Anti-oxidant and anti-bacterial activities of novel N′-arylmethylidene-2-(3, 4-dimethyl-5, 5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl) acetohydrazides

Article abstract of DOI:10.1016/j.ejmech.2009.11.016

A series of potential anti-oxidant and anti-bacterial N′-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetohydrazides was synthesized in a facile way starting from commercially available saccharine. 3,4-Dimethyl-2,4

Full text of DOI:10.1016/j.ejmech.2009.11.016

European Journal of Medicinal Chemistry 45 (2010) 698–704
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-oxidant and anti-bacterial activities of novel N0-arylmethylidene-2-
(3, 4-dimethyl-5, 5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)
acetohydrazides
,
,
a
c
Matloob Ahmad a b, Hamid Latif Siddiqui b , Muhammad Zia-ur-Rehman , Masood Parvez
*
a Applied Chemistry Research Centre, PCSIR Laboratories Complex, Ferozpur Road, Lahore-54600, Pakistan
b Institute of Chemistry, University of the Punjab, Lahore-54590, Pakistan
c Department of Chemistry, The University of Calgary, 2500 University Drive, NW, Calgary, Alberta, Canada T2N 1N4
a r t i c l e i n f o
a b s t r a c t
A series of potential anti-oxidant and anti-bacterial N0-arylmethylidene-2-(3,4-dimethyl-5,5-dioxido-
pyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetohydrazides was synthesized in a facile way starting from
commercially available saccharine. 3,4-Dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-
dioxide was obtained by ring expansion of 2-(2-oxopropyl)-1,2-benzisothiazol-3(2H)-one 1,1-dioxide
followed by N-methylation and cyclization with hydrazine using ultrasonic irradiation. N-alkylation of
the cyclized product with methyl chloroacetate followed by its reaction with hydrazine and subsequent
ultrasonic mediated condensations with aromatic aldehydes afforded the title compounds. All the
synthesized compounds were subjected to the preliminary evaluation for their anti-oxidant and anti-
bacterial activities.
Article history:
Received 8 September 2009
Received in revised form
2 November 2009
Accepted 4 November 2009
Available online 11 November 2009
Keywords:
Pyrazolo[4,3-c][1,2]benzothiazine
5,5-dioxide
Ó 2009 Elsevier Masson SAS. All rights reserved.
1,2-Benzothiazine 1,1-dioxides
Anti-oxidant activity
Anti-bacterial activity
1. Introduction
synergism of both the heterocyclic moieties in a single nucleus may
result in the formation of some worthwhile molecules from
Pharmacologically, pyrazole and its derivatives represent one of
the most important class of organic heterocyclic compounds, pos-
sessing anti-bacterial, anti-fungal [1], herbicidal [2] and anti-viral
activities [3]. Some of its derivatives have been reported to exhibit
significant anti-arrhythmic & sedative [4], hypoglycemic [5] and
anti-inflammatory activities [6]. On the other hand, 1,2-benzo-
thiazine 1,1-dioxides are also known as potentially biologically
active molecules e.g., 1,2-benzothiazine-3-carboxamide 1,1-dioxide
derivatives belonging to oxicams (Fig. 1) are well known as
analgesic and anti-inflammatory compounds. In addition, benzo-
thiazines are known as potent calpain I inhibitors [7], while its 3-
aryl-quinazolin-4-one derivatives showed marked activity against
Bacillus subtilis [8]. They have also been found as anti-oxidants [9]
and for the treatment of rheumatoid arthritis, ankylosing spondy-
litis, osteoarthrosis and other inflammatory rheumatic and non-
rheumatic processes, including onsets and traumatologic lesions
[10]. Keeping in view the potential biological activities of 1,2-ben-
zothiazine-1,1-dioxides and various pyrazoles, it was perceived that
the biological point of view. In the present paper, we report
the synthesis of novel N0-arylmethylidene-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetohydrazides
8a–q from commercially available saccharine along with their anti-
oxidant and anti-bacterial activities.
2. Results and discussion
2.1. Chemistry
1-(4-Hydroxy-1,1-dioxido-2H-1,2-benzothiazin-3-yl)ethanone
3 was synthesized by literature procedure [11] from commercially
available sodium saccharin [Scheme 1]. N-Methylation of 3 was
carried out using dimethyl sulphate in acetone to yield 1-(4-hydroxy-
2-methyl-1,1-dioxido-2H-1,2-benzothiazin-3-yl)ethanone 4, which
was further reacted with hydrazine hydrate in ultrasonic bath to
get 3,4-dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]benzothiazine 5,5-
dioxide 5. Reaction of 3,4-dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]-
benzothiazine 5,5-dioxide 5 with methyl chloroacetate in acetonitrile
afforded methyl (3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]-
benzothiazin-2(4H)-yl)acetate 6 which was then reacted with
hydrazine hydrate to get 2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
* Corresponding author. Tel.: þ92 425830503; fax: þ92 429231169.
E-mail address: drhamidlatif@yahoo.com (H.L. Siddiqui).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.11.016

M. Ahmad et al. / European Journal of Medicinal Chemistry 45 (2010) 698–704
699
2.3. Biological activities
OH
O
OH
O
N
CH3
2.3.1. Anti-oxidant studies
NH
N
S
NH
Generation of reactive oxygen species (ROS) and free radicals in-
vivo is involved in a wide range of human diseases [13]. ROS,
including super oxide anion, hydrogen peroxide and hydroxyl
radical are byproducts of a variety of pathways of aerobic metab-
olism [14]. These are unstable and react readily with a wide range of
biological substrates, such as lipids, DNA and protein molecules,
consequently resulting in the cell damage [15]. All the synthesized
compounds were assessed for their superoxide anion radical
scavenging activity.
Anti-oxidant (super oxide) activities of the synthesized
compounds along with the standard (n-propyl gallate) are pre-
sented in Table 4. It was found that almost all the newly synthesized
compounds have exhibited moderate to good activity. An insight to
the structure–activity relationship gives an idea that activity
generally increases with number and strength of oxygen containing
functional groups. Compounds having hydroxy and alkoxy groups in
the benzene ring of the imines are found relatively more active e.g.,
compound 8k (dihydroxy derivative; 98.34%) is more active than 8j
(monomethoxy derivative; 44.90%) which itself is more active than
8a (bearing no substituent). Among the compounds bearing alkoxy
groups, 3-ethoxy-4-hydroxy derivative 8l is more active (71.08%)
than its 2,4-dimethoxy analogue 8m indicating that the substitution
of an alkoxy group with hydroxyl group on the ring increases the
activity and vice-versa perhaps due to the steric reasons. For the
nitro and chloro derivatives, compounds show percentage radical
scavenging activity in the following order: m > o > p.
