Hybrid compounds from chalcone and 1,2-benzothiazine pharmacophores as selective inhibitors of alkaline phosphatase isozymes

Article abstract of DOI:10.1016/j.ejmech.2018.09.063

Chalcones and 1,2-benzothiazines are two important classes of bioactive compounds, each scaffold endowed with diverse pharmacological activities. Combining both of these pharmacophores in a single molecule was aimed to yield multi-modal agents. Herein, we report a series of hybrid compounds 3a–3o derived from chalcones and 1,2-benzothiazine cores. They were synthesized from commercially available sodium saccharin, and the resulting 1,2-benzothiazine-derived ketone was then condensed with aromatic aldehydes in an aldol condensation to obtain the respective chalcones. The compounds were characterized using different analytical techniques including FT-IR, NMR spectroscopy, mass spectrometry and X-ray crystallography. Some synthesized chalcones revealed potent and/or selective inhibitory properties towards alkaline phosphatase isozymes transiently expressed in COS-7 cells. A detailed structure-activity and selectivity study was carried out with regard to the effect of different substituents at ortho-, meta- and para-positions of the phenyl residue. Compound 3c was the most effective human intestinal alkaline phosphatase (h-IAP) inhibitor (IC₅₀ value of 1.04 μM), while it was not active against human tissue non-specific alkaline phosphatase (h-TNAP) isozyme. In contrast, 3i was a selective inhibitor of h-TNAP with IC₅₀ values of 0.25 ± 0.01 μM. The possible binding interactions of the most effective inhibitors of h-TNAP and h-IAP were obtained from molecular docking studies.

Full text of DOI:10.1016/j.ejmech.2018.09.063

Accepted Manuscript
Hybrid compounds from chalcone and 1,2-benzothiazine pharmacophores as
selective inhibitors of alkaline phosphatase isozymes
Adnan Ashraf, Syeda Abida Ejaz, Shafiq Ur Rahman, Waseeq Ahmad Siddiqui,
Muhammad Nadeem Arshad, Joanna Lecka, Jean Sévigny, Mohie E. Moustafa
Zayed, Abdullah M. Asiri, Jamshed Iqbal, Christian G. Hartinger, Muhammad Hanif
PII:
S0223-5234(18)30841-9
10.1016/j.ejmech.2018.09.063
EJMECH 10774
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2018
Revised Date: 17 September 2018
Accepted Date: 25 September 2018
Please cite this article as: A. Ashraf, S.A. Ejaz, S.U. Rahman, W.A. Siddiqui, M.N. Arshad, J.
Lecka, J. Sévigny, M.E. Moustafa Zayed, A.M. Asiri, J. Iqbal, C.G. Hartinger, M. Hanif, Hybrid
compounds from chalcone and 1,2-benzothiazine pharmacophores as selective inhibitors of alkaline
phosphatase isozymes, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.063.
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ACCEPTED MANUSCRIPT
Graphical Abstract

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Hybrid Compounds from Chalcone and 1,2-Benzothiazine
Pharmacophores as Selective Inhibitors of Alkaline Phosphatase
Isozymes
Adnan Ashraf,a,b Syeda Abida Ejaz,c Shafiq Ur Rahman,c Waseeq Ahmad Siddiqui,b*
Muhammad Nadeem Arshad,d,e Joanna Lecka,f,g Jean Sévigny,f,g Mohie E. Moustafa
Zayed,d,e Abdullah M. Asiri,d,e Jamshed Iqbal,c Christian G. Hartinger,a Muhammad
Hanifa*
a School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland
1142, New Zealand
b Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan
c Centre for Advanced Drug Research, COMSATS University of Islamabad,
Abbottabad Campus, Abbottabad 22060, Pakistan
d Department of Chemistry, Faculty of Science, King Abdul Aziz University, P.O. Box
80203, Jeddah 21589, Saudi Arabia
e Centre of Excellence for Advanced Materials Research (CEAMR), King Abdul Aziz
University, P.O.Box 80203, Jeddah 21589, Saudi Arabia
f Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine,
Université Laval, Québec, QC, G1V 0A6, Canada
g Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, G1V
4G2, Canada
* School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland
1142, New Zealand.
E-mail: m.hanif@auckland.ac.nz; Fax: (+64)9 373 7599 ext 86833
Department of Chemistry, University of Sargodha, Sargodha 40100, Pakistan.
Email: waseeq.ahmad@uos.edu.pk
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ABSTRACT
Chalcones and 1,2-benzothiazines are two important classes of bioactive
compounds, each scaffold endowed with diverse pharmacological activities.
Combining both of these pharmacophores in a single molecule was aimed to yield
multi-modal agents. Herein, we report a series of hybrid compounds 3a–3o derived
from chalcones and 1,2-benzothiazine cores. They were synthesized from
commercially available sodium saccharin, and the resulting 1,2-benzothiazine-
derived ketone was then condensed with aromatic aldehydes in an aldol
condensation to obtain the respective chalcones. The compounds were
characterized using different analytical techniques including FT-IR, NMR
spectroscopy, mass spectrometry and X-ray crystallography. Some synthesized
chalcones revealed potent and/or selective inhibitory properties towards alkaline
phosphatase isozymes transiently expressed in COS-7 cells. A detailed structure-
activity and selectivity study was carried out with regard to the effect of different
substituents at ortho-, meta- and para-positions of the phenyl residue. Compound 3c
was the most effective human intestinal alkaline phosphatase (h-IAP) inhibitor (IC50
value of 1.04 ꢀM), while it was not active against human tissue non-specific alkaline
phosphatase (h-TNAP) isozyme. In contrast, 3i was a selective inhibitor of h-TNAP
with IC50 values of 0.25 ± 0.01 µM. The possible binding interactions of the most
effective inhibitors of h-TNAP and h-IAP were obtained from molecular docking
studies.
Keywords
Chalcone; 1,2-benzothiazine; enzyme inhibition; alkaline phosphatase; aldol
condensation.
