Synthesis, monoamine oxidase inhibition activity and molecular docking studies of novel 4-hydroxy-N′-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides

Article abstract of DOI:10.1016/j.ejmech.2017.10.036

Three series of 4-hydroxy-N^′-[benzylidene/1-phenylethylidene]-2-H/methyl/benzyl-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides (9–11)a-l were synthesized and unraveled to be highly potent dual inhibitors of monoamine oxidases (MAO-A and MAO-B). All the examined compounds demonstrated IC₅₀ values in lower micro-molar range for both MAO-A as well as MAO-B. The most active MAO-A inhibitor was 4-hydroxy-N′-(1-phenylethylidene)-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide (9i) with an IC₅₀ value of 0.11 ± 0.005 μM, whereas, methyl 4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxylate 1,1-dioxide (3) was the most active MAO-B inhibitor with an IC₅₀ value of 0.21 ± 0.01 μM. Enzyme kinetics studies revealed that the most potent compounds inhibited both MAO enzymes (A & B) in a competitive fashion. Molecular docking studies were also performed to obtain an intuitive picture of inhibition potential for potent inhibitors. The high potency of these compounds is optimally combined with highly favorable ADME profile with predicted good oral bioavailability.

Full text of DOI:10.1016/j.ejmech.2017.10.036

Accepted Manuscript
Synthesis, monoamine oxidase inhibition activity and molecular docking studies
of novel 4-hydroxy-N′-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl/-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxides
Furqan Ahmad Saddique, Sumera Zaib, Saquib Jalil, Sana Aslam, Matloob Ahmad,
Sadia Sultan, Humera Naz, Mazher Iqbal, Jamshed Iqbal
PII:
S0223-5234(17)30828-0
10.1016/j.ejmech.2017.10.036
EJMECH 9828
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 August 2017
Revised Date: 11 October 2017
Accepted Date: 12 October 2017
Please cite this article as: F.A. Saddique, S. Zaib, S. Jalil, S. Aslam, M. Ahmad, S. Sultan, H. Naz,
M. Iqbal, J. Iqbal, Synthesis, monoamine oxidase inhibition activity and molecular docking studies
of novel 4-hydroxy-N′-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl/-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxides, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.10.036.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis, monoamine oxidase inhibition activity and molecular docking studies of novel 4-
hydroxy-N'-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl/-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxides
Furqan Ahmad Saddique, Sumera Zaib, Saquib Jalil, Sana Aslam, Matloob Ahmad, Sadia Sultan,
Humera Naz, Mazher Iqbal, Jamshed Iqbal*

ACCEPTED MANUSCRIPT
Synthesis, monoamine oxidase inhibition activity and molecular docking studies of novel 4-
hydroxy-N'-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl/-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxides
Furqan Ahmad Saddiquea, Sumera Zaibb, Saquib Jalilb, Sana Aslamc, Matloob Ahmada,*, Sadia
Sultand,e, Humera Nazd,e, Mazher Iqbalf, Jamshed Iqbalb,*
aDepartment of Chemistry, Government College University, Faisalabad-38000, Pakistan.
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan.
cDepartment of Chemistry, Government College Women University, Faisalabad-38000,
Pakistan.
dFaculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor Darul Ehsan, Malaysia.
eAtta-ur-Rahman Institute for Natural Products Discovery (AuRIns) , Universiti Teknologi
MARA, Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia.
fDrug Discovery and Structural Biology Group, Health Biotechnology Division, National
Institute for Biotechnology and Genetic Engineering, Faisalabad 38000, Pakistan.
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
Three
series
of
4-hydroxy-N׳-[benzylidene/1-phenylethylidene]-2-H/methyl/benzyl-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxides (9-11)a-l were synthesized and unraveled to be
highly potent dual inhibitors of monoamine oxidases (MAO-A and MAO-B). All the examined
compounds demonstrated IC50 values in lower micro-molar range for both MAO-A as well as
MAO-B. The most active MAO-A inhibitor was 4-hydroxy-N'-(1-phenylethylidene)-2H-
benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide (9i) with an IC50 value of 0.11 ± 0.005 µM,
whereas, methyl 4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxylate 1,1-dioxide (3) was the most
active MAO-B inhibitor with an IC50 value of 0.21 ± 0.01 µM. Enzyme kinetics studies revealed

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that the most potent compounds inhibited both MAO enzymes (A & B) in a competitive fashion.
Molecular docking studies were also performed to obtain an intuitive picture of inhibition
potential for potent inhibitors. The high potency of these compounds is optimally combined with
highly favorable ADME profile with predicted good oral bioavailability.
Key words: Benzothiazine, monoamine oxidase; Molecular docking; Bioavailability
1. Introduction
Monoamine oxidases (МАО, EC 1.4.3.4) are class of enzymes bound to mitochondrial outer
membrane. MAO is a flavoprotein and belongs to a family of flavin-containing amine
oxidoreductases. The catalytic activity of monoamine oxidase depends on a co-factor flavin
adenine dinucleotide (FAD), bound covalently to a cysteine amino acid (Cys406 in MAO-A and
Cys397 in MAO-B) [1-3]. In the brain, MAO catalyzes the oxidative deamination of neuroactive
and vasoactive amines as well as the oxidation of several xenobiotics [4]. MAOs are mostly
found in the central nervous system (CNS), however their expression can be found in liver,
gastrointestinal tract and platelets. There are two types of monoamine oxidases in mammals,
MAO-A and MAO-B. Despite sharing 70% sequence identity, flavin adenine dinucleotide
(FAD)-binding site structures, MAO-A differs from MAO-B in its substrate and inhibitor
specificities, i-e., serotonin is the preferred substrate for MAO-A, whereas, dopamine for MAO-
B. While, selective inhibitor for MAO-A is clorgyline and that of MAO-B is deprenyl [5-6].
MAO-A, the primary type in fibroblasts, preferentially degrades serotonin and norepinephrine,
whereas, MAO-B, the primary type found in platelets and leukocytes, preferentially degrades
phenylethylamine and benzylamine [4]. In addition, MAO-A also demonstrates a markedly
different monopartite cavity (~550 Å), compared to bipartite cavity (290 Å) in MAO-B. When
bound to membrane, both the proteins exist in dimeric form [7].
Since, MAOs are responsible for degradation of neurotransmitters, low levels of these
neurotransmitters by virtue of increased MAO activity, have been linked with depression,
anxiety and even Parkinson’s disease [8]. Monoamine oxidase inhibitors (MAOIs) are used to
cause an increase in the level in these neurotransmitters and hence constitute an effective
therapeutic remedy against a variety of neurological and psychiatric disorders [9,10]. Some
specific and reversible inhibitors are under trial for the treatment of Parkinson’s disease.

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Therefore, MAO inhibitors are attractive drug targets for the treatment of psychiatric and
neurological disorders including Parkinson’s disease.
1,2-Benzothiazines are well recognized templates in heterocyclic chemistry due to their
diversified pharmacological applications such as sudoxicam and its structural analog, isoxicam,
having extended plasma half-life [11-12]. Similarly, piroxicam is also employed to treat
rheumatoid arthritis, osteoarthritis and other inflammations [13]. Meloxicam is another potent
1,2-benzothiazine derivative that reduces pain and stiffness in patients and shows low
gastrointestinal disorders [14,15] (Fig. 1). Many other 1,2-benzothiazine derivatives demonstrate
potent activities like hypoglycemic [16], antileukemic [17-20], antipyretic [21], antidiabetic [22],
anti-HIV, anti-HCV [23-24], 11β-HSD1 inhibitors [25], endotheline receptor antagonists [26],
antimalarial [27], anti-allergic [28], anti-thrombotic [29], central nervous system depressants
[30], antimicrobials [31], antioxidants [32] and calpain I inhibitors [33].
Fig. 1. Structures of potent 1,2-benzothiazine derivatives.
The demand of carbohydrazides for the drug development is increasing day by day due to ease of
their condensation reactions [34,35]. At present, a variety of such type of compounds has been
reported in the literature with anti-malarial [36], anti-microbial [37], anti-bacterial [38], anti-
tubercular [39], anti-fungal [40] and anti-inflammatory [41] activities. In this context, we herein
report the synthesis and pharmacological potential of various 1,2-benzothiazine-3-
carbohydrazide 1,1-dioxides as new and efficient MAO inhibitors.