N
N
SO2 CH3
Piroxicam
SO2 CH3
Meloxicam
Fig. 1. Structure of two oxicams.
c][1,2]benzothiazin-2(4H)-yl)acetohydrazide 7 [Scheme 2]. Same
reaction was attempted in ultrasonic mediated conditions and the
reaction was completed in very good yield (86%) and reduction
of reaction time (13 min instead of 180 min). Product 7 was then
reacted with different aldehydes in ultrasonic bath to get the N0-
arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]be-
nzothiazin-2(4H)-yl)acetohydrazides 8a–q in excellent yields (See
Table 1). All of the newly synthesized compounds were characterized
through spectroscopic techniques along with their elemental anal-
yses which were found in accordance with the calculated values
[Table 1].
2.2. Stereochemistry
Configuration of C]N bond of N0-arylmethylidene-2-(3,4-
dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl) ace-
tohydrazides 8a–q was accomplished as E on the basis of singlecrystal
X-rayanalysis of 2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]ben-
zothiazin-1(4H)-yl)-N0-[phenylmethylidene]acetohydrazide 8a, as
a representative compound of the series. However, it was interesting
to know that on the basis of the structure confirmed by single crystal
X-ray diffractometer of compound 8a, compound 5, previously
reported as 3,4-dimethyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothia-
zine 5,5-dioxide [12] was in fact 3,4-dimethyl-2,4-dihydropyr-
azolo[4,3-c][1,2]benzothiazine 5,5-dioxide, an isomer of the former.
Crystal data and structure refinement parameters for compound 8a
have been provided in Table 2 while selected molecular dimensions
have been given in Table 3.
The heterocyclic thiazine ring in 8a (Fig. 2) adopts a half-chair
conformation wherein S1 is displaced by 0.699(2) Å from the plane
defined by the remaining atoms in the ring. The acetohydrazide
moiety composed of atoms N4/N5/C13/C14/O3 is almost planar and
the mean planes of pyrazole and phenyl rings are inclined at
87.19(9) and 25.54(12)ꢀ, respectively, with the mean plane of the
acetohydrazide moiety. Crystallographic data for 8a have been
deposited with the Cambridge Crystallographic Data Center (CCDC
deposition Number: 745411).
2.3.2. Antibacterial studies
The MICs of the active compounds against the susceptible patho-
genic organisms are presented in Table 5. It was found that
compoundsbearinghydroxylandmethoxygroupsatthebenzenering
of the N0-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4H)-yl)acetohydrazides possess anti-bacterial
activities against B. subtillis and Escherichia coli; the maximum activity
was shown by dihydroxy derivative 8k (MIC ¼ 5.0
m
g mlꢁ1). These are
perhaps attributed to the presence of lone pairs on oxygen atoms of
hydroxyl and methoxygroups. Besides, Compounds possessinghetero
atoms (nitrogen & sulfur) in the benzene ring were found moderately
to highly active against all the bacterial strains. Rest of the compounds
were found either inactive or presented a MIC value more than
200 m .
g mlꢁ1
3. Conclusion
The present study revealed that the compounds obtained by
synergism of the dihydropyrazolo[4,3-c][1,2]benzothiazine
5,5-dioxide moiety with different carbohydrazides were found to
possess anti-oxidant & anti-bacterial activities and could be useful
as a template for future development through modification or
derivatization to design more potent biologically active
compounds. The new skeleton may also possess other biological
activities of the parent ring systems.
O
O
O
CH3
ClCH2COCH3
DMF
N Na
SO2
N
SO2
1
2
NaOMe/ MeOH
4. Experimental
O
OH
OH
O
4.1. Chemistry
CH3
CH3
CH3
(Me)2SO4
Acetone
NH
SO2
N
All the chemicals were purchased from E. Merck, BDH or Fluka
and used without purification. However, solvents were purified
through distillation. 1H NMR spectra were recorded on a Bruker
DPX-400 instrument at 400 MHz. Chemical shifts are reported in
ppm referenced to the residual solvent signal. FT-IR spectra were
SO2
4
3
Scheme 1. Synthesis of 1-(4-hydroxy-2-methyl-1,1-dioxido-2H-1,2-benzothiazin-3-yl)-
ethanone from commercially available saccharine.

700
M. Ahmad et al. / European Journal of Medicinal Chemistry 45 (2010) 698–704
O
CH3
H
OH
O
N
N
N
O
N
CH3
CH3
CH3
SO2 CH3
i
ii
N
N
N
SO2 CH3
SO2 CH3
5
4
6
Reaction conditions:
R
i- NH2NH2.H2O/ Reflux ii- Methyl Chloroacetate / DMF
iii- NH2NH2.H2O/ Reflux iv- RCHO
iii
NH N
NH NH2
O
N
O
N
N
N
CH3
CH3
iv
N
N
SO2 CH3
8 a-q
SO2 CH3
7
Scheme 2. Conversion of 1-(4-hydroxy-1,1-dioxido-2H-1,2-benzothiazin-3-yl)ethanone 4 to N0-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4, 3-c][1,2]benzothiazin-
2(4H)-yl) acetohydrazides 8a–q.
recorded on a Thermo Nicolet IR 200 spectrometer. Mass spectra
were recorded on Agilent 5973N instrument using EI mode.
Melting points were recorded on a Gallenkamp melting point
apparatus and are uncorrected. Ultrasonic mediated reactions were
carried out in Clifton Ultrasonic Bath (2 x T2A, 300W, DU-4) made
by Nickel Electro Ltd, Weston-S-Mare Somerset, England. X-ray
crystallography was carried out on Bruker Nonius Kappa CCD
diffractometer with graphite monochromated Mo-Ka radiation and
the data were corrected for Lorentz and polarization effects and for
absorption using multi-scan method [16,17].