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INTRODUCTION
Alkaline phosphatases (APs, E.C. 3.1.3.1) are membrane-bound metalloenzymes
which have an active site facing the extracellular space [1]. They hydrolyze
phosphate monoesters and are involved in several cellular events including protein
phosphorylation, cell growth, and apoptosis. Based on their tissue distribution, APs
are classified into four isozymes; tissue specific alkaline phosphatase including
placental (PLAP), germ cell (GCAP), intestinal (IAP) and tissue non-specific alkaline
phosphatase (TNAP) [2-4]. TNAP is expressed in various tissues through all the
stages of development. In bone tissue, it is associated with the hydrolysis of
pyrophosphate. By maintaining an optimum level of pyrophosphate, TNAP maintains
proper bone mineralization [5]. According to recent reports, TNAP overexpression is
observed in renal and bone diseases [1]. Among tissue-specific APs, IAP is
responsible for regulating bicarbonate secretion, detoxification of bacterial
lipopolysaccharides, lipid intestinal absorption as well as the maintenance of the pH
value at the duodenal surface [6].
APs have been reported to be overexpressed in solid and metastasized tumors
including breast, esophageal, liver, colon, intestinal, prostate and ovarian cancer [7].
The elevated level of TNAP and IAP in many cancers and other pathologies makes
them an interesting molecular target in drug discovery [8, 9]. There is an increasing
interest in the design of effective and selective inhibitors of AP isozymes [10]. In
recent years, different inhibitors of APs based on diaryl sulfonamides, coumarin
sulfonates, triazole, chalcones and chromones have been reported [11-16]. Some
compounds were found to be effective inhibitors of TNAP and IAP but most of them
inhibited APs non-selectively [11-15].
With the aim to find selective inhibitors of APs, we report the synthesis of a series of
compounds based on two pharmaceutically-active scaffolds, namely chalcones and
benzothiazines. The chalcone scaffold is considered a privileged structure and
represents the key structural motif in a plethora of bioactive synthetic and natural
compounds, widely distributed across fruits, vegetables, and other plants [17].
Chalcones are the precursors in the biosynthesis of flavonoids in plants and the core
of many biologically-active hybrid natural compounds such as coumarin-chalcone
hybrids [18]. Chalcones and their derivatives have pharmacological properties such
as anticancer, antioxidative, antibacterial, antiviral, insecticidal, antiprotozoal,
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antiulcer and immunosuppressive activity [17, 19, 20], and several examples have
been approved for clinical use (Chart 1). They exert their cytotoxic effects through a
number of mechanisms, such as inhibition of receptor tyrosine kinases including the
epidermal growth factor receptor (EGFR) [21], and the vascular endothelial growth
factor receptor 2 (VEGFR-2) [21, 22]. They have also been shown to interfere with
the nuclear factor NF-κB signaling pathway [23], tubulin polymerization [24],
thioredoxin reductase (TrxR) and dihydrofolate reductase (DHFR) [25, 26].
Benzothiazines represent another interesting scaffold. Their structures feature a non-
aromatic heterocyclic core and an all-carbon aromatic fragment [27]. They are key
components of important pharmaceuticals such as anti-inflammatory drugs (Chart 1)
[28, 29], and have been extensively studied for biological applications including as
antibacterial [30], antiviral [31, 32], antifungal [33], antioxidant [34, 35], and antitumor
agents [36, 37]. Several recent studies highlighted their role as potent inhibitors of
11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) [38], calpain I [39], and
aldose reductase [40-42]. These diverse biological applications of 1,2-
benzothiazines have attracted an increasing interest in the structural modification of
this scaffold [43-46].
Chart 1. Chemical structures of the clinical approved chalcone-based drugs metochalcone
and sofalcone, and the 1,2-benzothiazine-derived anti-inflammatory agents piroxicam and
meloxicam.
In our continued interest in developing 1,2-benzothiazine and sulfonamide
heterocyclic compounds [29, 37, 47], we report a series of hybrid compounds based
on the 1,2-benzothiazine core and a chalcone scaffold to harvest the biological
properties of both motifs. Several compounds revealed to be either potent and/or
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specific inhibitor of APs. This evaluation was complemented with molecular docking
studies of the molecules with the highest potential for specificity and potency to
further understand the mechanisms of their interaction with the AP enzymes.
Results and discussion
Synthesis and characterization
We prepared compounds 5a–5o starting from methyl 4-hydroxy-2H,1,2-
benzothiazine-3-carboxylate 1,1-dioxide 3, which was obtained from sodium
saccharin 1 in a reaction with methyl chloroacetate under mild conditions. A Gabriel–
Colman type ring expansion converted the 5-membered isothiazole ring in 2 to a 6-
membered thiazine ring to give 3. Hydrolysis and decarboxylation of 3 in refluxing
concentrated hydrochloric acid yielded 1,1-dioxo-2H-1,2-benzothiazine-4-one 4 [48-
51]. An aldol condensation of 4 with the respective aldehydes was carried out in
alkaline solution at ambient temperature. After completion of the reaction, the
mixture was acidified which resulted in the formation of yellow precipitates. The
solids formed were recrystallized from methanol to obtain the pure compounds 5a–
5o.The preparation of 5a has been reported by Zinnes and co-workers under
relatively harsh conditions using sodium borohydride or sodium hydride at
temperatures as low as -20 °C [48, 49].
The compounds were characterized by FT-IR and NMR spectroscopy, high
resolution mass spectrometry and X-rays crystallographic techniques. In the IR
spectra of 5a–5o, the NH signal of the thiazine ring appeared in the range of 3230–
3078 cm-1 while this signal was present at 3269 cm-1 for precursor 4. The α,β-
unsaturated carbonyl stretching vibrations were detected in the range of 1689–1651
cm-1. A new alkene group (C=C) stretching frequency appeared in the range of
1600–1581 cm-1, indicative of the formation of the desired product. The sulfonyl
group asymmetric and symmetric stretching frequencies appeared in the range of
1375–1290 cm-1 and 1174–1156 cm-1, respectively.
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Scheme 1. Synthesis of the hybrid compounds 5a–5o based on the 1,2-benzothiazine and
chalcone scaffolds; i) ClCH2COOCH3, DMF, reflux; ii) NaOMe, MeOH, 60 °C; iii) HCl, re flux;
iv) NaOH, MeOH/H2O, 25 °C.
1
The H NMR spectra of 5a–5o revealed a characteristic singlet for the vinylic
hydrogen which appeared between 7.77 and 7.62 ppm and was assigned to H-11.
The CH2 signal at 4.29 ppm in precursor ketone 4 was absent in 5a–5o. The most
downfield signal was a singlet in the range 11.03–10.48 ppm which was assigned to
H-2 (NH). The proton signals for the benzothiazine aromatic ring were not impacted
by the introduction of substituents R1–R4. The aromatic proton signals from the
benzaldehyde fragment (H-13 to H-17) however appeared between 8.19 and 6.92
ppm. These signals were shifted in the presence of different electron-donating and -
withdrawing groups attached to the aromatic ring system.
