Microwave assisted synthesis of biologically active 4-hydroxy-N′- (phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxide derivatives

Article abstract of DOI:10.4067/S0717-97072012000400031

Microwave assisted synthesis of a series of 4-hydroxy-N′- (phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides from sodium saccharin is reported. Sodium o-benzosulfimide was N-alkylated with ethyl chloroacetate followed by base catalyzed ring expansion to six membered 1,2-benzothiazine through Gabriel-Coleman type rearrangement yielding ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide. It was later condensed with a series of benzohydrazides synthesized from various carboxylic acids to get the title compounds. All the synthesized compounds were subjected to preliminary evaluation for their biological activity against series of Gram positive, Gram negative bacteria and fungi. Some of the compounds showed marked activity against the selected microorganisms.

Full text of DOI:10.4067/S0717-97072012000400031

J. Chil. Chem. Soc., 57, Nº 4 (2012)
MICROWAVE ASSISTED SYNTHESIS OF BIOLOGICALLY ACTIVE 4-HYDROXY-N’-(PHENYLCARBONYL)-
2H-1,2-BENZOTHIAZINE-3-CARBOHYDRAZIDE 1,1-DIOXIDE DERIVATIVES
MUHAMMAD ZAHEER1,2, MUHAMMAD ZIA-UR-REHMAN1, SALMA RAHMAN1, NAVEED AHMED3,
AND MUHAMMAD NAWAZ CHAUDHARY2
1Applied Chemistry Research Centre, PCSIR Laboratories Complex, Ferozpur Road, Lahore-54600, Pakistan
2Centre for Envoirnment & Earth Sciences, University of the Punjab, Lahore-54590, Pakistan
3Forman Christian College – A Chartered University, Canal Road, Lahore-54600, Pakistan
(Received: November 22, 2011 - Accepted: September 18, 2012)
ABSTRACT
Microwave assisted synthesis of a series of 4-hydroxy-N’-(phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides from sodium saccharin
is reported. Sodium o-benzosulfimide was N-alkylated with ethyl chloroacetate followed by base catalyzed ring expansion to six membered 1,2-benzothiazine
through Gabriel-Coleman type rearrangement yielding ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide. It was later condensed with a series of
benzohydrazides synthesized from various carboxylic acids to get the title compounds. All the synthesized compounds were subjected to preliminary evaluation
for their biological activity against series of Gram positive, Gram negative bacteria and fungi. Some of the compounds showed marked activity against the selected
microorganisms.
Key Words: Microwaves; Benzohydrazide; Carbohydrazides; 1,2-Benzothiazine; Anti-bacterial; Anti-fungal.
INTRODUCTION
There is a need for development of clean chemical processes including
synthetic procedures, catalysis and reaction conditions to reduce the
environmental impact of chemical & pharmaceutical industry. To achieve
this, innovative and unconventional synthetic procedures using water as
solvent1 under normal2 and super heated conditions3, in supercritical fluids4,
ionic liquids5 and micro-emulsions6 are being used now a days. Besides these,
ultrasound7 and microwaves8 assisted reactions both in solvent as well as in
solventless conditions are becoming popular due to their shorter reaction times
and greater yields.
Hydrazides and related compounds are known as useful building
blocks for the synthesis of various heterocyclic rings9. A large number
of carbohydrazides and their derivatives have been reported in literature
focusing their biological activities which include anti-fungal, anti-viral &
bacteriostatic11, anti-parasite12, anti-tuberculous13 and insecticidal14 activities.
A large number of carbohydrazides and related compounds which are usually
synthesized by hydrazinolysis of carboxylic acid esters are found to possess
versatile activities.15,16
1,2-Benzothiazine 1,1-dioxide nucleus, firstly synthesized by Braun et
al17 is familiar for its various derivatives like Sudoxicam®, Ampiroxicam®,
EXPERIMENTAL
Meloxicam® and Lornoxicam®. These are known as oxicams and are being
used worldwide as non-steroidal anti-inflammatory drugs (NSAIDs)18 [Figure
Chemistry:
1]. Benzothiazine derivativesare also familiar for theirother biological activities
All the chemicals were purchased from E. Merck, BDH or Fluka and used
e.g., 3,4-dihydro-1,2-benzothiazine-3-carboxylate 1,1-dioxide α-ketomide and
without purification. However, solvents were purified through distillation.1H-
P(2)–P(3) peptide mimetic aldehyde compounds are reported as potent calpain
NMR spectra were recorded on a Bruker DPX-400 instrument at 400 MHz.
