Synthesis of novel 1,2-benzothiazine 1,1-dioxide-3-ethanone oxime N-aryl acetamide ether derivatives as potent anti-inflammatory agents and inhibitors of monocyte-to-macrophage transformation

Article abstract of DOI:10.1016/j.ejmech.2013.12.053

A series of novel 1,2-benzothiazine 1,1-dioxide-3-ethanone oxime N-aryl acetamide ether derivatives 7a-h and 9a-h were synthesized starting from sodium salt of saccharin 1 in series of steps. Final compounds 7a-h and 9a-h were evaluated for the anti-inflammatory activity and their ability to inhibit monocyte-to-macrophage transformation. Compounds 7e, 9b, 9e and 9h showed impressive anti-inflammatory activities (TNF-α, IL-8 and MCP-1) at micro molar concentration which was found to be better than positive control i.e., piroxicam. Compound 9e marginally and compound 9h significantly inhibited PMA-induced MMP-9 gelatinase activity. Also compounds 9e and 9h greatly inhibited the PMA-induced monocyte-to-macrophage transformation, a pre-requisite step in the formation of atheroma.

Full text of DOI:10.1016/j.ejmech.2013.12.053

European Journal of Medicinal Chemistry 75 (2014) 143e150
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 1,2-benzothiazine 1,1-dioxide-3-ethanone oxime
N-aryl acetamide ether derivatives as potent anti-inflammatory agents
and inhibitors of monocyte-to-macrophage transformation
a
b
a
Malla Reddy Gannarapu , Sathish Babu Vasamsetti , Nagender Punna ,
a
a
c
Naresh Kumar Royya , Shanthan Rao Pamulaparthy , Jagadeesh Babu Nanubolu ,
Srigiridhar Kotamraju , Narsaiah Banda
Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
Centre for X-ray Crystallography, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500607, India
b,
a,
*
*
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 August 2013
Received in revised form
A series of novel 1,2-benzothiazine 1,1-dioxide-3-ethanone oxime N-aryl acetamide ether derivatives 7a
eh and 9aeh were synthesized starting from sodium salt of saccharin 1 in series of steps. Final com-
pounds 7aeh and 9aeh were evaluated for the anti-inflammatory activity and their ability to inhibit
monocyte-to-macrophage transformation. Compounds 7e, 9b, 9e and 9h showed impressive anti-
18 November 2013
Accepted 25 December 2013
Available online 8 January 2014
inflammatory activities (TNF-a, IL-8 and MCP-1) at micro molar concentration which was found to be
better than positive control i.e., piroxicam. Compound 9e marginally and compound 9h significantly
inhibited PMA-induced MMP-9 gelatinase activity. Also compounds 9e and 9h greatly inhibited the PMA-
induced monocyte-to-macrophage transformation, a pre-requisite step in the formation of atheroma.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
N-aryl acetamide
Oxime ether
Anti-inflammatory activity
PMA-phorbol 12-myristate 13-acetate
1. Introduction
potential antigenicity and low tissue penetrating capability.
Though several efforts have been made on synthesis of new mol-
Inflammation is a multi-factorial process involved in many
pathological disorders. In response to infection stimulus, endo-
thelial activation and metabolic disorders like diabetes myeloid
lineage cells activate and generate an inflammatory environment
by secreting many pro-inflammatory cytokines like interleukins,
ecules to inhibit/regulate TNF-a, no small molecules have been
approved as reliable inhibitors till now.
The human chemokine Interleukin-8 (IL-8) in particular, a
member of the CXC chemokine family produced by stimulated
monocytes [8], activates adhesion molecules expression on endo-
thelial cells [9] and also serves as an important activator and che-
moattractant for neutrophils [10], as well as T-cells [11]. Monocyte
chemoattractant protein-1 (MCP-1) levels are known to be elevated
in various disorders including rheumatoid arthritis, insulin-
resistant diabetes etc. [12,and13]. Higher levels of MCP-1 are re-
ported in patients with coronary lesions compared to patients with
normal coronary arteries (NCA) [14]. MCP-1 also contributes to
atherosclerosis by attracting monocytes into the sub-endothelium,
a key event in the process of atherogenesis [15].
Matrix metalloproteinases (MMPs) are large family of proteases
that degrade extracellular matrix, play a pivotal role in several
disorders including cancer by promoting invasion and metastasis of
tumors [16]. MMPs are highly expressed in atherosclerotic plaques
and have been implicated in atherogenesis and acute coronary
syndromes [17]. Increased MMP-9 expression and activity is
tumor necrosis factor-
produced mainly by macrophages and T-cells play a key role in
many physiological immune processes. Over activation of TNF- is
a (TNF-a), chemokines etc. The TNF-a is
a
correlated with a plethora of pathological conditions including
diabetes [1], atherosclerosis [2], multiple sclerosis [3], ulcerative
colitis [4], stroke [5] and cirrhosis [6]. There is a spectrum of drugs
currently available to inhibit TNF-a such as Etanercept, Infliximab
and Adalimumab, however they have been shown to cause adverse
side effects such as aplastic anaemia, pancytopenia, vasculitis and
congestive heart failure [7] with limitation of oral bio-availability,
*
Corresponding authors. Tel.: þ91 40 27193630, þ91 40 27191867; fax: þ91 40
7193185.
E-mail addresses: giridhar@iict.res.in (S. Kotamraju), narsaiah@iict.res.in,
narsaiahbanda84@gmail.com (N. Banda).
2
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.053

144
M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
reported in advanced atherosclerotic lesions followed by macro-
phage infiltration and in progression of atherosclerosis [18].
Macrophage derived MMPs play a potential role in the weakening
and ultimate rupture of plaque structure [19]. MMPs have been
considered as potential target in many types and stages of cancer
and also in the treatment of atherosclerosis.
4-hydroxy benzothiazin-1,1-dioxide 3. Compound 3 was further
N-alkylated using dimethyl sulfate and obtained 3-acetyl-4-
hydroxy-2-N-methyl benzothiazin-1,1-dioxide 4. Compound
4 was O-alkylated with ethyl iodide to obtain product 1-(4-
6
ethoxy-2-methyl-1,1-dioxo-1,2-dihydro-1
l
-benzo[e][1,2]thiazin-3-
yl)ethanone 5 and was further reacted with hydroxylamine hy-
During the transformation, along with inflammatory property,
monocytes acquire a number of specialized functions, including the
potential for invasion into sub-endothelial space and also increased
phagocytosis activity promoting ingestion of lipid moieties from
surroundings and converts to foam cells which ultimately narrows
the aortal lumen causing ischemia/stroke. Strategies to prevent
monocyte recruitment or/and their transformation into macro-
phages could attenuate the development of atherogenesis.
drochloride in presence of piperidine, as a result the product
6
formed as (E)-1-(4-ethoxy-2-methyl-1,1-dioxo-1,2-dihydro-1
l
-
benzo[e][1,2] thiazin-3-yl)ethanone oxime 6. The oxime 6 was
reacted with 2-chloro-N-substituted acetamide and obtained (E)-
6
N-substituted-2-(1-(4-ethoxy-2-methyl-1,1-dioxo-1,2-dihydro-1
l
-
benzo[e][1,2]thiazin-3-yl)ethylideneaminooxy) acetamide 7. Alter-
natively, the 3-acetyl-4-hydroxy-2-N-methyl benzothiazin-1,1-
dioxide 4 was reacted with hydroxylamine hydrochloride and
Development of hybrids containing two or three biologically
active scaffolds possessing good anti-inflammatory and anti-
atherogenic activities provide a different biochemical profile.