In the 13C{1H} NMR spectra, the most deshielded carbon atoms C-4 and C-3 of
benzothiazine were found around 180 and 141 ppm in all compounds. The detection
of C-3 as quaternary carbon indicated the formation of the chalcones.
The chemical shift of the C-11 signals depends upon the substituents attached to the
ring. Electron donating groups, including methoxy, methyl and hydroxyl attached to
the benzaldehyde ring in 5b–5d, 5n and 5o, caused an upfield shift of C-11, whereas
electron withdrawing fluoro, chloro and bromo in compounds 5d–5m led to a
deshielding effect. In 5k, 5l and 5m, the fluorine couple with neighbouring carbon
atoms, with coupling constant 1J(C,F) = 252 Hz in case of 5l (Figure S27 and S29).
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The synthesized compounds were also analyzed by high resolution ESI-MS in
positive ion mode. All mass spectra featured the pseudomolecular [M + H]+ or [M +
Na]+ ions (100%) with very well matched calculated and observed m/z values.
The molecular structures of 5d, 5h, and 5n were determined by X-ray diffraction
analysis and all three compounds demonstrated (Z)-configuration. The
crystallographic data is provided in Table S1. The tetrahedral geometry found for the
sulfur atom was distorted with O=S=O angles of about 120°(Figure 1 and S1) [52-
54]. The substituted aromatic rings are oriented at dihedral angles of 10.832(1)°,
18.832(6)° and 13.128(5)° with respect to the thiaz ine ring in 5h, 5n and 5d,
respectively. The puckering parameters for the rings in all three molecules were
determined, and the thiazine rings were found to be non-planar.
Figure 1. Molecular structure of 5d drawn at 50% probability level. Intermolecular H bonds
are shown between the hydrogen of NH in position 2 and the oxygen of the methoxy group.
Intermolecular hydrogen bonding was observed in all three molecules (Figure 1 and
S1). In 5d intermolecular hydrogen bonds were formed between the NH hydrogen
and O4 of the methoxy group with N1-H···O4 distances of 2.242 Å (Figure 2). The N-
H···O interaction causes formation of an extended network of H bond-bridged
molecules (Figure 1). While in 5h and 5n intermolecular hydrogen bonds were
present between the N1H hydrogen and carbonyl oxygen O3 with N1H···O3
distances of 2.074(3) and 2.000(3) Å, respectively (Figure 1 and S1). In 5h and 5n,
N1H···O interactions connect the molecules to generate chain structures. The bond
lengths and bond angles were in the normal range (Figure 2 and S1) [52-54]. In all
three structures the C=O bond lengths were between 1.229(3) and 1.217(13) Å,
while the C=C distances were in the range of 1.342(3)-1.316(17) Å.
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Enzyme inhibitory activity
The 1,2-benzothiazine-based chalcones 5a–5o were evaluated for alkaline
phosphatase inhibitory activity against selected isozymes of alkaline phosphatase,
i.e., h-TNAP and h-IAP. The introduction of substituents on the phenyl ring of 5a
resulted in promising inhibitory effects on the selected isozymes of alkaline
phosphatase; h-TNAP and h-IAP. It appeared that para-substituted derivatives
exhibited more promising inhibitory effects on h-TNAP while the ortho- and meta-
substituted derivatives exhibited more selective inhibition of h-IAP. Derivative 5a,
having an unsubstituted phenyl ring, showed inhibition of h-TNAP with an IC50 value
of 0.66 µM (Table 1). The most potent inhibitor of h-TNAP was 5i bearing chloro
group at para position with an IC50 value of 0.25 µM. It exhibited approximately 80
fold greater inhibition compared to the standard reference compound levamisole
(IC50 = 20.2 µM) [55].
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Table 1. In vitro alkaline phosphatase (h-TNAP and h-IAP) inhibitory activities of 5a–
5o expressed as IC50 (µM) and their cytotoxic activities against HeLa cervical
carcinoma cells after treatment with 100 ꢀM of the compound.
h-TNAP
h-IAP
selectivity index [a] % growth inhibition
Compounds
IC50[b] / µM
5a
0.66 ± 0.02
2.59 ± 0.22
> 200 [b]
1.78 ± 0.22
1.24 ± 0.42
1.04 ± 0.11
1.53 ± 0.11
7.43 ± 0.26
2.55 ± 0.72
3.69 ± 0.51
3.77 ± 0.38
4.35 ± 0.39
4.96 ± 0.31
9.93 ± 1.03
3.06 ± 0.35
10.6 ± 1.2
3.75 ± 0.38
1.21 ± 0.13
> 200 [c]
0.4
2.1
19 ± 2
17 ± 0.3
39 ± 3
11 ± 2
53 ± 1
65 ± 1
59 ± 3
64 ± 1
55 ± 3
59 ± 4
53 ± 2
41 ± 6
17 ± 1
7.0 ± 0.9
15 ± 2
–
5b
5c
> 190
2.6
5d
4.04 ± 0.21
> 200 [b]
> 200 [b]
5e
> 1
5f
> 26
0.4
5g
1.43 ± 0.13
> 200 [b]
5h
> 50
0.1
5i
0.25 ± 0.01
> 200 [b]
5j
> 53
0.3
5k
3.19 ± 0.74
1.35 ± 0.11
1.53 ± 0.34
2.36 ± 0.19
3.57 ± 0.24
20.2 ± 1.9
> 200 [c]
5l
0.4
5m
0.1
5n
0.6
5o
3.0
levamisole
< 0.1
> 2
L
-phenylalanine
100 ± 3
–
cisplatin
–
–
–
89 ± 2
[a] a selectivity index > 1 indicates selectivity for h-IAP and < 1 for h-TNAP; [b] mean ± SEM (Standard
error mean; n = 3); [c] highest conc. used.
The substitution of a fluoro with a chloro atom at para position in 5l, resulted in
relatively reduced inhibitory effects (IC50 = 1.35 µM) on h-TNAP as compared to 5i,
but it was still more selective for h-TNAP. The derivatives 5c, 5e, 5f, 5h and 5j
exhibited less than 50% inhibitory activity against h-TNAP isozyme at the highest
used concentration of 200 µM and therefore these derivatives were not selected for
the evaluation of their IC50 values.