J ¼ 7.8 Hz, ArH), 8.43 (1H,br s, NH), 12.29 (1H, s, OH enolic). 13C
NMR: 21.5, 69.2, 122.5, 123.4, 124.4, 127.2, 129.2, 133.4, 136.7, 167.2.
MS m/z: 239.0 [Mþ].
4.1.2. 1-(4-Hydroxy-2-methyl-1,1-dioxido-2H-1,2-benzothiazin-3-yl)-
ethanone (4)
A mixture of 1-(4-hydroxy-1,1-dioxido-2H-1,2-benzothiazin-3-
yl)ethanone (3) (5.0 g; 20.9 mmol), 20% aqueous sodium hydroxide
(8.4 ml) and acetone (50 ml) was stirred at room temperature for
5 min. Dimethyl sulfate (5.9 ml) was added to the mixture drop wise
overa periodof 5 min. Themixturewasstirred for furtherhalfanhour
followed by the careful addition of dilute HCl (20 ml; 5%) to get the
white precipitates which were filtered, washed with water and dried.
Yield: 88%.; mp 151–152 ꢀC. IR (KBr) cmꢁ1: 3470, 1598, 1586, 1345,
4.1.1. 1-(4-Hydroxy-1,1-dioxido-2 H-1,2-benzothiazin-3-yl)-
ethanone (3)
The compound was synthesized according to the literature
procedure [11].
A
mixture of N-acetonyl saccharine (5.0 g,
1187. 1H NMR (CDCl3) (400 MHz)
d: 2.23 (3H, s, CH3), 3.09 (3H, s,
20.9 mmol), sodium methoxide (7.89 g, 146.1 mmol) and dry
methanol (50 ml) was refluxed for a period of 25 min till the
formation of red colored suspension which was poured over ice
cold solution of hydrochloric acid (50 ml; 10% v/v). Yellowish brown
precipitates thus obtained were washed with excess water and
dried. Yield: 62%; m.p 155–156 ꢀC. IR (KBr) cmꢁ1: 3467, 3210, 1598,
NCH3), 7.55 (1H, t, J ¼ 7.3 Hz, ArH), 7.66 (1H, t, J ¼ 7.2 Hz, ArH), 7.96
(2H, t, J ¼ 7.7 Hz, ArH), 12.22 (1H, s, OH). 13C NMR: 21.5, 38.7, 117.9,
122.5,123.4,124.4,127.2,129.2,133.4,136.7,167.2. MSm/z: 253.0 [Mþ].
4.1.3. Synthesis of 3,4-dimethyl-2,4-dihydropyrazolo[4,3-
c][1,2]benzothiazine 5,5-dioxide (5)
A mixture of 1-(4-hydroxy-2-methyl-1,1-dioxido-2H-1,2-ben-
zothiazin-3-yl)ethanone (4) (5.0 g, 19.8 mmol) and hydrazine
1586, 1345, 1187. 1H NMR (CDCl3) (400 MHz)
d: 2.22 (3H, s, CH3),
7.55 (1H, t, J ¼ 7.4 Hz, ArH), 7.67 (1H, t, J ¼ 7.2 Hz, ArH), 7.98 (2H, t,
Table 1
Reaction parameters and CHN analysis of N0-arylmethylidene-2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetohydrazides (8a–q).
S. No.
Product
R
Reaction time
(minutes)
Yield %a
Analysis %
Calculated (Found)
C
H
N
1
2
3
4
5
6
7
8
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
Phenyl
2-Nitrophenyl
3-Nitrophenyl
4-Nitrophenyl
3
2
2.5
2
2.5
2
2.5
3
2
3.5
3.5
3.0
2.5
3.5
3.5
4.0
3.0
67
79
84
88
81
90
85
83
89
87
78
82
83
76
76
75
76
58.67 (58.68)
52.86 (52.87)
52.86 (52.86)
52.86 (52.87)
54.11 (54.10)
54.11 (54.09)
50.22 (50.20)
50.22 (50.19)
56.20 (56.18)
57.39 (57.41)
54.41 (54.43)
56.28 (56.30)
56.28 (56.27)
52.03 (51.99)
52.03 (52.01)
55.60 (55.58)
55.60 (55.62)
4.68 (4.67)
3.99 (4.01)
3.99 (3.98)
3.99 (4.00)
4.09 (4.07)
4.09 (4.08)
3.58 (3.60)
3.58 (3.57)
4.24 (4.22)
4.82 (4.80)
4.34 (4.32)
4.94 (4.94)
4.94 (4.96)
4.12 (4.11)
4.12 (4.13)
4.42 (4.41)
4.42 (4.39)
17.10 (17.11)
18.49 (18.51)
18.49 (18.47)
18.49 (18.50)
15.78 (15.78)
15.78 (15.80)
14.64 (14.65)
14.64 (14.63)
16.38 (16.40)
15.94 (15.92)
15.86 (15.84)
14.92 (14.90)
14.92 (14.94)
16.86 (16.88)
16.86 (16.85)
20.48 (20.50)
20.48 (20.49)
2-Chlorophenyl
4-Chlorophenyl
2,4-Dichlorophenyl
3,4-Dichlorophenyl
4-Fluorophenyl
3-Methoxyphenyl
2,4-Dihydroxyphenyl
3-Ethoxy-4-hydroxyphenyl
2,4-Dimethoxyphenyl
Thiophen-2-yl
9
10
11
12
13
14
15
16
17
Thiophen-3-yl
Pyridin-2-yl
Pyridin-3-yl
a
Isolated yields based on 2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-yl)acetohydrazide.

M. Ahmad et al. / European Journal of Medicinal Chemistry 45 (2010) 698–704
701
Table 2
a period of 5 h followed by the removal of unreacted hydrazine and
ethanol under vacuum. The crude product obtained was washed
Crystal data and structure refinement for compound 6a.
with water and dried. White powder; mp 187 ꢀC. IR (KBr) cmꢁ1
:
Structural formula C20 H19 N5 O3
S
Cell volume
Z
1941.9(13) Å3
4
3451, 1678,1346, 1150. 1H NMR (DMSO-d6) (300 MHz)
d: 2.26 (3H, s,
Formula weight
Crystal system
Space group
T (K)
a (Å)
b (Å)
409.46
Orthorhombic
P 21 21 21
173(2) K
8.101(2)
Calculated density
1.401 Mg/m3
CH3), 2.95 (3H, s, NCH3), 4.35 (2H, s, NH2), 4.83 (2H, s, NCH2), 7.62
(1H, t, J ¼ 7.5 Hz, ArH), 7.77 (1H, t, J ¼ 7.5 Hz, ArH), 7.85–7.92 (2H, dd,
J ¼ 13.8, 7.78 Hz, ArH), 9.45 (1H, br s, NH). 13C NMR: 8.7, 38.9, 51.3,
122.3, 123.3, 124.3, 127.9, 128.8, 129.0, 131.4, 133.3, 136.4, 165.4. MS
m/z: 321.0(Mþ).
