Microwave assisted synthesis and structure-activity relationship of 4-hydroxy-N′-[1-phenylethylidene]-2H/2-methyl-1,2-benzothiazine-3- carbohydrazide 1,1-dioxides as anti-microbial agents

Article abstract of DOI:10.1016/j.ejmech.2011.03.020

A series of 4-hydroxy-N′-[1-phenylethylidene]-2H/2-methyl, 1,2-benzothiazine-3-carbohydrazide 1,1-dioxides was synthesized from commercially available sodium saccharin. Base catalyzed ring expansion of methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)acetate followed by ultrasound mediated hydrazinolysis and subsequent reaction with 1-phenylethanones under the influence of microwaves yielded the title compounds. Besides, microwave assisted synthesis of 1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide and 4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide is also discussed. Most of the synthesized compounds were found to possess moderate to significant anti-microbial (anti-bacterial and anti-fungal) activities. It is found that compounds with greater lipophilicity (N-methyl analogues) possessed higher anti-bacterial activities.

Full text of DOI:10.1016/j.ejmech.2011.03.020

European Journal of Medicinal Chemistry 46 (2011) 2368e2377
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Microwave assisted synthesis and structureeactivity relationship of 4-hydroxy-
N0-[1-phenylethylidene]-2H/2-methyl-1,2-benzothiazine-3-carbohydrazide
1,1-dioxides as anti-microbial agents
,
a
a
Naveed Ahmad a, Muhammad Zia-ur-Rehman b , Hamid Latif Siddiqui , Muhammad Fasih Ullah ,
*
Masood Parvez c
a Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan
b Applied Chemistry Research Centre, PCSIR Laboratories Complex, Ferozpur Road, Lahore 54600, 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 4-hydroxy-N0-[1-phenylethylidene]-2H/2-methyl, 1,2-benzothiazine-3-carbohydrazide 1,1-
dioxides was synthesized from commercially available sodium saccharin. Base catalyzed ring expansion
of methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)acetate followed by ultrasound mediated
hydrazinolysis and subsequent reaction with 1-phenylethanones under the influence of microwaves
yielded the title compounds. Besides, microwave assisted synthesis of 1,4-dihydropyrazolo[4,3-c][1,2]
benzothiazin-3-ol 5,5-dioxide and 4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-
dioxide is also discussed. Most of the synthesized compounds were found to possess moderate to
significant anti-microbial (anti-bacterial and anti-fungal) activities. It is found that compounds with
greater lipophilicity (N-methyl analogues) possessed higher anti-bacterial activities.
Article history:
Received 17 October 2010
Received in revised form
23 February 2011
Accepted 10 March 2011
Available online 17 March 2011
Keywords:
1,2-Benzothiazine 1,1-dioxide
Benzothiazine carbohydrazides
Microwaves assisted synthesis
1,4-Dihydropyrazolo[4,3-c][1,2]
benzothiazin-3-ol 5,5-dioxide
Sodium saccharin
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
Carbohydrazides are becoming popular day by day in the field of
drug development due to their condensation and cyclization
Among condensed sulfurenitrogen heterocycles, sulfones and
sultams (cyclic sulfonamides) possess enormous potential as
pharmaceutical and agricultural agents [1e3], chiral auxiliaries
[4,5] and therapeutic compounds [6]. Benzothiazepines and ben-
zothiazines are the most familiar classes of such compounds and
are under investigation since a long. The six-membered homo-
logues, 1,4-benzothiazine 1,1-dioxides, generally possess significant
anti-fungal activities [7], while 1,2-benzothiazine 1,1-dioxides [8]
correspond to the most familiar class of non-steroidal anti-
inflammatory drugs (NSAIDs) available in the market (Fig. 1). Some
reactions [12,13]. Up till now, a large number of such compounds
have been synthesized with significant anti-microbial [14], anti-
tubercular [15], anti-inflammatory [16], anti-cancer [17], anti-
fungal [18], anti-bacterial [19] and anti-malarial [20] activities.
In continuation of our on-going research on various biologically
active benzothiazine derivatives [21,22] and due to versatile anti-
microbial activities of carbohydrazides, synthesis of a series of novel
4-hydroxy-N0-[1-phenylethylidene]-2H/2-methyl-1,2-benzothia-
zine-3-carbohydrazide 1,1-dioxides has been contemplated with
the perception of getting biologically active compounds. Besides the
characterization of newly synthesized compounds through spec-
troscopic techniques and single crystal X-ray analysis, these have
been evaluated for their anti-bacterial and anti-fungal potential. In
addition to synthesis of the title compounds, microwave assisted
synthesis of novel cyclized products [1,4-dihydropyrazolo[4,3-c]
of the 3,4-dihydro-1,2-benzothiazine-3-carboxylate 1,1-dioxide a-
ketomide and P(2)eP(3) peptide mimetic aldehydes act as potent
calpain I inhibitors [9,10]. These are also found useful for the
treatment of rheumatoid arthritis, ankylosing spondylitis and
osteoarthritis [11].
[1,2]benzothiazin-3-ol 5,5-dioxide
5
and 4-methyl-1,4-dihy-
dropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide (10)] is also
described.
* Corresponding author. Tel.: þ92 333 4571977.
E-mail address: rehman_pcsir@hotmail.com (M. Zia-ur-Rehman).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.020

N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
2369
2. Results and discussion
2.1. Chemistry
4-Hydroxy-N0-[1-phenylethylidene]-2H-1,2-benzothiazine-3-ca
-rbohydrazide 1,1-dioxides (6aen) and 4-hydroxy-2-methyl-N0-[1-
phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dio
-xides (9aen) were synthesized from commercially available
sodium 3-oxo-3H-1,2-benzisothiazol-2-ide 1,1-dioxide 1. The later,1,
was reacted with methyl chloroacetate in microwave conditions to
get methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl) acetate
2 in excellent yield. GabrieleColman type ring expansion of five
membered isothiazole ring of 2 to the six-membered thiazine ring,
having synchronous ring cleavage and ring closure steps, in an inert
(nitrogen) atmosphere yielded 1,2-benzothiazine 1,1-dioxide [23].
Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide
3, synthesized as outlined in Scheme 1, was reacted with hydrazine
hydrate in methanol to get 4-hydroxy-2H-1,2-benzothiazine-3-car-
bohydrazide 1,1-dioxide 4. However, it was found that prolonged
induction of microwave radiations to this reaction facilitated the
intramolecular cyclization yielding 1,4-dihydropyrazolo[4,3-c][1,2]
benzothiazin-3-ol 5,5-dioxide 5, which may be attributed to greater
energy associated with microwaves. 4-Hydroxy-2H-1,2-benzothia-
zine-3-carbohydrazide 1,1-dioxide 4 and its 2-methyl analogue 7,
synthesized by N-methylation of 3 with dimethyl sulfate using
literature procedure [18], were then reacted with a number of 1-
phenylethanones to get series of novel 4-hydroxy-N0-[1-phenyl-
ethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides
6aen and 4-hydroxy-2-methyl-N0-[1-phenylethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxides 9aen [Scheme 2]. All
the condensation reactions were also attempted under microwaves
and were found more effective than in thermal conditions due to
greater yields and lesser reaction times [Table 1]. Induction of
microwaves to the reaction of methyl 4-hydroxy-2-methyl-2H-1,2-
benzothiazine-3-carboxylate 1,1-dioxide facilitated the formation of
Fig. 1. Structures of some 1,2-benzothiazines 1,1-dioxide based drugs.
CH3
OH
O
O
O
O
O
OCH3
NNa
N
ii
i
NH
S
S
S
O
O
2
1
3
O
O
O
O
Reaction conditions: i-Methyl chloroacetate/ DMFii- Sodium methoxide/ methanol
Scheme 1. Synthesis of methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-
dioxide from commercially available sodium saccharin.
N
OH
S
O
OH
O
NH
NH2
OH
OCH3
NH
i
ii
NH
NH
NH
S
S
5
3
4
O
O
O
N
O
O
O
O
iv
iii
R3
R2
OH
O
OH
S
O
O
OCH3
NH
N
NH
CH3 R1
S
CH3
OH
S
O
7
O
v
6
O
O
NH2
NH
CH3
vi
R3
R2
N
N
OH
NH
8
O
O
vii
N
OH
CH3
NH
CH3
N
N
CH3 R1
S
S
10
9
O
O
O O
Reaction conditions: i- NH2NH2/ MeOH; reflux ii- NH2NH2/ microwaves iii- 1-Phenyl ethanones/ MeOH/
H3PO4 / microwaves iv- (Me)2 SO4 / CH3 COCH3 v- NH2 NH2/ MeOH; reflux
vii- 1-Phenyl ethanones/ MeOH/ H3PO4 / microwaves
vi- NH2 NH2 / microwaves
Scheme 2. Conversion of methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide to 4-hydroxy-N0-[1-phenylethylidene]-2H/2-methyl-1,2-benzothiazine-3-carbohy-
drazide 1,1-dioxides, 1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide and 4-methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide.

2370
N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
cyclized product 10 as in the previous reaction for the synthesis of 5.