All compounds showed moderate to excellent inhibitory effects on h-IAP depending
on the type and position of the attached substituent. The derivatives having electron
donating group (5b, 5c, 5d and 5o) exhibited greater inhibitory potential as
compared to other derivatives with unsubstituted phenyl ring 5a or carrying electron
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withdrawing group substitution. Compound 5c (IC50 = 1.04 ± 0.11 µM) exhibited the
highest selectivity and strongest inhibitory effects on h-IAP, while 5i was the most
potent and selective h-TNAP inhibitor of the series of compounds investigated.
For the investigation of the kinetics of the enzyme inhibition of the most potent
inhibitors 5c against h-IAP and 5i against h-TNAP, double reciprocal plots of initial
velocity and substrate CDP-Star® concentrations were prepared. The enzyme kinetic
studies were performed for 5i and 5c at different compound concentrations (Figures
2 and S3). The selective inhibitor of h-IAP, i.e., 5c, exhibited competitive inhibition of
this isozyme as the intercept of the curves is on the y-axis for uninhibited and
inhibited enzymes (Figure 2). In contrast, the selective h-TNAP inhibitor 5i exhibited
non-competitive inhibition (Figure S3).
Figure 2. Double reciprocal plot of the inhibition kinetics of h-IAP by the most potent
compound 5c, showing competitive inhibition.
Molecular Docking Studies
Molecular docking studies were carried out to identify plausible binding interactions
of the most potent derivatives against h-IAP and h-TNAP, i.e., 5c and 5i,
respectively, with the modeled structures of the respective isozymes. For validation
of the docking studies, the standard inhibitors levamisole and L-phenylalanine, which
were also used in the inhibition assays for h-TNAP and h-IAP, respectively, were
docked in the active pockets of the enzymes.
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Docking studies of 5c into the active pocket of h-IAP revealed several interactions
(Figure 3). One of the oxygen atoms of the sulfur dioxide group was involved in H
bonds with Arg166 and His432 and the sulfur atom is involved in a π-interaction with
His432, while the other oxygen atom demonstrated binding to the zinc(II) ion within
the active site of h-IAP (Figure 3). The chalcone carbonyl oxygen formed two H
bonds with Arg166 and Gln108. The benzene ring adjacent to the thiazine ring was
involved in a π-interaction with Thr431. Notably, when docking 5i into h-IAP, different
interactions were observed. These included coordination of the sulfoxide oxygen
atoms to Zn and π-interactions with His320 as well as between chlorophenyl group
and His432 and Phe107, while no strong hydrogen bonds were observed
(Figure S4).
Figure 3. Docking study of 5c into the active site of h-IAP. Hydrogen bonds and π-
interactions between compound and amino acid residues are shown as dashed lines.
The docking of 5i with h-TNAP revealed H bond formation between the chalcone
oxygen atom and His154 and Arg151, while π-interactions were formed between the
condensed aromatic system of 5i and His321 (Figure 4). His324 was involved in a π-
interaction with the sulfur atom and an oxygen of the sulfur dioxide moiety
coordinated to the Zn in the active site (Figure 4). Compound 5c formed very
different interactions with h-TNAP involving interactions between an SO2 oxygen
atom and the two adjacent His residues, and π-interactions with Arg167 and His321
(Figure S5), while H bonds were absent.
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Figure 4. Docking study of 5i into the active site of h-TNAP. Hydrogen bonds and π-
interactions between compound and amino acid residues are shown as dashed lines.
In vitro cytotoxicity against HeLa cervical carcinoma cells
The cytotoxic activity of 5a–5o against cervical carcinoma cells HeLa was evaluated
by means of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay. The percentage inhibition of all the derivatives was investigated at 100 µM
(Table 1). Cisplatin was used as a positive control and it showed a 89% growth
inhibition at the same concentration level (100 µM). As cisplatin is known to be highly
cytotoxic, it is not surprising that most of the derivatives showed lower
antiproliferative activity towards the cancer cells than cisplatin. Compounds 5e–5k
were the most cytotoxic derivatives with 53 to 65% growth inhibition of HeLa cells. In
contrast, 5a, 5b, 5d and 5m–5o exhibited very low toxicity towards HeLa cells.
Conclusions
A series of heterocyclic compounds 5a–5o based on chalcone and 1,2-
benzothiazine scaffolds was developed by condensation of different aldehydes with
the 1,2-benzothiazine core. All compounds were evaluated for their inhibitory
potential against the alkaline phosphatase isoforms h-TNAP and h-IAP and the latter
was more responsive to inhibition by the compound class. The positioning and
nature of the functional groups impacted the activity as well as the isozyme
selectivity of the compounds. All the para-substituted compounds showed activity in
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the low ꢀM concentration range against both isoforms but with limited selectivity. The
notable exception is the p-Cl derivative 5i which was the most potent inhibitor of h-
TNAP at 0.25 ꢀM and a selective index of 0.1. For the meta-substituted compounds
5c, 5f, and 5h high selectivity for h-IAP was observed with 5c being most potent
(1.04 ꢀM). Molecular docking studies revealed different interaction modes for
compounds 5c and 5i within the active site of the two AP isoforms for h-IAP and h-
TNAP, respectively. While the compounds interacted with the Zn ions in both
isoforms, they lacked the formation of H bonds with the isoform they showed lower
activity in. Therefore, these observations are in line with the data collected in the
enzyme inhibition studies and support the high selectivity of 5c for h-IAP over h-
TNAP and vice versa for 5i.
Conflicts of interest
There are no conflicts to declare.
Acknowledgments
We thank the Higher Education Commission of Pakistan for research project NRPU
20-1582 and indigenous/IRSIP scholarship (A.A.). J. I. is thankful to the Organization
for the Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands and
Higher Education Commission of Pakistan for the financial support through Project
No. 20–3733/NRPU/R&D/14/520. J.S. received support from the Canadian Institutes
of Health Research (CIHR) and was the recipient of a “Chercheur National” research
award from the Fonds de recherche du Québec – Santé (FRQS).