Crystal size (mm3) 0.20 ꢂ 0.12 ꢂ 0.04
Reflections collected 4291
s
min;
s
max
3.7; 27.5
1.08
9.221(4)
Goodness-of-fit
on F2
F(000)
c (Å)
Final R indices
[I > 2sigma(I)]
25.996(12)
R1 ¼ 0.042,
wR2 ¼ 0.080
856
Absolute structure ꢁ0.02(8)
4.1.6. General procedure for the synthesis of N0-arylmethylidene-2-
(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-2(4H)-
yl)acetohydrazides (8a–q)
parameter
monohydrate (4.8 ml, 99.0 mmol) was irradiated with ultrasonic
waves for 10 min at 65 ꢀC. Un-reacted hydrazine was removed
under vacuum and the residue obtained was poured over hydro-
chloric acid (20 ml, 10%). Precipitates obtained were filtered,
washed with excess water and cold ethanol. Light yellow solid; mp
230 ꢀC. IR (KBr) cmꢁ1: 3359, 1599, 1322, 1142. 1H NMR (CDCl3)
A
mixture of of 2-(3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4H)-yl)acetohydrazide (7) (20.0 mmol), cor-
responding aromatic aldehydes 20.0 mmol, methanol (50 ml) and
glacial acetic acid (2 drops) was subjected to ultrasonic irradiation
for 3–5 min. The precipitates formed were collected and washed
with methanol.
(400 MHz) d: 2.39 (3H, s, CH3), 3.06 (3H, s, NCH3), 7.53 (1H, t,
J ¼ 7.4 Hz, ArH), 7.66 (1H, t, J ¼ 7.2 Hz, ArH), 7.94 (2H, t, J ¼ 7.8 Hz,
ArH), 10.09 (1H, br s, NH). 13C NMR: 8.6, 38.8, 121.8, 123.3, 124.2,
128.8, 129.3, 131.1, 132.9, 133.2, 134.5. MS m/z: 249.1(Mþ).
4.1.6.1. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[phenylmethylidene]acetohydrazide (8a). White pow-
der; mp 287–288 ꢀC. IR (KBr) cmꢁ1: 3208, 1698, 1599, 1376, 1124. 1H
NMR (DMSO-d6) (500 MHz) d: 2.32 (3H, s, CH3), 2.98 (3H, s, NCH3),
4.1.4. Methyl (3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4 H)-yl)acetate (6)
5.51 (2H, s, NCH2), 7.44 (4H, d, J ¼ 6.5 Hz, ArH), 7.63 (1H, t, J ¼ 7.6 Hz,
ArH), 7.70–7.79 (2H, m, ArH), 7.87 (1H, d, J ¼ 7.8 Hz, ArH), 7.93 (1H,
d, J ¼ 7.7 Hz, ArH), 8.07 (1H, s, NCH), 11.78 (1H, br s, NH). 13C NMR:
8.7, 38.8, 51.9, 122.3, 122.7, 123.0, 123.4, 123.6, 123.8, 124.1, 125.3,
126.9, 127.4, 128.1, 128.9, 130.7, 133.3, 135.6, 135.9, 165.8. MS m/z:
409.0(Mþ).
A mixture of 3,4-dimethyl-2,4-dihydropyrazolo[4,3-c][1,2]ben-
zothiazine 5,5-dioxide (5) (5.0 g, 20.0 mmol), anhydrous potassium
carbonate (3.31 g, 24.0 mmol), methyl chloroacetate (2.60 g,
24.0 mmol) and acetonitrile (30 ml) was refluxed for a period of
10 h followed by the removal of solvent under vacuum. Residue
obtained was washed with cold water to get the white crystalline
4.1.6.2. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[(2-nitrophenyl)methylidene]acetohydrazide (8b). Off-
white powder; mp 205–207 ꢀC. IR (KBr) cmꢁ1: 3203, 1681, 1565,
product. Yield: 80%; mp 180 ꢀC. 1H NMR (CDCl3) (400 MHz)
d: 2.32
(3H, s, CH3), 3.04 (3H, s, NCH3), 3.79 (3H, s, OCH3), 4.91 (2H, s,
NCH2), 7.51 (1H, t, J ¼ 7.7 Hz, ArH), 7.63 (1H, t, J ¼ 7.7 Hz, ArH), 7.90
(1H, d, J ¼ 7.7 Hz, ArH), 7.97 (1H, d, J ¼ 7.7 Hz, ArH). 13C NMR: 8.6,
38.7, 51.3, 52.4, 122.5, 123.4, 124.4, 127.7, 129.2, 131.5, 133.4, 134.4,
136.8, 167.6. MS m/z: 321.2(Mþ).
1325, 1152. 1H NMR (DMSO-d6) (400 MHz)
d 2.32 (3H, s, CH3), 2.98
(3H, s, NCH3), 5.50 (2H, s, NCH2), 7.62–7.69 (2H, m, ArH), 7.76–7.81
(2H, q, J ¼ 17.9, 7.6 Hz, ArH), 7.87 (1H, d, J ¼ 7.80 Hz, ArH), 7.93 (1H, d,
J ¼ 7.7 Hz, ArH), 8.06 (1H, d, J ¼ 8.2 Hz, ArH), 8.16 (1H, d, J ¼ 8.0 Hz,
ArH), 8.44 (1H, s, N]CH), 12.06 (1H, br s, NH). 13C NMR: 8.6, 39.0,
51.9, 121.9, 122.7, 123.4, 124.1, 124.5, 126.3, 126.9, 128.1, 128.5, 128.9,
130.1, 130.7, 133.3, 135.9, 136.7, 143.1, 165.8. MS m/z: 454.0(Mþ).