I inhibitors19 while 1,2-benzothiazin-3-yl-quinazolin-4(3H)-ones possess anti-
Chemical shifts are reported in ppm referenced to the residual solvent signal.
bacterial properties20. They have also been found as anti-oxidants21-24 and for
FT-IR spectra were recorded on a Bruker Tensor 27 spectrometer. Mass spectra
the treatment of rheumatoid arthritis, ankylosing spondylitis, osteoarthrosis
were recorded on Agilent 5973 N instrument using EI mode. Melting points
and other inflammatory rheumatic and non-rheumatic processes25.
were recorded on a Mettller Toledo FP 62 melting point apparatus and are
uncorrected. Microwave assisted reactions were carried out in a household
Keeping in view the potential biological activities of 1,2-benzothiazine-
Microwave oven (Orient eNNe781JF) equipped with inverter technology
1,1-dioxides and carbohydrazides, it was perceived that if both the heterocyclic
(generating fixed frequency throughout the required time) for realistic control
moieties are synergized in a single nucleus, the newly obtained compounds
of the microwaves operating at multiples of 100 Watts up to 1000 Watts
may possess significant biological activities of individual compounds. In
generating 2450 MHz frequency. The apparatus was modified for laboratory
this quest, novel 4-hydroxy-N’-(phenylcarbonyl)-2H-1,2-benzothiazine-3-
applications, equipped with magnetic stirrer and an external reflux condenser.
carbohydrazide 1,1-dioxides derivatives (8a-l) are synthesized by microwave
Synthesis of ethyl (1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-
assisted reactions of various benzohydrazides (7a-l) and ethyl 4-hydroxy-2H-
acetate (3)
1,2-benzothiazine-3-carboxylate 1,1-dioxide (4) along with their preliminary
A mixture of ethylchloroacetate (1.19 g, 9.71 mmoles), sodium saccharin
assay for anti-bacterial and anti-fungal activities [Scheme 1].
(1.50 g, 7.31 mmoles) and dimethylformamide (10 ml) was taken in a round
bottom flask. The mixture was heated at water bath for 4 hours. The reaction
mixture was cooled to room temperature and poured into ice cold water (60
ml) to get white solid, which was filtered and washed with water. The solid was
o
o
dried to get white crystalline solid mp 105-106 C (Lit. mp 104-106 C).17 IR
(KBr):1752, 1743, 1340, 1181 cm-1. 1H-NMR (CDCl3), δ: 1.29 (t, J = 7.2 Hz,
1492

J. Chil. Chem. Soc., 57, Nº 4 (2012)
3H), 4.30 (q, J = 7.2Hz, 2H), 4.41 (s, 2H), 7.74 (dd, J = 5.6, 3.2 Hz, 2H), 7.80
(dd, J = 5.6, 3.2Hz, 2H). MS m/z: 269 [M+], 224 [M+-OC2H ]. Anal. Calcd for
C H11NO S: C, 49.06; H, 4.12; N, 5.20; Found: C, 49.08;5H, 4.11; N, 5.19.
H1R1 MS: C5alcd. 269.0358, Found 269.0360.
Synthesis of ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate
1,1-dioxide (4)
of reaction. After the completion of reaction, excess ethanol was distilled off
and acidified with HCl (5%). The product obtained was washed with ice cold
water and dried in oven.
4-Hydroxy-N’-[(2-hydroxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8a)
Pale yellow powder; mp: 116˚C. IR (KBr) Cm-1 : 3749, 3653, 3562,
1
Sodium metal (2.7 g, 115 mmoles) was added to dry ethanol (60 ml)
in small portions. On the complete dissolution of sodium, the solution was
concentrated in vacuum for the synthesis of sodium ethoxide suspension. To
it ester (4) (10.0g, 37.17 mmol) and absolute ethanol (30 ml) were added and
sonicated for 4 minutes in ultrasonic bath at room temperature. Hydrochloric
acid (430 ml) was added to the reaction mixture to precipitate the product
which was filtered and washed with water.