Therefore, there is an urgent need to synthesize hybrid molecules
with appreciable biological activity. Benzothiazine derivatives are
known to possess versatile biological activities [20,21]. Among
them, 1,2-benzothiazine-3-carboxamide-1,1-dioxide derivatives
such as piroxicam, ampiroxicam and meloxicam are promising
anti-inflammatory agents used worldwide as non-steroidal anti-
inflammatory drugs (NSAIDs). Recent findings proved that the 1,2-
benzothiazine 1,1-dioxide derivatives as excellent antibacterial,
formed exclusively (E)-1-(4-hydroxy-2-N-methyl-1,1-dioxo-1,2-
6
dihydro-1
l
-benzo[e][1,2]thiazin-3-yl)ethanone oxime 8. The
oxime 8 was further reacted with 2-chloro-N-substituted acet-
amide and obtained (E)-2-(((1-(4-hydroxy-2-methyl-1,1-dioxido-
2H-benzo[e][1,2]thiazin-3-yl)ethylidene) amino)oxy)-N-substituted
acetamide 9. The structure of the compound 9e was established
by single crystal X-ray crystallography as shown in Fig. 1 and
deposited in data bank as CCDC 952998 (e-mail: deposit@ccdc.
cam.ac.uk). The reactions are outlined in Scheme 1.
antifungal, anticancer, antioxidant agents and 11
b-HSD1 inhibitors.
2.2. Pharmacology
Synthetic modifications of 1,2-benzothiazine 1,1-dioxides to
enhance their bio-activity and to develop better anti-inflammatory
agents have been intensively studied [22]. In the present work, we
have selected 1,2-benzothiazine as a core moiety which was func-
tionalized at 3rd position due to its biological importance of oxime
ether link and accomplished a series of novel compounds. Final
compounds were screened for anti-inflammatory and their ability
to inhibit PMA-induced monocyte-to-macrophage transformation.
The promising compounds were further screened for PMA induced
MMP-9 gelatinase activity. It was found that compounds 9e and 9h
significantly inhibited PMA-induced monocyte-to-macrophage
transformation and compound 9h, also greatly inhibited MMP-9
activity.
2.2.1. Anti-inflammatory activity
All the final compounds, 1,2-benzothiazine 1,1-dioxide-3-
ethanone oxime N-aryl acetamide ether derivatives 7aeh and
9aeh were screened for anti-inflammatory activity in the presence
of PMA-induced inflammation, by measuring TNF-a, IL-8 and MCP-
1 levels using THP-1 monocyte cell system with reference to pir-
oxicam and pioglitazone. It was found that compounds 9e and 9h
inhibited both TNF-a and MCP-1 activities. Compound 7e inhibited
both IL-8 and MCP-1 activities and compound 9b inhibited only
MCP-1 activity. It is interesting to note that compound 9e and 9h
showed potent activity against MCP-1 with IC50 of 4.0
2.5 M respectively. The IC50 values of these compounds are much
lower than the standard pioglitazone which was found to be
18.6 M (Table 1). None of the compounds showed a significant
effect on viability of cells even at 20 M concentration as shown in
mM and
m
2
. Results and discussion
m
m
2
.1. Chemistry
Table 1. This clearly indicates that the anti-inflammatory activities
of these compounds are not due to the induction of cytotoxicity in
these cells. The structure verses activity data revealed that, the
presence of trifluoromethyl group on phenyl ring in combination
with presence of hydroxyl on C-4 position appears to be crucial for
the observed activity.
The N-methyl-3-acetyl-4-hydroxy benzothiazin-1,1-dioxide
4
[
23] was prepared starting from sodium salt of saccharin 1, via N-
alkylation with -haloketone to obtain compound 2 followed by
ring expansion with sodium ethoxide in ethanol resulted 3-acetyl-
a
Fig. 1. Crystal structure of compound 9e. The molecular structure of 9e, with the atom-numbering scheme: displacement ellipsoids are drawn at the 30% probability level and H
0
atoms are shown as small spheres of arbitrary radius in the ORTEP picture. The crystal structure contains two symmetry independent molecules in the asymmetric unit (Z ¼ 2),
however, a single molecule is shown for clarity.

M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
145
ꢀ
Scheme 1. Preparation of target compounds 7aeh and 9aeh. Reagents and conditions: (i) chloroacetone, DMF, 100 C, 4 h (ii) NaOEt, RT, 5 min (iii) dimethyl sulfate, 20% aq. NaOH,
ꢀ
ꢀ
RT, 30 min (iv) K
NH OH.HCl, aq NaOH, EtOH, reflux, 2 h.
2
CO , EteI, DMF, 50 C, 24 h (v) NH OH.HCl, piperidine, EtOH, reflux, 2 h (vi) K CO , 2-chloro-N-substituted acetamide, dry DMF, 0 C for 30 min, RT for 12e20 h (vii)
3
2
2
3
2
2.2.2. MMP-9 gelatinase activity
this, THP-1 cells were pretreated with 10 mM concentration of
As compounds 9e and 9h showed excellent anti-inflammatory
compounds for 2 h followed by PMA stimulation for 48 h. MMP-9
activity was measured in conditioned medium as described in
Materials and Methods. It was observed that compound 9h
inhibited MMP-9 activity by 75% (Fig. 2).
activity, we further examined the effects of these compounds on
PMA-induced MMP-9 activity during monocyte-to-macrophage
transformation by performing in-gel MMP-9 activity assay. For
Table 1
2
.2.3. Inhibition of PMA-induced monocyte to macrophage
Secretion inhibition efficacy of the synthesized compounds 7aeh and 9aeh for TNF-
,IL-8 and MCP-1.a
transformation
a
Monocyte-to-macrophage transformation is one of the pre-
requisite steps in the progression of atherosclerosis. Transformed
monocytes initiate inflammatory responses by secreting various
pro-inflammatory cytokines. This in turn, initiates the migration
and proliferation of smooth muscle cells and eventually the plaque
formation as seen in atherosclerosis. PMA treatment induces a
greater degree of THP-1 monocyte differentiation into macro-
phages as reflected by increased cell adherence and increase in cell
size along with increased number of cellular organelles including
mitochondria [24], upregulation of scavenger receptors like LOX-1,
CD-36 etc, which are involved in foam cell formation by enhancing
the uptake of LDL-cholesterol [25]. To this angle, we next examined
the effect of these compounds on PMA-induced monocytes-to-
macrophage transformation. It was found that, pretreatment of
S. no
Compound
IC50
(
m
M)b
Cell viabilityd
% control)
(
TNF-
a
IL-8
MCP-1
secretion
inhibition
efficacy
secretion
inhibition
efficacy
secretion
inhibition
efficacy
1
2
3
4
5
6
7
8
9
7a
7b
7c
7d
7e
7f
7g
7h
9a
9b
9c
9d
9e
9f
9g
9h
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
93.05 ꢁ 1.09
91.95 ꢁ 2.09
96.25 ꢁ 1.22
94.23 ꢁ 3.25
93.00 ꢁ 1.26
97.50 ꢁ 1.29
92.34 ꢁ 2.01
90.25 ꢁ 2.87
94.12 ꢁ 1.69
95.50 ꢁ 3.11
92.45 ꢁ 1.65
97.50 ꢁ 1.73
93.00 ꢁ 1.41
95.12 ꢁ 2.51
92.15 ꢁ 1.52
91.25 ꢁ 1.26
e
16.7 ꢁ 1.9
5.7 ꢁ 1.1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NT
NT
NA
NA
NA
10
11
12
13
14
15
16
9.4 ꢁ 0.7
NA
compounds 9e and 9h at 10 mM concentration significantly
inhibited the PMA-induced increase in cell adherence, cell size,
mitochondrial number, LOX-1 and CD-36 expressions in THP-1 cells
NA
12.1 ꢁ 1.1
4.0 ꢁ 1.0
NA
NA
(Fig. 3). These results confirmed that compound 9e and 9h
NA
NA
6.0 ꢁ 0.7
2.5 ꢁ 0.9
Piroxicamc
Pioglitazone
NA
NT
NT
18.6 ꢁ 2.1
e
a
THP-1 monocytes are pre-treated with 5, 10 and 20
m
M concentrations of the
above mentioned benzothiazine derivatives (7aeh and 9aeh) for 2 h before
simulation with 100 nM of phorbol 13-myristate 12-acetate (PMA) to induce
inflammation for a period of 48 h. At the end of the treatment, conditioned media
was collected and levels of TNF-a, IL-8 and MCP-1 were measured by ELISA as
described in the Materials and methods (Section 4.3.3).