4.1.5. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4 H)-yl)acetohydrazide (7)
A mixture of methyl (3,4-dimethyl-5,5-dioxidopyrazolo[4,3-
c][1,2]benzothiazin-2(4H)-yl)acetate (6) (16.07 g; 50 mmol),
hydrazine hydrate (15 ml) and ethanol (200 ml) was refluxed for
4.1.6.3. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[(3-nitrophenyl)methylidene]acetohydrazide (8c). Whi-
te powder; mp 211–212 ꢀC. IR (KBr) cmꢁ1: 3448, 3088, 1697, 1613,
1327, 1151. 1H NMR (DMSO-d6) (400 MHz)
d 2.33 (3H, s, CH3), 2.98
Table 3
Selected molecular dimensions for C20H19N5O3S.
(3H, s, NCH3), 5.57 (2H, s, NCH2), 7.62 (1H, t, J ¼ 7.64 Hz, ArH), 7.72–
7.80 (2H, m, ArH), 7.87 (1H, d, J ¼ 7.80 Hz, ArH), 7.92 (1H, d,
J ¼ 7.7 Hz, ArH), 8.19 (1H, s, ArH), 8.21 (1H, d, J ¼ 7.8 Hz, ArH), 8.25
(1H, d, J ¼ 8.2 Hz, ArH), 8.55 (1H, s, NCH), 12.05 (1H, br s, NH). 13C
NMR: 8.7, 39.0, 51.7, 118.1, 121.4, 123.1, 123.6, 124.0, 124.7, 126.4,
127.9, 128.9, 129.3, 130.1, 130.8, 133.4, 136.1, 137.3, 142.9, 166.5. MS
m/z: 454.0(Mþ).
a. Bond lengths [Å].
S(1)–O(2)
S(1)–O(1)
S(1)–N(1)
S(1)–C(1)
O(3)–C(13)
N(1)–C(8)
N(1)–C(9)
1.4291(18)
1.4346(17)
1.648(2)
1.766(2)
1.225(3)
1.434(3)
1.466(3)
N(2)–N(3)
N(3)–C(10)
N(3)–C(12)
N(4)–C(13)
N(4)–N(5)
N(5)–C(14)
N(2)–C(7)
1.360(2)
1.359(3)
1.450(3)
1.341(3)
1.400(3)
1.274(3)
1.337(3)
b. Bond angles [ꢀ]
O(2)–S(1)–O(1)
O(1)–S(1)–N(1)
O(1)–S(1)–C(1)
C(8)–N(1)–C(9)
C(9)–N(1)–S(1)
C(10)–N(3)–N(2)
N(2)–N(3)–C(12)
4.1.6.4. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[(4-nitrophenyl)methylidene]acetohydrazide (8d). White
crystalline solid; mp 241–242 ꢀC. IR (KBr) cmꢁ1: 3428, 1702, 1587,
119.32(10)
107.83(10)
109.46(10)
115.8(2)
117.10(17)
113.87(17)
120.24(17)
O(2)–S(1)–N(1)
O(2)–S(1)–C(1)
N(1)–S(1)–C(1)
C(8)–N(1)–S(1)
C(7)–N(2)–N(3)
C(10)–N(3)–C(12)
C(13)–N(4)–N(5)
106.94(10)
107.67(10)
104.65(10)
110.11(15)
103.64(16)
125.89(18)
120.69(18)
1342, 1158. 1H NMR (DMSO-d6) (300 MHz)
d: 2.32 (3H, s, CH3), 2.98
(3H, s, NCH3), 5.57 (2H, s, NCH2), 7.64 (1H, t, J ¼ 7.58 Hz, ArH), 7.82
(1H, t, J ¼ 7.5 Hz, ArH), 7.87 (1H, d, J ¼ 7.82 Hz, ArH), 7.92 (1H, d,
J ¼ 7.8 Hz, ArH), 7.97 (1H, d, J ¼ 8.7 Hz, ArH), 8.02 (1H, d, J ¼ 8.7 Hz,
ArH), 8.14 (1H, d, J ¼ 8.8 Hz, ArH), 8.28 (1H, d, J ¼ 8.7 Hz, ArH), 8.35
(1H, s, NCH), 12.11 (1H, br s, NH). 13C NMR: 8.7, 39.1, 51.4, 121.3, 121.4,
123.1, 123.6, 124.0, 125.6, 125.7, 126.4, 127.9, 128.9, 130.8, 133.4, 134.3,
136.1, 136.9, 145.7, 166.1. MS m/z: 454.0(Mþ).
c. Torsion angles [ꢀ].
C(1)–S(1)–N(1)–C(8)
S(1)–C(1)–C(6)–C(7)
C(6)–C(7)–C(8)–N(1)
ꢁ49.3(2)
ꢁ7.9(3)
1.3(3)
N(1)–S(1)–C(1)–C(6)
C(1)–C(6)–C(7)–C(8)
S(1)–N(1)–C(8)–C(7)
39.2(2)
ꢁ15.8(3)
34.8(3)

702
M. Ahmad et al. / European Journal of Medicinal Chemistry 45 (2010) 698–704
Fig. 2. ORTEP II diagram of compound 8a with the numbering scheme. Displacement ellipsoids are drawn at the 50% probability level; H atoms are represented by circles of
arbitrary radii.
4.1.6.5. N0-[(2-Chlorophenyl)methylidene]-2-(3,4-dimethyl-5,5-dioxido-
pyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide (8e). White
crystals; mp 209–211 ꢀC. IR (KBr) cmꢁ1: 3478, 1699, 1595, 1340, 1155.