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Our research group has actively been involved in design of new molecules with potent MAO
inhibitory activity [42-45]. In continuation of our efforts, three series of 1,2-benzothiazine-3-
carbohydrazide 1,1-dioxides (9-11)a-l were synthesized and evaluated for their inhibitory
potential against monoamine oxidase (MAO-A and MAO-B). All compounds exhibited excellent
MAO inhibition activity. To investigate mode of inhibition, enzyme kinetics studies were carried
out followed by detailed molecular docking studies. In order to evaluate drug likeness of these
newly synthesized molecules, in-silico ADME properties were evaluated.
2. Results and Discussion
2.1. Chemistry
Three
series
of
4-hydroxy-N׳-[benzylidene/1-phenylethylidene]-2-H/methyl/benzyl-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxides (9-11)a-l were synthesized starting from
commercially available sodium saccharin 1 which was N-alkylated with methyl chloroacetate to
afford 2. Subsequent Gabriel-Colman rearrangement of N-alkylated saccharin 2 produced methyl
4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide 3. It was observed that high yield of
ester 3 was obtained when the reaction was performed under nitrogen and in the absence of
moisture.
N-methylation of ester 3 with dimethyl sulfate provided the corresponding N-methylated product
4 while N-benzylation of methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-
dioxide 3 was carried using benzyl chloride. Reaction was performed at different temperatures
for different time intervals and finally it was observed that maximum yield (87.6%) of methyl 2-
benzyl-4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide 5 was attained at 80-100 °C
in 4 h. After the completion of reaction, addition of cold water in the reaction mixture
precipitated the desired N-benzylated product 5. Precipitates were washed with excess of water
and re-crystallized with methanol to afford colorless pure crystalline product 5. This protocol is
superior to the previously reported method [46] as it employs mild reaction conditions, cheaper
base (sodium carbonate), not moisture sensitive and provides significant yield of product.
The hydrazinolysis of esters 3-5 under refluxing conditions furnished the corresponding
hydrazides 6-8 which were effectively condensed with diversified aldehydes and ketones in the
presence of glacial acetic acid as a catalyst to achieve targeted series of 1,2-benzothiazine-3-

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carbohydrazide 1,1-dioxides (9-11)a-l (Scheme 1). Structural parameters and reaction time for
the synthesized 1,2-benzothiazine derivatives 9-11 (a-l) are shown in Table 1. All the
1
13
synthesized derivatives were well characterized with H NMR and C NMR spectroscopy and
LC-MS technique.
O
OH
O
OH
O
O
O
OMe
i
OMe
ii
OMe
NNa
iii
N
NH
S
N
S
S
O2
S
R1
O2
O2
O2
1
2
3
4: R1 = Me
5: R1 = Bn
iv
iv
OH
O
OH
O
N
R2
NH2
N
v
N
H
N
R3
H
N
S
R1
S
R1
O2
O2
9(a-l): R1 = H
10(a-l): R1 = Me
11(a-l): R1 = Bn
6: R1 = H
7: R1 = Me
8: R1 = Bn
Reaction Conditions: i. Methyl chloroacetate, DMF, 110-120 °C; ii. NaOMe/MeOH, reflux; iii. Me2SO4, aq. NaOH,
Acetone, r.t (for 4); PhCH2Cl, Na2CO3, DMF, 80 °C, 4 h (for 5); iv. N2H4.H2O, MeOH, reflux; v. aldehydes/ketones
(R2COR3), MeCO2H/MeOH, reflux.
Scheme 1. Synthesis of 4-hydroxy-N'-[benzylidene or 1-phenylethylidene]-2-H/methyl/benzyl-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxides.
Table 1. Structural parameters and reaction time for the synthesized 1,2-benzothiazine
derivatives 9-11 (a-l).
Reaction
Time (h)
Reaction
Compd.
R2
R3
Compd.
R2
R3
Time (h)
1.5
phenyl
H
H
H
1.0
1.0
1.0
2-hydroxyphenyl
3-chlorophenyl
phenyl
H
H
9a
9b
9c
10g
10h
10i
4-methoxyphenyl
4-chlorophenyl
2.0
CH3
7.5

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4-methylphenyl
4-ethoxyphenyl
4-isopropylphenyl
2-hydroxyphenyl
3-chlorophenyl
phenyl
H
H
2.0
2.0
2.0
0.5
1.0
8.0
6.5
7.0
2.0
1.0
1.5
1.5
2.0
2.0
1.0
4-chlorophenyl
4-flourophenyl
CH3
CH3
7.5
8.0
2.0
1.5
1.0
1.0
1.5
2.0
2.0
1.5
1.5
8.0
8.0
8.0
2.0
9d
9e
10j
10k
10l
H
-CH2CH2CH2CH2-
phenyl
9f
H
H
H
H
H
H
H
H
H
9g
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
4-methoxyphenyl
4-chlorophenyl
4-methylphenyl
4-ethoxyphenyl
4-isopropylphenyl
2-hydroxyphenyl
3-chlorophenyl
9h
9i
CH3
CH3
CH3
4-chlorophenyl
4-flourophenyl
9j
9k
9l
-CH2CH2CH2CH2-
phenyl
H
H
H
H
H
H
10a
10b
10c
10d
10e
10f
4-methoxyphenyl
4-chlorophenyl
4-methylphenyl
4-ethoxyphenyl
4-isopropylphenyl
phenyl
CH3
CH3
CH3
4-chlorophenyl
4-flourophenyl
11j
11k
11l
-CH2CH2CH2CH2-
2.2. Stereochemistry and X-ray crystallography
In the crystal structure of 9f (Fig. 2), two fused rings (benzene & thiazine) are twisted with a
dihedral angle of 7.07 (8)° while the thiazine ring (C8/C3/C2/C1/N1/S1) adopts half chair
conformation [nitrogen (-0.2725 (18)Å and sulfur 0.3091 (9)Å) atoms show maximum deviation
from the plane defined by the remaining atoms in the ring]. The isopropylphenyl ring (C11—
C16) is almost planar and the mean planes of thiazine and aromatic ring (C3—C8) are inclined at
17.25 (11)° and 10.65 (13)°, respectively with respect to the this ring (C11—C16).
In the crystal structure of 11h (Fig. 2), the benzene and thiazine rings are twisted with a dihedral
angle of 8.93 (11)° while the thiazine ring (C8/C3/C2/C1/N1/S1) adopts screw boat

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conformation [nitrogen 0.325(2)Å and sulfur -0.3261(13)Å) atoms show maximum deviation
from the plane defined by the remaining atoms in the ring]. The benzyl (C17—C23) and 3-
chlorophenyl rings (C11—C16) are almost planar (Table 2).
Fig. 2. Molecular structures of 9f and 11h with the atom numbering scheme. Displacement
ellipsoids are drawn at the 50 % probability level. H atoms are presented as small spheres of
arbitrary radius.

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Table 2. Crystal data and structure refinement parameters of compounds 9f and 11h.
Parameters
CCDC No.
Compound 9f
1537118
Compound 11h
1537538
Molecular Formula
Formula mass (g mol-1)
Crystal system
Space group
Unit cell dimensions
a (Å)
C19H19N3O4S.H2O
403.45
C23H18ClN3O4S
467.91
Monoclinic
P21/c
Monoclinic
C2/c
9.2159(17)
16.981(3)
b (Å)
11.097(2)
24.490(4)
c (Å)
19.427(4)
11.465(18)
α (˚)
90
90
β (˚)
92.178(6)
116.294(5)
γ(˚)
90
90
V (Å 3)
1985.4(6)
4274.7(11)
µ(Mo Kα) (mm-1)
0.192
0.313
Z
4
302(2)
8
302(2)
T (K)
Completeness
Final R indices[1 >/2σ(I)]
Largest diff, peak and hole
99.9%
99.9%
R1 = 0.0477, wR2 = 0.1177
0.426/ -0.527 e Å-3
R1 = 0.0502,wR2 = 0.1312
0.302/ -0.328 e Å-3
2.3. Biological activity
Monoamine oxidase inhibitory activity
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 synthesized compounds and reference inhibitors were summarized in Table

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3. Selectivity for MAO-A increases as the SI decreases while selectivity towards MAO-B
isoform increases as the SI increases. All compounds exhibited excellent MAO inhibition in the
lower micromolar range. By examining the MAO-A and MAO-B inhibition potencies of the
synthesized benzothiazine-3-carbohydrazide, it was revealed that the unsubstituted
benzothiazines were also potent inhibitors of MAO-B. The compounds were found to be dual
inhibitors of MAO-A and MAO-B, and were able to inhibit both enzymes efficiently. Most of
the compounds were found to be selective toward MAO-B, while few are selective towards
MAO-A.
4-Hydroxy-N'-(1-phenylethylidene)-2H-benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide (9i),
having an IC50 value of 0.11 ± 0.005 µM was found to be the most potent MAO-A inhibitor,
while the other compounds like 9a, 9g, 9h, 10b, 10c, 10d, 10e, 10f, 10h, 10i, 10k, 10l, 11f, 11h,
11i and 11j exhibited moderate inhibition potential. Similarly, compound 3 (methyl 4-hydroxy-
2H-benzo[e][1,2]thiazine-3-carboxylate 1,1-dioxide) having an IC50 value of 0.21 ± 0.01 µM
was found to be the most active MAO-B inhibitor, whereas, 2, 5, 8, 9b, 9d, 9e, 9f, 9g, 9j, 9l, 10a,
10g, 10j and 11a, 11c, 11d, 11e, 11g, 11k and 11l showed significant and comparable IC50
values.
Compound
11b,
(2-benzyl-4-hydroxy-N'-(4-methoxybenzylidene)-2H-
benzo[e][1,2]thiazine-3-carbohydrazide 1,1-dioxide) showed dual inhibition for MAO-A and
MAO-B with the IC50 values of 0.24 ± 0.007 and 0.29 ± 0.005 µM, respectively.
Structure-activity relationships
Among the three series, presence of different substituents plays an important role in determining
the potency of the inhibitors and their specificities towards MAOs. The 4-flouro-
phenylethylidene ring in compounds 11k and 9k exhibited selectivity towards MAO-B, but 10k
in the presence of same substituent was found to be more selective towards MAO-A. Similarly,
chloro group has different influence when attached at varied position on the same ring. For
example, when chloro group was attached at position 3 of the benzylidene ring, the compound
10h was found less active against any of the isoforms, but 11h and 9h showed selective
inhibition for MOA-A, while less activity for MAO-B. Similarly, for 4-chloro benzylidene ring,
11c was found selective for MAO-B, while, 10c showed selectivity for MAO-A. However, when
4-chloro phenylethylidene substituents were examined, 11j was found selective towards MAO-
A, while 9j and 10j showed selectivity for MAO-B. The presence of N'-cyclopentylidene as a

