Synthesis and biological activity of substituted 4H-1,4-benzothiazines, their sulfones, and ribofuranosides

Article abstract of DOI:10.1080/15257770.2013.765012

The present article describes the synthesis of new 4H-1,4-benzothiazines via condensation and oxidative cyclization of substituted 2-aminobenzenethiols with β-diketones/β-ketoesters in dimethyl sulfoxide. The oxidation of these synthesized 4H-1,4-benzothi

Full text of DOI:10.1080/15257770.2013.765012

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Synthesis And Biological Activity Of
Substituted 4H-1,4-Benzothiazines,
Their Sulfones, And Ribofuranosides
Kshamta Goyal a , Naveen Gautam b , Nishidha Khandelwal a & D. C.
Gautam a
a Department of Chemistry, University of Rajasthan, Jaipur, India
b L.B.S. Govt. P.G. College, Kotputli, Jaipur, India
Version of record first published: 28 Feb 2013.
To cite this article: Kshamta Goyal , Naveen Gautam , Nishidha Khandelwal & D. C. Gautam
(2013): Synthesis And Biological Activity Of Substituted 4H-1,4-Benzothiazines, Their Sulfones, And
Ribofuranosides, Nucleosides, Nucleotides and Nucleic Acids, 32:2, 81-97
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Nucleosides, Nucleotides and Nucleic Acids, 32:81–97, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.765012
SYNTHESIS AND BIOLOGICAL ACTIVITY OF SUBSTITUTED
4H-1,4-BENZOTHIAZINES, THEIR SULFONES,
AND RIBOFURANOSIDES
Kshamta Goyal,1 Naveen Gautam,2 Nishidha Khandelwal,1
and D. C. Gautam1
1Department of Chemistry, University of Rajasthan, Jaipur, India
2L.B.S. Govt. P.G. College, Kotputli, Jaipur, India
The present article describes the synthesis of new 4H-1,4-benzothiazines via condensation and
2
oxidative cyclization of substituted 2-aminobenzenethiols with β-diketones/β-ketoesters in dimethyl
sulfoxide. The oxidation of these synthesized 4H-1,4-benzothiazines with 30% hydrogen peroxide
in glacial acetic acid yielded 4H-1,4-benzothiazine sulfones and the reaction of these synthesized
benzothiazines with sugar (β-D-ribofuranose-1-acetate-2,3,5-tribenzoate) afforded the new ribofura-
nosides. These compounds were evaluated for their antioxidant and antimicrobial activities (using
broth microdilution method). The structural assignments of the synthesized compounds were made
on the basis of elemental analyses and spectroscopic data.
Keywords Benzothiazines; ribofuranosides; sulfones; antioxidant activities; antimicro-
bial activities
INTRODUCTION
The synthesis of heterocyclic moieties such as 4H-1,4-benzothiazines,
their sulfones and ribofuranosides by molecular modification has been of
tremendous interest in recent years because of their biological, pharmaco-
logical, and industrial significance and they are an integral part of many
naturally occurring and biologically active compounds.[1–9]
Received 24 October 2012; accepted 7 January 2013.
The authors are grateful to the Department of Chemistry, University of Rajasthan, Jaipur for pro-
viding necessary facilities. UGC, New Delhi is duly acknowledged for financial support. Authors are also
thankful to Institute of Applied Sciences and Biotechnology (Chemind Biosolutions), Jaipur and De-
partment of Zoology, University of Rajasthan for assistance in carrying out antimicrobial and antioxidant
activities respectively.
Address correspondence to Kshamta Goyal, W/o Sunil Kumar Agrawal, 230, Rameshward-
ham, Murlipura Scheme, Jaipur-302013, Rajasthan, India. E-mail: kshamtaa@gmail.com; drdcgautam@
yahoo.co.in
81