4.1.6.8. N0-[(3,4-Dichlorophenyl)methylidene]-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide
(8h). White amorphous powder; mp 249–250 ꢀC. IR (KBr) cmꢁ1
:
1H NMR (DMSO-d6) (400 MHz)
d
2.32 (3H, s, CH3), 2.98 (3H, s, NCH3),
3412, 1682, 1597, 1325, 1147. 1H NMR (DMSO-d6) (400 MHz)
d 2.28
5.52 (2H, s, NCH2), 7.63–7.69 (2H, m, ArH), 7.76–7.80 (2H, q, J ¼ 17.9,
7.6 Hz, ArH), 7.86 (1H, d, J ¼ 7.80 Hz, ArH), 7.93 (1H, d, J ¼ 7.7 Hz, ArH),
8.08 (1H, d, J ¼ 8.2 Hz, ArH), 8.16 (1H, d, J ¼ 8.0 Hz, ArH), 8.46 (1H, s,
NCH), 12.09 (1H, br s, NH). 13C NMR: 8.5, 38.9, 51.3, 121.4, 122.4, 123.1,
124.6, 124.7, 124.9, 126.2, 126.4, 127.5, 127.7, 127.8, 128.1, 130.6, 132.9,
136.2, 136.8, 166.3. MS m/z: 444.0(Mþ).
(3H, s, CH3), 2.97 (3H, s, NCH3), 5.55 (2H, s, NCH2), 7.63 (1H, t,
J ¼ 7.6 Hz, ArH), 7.72 (1H, s, ArH), 7.74 (1H, d, J ¼ 1.48 Hz, ArH), 7.78
(1H, t, J ¼ 7.65 Hz, ArH), 7.87 (1H, d, J ¼ 7.83 Hz, ArH), 7.94 (1H, d,
J ¼ 7.8 Hz, ArH), 8.03 (1H, s, NCH), 8.06 (1H, d, J ¼ 1.41 Hz, ArH), 11.97
(1H, br s, NH). 13C NMR: 8.7, 38.8, 51.7,123.3,123.7,124.1,124.6,125.4,
125.7, 126.3, 127.3, 128.1, 128.4, 130.5, 130.7, 133.3, 134.3, 136.8, 137.9,
165.7. MS m/z: 479.0(Mþ).
4.1.6.6. N0-[(4-Chlorophenyl)methylidene]-2-(3,4-dimethyl-5,5-dioxido-
pyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide (8f). White
crystals; mp 226–227 ꢀC. IR (KBr) cmꢁ1: 3445, 1690, 1598, 1332. 1H
4.1.6.9. -(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[(4-fluorophenyl)methylidene]acetohydrazide (8i). White
powder; mp 256–257 ꢀC. IR (KBr) cmꢁ1: 3416, 3194, 3045, 1699,
NMR (DMSO-d6) (300 MHz) d 2.31 (3H, s, CH3), 2.97 (3H, s, NCH3), 5.52
(2H, s, NCH2), 7.49 (2H, d, J ¼ 8.4 Hz, ArH), 7.63 (1H, t, J ¼ 7.6 Hz, ArH),
7.76 (3H, m, ArH), 7.86–7.94 (2H, dd, J ¼ 16.5, 7.7 Hz, ArH), 8.05 (1H, s,
NCH), 11.85 (1H, br s, NH). 13C NMR: 8.6, 38.9, 51.2, 123.3, 124.1, 124.9,
125.2, 125.3, 126.3, 127.5, 127.7, 127.9, 128.4, 128.6, 129.6, 133.1, 133.3,
136.1, 139.1, 165.5. MS m/z: 444.0(Mþ).
1599, 1333, 1154. 1H NMR (DMSO-d6) (500 MHz)
d 2.32 (3H, s, CH3),
2.98 (3H, s, NCH3), 5.51 (2H, s, NCH2), 7.26–7.30 (2H, m, ArH), 7.63
(1H, t, J ¼ 7.7 Hz, ArH), 7.76–7.83 (3H, m, ArH), 7.87 (1H, d, J ¼ 7.8 Hz,
ArH), 7.92 (1H, d, J ¼ 7.8 Hz, ArH), 8.06 (1H, s, NCH), 11.78 (1H, br s,
NH). 13C NMR: 8.7, 38.9, 51.7, 111.6, 111.7, 123.1, 124.1, 124.5, 125.1,
125.4, 126.4, 127.3, 128.3, 130.2, 133.3, 134.1, 136.8, 138.7, 160.1, 165.6.
MS m/z: 427.0(Mþ).
4.1.6.7. N0-[(2,4-Dichlorophenyl)methylidene]-2-(3,4-dimethyl-5,5-dio-
xidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide (8g). Whi-
te crystals; mp 218–219 ꢀC. IR (KBr) cmꢁ1: 3429, 1700, 1590, 1333, 1155.
4.1.6.10. N0-[(3-Methoxyphenyl)methylidene]-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide
(8j). White powder; mp 222–223 ꢀC. IR (KBr) cmꢁ1: 3449, 3364,
1H NMR (DMSO-d6) (300 MHz)
d 2.31 (3H, s, CH3), 2.97 (3H, s, NCH3),
5.54 (2H, s, NCH2), 7.51–7.53 (1H, dd, J ¼ 8.56, 1.92 Hz, ArH), 7.64 (1H, t,
J ¼ 7.6 Hz, ArH), 7.73 (1H, d, J ¼ 1.98 Hz, ArH), 7.78 (1H, t, J ¼ 7.7 Hz,
ArH), 7.87 (1H, d, J ¼ 7.8 Hz, ArH), 7.92 (1H, d, J ¼ 7.7 Hz, ArH), 8.08 (1H,
d, J ¼ 8.6 Hz, ArH), 8.38 (1H, s, NCH), 12.02 (1H, br s, NH). 13C NMR: 8.6,
38.8, 51.7,122.0,123.3, 124.2,124.6, 125.2, 126.1,126.4,127.3,127.5,128.1,
130.1, 130.6, 133.3, 133.5, 136.5, 137.4, 165.7. MS m/z: 478.0(Mþ)
3033, 1692, 1616, 1310, 1164. 1H NMR (DMSO-d6) (500 MHz)
d: 2.32
(3H, s, CH3), 2.78 (3H, s, OCH3), 2.98 (3H, s, NCH3), 5.52 (2H, s, NCH2),
6.99–7.02 (1H, dd, J ¼ 8.2, 2.0 Hz, ArH), 7.26–7.38 (3H, m, ArH), 7.63
Table 5
Anti-bacterial activity (MIC,m
g mlꢁ1).
Table 4
Percent radical scavenging activity.