ACCEPTED MANUSCRIPT
substituent, as in 11l and 9l, selective inhibition of MAO-B was observed, whereas, 10l showed
more specificity for MAO-A. When N'-cyclopentylidene group was replaced by N-
ethyoxybenzylidene, the inhibition activity of 11e and 9e was found to be reduced, but enhanced
inhibition of 10e was noticed and exhibited selective inhibition for MAO-A. In general, different
substituents showed various trends and could lead to more effective and selective inhibitors for
both the isoforms.
Table 3. IC50 values of synthesized compounds 2-8 & 9-11(a-l) against monoamine oxidases
SIc
Codes
MAOA
MAOB
MAO Selectivity
IC50 ± SEMa (µM)
2
3
1.11 ± 0.02
2.97 ± 0.06
26.6 ± 2.79
9.51 ± 0.08
1.58 ± 0.02
0.12 ± 0.01
≥100
0.27 ± 0.008
0.21 ± 0.01
23.1 ± 1.12
0.29 ± 0.04
4.33 ± 0.06
1.50 ± 0.11
10.6 ± 0.05
9.52 ± 0.07
0.29 ± 0.005
2.08 ± 0.003
89.1 ± 2.46
5.78 ± 0.08
36.8 ± 1.58
4.24 ± 0.01
36.4 ± 1.26
2.26 ± 0.02
10.1 ± 0.02
6.04 ± 0.03
0.99 ± 0.01
8.86 ± 0.79
1.91 ± 0.03
4.11
14.1
1.15
32.8
0.36
0.08
9.43
10.5
0.83
48.1
1.12
4.89
0.13
4.41
0.02
0.54
0.03
16.5
101.0
0.21
3.02
MAO-B
MAO-B
MAO-B
MAO-B
MAO-A
MAO-A
MAO-B
MAO-B
MAO-A
MAO-B
MAO-B
MAO-B
MAO-A
MAO-B
MAO-A
MAO-A
MAO-A
MAO-B
MAO-B
MAO-A
MAO-B
4
5
6
7
8
≥100
11a
11b
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
9a
0.24 ± 0.007
≥100
≥100
28.3 ± 0.67
5.07 ± 0.02
18.7 ± 0.02
0.96 ± 0.02
1.22 ± 0.006
0.33 ± 0.01
≥100
≥100
1.91 ± 0.12
5.76 ± 0.06
9b

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0.177 ± 0.04
7.48 ± 0.12
16.9 ± 0.45
≥100
0.31 ± 0.01
0.64 ± 0.02
9.31 ± 0.15
38.1 ± 1.26
9.47 ± 0.55
39.3 ± 2.58
3.33 ± 0.32
10.9 ± 0.78
40.3 ± 2.17
0.92 ± 0.05
1.76 ± 0.21
23.1 ± 1.78
15.5 ± 0.66
24.1 ± 2.17
0.76 ± 0.03
18.2 ± 1.43
3.41 ± 0.01
38.3 ± 2.45
7.14 ± 0.11
0.76 ± 0.07
25.1 ± 2.79
8.61 ± 0.02
0.57
11.7
1.81
2.62
0.53
0.01
0.03
9.17
1.32
108.6
11.9
0.82
0.39
0.20
0.42
0.02
3.34
0.78
0.93
14.7
0.43
0.04
0.00007
3431.1
MAO-A
MAO-B
MAO-B
MAO-B
MAO-A
MAO-A
MAO-A
MAO-B
MAO-B
MAO-B
MAO-B
MAO-A
MAO-A
MAO-A
MAO-A
MAO-A
MAO-B
MAO-A
MAO-A
MAO-B
MAO-A
MAO-A
MAO-A
MAO-B
9c
9d
9e
9f
5.07 ± 0.06
0.35 ± 0.01
0.11 ± 0.005
≥100
9g
9h
9i
9j
53.4 ± 1.68
≥100
9k
9l
21.1 ± 1.67
18.9 ± 0.89
6.12 ± 0.23
4.85 ± 0.06
0.32 ± 0.01
0.44 ± 0.09
11.4 ± 0.19
29.8 ± 2.55
6.67 ± 0.04
11.2 ± 0.21
10.8 ± 0.32
0.38 ± 0.01
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
Clorgylineb
Deprenylb
0.0045 ± 0.0003
61.35 ± 1.13
67.25 ± 1.02
0.0196 ± 0.001
aAll the values are expressed as the mean ± SEM of triplicate determinations; bPositive control;
cSI = [IC50 MAO-A(µM)]/[IC50 MAO-B (µM)].
Kinetics Studies
In order to investigate the mode of inhibition, enzyme kinetics studies were carried out for most
active inhibitors of MAO-A (9i) and MAO-B (3). The results plotted in Figure 3 revealed that

ACCEPTED MANUSCRIPT
the compounds inhibited both MAO-A and MAO-B in a competitive fashion. The Lineweaver-
Burk plots of compounds 9i and 3 were shown in Figure 3.
Fig. 3. Lineweaver-Burk plots of MAO-A (left) and MAO-B (right) activity in the absence and
presence of various concentrations of potent inhibitors, 9i for MAO-A and 3 for MAO-B.
Molecular docking study
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) [47]. 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 11b (2-benzyl-4-hydroxy-N'-(4-methoxybenzylidene)-2H-benzo[e][1,2]thiazine-3-
carbohydrazide 1,1-dioxide), π‒π interactions were observed between 4-methoxybenzilidine
moiety and Tyr407 residue. The same moiety was oriented towards flavin cofactor, however, no
significant interactions were observed. The 2-benzyl part was found towards amino acid residues
Ile325 and Cys323.

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Compound 9i (4-hydroxy-N'-(1-phenylethylidene)-2H-benzo[e][1,2]thiazine-3-carbohydrazide
1,1-dioxide), showed non-covalent molecular interactions with the active site residues which are
consistent to those observed previously [48]. The phenylethylidene moiety was found towards
amino acid residues Ile325 and Cys323, whereas, benzothiazine part was deeply oriented inside
the active pocket and maybe possible reason for the potential inhibition of MAO-A. The possible
binding modes of compound 9i, 11b (dual inhibitor) and the crystallographic inhibitor harmine
were selected after Hyde assessment and visualization and shown in Fig. 4.
Fig. 4. Putative binding mode of 9i (left) and 11b (right) and the crystallographic inhibitor
harmine (blue) bound to the active site of MAO-A.
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
[1,47]. 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.
For compound 3 (methyl 4-hydroxy-2H-benzo[e][1,2]thiazine-3-carboxylate 1,1-dioxide),
carboxylate moiety having methyl group was found more towards entrance cavity whereas,

ACCEPTED MANUSCRIPT
benzothiazine part was located near Ile316 and Leu164. In 11b, (2-benzyl-4-hydroxy-N'-(4-
methoxybenzylidene)-2H-benzo[e][1,2]thiazine-3-carbohydrazide1,1-dioxide), 4-methoxybenzy-
lidene moiety was oriented towards Tyr326 and making π-π interaction, while benzothiazine part
is directed towards Ile316 and Leu164. Amino acid residues involved in electrostatic interactions
were Tyr60, Leu164, Tyr188, Gln206, Tyr435, Ile316, Tyr326 and Cys172. Additionally, π‒π
interactions were observed between 2-benzyl ring and residue Tyr398 located in hydrophilic
region, as hydrophilic region lies between Tyr398 and Tyr435 [49]. Figure 5 reports prioritized
binding poses of the most potent MAO-B inhibitor 3, 11b (dual inhibitor) and crystallographic
inhibitor safinamide which were selected after Hyde assessment and visualization.
Fig. 5. Putative binding mode of 3 (left) and11b (right) and the crystallographic inhibitor
safinamide (pink) inside the binding site of MAO-B.
In silico ADME properties
Drug-likeness and in silico ADME properties for all the tested compounds were predicted using
MedChem Designer3.0 [50]. 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 [51]. 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 [52]. Herein, we determined the octanol-water
distribution and partition coefficients (S + log D), octanol-water partition coefficient (S + log P),