82
K. Goyal et al.
4H-1,4-benzothiazines constitute an important class of heterocycles con-
taining 1,4-thiazine ring fused to benzene ring. These compounds are poten-
tial pharmacological and industrially useful agents and are extensively used
as tranquilizer, sedative, antispasmodic, CNS depressant, antiulcer, antibac-
terial, antifungal, antioxidant, anticancer agents, etc. A slight change in the
substitution pattern in benzothiazine nucleus cause distinguishable differ-
ence in their biological activities. Therefore, these observations prompted
us to synthesize new 4H-1,4-benzothiazines, their sulfones and ribofurano-
sides, which would help to design new and improved pharmacological ap-
proaches based on a better understanding of the correlation between chem-
ical structures. On refluxing with 30% hydrogen peroxide in glacial acetic
acid, 4H-1,4-benzothiazines yielded 4H-1,4-benzothiazine-1,1-dioxides. Ben-
zothiazines surve as heterocyclic base for the formation of ribofuranosides
on treatment with sugar. These prepared sulfones and ribofuranosides too
possess similar chemotherapeutic activities. All the synthesized compounds
were screened for their antioxidant[10–13] and antimicrobial activities.[14–17]
RESULTS AND DISCUSSION
Substituted 4H-1,4-benzothiazines were prepared by condensation of
substituted 2-aminobenzenethiols 1a,b with β-diketones/β-ketoesters 3a–d
in the presence of dimethyl sulfoxide which cause oxidative cyclization.
The mechanism of the reaction involves an intermediate, that is, bis-(2-
aminophenyl) disulfides 2a,b which were obtained by the readily oxidation
of substituted 2-aminobenzenethiols 1a,b. The bis-(2-aminophenyl) disul-
fides underwent cyclization by the scission of the sulfur–sulfur bond due to
high reactivity of α-position of the enaminoketone system 4a–d toward nucle-
ophilic attack leading to the formation of substituted 4H-1,4-benzothiazines
5a–d. Oxidation of 4H-1,4-benzothiazines 5a–d with 30% hydrogen perox-
ide in glacial acetic acid yielded their corresponding sulfones 6a–d. Syn-
thesized benzothiazines 5a–b were treated with β-D- ribofuranose-1-acetate-
2,3,5-tribenzoate in toluene stirred in vacuum on an oil bath at 155–160◦C
for 10 hours to form ribofuranosides 7a–b. β-diketones/β-ketoesters are ca-
pable of forming two isomeric enol tautomers both reacting as nucleophiles
(Scheme 1).
SCHEME 1 Tautomerism in β-Diketones/β-Ketoesters.
Hence, there is a possibility for the formation of two types of 4H-1,4-
benzothiazines 5a–d and 5aꢁ–dꢁ, but only one type of 4H-1, 4-benzothiazines
5a–d were isolated. Mass spectral investigation discarded the possibility

Benzothiazines, Their Sulfones, and Ribofuranosides
83
of 4H-1,4-benzothiazines 5aꢁ–dꢁ since spectral investigations proved that
the benzothiazines obtained contain COR3 linkage (appearing as base
peak), which suggested that under the experimental conditions (employing
dimethyl sulfoxide, a polar solvent) the tautomers 3a–d were involved and
SCHEME 2 Mechanism for the synthesis of 4H-1,4-benzothiazines, their sulfones and ribofuranosides.

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K. Goyal et al.
resulted in the formation of 4H-1, 4-benzothiazines 5a–d via the formation
of intermediate enaminoketones 4a–d (Scheme 2). The structures proposed
for the synthesized compounds were elucidated by spectroscopic data and
elemental analyses. All the synthesized compounds were also screened for
their antioxidant and antimicrobial activities.
Antioxidant Activity
The present study revealed that the synthesized compounds showed
mixed radical scavenging activity in both 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging assay and 2,2-azinobis (3-ethylbenzothiazoline-6-
sulfonic acid) (ABTS) radical cation decolorization assay. These compounds
showed good chemopreventive and antigenotoxic effect.
a. DPPH stable free radical method is an easy, rapid, and sensitive way
to survey the antioxidant activity of a specific compound. Since IC50
value is inversely related to the antioxidant activity, so the highest
radical scavenging activity was showed by compound 5b with IC50 value
(71.14 μg/mL) and the lowest radical scavenging activity was exhibited
by compounds 5d and 6d with IC50 values (>1000 μg/mL). The IC50
values of ascorbic acid was about 50.00 μg/mL. Thus, the result of
the present study demonstrated that compounds 5a–c, 6a–c, and 7a–b
exhibited antioxidant activity (i.e., inhibitory activity against the DPPH
radical) but lower than that of ascorbic acid.
b. Compounds 5a, 5b, 5c, 6b, 7a, and 7b were found to be more active in
ABTS+ assays which showed much decline in graph.
ANTIMICROBIAL ACTIVITY
The present paper is focused on the synthesis of novel benzothiazine
derivatives as possible antibacterial and antifungal agents. A series of novel
compounds 5a–d, 6a–d, and 7a–b were prepared and tested for their in vitro
antibacterial activity against the four strains of bacteria (two gram positive
and two gram negative) and antifungal activity against the four strains of
fungi. Four compounds (6a–d) of the obtained series showed high in vitro
antimicrobial activity.
Compounds 6a–d showed excellent activity against all the four strains
of bacteria (Bacillus cereus, Staphylococcus aureus, E. coli DH 5 alpha and Pseu-
domonas aeruginosa). This indicated that all these four compounds showed
in vitro antibacterial activity lower than that of ampicillin sodium salt. Com-
pounds 5c and 5d showed good activity against all the four strains of bacteria
indicating in vitro antibacterial activity slightly higher than that of ampicillin
sodium salt. Compounds 5a, 7a and 5b, 7b showed moderate activity against