All of the newly synthesized compounds were characterized
through spectroscopic techniques (FT-IR,1H NMR,13C NMR and mass
spectrometry) along with their elemental analyses and were found
in accordance with the calculated values [Table 2].
thuringiensis and Staphylococcus aureus) and three gram negative
bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi)
by determining their minimum inhibitory concentrations (MICs)
and minimum bactericidal concentrations (MBCs) using the agar
dilution technique [24] and streptomycin as reference drug. The
choice of streptomycin as a clinical standard was based on the fact
that at low concentrations, it only inhibits growth of the bacteria
through induction of prokaryotic ribosomes to misread mRNA [25].
Streptomycin also prevents initiation of protein synthesis and leads
to death of microbial cells. Also, in humans, they have structurally
different ribosomes from bacteria, thereby allowing the selectivity
of this antibiotic for bacteria.
2.2. Stereochemistry and X-ray crystallography
To explore the stereochemistry (E or Z configuration) of C]N
bond of the compounds under investigation 6aen and 9aen,
a single crystal of N0-[1-(4-chlorophenyl)ethylidene]-4-hydroxy-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide 6b (as a repre-
sentative compound) was grown by dissolving the compound in
90% ethanol and was studied by single crystal X-ray crystallog-
raphy. It was found that C]N bond exhibits E configuration and the
compound 6b crystallizes in an orthorhombic space group Pccn
with Z ¼ 8. The heterocyclic thiazine ring (Fig. 2) adopts half-chair
conformation wherein S1 and N1 are displaced by 0.467 (5) Ǻ and
0.255 (5) Ǻ respectively from the plane defined by the remaining
atoms of the ring. Rest of the molecule is more or less planar.
Supplementary crystallographic data have been deposited with the
CCDC number 785553. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif. Details of crystal data and structure
refinement have been provided in Table 3.
The MICs of the active compounds against a panel of selected
Gram positive and Gram negative bacteria are presented in Table 4.
Results of anti-bacterial activity of compounds show that the
minimum inhibitoryconcentration (MIC) of compounds varies in the
range of 4.0e33.4 ꢀ 10ꢁ2
mmol/ml, while minimum bactericidal
concentration (MBC) varies between 7.3e67.2 ꢀ10ꢁ2 mmol/ml. It was
found that the title compounds were found more active against gram
positive bacteria than gram negative bacteria. The most sensitive
bacterial species on these compounds is B. cereus, while S. typhi is the
most resistant species. An insight to the structureeactivity rela-
tionship gives an idea that activity generally increases with the
incorporation of hydroxyl and methoxygroups at the benzene ringof
the 4-hydroxy-N0-[1-phenylethylidene]-2H/2-methyl, 1,2-benzo-
thiazine-3-carbohydrazide 1,1-dioxides. Hydroxy derivatives (6iek
and9iek) were foundthemost activewith MICvalues comparable to
the standard and this may be attributed to the presence of lone pairs
on oxygen atoms of hydroxyl and methoxy groups perhaps by
blocking the active sites through hydrogen bonding. Compounds
possessing amino (6n and 9n) and fluoro (6h and 9h) groups were
found moderately active against the gram positive strains. It was
found that incorporation of second chlorine atom to moderately
active monosubstituted chloro compounds (6a, 6b and 9a, 9b)
decreased the activity making the compounds (6c, 6d and 9c, 9d)
weakly active. Further, it is observed that bromo substituted
compounds (6e, 6f and 9e, 9f) were found inactive probably due to
steric hindrance, while the rest of compounds were found weakly
active. Groups substituted on the benzene ring may be arranged in
the following order with respect to their anti-bacterial activities:
OH > OCH3 > NH2 > F > Br > Cl > I while activities for the same
substituents follow the order: ortho > meta > para. It is found that
higher the ClogP values of the compounds under investigation,
greater are the anti-bacterial activities [Table 4]. Series of more active
compounds, with N-methyl group on thiazine ring, (9aen) have
higher ClogP values than the un-substituted ones (6aen) supporting
the fact that increase in activity goes in parallel with their lipophilic
character [26].
2.3. Biological activities
2.3.1. Anti-bacterial activities
All the newly synthesized compounds (dissolved in dime-
thylformamide) were subjected to anti-bacterial screening against
four gram positive bacteria (Bacillus cereus, Bacillus subtilis, Bacillus
Table 1
Synthesis of 4-hydroxy-2H/2-methyl-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxides and various 1-phenylethanones under normal and microwave conditions.
Entry Reactant Product Conventional
Microwave
Reaction
conditions
Yield (%)a Reaction
conditions
Yield (%)a
1
2
3
4
5
6
7
8
4
4
4
4
4
4
4
4
4
4
4
4
4
4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6a
6b
6c
6d
6e
6f
6g
6h
6i
Reflux; 6 h
Reflux; 6 h
Reflux; 7 h
78
75
80
200 W; 5 min 92
200 W; 8 min 90
200 W; 8 min 93
200 W; 7 min 90
200 W; 7 min 92
200 W; 8 min 92
200 W; 8 min 91
200 W; 7 min 90
200 W; 9 min 89
200 W; 7 min 94
200 W; 8 min 93
200 W; 7 min 91
200 W; 5 min 94
200 W; 7 min 92
200 W; 10 min 90
200 W; 7 min 94
200 W; 10 min 92
200 W; 6 min 89
200 W; 10 min 93
200 W; 8 min 95
200 W; 10 min 92
200 W; 8 min 90
200 W; 8 min 90
200 W; 10 min 87
200 W; 10 min 91
200 W; 8 min 86
200 W; 10 min 87
200 W; 9 min 89
Reflux; 6.5 h 77
Reflux; 7 h
Reflux; 7 h
Reflux; 6 h
Reflux; 6 h
Reflux; 6 h
Reflux; 6 h
80
82
80
75
75
80
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
6J
6k
6l
Reflux; 6.5 h 77
Reflux; 7 h
Reflux; 7 h
Reflux; 7 h
Reflux; 8 h
82
80
82
78
2.3.2. Anti-fungal activities
6m
6n
9a
9b
9c
9d
9e
9f
9g
9h
9i
9J
9k
9l
All the newly synthesized compounds 6aen and 9aen (dissolved
in dimethylsulfoxide) along with (4-hydroxy-2H-1,2-benzothiazine-
3-carbohydrazide 1,1-dioxide 4 and 4-hydroxy-2-methyl-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide 8), bifonazole (refer-
ence drug) and control 0 were subjected to in vitro anti-fungal
screening by determining the minimum inhibitory concentration
(MICs) and minimum fungicidal concentrations (MFCs)through
microdilution technique [27] against a panel of six strains of fungi
i.e., Aspergillus niger, Aspergillus flavus, Aspergillus fumigates, Tricho-
derma viride, Trichoderma reesei and Drechslera australiensis.
Results of anti-fungal activity of compounds show that the
minimum inhibitory concentration (MIC) of compounds varies in the
Reflux; 7.5 h 82
Reflux; 8 h
Reflux; 8 h
Reflux; 8 h
70
77
79
Reflux; 7.5 h 84
Reflux; 8 h
Reflux; 8 h
Reflux; 8 h
Reflux; 6 h
Reflux; 6 h
Reflux; 7 h
Reflux; 7 h
Reflux; 7 h
75
80
80
75
80
78
77
76
9m
9n
range of 59.0e131 ꢀ 10ꢁ2
mmol/ml, while minimum fungicidal
concentration (MFC) varies between 117.2e261 ꢀ 10ꢁ2
mmol/ml.
a
Isolated yields based on 4-hydroxy-2H/2-methyl-1,2-benzothiazine-3-carbo-
hydrazide 1,1-dioxides.
Compounds 6c, 6d, 9c and 9d showed the best activity against all the

N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
2371
Table 2
Reaction parameters and CHN analysis of 4-hydroxy-N0-[(1E)-1-phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides 6aen and 4-hydroxy-2-methyl-N0-
[(1E)-1-phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides 9aen.