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EXPERIMENTAL SECTION
Materials and Methods
All the reactions were carried out at ambient temperature. All the chemicals were of
analytical grade and used without further purification. Food grade sodium saccharin
(cooko) was purchased from a local market. Chloroacetic acid, sodium,
benzaldehyde, 2-methoxy benzaldehyde, 3-methoxy benzaldehyde, 4-methoxy
benzaldehyde, 2-bromo benzaldehyde, 3-bromo benzaldehyde, 4-chloro
benzaldehyde, 2,4-dichloro benzaldehyde, 2- methyl benzaldehyde and 2-hydroxy
benzaldehyde were purchased from Scharlau (United Kingdom) and 2-bromo
benzaldehyde, 4-bromo benzaldehyde, 3-chloro benzaldehyde, 3-fluoro
benzaldehyde, 4-fluoro benzadehyde and 2,6-difluro benzaldehyde were purchased
from Sigma-Aldrich (United Kingdom). 4-Hydroxy-1,1-dioxo-1,2-dihydro-1λ6-
benzo[e][1,2]thiazine-3-carboxylic acid methyl ester 3 and 1,1- dioxo-2,3-dihydro-1H-
1λ6-benzo[e][1,2]thiazin-4-one 4 were prepared following reported procedures [48,
49, 56, 57].
1H and 13C{1H} (DEPTQ) NMR spectra were recorded on a Bruker Avance AVIII 400
MHz NMR spectrometers at ambient temperature. High resolution mass spectra data
recorded on a Bruker micrOTOF-Q II mass spectrometer in positive electrospray
ionization (ESI) mode. Thin layer chromatography (TLC) was performed on
aluminum sheets pre-coated with Merck silica gel 60 F254 and detection was
achieved under ultraviolet (UV) light.
General Procedure for the Synthesis of 5a–5o
Compound 4 (5.00 g, 25.4 mmol) was dissolved in methanol (50 mL) and aqueous
NaOH (3.04 g, 76.1 mmol) was added to get a brownish yellow solution. Then a
methanolic solution of the corresponding aldehyde was added dropwise and stirred
at room temperature for 2 h. After completion of the reaction as monitored by TLC,
acidified the reaction mixture with 10% HCl to get the yellowish precipitates, which
was filtered, washed with water (3 × 25 mL) and recrystallised from methanol to
obtain pure 5a-5o [48, 49].
(Z)-3-Benzylidene-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-one, 5a
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Compound 5a was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and benzaldehyde (1.55 mL, 15.21 mmol). Yield: 68% (2.95 g, light
yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H11NNaO3S 308.0357, obs:
308.0347. FT-IR (KBr, cm-1): 3099 (NH), 1651 (C=O), 1570 (C=C), 1492 (NHdef.),
1446 (CHdef.), 1319, 1172 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.72
(s, 1H, H-2), 8.15-8.13 (m, 1H, H-5), 8.03-8.01 (m, 2H, H-13, H-17), 7.96-7.88 (m,
3H, H-6, H-8, H-7), 7.67 (s, 1H, H-11), 7.55-7.48 (m, 3H, H-14, H-15, H-16) ppm.
13C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 180.0 (C-4), 141.5 (C-3), 134.7
(C-6), 133.4 (C-7), 132.7 (C-12), 132.3 (C-11), 131.6 (C-10), 131.5 (C-14, C-16),
130.9 (C-15), 130.4 (C-9), 129.0 (C-5), 128.9 (C-13, C-17), 122.16 (C-8) ppm.
(Z)-3-(2-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5b
Compound 5b was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 2-methoxybenzaldehyde (1.84 mL, 15.21 mmol). Yield: 73% (3.50
g, light orange powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C16H13NNaO4S
338.0463, obs: 338.0496. FT-IR (KBr, cm-1): 3230 (NH), 1670 (C=O), 1585 (C=C),
1463 (CHdef.), 1290, 1168 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.61
3
4
(s, 1H, H-2), 8.19 (dd, J(H17,H16) = 8 Hz, J(H17,H15) = 2 Hz, 1H, H-17), 8.14-8.12 (m,
1H, H-5), 7.94-7.87 (m, 4H, H-11, H-6, H-8, H-7), 7.52-7.47 (m, 1H, H-15), 7.15-7.09
(m, 2H, H-16, H-14), 3.89 (s, 1H, H-18) ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO,
25 °C): δ = 180.0 (C-4), 158.7 (C-13), 141.5 (C-3), 134.7 (C-6), 133.4 (C-7), 132.7
(C-15), 131.3 (C-10), 130.5 (C-9), 130.3 (C-17), 129.0 (C-5), 126.2 (C-16), 122.1 (C-
8), 121.0 (C-12), 120.5 (C-11), 111.7 (C-13), 55.9 (C-18) ppm.
(Z)-3-(3-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5c
Compound 5c was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 3-methoxybenzaldehyde (1.86 mL, 15.21 mmol). Yield: 67% (3.21
g, light yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C16H13NNaO4S
338.0463, obs: 338.0458. FT-IR (KBr, cm-1): 3128 (NH), 1658 (C=O), 1589 (C=C),
1454 (CHdef.), 1303, 1166 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.74
(s, 1H, H-2), 8.15-8.12 (m, 1H, H-5), 7.97-7.88 (m, 3H, H-6, H-8, H-7), 7.64 (s, 1H,
3
H-11), 7.60-7.59 (m, 2H, H-13, H-17), 7.10 (t, J(H16,H17) = 8Hz, 1H, H-16), 7.10-7.08
15

ACCEPTED MANUSCRIPT
(m, 1H, H-15), 3.82 (s, 1H, H-18) ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO, 25
°C): δ = 180.0 (C-4), 159.3 (C-14), 141.3 (C-3), 134.7 (C-6), 133.8 (C-12), 133.4 (C-
7), 132.1 (C-16), 131.7 (C-10), 130.4 (C-9), 129.9 (C-17), 129.0 (C-5), 123.9 (C-11),
122.1 (C-8), 116.7 (C-15), 116.5 (C-13), 55.3 (C-18) ppm.
(Z)-3-(4-Methoxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5d
Compound 5d was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 4-methoxybenzaldehyde (1.85 mL, 15.21 mmol). Yield: 70% (3.36
g, light yellow crystalline solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C16H13NNaO4S
338.0463, obs: 338.0458. FT-IR (KBr, cm-1): 3196 (NH), 1664 (C=O), 1575 (C=C),
1508 (CHdef.), 1309, 1172 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.48
(s, 1H, H-2), 8.14-8.11 (m, 1H, H-5), 8.05-8.02 (m, 2H, H-13, H-17), 7.94-7.86 (m,
3H, H-6, H-8, H-7), 7.66 (s, 1H, H-11), 7.11-7.09 (m, 2H, H-14, H-16), 3.85 (s, 1H, H-
13
18) ppm. C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.8 (C-4), 161.5 (C-
15), 141.5 (C-3), 134.5 (C-6), 133.8 (C-13, C-17), 134.0 (C-7), 133.3 (C-11), 130.6
(C-9), 129.5 (C-10), 128.9 (C-5), 125.3 (C-12), 122.2 (C-8), 114.5 (C-14, C-16), 55.5
(C-18) ppm.