ACCEPTED MANUSCRIPT
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 [53]. 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 [54-55]. 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 9-11(a-l) derivatives are given in Table 4.
Table 4. Calculated ADME properties of 2-8 & 9-11(a-l) derivatives.
Code
M
log
P
S
+ log
P
S
+ log
D
Mol. Wt.
MNO
TPSA
HBDH
-0.109
-0.004
0.287
1.625
-0.255
0.049
1.435
1.630
1.393
2.142
1.874
1.631
2.343
0.591
0.858
1.285
2.807
-0.144
0.265
1.732
3.200
3.269
3,794
3.592
3.640
4.259
0.591
-0.647
0.650
2.083
-0.968
0.039
1.286
1.313
1.376
1.910
1.718
1.784
2.644
255.250
255.250
269.277
345.376
255.253
269.280
345.379
343.363
373.389
377.808
357.390
387.416
385.444
6
6
6
6
7
7
7
7
8
7
7
8
7
80.750
92.700
0
2
1
1
5
4
4
3
3
3
3
3
3
2
3
83.910
4
83.91
5
121.520
112.730
112.73
6
7
8
107.860
117.090
107.860
107.860
117.0090
107.860
9a
9b
9c
9d
9e
9f

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1.150
2.142
1.874
2.379
2.261
1.079
1.874
1.631
2.379
2.111
1.863
2.571
1.393
2.379
2.111
2.611
2.496
1.335
3.017
2.748
3.497
3.229
2.956
3.641
2.537
3.046
3.725
3.342
3.948
3.655
2.302
3.490
3.578
4.081
3.905
3.955
4.596
3.420
3.999
3.642
4.240
3.955
2.698
4.613
4.734
5.240
5.011
5.082
5.659
4.564
1.186
1.744
1.326
1.894
1.405
0.603
2.387
2.114
2.679
2.825
2.565
3.628
2.005
2.541
2.265
2.668
2.268
1.633
2.936
2.738
3.260
3.353
3.150
4.200
2.594
359.362
377.808
357.390
391.835
375.380
321.357
357.390
387.416
391.835
371.417
401.443
399.471
373.389
391.835
371.417
405.862
389.407
335.384
433.488
463.515
467.933
447.515
477.542
475.570
449.488
8
7
7
7
7
7
7
8
7
7
8
7
8
7
7
7
7
7
7
8
7
7
8
7
8
128.090
107.860
107.860
107.860
107.860
107.860
99.070
108.300
99.070
99.070
108.300
99.070
119.300
99.070
99.070
99.070
99.070
99.070
99.070
108.300
99.070
99.070
108.300
99.070
119.300
4
3
3
3
3
3
2
2
2
2
2
2
3
2
2
2
2
2
2
2
2
2
2
2
3
9g
9h
9i
9j
9k
9l
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
11a
11b
11c
11d
11e
11f
11g

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3.497
3.229
3.704
3.601
5.169
4.760
5.383
5.108
3.126
2.925
3.254
2.864
467.933
447.515
481.960
465.506
7
7
7
7
99.070
99.070
99.070
99.070
2
2
2
2
11h
11i
11j
11k
3. Conclusion
In continuation of our efforts to design and evaluate new and effective inhibitors of MAO-A and
B, herein we synthesized three series of 4-hydroxy-N׳-[benzylidene/1-phenylethylidene]-2-
H/methyl/benzyl-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides (9-11)a-l. These compounds
were evaluated for their inhibitory potential against rat monoamine oxidase A and B (MAO-A
and MAO-B). All compounds exhibited excellent MAO inhibition activity in the lower micro-
molar range. Compounds were found to act as dual inhibitors of both MAO-A and MAO-B.
Compound 9i, having an IC50 value of 0.11±0.005 µM, was the most active MAO-A inhibitor,
whereas compound 3, with an IC50 value of 0.21±0.01 µM, was the most active MAO-B
inhibitor. Enzyme kinetics studies were carried out to investigate the mode of inhibition and it
was found that potent compounds act as competitive inhibitors for MAO-A and MAO-B. The
docking studies further unlocked the binding site interactions of the potent inhibitors. In order to
evaluate drug likeness of these newly synthesized molecules, in-silico ADME evaluation was
carried out. All compounds were found to have a favorable ADME profile, and were predicted to
have good oral bioavailability. Furthermore, the pivotal role played by this target in PD
pathogenesis suggests that potent compounds may act as promising new chemical entity in the
design of multi-target-directed-ligands.
4. Experimental
4. 1. Chemistry
4.1.1. Synthesis of methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)acetate (2)
Sodium saccharin (100 g, 488 mmol) and methyl chloroacetate (38.87 ml, 444 mmol) were
mixed in N,N-dimethylformamide (150 mL) and mixture was stirred at 110-120 °C for 3 h. After
completion of reaction, mixture was cooled to room temperature and poured into ice cooled

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water (300 mL) to precipitate the final product that was filtered, washed with cold water (× 3)
1
and finally dried. Yield: 96%; white crystalline solid; m.p 115-117 °C; H NMR (DMSO-d6) δ:
3.719 (s, 3H, OCH3), 4.653 (s, 2H, CH2), 8.027-8.057 (t, J = 7.5 Hz, 1H, ArH), 8.092-8.121 (t, J
= 7.5 Hz, 1H, ArH), 8.154-8.169 (d, J = 7.5 Hz, 1H, ArH), 8.359-8.374 (d, J = 7.5 Hz, 1H, ArH);
13C NMR: 53.05, 122.29, 125.74, 126.40, 135.91, 136.65, 137.50, 158.98 (2C), 167.28 (2C); LC-
MS (m/z): calcd: 254 [M-H]-, 256 [M+H]+; found: 254 [M-H]-, 256 [M+H]+.
4.1.2. Synthesis of methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (3)
Sodium metal (11.27 g, 490 mmol) was dissolved completely in freshly dried methanol under
reflux (150 mL) for the preparation of fresh sodium methoxide. Then, methyl (1,1-dioxido-3-
oxo-1,2-benzisothiazol-2(3H)-yl)acetate (2) (25 g, 98 mmol) was added in a single portion to the
mixture of sodium methoxide and methanol under reflux. Orange colored suspension was
produced immediately. The reaction mixture was heated for 35 minutes and then it was cooled to
room temperature. The mixture was treated with ice cold 15% HCl to precipitate completely.
These precipitates were filtered, washed with water (× 3) and dried [56]. Yield: 70 %; white
crystalline solid; m.p 172-173 °C; 1H NMR (DMSO-d6) δ: 3.893 (s, 3H, OCH3), 7.830-7.895 (m,
3H, ArH), 8.043-8.059 (d, J = 8.0 Hz, 1H, ArH), 10.072 (s, 1H, NH), 11.451 (s, 1H, OHenolic);
13C NMR: 53.26, 105.87, 122.01, 126.31, 128.24, 132.94, 133.00, 137.92, 154.40, 168.87; LC-
MS (m/z): calcd: 254 [M-H]-, 256 [M+H]+; found: 254 [M-H]-, 256 [M+H]+.
4.1.3. Synthesis of methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide
(4)
Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (3) (5.0 g, 19.6 mmol) was
added to 50 mL acetone. 8.4 mL of 20% aqueous solution of sodium hydroxide was added and
mixture was stirred at room temperature for 5 minutes. Drop wise addition of dimethyl sulfate
(7.44 mL, 78.4 mmol) was executed for 5 minutes and stirring was continued for further 35
minutes. Then, the reaction mixture was treated with 5% HCl (20 mL) to afford white colored
precipitates that were filtered, washed with water and dried. Yield: 90%; white crystalline solid;
m.p 167 °C; 1H NMR (DMSO-d6) δ: 2.862 (s, 3H, NCH3), 3.919 (s, 3H, OCH3), 7.901-7.945 (m,
13
3H, ArH), 8.054-8.073 (m, 1H, ArH); C NMR: 38.61, 53.39, 110.62, 124.08, 126.98, 127.86,
133.80, 133.82, 135.30, 157.54, 168.94; LC-MS (m/z): calcd: 268 [M-H]-, 270 [M+H]+; found:
268 [M-H]-, 270 [M+H]+.