Compd
R1
R2
R3
Mol. formula
Analysis %
Calculated (found)
C
H
N
6a
6b
6c
6d
6e
6f
6g
6h
6i
Cl
H
Cl
H
Br
H
H
F
OH
H
H
H
H
H
Cl
H
Cl
H
Br
H
H
F
H
H
H
Cl
H
H
H
H
H
OH
H
OCH3
H
H
H
H
H
Cl
H
H
H
Cl
Cl
Cl
H
Br
I
F
H
H
OH
H
OCH3
NH2
H
Cl
Cl
Cl
H
Br
I
F
H
H
OH
H
OCH3
NH2
C17H14ClN3O4S
C17H14ClN3O4S
52.11 (52.08)
52.11 (52.09)
47.90 (47.88)
47.90 (47.87)
46.80 (46.82)
46.80 (46.77)
42.25 (42.27)
51.91 (51.89)
54.68 (54.70)
54.68 (54.66)
54.68 (54.71)
55.80 (55.76)
55.80 (55.77)
54.83 (54.86)
53.27 (53.31)
53.27 (53.29)
49.10 (49.07)
49.10 (49.08)
48.01 (47.97)
48.01 (48.03)
43.47 (43.43)
53.07 (53.08)
55.80 (55.79)
55.80 (55.77)
55.80 (55.83)
56.85 (56.84)
56.85 (56.86)
55.95 (55.93)
3.60 (3.62)
3.60 (3.61)
3.07 (3.08)
3.07 (3.09)
3.23 (3.22)
3.23 (3.25)
2.92 (2.94)
3.33 (3.34)
4.05 (4.04)
4.05 (4.05)
4.05 (4.03)
4.42 (4.43)
4.42 (4.44)
4.33 (4.31)
3.97 (3.95)
3.97 (3.95)
3.43 (3.45)
3.43 (3.45)
3.58 (3.60)
3.58 (3.58)
3.24 (3.25)
3.71 (3.73)
4.40 (4.43)
4.40 (4.42)
4.40 (4.38)
4.77 (4.75)
4.77 (4.76)
4.70 (4.74)
10.72 (10.73)
10.72 (10.73)
9.86 (9.87)
9.86 (9.87)
9.63 (9.62)
C17H13Cl2N3O4S
C17H13Cl2N3O4S
C17H14BrN3O4S
C17H14BrN3O4S
C17H14IN3O4S
C17H13F2N3O4S
C17H15N3O5S
9.63 (9.64)
8.69 (8.69)
10.68 (10.69)
11.25 (11.24)
11.25 (11.27)
11.25 (11.24)
10.85 (10.87)
10.85 (10.86)
15.04 (15.03)
10.35 (10.33)
10.35 (10.35)
9.54 (9.55)
9.54 (9.54)
9.33 (9.35)
9.33 (9.35)
8.45 (8.48)
10.31 (10.28)
10.85 (10.83)
10.85 (10.86)
10.85 (10.84)
10.47 (10.50)
10.47 (10.47)
14.50 (14.48)
6J
6k
6l
C17H15N3O5S
C17H15N3O5S
C18H17N3O5S
C18H17N3O5S
C17H16N4O4S
C18H16ClN3O4S
C18H16ClN3O4S
C18H15Cl2N3O4S
6m
6n
9a
9b
9c
9d
9e
9f
9g
9h
9i
9J
9k
9l
C18H15Cl2N3O4S
C18H16BrN3O4S
C18H16BrN3O4S
C18H16IN3O4S
C18H15F2N3O4S
C18H17N3O5S
C18H17N3O5S
H
H
H
OH
H
OCH3
H
H
OH
H
H
H
H
H
C18H17N3O5S
C19H19N3O5S
C19H19N3O5S
C18H18N4O4S
9m
9n
fungi, with 9d exhibiting the highest anti-fungal potential (MIC
the standard, while the compounds (6a, 6b, 6eeh and 9a, 9b, 9eeh)
were found significantly active with MIC values comparable to the
standard drug; Rest of the compounds were found moderately active.
59.0 ꢀ 10ꢁ2
m
mol/ml and MFC 158.1 ꢀ 10ꢁ2
mmol/ml, while
compounds 6i, 6j, 6k, 9i, 9j and 9k were found almost inactive (with
MIC>100 ꢀ10ꢁ2 mmol/ml). Acloserlookintotheanti-fungalactivities
of the compounds (Table 5) indicates that, in general, the compounds
bearing methyl group at 2-position of benzothiazine nucleus (9aen)
are more active thanwiththosehavingnosubstituent(6aen) andthis
is of same pattern previously reported activities of 1,2-benzothiazine
1,1-dioxide based oxicams [17]. Results showed moderate to signifi-
cant activity of almost all the compounds against T. viride, T. reesei and
D. australiensis while these were found less active against Aspergillus
species except compounds bearing hydroxyl and methoxy groups on
benzene ring (6iem and 9iem), which were either found inactive or
with MIC greater than 100. The majority of compounds showed the
bestactivityagainstT. viride whileA. nigeristhemostresistantspecies.
An insight to the structureeactivity relationship gives an idea that
activity generally increases with number and strength of electron
withdrawing groups. Compounds bearing two chloro groups (6c, 6d
and 9c, 9d) were found highly active with MIC values even lower than
3. Conclusion
Prompted by the well established anti-bacterial and anti-fungal
properties of carbohydrazides, series of novel 4-hydroxy-N0-[1-phe-
nylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides
(6aen) and 4-hydroxy-2-methyl-N0-[1-phenylethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxides (9aen) are synthe-
sized emphasizing, in particular, on the strategy of combining two
chemically different but pharmacologically compatible molecules
(1,2-benzothiazine 1,1-dioxide nucleus and 1-phenylethylidene car-
bohydrazides) in one frame. The title compounds were synthesized
under the influence of microwaves and assayed in vitro for the
evaluation of their anti-microbial activity against Gram positive,
Gram negative bacteria and fungi. It revealed that the compounds
obtained by the synergism were found biologically active (anti-
fungal, anti-bacterial) and could be useful as a template for further
development through modification or derivatization to design more
potent biologically active compounds. Facilitation of the syntheses
with microwaves was found quite useful to obtain higher yields and
purity than those which were carried out by simple thermal
condensation in methanol and the duration of reactions was reduced
considerably (from 6 to 8 h to 5e10 min).
4. Experimental
4.1. Chemistry
Fig. 2. ORTEP3 diagram of compound 6b with the numbering scheme. Displacement
ellipsoids are drawn at the 50% probability level; H atoms are represented by circles of
arbitrary radii.
All the chemicals were purchased from E. Merck, BDH or Fluka
and used without purification. However, solvents were purified

2372
N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
Table 3
frequency. The apparatus was modified for laboratory applications,
equipped with magnetic stirrer and an external reflux condenser. X-
Crystallographic parameters for compound 6a.
Structural
formula
C17H14ClN3O4S$H2 O$CH3O H Cell volume
3851. 2 (3) Å3
8
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.
Formula
weight
Crystal
system
Space
441.88
Z
Orthorhombic
Pnnc
Absorption
correction
Calculated
density
Multi-scan
method
1.524 Mg/m3
4.1.1. Methyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl) acetate
(2)
group
T (K)
173 (2) K
Crystal size
0.12 ꢀ 0.10 ꢀ
0.06 mm3
7472
A mixture of sodium saccharin (7.50 g; 36.55 mmol), N,N-
dimethylformamide (50 ml) and methyl chloroacetate (3.942 g;
36.55 mmol) was allowed to react in microwave oven at 200 W for
a period of 3 min. Contents were then cooled to room temperature
and poured over ice cooled water (300 ml) resulting in the forma-
tion of a white solid, which was filtered and washed with cold water.
The solid was dried and recrystallized from methyl alcohol to get
the product (8.80 g; 96.2%); m.p. 116e117 ꢂC (lit. m.p. 115e116 ꢂC)
a (Å);
b (Å);
c (Å);
a
a
g
15.7936 (6); 90ꢂ
30.5165 (13); 90ꢂ
7.9907 (3); 90ꢂ
Reflections
collected
Independent
reflections
Goodness-of-fit 1.094
on F2
4283
[R(int) ¼ 0.0264]
q
min;
q
max 2.90ꢂ; 27.47ꢂ
F(000)
1840
[18]; IR (KBr) 1754,1671, 1344,1189 cmꢁ1. 1H NMR (CDCl3)
d: 3.81 (s,
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 recorded
on a Thermo Nicolet IR 200 spectrometer. Mass spectra were recor-
dedonAgilent5973NinstrumentusingEImode. Meltingpointswere
recorded on a Gallenkamp melting point apparatus and are uncor-
rected. Ultrasonic mediated reactions were carried out in Clifton
Ultrasonic Bath (2 ꢀ T2A, 300W, DU-4) made by Nickel Electro Ltd,
Weston-S-Mare Somerset, England. Microwave assisted reactions
were carried out in a household MW oven (OrienteNNe781JF)
equipped with inverter technology (generating fixed frequency
throughout the required time) for realistic control of the microwaves
operating at multiples of 100 W up to 1000 W generating 2450 MHz
3H, OCH3), 4.42 (s, 2H, CH2), 7.73e7.79 (m, 4H, ArH).
4.1.2. Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-
dioxide (3)
Compound 3 was synthesized according to literature procedure
[21]. Sodium metal (2.3 g; 100 mmol) and dry methanol (125 ml) was
allowed torefluxuntilallthemetaldissolved. Tothis solution, methyl
(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl) acetate (2) (10.2 g;
40.0 mmol) was added in a single portion under inert conditions.
Temperature of the mixture was maintained at 55 ꢂC for 30 min till
thecompletionofreaction. Thecontents were then cooledto5 ꢂC and
poured over an ice-water mixture. HCl (15%) was added to the
mixture till the pH became approximately 3. The precipitates formed
Table 4
Anti-bacterial activity of compounds 6aen and 9aen tested by Micro dilution method (MIC and MFC
m
mol ꢀ 10ꢁ2).