(Z)-3-(2-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5e
Compound 5e was synthesized following general procedure using 4 (3.00 g, 15.21
mmol) and 2-bromobenzaldehyde (1.78 mL, 15.21 mmol). Yield: 66% (3.66 g, light
yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10BrNNaO3S 385.9462,
obs: 385.9458. FT-IR (KBr, cm-1): 3156 (NH), 1638 (C=O), 1594 (C=C), 1435
1
(CHdef.), 1313, 1156 (SO2). H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.91 (s,
1H, H-2), 8.16-8.11 (m, 2H, H-5, H-14), 7.99-7.89 (m, 3H, H-6, H-8, H-7), 7.79 (dd,
4
3J(H17,H16) = 8 Hz, J(H17,H15) = 1 Hz, 1H, H-17), 7.71 (s, 1H, H-11), 7.59-7.55 (m, 1H,
3
4
H-16), 7.41 (td, J(H15,H16)/(H15,H14) = 8 Hz, J(H15,H17) = 2 Hz, 1H, H-15) ppm. 13C{1H}
NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.9 (C-4), 141.4 (C-3), 135.0 (C-12),
133.5 (C-6), 133.3 (C-7), 133.2 (C-14), 132.3 (C-10), 131.9 (C-15), 131.3 (C-17),
130.2 (C-9), 129.1 (C-5), 128.6 (C-11), 128.0 (C-16), 125.7(C-13), 122.1 (C-8) ppm.
16

ACCEPTED MANUSCRIPT
(Z)-3-(3-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5f
Compound 5f was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 3-bromobenzaldehyde (1.77 mL, 15.21 mmol). Yield: 64% (3.55 g,
yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10BrNNaO3S 385.9462,
obs: 385.9454. FT-IR (KBr, cm-1): 3186 (NH), 1660 (C=O), 1579 (C=C), 1469
1
(CHdef.), 1298, 1165 (SO2). H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.89 (s,
4
1H, H-2), 8.21 (t, 1H, J(H13,H15)/(H13,H17) = 2 Hz, H-13), 8.14-8.12 (m, 1H, H-5), 7.99-
7.89 (m, 4H, H-17, H-8, H-6, H-7), 7.70-7.68 (m, 1H, H-15), 7.62 (s, 1H, H-11), 7.50-
7.46 (m, 1H, H-16) ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.8
(C-4), 141.3 (C-3), 134.9 (C-12), 134.9 (C-6), 133.5 (C-7), 133.3 (C-13), 133.2 (C-
15), 132.6 (C-10), 130.9 (C-17), 130.4 (C-16), 130.3 (C-9), 129.9 (C-11), 129.1 (C-
5), 122.2 (C-8), 122.0 (C-14) ppm.
(Z)-3-(4-Bromo-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5g
Compound 5g was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 4-bromobenzaldehyde (2.82 g, 15.21 mmol). Yield: 69% (3.82 g,
light yellow solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10BrNNaO3S 385.9462,
obs: 385.9457. FT-IR (KBr, cm-1): 3059 (NH), 1655 (C=O), 1580 (C=C), 1485
1
(CHdef.), 1320, 1174 (SO2). H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.76 (s,
1H, H-2), 8.14-8.12 (m, 1H, H-5), 7.95-7.88 (m, 5H, H-13, H-17, H-6, H-8, H-7), 7.75-
7.73 (m, 2H, H-14, H-16), 7.62 (s, 1H, H-11) ppm. 13C{1H} NMR (100.61 MHz,
[d6]DMSO, 25 °C): δ = 179.9 (C-4), 141.3 (C-3), 134.8 (C-6), 133.5 (C-7), 133.2 (C-
14, C-16), 132.1 (C-10), 132.0 (C-13, C-17), 131.9 (C-12), 130.6 (C-11), 130.4 (C-9),
129.0 (C-5), 124.4 (C-15), 122.2 (C-8) ppm.
(Z)-3-(3-Chloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5h
Compound 5h was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 3-chlorobenzaldehyde (2.14 g, 15.21 mmol). Yield: 73% (3.65 g,
light yellow crystaline solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10ClNNaO3S
341.9967, obs: 341.9962. FT-IR (KBr, cm-1): 3167 (NH), 1655 (C=O), 1578 (C=C),
1444 (CHdef.), 1375, 1166 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.87
17

ACCEPTED MANUSCRIPT
(s, 1H, H-2), 8.13 (d, 3J(H5,H6) = 8 Hz, 1H, H-5), 8.08 (s, 1H, H-13), 7.97-7.89 (m, 4H,
H-17, H-6, H-8, H-7), 7.63 (s, 1H, H-11), 7.56-7.55 (m, 2H, H-15, H-16) ppm. 13C{1H}
NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.8 (C-4), 141.3 (C-3), 134.9 (C-6),
134.7 (C-12), 133.5 (C-7), 133.4 (C-14), 132.6 (C-10), 130.7 (C-11), 130.03 (C-13),
130.4 (C-15), 130.2 (C-9), 130.2 (C-16), 130.1 (C-17), 129.0 (C-5), 122.2 (C-8) ppm.
(Z)-3-(4-Chloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5i
Compound 5i was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 4-chlorobenzaldehyde (2.14 g, 15.21 mmol). Yield: 71% (3.45 g,
yellow crystaline solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10ClNNaO3S
341.9967, obs: 341.9959. FT-IR (KBr, cm-1): 3078 (NH), 1654 (C=O), 1577 (C=C),
1489 (NHdef.), 1400 (CHdef.), 1300, 1161 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25
°C): δ = 10.76 (s, 1H, H-2), 8.14-8.12 (m, 1H, H-5), 8.04-8.02 (m, 2H, H-13, H-17),
7.96-7.89 (m, 3H, H-6, H-8, H-7), 7.65 (s, 1H, H-11), 7.62-7.59 (m, 2H, H-14, H-16)
ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.9 (C-4), 141.4 (C-3),
135.4 (C-15), 134.8 (C-6), 133.5 (C-7), 133.1 (C-14, C-16), 132.0 (C-12), 131.6 (C-
10), 130.5 (C-11), 130.4 (C-9), 129.0 (C-11), 129.0 (C-13, C-17), 122.3 (C-8) ppm.