2365, 1643, 1348, 1177, 1069. H NMR (DMSO-d6)(400MHz) δ: 3.43(1H,
s, SO2NH),6.90-6.97(4H, m, ArH), 7.75-7.89 (4H, m, ArH), 8 .18(1H, s,
-OH)9.72(1H, s,-NH), 9.94(1H, s,-NH),13.55(1H, s, -OH). MS m/z 375.0 [M+]
4-Hydroxy-N’-[(3-hydroxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8b)
Dirty yellow powder; mp: 150˚C. IR (KBr) Cm-1 : 3760, 3657, 3568,
1
2366, 1642, 1349, 1180, 1070. H NMR (DMSO-d6)(400MHz) δ: 3.31(1H, s,
White crystalline solid; mp 138-139 oC (Lit. mp 136-139 oC).17 IR (KBr):
SO2NH), 6.96(1H, d, J=7.6 Hz, ArH), 7.12 (1H, d, J=8.0 Hz, ArH), 7.25-
7.42 (4H, m, ArH), 7.77(1H, s, ArH), 7.87(1H, d, J=7.2 Hz, ArH), 8 .19(1H, s,
-OH)9.72(1H, s,-NH), 9.94(1H, s,-NH), 13.55(1H, s, -OH). MS m/z 375.0 [M+]
4-Hydroxy-N’-[(4-hydroxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8c)
1
3180, 1667, 1338, 1179 cm−1. H-NMR (CDCl3), δ: 1.33 (t, J = 7.2 Hz, 3H),
4.25 (q, J = 7.2 Hz, 2H), 6.42 (br s, NH), 7.74 (dd, J = 5.6, 3.2 Hz, 2H), 7.80
(dd, J = 5.6, 3.2Hz, 2H), 12.38 (s, 1H). MS m/z: 269 [M+], 268 [M+-H], 224
[M+-OC2H5]. HRMS: Calcd: 269.0358, Found: 269.0359.
General procedure for the synthesis of alkyl benzoates compounds
(6a-l)
A mixture of substituted benzoic acid in methanol (50ml) and concentrated
sulphuric acid (2-3 drops) was refluxed in microwave oven till the completion
of reaction as indicated by TLC. After completion of reaction; excess methanol
was distilled off and the content were poured in ice cold aqueous sodium
bicarbonate (4%). The product obtained was washed with ice cold water and
dried in oven. Experimental details are given in Table 1.
Dirty yellow powder; mp: 185˚C. IR (KBr) Cm-1 : 3753, 3655, 3568, 2365,
1648, 1345, 1189, 1078. 1H NMR (DMSO-d )(400MHz) δ: 3.36(1H, s, SO2NH
), 6.92(2H, d, J=8.0 Hz, ArH), 7.36 (2H, d,6 J=8.4 Hz, ArH), 7.97-8.05 (4H,
m, ArH), 8 .20(1H, s, -OH),9.78(1H, s,-NH), 9.99(1H, s,-NH), 13.57(1H, s,
-OH). MS m/z 375.0 [M+]
4-Hydroxy-N’-[(2-methoxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8d)
Brownish yellow powder; mp: 112˚C. IR (KBr) Cm-1 : 3751, 3654, 3564,
1
General procedure for the synthesis of substituted benzohydrazides
(7a-l)
2364, 1642, 1349, 1178, 1069. H NMR (DMSO-d6)(400MHz) δ: 3.49(1H,
s, SO NH), 3.90(3H, s, -OCH3 ),6.89-6.95(4H, m, ArH), 7.77-7.90 (4H, m,
ArH),210.18(1H, s,-NH),-10.31(1H, s,-NH), 13.60(1H, s, -OH). MS m/z 389.3
[M+]
4-Hydroxy-N’-[(3-methoxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8e)
A mixture of substituted alkyl benzoate (6a-1) (0.001moles) and hydrazine
hydrate (0.001moles) in ethanol (50ml) was refluxed in a microwave oven
till the completion of reaction as indicated by TLC. After the completion of
reaction; excess ethanol was distilled off and acidified with hydrochloric acid
(5%). The product obtained was washed with ice cold water and dried in oven.
Experimental details are given in Table 1.