Benzothiazines, Their Sulfones, and Ribofuranosides
85
all the four strains of bacteria. This indicated that these compounds showed
in vitro antibacterial activity higher than that of ampicillin sodium salt.
Compounds 6a–d showed excellent activity against all the four strains
of fungi (Aspergillus niger, Aspergillus flavus, Fusarium oxysporum, and Rhizopus
stolonifer). This indicated that all these four compounds showed in vitro
antifungal activity lower than that of ketoconazole. Compounds 5c and 5d
showed good activity against all the four strains of fungi indicating in vitro
antifungal activity slightly higher than that of ketoconazole. Compounds 5a,
7a and 5b, 7b showed moderate activity against all the four strains of fungi.
This indicated that these compounds showed in vitro antifungal activity
higher than that of ketoconazole.
STRUCTURE-ACTIVITY RELATIONSHIPS
Structure-activity relationships between benzothiazines, their sulfones
and ribofuranosides have been expressed in terms of MIC. Benzothiazine’s
sulfones (6a–d) have superior antimicrobial properties (i.e., biologically
more active) than their corresponding benzothiazines (5a–d) and their
ribofuranosides (7a–b) as it is evidenced by their antimicrobial activities
expressed in terms of MIC (Table 1).
CONCLUSION
The structures proposed for the synthesized compounds were well sup-
ported by elemental analysis and spectroscopic data. Regarding both DPPH
and ABTS assays, compounds 5b and 7b showed excellent activity. Com-
pounds 6a–c showed moderate activity as compared to their benzothiazines
5a–c respectively due to electron withdrawing effect of oxygen in 6a–c. Com-
pounds 5a–b showed good antioxidant activity as compared to their ribo-
furanosides 7a–b respectively due to slight electron withdrawing effect of
ribofuranosyl moiety in the framework of 7a–b. In general, the presence
of electron donating groups on the aromatic ring increase the antioxidant
activities of tested compounds as compared to compounds having electron
withdrawing groups.
Regarding antimicrobial activity, the minimum inhibitory concentra-
tions (MICs, μg mL−1) values of antibacterial and antifungal screening
suggest that compounds 6a–d exhibited significant antibacterial and anti-
fungal activities as compared to their benzothiazines 5a–d respectively due
to electron withdrawing effect of oxygen in 6a–d. Compounds 5a, 7a, and 5b,
7b bearing electron donating group like methoxy group exhibited moderate
antibacterial and antifungal activities as compared to compounds 5c and 5d
which showed good activity due to electron withdrawing effect of chlorine
against all the four strains of bacteria and fungi respectively. In general,
the presence of electron withdrawing group on the aromatic ring increases