Entry
Compound
Susceptible microorganism
ClogP
B. cereus
B. subtilis
B. thuringiensis
S. aureus
E. coli
P. aeruginosa
S. typhi
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
MIC/MBC
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
4
8
11.5/23.1
11.7/23.1
e
11.4/22.6
12.5/24.8
e
12.1/24.1
12.3/24.3
e
31.3/59.2
30.8/58.7
e
32.5/63.6
31.2/62.4
e
e
e
1.97
2.55
2.74
3.23
2.14
2.74
3.00
1.68
2.06
1.45
1.43
1.52
1.96
1.04
3.46
4.05
4.24
4.72
3.64
4.23
4.49
3.17
3.55
2.94
2.92
3.02
3.45
2.53
32.4/64.8
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
9.1/18.0
7.8/14.9
4.2/7.8
4.5/9.4
4.8/8.9
7.2/14.2
7.8/15.5
6.2/12.6
10.8/19.1
11.0/20.2
e
9.5/18.6
8.0/14.8
4.4/9.3
4.6/7.3
4.9/9.4
7.5/15.0
7.5/14.7
6.0/11.8
11.0/20.2
11.6/22.7
e
9.4/18.8
8.2/16.4
4.1/7.6
4.4/8.1
5.0/8.7
7.7/15.9
7.8/15.3
6.1/12.2
12.0/23.6
12.3/24.3
e
28.2/56.0
28.1/55.9
17.8/33.2
18.4/37.0
19.0/38.6
24.5/42.9
26.7/53.4
20.5/41.1
30.7/56.1
30.9/57.4
e
29.5/58.9
29.2/58.4
19.9/42.1
19.7/39.4
21.3/42.6
25.6/46.5
27.8/54.5
22.9/46.5
31.7/58.3
31.0/48.4
e
30.9/61.8
30.6/61.6
20.6/39.5
21.2/39.7
22.6/43.2
26.7/52.1
29.3/58.5
24.5/49.8
32.6/56.8
32.3/64.3
e
33.4/57.3
33.2/71.5
22.7/45.3
23.5/46.3
25.2/50.3
29.3/63.1
32.7/60.2
27.7/57.5
e
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
9.0/18.0
7.5/14.4
4.0/7.7
4.2/8.2
4.6/9.1
7.0/14.7
7.9/15.8
6.2/10.9
e
9.3/18.2
7.8/15.2
4.2/8.4
4.5/8.9
4.8/9.4
7.7/13.1
7.6/15.0
6.2/12.1
e
9.2/17.8
8.0/15.2
4.0/7.8
4.3/8.6
5.0/9.9
7.8/14.1
7.8/15.1
6.3/12.6
e
28.0/48.0
27.6/47.6
17.7/33.2
18.2/36.4
18.9/32.4
24.1/40.6
26.3/52.6
20.4/35.2
e
29.3/50.9
28.9/58.7
19.6/39.0
19.3/38.6
21.0/42.9
25.6/54.7
27.9/49.7
22.4/39.2
e
30.5/56.0
30.5/57.3
20.2/38.3
21.0/41.2
22.1/44.8
26.8/58.4
29.2/47.6
24.3/51.9
e
33.2/56.2
33.1/64.3
22.3/44.3
23.1/39.6
25.1/58.2
29.4/61.8
32.4/67.2
27.5/58.3
e
e
e
e
e
e
e
e
Control
Streptomycin
0
0
0
0
0
0
0
4.3/8.6
4.2/8.3
4.4/8.8
16.5/33.0
16.5/33.0
16.5/33.0
16.5/33.0

N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
2373
Table 5
Anti-fungal activity of compounds 6aen and 9aen tested by microdilution Method (MIC and MBC in
m
mol ꢀ 10ꢁ2).
Entry
Compound
Susceptible microorganism
Aspergillus niger
Aspergillus flavus
Aspergillus fumigatus
Trichoderma viride
Trichoderma reesei
Drechslera australiensis
MIC/MFC
MIC/MFC
MIC/MFC
MIC/MFC
MIC/MFC
MIC/MFC
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
89.6/179.1
82.9/145.4
74.6/149.0
75.2/150.2
93.2/186.8
96.4/188.5
91.5/166.1
76.6/160.3
>100/>200
>100/>200
>100/>200
90.1/176.3
87.3/179.5
83.8/177.5
88.6/167.9
82.0/166.4
74.2/148.3
75.0/149.5
93.1/186.0
96.0/>200
91.4/198.4
76.5/143.6
>100/>200
>100/>200
>100/>200
90.1/190.0
87.2/180.7
83.7/167.0
>100/>200
>100/>200
0
87.3/164.6
81.9/163.0
71.8/143.5
75.2/150.3
90.0/177.7
93.7/173.2
89.9/179.8
76.4/168.7
>100/>200
>100/>200
>100/>200
92.7/198.4
86.2/169.3
82.9/175.9
87.1/170.2
81.7/162.3
71.6/143.1
75.0/150.0
89.4/168.5
93.4/189.8
89.8/190.7
76.3/149.4
>100/>200
>100/>200
>100/>200
92.6/188.9
86.1/170.0
82.9/166.3
>100/>200
>100/>200
0
76.2/150.4
72.7/140.1
67.4/142.5
68.1/136.2
83.8/157.3
85.7/191.0
82.0/159.2
69.8/118.2
>100/>200
>100/>200
>100/>200
85.9/181.2
77.7/153.6
74.0/150.1
76.1/144.6
72.6/143.6
67.3/134.7
68.1/136.3
83.6/163.6
85.5/177.0
81.9/159.8
69.6/140.1
>100/>200
>100/>200
>100/>200
85.7/168.2
77.6/149.5
73.6/151.6
>100/>200
>100/>200
0
66.8/123.9
67.3/114.7
62.9/125.7
61.3/118.7
75.6/142.4
74.4/188.2
71.3/139.7
72.0/139.6
>100/>200
>100/>200
>100/>200
80.7/149.9
80.5/158.8
71.3/142.8
66.5/133.1
67.2/134.4
62.7/125.0
61.2/122.5
75.4/149.9
74.3/139.4
71.1/139.2
72.1/143.0
>100/>200
>100/>200
>100/>200
80.5/160.4
79.2/158.4
71.1/142.0
>100/>200
>100/>200
0
61.4/122.9
62.5/124.8
59.4/118.6
59.2/108.0
74.8/146.6
74.9/129.7
70.3/133.5
69.0/138.0
>100/>200
>100/>200
>100/>200
80.0/160.7
79.9/159.8
70.3/138.4
61.1/119.2
62.2/124.4
59.1/118.1
59.0/158.1
74.5/151.2
74.6/142.5
70.1/122.7
68.4/130.2
>100/>200
>100/>200
>100/>200
79.6/159.2
78.9/157.6
68.0/135.8
>100/>200
>100/>200
0
63.8/125.1
64.4/130.5
62.0/123.9
61.8/123.4
76.3/158.4
77.5/143.9
74.0/145.8
72.3/145.7
>100/>200
>100/>200
>100/>200
82.0/162.7
81.1/159.6
70.0/129.5
63.6/126.5
64.3/128.6
61.5/122.7
61.5/122.6
76.2/151.7
77.3/148.4
74.0/150.6
72.1/144.2
>100/>200
>100/>200
>100/>200
81.7/154.8
80.0/159.9
69.8/133.7
>100/>200
>100/>200
0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
6j
6k
6l
6m
6n
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
4
8
0
Bifonazole
48.0/64.0
48.0/64.0
48.0/64.0
64.0/80.0
60.2/78.0
57.5/75.0
were filtered and dried at 70ꢂC to get the product as white crystalline
solid (8.46 g, 82.9%); m.p. 172e173 ꢂC; IR (KBr): 3182, 1668, 1340,
s, NH), 10.02 (1H, br s, NH), 11.16 (1H, s, OH), 13C NMR: 121.8, 127.3,
127.6, 128.8, 132.3, 133.5, 134.5, 137.7, 151.9. MS m/z: 237 (Mþ).
1182 cmꢁ1; 1H NMR (CDCl3)
d
: 3.84 (s, 3H, OCH3), 7.77 (dd, J ¼ 5.6,
3.2 Hz, 2H, ArH), 7.83 (dd, J ¼ 5.6, 3.2 Hz, 2H, ArH), 12.33 (s, 1H,
4.2. General procedure for the synthesis of 4-hydroxy-N0-[1-
phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides
(6aen)
OHenolic), 8.33 (br s, 1 H, NH); MS m/z (%): 255 [Mþ].
4.1.3. 4-Hydroxy-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (4)
A
mixture of methyl 4-hydroxy-2H-1,2-benzothiazine-3-
4.2.1. Using conventional procedure
carboxylate 1,1-dioxide (3) (5.1 g; 20.0 mmol), hydrazine hydrate
(1.25 g, 25 mmol) and methyl alcohol (30 ml) was stirred and
refluxed for a period of 35 min. After completion of the reaction (as
indicated by TLC), solvent was removed under vacuum and the
residue obtained was treated with cold hydrochloric acid (5%) fol-
lowed by washing with water to get the product (4.89 g; 96.4%);
m.p. 292 ꢂC decomp; IR (KBr) 3409, 3266, 1674, 1326, 1128,
A mixture of 4-hydroxy-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxide (4) (2.0 mmol), 1-phenylethanone (2.0 mmol), ortho
phosphoric acid (2 drops) and methanol (50 ml) was refluxed till
completion of the reaction (for yields, reaction conditions and
reaction times, see Table 1). The contents were cooled to 5 ꢂC in an
ice bath, filtered and the solids were washed with cold methanol to
get the pure compound.