b
IC50 values are mean ꢁ SD of three independent experiments.
Fig. 2. Compounds 9e and 9h inhibit PMA-induced MMP-9 gelatinase activity. Cells
were treated with 10 M concentrations of compounds for 2 h followed by stimulation
c
Piroxicam, a known anti-inflammatory agent was used as a positive control; NA:
m
denotes activity >20
m
M; NT: indicates ‘not tested’.
with PMA for 48 h. MMP-9 activity was performed in conditioned media using gelatin
loaded gels.
d
THP-1 cell viability with synthesized compounds (20
m
M).

146
M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
Fig. 3. Compounds 9e and 9h inhibit PMA-induced monocyte-to-macrophage transformation. THP-1 cells were pretreated with 10
2
9
m
M concentrations of compound 9e and 9h for
h followed by stimulation with PMA (100 nM) for 48 h. A) Shows phase contrast images indicating the inhibition of PMA-induced monocyte transformation by compounds 9e and
h. B) Same as A, except that differentiation markers LOX-1 and CD-36 transcript levels were measured by RT-PCR. C) Same as A, except that mitochondrial staining was performed
using mitotracker dye by confocal microscopy.
inhibited PMA-induced monocyte-to-macrophage transformation,
a key step during progression of atherogenesis.
ammonium acetate: acetonitrile (40:60) on Zodiac C18 Column
(150 mm ꢂ 4.6 mm, 5 M) in an isocratic mode at a flow rate of
.0 ml/min with Photo-diode Array (PDA) detection at 254 nm and
purity exceeded 95%.
m
1
3
. Conclusion
A series of novel 1,2-benzothiazine 1,1-dioxide-3-ethanone
4
[
.1.1. Preparation of 1-(4-ethoxy-2-methyl-1,1-dioxido-2H-benzo
e][1,2]thiazin-3-yl)ethanone (5)
.1.1.1. Procedure. The 3-acetyl-4-hydroxy-2-methyl benzothiazin-
,1-dioxide 4 (1.0 g, 4 mmol) and K CO (0.83 g, 6 mmol) were taken
oxime N-aryl acetamide ether derivatives were prepared,
screened for anti-inflammatory activity in vitro. The promising
compounds further inhibited MMP-9 gelatinase activity. Com-
pounds 9e and 9h even inhibited PMA-induced monocyte-to
macrophage-transformation. The structure verses activity data
revealed that, the presence of trifluoromethyl group on phenyl ring
in combination with presence of hydroxyl on C-4 position appears
to be crucial for the observed activity. Therefore, the results of this
study suggests that compound 9e and 9h can be further studied in-
depth in future studies for their vasculo-protective effects.
4
1
2
3
in dry DMF and stirred at room temperature for 30 min, then
iodoethane (0.935 g, 6 mmol) was added. Reaction mixture was
heated to 50 C for 24 h. After completion of reaction, the reaction
mixture was poured into ice cold water then solid was filtered,
washed with water and dried.
ꢀ
ꢀ
ꢃ1
White solid, Yield: 86%; mp: 77e79 C; I.R. (KBr, cm ): 1667,
1
1441, 1335; H NMR (CDCl
3
, 300 MHz):
d
1.40 (t, 3H, CH
), 4.04 (q, 2H, CH
2
CH
CH
3
,
,
J ¼ 6.8 Hz), 2.62 (s, 3H, CH ), 2.94 (s, 3H, NCH
3
3
2
3
4
. Experimental protocols
J ¼ 6.8 Hz), 7.66e7.76 (m, 2H, AreH), 7.78e7.83 (m, 1H, AreH),
7.86e7.91 (m, 1H, AreH); MS (ESI, 70 eV): m/z: 282 (M þ 1).
4
.1. Synthetic chemistry
4
.1.2. Preparation of (E)-4-ethoxy-3-(1-(hydroxyimino) ethyl)-2-
Melting points were recorded on Casia-Siamia (VMP-AM)
melting point apparatus and are uncorrected. IR spectra were
methyl-2H-benzo[e][1,2] thiazine 1,1-dioxide (6)
recorded on a PerkineElmer FT-IR 240-C spectrophotometer using
4.1.2.1. Procedure. The
1-(4-ethoxy-2-methyl-1,1-dioxido-2H-
1
KBr optics. H NMR spectra were recorded on Bruker AV 300 MHz
benzo[e][1,2]thiazin-3-yl)ethanone 5 (1.12 g, 4 mmol) and hy-
droxylamine hydrochloride (0.55 g, 8 mmol) were taken in ethanol
(10 mL) and piperidine (0.4 mL, 4 mmol) was added. The reaction
mixture was refluxed for 2 h, and after completion of the reaction,
solvent was removed under vacuum. The residue was treated with
ice cold water, the separated solid was filtered, washed with water
and dried.
1
3
and INOVA 500 MHz in CDCl
3
,
C NMR spectra were recorded on
using TMS as an internal
Bruker AV 75 MHz in CDCl & DMSO-d
3
6
standard. All high-resolution spectra were recorded on QSTARXL
hybrid MS/MS system (Applied Biosystems, USA) under electro
spray ionization. All the reactions were monitored by thin layer
chromatography (TLC) on pre-coated silica gel 60 F254 (mesh); spots
were visualized with UV light. Merck silica gel (60e120 mesh) was
used for column chromatography. All the final compounds were
analyzed by HPLC to evaluate purity using SHIMADZU LC-20 in-
strument. Separation was carried out by using a mobile phase of
ꢀ
ꢃ1
White solid, Yield: 83%; mp: 165e167 C; I.R. (KBr, cm ): 3290,
1
1591, 1444, 1345; H NMR (CDCl
3
, 300 MHz):
d
1.27 (t, 3H, CH
2
CH
CH
3
,
,
J ¼ 6.8 Hz), 2.26 (s, 3H, CH ), 2.95 (s, 3H, NCH
3
3
), 3.89 (q, 2H, CH
2
3
J ¼ 6.8 Hz), 7.56e7.63 (m, 1H, AreH), 7.64e7.72 (m, 1H, AreH),

M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
147
ꢃ1
7
.75e7.80 (m, 1H, AreH), 7.83e7.89 (m, 1H, AreH), 8.15 (brs, 1H,
cm ): 3362, 1685, 1609, 1528, 1342; 1H NMR (CDCl
1.20 (t, 3H, CH CH
, J ¼ 7.02 Hz), 2.33 (s, 3H, CH
NCH ), 3.79 (s, 3H, OCH ), 3.86 (q, 2H, CH CH
, J ¼ 7.02 Hz), 4.80 (s,
2H, OCH ), 6.65e6.71 (m, 1H, AreH), 7.03e7.08 (m, 1H, AreH),
3
, 500 MHz):
3
), 2.95 (s, 3H,
NH); MS (ESI, 70 eV): m/z: 297 (M þ 1).