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4.1.4. Synthesis of methyl 2-benzyl-4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide
(5)
A mixture of methyl 4-hydoxy-2H-1,2-benzothiazine-3-carboxylate1,1-dioxide (3) (5 g, 19
mmol), benzyl chloride (2.25 ml, 19 mmol) and sodium carbonate (4.15 g, 39 mmol) in DMF (30
mL) was stirred at 70 °C for 4 h. After the completion of reaction, mixture was cooled to room
temperature and cold water was added with stirring. Then, it was acidified with cold 15% HCl
and allowed to stand for 30 minutes in crushed ice mixture. Precipitates formed were filtered and
washed with excess of cold water and re-crystallized with methanol. Yield: 87.6%; colorless
1
crystals; m.p 150-152 °C (lit. m.p. 148-150 ºC [32]); H NMR (DMSO-d6) δ: 43.975 (s, 3H,
OCH3), 4.690 (br-s, 2H, CH2), 6.913-6.926 (d, J = 7.8 Hz, 2H, ArH), 6.969-6.994 (t, J = 7.5 Hz,
2H, ArH), 7.030-7.075 (m, 1H, ArH), 7.507-7.535 (td, J = 7.8, 1.2 Hz, 1H, ArH), 7.583-7.611
(td, J = 7.8, 1.2 Hz, 1H, ArH), 7.675-7.688 (d, J = 7.8 Hz, 1H, ArH), 7.766-7.779 (d, J = 7.8 Hz,
1H, ArH), 12.199 (s, 1H, OHenolic); 13C NMR: 52.80, 55.09, 106.55, 122.72, 126.21, 127.69
(2C), 127.96, 128.03, 129.41 (2C), 131.85, 132.03, 132.40, 138.09, 160.67, 169.92; LC-MS
(m/z): calcd: 344 [M-H]-, 368 [M+Na]+; found: 344.17 [M-H]-, 368.17 [M+Na]+.
4.1.5. General procedure for the synthesis of N-alkyl-4-hydroxy-2H-1,2-benzothiazine-3-
carbohydrazides 1,1-dioxides (6-8)
4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (3)/methyl 4-hydroxy-2-methyl-2H-1,2-
benzothiazine-3-carboxylate 1,1-dioxide (4)/methyl 4-hydroxy-2-benzyl-2H-1,2-benzothiazine-3-
carboxylate 1,1-dioxide (5) (20 mmol) were dissolved in methanol (100 mL) followed by the
addition of 80% hydrazine monohydrate (25 mmol). The reaction mixture was heated at reflux until
completion (monitored by TLC). The reaction mixture was concentrated under vacuum and acidified
with cold dilute HCl (5%) till turbidity or precipitates were formed. It was allowed to stand for 1 h at
5 °C. Precipitates of the corresponding carbohydrazides 6-8 formed were filtered, washed with water
and dried.
4.1.5.1. 4-Hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (6)
Yield: 94%; pale yellow crystals; m.p 292 °C decomp.; 1H NMR (DMSO-d6) δ: 5.561 (s, 2H,
NH2), 7.726-7.844 (m, 3H, ArH), 7.935-7.966 (m, 1H, ArH), 9.886 (br-s, 1H, NH), 13.927(br-s,
1H, OHenolic); 13C NMR: 105.70, 122.30, 125.76, 129.11, 131.98, 132.99, 137.67, 153.12,
166.81.; LC-MS (m/z): calcd: 254 [M-H]-, 256 [M+H]+; found: 254.17 [M-H]-, 256.17 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.5.2. 4-Hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (7)
1
Yield: 90%; light yellow crystals; m.p 222 °C. H NMR (DMSO-d6) δ: 2.734 (s, 3H, NCH3),
4.688 (br-s, 2H, NH2), 7.810-7.866 (m, 3H, ArH), 7.952-7.971 (d, J = 7.6 Hz, 1H, ArH), 10.048
(s, 1H, NH), 14.230 (s, 1H, OHenolic); 13C NMR: 110.95, 124.40 (2C), 126.36 (2C), 132.82,
133.75 (2C), 134.55, 166.35; LC-MS (m/z): calcd: 268 [M-H]-, 270 [M+H]+; found: 268.17 [M-
H]-, 270.17 [M+H]+.
4.1.5.3. 2-Benzyl-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (8)
1
Yield: 87%; light brown crystals; m.p 201-203 °C; H NMR (DMSO-d6) δ: 4.516 (br-s, 2H,
NH2), 4.739 (br-s, 2H, CH2), 6.816-6.828 (d, J = 7.2 Hz, 2H, ArH), 6.903-6.928 (t, J = 7.5 Hz,
2H, ArH), 6.984-7.008 (t, J = 7.2 Hz, 1H, ArH), 7.453-7.466 (d, J = 7.8 Hz, 1H, ArH), 7.537-
7.562 (t, J = 7.5 Hz, 1H, ArH), 7.600-7.622 (br-t, J = 6.6 Hz, 1H, ArH), 7.658-7.669 (br-d, J =
6.6 Hz, 1H, ArH), 10.179 (s, 1H, NH), 14.225 (s, 1H, OHenolic); 13C NMR: 54.57, 122.23,
125.18, 127.19 (4C), 127.85, 129.54 (2C), 131.60, 132.25, 137.05; LC-MS (m/z): calcd: 344 [M-
H]-, 346 [M+H]+, 368 [M+Na]+; found: 344.17 [M-H]-, 346.17 [M+H]+, 368.17 [M+Na]+.
4.1.6. General procedure for the synthesis of 4-hydroxy-N׳-[benzylidene or 1-
phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide series (9a-l)
4-Hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide 6 (2 mmol) was dissolved in
methanol (50 mL) followed by the addition of 2 drops of glacial acetic acid. Ketones or aldehydes (2
mmol) were added to the mixture and allowed to reflux till completion of reaction (for reaction time
see Table S1). The reaction mixture was cooled to 5 °C in an ice bath. Precipitates were filtered,
washed with cold methanol and dried.
4.1.6.1.N'-Benzylidene-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9a)
[32]
Yield: 78%; light yellow crystals; m.p. 251 °C decomp; 1H NMR (DMSO-d6) δ: 7.487-7.500 (m,
3H, ArH), 7.738-7.757 (m, 2H, ArH), 7.827-7.918 (m, 3H, ArH), 8.044-8.060 (d, J = 8.0 Hz, 1H,
ArH), 8.685 (s, 1H, NCH), 9.612 (s, 1H, SO2NH), 11.893 (s, 1H, CONH), 14.027 (s, 1H,
13
OHenolic); C NMR: 105.55, 122.48, 126.17, 127.88 (2C), 128.78, 129.39 (2C), 131.06, 132.68,
133.23, 134.44, 138.05, 151.12, 156.13, 166.16; LC-MS (m/z): calcd: 342 [M-H]-, 344 [M+H]+;
found: 342.17 [M-H]-, 344.25 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.6.2.4-Hydroxy-N'-(4-methoxybenzylidene)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-
dioxide (9b) [32]
1
Yield: 81%; yellow crystals; m.p. 250-251 °C; H NMR (DMSO-d6) δ: 3.622 (s, 3H, OCH3),
7.152-7.068(d, J = 8.4.0 Hz, 2H, ArH), 7.595-7.721 (d, J = 8.4 Hz, 2H, ArH), 7.881-7.910 (m,
1H, ArH), 7.919-7.953 (m, 2H, ArH), 8.032-8.048 (m, 1H, ArH), 8.619 (s, 1H, NCH), 9.586 (s,
1H, SO2NH), 11.651 (s, 1H, CONH), 14.310 (br-s, 1H, OHenolic); 13C NMR: 54.33, 114.44,
126.06, 126.16 (2C), 126.45, 127.78, 129.11 (3C), 132.96, 132.53, 135.13, 151.76, 158.28,
161.28, 165.71; LC-MS (m/z): calcd: 374 [M-H]-, 376 [M+H]+; found: 374.17 [M-H]-, 376.25
[M+H]+.
4.1.6.3.N'-(4-chlorobenzylidene)-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide
1,1-
dioxide (9c) [32]
1
Yield: 84%; yellow powder; m.p. 255-256 °C; H NMR (DMSO-d6) δ: 7.551-7.565 (d, J = 8.4
Hz, 2H, ArH), 7.662-7.776 (d, J = 8.4 Hz, 2H, ArH), 7.877-7.915 (m, 3H, ArH), 8.045-8.058 (d,
J = 7.8 Hz, 1H, ArH), 8.669 (s, 1H, ArH), 9.596 (br-s, 1H, SO2NH), 11.949 (s, 1H, CONH),
13
13.979 (br-s, 1H, OHenolic); C NMR: 104.99, 122.00, 125.71, 128.21, 129.00, 129.02, 129.05,
129.99, 132.26, 132.77, 132.85, 135.04, 137.52, 149.23, 155.77, 165.73; LC-MS (m/z): calcd:
376.5 [M-H]-, 378.5 [M+H]+; found: 376.5 [M-H]-, 378.5 [M+H]+.
4.1.6.4.4-Hydroxy-N'-(4-methylbenzylidene)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (9d)
Yield: 77%; light yellow powder; m.p. 247-249 °C; 1H NMR (DMSO-d6) δ: 2.368 (s, 3H, CH3),
7.301-7.314 (d, J = 7.8 Hz, 2H, ArH), 7.632-7.646 (d, J = 8.4 Hz, 2H, ArH), 7.828- 7.855 (td, J
= 7.8, 1.2 Hz, 1H, ArH), 8.040-8.054 (d, J = 7.8 Hz, H, ArH), 8.640 (s 1H, NCH), 9.604 (s, 1H,
SO2NH), 11.833 (s, 1H, CONH), 14.062 (s, 1H, OHenolic); 13C NMR: 21.05, 105.01, 121.99,
125.65, 127.38 (2C), 128.26, 129.51 (2C), 131.21, 132.18, 132.76, 137.47, 140.53, 150.62,
155.52, 165.55; LC-MS (m/z): calcd: 356 [M-H]-; found: 356.17 [M-H]-.
4.1.6.5.N'-(4-ethoxybenzylidene)-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (9e)
1
Yield: 83%; yellow powder; m.p. 244-245 °C; H NMR (DMSO-d6) δ: 1.346-1.369 (t, J = 6.9
Hz, 3H, CH3), 4.083-4.118 (q, J = 7.0 Hz, 2H, OCH2), 7.022-7.045 (dt, J = 9.0, 2.1 Hz, 2H,