1030 cmꢁ1 1H NMR (CDCl3)
; d: 2.49 (br s, 2H, NH2), 7.55e7.87 (m,
4H, ArH), 9.61 (br s, 1H, NH),10.28 (br s, 1H, NH), 12.64 (s, 1H,
OHenolic); HRMS/EI: calcd for C9H9N3O4S 255.2505, found 255.2511.
4.2.2. Using microwaves
A
mixture of 4-hydroxy-2H-1,2-benzothiazine-3-carbohy-
drazide 1,1-dioxide (4) (2.0 mmol), 1-phenylethanone (2.0 mmol),
ortho phosphoric acid (2 drops) and methanol (50 ml) was heated
at 200 W till completion of the reaction (for yields, reaction
conditions and reaction times, see Table 1). The contents were
cooled to 5 ꢂC in an ice bath, filtered and the solids were washed
with cold methanol to get the pure compound.
4.1.4. 1,4-Dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol 5,5-dioxide (5)
A mixture of methyl 4-hydroxy-2H-1,2-benzothiazine-3-carbox-
ylate 1,1-dioxide (3) (2.55 g; 10.0 mmol), hydrazine hydrate (2.50 g;
50.0 mmol), acetic acid (3.0 ml) and methyl alcohol (50 ml) was
stirred and heated in microwave oven for a period of 4 min. After
coolingtoroomtemperature, thecontentswere poured overcrushed
ice and pH was maintained at 1 with conc. hydrochloric acid.
Precipitates obtained were filtered, washed with water and dried
(1.82 g; 77%); Light yellow solid; m.p. 230 ꢂC. IR (KBr) cmꢁ1: 3355,
4.2.3. N0-[1-(2-Chlorophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6a)
Light yellow powder; m.p. 146e147 ꢂC. IR (KBr) cmꢁ1: 3652,
1599,1322,1142. 1H NMR (CDCl3) (400 MHz)
ArH), 7.65 (1H, t, J ¼ 7.2 Hz, ArH), 7.91 (2H, t, J ¼ 7.8 Hz, ArH), 8.16 (1H,
d: 7.55 (1H, t, J ¼ 7.4 Hz,
3550, 2364, 1643,1376,1181,1061. 1H NMR (DMSO-d6)
d: 2.37 (3H, s,
CH3), 7.52 (2H, d, J ¼ 8.7 Hz, ArH), 7.85e7.90 (5H, m, ArH), 8.03 (1H,

2374
N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
dd, J ¼ 6.4, 1.2 Hz, ArH), 8.57 (1H, br s, NH), 9.79 (1H, br s, CONH),
13.73 (1H, br s, OHenolic). 13C NMR: 16.2, 106.4, 123.5, 126.7, 128.8,
128.9, 130.1, 130.3, 130.6, 131.8, 132.4, 132.5, 137.2, 137.3, 155.1, 168.7,
168.8. MS m/z: 391 [Mþ], 393 [Mþþ2].
4.2.10. N0-[1-(2,4-Difluorophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6h)
Off white powder; m.p. 140 ꢂC. IR (KBr) cmꢁ1: 3670, 3531, 2362,
1616, 1350, 1182, 1068. 1H NMR (DMSO-d6)
d: 2.30 (3H, s, CH3), 6.89
(1H, m, ArH), 7.27 (2H, m, ArH), 7.59 (2H, t, J ¼ 7.8 Hz, ArH), 7.77 (2H,
t, J ¼ 7.8 Hz, ArH), 8.58 (1H, br s, NH), 9.95 (1H, br s, CONH), 13.74
(1H, br s, OHenolic); 13C NMR: 16.6, 106.5, 111.3, 112.7, 113.8, 123.6,
126.7, 128.9, 131.9, 132.4, 132.7, 137.3, 155.3, 161.4, 163.5, 168.7,
168.9; MS m/z: 393 [Mþ].
4.2.4. N0-[1-(4-Chlorophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6b)
Off white crystalline powder; m.p. 140e141 ꢂC. IR (KBr) cmꢁ1
:
3674, 3566, 2363, 1639, 1399, 1172,1094. 1H NMR (DMSO-d6)
d: 2.35
(3H, s, CH3), 7.62 (2H, d, J ¼ 8.7 Hz, ArH), 7.83e7.89 (5H, m, ArH),
8.06 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 8.64 (1H, br s, NH), 9.86 (1H, br s,
CONH), 13.77 (1H, br s, OHenolic). 13C NMR: 16.6, 106.3, 123.5, 126.8,
128.2, 128.3, 128.6, 128.9, 130.0, 131.7, 132.4, 135.6, 136.5, 137.1,147.7,
155.0, 168.7. MS m/z 391 [Mþ], 393 [Mþþ2].
4.2.11. 4-Hydroxy-N0-[1-(2-hydroxyphenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6i)
Off white crystals; m.p. 263e265 ꢂC. IR (KBr) cmꢁ1: 3674, 3560,
2364, 1648, 1387, 1185, 1074. 1H NMR (DMSO-d6)
d: 2.30 (3H, s, CH3),
5.21 (1H, s, OH), 6.9e6.95 (2H, m, ArH), 7.3e7.37 (1H, m, ArH), 7.65e7.68
(1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 7.80e7.92 (3H, m, ArH), 8.05 (1H, m, ArH),
4.2.5. N0-[1-(2,4-Dichlorophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6c)
8.59 (1H, br s, NH), 9.98 (1H, br s, CONH), 13.68 (1H, br s, OHenolic). 13
C
Brownish gray powder; m.p. 216e217 ꢂC. IR (KBr) cmꢁ1: 3676,
NMR: 17.0, 106.3, 117.6, 118.8, 121.3, 123.5, 126.6, 128.8, 131.7, 132.0,
3597, 2364,1642,1378,1171,1059. 1H NMR (DMSO-d6)
d
: 2.28 (3H, s,
132.4, 132.5, 137.2, 155.0, 162.4, 168.6, 168.8; MS m/z: 373 [Mþ].
CH3), 6.85 (1H, m, ArH), 7.24 (2H, m, ArH), 7.59 (2H, t, J ¼ 7.8 Hz,
ArH), 7.76 (2H, t, J ¼ 7.8 Hz, ArH), 8.58 (1H, br s, NH), 9.99 (1H, br s,
CONH), 13.73 (1H, br s, OH). 13C NMR: 16.3, 106.4,123.4, 126.6, 126.7,
128.4, 128.8,129.0,131.8,132.3,133.4,134.7,135.3,137.1,155.1,168.6,
168.8; MS m/z 425 [Mþ], 427 [Mþþ2], 429 [Mþþ4].
4.2.12. 4-Hydroxy-N0-[1-(3-hydroxyphenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6j)
Light yellow powder; m.p. 253e255 ꢂC. IR (KBr) cmꢁ1: 3674,
3550, 2363,1657, 1354,1190,1062. 1H NMR (DMSO-d6)
d: 2.33 (3H, s,
CH3), 5.24 (1H, s, OH), 6.85 (1H, m, ArH), 7.24 (2H, m, ArH), 7.59 (2H,
t, J ¼ 7.8 Hz, ArH), 7.76 (2H, t, J ¼ 7.8 Hz, ArH), 8.03 (1H, m, ArH), 8.57
4.2.6. N0-[1-(3,4-Dichlorophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6d)
(1H, br s, NH), 9.96 (1H, br s, CONH), 13.70 (1H, br s, OHenolic). 13
C
Light yellow powder; m.p. 254 ꢂC. IR (KBr) cmꢁ1: 3674, 3560,
NMR: 16.8, 106.4, 114.9, 118.3, 120.9, 123.6, 126.8, 128.9, 130.3, 131.8,
2363,1619,1379, 1176,1066. 1H NMR (DMSO-d6)
d
: 2.29 (3H, s, CH3),
132.6, 135.4, 137.4, 147.8, 155.2, 158.7, 168.8; MS m/z: 373 [Mþ].
6.88 (1H, m, ArH), 7.31 (2H, m, ArH), 7.61 (2H, t, J ¼ 7.8 Hz, ArH), 7.77
(2H, t, J ¼ 7.8 Hz, ArH), 8.55 (1H, br s, NH), 9.89 (1H, br s, CONH),
13.77 (1H, br s, OHenolic). 13C NMR: 16.7, 106.3, 123.6, 126.4, 126.6,
129.0, 130.3, 130.5, 131.7, 132.4, 133.5, 133.6, 135.6, 137.2, 147.8,
155.2, 168.8; MS m/z: 425 [Mþ], 427 [Mþþ2], 429 [Mþþ4].
4.2.13. 4-Hydroxy-N0-[1-(4-hydroxyphenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6k)
Off white powder; m.p. 268e269 ꢂC. IR (KBr) cmꢁ1: 3674, 3547,
2363, 1642, 1379, 1180, 1065. 1H NMR (DMSO-d6)
d: 2.30 (3H, s,
CH3), 5.22 (1H, s, OH), 6.91e6.94 (2H, m, ArH), 7.29e7.34 (1H, m,
ArH), 7.63e7.66 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 7.82e7.90 (3H, m,
ArH), 8.02 (1H, m, ArH), 8.59 (1H, br s, NH), 9.97 (1H, br s, CONH),
13.68 (1H, br s, OHenolic). 13C NMR: 16.7, 106.3, 115.9, 116.0, 123.4,
126.7, 128.7, 128.8, 129.0, 130.0, 131.7, 132.4, 137.1, 147.6, 155.0, 160.7,
168.7; MS m/z: 373 [Mþ].