d
2
3
3
3
2
3
4
.1.3. Preparation of (E)-4-hydroxy-3-(1-(hydroxyimino)ethyl)-2-
methyl-2H-benzo[e][1,2] thiazine 1,1-dioxide (8)
.1.3.1. Procedure. The 3-acetyl-4-hydroxy-2-methyl benzothiazin-
,1-dioxide 4 (1.0 g, 4 mmol) and hydroxylamine hydrochloride
2
7.19e7.26 (m, 1H, AreH), 7.28e7.35 (m, 1H, AreH), 7.57e7.63 (m,
1H, AreH), 7.65e7.78 (m, 2H, AreH), 7.81e7.87 (m, 1H, AreH), 8.25
(brs, 1H, NH); C NMR (CDCl , 75 MHz): d 14.5, 14.8, 35.1, 54.9, 69.1,
3
4
1
13
(
(
0.55 g, 8 mmol) were taken in ethanol (10 mL) and aq NaOH
0.1 mL, 20%) was added. The reaction mixture was refluxed for 2 h,
73.2, 105.7, 109.9, 112.0, 122.5, 124.9, 128.2, 129.3, 129.5, 129.7, 131.9,
133.5, 138.1, 144.1, 152.7, 159.7, 167.3; HRMS m/z Calcd. for
þ
and after completion of the reaction, solvent was removed under
vacuum. The residue was treated with ice cold water and separated
solid was filtered, washed with water and dried.
C
22
H
26
O
6
N
3
S ([M þ H] ): 460.1537, found 460.1523.
4.1.4.5. (E)-2-(((1-(4-Ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]
ꢀ
ꢃ1
White solid, Yield: 84%; mp: 173e175 C; I.R. (KBr, cm ): 3386,
thiazin-3-yl)ethylidene) amino)oxy)-N-(4-methoxyphenyl)acetamide
1
1
(
8
(
638,1547,1331; H NMR (CDCl
s, 3H, NCH ), 7.51 (brs, 1H, NeOH), 7.58e7.76 (m, 2H, AreH), 7.80e
.00 (m, 2H, AreH), 11.84 (brs, 1H, OH); MS (ESI, 70eV): m/z: 269
M þ 1).
3
, 300 MHz):
d
2.24 (s, 3H, CH
3
), 2.84
ꢀ
(
(
7d). White solid, Purity: 98.6%; Yield: 70%; mp: 95e97 C; I.R.
ꢃ1
1
3
KBr, cm ): 3368, 1689, 1613, 1529, 1340; H NMR (CDCl ,
3
5
(
4
00 MHz):
s, 3H, NCH
.81 (s, 2H, OCH
J ¼ 8.85 Hz), 7.58e7.64 (m, 1H, AreH), 7.67e7.77 (m, 2H, AreH),
d
1.18 (t, 3H, CH
), 3.80 (s, 3H, OCH
2
CH
3
, J ¼ 7.02 Hz), 2.32 (s, 3H, CH
3
), 2.95
, J ¼ 7.02 Hz),
3
3
), 3.85 (q, 2H, CH CH
2
3
2
), 6.85 (d, 2H, AreH, J ¼ 9.00 Hz), 7.47 (d, 2H, AreH,
4
2
.1.4. Preparation of (E)-2-(((1-(4-ethoxy-2-methyl-1,1-dioxido-
H-benzo[e][1,2]thiazin-3-yl) ethylidene)amino)oxy)-N-substituted
13
7.84e7.88 (m, 1H, AreH), 8.07 (brs, 1H, NH); C NMR (CDCl
3
,
acetamide (7) and (E)-2-(((1-(4-hydroxy-2-methyl-1,1-dioxido-2H-
benzo[e][1,2]thiazin-3-yl)ethylidene)amino) oxy)-N-substituted
acetamide (9)
4
(
7
5 MHz): 14.5, 14.8, 35.2, 55.1, 69.2, 73.3, 113.8, 121.9, 122.7, 125.0,
d
1
28.2, 129.5, 129.8, 130.0, 132.0, 133.6, 144.1, 152.6, 156.4, 167.2;
þ
HRMS m/z Calcd. for C22
4
H
25
O
6
N
3
NaS ([M þ Na] ): 482.1356, found
.1.4.1. General procedure. The oxime 6 or 8 (0.7 mmol) and K
0.116 g, 0.84 mmol) were taken in dry DMF (5 mL), cooled to 0 C
2
CO
3
82.1340.
ꢀ
and stirred for 30 min, then N-substituted acetamide (0.77 mmol)
was added. The total reaction mixture was stirred at room tem-
perature for 12e20 h. The reaction was monitored by TLC and after
completion of the reaction, treated with ice cold water. Aqueous
solution was extracted thrice with ethylacetate, combined extracts
were washed with water till washings are neutral to pH, dried over
anhydrous sodium sulfate and concentrated. The residue was pu-
rified by passing through a column packed with silica gel using
petroleum ether/EtOAc (8:2) as eluent.
4
.1.4.6. (E)-2-(((1-(4-Ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]
thiazin-3-yl)ethylidene) amino)oxy)-N-(4-(trifluoromethyl)phenyl)
acetamide (7e). White solid, Purity: 96.9%; Yield: 68%; mp: 82e
ꢀ
ꢃ1
1
8
4
C; I.R. (KBr, cm ): 3362, 1689, 1612, 1528, 1343; H NMR
CDCl , 300 MHz): 1.20 (t, 3H, CH CH
, J ¼ 6.80 Hz), 2.34 (s, 3H,
CH ), 2.96 (s, 3H, NCH ), 3.87 (q, 2H, CH CH
, J ¼ 6.80 Hz), 4.82 (s,
H, OCH ), 7.49e8.09 (m, 8H, AreH), 8.54 (brs, 1H, NH); C NMR
CDCl , 75 MHz): 14.7, 15.1, 35.5, 69.5, 73.6, 119.8, 123.1, 124.0 (q,
J ¼ 271.57 Hz), 125.2, 126.2, 126.5, 128.4, 129.6, 130.1, 132.2, 134.0,
(
3
d
2
3
3
3
2
3
13
2
(
2
3
d
140.3, 144.7, 153.4, 167.9; HRMS m/z Calcd. for C22
H
25
O
6
N
3
NaS
4
.1.4.2. (E)-2-(((1-(4-Ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]
þ
([M þ Na] ): 482.1356, found 482.1340.
thiazin-3-yl)ethylidene)
amino)oxy)-N-phenylacetamide
(7a).