Yellow powder; mp: 152˚C. IR (KBr) Cm-1 : 3754, 3653, 3564, 2364,
1645, 1352, 1182, 1071. 1H NMR (DMSO-d6)(400MHz) δ: 3.37(1H, s,
SO NH), 3.93(3H, s, -OCH ), 6.95(1H, d, J=7.6 Hz, ArH), 7.16 (1H, d, J=8.0
Hz,2 ArH), 7.26-7.45 (4H, 3m, ArH), 7.75(1H, s, ArH), 7.88(1H, d, J=7.2 Hz,
ArH),9.79(1H, s,-NH), 9.96(1H, s,-NH), 13.61(1H, s, -OH).MS m/z 389.3 [M+]
4-Hydroxy-N’-[(4-methoxyphenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8f)
General procedure for the synthesis of derivatives of 4-hydroxy-N’-
(phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxides
(8a-l)
A mixture of substituted benzohydrazide [7a-1] (0.001moles) and ethyl
4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide (4) (0.001moles;
0.269g) in ethanol (50ml) was refluxed in a microwave oven till the completion
Brownish yellow powder; mp: 119˚C. IR (KBr) Cm-1 : 3751, 3654, 3564,
1493

J. Chil. Chem. Soc., 57, Nº 4 (2012)
1
2364, 1642, 1349, 1178, 1069. H NMR (DMSO-d6)(400MHz) δ: 3.39(1H, s,
RESULTS AND DISCUSSION
SO NH), 3.91(3H, s, -OCH3 ),6.96(2H, d, J=8.0 Hz, ArH), 7.29 (2H, d, J=8.4
Hz,2 ArH), 7.90-8.01 (4H, m, ArH), 10.19(1H, s,-NH), 10.32(1H, s,-NH),
13.56(1H, s, -OH). MS m/z 389.3 [M+].
During the course of the work, using the green innovative technology ethyl
(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)- acetate (3) was synthesized
by the reaction of sodium saccharin (1) and ethyl chloroacetate (2) followed
by its ring expansion to six membered benzothiazine through Gabriel-Coleman
type rearrangement yielding ethyl 4-hydroxy-2H-1,2-benzothiazine-3-
carboxylate 1,1-dioxide (4). The formation of ethyl (1,1-dioxido-3-oxo-1,2-
benzisothiazol-2(3H)-yl)- acetate (3) was carried out on water bath and the
formation of ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide
(4) was carried out using ultrasonic waves.
Synthesis of alkyl benzoates (6a-l), substituted benzohydrazides (7a-l)
and 4-hydroxy-N’-(phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide
1,1-dioxides compounds (8a-l) were synthesized as per scheme 1 through
environment friendly fashion by the use of microwaves. Alkyl benzoate
derivatives (6a-l), were synthesized by acid catalysis of respective benzoic
acid under microwaves in excellent yields (92 - 97%; Table 1) which were
converted to the corresponding benzohydrazide derivatives (7a-l) by
microwave induced hydrazinolyses in very good to excellent yields (81-96%).
These benzohydrazides were then reacted with ethyl 4-hydroxy-2H-1,2-
benzothiazine-3-carboxylate 1,1-dioxide (4) to get a series of 4-hydroxy-N’-
(phenylcarbonyl)-2H-1,2-benzothiazine-3-carbohydrazide 1,1-dioxides (8a-
l). These reactions were carried out under the influence of microwaves and
were completed in good yields with in shorter reaction times (Table 1). All of
the newly synthesized compounds were characterized through spectroscopic
techniques and their data was found in accordance to the proposed structures.