(Z)-3-(2,4-Dichloro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5j
Compound 5j was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 2,4-dichlorobenzaldehyde (2.7 g, 15.21 mmol). Yield: 67% (3.61 g,
yellow crystaline solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H9Cl2NNaO3S
375.9578, obs: 375.9461. FT-IR (KBr): 3163 (NH), 1689 (C=O), 1600 (C=C), 1465
(NHdef.), 1392 (CHdef.), 1303, 1161 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ
= 10.98 (s, 1H, H-2), 8.17-8.13 (m, 2H, H-5, H-17), 7.99-7.89 (m, 3H, H-6,H-8, H-7),
4
7.81 (d, J(H14,H16) = 2 Hz, 1H, H-14), 7.67-7.64 (m, 2H, H-11, H-16) ppm. 13C{1H}
NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.7 (C-4), 141.3 (C-3), 135.6 (C-15),
135.3 (C-13), 135.1 (C-6), 133.9 (C-12), 133.5 (C-7), 132.2 (C-11), 130.1 (C-10),
129.7 (C-9), 129.5 (C-17), 129.1 (C-5), 127.8 (C-16), 124.2 (C-14), 122.1 (C-8) ppm.
(Z)-3-(3-Fluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5k
18

ACCEPTED MANUSCRIPT
Compound 5k was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 3-fluorobenzaldehyde (1.89 mL, 15.21 mmol). Yield: 85% (3.92 g,
brownish yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10FNNaO3S
326.0263, obs: 326.0257. FT-IR (KBr, cm-1): 3116 (NH), 1685 (C=O), 1590 (C=C),
1448 (CHdef.), 1372, 1160 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.85
(s, 1H, H-2), 8.15-8.13 (m, 1H, H-5), 7.96-7.82 (m, 4H, H-6, H-8, H-7, H-13, H-17),
7.65 (s, 1H, H-11), 7.59-7.54 (m, 1H, H-16), 7.38-7.32 (m, 1H, H-15) ppm. 13C{1H}
NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.9 (C-4), 162.2 (d, 1J(C,F) = 243 Hz, C-
14), 141.4 (C-3), 134.9 (C-6), 134.8 (C-10), 133.5 (C-7), 132.5 (C-12), 130.8 (d,
4
3J(C,F) = 8 Hz, C-16), 130.3 (C-9), 130.2 (C-17), 129.1 (C-5), 127.8 (d, J(C,F) = 2 Hz,
2
2
C-11), 122.2 (C-8), 117.4 (d, J(C,F) = 23 Hz, C-15), 117.2 (d, J(C,F) = 23 Hz, C-13)
ppm.
(Z)-3-(4-Fluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5l
Compound 5l was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 4-fluorobenzaldehyde (1.63 mL, 15.21 mmol). Yield: 75% (3.42 g,
light yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H10FNNaO3S 326.0263,
obs: 326.0259. FT-IR (KBr, cm-1): 3187 (NH), 1661 (C=O), 1577 (C=C), 1443
1
(CHdef.), 1320, 1153 (SO2). H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.69 (s,
1H, H-2), 8.14-8.09 (m, 3H, H-5, H-13, H-17), 7.96-7.88 (m, 3H, H-6,H-8, H-7), 7.68
(s, 1H, H-11), 7.41-7.36 (m, 2H, H-14, H-16) ppm. 13C{1H} NMR (100.61 MHz,
1
[d6]DMSO, 25 °C): δ = 179.9 (C-4), 163.2 (d, J(C,F) = 252 Hz, C-15), 141.4 (C-3),
134.7 (C-6), 134.1 (C-13/C-17), 134.0 (C-13/C-17), 133.5 (C-7), 131.2 (C-10), 131.1
(C-11), 130.4 (C-9), 129.3 (C-12), 129.0 (C-5), 122.2 (C-8), 116.2 (C-14/C-16), 115.9
(C-14/C-16) ppm.
(Z)-3-(2,6-Difluoro-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5m
Compound 5m was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 2,6-difluorobenzaldehyde (1.64 mL, 15.21 mmol). Yield: 76% (3.71
g, light yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H9F2NNaO3S
344.0169, obs: 344.0163. FT-IR (KBr, cm-1): 3085 (NH), 1655 (C=O), 1571 (C=C),
1490 (NHdef.), 1446 (CHdef.), 1319, 1170 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25
19

ACCEPTED MANUSCRIPT
°C): δ = 11.03 (s, 1H, H-2), 8.14 (dd, 3J(H5,H6) = 7 Hz, 4J(H5,H7) = 2 Hz, 1H, H-5), 7.98-
7.88 (m, 3H, H-6, H-8, H-7), 7.79-7.52 (m, 1H, H-15), 7.25-7.19 (m, 3H, H-11, H-14,
H-16) ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 179.0 (C-4), 161.2 (d,
3J(C,F) = 7 Hz, C-13/C-17), 158.8 (d, 3J(C,F) = 7 Hz, C-13/C-17), 141.6 (C-3), 135.9 (C-
10), 135.0 (C-6), 133.3 (C-7), 132.1 (d, 2J(C,F) = 11 Hz, C-15), 130.1 (C-9), 129.1 (C-
5), 121.6 (C-8), 114.4 (C-11), 112.1 (C-14), 111.9 (C-16), 130.4 (t, 2J(C,F) = 19 Hz, C-
12) ppm.
(Z)-3-(2-Methyl-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5n
Compound 5n was synthesized following the general procedure using 4 (3.00 g,
15.21 mmol) and 2-methylbenzaldehyde (1.76 mL, 15.12 mmol). Yield: 76% (3.46 g,
light yellow crystaline solid). MS (ESI+): m/z [M+Na]+ = Calcd. for C16H13NNaO3S
322.0514, obs: 322.0504. FT-IR (KBr, cm-1): 3176 (NH), 1666 (C=O), 1575 (C=C),
1458 (CHdef.), 1301, 1165 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ = 10.66
(s, 1H, H-2), 8.14 (dd, 3J(H5,H6) = 8 Hz, 4J(H5,H7) = 1 Hz, 1H, H-5), 8.0-7.88 (m, 4H, H-
17, H-6, H-8, H-7), 7.77 (s, 1H, H-11), 7.37-7.32 (m, 3H, H-15, H-16, H-14), 2.39 (s,
1H, H-18) ppm. 13C{1H} NMR (100.61 MHz, [d6]DMSO, 25 °C): δ = 180.1 (C-4),
141.5 (C-3), 138.9 (C-13), 134.8 (C-6), 133.4 (C-7), 132.1 (C-12), 131.5 (C-10),
130.6 (C-11), 130.4 (C-9), 130.2 (C-14), 129.6 (C-17), 129.4 (C-15), 129.0 (C-5),
126.3 (C-16), 122.1 (C-8), 19.7 (C-18) ppm.