ꢀ
White solid, Purity: 98.0%; Yield: 80%; mp: 124e126 C; I.R. (KBr,
ꢃ1
1
4.1.4.7. (E)-N-(3-Chloro-4-fluorophenyl)-2-(((1-(4-ethoxy-2-methyl-
,1-dioxido-2H-benzo[e] [1,2]thiazin-3-yl)ethylidene)amino)oxy)
acetamide (7f). White solid, Purity: 98.2%; Yield: 75%; mp: 142e
cm ): 3363, 1678, 1598, 1534, 1340; H NMR (CDCl
1.18 (t, 3H, CH CH ), 2.94 (s, 3H,
, J ¼ 6.99 Hz), 2.32 (s, 3H, CH
NCH ), 3.85 (q, 2H, CH CH ), 7.10e
, J ¼ 6.99 Hz), 4.80 (s, 2H, OCH
.16 (m, 1H, AreH), 7.30e7.37 (m, 2H, AreH), 7.53e7.63 (m, 3H, Are
H), 7.65e7.71 (m, 1H, AreH), 7.72e7.76 (m, 1H, AreH), 7.82e7.87
3
, 500 MHz):
1
d
2
3
3
3
2
3
2
ꢀ
ꢃ1
1
144 C; I.R. (KBr, cm ): 3363, 1682, 1608, 1529, 1341; H NMR
7
(CDCl , 300 MHz): 1.19 (t, 3H, CH CH
3
d
2
3
, J ¼ 6.80 Hz), 2.31 (s, 3H,
13
CH ), 2.96 (s, 3H, NCH ), 3.87 (q, 2H, CH CH , J ¼ 6.80 Hz), 4.79 (s,
(
m, 1H, AreH), 8.15 (brs, 1H, NH); C NMR (CDCl
3
, 75 MHz):
d
14.7,
3
3
2
3
2
2
H, OCH ), 7.08e7.17 (m,1H, AreH), 7.45e7.56 (m, 1H, AreH), 7.58e
15.0, 35.4, 69.3, 73.5, 120.1, 122.8, 124.6, 125.2, 128.3, 128.9, 129.6,
13
7.81 (m, 4H, AreH), 7.84e7.92 (m, 1H, AreH), 8.40 (brs, 1H, NH);
C
1
C
29.9, 132.1, 133.8, 137.0, 144.4, 152.9, 167.4; HRMS m/z Calcd. for
þ
NMR (CDCl , 75 MHz):
d
14.6, 15.1, 35.4, 69.4, 73.4, 116.6 (d,
23
H O
5
N
3
NaS ([M þ Na] ): 452.1250, found 452.1235.
3
21
J ¼ 21.80 Hz), 119.9 (d, J ¼ 7.26 Hz), 121.4 (d, J ¼ 18.16 Hz), 122.4,
123.0, 125.2, 128.4, 129.5, 130.1, 132.2, 133.9, 134.0, 144.5, 153.2,
4
.1.4.3. (E)-2-(((1-(4-Ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]
1
54.8 (d, J ¼ 247.05 Hz), 167.7; HRMS m/z Calcd. for C21
21 5 3-
H O N
thiazin-3-yl)ethylidene)
amino)oxy)-N-(3-fluorophenyl)acetamide
þ
ꢀ
ClFNaS ([M þ Na] ): 504.0766, found 504.0754.
(
(
3
(
6
4
7b). White solid, Purity: 96.9%; Yield: 76%; mp: 115e117 C; I.R.
ꢃ1
1
KBr, cm ): 3356, 1682, 1613, 1533, 1341; H NMR (CDCl
00 MHz): 1.19 (t, 3H, CH CH ), 2.95
, J ¼ 6.99 Hz), 2.33 (s, 3H, CH
s, 3H, NCH ), 3.87 (q, 2H, CH CH ),
, J ¼ 6.99 Hz), 4.80 (s, 2H, OCH
.78e6.90 (m, 1H, AreH), 7.20e7.34 (m, 2H, AreH), 7.47e7.80 (m,
3
,
d
2
3
3
4.1.4.8. (E)-N-(2,6-Difluorophenyl)-2-(((1-(4-ethoxy-2-methyl-1,1-
dioxido-2H-benzo[e][1,2]
amide (7g). White solid, Purity: 97.9%; Yield: 70%; mp: 132e134 C;
I.R. (KBr, cm ): 3366, 1689, 1611, 1533, 1342; H NMR (CDCl
, J ¼ 7.02 Hz), 2.31 (s, 3H, CH
500 MHz): 1.16 (t, 3H, CH CH ), 2.97
(s, 3H, NCH ), 3.81 (q, 2H, CH CH ),
3
2
3
2
thiazin-3-yl)ethylidene)amino)oxy)acet-
ꢀ
13
ꢃ1
1
H, AreH), 7.82e7.90 (m, 1H, AreH), 8.34 (brs, 1H, NH); C NMR
, 75 MHz):
14.7, 15.0, 35.4, 69.4, 73.5, 107.6 (d, J ¼ 26.3 Hz),
11.3 (d, J ¼ 20.9 Hz), 115.3 (d, J ¼ 2.7 Hz), 123.0, 125.2, 128.3, 129.6,
30.0, 130.1 (d, J ¼ 9.1 Hz), 132.2, 133.9, 138.6 (d, J ¼ 10.9 Hz), 144.5,
3
,
(
CDCl
3
d
d
2
3
3
1
1
1
3
2
3
, J ¼ 7.02 Hz), 4.91 (s, 2H, OCH
2
6.95e7.03 (m, 2H, AreH), 7.20e7.26 (m, 1H, AreH), 7.58e7.63 (m,
53.2, 162.9 (d, J ¼ 245.2 Hz), 167.6; HRMS m/z Calcd. for
1H, AreH), 7.65e7.75 (m, 2H, AreH), 7.83e7.91 (m, 2H, AreH&NH);
þ
13
C
21
22
H O
5
N
3
FNaS ([M þ Na] ): 470.1156, found 470.1142.
3
C NMR (CDCl , 75 MHz): d 14.5, 14.9, 35.3, 69.3, 73.4, 111.7 (dd,
J ¼ 19.1 and 3.6 Hz), 113.2 (t, J ¼ 16.35 Hz), 122.9, 125.2, 127.8 (t,
4
.1.4.4. (E)-2-(((1-(4-Ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]
J ¼ 9.53 Hz), 128.3, 129.7, 129.9, 132.1, 133.9, 144.2, 152.8, 157.7 (dd,
thiazin-3-yl)ethylidene) amino)oxy)-N-(3-methoxyphenyl)acetamide
J ¼ 251.5 and 3.6 Hz), 167.9; HRMS m/z Calcd. for C21
21 5 3 2
H O N F NaS
ꢀ
þ
(
7c). White solid, Purity: 96.6%; Yield: 78%; mp: 62e64 C; I.R. (KBr,
([M þ Na] ): 488.1062, found 488.1048.