N’-[(2-Bromophenyl)carbonyl]-4-hydroxy-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8g)
Light yellow powder; mp: 116˚C. IR (KBr) Cm-1 : 3751, 3654, 3564,
1
2364, 1642, 1349, 1178, 1069. H NMR (DMSO-d6)(400MHz) δ: 3.49(1H,
s, SO2NH),6.90-6.95(4H, m, ArH), 7.78-7.89 (4H, m, ArH),10.17(1H, s,-
NH),10.30(1H, s,-NH), 13.61 (1H, s, -OH). MS m/z 438[M+]
N’-[(3-Bromophenyl)carbonyl]-4-hydroxy-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8h)
Light brown powder; mp: 216˚C. IR (KBr) Cm-1 : 3754, 3656, 3563,
1
2365, 1643, 1348, 1180, 1072. H NMR (DMSO-d6)(400MHz) δ: 3.47(1H, s,
SO2NH), 6.95(1H, d, J=7.6 Hz, ArH), 7.19 (1H, d, J=8.0 Hz, ArH), 7.25-
7.44 (4H, m, ArH), 7.78(1H, s, ArH), 7.89(1H, d, J=7.2 Hz, ArH),10.19(1H,
s,-NH), 10.27(1H, s,-NH), 13.59(1H, s, -OH).MS m/z 438[M+]
N’-[(4-Bromophenyl)carbonyl]-4-hydroxy-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8i)
Light brownish powder; mp: 260˚C. IR (KBr) Cm-1 : 3751, 3654, 3564,
1
2364, 1642, 1349, 1178, 1069. H NMR (DMSO-d6)(400MHz) δ: 3.39(1H, s,
SO2NH ), 6.96(2H, d, J=8.0 Hz, ArH), 7.30 (2H, d, J=8.4 Hz, ArH), 7.92-
8.02 (4H, m, ArH), 10.19(1H, s,-NH), 10.34(1H, s,-NH), 13.60(1H, s, -OH).
MS m/z: 438[M+], 440[M++2].
4-Hydroxy-N’-[(2-nitrophenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8j)
Pale yellow powder; mp: 155˚C. IR (KBr) Cm-1 : 3754, 3651, 3563,
1
2363, 1644, 1347, 1182, 1071. H NMR (DMSO-d )(400MHz) δ: 3.50(1H, s,
Anti-bacterial activity
SO2NH),6.90-6.96(4H, m, ArH), 7.80-7.91 (4H, m6, ArH),10.16(1H, s,-NH),-
All the newly synthesized carbohydrazide derivatives (dissolved in
dimethylformamide) were subjected to anti-bacterial screening against
three gram positive bacteria (Bacillus cereus, Bacillus subtilis and Bacillus
thuringiensis) and three gram negative bacteria (Escherichia coli, Pseudomonas
aeruginosa, Salmonella typhi) by determining their minimum inhibitory
concentrations (MICs) using agar dilution technique29 and streptomycin as a
reference drug. The choice of streptomycin as a clinical standard was made due
to the fact that at lower concentrations, it only inhibits growth of the bacteria
through induction of prokaryotic ribosomes to misread mRNA. It 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 bacteria27.
The Minimum inhibitory concentrations of the synthesized compounds
against selected bacteria are presented in Table 2. Results of anti-bacterial
activity of compounds show that the minimum inhibitory concentration
(MIC) of compounds varies in the range of 6.0 - 67.0 x 10-2 µmol/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 Bacillus subtilis, while Pseudomonas aeruginosa is the
most resistant species. An insight to the structure-activity relationship gives
an idea that activity generally increases with the incorporation of hydroxyl
and methoxy groups at the benzene ring of the title compounds. Hydroxy
derivatives (8a-8c) were found the most active with MIC values comparable
to the standard and this may be attributed to the presence of lone pairs on
oxygen atoms of hydroxyl groups perhaps by blocking the active sites through
hydrogen bonding. Compounds having methoxy groups are found weaker
than the hydroxyl analogues probably due to steric hindrance of methyl group.
Compounds possessing nitro groups (8j-8l) were found least active against
both the gram positive and gram negative strains.
10.29(1H, s,-NH), 13.62(1H, s, -OH). MS m/z 403.9 [M+]
4-Hydroxy-N’-[(3-nitrophenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8k)
Yellow powder; mp: 230˚C. IR (KBr) Cm-1 : 3751, 3654, 3564, 2364, 1642,
1349, 1178, 1069. 1H NMR (DMSO-d )(400MHz) δ: 3.31(1H, s, SO2NH),
6.98(1H, d, J=7.6 Hz, ArH), 7.14 (1H,6d, J=8.0 Hz, ArH), 7.23-7.41 (4H,
m, ArH), 7.79(1H, s, ArH), 7.88(1H, d, J=7.2 Hz, ArH),9.77(1H, s,-NH),
9.98(1H, s,-NH),13.59(1H, s, -OH). MS m/z 403.9 [M+]
4-Hydroxy-N’-[(4-nitrophenyl)carbonyl]-2H-1,2-benzothiazine-3-
carbohydrazide 1,1-dioxide (8l)
Pale yellow powder; mp: 155˚C. IR (KBr) Cm-1 : 3754, 3651, 3563,
1
2363, 1644, 1347, 1182, 1071. H NMR (DMSO-d6)(400MHz) δ: 3.49(1H, s,
SO2NH ),6.87-6.97(4H, m, ArH), 7.67-7.82 (4H, m, ArH),10.17(1H, s,-NH),-
10.33(1H, s,-NH), 13.62(1H, s, -OH). MS m/z 403.9 [M+]
ANTI-MICROBIAL ACTIVITIES:
Anti-Bacterial Activity: The anti-bacterial assay was carried out by
microdilution method26,27 against human pathogenic bacteria. The bacterial
suspensions were adjusted with sterile saline to a concentration of 1.0 x 105
colony forming unit (CFU)/ml. The inocula were prepared daily and stored
at +4°C 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) were determined using 96-
well microtitre plates. Compounds under investigation were dissolved in broth
LB medium (100 µl) with bacterial inoculum (1.0 x 104 cfu per well) to achieve
the desired concentrations (1mg/ ml). All the microplates were incubated for 24
hours at 48°C. 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).