Sr. No
Compound
Susceptible micro organism
S. No.
Compound
% RSA
B. subtillis
E. coli
S. aureus
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
33.9
28.8
52.2
23.7
44.2
23.3
48.7
51.5
17.4
44.9
98.34
71.08
53.09
14.6
16.5
24.01
25.1
91.3
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
69
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9
9
–
10
11
12
13
14
15
16
17
18
10
11
12
13
14
15
16
17
18
23
5
69
39
47
–
77
82
88
95
0.35
11
17
23
24
28
27
0.25
–
55
61
–
–
0.78
6q
n-Propyl gallate
Tetracycline

M. Ahmad et al. / European Journal of Medicinal Chemistry 45 (2010) 698–704
703
(1H, t, J ¼ 7.8 Hz, ArH), 7.76 (1H, t, J ¼ 7.6 Hz, ArH), 7.87 (1H, d,
J ¼ 7.8 Hz, ArH), 7.93 (1H, d, J ¼ 7.7 Hz, ArH), 8.03 (1H, s, NCH), 11.79
(1H, br s, NH). 13C NMR: 8.5, 38.9, 47.3, 51.6, 110.5, 113.6, 117.8, 123.1,
124.1, 124.5, 126.2, 126.7, 127.5, 128.3, 130.1, 131.8, 133.4, 136.9, 139.3,
157.6, 165.7. MS m/z: 439.0(Mþ).
powder; mp 247–248 ꢀC. IR (KBr) cmꢁ1: 3467, 3015,1697,1612,1353,
1159. 1H NMR (DMSO-d6) (300 MHz)
2.33 (3H, s, CH3), 2.98 (3H, s,
d
NCH3), 5.55 (2H, s, NCH2), 7.42 (1H, t, J ¼ 6.47 Hz, ArH), 7.64 (1H, t,
J ¼ 7.6 Hz, ArH), 7.82 (1H, t, J ¼ 7.6 Hz, ArH), 7.87–7.95 (3H, m, ArH),
8.03 (1H, d, J ¼ 7.9 Hz, ArH), 8.10 (1H, s, NCH), 8.51 (1H, d, J ¼ 4.5 Hz,
ArH), 11.99 (1H, s, NH). 13C NMR: 8.6, 38.9, 51.3, 121.5, 123.1, 123.4,
124.2,124.7, 126.3, 127.6, 128.5, 130.3, 133.2, 133.9, 135.6, 136.7, 144.5,
147.3, 165.6. MS m/z: 410.0(Mþ).
4.1.6.11. N0-[(2,4-Dihydroxyphenyl)methylidene]-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide
(8k). Light orange crystals; mp 190–191 ꢀC. IR (KBr) cmꢁ1: 3562,
1
3226, 2988, 1680, 1621, 1362, 1127. H NMR (DMSO-d6) (300 MHz)
4.1.6.17. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
d
2.31 (3H, s, CH3), 2.97 (3H, s, NCH3), 5.02 (2H, s, OH), 5.42 (2H, s,
1(4H)-yl)-N0-[pyridin-3-ylmethylidene]acetohydrazide
(8q). White
powder; mp 259–260 ꢀC. IR (KBr) cmꢁ1: 3467, 3015,1697,1612,1353,
1159. 1H NMR (DMSO-d6) (300 MHz)
2.32 (3H, s, CH3), 2.98 (3H, s,
NCH2), 7.31 (1H, d, J ¼ 8.5 Hz, ArH), 7.54 (1H, d, J ¼ 8.3 Hz, ArH), 7.63
(1H, t, J ¼ 7.6 Hz, ArH), 7.78 (1H, t, J ¼ 7.35 Hz, ArH), 7.86–7.94 (2H, dd,
J ¼ 17.0, 7.8 Hz, ArH), 8.23 (1H, s, ArH). 8.33 (1H, s, NCH),11.90 (1H, br
s, NH). 13C NMR: 8.6, 38.7, 51.6, 103.2, 107.8, 111.1, 123.1, 124.2, 124.7,
126.2, 127.4, 128.4, 130.1, 131.9, 133.5, 136.6, 139.6, 158.7, 158.9, 165.8.
MS m/z: 441.0(Mþ).
d
NCH3), 5.54 (2H, s, NCH2), 7.45–7.50 (1H, dd, J ¼ 7.76, 4.89 Hz, ArH),
7.63 (1H, t, J ¼ 7.6 Hz, ArH), 7.82 (1H, t, J ¼ 7.6 Hz, ArH), 7.87–7.95 (2H,
dd, J ¼ 16.8, 7.6 Hz, ArH), 8.1 (1H, s, NCH). 8.16 (1H, d, J ¼ 8.0 Hz, ArH),
8.60 (1H, d, J ¼ 4.7 Hz, ArH), 8.91 (1H, d, J ¼ 1.4 Hz, ArH), 11.95 (1H, br
s, NH). 13C NMR: 8.7, 38.9, 51.2, 119.3, 123.5, 124.1, 124.7, 126.2, 127.4,
127.6, 128.4, 130.2, 133.1, 133.6, 136.6, 137.4, 147.5, 147.7, 165.5. MS m/
z: 410.0(Mþ).
4.1.6.12. N0-[(3-Ethoxy-4-hydroxy)methylidene]-2-(3,4-dimethyl-5,5-
dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide
(8l). White powder; mp 180–181 ꢀC. IR (KBr) cmꢁ1: 3405,1677,1598,
1339, 1166. 1H NMR (DMSO-d6) (400 MHz)