4.2.7. N0-[1-(2-Bromophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6e)
Pale yellow powder; m.p. 262e264 ꢂC. IR (KBr) cmꢁ1: 3674,
3566, 2363, 1639, 1399, 1172, 1078. 1H NMR (DMSO-d6)
d: 2.34 (3H,
s, CH3), 7.55 (2H, d, J ¼ 8.7 Hz, ArH), 7.88e7.90 (5H, m, ArH), 8.06
(1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 8.67 (1H, br s, NH), 9.89 (1H, br s,
CONH), 13.78 (1H, br s, OHenolic). 13C NMR: 16.6, 106.2, 122.1, 123.5,
126.7, 127.7, 128.8, 130.1, 131.8, 132.3, 132.8, 134.6, 135.5, 137.2, 155.1,
168.6, 168.7; MS m/z: 435 [Mþ], 437 [Mþþ2].
4.2.14. 4-Hydroxy-N0-[1-(3-methoxyphenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6l)
Off white powder; m.p. 205e207 ꢂC. IR (KBr) cmꢁ1: 3674, 3547,
2363,1642,1379,1180,1068. 1H NMR (DMSO-d6)
d: 2.33 (3H, s, CH3),
4.2.8. N0-[1-(4-Bromophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6f)
3.94 (3H, s, OCH3), 6.78 (1H, m, ArH), 7.34 (2H, m, ArH), 7.59 (2H, t,
J ¼ 7.8 Hz, ArH), 7.76 (2H, t, J ¼ 7.8 Hz, ArH), 8.03 (1H, m, ArH), 8.57
Light yellow powder; m.p. 272e274 ꢂC. IR (KBr) cmꢁ1: 3675,
(1H, br s, NH), 9.96 (1H, br s, CONH), 13.73 (1H, br s, OHenolic). 13
C
3539, 2364, 1632, 1355, 1182, 1069. 1H NMR (DMSO-d6)
d
: 2.35 (3H,
NMR: 16.8, 55.9, 106.5, 113.4, 116.7, 120.6, 123.5, 126.8, 128.9, 129.7,
s, CH3), 7.53 (2H, d, J ¼ 8.7 Hz, ArH), 7.83e7.89 (5H, m, ArH), 8.04
(1H, d, J ¼ 6.4,1.2 Hz, ArH), 8.70 (1H, br s, NH), 9.87 (1H, br s, CONH),
13.74 (1H, br s, OHenolic). 13C NMR: 16.6, 106.5, 123.7, 125.5, 126.7,
128.6, 128.7, 130.0, 131.6, 131.8, 131.9, 132.4, 136.6, 137.3, 147.7, 155.0,
168.8; MS m/z: 435 [Mþ], 437 [Mþþ2].
131.7,132.4,135.2,137.1,147.8,155.3,160.8,168.8; MS m/z: 387 [Mþ].
4.2.15. 4-Hydroxy-N0-[1-(4-methoxyphenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6m)
Off white powder; m.p. 208e210 ꢂC. IR (KBr) cmꢁ1: 3676, 3560,
2364, 1638, 1380, 1170, 1066. 1H NMR (DMSO-d6)
d: 2.34 (3H, s,
4.2.9. 4-Hydroxy-N0-[1-(4-iodophenyl)ethylidene]-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6g)
CH3), 3.93 (3H, s, OCH3), 7.62 (2H, d, J ¼ 8.7 Hz, ArH), 7.83e7.89 (5H,
m, ArH), 8.06 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 8.56 (1H, br s, NH), 9.89
(1H, br s, CONH), 13.75 (1H, br s, OHenolic). 13C NMR: 16.8, 60.0,
106.5, 114.4, 114.6, 123.5, 126.8, 128.7, 128.8, 128.9, 129.9, 131.9,
132.5, 137.3, 147.8, 155.2, 162.9, 168.9; MS m/z: 387 [Mþ].
Pale yellow powder; m.p. 220 ꢂC. IR (KBr) cmꢁ1: 3673, 3570,
2363, 1644, 1389, 1183, 1062. 1H NMR (DMSO-d6)
d: 2.34 (3H, s,
CH3), 7.65 (2H, d, J ¼ 8.4 Hz, ArH), 7.82e7.84 (2H, d, J ¼ 8.4 Hz, ArH),
7.86e7.91 (3H, m, ArH),8.07 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 8.66 (1H,
br s, NH), 9.80 (1H, br s, CONH), 13.72 (1H, br s, OHenolic); 13C NMR:
16.5, 96.5, 106.3, 123.3, 126.5, 128.8, 130.7, 131.0, 131.6, 132.3, 136.2,
137.1, 137.5, 137.6, 147.5, 155.1, 168.6; MS m/z: 483 [Mþ], 485
[Mþþ2].
4.2.16. N0-[1-(4-Aminophenyl)ethylidene]-4-hydroxy-2H-1,2-
benzothiazine-3-carbohydrazide 1,1-dioxide (6n)
Yellow powder; m.p. 239e240 ꢂC. IR (KBr) cmꢁ1: 3674, 3560,
2363, 1638, 1380, 1183, 1059. 1H NMR (DMSO-d6)
d: 2.35 (3H, s,

N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
2375
CH3), 7.60 (2H, d, J ¼ 8.7 Hz, ArH), 7.81e7.88 (5H, m, ArH), 8.01 (1H,d
4.3.3. N0-[1-(2,4-Dichlorophenyl)ethylidene]-4-hydroxy-2-methyl-
d, J ¼ 6.4, 1.2 Hz, ArH), 8.58 (1H, br s, NH), 9.93 (1H, br s, CONH),
13.76 (1H, br s, OHenolic). 13C NMR: 16.6, 106.3, 114.1, 114.2, 123.4,
125.2, 126.5, 128.8, 130.0, 130.0, 131.6, 132.3, 137.1, 147.6, 150.5,
155.0, 168.5; MS m/z: 372 [Mþ].
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9c)
Yellow powder; m.p. 178e180 ꢂC. IR (KBr) cmꢁ1: 3678, 3572,
2929, 2365, 1656, 1423, 1122, 1042. 1H NMR (DMSO-d6)
d: 2.28 (3H,
s, CH3), 2.95 (3H, s, NCH3), 6.85 (1H, m, ArH), 7.24 (3H, m, ArH), 7.59
(2H, t, J ¼ 7.8 Hz, ArH), 7.76 (1H, s, ArH), 9.98 (1H, br s, NH), 13.74
(1H, br s, OHenolic). 13C NMR: 16.2, 40.7, 111.2, 123.4, 126.4, 126.6,
128.2, 128.9, 129.2, 131.9, 132.5, 133.5, 134.4, 134.7, 135.5, 156.6,
168.7, 168.9; MS m/z 439 [Mþ], 441 [Mþþ2], 443 [Mþþ4].
4.2.17. Methyl 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-
carboxylate-1,1-dioxide (7)
Compound 7 was synthesized according to literature procedure
[21]. A mixture of Methyl 4-hydroxy-2H-1,2-benzothiazine-3-carbox-
ylate 1,1-dioxide (3) (5.0 g; 19.6 mmol), 20% aqueous sodium hydroxide
(8.4 ml) and acetone (50 ml) was stirred at room temperature for 5min.
Dimethyl sulfate (5.9 ml) was added to the mixture drop wise over
a period of 5 min. The mixture was stirred for further half an hour
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: 91%; m.p. 167 ꢂC. IR (KBr): 3439, 1667, 1319, 1160 cmꢁ1. 1H NMR
4.3.4. N0-[1-(3,4-Dichlorophenyl)ethylidene]-4-hydroxy-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9d)
Light yellow powder; m.p. 222e224 ꢂC. IR (KBr) cmꢁ1: 3678,
3573, 2932, 2364,1653,1424,1178,1062. 1H NMR (DMSO-d6)
d: 2.30
(3H, s, CH3), 2.94 (3H, s, NCH3), 6.87 (1H, m, ArH), 7.32e7.61 (4H, m,
ArH), 7.70 (1H, m, ArH), 7.78 (1H, t, J ¼ 7.8 Hz, ArH), 9.94 (1H, br s,
NH),13.75 (1H, br s, OHenolic). 13C NMR: 16.7, 40.9,111.4,123.6,126.3,
126.8, 128.9, 130.3, 130.7, 131.8, 132.5, 133.5, 133.7, 134.6, 135.8,
147.8, 156.6, 168.7; MS m/z 439 [Mþ], 441 [Mþþ2], 443 [Mþþ4].
(CDCl3), d:2.95(3H, s, NCH3), 3.95 (3H, s, OCH3), 7.75e8.12 (4H, m, ArH),
12.09 (1H, s, OH). MS m/z: 269 [Mþ], 254 [Mþ ꢁ CH3]. Anal. Calcd for
C11H11NO5S: C, 49.06; H, 4.12; N, 5.20; Found: C, 49.11; H, 4.20; N, 4.19.