148
M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
4
.1.4.9. (E)-N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-(((1-(4-
4.1.4.14. (E)-2-(((1-(4-Hydroxy-2-methyl-1,1-dioxido-2H-benzo[e]
[1,2]thiazin-3-yl)ethylidene) amino)oxy)-N-(4-(trifluoromethyl)
phenyl)acetamide (9e). White solid, Purity: 99.3%; Yield: 69%; mp:
ethoxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]thiazin-3-yl)ethyl-
idene)amino)oxy)acetamide (7h). White solid, Purity: 98.0%; Yield:
ꢀ
ꢃ1
ꢀ
ꢃ1
1
7
2%; mp: 68e70 C; I.R. (KBr, cm ): 3364, 1682, 1610, 1532, 1339;
166e168 C; I.R. (KBr, cm ): 3378, 1698, 1618, 1524, 1342; H NMR
(CDCl , 300 MHz): 2.37 (s, 3H, CH ), 2.85 (s, 3H, NCH ), 4.77 (s, 2H,
OCH ), 7.57e7.75 (m, 6H, AreH), 7.82e7.87 (m, 1H, AreH), 7.91e
7.96 (m, 1H, AreH), 8.02 (brs, 1H, NH), 11.21 (s, 1H, OH); C NMR
(CDCl , 75 MHz): 12.1, 39.4, 73.6, 114.6, 119.9, 123.8 (q,
1
H NMR (CDCl
s, 3H, CH ), 2.97 (s, 3H, NCH
.79 (s, 2H, OCH ), 7.46e7.51 (m, 1H, AreH), 7.60e7.67 (m, 1H, Are
H), 7.70e7.78 (m, 2H, AreH), 7.83e7.90 (m, 2H, AreH), 7.95e8.00
3
, 500 MHz):
d
1.16 (t, 3H, CH
2
CH
3
, J ¼ 7.02 Hz), 2.31
3
d
3
3
(
4
3
3
), 3.87 (q, 2H, CH
2
CH
3
, J ¼ 7.02 Hz),
2
13
2
3
d
13
(
1
1
m, 1H, AreH), 8.69 (brs, 1H, NH); C NMR (CDCl
4.9, 35.4, 69.3, 73.3, 119.9 (q, J ¼ 5.50 Hz), 122.5 (q, J ¼ 273.4 Hz),
23.0, 124.3, 125.2, 127.0, 128.3, 128.4, 128.7, 130.1, 131.9, 132.3,
3
, 75 MHz):
d
14.6,
J ¼ 272.2 Hz), 124.0, 126.1 (q, J ¼ 4.4 Hz), 126.2, 126.4, 128.8, 131.3,
132.7, 132.9, 139.7, 150.8, 161.9, 166.7; HRMS m/z Calcd. for
þ
C
20
18
H O
5
N
3
F
3
NaS ([M þ Na] ): 492.0811, found 492.0809.
133.9, 136.2, 144.4, 153.3, 168.0; HRMS m/z Calcd. for
þ
C
H
22 22
O
5
N
3
ClF
3
S ([M þ H] ): 532.0915, found 532.0904.
4.1.4.15. (E)-N-(3-Chloro-4-fluorophenyl)-2-(((1-(4-hydroxy-2-
methyl-1,1-dioxido-2H-benzo[e] [1,2]thiazin-3-yl)ethylidene)amino)
oxy)acetamide (9f). White solid, Purity: 99.4%; Yield: 74%; mp:
4
.1.4.10. (E)-2-(((1-(4-Hydroxy-2-methyl-1,1-dioxido-2H-benzo[e]
ꢀ
ꢃ1
1
1
(
54e156 C; I.R. (KBr, cm ): 3382, 1696, 1621, 1520, 1339; H NMR
CDCl , 300 MHz): 2.34 (s, 3H, CH ), 2.86 (s, 3H, NCH ), 4.74 (s, 2H,
OCH
), 7.11 (t, 1H, AreH, J ¼ 8.68 Hz), 7.34e7.42 (m, 1H, AreH),
.60e7.79 (m, 3H, AreH), 7.81e7.88 (m, 2H, AreH & NH), 7.91e7.97
[1,2]thiazin-3-yl)ethylidene) amino) oxy)-N-phenylacetamide (9a).
3
d
3
3
ꢀ
White solid, Purity: 98.2%; Yield: 81%; mp: 150e152 C; I.R. (KBr,
cm ): 3376, 1702, 1620, 1518, 1344; 1H NMR (CDCl
2.35 (s, 3H, CH ), 2.84 (s, 3H, NCH ), 4.75 (s, 2H, OCH
m, 1H, AreH), 7.28e7.40 (m, 2H, AreH), 7.49e7.74 (m, 4H, AreH),
.78e7.86 (m, 1H, AreH), 7.89e7.98 (m, 2H, AreH&NH),11.27 (s, 1H,
ꢃ1
, 300 MHz):
2
), 7.09e7.20
2
3
7
(
d
3
3
1
3
m, 1H, AreH), 11.21 (s, 1H, OH); C NMR (DMSO-d
6
, 75 MHz):
(
7
d
11.1, 38.3, 72.5, 114.2, 115.4 (d, J ¼ 22.00 Hz), 119.1, 119.3, 119.4,
1
21.3, 122.9, 125.3, 128.2, 130.1, 131.8, 132.0, 134.0, 148.7, 153.3 (d,
13
OH); C NMR (CDCl
3
, 75 MHz): d 12.2, 39.5, 73.8, 114.6, 120.3, 124.1,
J ¼ 245.38 Hz), 159.2, 166.1; HRMS m/z Calcd. for C19
17 5 3
H O N ClFNaS
125.0, 126.5, 129.0, 131.2, 132.7, 133.1, 136.5, 150.8, 161.9, 166.3;
þ
(
[M þ Na] ): 476.0453, found 476.0456.
þ
HRMS m/z Calcd. for C19
4
19
H O
5
N
3
NaS ([M þ Na] ): 424.0937, found
24.0928.
4
.1.4.16. (E)-N-(2,6-Difluorophenyl)-2-(((1-(4-hydroxy-2-methyl-
thiazin-3-yl)ethylidene)amino)oxy)
acetamide (9g). White solid, Purity: 96.3%; Yield: 70%; mp: 178e
1,1-dioxido-2H-benzo[e][1,2]
4
.1.4.11. (E)-N-(3-Fluorophenyl)-2-(((1-(4-hydroxy-2-methyl-1,1-
thiazin-3-yl)ethylidene)amino)oxy)acet-
amide (9b). White solid, Purity: 96.3%; Yield: 78%; mp: 146e
ꢀ
ꢃ1
1
dioxido-2H-benzo[e][1,2]
180 C; I.R. (KBr, cm ): 3384, 1694, 1620, 1528, 1340; H NMR
(CDCl , 300 MHz): 2.36 (s, 3H, CH ), 2.85 (s, 3H, NCH ), 4.86 (s, 2H,
OCH ), 6.92e7.04 (m, 2H, AreH), 7.19e7.29 (m, 1H, AreH), 7.49 (brs,
1H, NH), 7.60e7.76 (m, 2H, AreH), 7.81e7.88 (m, 1H, AreH), 7.94e
3
d
3
3
ꢀ
ꢃ1
1
1
48 C; I.R. (KBr, cm ): 3377, 1698, 1618, 1520, 1341; H NMR
CDCl , 500 MHz): 2.37 (s, 3H, CH ), 2.84 (s, 3H, NCH ), 4.76 (s, 2H,
OCH ), 6.81e6.90 (m, 1H, AreH), 7.16e7.33 (m, 2H, AreH), 7.50e
.57 (m, 1H, AreH), 7.61e7.75 (m, 2H, AreH), 7.81e7.98 (m, 3H, Are
2
(
3
d
3
3
13
2
6
8.03 (m, 1H, AreH), 11.20 (s, 1H, OH); C NMR (DMSO-d , 75 MHz):
7
d
11.3, 38.4, 72.3, 110.8 (d, J ¼ 22.56 Hz), 112.9 (t, J ¼ 16.50 Hz), 114.2,
13
H&NH), 11.20 (s, 1H, OH); C NMR (CDCl
3
, 75 MHz):
d
12.1, 39.4,
123.0, 125.5, 127.1 (t, J ¼ 9.35 Hz), 128.4, 130.2, 131.9, 132.1, 148.8,
7
3.7, 107.7 (d, J ¼ 26.4 Hz), 111.6 (d, J ¼ 21.4 Hz), 114.6, 115.5, 124.0,
157.3 (dd, J ¼ 250.9 and 4.9 Hz), 159.7, 166.8; HRMS m/z Calcd. for
þ
1
26.5, 128.9, 130.1 (d, J ¼ 9.3 Hz), 131.2, 132.7, 133.0, 138.1 (d,
C
19
17
H O
5
N
3
F
2
NaS ([M þ Na] ): 460.0749, found 460.0735.