ACCEPTED MANUSCRIPT
ArH), 7.660-7.684 (dt, J = 9.0, 2.4 Hz, 2H, ArH), 7.823-7.850 (td, J = 8.6, 1.8 Hz, 1H, ArH),
7.870-7.907 (m, 2H, ArH), 8.034-8.048 (d, J = 7.8 Hz, 1H, ArH), 8.601 (s, 1H, NCH), 9.589 (s,
13
1H, SO2NH), 11.756 (s, 1H, CONH), 14.098 (s, 1H, OHenolic); C NMR: 14.53, 63.31, 105.04,
114.83, 121.97, 125.61, 126.29, 128.30, 129.07 (3C), 132.11, 132.73, 137.45, 150.49, 155.35,
160.53, 165.37; LC-MS (m/z): calcd: 386 [M-H]-; found: 386.17 [M-H]-.
4.1.6.6.4-Hydroxy-N'-(4-isopropylbenzylidene)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (9f)
1
Yield: 74%; off white powder; m.p. 188-168 °C; H NMR (DMSO-d6) δ: 1.226-1.254 (d, J =
16.8 Hz, 6H, 2CH3), 2.924-2.969 (septet, J = 6.7 Hz, 1H, CH), 7.361-7.375 (d, J = 7.8 Hz, 2H,
ArH), 7.665-7.679 (d, J = 7.8 Hz, 2H, ArH), 7.829-7.914 (m, 3H, ArH), 8.045-8.058 (d, J = 7.8
Hz, 1H, ArH), 8.651 (s, 1H, NCH), 9.586 (s, 1H, SO2NH), 11.815 (s, 1H, CONH), 14.053 (s,
13
1H, OHenolic); C NMR: 23.60 (2C), 33.40, 105.01, 121.98, 125.66, 126.88 (2C), 127.50 (2C),
128.28, 131.61, 132.17, 132.74, 137.50, 150.64, 151.30, 165.54; LC-MS (m/z): calcd: 384 [M-
H]-, 408 [M+Na]+, 424 [M+K]+; found: 384.17 [M-H]-, 408.17 [M+Na]+, 424.08 [M+K]+.
4.1.6.7.4-Hydroxy-N'-(2-hydroxybenzylidene)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-
dioxide (9g) [32]
1
Yield: 79%; yellow crystals; m.p. 262-263 °C; H NMR (DMSO-d6) δ: 6.932-6.969 (m, 2H,
ArH), 7.324-7.358 (t, J = 8.5 Hz, 1H, ArH), 7.571-7.589 (d, J = 8.0 Hz, 1H, ArH), 7.831-7.924
(m, 3H, ArH), 8.049-8.065 (d, J = 8.0 Hz, 1H, ArH), 8.896 (s, 1H, NCH), 9.644 (s, 1H, SO2NH),
11.061 (s, 1H, CONH), 12.195 (s, 1H, AOH), 13.809 (s, 1H, OHenolic); 13C NMR: 105.39,
116.99, 119.15, 119.94, 122.51, 126.24, 128.70, 129.95, 132.41, 132.77, 133.26, 138.08, 151.12,
156.17, 158.13, 165.81; LC-MS (m/z): calcd: 358 [M-H]-, 360 [M+H]+; found: 358.17 [M-H]-,
360.17 [M+H]+.
4.1.6.8.N'-(3-chlorobenzylidene)-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide
1,1-
dioxide (9h) [32]
1
Yield: 83%; yellow powder; m.p. 253-254 °C; H NMR (DMSO-d6) δ: 7.506-7.557 (m, 2H,
ArH), 7.693-7.707 (d, J = 7.0 Hz, 1H, ArH), 7.785 (s, 1H, ArH), 7.785-7.899 (m, 3H, ArH),
8.032-8.047 (d, J = 7.5 Hz, 1H, ArH), 8.647 (s, 1H, NCH), 9.616 (s, 1H, SO2NH), 12.005 (s, 1H,
13
CONH), 13.865 (s, 1H, OHenolic); C NMR: 107.15, 121.18, 127.27, 127.88, 128.13, 128.68,

ACCEPTED MANUSCRIPT
129.39, 129.44, 131.16, 133.08, 133.20, 135.64, 138.55, 152.02, 157.03, 167.06; LC-MS (m/z):
calcd: 376.5 [M-H]-, 378.5 [M+H]+; found: 376.5 [M-H]-, 378.5 [M+H]+.
4.1.6.9.4-Hydroxy-N'-(1-phenylethylidene)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (9i)
1
Yield: 72%; yellow powder; m.p. 235-237 °C; H NMR (DMSO-d6) δ: 2.406 (s, 3H, CH3),
7.464-7.499 (m, 3H, ArH), 7.829-7.856 (td, J = 7.5, 1.2 Hz, 1H, ArH), 7.875-7.909 (m, 3H,
ArH), 7.918-7.932 (d, J = 7.8 Hz, 1H, ArH), 8.047-8.061 (d, J = 7.8 Hz, 1H, ArH), 9.801 (br-s,
1H, SO2NH), 10.565 (s, 1H, CONH), 13.801 (s, 1H, OHenolic); 13C NMR: 14.72, 105.55, 121.83,
125.58, 126.67, 128.30, 128.45 (2C), 130.11, 132.07, 132.70 137.25, 137.33, 154.30, 159.31,
164.91; LC-MS (m/z): calcd: 356 [M-H]-, 358 [M+H]+, 380 [M+Na]+, 396 [M+K]+; found:
356.17 [M-H]-, 358.17 [M+H]+, 380.17 [M+Na]+, 396.08 [M+K]+.
4.1.6.10.
N'-(1-(4-chlorophenyl)ethylidene)-4-hydroxy-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (9j) [43]
1
Yield: 69%; off white crystalline powder; m.p. 140-141 °C. H NMR (DMSO-d6) δ: 2.511 (s,
3H, CH3), 7.535-7.552 (d, J = 8.5 Hz, 1H, ArH), 7.764-7.924 (m, 6H, ArH), 8.041-8.056 (d, J =
7.5 Hz, 1H, ArH), 10.581 (s, 1H, SO2NH), 12.646 (br-s, 1H, CONH), 13.735 (br-s, 1H, OHenolic);
13C NMR: 15.02, 106.11, 122.31, 123.28, 123.36, 126.09, 128.81, 128.93, 129.12, 132.57,
133.00, 133.17, 135.34, 136.70, 137.78, 154.87, 165.45; LC-MS (m/z): calcd: 390.5 [M-H]-,
392.5 [M+H]+; found: 390.5 [M-H]-, 392.5 [M+H]+.
4.1.6.11.
4-Hydroxy-N'-[1-(4-flourophenyl)ethylidene]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (9k)
1
Yield: 76%; off-white powder; m.p. 251-253 °C; H NMR (DMSO-d6) δ: 2.393 (s, 3H, CH3),
7.300-7.329 (t, J = 8.7 Hz, 2H, ArH), 7.602-7.627 (t, J = 7.5 Hz, 1H, ArH), 7.853-7.913 (m, 1H,
ArH), 7.934-7.968 (m, 2H, ArH), 8.042-8.055 (d, J = 7.2 Hz, 1H, ArH), 9.672 (s, 1H, SO2NH),
10.591 (s, 1H, CONH), 13.792 (s, 1H, OHenolic); 13C NMR: 14.75, 105.56, 115.31, 115.45,
121.82, 122.88, 125.57, 128.30, 128.65, 129.05, 132.06, 132.54, 132.69, 133.82, 137.23, 154.29,
158.37, 164.92; LC-MS (m/z): calcd: 374 [M-H]-, 376 [M+H]+; found: 374.17 [M-H]-, 376.33
[M+H]+.