4.3.5. N0-[1-(2-Bromophenyl)ethylidene]-4-hydroxy-2-methyl-2H-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9e)
4.2.18. 4-Hydroxy-2-methyl-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8)
Off white powder; m.p. 219e220 ꢂC. IR (KBr) cmꢁ1: 3677, 3574,
A mixture of methyl 4-hydroxy-2H-1,2-benzothiazine-3-carbox-
ylate 1,1-dioxide (7) (5.38 g; 20.0 mmol), hydrazine hydrate (1.25 g,
25.0 mmol) and methyl alcohol (50 ml) was stirred and refluxed for
a period of 40 min. After completion of the reaction (as indicated by
TLC), solvent was removed under vacuum and the residue obtained
was treated with cold hydrochloric acid (5%) followed by washing with
water to get the product (4.79 g; 93.2%); m.p. 222 ꢂC; IR (KBr) 3467;
2923, 2364, 1653, 1424, 1167, 1067. 1H NMR (DMSO-d6)
d: 2.34 (3H,
s, CH3), 2.93 (3H, s, NCH3), 7.54 (2H, d, J ¼ 8.7 Hz, ArH), 7.86e7.90
(5H, m, ArH), 8.05 (1H, d, J ¼ 6.4, 1.2 Hz, ArH), 9.96 (1H, br s, NH),
13.73 (1H, br s, OHenolic). 13C NMR: 16.1, 40.8, 111.3, 122.3, 123.6,
126.7, 127.9, 128.9, 130.2, 131.9, 132.5, 132.8, 134.5, 134.7, 135.6,
156.7, 168.6, 168.8; MS m/z 449 [Mþ], 451 [Mþþ2].
3364; 2982; 1615; 1331; 1171 cmꢁ1; 1H NMR
d
2.73 (3H, s, NCH3), 4.69
4.3.6. N0-[1-(4-Bromophenyl)ethylidene]-4-hydroxy-2-methyl-2H-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9f)
(2H, br s, NH2), 7.81e7.86 (3H, m, ArH), 7.95 (1H, d, J ¼ 7.6 Hz, ArH),
10.04 (1H, br s, NH), 14.23 (s, 1H, OHenolic); MS m/z 255 [Mþ].
Off white powder; m.p. 231e233 ꢂC. IR (KBr) cmꢁ1: 3677, 3574,
2923, 2364, 1653, 1424, 1167, 1067. 1H NMR (DMSO-d6)
d: 2.35 (3H,
4.3. General procedure for the synthesis of 4-hydroxy-N0-[1-
phenylethylidene]-2H-1,2-benzothiazine-3-carbohydrazide 1,1-
dioxides 9aen
s, CH3), 2.95 (3H, s, NCH3), 7.52 (2H, d, J ¼ 8.7 Hz, ArH), 7.85e7.89
(5H, m, ArH), 8.01 (1H, d, J ¼ 6.4, 1.2 Hz, ArH), 9.91 (1H, br s, NH),
13.74 (1H, br s, OHenolic). 13C NMR: 16.8, 40.9, 111.5, 123.7, 125.4,
126.8, 128.5, 128.8, 128.9, 131.6, 131.7, 131.9, 132.4, 134.5, 136.5,
147.8, 156.7, 168.9; MS m/z 449 [Mþ], 451 [Mþþ2].
A mixture of 4-hydroxy-2-methyl-2H-1,2-benzothiazine-3-car-
bohydrazide 1,1-dioxide (8) (2.0 mmol), 1-phenylethanone
(2.0 mmol), ortho phosphoric acid (2 drops) and methanol (50 ml)
was heated at 200 W till completion of the reaction (for yields,
reaction conditions and reaction times, see Table 1). The contents
were cooled to 5 ꢂC in an ice bath, filtered and the solids were
washed with cold methanol to get the pure compound.
4.3.7. 4-Hydroxy-N0-[1-(4-iodophenyl)ethylidene]-2-methyl-2H-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9g)
Pale yellow powder; m.p. 239e240 ꢂC. IR (KBr) cmꢁ1: 3677,
3594, 2935, 2364,1652,1423,1175,1058. 1H NMR (DMSO-d6)
d: 2.34
(3H, s, CH3), 2.96 (3H, s, NCH3), 7.65 (2H, d, J ¼ 8.4 Hz, ArH),
7.84e7.91 (5H, m, ArH), 8.09 (1H, d, J ¼ 6.4, 1.2 Hz, ArH), 9.94 (1H, br
s, NH), 13.72 (1H, br s, OHenolic). 13C NMR: 16.8, 40.9, 96.5, 111.4,
123.5, 126.7, 128.9, 130.8, 130.8, 131.9, 132.4, 134.5, 136.4, 137.8,
137.8, 147.8, 156.5, 168.8; MS m/z 497 [Mþ], 499 [Mþþ2].
4.3.1. N0-[1-(2-Chlorophenyl)ethylidene]-4-hydroxy-2-methyl-2H-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9a)
Pale yellow powder; m.p. 235e236 ꢂC. IR (KBr) cmꢁ1: 3678,
3573, 2926, 2365, 1657, 1420, 1173, 1067. 1H NMR (DMSO-d6)
d: 2.37
(3H, s, CH3), 2.92 (3H, s, NCH3), 7.52 (2H, d, J ¼ 8.7 Hz, ArH),
7.85e7.90 (5H, m, ArH), 8.03 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 9.79 (1H,
br s, NH), 13.73 (1H, br s, OHenolic). 13C NMR: 16.1, 40.7, 111.2, 123.4,
126.7, 128., 128.9, 130.0, 130.3, 130.6, 131.9, 132.3, 132.4, 134.3, 137.1,
156.5, 168.6, 168.7; MS m/z 405 [Mþ], 407 [Mþþ2].
4.3.8. N0-[1-(2,4-Difluorophenyl)ethylidene]-4-hydroxy-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9h)
Light yellow powder; m.p. 251 ꢂC. IR (KBr) cmꢁ1: 3678, 3570,
2932, 2362, 1656, 1435, 1178, 1059. 1H NMR (DMSO-d6)
d: 2.30 (3H,
s, CH3), 2.93 (3H, s, NCH3), 6.89 (1H, m, ArH), 7.27 (3H, m, ArH), 7.61
(2H, t, J ¼ 7.8 Hz, ArH), 7.78 (1H, s, ArH), 9.95 (1H, br s, NH), 13.73
(1H, br s, OHenolic). 13C NMR: 16.7, 40.9, 111.3, 111.3, 112.7, 113.9,
123.5, 126.9, 128.9, 131.9, 132.6, 132.7, 134.4, 156.5, 161.4, 163.4,
168.7, 168.9; MS m/z 407 [Mþ], 409 [Mþþ2].
4.3.2. N0-[1-(4-Chlorophenyl)ethylidene]-4-hydroxy-2-methyl-2H-
1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9b)
Light yellow powder; m.p. 240e242 ꢂC. IR (KBr) cmꢁ1: 3677,
3573, 2923, 2365, 1657, 1409, 1170, 1070. 1H NMR (DMSO-d6)
d: 2.35
(3H, s, CH3), 2.93 (3H, s, NCH3), 7.62 (2H, d, J ¼ 8.7 Hz, ArH),
7.83e7.89 (5H, m, ArH), 8.06 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH), 9.86 (1H,
br s, NH), 13.77 (1H, br s, OHenolic). 13C NMR: 16.8, 40.9, 111.5, 123.6,
126.8, 128.3, 128.5, 128.8, 128.9, 129.2, 131.8, 132.5, 134.4, 135.7,
136.8, 147.8, 156.6, 168.8; MS m/z 405 [Mþ], 407 [Mþþ2].
4.3.9. 4-Hydroxy-N0-[1-(2-hydroxyphenyl)ethylidene]-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9i)
Pale yellow powder; m.p. 255e256 ꢂC. IR (KBr) cmꢁ1: 3677, 3573,
2935, 2361, 1658, 1429, 1176, 1060. 1H NMR (DMSO-d6)
d: 2.30 (3H, s,
CH3), 2.94 (3H, s, NCH3), 5.20 (1H, s, OH), 6.89e6.94 (2H, m, ArH),

2376
N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
7.31e7.35 (1H, m, ArH), 7.64e7.67 (1H, dd, J ¼ 6.4, 1.2 Hz, ArH),
7.82e7.92 (3H, m, ArH), 8.04 (1H, m, ArH), 9.97 (1H, br s, NH),13.69(1H,
br s, OHenolic). 13C NMR: 17.0, 40.9,111.4,117.9,118.6,121.5,123.4,126.9,
128.7,131.9,132.0,132.3,132.4,134.4,156.7,162.8,168.9,168.7; MS m/z
387 [Mþ].
water and dried (1.79 g; 75%); Light yellow solid; m.p. 258 ꢂC. IR
(KBr) cmꢁ1: 3353, 1601, 1319, 1140. 1H NMR (CDCl3) (400 M Hz)
2.95 (3H, s, NCH3), 7.55 (1H, t, J ¼ 7.4 Hz, ArH), 7.65 (1H, t, J ¼ 7.2 Hz,
ArH), 7.91 (2H, t, J ¼ 7.8 Hz, ArH), 9.99 (1H, br s, NH), 11.13 (1H, s,
OH), 13C NMR: 31.7, 122.8, 127.5, 127.6, 128.8, 132.3, 133.6, 134.2,
137.7, 151.2; MS m/z: 251 (Mþ).
d
:
4.3.10. 4-Hydroxy-N0-[1-(3-hydroxyphenyl)ethylidene]-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9j)
4.4. Biological activities
Pale yellow powder; m.p. 260e262 ꢂC. IR (KBr) cmꢁ1: 3678,
3573, 2935, 2361, 1658, 1425, 1174, 1059. 1H NMR (DMSO-d6)
d: 2.33
4.4.1. Anti-bacterial activity
(3H, s, CH3), 2.93 (3H, s, NCH3), 5.23 (1H, s, OH), 6.85 (1H, m, ArH),
7.24 (2H, m, ArH), 7.67e7.75 (4H, m, ArH), 8.03 (1H, m, ArH), 9.96
(1H, br s, NH), 13.70 (1H, br s, OHenolic). 13C NMR: 16.6, 40.8, 111.3,
115.0, 118.2, 120.9, 123.5, 126.7, 128.8, 130.3, 131.9, 132.5, 134.3,
135.6, 147.7, 156.6, 158.9, 168.8; MS m/z 387 [Mþ].