J ¼ 11.0 Hz), 150.8, 161.9, 162.8 (d, J ¼ 245.4 Hz), 166.5; HRMS m/z
þ
Calcd. for C19
H
18
O
5
N
3
FNaS ([M þ Na] ): 442.0843, found 424.0835.
4.1.4.17. (E)-N-(4-Chloro-3-(trifluoromethyl)phenyl)-2-(((1-(4-
hydroxy-2-methyl-1,1-dioxido-2H-benzo[e][1,2]thiazin-3-yl)ethyl-
idene)amino)oxy)acetamide (9h). White solid, Purity: 98.3%; Yield:
4
.1.4.12. (E)-2-(((1-(4-Hydroxy-2-methyl-1,1-dioxido-2H-benzo[e]
ꢀ
ꢃ1
7
2%; mp: 132e134 C; I.R. (KBr, cm ): 3386,1698,1618,1526,1342;
[1,2]thiazin-3-yl)ethylidene) amino) oxy)-N-(3-methoxyphenyl)acet-
1
H NMR (CDCl
.78 (s, 2H, OCH
H), 7.70e7.74 (m, 1H, AreH), 7.77e7.80 (m, 1H, AreH), 7.84e7.86
m, 1H, AreH), 7.90e7.98 (m, 3H, AreH&NH), 11.20 (s, 1H, AreH);
3
, 500 MHz):
3 3
d 2.37 (s, 3H, CH ), 2.85 (s, 3H, NCH ),
ꢀ
amide (9c). White solid, Purity: 96.1%; Yield: 76%; mp: 64e66 C;
I.R. (KBr, cm ): 3378, 1702, 1622, 1524, 1340; H NMR (CDCl ,
3
3
4
4
2
), 7.47e7.50 (m, 1H, AreH), 7.63e7.68 (m, 1H, Are
ꢃ1
1
00 MHz):
d
2.36 (s, 3H, CH
3 3 3
), 2.85 (s, 3H, NCH ), 3.82 (s, 3H, OCH ),
(
.75 (s, 2H, OCH
2
), 6.62e6.77 (m, 1H, AreH), 6.94e7.35 (m, 4H, Are
1
3
C NMR (CDCl
3
, 75 MHz): d 12.3, 39.5, 73.6, 114.6, 119.3 (q,
H), 7.57e7.75 (m, 2H, AreH), 7.80e7.98 (m, 2H, AreH&NH), 11.25 (s,
H, OH); 13C NMR (CDCl
, 75 MHz): 12.7, 39.5, 55.3, 73.8, 106.1,
10.7, 112.4, 114.7, 124.1, 126.4, 129.0, 129.7, 131.2, 132.7, 133.1, 137.8,
J ¼ 5.4 Hz), 122.4 (q, J ¼ 273.4 Hz), 124.1, 124.3, 126.5, 127.6, 128.6,
1
1
3
d
1
28.8, 131.3, 132.0, 132.8, 133.0, 135.5, 151.0, 162.2, 166.7; HRMS m/z
þ
Calcd. for C20
18
H O
5
N
3
ClF
3
S ([M þ H] ): 504.0602, found 504.0596.
150.7, 160.1, 161.7, 166.3; HRMS m/z Calcd. for C20
H
21
O
6
N
3
NaS
þ
(
[M þ Na] ): 454.1043, found 454.1032.
4.2. Crystal structure of compound 9e
4
.1.4.13. (E)-2-(((1-(4-Hydroxy-2-methyl-1,1-dioxido-2H-benzo[e]
In addition to the spectral characterization, a single crystal of
[1,2]thiazin-3-yl)ethylidene) amino)oxy)-N-(4-methoxyphenyl)acet-
compound 9e is developed from methanol/water (80:20). X-ray
amide (9d). White solid, Purity: 99.2%; Yield: 72%; mp: 190e
data for the compound were collected at room temperature using a
ꢀ
ꢃ1
1
1
92 C; I.R. (KBr, cm ): 3383, 1694, 1620, 1519, 1341; H NMR
CDCl , 500 MHz): 2.36 (s, 3H, CH ), 2.84 (s, 3H, NCH ), 3.80 (s, 3H,
OCH ), 4.76 (s, 2H, OCH
), 6.88 (d, 2H, AreH, J ¼ 8.85 Hz), 7.45 (d,
H, AreH, J ¼ 9.00 Hz), 7.62e7.74 (m, 2H, AreH), 7.78 (brs, 1H, NH),
.82e7.87 (m,1H, AreH), 7.92e7.97 (m,1H, AreH),11.28 (s,1H, OH);
Bruker Smart Apex CCD diffractometer with graphite mono-
ꢀ
(
3
d
3
3
chromated Mo-K
a
radiation (
l
¼ 0.71073 A) with
u-scan method
3
2
[26]. Preliminary lattice parameters and orientation matrices were
obtained from four sets of frames. Unit cell dimensions were
determined using 3910 reflections for compound 9e data. Integra-
tion and scaling of the intensity data was accomplished using SAINT
program [26]. The structures were solved by Direct Methods using
SHELXS97 [27] and refinement was carried out by full-matrix least-
squares technique using SHELXL97 [27]. Anisotropic displacement
2
7
13
6
C NMR (DMSO-d , 75 MHz): d 11.6, 38.6, 54.8, 73.1, 113.5, 115.0,
1
21.3, 123.4, 125.7, 128.6, 131.0, 131.1, 132.4, 132.7, 148.5, 155.5, 158.7,
þ
166.0; HRMS m/z Calcd. for C20
H
21
O
6
N
3
NaS ([M þ Na] ): 454.1043,
found 454.1040.

M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
149
parameters were included for all non-hydrogen atoms. All H atoms
values from the standards, and were used to quantify the amount of
cytokines released by the cells.
were positioned geometrically and treated as riding on their parent
ꢀ
C
U
atoms, with CeH distances of 0.93e0.97 A, and with
iso(H) ¼ 1.2Ueq (C) or 1.5Ueq for methyl atoms. Two fluorine atoms
4.3.4. Gelatin zymography
3
of eCF group in the first symmetry independent molecule were
THP-1 monocytes were seeded in 24 well plates for studying
disordered over two sites [F1A(0.53) & F1B(0.47); F3A(0.52) &
F3B(0.48)]. Similarly, a single fluorine atom of eCF group in the
MMP-9 activity by gelatin zymography. Cells were pretreated for
h with 10 M concentrations of compounds 9e and 9h followed
by stimulation with 100 nm PMA. After 48 h of treatment, 25 l of
conditioned media was mixed with 25 l of Laemmli buffer without
-mercaptoethanol and ran the 10% SDS-PAGE containing 0.1%
gelatin. The gel was incubated in Zymogram renaturing Buffer
0.25% Triton X-100 solution) with gentle agitation for 30 min at
3
2
m
second symmetry independent molecule was disordered over two
sites [F6A(0.56) & F6B(0.44)]. PART and ISOR instructions were used
for disorder modeling of the fluorine atoms and DFIX was used for
m
m
b
ꢀ
restraining all the CeF bond distances to 1.295 A with e.s.d 0.002 in
the crystal structure refinement.