ACCEPTED MANUSCRIPT
4.1.6.12.
N'-cyclopentylidene-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (9l)
1
Yield: 77%; light yellow powder; m.p. 229-232 °C; H NMR (DMSO-d6) δ: 1.724-1.770
(quintet, J = 6.9 Hz, 2H, cyclo-H), 1.786-1.831 (quintet, J = 6.75 Hz, 2H, cyclo-H), 2.435-2.470
(q, J = 7 Hz, 4H, cyclo-H), 7.805-7.832 (td, J = 7.8, 1.0 Hz, 1H, ArH), 7.854-7.897 (m, 2H,
ArH), 8.005-8.020 (dd, J = 7.8, 1.2 Hz, 1H, ArH), 9.716 (s, 1H, SO2NH), 9.995 (s, 1H, CONH),
13
13.843 (s, 1H, OHenolic); C NMR: 24.17, 24.18, 28.66, 33.05 105.31, 121.79, 125.45, 128.33,
131.92, 132.65, 137.14, 153.99, 164.34, 174.36; LC-MS (m/z): calcd: 320 [M-H]-, 322 [M+H]+,
344 [M+Na]+, 360 [M+K]+; found: 320.17 [M-H]-, 322.17 [M+H]+, 344.17 [M+Na]+, 360.08
[M+K]+.
4.1.7. General procedure for the synthesis of 4-hydroxy-N׳-[benzylidene or 1-
phenylethylidene]-2-methyl-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide series (10a-l)
4-Hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide 7 (2 mmol) was dissolved
in methanol (50 mL) followed by the addition of 2 drops of glacial acetic acid. Ketones or aldehydes
(2 mmol) were added to the mixture and allowed to reflux till completion of reaction (for reaction
time see Table S1). The reaction mixture was cooled to 5 °C in an ice bath. Precipitates were
filtered, washed with cold methanol and dried.
4.1.7.2.N'-Benzylidene-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide
1,1-
dioxide (10a) [57]
1
Yield: 74%; white powder; m.p. 223-224 °C. H NMR (DMSO-d6) δ: 2.860 (s, 3H, NCH3),
7.487-7.500 (m, 3H, ArH), 7.749-7.768 (m, 2H, ArH), 7.894-7.950 (m, 3H, ArH), 8.040-8.058
(dd, J = 7.0, 2.0 Hz, 1H, ArH), 8.692 (s, 1H, NCH), 11.966 (s, 1H, CONH), 14.230 (s, 1H,
13
OHenolic); C NMR: 111.11, 124.66, 126.70, 127.89 (3C), 128.28, 129.40 (2C) 131.08, 133.52,
134.00, 134.40, 134.73, 151.42, 158.03, 165.56; LC-MS (m/z): calcd: 356 [M-H]-, 358 [M+H]+;
found: 356.17 [M-H]-, 358.25 [M+H]+.
4.1.7.3.4-Hydroxy-N'-(4-methoxybenzylidene)-2H-2-methyl-y1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (10b)
1
Yield: 86%; yellow powder; m.p. 235-236 °C; H NMR (DMSO-d6) δ: 2.848 (s, 3H, NCH3),
3.832 (s, 3H, OCH3), 7.051-7.066 (d, J = 9.0 Hz, 2H, ArH), 7.696-7.711 (d, J = 9.0 Hz, 2H,

ACCEPTED MANUSCRIPT
ArH), 7.880-7.906 (m, 1H, ArH), 7.926-7.951 (m, 2H, ArH), 8.036-8.050 (m, 1H, ArH), 8.617
(s, 1H, NCH), 11.642 (s, 1H, CONH), 14.301 (br-s, 1H, OHenolic); 13C NMR: 55.34, 110.56,
114.44, 124.16, 126.16 (2C), 126.41, 127.79, 129.08 (3C), 132.98, 133.53, 134.13, 150.77,
157.29, 161.26, 164.75; LC-MS (m/z): calcd: 386 [M-H]-, 388 [M+H]+, 410 [M+Na]+; found:
386.17 [M-H]-, 388.25 [M+H]+, 410.08 [M+Na]+.
4.1.7.1. N'-(4-chlorobenzylidene)-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxide (10c)
1
Yield: 80%; off-white powder; m.p. 252-253 °C; H NMR (DMSO-d6) δ: 2.853 (s, 3H, NCH3),
7.557-7.579 (dt, J = 7.8, 2.1 Hz, 2H, ArH), 7.770-7.792 (dt, J = 9.0, 1.9 Hz, 2H, ArH), 7.889-
7.958 (m, 3H, ArH), 8.045-8.059 (m, 1H, ArH), 8.670 (s, 1H, NCH), 12.038 (s, 1H, CONH),
13
14.181 (br-s, 1H, OHenolic); C NMR: 110.54, 124.18, 126.24 (2C), 129.01 (2C), 129.04 (3C),
132.83, 133.10, 133.56, 134.19, 135.07, 149.50, 157.62, 165.11; LC-MS (m/z): calcd: 390 [M-
H]-, 392 [M+H]+, 414 [M+Na]+; found: 390.08 [M-H]-, 392.17 [M+H]+, 414.17 [M+Na]+.
4.1.7.4.4-Hydroxy-2-methyl-N'-(4-methylbenzylidene)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxide (10d)
1
Yield: 80%; off-white powder; m.p. 237-238 °C; H NMR (DMSO-d6) δ: 2.368 (s, 3H, CH3),
2.852 (s, 3H, NCH3), 7.302-7.315 (d, J = 7.8 Hz, 2H, ArH), 7.644-7.315 (d, J = 8.4 Hz, 2H,
ArH), 7.884-7.908 (m, 1H, ArH), 7.927-7.950 (t, J = 6.9 Hz, 2H, ArH), 8.040-8.053 (d, J = 7.8
Hz, 1H, ArH), 8.645 (s, 1H, NCH), 11.910 (s, 1H, CONH), 14.268 (br-s, 1H, OHenolic); 13C
NMR: 21.06, 110.55, 124.17, 126.19, 127.38 (2C), 127.76, 129.53 (3C), 131.18, 133.02, 133.54,
134.14, 140.56, 150.90, 157.43, 164.92; LC-MS (m/z): calcd: 370 [M-H]-, 372 [M+H]+, 394
[M+Na]+, 410 [M+K]+; found: 370.17 [M-H]-, 372.17 [M+H]+, 394.17 [M+Na]+, 410.08
[M+K]+.
4.1.7.5.N'-(4-ethoxybenzylidene)-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxide (10e)
1
Yield: 70%; light yellow amorphous solid; m.p. 216-218 °C; H NMR (CDCl3) δ: 1.440-1.475
(q, J = 7.0 Hz, 3H, CH3), 2.953 (s, 3H, NCH3), 4.076-4.119 (m, 2H, OCH2), 6.930-6.949 (m, 2H,
ArH), 7.721-7.817 (m, 2H, ArH), 7.916-7.930 (d, J = 7.5 Hz, 1H, ArH), 8.084-8.098 (d, J = 7.8