Anti-fungal Activity: Anti-fungal screening of the newly synthesized
compounds was carried out by micro dilution technique26 against Aspergillus
flavus, Trichoderma reesei, Drechslera australiensis, Trichoderma viride,
Alternaria alternate and Macrophomina phaseolina. The micromycetes were
maintained on malt agar and the cultures were stored at 4°C and sub-cultured
once a month28. Fungal spores were washed from the surface of agar plates with
sterile 0.85% saline containing 0.1% Tween 80 (v/v) and the spore suspension
was adjusted with sterile saline to a concentration of approximately 1.0 x 105
in a final volume of 100 µl per well. Minimum inhibitory concentration (MIC)
determinations were performed by a serial dilution technique using 96-well
microtiter plates. The compounds investigated were dissolved in DMSO (1 mg/
ml) and were added in broth malt medium with inoculum. The microplates
were incubated for 72 hours at 28°C. The lowest concentrations without visible
growth (at the binocular microscope) were defined as MICs.
1494

J. Chil. Chem. Soc., 57, Nº 4 (2012)
Table 1. Data of the synthesized compounds (6a- 8l).
Reaction
time
Entry
Compound
R1
R2
R3
M.P/ B.P
Yield
6a
6b
6c
6d
6e
6f
OH
H
H
OH
H
H
H
224˚C*
70˚C
17 minutes
94%
93%
96%
94%
95%
96%
95%
94%
96%
92%
97%
95%
85%
81%
93%
87%
86%
96%
89%
83%
91%
88%
95%
91%
81%
79%
84%
81%
77%
85%
75%
73%
80%
82%
86%
74%
19 minutes
13 minutes
18 minutes
20 minutes
15 minutes
15 minutes
18 minutes
12 minutes
19 minutes
14 minutes
16 minutes
10 minutes
15 minutes
12 minutes
13 minutes
16 minutes
11 minutes
14 minutes
15 minutes
14 minutes
18 minutes
12 minutes
15 minutes
21minutes
23 minutes
17 minutes
15 minutes
18 minutes
12 minutes
20 minutes
25 minutes
15 minutes
22 minutes
14 minutes
20 minutes
H
OH
H
126˚C
247˚C*
238˚C*
50˚C
OCH3
H
H
OCH3
H
H
H
OCH3
H
6g
6h
6i
Br
H
H
241˚C*
140˚C*
78˚C
-Br
H
H
H
Br
H
6j
NO2
H
H
275˚C*
78˚C
6k
6l
NO2
H
H
H
NO2
H
96˚C
7a
7b
7c
7d
7e
7f
OH
H
H
148˚C
88˚C
OH
H
H
H
OH
H
266˚C dec.
78˚C
OCH3
H
H
OCH3
H
H
92˚C
H
OCH3
H
137˚C
152˚C
155˚C
168˚C
120˚C
152˚C
215˚C
116˚C
150˚C
185˚C
112˚C
152˚C
119˚C
116˚C
216˚C
260˚C
155˚C
230˚C
155˚C
7g
7h
7i
Br
H
H
Br
H
H
H
Br
H
7j
NO2
H
H
7k
7l
NO2
H
H
H
NO2
H
8a
8b
8c
8d
8e
8f
OH
H
H
OH
H
H
H
OH
H
OCH3
H
H
OCH3
H
H
H
OCH3
H
8g
8h
8i
Br
H
H
Br
H
H
H
Br
H
8j
NO2
H
H
8k
8l
NO2
H
H
H
NO2
* = Boiling point
Dec. = Decomposition
Table 2. Anti-bacterial activity of compounds (8a-l) tested by micro dilution method (MIC µmol x 10-2).