d: 1.32 (3H, t, J ¼ 2.64 Hz,
4.2. Superoxide scavenging assay
CH3), 2.32 (3H, s, CH3), 2.97 (3H, s, NCH3), 4.05(2H, m, OCH2), 5.01 (1H,
s, OH), 5.48 (2H, s, NCH2), 6.82 (1H, d, J ¼ 8.2 Hz, ArH), 7.08–7.10 (1H,
dd, J ¼ 8.2, 1.6 Hz, ArH), 7.33 (1H, d, J ¼ 1.52 Hz, ArH), 7.63 (1H, t,
J ¼ 7.6 Hz, ArH), 7.78 (1H, t, J ¼ 7.6 Hz, ArH), 7.87 (1H, d, J ¼ 7.8 Hz,
ArH), 7.92 (1H, d, J ¼ 8.2 Hz, ArH), 9.43 (1H, s, NCH), 11.62 (1H, br s,
NH).13C NMR: 8.7, 12.3, 38.9, 51.2, 61.8, 113.1, 114.5,118.5, 123.0, 123.4,
124.4, 124.8, 126.3, 127.5, 128.5, 130.2, 133.3,136.8, 139.2, 145.7, 145.9,
166.0. MS m/z: 469.0(Mþ).
Compounds were assessed by the method used by literature
method [18]. The reaction mixture comprises 40
nicotinamide adenine dinucleotide reduced form (NADH), 40
80 M nitro blue tetrazolium (NBT), 20 l of 8 M phenazine
methosulphate (PMS) 10 l of 1 mM sample and 90 l of 0.1 M
ml of 280 mM b-
ml of
m
m
m
m
m
phosphate buffer (pH 7.4). The reagents were prepared in buffer
and sample in DMSO. The reaction was performed in 96-well
microtitre plate at room temperature and absorbance was
measured at 560 nm. The formation of superoxide was monitered
by measuring the formation of water soluble blue formazan dye. A
lower absorbance of reaction mixture indicates a higher scavenging
activity of sample. Percent Radical Scavenging Activity was deter-
mined in comparison with control using the formula given below
and are presented in Table 4.
4.1.6.13. N0-[(2,4-Dimethoxyphenyl)methylidene]-2-(3,4-dimethyl-
5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-1(4H)-yl)acetohydrazide
(8m). White crystals; mp 207–208 ꢀC. IR (KBr) cmꢁ1: 3422, 1699,
1614, 1325, 1157. 1H NMR (DMSO-d6) (300 MHz)
d 2.31 (3H, s, CH3),
2.97 (3H, s, NCH3), 3.81 (3H, s, OCH3), 3.85 (3H, s, OCH3), 5.46 (2H, s,
NCH2), 6.59 (1H, d, J ¼ 2.0 Hz, ArH), 6.63 (1H, s, ArH), 7.62 (1H, t,
J ¼ 7.6 Hz, ArH), 7.70–7.95 (4H, m, ArH), 8.30 (1H, s, NCH). 11.70 (1H,
br s, NH). 13C NMR: 8.6, 38.8, 51.1, 54.7, 54.8, 101.5, 104.7, 108.3,
123.4,124.5,124.9,126.4,127.4,127.9,128.5,130.1,133.1,136.7,139.0,
159.8, 160.3, 166.1. MS m/z: 469.0(Mþ).
%RSA [ 100 L ðOD test compound=OD controlÞ3 100
4.3. Anti-bacterial testing
4.1.6.14. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[thiophen-2-ylmethylidene]acetohydrazide (8n). Light
brown powder; mp 283–284 ꢀC. IR (KBr) cmꢁ1: 3464, 1678, 1571,
All the newly synthesized compounds (dissolved in dime-
thylformamide) were subjected to antimicrobial screening by
determining the minimum inhibitory concentration (MIC) using
the agar dilution technique [19]. The in vitro antimicrobial activity
of the prepared compounds (8a–q) against three strains of bacteria
i.e., E. coli, B. subtilis and Staphylococcus aureus was determined by
preparing suspensions of each microorganism to contain approxi-
mately 105–106 CFU (colony forming units)/well. The test
compounds were applied to the wells at concentrations ranging
1331, 1152. 1H NMR (DMSO-d6) (400 MHz)
d 2.32 (3H, s, CH3), 2.97
(3H, s, NCH3), 5.40 (2H, s, NCH2), 7.13 (1H, t, J ¼ 4.28 Hz, ArH), 7.47
(1H, d, J ¼ 3.8 Hz, ArH), 7.61 (1H, d, J ¼ 7.70 Hz, ArH), 7.66 (1H, t,
J ¼ 4.2 Hz, ArH), 7.78 (1H, t, J ¼ 7.6 Hz, ArH), 7.86 (1H, d, J ¼ 7.8 Hz,
ArH), 7.92 (1H, d, J ¼ 7.8 Hz, ArH), 8.24 (1H, s, NCH), 11.77 (1H, s, NH).
13C NMR: 8.7, 38.8, 51.2, 121.4, 122.4, 123.3, 123.4, 123.5, 124.1, 124.6,
126.2, 127.4, 128.4, 130.2, 133.3, 136.6, 138.1, 165.8. MS m/z: 415.0(Mþ).
from 200 to about 3.0 m
g mlꢁ1 in dimethyl formamide solution, in
addition to the 0 (control) and the standard, tetracycline. The plates
were incubated for 24 h at 37 ꢀC and growth assessed by visual
inspection. The minimum inhibitory concentration (MIC) was
defined as the lowest concentration of inhibitor at which microbial
growth was not apparent disregarding a single colony or a faint
haze caused by the inoculums.
4.1.6.15. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[thiophen-3-ylmethylidene]acetohydrazide (8o). White
powder; mp 265–266 ꢀC. IR (KBr) cmꢁ1: 3198, 1696, 1599, 1341, 1158.
1H NMR (DMSO-d6) (400 MHz)
d 2.31 (3H, s, CH3), 2.97 (3H, s, NCH3),
5.46(2H, s, NCH2), 7.54 (1H, d, J ¼ 4.62 Hz, ArH), 7.63 (2H, m, ArH), 7.78
(1H, m, ArH), 7.87 (1H, d, J ¼ 7.8 Hz, ArH), 7.92 (2H, d, J ¼ 7.6 Hz, ArH),
8.09 (1H, s, NCH), 11.70 (1H, br s, NH). 13C NMR: 8.8, 38.9, 51.2, 121.3,
121.7, 122.0, 123.4, 123.7, 124.2, 124.6, 126.1, 127.3, 128.1, 130.2, 133.2,
136.6, 137.3, 165.7. MS m/z: 415.0(Mþ).
Acknowledgements
The authors are grateful to the Higher Education Commission,
Pakistan and Institute of Chemistry, University of the Punjab,
Lahore, for financial assistance. We are also thankful to Interna-
tional Centre for Chemical and Biological Sciences, HEJ Research
4.1.6.16. 2-(3,4-Dimethyl-5,5-dioxidopyrazolo[4,3-c][1,2]benzothiazin-
1(4H)-yl)-N0-[pyridin-2-ylmethylidene]acetohydrazide (8p). Off-white

704
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