(
Crystal data for 9e: C20
H
18
F
3
N
3
O
5
S, M ¼ 469.43, colorless needle,
room temperature and allowed to develop using Zymogram
developing buffer (50 mM Tris base, 50 mM TriseHCl, 0.2 mM NaCl,
3
0
.28 ꢂ 0.14 ꢂ 0.09 mm , monoclinic, space group Pn (No. 7),
ꢀ
ꢀ
a ¼ 7.7781(9), b ¼ 27.127(3), c ¼ 10.1351(11) A,
b
¼ 99.674(2) ,
5
2
mM CaCl and 0.02% Brij 35) at room temperature for overnight.
ꢀ
3
3
V ¼ 2108.1(4) A , Z ¼ 4, D
c
¼ 1.479 g/cm , F000 ¼ 968, CCD area de-
Gels were stained with Coomassie Blue R-250 solution to get clear
bands against a dark blue background where the protease had
digested the substrate.
ꢀ
ꢀ
tector, Mo-K radiation,
a
l
¼ 0.71073 A, T ¼ 293(2)K, 2
q
max ¼ 50.0 ,
2
0,239 reflections collected, 7409 unique (Rint ¼ 0.0369), Final
GooF ¼ 1.058, R
1
¼ 0.0709, wR ¼ 0.1732, R indices based on 5665
2
2
reflections with I > 2
s
(I) (refinement on F ), 619 parameters, 60 re-
ꢃ1
straints,
m
¼ 0.218 mm . CCDC 952998 contains the supplementary
4.3.5. Qualitative analysis of monocyte/macrophage transformation
by light microscopy
crystallographicdata. Thedatacanbeobtainedfreeofchargeatwww.
ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crys-
tallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ,
UK; fax: þ44(0) 1223 336 033; email: deposit@ccdc.cam.ac.uk].
THP-1 cells were pretreated with 10 mM concentration of com-
pounds 9e and 9h for 2 h followed by PMA stimulation. After 48 h,
medium was removed, washed twice with warm DPBS and phase
contrast pictures were taken by light microscopy to study the
monocyte/macrophage adherence and transformation.
4
4
.3. Biological experiments
.3.1. THP-1 cell culture
4
.3.6. Measurement of mitochondrial biogenesis by confocal
microscopy
THP-1 monocytes were pretreated with 10
THP-1 monocytes were obtained from ATCC and grown in RPMI
1
640 (Sigma) containing 10% FBS (Lonza), non essential amino
acids, penicillin (100 U/mL), and streptomycin (100 g/mL) in 75-
cm filter vent flasks (Costar), incubated at 37 C in a humidified
atmosphere containing 5% CO and 95% air.
m
M concentration of
m
compounds 9e and 9h for 2 h in cover glass bottom dishes (SPL life
science) and stimulated with 100 nM PMA. After 48 h, media was
removed and incubated in 1 mL of HBSS (Hanks balanced salt so-
lution) containing 200 nM mitotracker solution CMXROS (molec-
ular probes) for 1 h followed by counter staining of nucleus with
DAPI (sigma). Images were taken using an Olympus confocal mi-
croscope equipped with Rhodamine and DAPI filter settings. Data
are representative of five different fields of view. After every step,
cells were washed with gentle shaking for 5 min at very low RPM
speed to prevent the loss of weakly adherent cells.
2
ꢀ
2
4.3.2. Measurement of cell viability
The effect of synthesized compounds on cell viability was
determined by Trypan blue dye exclusion assay. THP-1 cells were
5
seeded at a density of 2 ꢂ 10 cells/ml in 24-well plates in triplicates
and were treated with compounds (20 mM) for 72 h. At the end of
the treatments, cells were harvested and re-suspended in 0.4%
Trypan blue (Invitrogen) and cell viability percentage was counted
using cell countess chamber (Invitrogen).
4
.3.7. Measurement of LOX-1, CD-36 gene expression levels by RT-
PCR
THP-1 monocytes were pretreated for 2 h with 10 mM of 9e and
4.3.3. Enzyme-linked immunosorbent assay for TNF-
a, IL-8 and
MCP-1 inhibition
9h compounds followed by stimulation with 100 nM of PMA for a
period of 36 h. At the end of the treatment, cells were collected in
TRIzol (sigma), to extract total RNA. Subsequently, cDNA was pre-
pared using Fermentas cDNA synthesis kit. LOX-1 and CD-36 ex-
pressions were measured by RT-PCR using respective gene specific
primers. GAPDH was used as a loading control. The primers se-
quences used are given in Table 2.
To check the inhibitory effects of 1,2-benzothiazine 1,1-dioxide-
3
-ethanone oxime N-aryl acetamide ether derivatives (7aeh and
9
aeh) on PMA-induced inhibition of inflammation, THP-1 mono-
5
cytes were seeded at a density of 2 ꢂ 10 /ml in 24 well plates. Cells
were pre-treated with 5, 10 and 20
mM concentrations of com-
pounds for 2 h before they were stimulated with 100 nM of PMA.
After 48 h of PMA stimulation supernatants were harvested and
assayed for TNF-a, IL-8 and 72 h for MCP-1 estimation using an
enzyme-linked immunosorbent assay (ELISA) kit following the
manufacturer’s instructions (eBiosciences, San Diego, CA, USA). The
absorbance in each well was measured with a microplate reader at
Table 2
The sequences of LOX-1, CD-36 primers.
Gene
Primer
Sequence
Product
size (bp)
4
50 nm and corrected at 570 nm. Concentrations of cytokines
released were obtained and the percentage of inhibition of cyto-
kines production was calculated as compared to control conditions.
IC50 was also obtained as a mean of anti-inflammatory potency,
expressing the predicted concentration of the correspondent
compound able to reach 50% inhibition. The standard curves were
calculated by plotting the pg/ml concentrations versus absorbance
LOX-1
CD-36
GAPDH
Forward
Reverse
Forward
Reverse
Forward
Reverse
ACTCTCCATGGTGGTGCCTGG
CATTCAGCTTCCGAGCAAGGG
TGCAAAACGGCTGCAGGTCAA
GAGGATGACAGGAATGCAGGGC
GCACCACCAACTGCTTAGCAC
CCCTGTTGCTGTAGCCAAAT
252
394
516

1
50
M.R. Gannarapu et al. / European Journal of Medicinal Chemistry 75 (2014) 143e150
atherosclerotic burden and collateral degree in stable coronary artery disease,
Acknowledgments
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Authors are thankful to the Council of Scientific & Industrial
Research, New Delhi, India for the financial support through XII five
year plan projects DITSF code: CSC-0204 and “Small Molecules in
Lead Exploration” (SMiLE). Authors (GMR, PN, RNR and SBV) are
also thankful to Council of Scientific and Industrial Research (CSIR),
India for providing financial assistance in the form of Research
Fellowship and contingency grant.
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