file
was retrieved from NCBI Pubchem. After this, 3D protonation and energy minimizations
of all these compounds along with the reference acarbose were executed under default
parameters in the MOE.
3.5.2. Protein Preparation
The 3D structure of Saccharomyces cerevisiae α-glucosidase (PDB ID: 2QMJ) was down-
loaded from the Protein Data Bank (http://www.rcsb.org/pdb; accessed on 10 October
2020). Binding interactions of the ligand (acarbose) with the protein residues were checked
(Asp203, Asp542, Asp327, His600, Arg526) and then the ligand, solvent molecules, and
unknown atoms from the receptor protein were removed. With the help of the MOE site
finder module, the active site from the 3D structure of the protein was computed. The
ligand site was found surrounded by Asp203, Tyr299, Asp327, Ile328, Asp366, Trp406,
Trp441, Asp443, Met444, Arg526, Trp539, Gly541, Asp542, Asp571, Phe575, Arg598, and
His600 residues. Later on, the site was occupied by dummy atoms and the protein was
3D-protonated and energy minimized with default parameters set in the MOE software
(Ver.2014.0901).
3.5.3. Molecular Docking
To access the binding modes of all the ligands with
α-glucosidase enzymes, ten
conformations of all the ligands along with acarbose were docked into the selected site of
enzymes with the help of the MOE dock module under the parameters fixed as, placement:
triangle matcher, rescoring: London dG, refinement: forcefield and retain: 10. Finally, 2D
and 3D maps of ligands were obtained. Binding modes of ligands were studied using rmsd
values and docking scores of their top-ranked conformations. Results of molecular docking
are depicted in Table 1, Table S1, and Figure 2, Figure 3, and Figure S1.
3.6. In Vitro α-Glucosidase Inhibition
A general spectrophotometric technique was used to perform the
tion assay with slight modifications [69]. All the compounds were dissolved in a dimethyl
sulfoxide (DMSO) solvent while -glucosidase enzymes (obtained from Saccharomyces
cerevisiae (Sigma-Aldrich, Munich, Germany) were dissolved in a phosphate buffer (pH 6.8,
100 mM). Twelve and a half microliters of each test compound (dissolved in DMSO), 40
of enzymes (0.5 U/mL), and 120 L of phosphate buffer (100 mM) were added in 96-well
microliter plates. The plates were incubated for 5 min at 37 ◦C. After incubation, 40 µL of
5 mM p-nitrophenyl- -D-glucopyranoside (Sigma Aldrich, Munich, Germany) was added
α-glucosidase inhibi-
α
µL
µ
α

Molecules 2021, 26, 3043
15 of 18
in each sample solution and incubated for a further 30 min. Afterward, absorbance of
◦
released 4-nitrophenol was measured at 405 nm and 37 C using a microplate reader.
DMSO and acarbose (Sigma-Aldrich, Munich, Germany) were used as a negative control
and reference inhibitor, respectively. The experiment was repeated thrice and inhibition
percentages were calculated using the following formula.
% Inhibition = [(A control − A sample) / A control] × 100
where ‘A’ stands for absorbance.
(1)
Finally, IC50 values were also determined for the compounds having 50% or above
inhibition values.
4. Conclusions
Novel 1,2-benzothiazine-N-arylacetamides have been discovered as inhibitors of
glucosidase enzymes for the first time. The 1,2-benzothiazine-N-arylacetamides 12a
proved better inhibitors compared to derivatives 11a . The compounds 11c 11h 11i 12a
12d 12e 12g, and 12m exhibited low IC50 values compared to acarbose (IC50 = 58.8 µM).
However, the derivatives 11c 12a 12d 12e, and 12g were envisioned as the most potent
α
-
–
m
–
m
,
,
,
,
,
,
,
,
,
inhibitors as these templates showed good docking scores, low rmsd values and significant
interactions with the targeted receptor pockets during in silico experiments. Theses deriva-
tives also showed low IC50 values of 30.65, 18.25, 20.76, 35.14, and 24.24 µM, respectively,
during in vitro α-glucosidase inhibition. We hope the present work will benefit researchers
to develop more active antidiabetic agents with no side effects.
1
Supplementary Materials: The following are available online: H-NMR, 13C-NMR, and MS spec-
tra of potent α-glucosidase inhibitors. Molecular docking 2D/3D maps (Figure S1, Table S1) of
compounds 9a, 9b, 11(a,b,d–m), and 12(b,c,f,h–m).
Author Contributions: Conceptualization, S.A., M.A., U.A.A.; methodology, F.A.S., S.T., M.H.;
synthesis, F.A.S., S.A., M.A.; characterization, F.A.S., S.A., M.A., S.S., M.H., D.S.L., M.E.A.Z.; en-
zyme inhibition studies, F.A.S., U.A.A., S.T., M.M.; manuscript writing, review and editing, F.A.S.,
M.A., M.M., D.S.L., M.E.A.Z.; supervision, M.A., M.E.A.Z. All authors have read and agreed to the
published version of the manuscript.
Funding: The research work was supported by the ‘Higher Education Commission of Pakistan’
through Research Grant NRPU-3715.
Data Availability Statement: The data presented in this study are available in the Supplemen-
tary Materials.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.
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