ACCEPTED MANUSCRIPT
13
Hz, 1H, ArH), 8.190 (s, 1H, NCH), 9.722 (s, 1H, CONH), 13.489 (s, 1H, OHenolic); C NMR:
14.74, 40.06, 63.66, 110.64, 114.73, 114.78, 124.74, 125.49, 126.76, 128.48, 129.72, 130.12,
132.40, 133.20, 134.32, 150.11, 158.13, 161.10, 164.53; LC-MS (m/z): calcd: 400 [M-H]-, 402
[M+H]+, 424 [M+Na]+, 440 [M+K]+; found: 400.17 [M-H]-, 402.25 [M+H]+, 424.25 [M+Na]+,
440.17 [M+K]+.
4.1.7.6.4-Hydroxy-N'-(4-isopropylbenzylidene)-2-methyl-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (10f) [44]
Yield: 70%; white powder; m.p. 228-229 °C. 1H NMR (DMSO-d6) δ: 1.221-1.235 (d, J = 7.0 Hz,
6H, CH(CH3)3), 2.850 (s, 3H, NCH3), 2.914-2.990 (septet, 1H, J = 7.0 Hz, CHMe2), 7.373-7.357
(d, J = 8.0 Hz, 2H, ArH), 7.669-7.685 (d, J = 8.0 Hz, 2H, ArH), 7.875-9.942 (m, 3H, ArH),
8.038-8.053 (d, J = 7.5 Hz, 1H, ArH), 8.649 (s, 1H, NCH), 11.898 (s, 1H, CONH), 14.254 (br-s,
13
1H, OHenolic); C NMR: 24.07 (2C), 33.88, 111.10, 124.64, 126.68, 127.37 (2C), 128.01 (2C)
128.32, 132.09, 133.48, 133.99, 134.72, 151.46, 151.82, 157.97, 165.44; LC-MS (m/z): calcd:
398 [M-H]-, 400 [M+H]+; found: 398.17 [M-H]-, 400.17 [M+H]+.
4.1.7.7.4-Hydroxy-2-methyl-N'-(2-hydroxybenzylidene)-2H-benzothiazine-3-carbohydrazide
1,1-dioxide (10g)
1
Yield: 81%; light yellow powder; m.p. 256-257 °C; H NMR (DMSO-d6) δ: 2.860 (s, 3H,
NCH3), 6.933-6.959 (m, 2H, ArH), 7.327-7.355 (t, J = 8.4 Hz, 1H, ArH), 7.597-7.612 (dd, J =
7.5, 1.5 Hz, 1H, ArH), 7.900-7.953 (m, 3H, ArH), 8.046-8.060 (m, 1H, ArH), 8.899 (s, 1H,
13
NCH), 11.018 (s, 1H, CONH), 12.217 (s, 1H, ArOH), 14.010 (br-s, 1H, OHenolic); C NMR:
110.43, 116.46, 118.70, 119.46, 124.19, 126.26, 129.12, 131.96, 133.56, 134.18, 150.45, 157.46,
157.58, 158.61, 162.76, 164.67; LC-MS (m/z): cacld: 372 [M-H]-, 396 [M+Na]+, 412 [M+K]+;
found: 372.08 [M-H]-, 396.08 [M+Na]+, 412.00 [M+K]+.
4.1.7.8.N'-(3-chlorobenzylidene)-4-hydroxy-2methyl-2H-benzothiazine-3-carbohydrazide 1,1-
dioxide (10h)
1
Yield: 82%; off-white powder; m.p. 246-248 °C; H NMR (DMSO-d6) δ: 2.856 (s, 3H, NCH3),
7.515-7.563 (m, 2H, ArH), 7.707-7.723 (dt, J = 7.5, 1.2 Hz, 1H, ArH), 7.796 (s, 1H, ArH),
7.891-7.918 (m 1H, ArH), 7.931-7.958 (m 1H, ArH), 8.046-8.060 (m, 1H, ArH), 8.659 (s, 1H,
13
NCH), 12.115 (s, 1H, CONH), 14.153 (br-s, 1H, OHenolic); C NMR: 110.52, 124.20, 126.16,

ACCEPTED MANUSCRIPT
126.26, 126.48, 127.67, 130.22, 130.85 (2C), 133.15, 133.58, 133.70, 134.17, 136.11, 149.08,
157.71, 165.23; LC-MS (m/z): calcd: 390 [M-H]-, 392 [M+H]+, 414 [M+Na]+, 430 [M+K]+;
found: 390.00 [M-H]-, 392.17 [M+H]+, 414.17 [M+Na]+, 430.08 [M+K]+.
4.1.7.9.4-Hydroxy-2-methyl-N'-(1-phenylethylidene)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxide (10i)
1
Yield: 70%; off-white powder; m.p. 189-191 °C; H NMR (DMSO-d6) δ: 2.406 (s, 3H, CH3),
2.909 (s, 3H, NCH3), 7.473-7.504 (dd, J = 12.9, 5.7 Hz, 2H, ArH), 7.886-7.946 (m, 5H, ArH),
8.038-8.051 (d, J = 7.8, 1H, ArH), 10.987 (s, 1H, CONH); 13C NMR: 15.39, 26.67, 110.91,
124.13, 126.00, 126.16, 126.73, 128.46, 128.65, 130.25, 132.87, 133.47, 134.12, 137.27, 156.95,
161.87, 164.49; LC-MS (m/z): calcd: 370 [M-H]-, 372 [M+H]+, 394 [M+Na]+, 410 [M+K]+;
found: 370.17 [M-H]-, 372.17 [M+H]+, 394.17 [M+Na]+, 410.08 [M+K]+.
4.1.7.10.
N'-(1-(4-chlorophenyl)ethylidene)-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (10j) [58]
1
Yield: 71%; light yellow powder; m.p. 240-242 °C. H NMR (DMSO-d6) δ: 2.393 (s, 3H,
CCH3), 2.901 (s, 3H, NCH3), 7.535-7.552 (d, J = 8.5 Hz, 2H, ArH), 7.870-7.943 (m, 5H, ArH),
8.033-8.048 (d, J = 7.5 Hz, 1H, ArH), 10.972 (s, 1H, CONH), 13.234 (s, 1H, OHenolic); 13C
NMR: 15.66, 111.45, 124.60, 126.69 (2C), 129.00 (6C), 133.37, 133.93, 134.69 135.49, 136.64,
157.60, 165.05; LC-MS (m/z): calcd: 404.5 [M-H]-, 406.5 [M+H]+; found: 404.5 [M-H]-, 406.5
[M+H]+.
4.1.7.11.
4-Hydroxy-N'-[1-(4-flourophenyl)ethylidene]-2H-2-methyl-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (10k)
Yield: 75%; light yellow crystals; m.p. 222-224 °C; 1H NMR (DMSO-d6) δ: 2.397 (s, 3H, CH3),
2.904 (s, 3H, NCH3), 7.305-7.334 (t, J = 8.7 Hz, 2H, ArH), 7.875-7.902 (dt, J = 6.9, 1.5Hz, 1H,
ArH), 7.919-7.946 (t, J = 7.5, 2H, ArH), 8.034- 8.048 (dd, J = 6.9, 2.1 Hz, 1H, ArH), 10.991 (br-
s, 1H, CONH); 13C NMR: 15.41, 110.89, 115.33, 115.47, 124.13, 126.16, 127.98, 129.09,
129.14, 132.89, 133.47, 133.76, 133.77, 134.12, 156.98, 162.56, 164.20, 164.51; LC-MS (m/z):
calcd: 380 [M-H]-, 390 [M+H]+, 412 [M+Na]+, 428 [M+K]+; found: 380.08 [M-H]-, 390.25
[M+H]+, 412.17 [M+Na]+, 428.17 [M+K]+.

ACCEPTED MANUSCRIPT
4.1.7.12.
N'-cyclopentylidene-4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (10l)
Yield: 73%; light brown powder; m.p. 188-190 °C; 1H NMR (DMSO-d6) δ: 1.764-1.803 (m, 4H,
cyclo-H), 2.453-2.476 (t, J = 6.9 Hz, 4H, cyclo-H), 2.856 (s, 3H, NCH3), 7.856-7.880 (t, J = 7.2
Hz, 1H, ArH), 7.905-7.929 (m, 2H, ArH), 8.001-8.015 (m, 1H, ArH), 10.481 (s, 1H, CONH),
14.221 (s, 1H, OHenolic); 13C NMR: 22.67, 24.16, 24.22, 29.30, 33.12, 37.75, 110.68, 124.05
126.01(2C), 132.75, 133.43, 134.03, 156.57, 163.89; LC-MS (m/z): calcd: 334 [M-H]-, 336
[M+H]+, 358 [M+Na]+, 374 [M+K]+; found: 334.17 [M-H]-, 336.17 [M+H]+, 358.17 [M+Na]+,
374.08 [M+K]+.
4.1.8. General procedure for the synthesis of 4-hydroxy-N׳-[benzylidene or 1-
phenylethylidene]-2-benzyl-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide series (11a-l)
2-Benzyl-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide 8 (2 mmol) was dissolved
in methanol (50 mL) followed by the addition of 2 drops of glacial acetic acid. Ketones or aldehydes
(2 mmol) were added to the mixture and allowed to reflux till completion of reaction (for reaction
time see Table S1). The reaction mixture was cooled to 5 °C in an ice bath. Precipitates were
filtered, washed with cold methanol and dried.
4.1.8.2.2-Benzyl-N'-benzylidene-4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxide (11a)
Yield: 71%; yellow crystals; m.p. 219-221 °C; 1H NMR (CDCl3) δ: 4.351-4.375 (d, J = 14.4 Hz,
1H, CH2), 4.691-4.715 (d, J = 14.4 Hz, 1H, CH2), 7.169-7.189 (m, 3H, ArH), 7.250-7.266 (q, J =
3.2 Hz, 2H, ArH), 7.406-7.446 (m, 3H, ArH), 7.610 (s, 1H, NCH), 7.620-7.665 (m, 2H, ArH),
7.669-7.715 (dd, J = 7.8, 1.8 Hz, 2H, ArH), 7.828-7.854 (td, J = 6.9, 2.0 Hz, 2H, ArH), 9.243 (s,
13
1H, CONH), 13.556 (s, 1H, OHenolic); C NMR: 57.83, 108.27, 123.71, 126.60, 127.90 (2C),
128.51 (2C), 128.58, 128.62, 128.76 (2C), 129.90 (3C), 130.96, 132.05, 132.70, 132.95, 136.83,
149.57, 159.95, 164.90; LC-MS (m/z): calcd: 432 [M-H]-, 434 [M+H]+, 456 [M+Na]+, 472
[M+K]+; found: 432.17 [M-H]-, 434.25 [M+H]+, 456.17 [M+Na]+, 472.17 [M+K]+.
4.1.8.3.2-Benzyl-4-hydroxy-N'-(4-methoxybenzylidene)-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (11b)