The anti-bacterial assay was carried out by microdilution method
[24,25] in order to determine the anti-bacterial activity of compounds
tested against the human pathogenic bacteria. The bacterial suspen-
sions were adjusted with sterile saline to a concentration of 1.0 ꢀ 105
colony forming unit (CFU)/ml. The inocula were prepared daily and
stored at þ4 ꢂC until use. Dilutions of the inocula were cultured on
solid medium to verify the absence of contamination and to check the
validity of the inoculum. The minimum inhibitory concentrations
(MICs) and minimum bactericidal concentrations (MIFs) were deter-
mined using 96-well microtitre plates. Compounds under investiga-
4.3.11. 4-Hydroxy-N0-[1-(4-hydroxyphenyl)ethylidene]-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9k)
Light yellow powder; m.p. 270e273 ꢂC. IR (KBr) cmꢁ1: 3677,
3573, 2926, 2364, 1658, 1446, 1174, 1060. 1H NMR (DMSO-d6)
d: 2.30
(3H, s, CH3), 2.95 (3H, s, NCH3), 5.24 (1H, s, OH), 6.90e6.95 (2H, m,
ArH), 7.29e7.33 (1H, m, ArH), 7.64e7.66 (1H, dd, J ¼ 6.4,1.2 Hz, ArH),
7.80e7.89 (3H, m, ArH), 8.01 (1H, m, ArH), 9.94 (1H, br s, NH), 13.68
(1H, br s, OHenolic). 13C NMR: 16.7, 40.7, 111.2, 115.9, 116.0, 123.2,
126.7, 128.7, 129.0, 129.1, 130.0, 131.8, 132.2, 134.3, 147.6, 156.4,
160.6, 168.6; MS m/z 387 [Mþ].
tion were dissolved in broth LB medium (100 ml) with bacterial
inoculum(1.0ꢀ104 cfuperwell)toachievethedesiredconcentrations
(1 mg/ml). All the microplates were incubated for 24 h at 48 ꢂC. The
lowest concentrations without visible growth (at the binocular
microscope)weredefinedasconcentrationsthatcompletelyinhibited
bacterial growth (MICs). Minimum bactericidal concentrations
(MBCs)weredeterminedbyserialsubcultivationof2
mlintomicrotitre
4.3.12. 4-Hydroxy-N0-[1-(3-methoxyphenyl)ethylidene]-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9l)
plates containing 100 l of broth per well followed by incubation at
m
and further incubation 48 ꢂC for 72 h. The lowest concentration with
no visible growth was defined as the MBC, indicating 99.5% killing of
the original inoculum. The optical density of each well was measured
at a wavelength of 655 nm and compared with a blank. Streptomycin
was used as a positive control (1 mg/ml DMSO).
Off white powder; m.p. 271e274 ꢂC. IR (KBr) cmꢁ1: 3678, 3571,
2927, 2364, 1659, 1437, 1171, 1064. 1H NMR (DMSO-d6)
d: 2.36 (3H, s,
CH3), 2.97 (3H, s, NCH3), 3.94(3H, s, OCH3), 6.77 (1H, m, ArH), 7.35 (2H,
m, ArH), 7.60 (2H, t, J ¼ 7.8 Hz, ArH), 7.76 (2H, t, J ¼ 7.8 Hz, ArH), 8.05
(1H, m, ArH), 9.97 (1H, br s, NH), 13.73 (1H, br s, OHenolic). 13C NMR:
16.6, 40.6, 55.6, 111.2, 113.2, 116.5, 120.5, 123.3, 126.7, 128.8, 129.8,
131.6, 132.2, 134.3, 135.0, 147.7, 156.4, 160.6, 168.6; MS m/z 401 [Mþ].
4.4.2. Anti-fungal activity
Anti-fungal screening of the newly synthesized compounds was
carried out by microdilution technique [24] against A. niger, A. flavus,
A. fumigates, T. viride, T. reesei and D. australiensis. The micromycetes
were maintained on malt agar and the cultures were stored at 4 ꢂC
and sub-cultured once a month [28]. The fungal spores were washed
from the surface of agar plates with sterile 0.85% saline containing
0.1% Tween 80 (v/v). The spore suspension was adjusted with sterile
saline to a concentration of approximately 1.0 ꢀ 105 in a final volume
4.3.13. 4-Hydroxy-N0-[1-(4-methoxyphenyl)ethylidene]-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9m)
Off white powder; m.p. 267e269 ꢂC. IR (KBr) cmꢁ1: 3679, 3573,
2927, 2363, 1661, 1435, 1169, 1085. 1H NMR (DMSO-d6)
d: 2.34 (3H,
s, CH3), 2.95 (3H, s, NCH3), 3.93 (3H, s, OCH3), 7.63 (2H, d, J ¼ 8.7 Hz,
ArH), 7.83e7.89 (5H, m, ArH), 8.07 (1H, d, J ¼ 6.4, 1.2 Hz, ArH), 9.93
(1H, br s, NH), 13.76 (1H, br s, OHenolic). 13C NMR: 16.7, 55.9, 40.9,
111.3,114.4,114.5,123.5,126.9,128.6,128.7,128.9,129.9,131.9,132.5,
134.6, 147.9, 156.6, 162.9, 168.8; MS m/z 401 [Mþ].
of 100 m
l per well. The inocula were stored at 4 ꢂC for further use.
Dilutions of the inocula were cultured on solid malt agar to verify the
absence of contamination and to check the validity of the inoculum.
Minimum inhibitory concentration (MIC) determinations were per-
formed by a serial dilution technique using 96-well microtiter plates.
The compounds investigated were dissolved in DMSO (1 mg/ml) and
added in broth Malt medium with inoculum. The microplates were
incubated for 72 h at 28 ꢂC, respectively. The lowest concentrations
without visible growth (at the binocular microscope) were defined as
MICs. Minimum fungicidal concentrations (MFCs) were determined
4.3.14. N0-[1-(4-Aminophenyl)ethylidene]-4-hydroxy-2-methyl-
2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide (9n)
Yellow powder; m.p. 258e260 ꢂC. IR (KBr) cmꢁ1: 3677, 3573,
2925, 2363,1657, 1425,1173,1059. 1H NMR (DMSO-d6)
d: 2.35 (3H, s,
CH3), 2.91 (3H, s, NCH3), 6.40 (2H, s, NH2), 7.61 (2H, d, J ¼ 8.7 Hz, ArH),
7.84e7.88 (5H, m, ArH), 8.04 (1H, m, ArH), 9.97 (1H, br s, NH), 13.72
(1H, br s, OHenolic).13C NMR: 16.8, 40.9,111.5,114.5,123.5,126.7,127.5,
128.8, 130.0, 131.9, 132.4, 134.5, 147.9, 156.7, 168.9; MS m/z 386 [Mþ].
by serial subcultivation of a 2
ml into microtiter plates containing
100
m
l of broth per well and were further incubated for 72 h at 28 ꢂC.
The lowest concentration with no visible growth was defined as MFC
indicating 99.5% killing of the original inoculum. DMSO was used as
a negative control while commercially available fungicide, bifona-
4.3.15. 4-Methyl-1,4-dihydropyrazolo[4,3-c][1,2]benzothiazin-3-ol
5,5-dioxide (10)
A mixture of methyl 4-hydroxy-2-methyl-2H-1,2-benzothia-
zine-3-carboxylate-1,1-dioxide (7) (2.69 g; 10.0 mmol), hydrazine
hydrate (2.50 g; 50.0 mmol), acetic acid (3.0 ml) and methyl alcohol
(50 ml) was stirred and heated in microwave oven for a period of
4 min. After cooling to room temperature, the contents were
poured over crushed ice and pH was maintained at 1 with conc.
hydrochloric acid. Precipitates obtained were filtered, washed with
zole, was used as positive controls (1e3000 mg/ml).
Acknowledgments
Authors are grateful to HEJ research institute Karachi, Pakistan,
and chemistry department, university of Calgary for the spectral,
biological and crystal analyses respectively.

N. Ahmad et al. / European Journal of Medicinal Chemistry 46 (2011) 2368e2377
[15] Y.L. Janin, Bioorg. Med. Chem. 15 (2007) 2479e2513.
2377
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