(Z)-3-(3-Hydroxy-benzylidene)-1,1-dioxo-2,3-dihydro-1H-1λ6-benzo[e][1,2]thiazin-4-
one, 5o
Compound 5o was synthesized the following general procedure using 4 (3.00 g,
15.21 mmol) and 3-hydroxybenzaldehyde (1.86 g, 15.21 mmol). Yield: 63% (2.89 g,
light yellow powder). MS (ESI+): m/z [M+Na]+ = Calcd. for C15H11NNaO4S 324.0306,
obs: 324.0309. FT-IR (KBr, cm-1): 3163 (NH), 3105-2725 (OH), 1637 (C=O), 1581
(C=C), 1450 (CHdef.), 1336, 1174 (SO2). 1H NMR (400.13 MHz, [d6]DMSO, 25 °C): δ
= 10.64 (s, 1H, H-2), 9.75 (s, 1H, H-18), 8.12 (d, 3J(H5,H6)= 8 Hz, 1H, H-5), 7.97-7.87
(m, 3H, H-6, H-8, H-7), 7.55-7.53 (s, 2H, H-11, H-13), 7.38-7.29 (m, 2H, H-15, H-16),
3
4
13
6.91 (dd, J(H17,H16) = 8 Hz, J(H17,H15) = 1 Hz, 1H, C-17) ppm. C{1H} NMR (100.61
MHz, [d6]DMSO, 25 °C): δ = 180.0 (C-4), 156.5 (C-14), 141.5 (C-3), 134.7 (C-6),
20

ACCEPTED MANUSCRIPT
133.8 (C-12), 133.4 (C-7), 132.4 (C-11), 131.4 (C-10), 130.5 (C-9), 129.9 (C-16),
129.0 (C-5), 123.2 (C-17), 122.2 (C-8), 118.3 (C-13), 117.4 (C-15)ppm.
Cell transfection and preparation of membrane fractions
Plasmids expressing human alkaline phosphatases (h-TNAP & h-IAP) were
transfected into COS-7 cells using lipofectamine following a published protocol [58,
59]. COS-7 cells at a confluence of 80-90% were incubated with 6 µg of plasmid
DNA and 24 µL of lipofectamine reagent for 5 h at 37 °C in Dulbecco’s modified
Eagle’s medium (DMEM) without fetal bovine serum (FBS). Transfection was
stopped by adding the same volume of DMEM/F-12 containing 20% FBS. The cells
were harvested 40 to 72 h later and were processed as reported [58]. Total protein
was estimated using the Bradford test and different aliquotes of the protein were
prepared with the addition of 7.5% glycerol and stored at -80 °C [60].
Inhibition of alkaline phosphatases h-TNAP and h-IAP
The inhibitory effect of the synthesized derivatives on h-TNAP and h-IAP was
determined at concentrations of up to 200 µM, using a method reported previously
[13, 61]. The solutions were prepared in buffer containing 3 M DEA (pH 9.8), 2.5 mM
MgCl2 and 0.05 mM ZnCl2. The compound samples (0.2 mM) were prepared in an
aqueous solution with a final DMSO content of 1% (v/v) and 10 µL of that solution
was mixed with 20 µL of a solution of h-TNAP (46 ng/well) or h-IAP (57 ng/well) cell
lysates in 384-well plates. The luminescence signals were recorded with a
microplate reader (BioTek FLx800, Instruments, Inc. USA) after incubating the plates
at 37 °C for 5–10 min. Then, CDP-star® substrate (2 0 µL; disodium 2-chloro-5-(4-
methoxyspiro[1,2-dioxetane-3,2′-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl
phosphate) was added and the luminescence was again measured after 15–20 min
of incubation. The data was evaluated with PRISM 5.0 (GraphPad, San Diego,
California, USA) software and the inhibitory concentration values at 50% inhibition
(IC50 values) were obtained [61].
Molecular docking studies
21

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The binding interactions of the most effective derivatives 5c and 5i within the
respective enzymes i.e., h-IAP and h-TNAP were studied with molecular docking
using modelled structures [13, 62]. For docking studies the chemical structure of
potent compounds were 3D optimized using ACD/Chem Sketch software. Energy
minimization and protonation of targeted proteins structures were carried out by
using Molecular Operating Environment (MOE 2014.009) software [63], as discussed
previously [13]. After the docking calculations, the configurations with the lowest
binding free energy were selected and further analyzed with Discovery Studio
Visualizer to determine their probable binding modes within the active site of the
target enzymes.
Cell Viability Assay (MTT Assay)
The antiproliferative effect of 5a–5o was investigated against HeLa cells with the
MTT assay following a reported protocol [64, 65]. Briefly, 90 ꢀL of the cell culture
medium containing 2.5 × 104 cells/mL was seeded overnight in 96-well plates. Then,
10 ꢀL (final concentration of 100 µM) of the test compound was added, followed by
an incubation for 24 h at 37 °C. Cisplatin (final c oncentration of 100 µM) was used as
reference drug while medium was used as the negative control. After 24 h, 10 ꢀL of
MTT reagent (final concentration of 5 ꢀg/well) was added to each well and incubated
for 4 h at 37 °C. The MTT activity on the cells was stopped by adding 100 µL/well of
an aqueous solution of 50% isopropanol and 10% sodium dodecyl sulfate, followed
by gentle agitation (for 30 minutes) at room temperature. The optical density was
measured using a microplate reader (Bio-Tek ELx 800TM, Instruments, Inc. Winooski,
VT, USA) at 570 nm along with the background at 690 nm. All experiments were
performed in triplicate. Results reported are means of three independent
experiments (± SEM) and expressed as percent inhibitions calculated by the
equation
ꢈꢃꢉꢅꢊꢃꢈꢁꢋꢌ ꢅꢍ ꢄℎꢌ ꢄꢌꢉꢄ ꢋꢅꢎꢏꢅꢐꢁꢑꢉ
ꢆ ꢇ
ꢀꢁℎꢂꢃꢂꢄꢂꢅꢁ % = 100 –
× 100
ꢈꢃꢉꢅꢊꢃꢈꢁꢋꢌ ꢅꢍ ꢄℎꢌ ꢋꢅꢁꢄꢊꢅꢒ
22

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