Susceptible Bacteria
Entry
Compound
E. coli
25
P. aeruginosa
S. typhi
22
B. cereus
B. subtilis
B. thuringiensis
1
2
8a
26
23
28
35
31
46
56
59
67
44
49
51
0
9
8
6
22
20
23
20
19
21
25
23
27
18
20
18
0
8b
21
21
11
14
17
16
19
22
18
20
20
20
23
0
3
8c
30
24
9
4
8d
39
38
14
18
15
22
24
20
17
19
24
0
5
8e
37
33
6
8f
40
42
7
8g
60
55
8
8h
66
51
9
8i
61
49
10
11
12
13
14
8j
8k
58
48
59
42
8l
63
52
Control
Streptomycin
0
0
16.5
16.5
16.5
4.3
4.2
4.4
1495

J. Chil. Chem. Soc., 57, Nº 4 (2012)
Anti-fungal activities
All the newly synthesized compounds 8a–8l (dissolved in dimethylsulfoxide) along with ethyl 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide
(4), bifonazole (reference drug) and control 0 were subjected to in-vitro anti-fungal screening by determining the minimum inhibitory concentration (MICs)
through micro dilution technique29 against a panel of six strains of fungi i.e., Aspergillus flavus, Trichoderma reesei, Drechslera australiensis, Trichoderma viride,
Alternaria alternate and Macrophomina phaseolina.
Results of anti-fungal activity of compounds show that the minimum inhibitory concentration (MIC) of compounds varies in the range of 68.1 – 124.0 x 10-2
µmol/ml [Table 3]. Compounds 8j, 8k and 8l showed the best activity against all the fungi, with 8i exhibiting the highest antifungal potential (MIC 68.1 x 10-2
µmol/ml, while compounds 8g, 8h and 8i were found moderately active. Rest of the compounds were found almost inactive (with MIC >100 x 10-2 µmol/ml).
Table 3. Anti-fungal activity of compounds (8a-l) tested by micro dilution method (MIC µmol x 10-2).
Susceptible Fungi
Entry
Compound
Aspergillus
flavus
Trichoderma
reesei
Drechslera
australiensis
Trichoderma
Alternaria
alternata
Macrophomina
phaseolina
viride
1
2
8a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0
98.0
96.5
99.6
81.3
80.6
79.9
77.4
74.9
72.7
72.5
70.1
68.4
0
>100
>100
>100
>100
92.8
89.9
99.7
98.8
95.4
92.0
90.9
87.0
0
97.3
94.2
97.9
98.8
97.6
95.1
75.6
74.4
71.6
71.9
69.6
68.1
0
98.0
>100
>100
>100
>100
93.3
81.5
78.9
77.3
80.1
77.8
74.8
0
96.5
95.2
>100
>100
>100
96.2
87.4
85.6
83.9
82.6
81.2
78.3
0
8b
3
8c
4
8d
5
8e
6
8f
7
8g
8
8h
9
8i
10
11
12
13
14
15
8j
8k
8l
0
4
>100
48.0
>100
60.2
>100
57.5
>100
64.0
>100
58.0
>100
58.0
Bifonazole
Results showed moderate to significant activity of almost all the compounds
against Trichoderma reesei, Drechslera australiensis, Trichoderma viride,
Alternaria alternate and Macrophomina phaseolina while these were found
inactive against Aspergillus flavus; all of the compounds were either found
inactive or with MIC greater than 100. Majority of the compounds showed
good activity against Trichoderma viride and Trichoderma reesei while
Aspergillus flavus is the most resistant species. An insight to the structure–
activity relationship gives an idea that activity generally increases with
substitution groups having electron withdrawing nature.
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CONCLUSION
The present study revealed that the compounds obtained by synergism
of 4-hydroxy-2H-1,2-benzothiazine-3-carboxylate 1,1-dioxide moiety with
different carbohydrazides in one frame were found to possess anti-bacterial &
anti-fungal 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
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