86

Benzothiazines, Their Sulfones, and Ribofuranosides
87
the antimicrobial activities of tested compounds as compared to compounds
having electron donating groups.
EXPERIMENTAL SECTION
All the melting points were determined in open capillary tubes and are
uncorrected. 1H NMR and 13C NMR spectra (by broad band proton decou-
pling technique) were recorded on JEOL AL-300 spectrometer at frequen-
cies of (300.40 and 75.45 MHz) respectively, in DMSO-d6 using TMS as an
internal standard. IR spectra were recorded in KBr on SHIMADZU 8400 S
FTIR spectrophotometer. The FAB (Fast Atom Bombardment) mass spectra
were recorded on a JEOL SX 102/DA-600 mass spectrometer using Ar/Xe
as FAB gas at an accelerating voltage of 10 kV. The purity of compounds was
checked by TLC using silica gel ‘G’ as adsorbent, visualizing these by UV
light or in an iodine chamber.
General Procedure for the Synthesis of Substituted
4H-1,4-benzothiazines 5a–d
2-Amino-5-methoxybenzenethiol 1a/2-amino-5-chlorobenzenethiol 1b
(0.01 mol) was added to a stirred suspension of β-diketones/β-ketoesters
3a–d (0.01 mol) in DMSO (5 mL) and the resulting mixture was refluxed
for 20–40 minutes. The reaction mixture was cooled down to room tempera-
ture. The solid separated out was filtered, washed with petroleum ether and
crystallized from MeOH.
2-Trifluoroacetyl-3-trifluoromethyl-7-methoxy-4H-1,4-
benzothiazine (5a)
m.p.: 45◦C; yield: 79%; IR (KBr): v 3320 (N–H), 1625 (C O), 1063
1
(C–S), 1235 and 1039 (C–O–C), 1325 and 1145 (–CF3) cm−1; H NMR
(300.40 MHz, DMSO-d6): δ 10.90 (s, 1H, N–H), 7.53–6.68 (m, 3H, Ar–H),
3.75 (s, 3H, CH3 of OCH3 at C7) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ
117.1 (C-2), 138.2 (C-3), 114.8 (C-5), 110.1 (C-6), 150.8 (C-7), 114.0 (C-8),
140.3 (C-4a), 119.5 (C-8a), 196.8 (C of COCF3 at C2), 128.9 (CF3 at C2),
114.6 (CF3 at C3), 55.7 (OCH3 at C7) ppm; MS (FAB), m/z (%): 343 (M+,
58), 327 (41), 299 (29), 246 (62), 230 (34), 135 (53), 97 (100); Anal. Calcd.
for C12H7NO2SF6 (343): C, 41.98, H, 2.04, N, 4.08. Found: C, 42.15, H, 2.09,
N, 4.13%.
2-Trifluoroacetyl-7-methoxy-3-methyl-4H-1,4-benzothiazine (5b)
m.p.: 80◦C; yield: 88%; IR (KBr): v 3310 (N–H), 1600 (C O), 1055
(C–S), 1220 and 1025 (C–O–C), 2925 and 2815 (–CH3 str.), 1420 and 1320
(–CH3 def.), 1320 and 1130 (–CF3) cm−1; 1H NMR (300.40 MHz, DMSO-d6):

88
K. Goyal et al.
δ 8.46 (s, 1H, N–H), 7.24–6.35 (m, 3H, Ar–H), 3.72 (s, 3H, CH3 of OCH3
at C7), 2.30 (s, 3H, CH3 at C3) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ
116.2 (C-2), 136.3 (C-3), 115.8 (C-5), 111.1 (C-6), 151.6 (C-7), 114.9 (C-8),
139.9 (C-4a), 119.8 (C-8a), 195.9 (C of COCF3 at C2), 128.4 (CF3 at C2), 16.4
(CH3 at C3), 56.0 (OCH3 at C7) ppm; MS (FAB), m/z (%): 289 (M+, 55),
273 (39), 245 (24), 192 (68), 176 (43), 135 (61), 97 (100); Anal. Calcd. for
C12H10NO2SF3 (289): C, 49.82, H, 3.46, N, 4.84. Found: C, 49.95, H, 3.52,
N, 4.81%.
7-Chloro-3-ethyl-2-propionyl-4H-1,4-benzothiazine (5c)
m.p.: 106◦C; yield: 73%; IR (KBr): v 3350 (N–H), 1650 (C O), 2940 and
2830 (–CH3 str.), 1435 and 1340 (–CH3 def.), 1072 (C–S), 770 (C–Cl) cm−1;
1H NMR (300.40 MHz, DMSO-d6): δ 8.91 (s, 1H, N–H), 7.79–6.80 (m, 3H,
3
3
Ar–H), 2.81 (q, J HH = 6.51 Hz, 2H, CH2 of C2H5 at C2), 2.62 (q, J HH
=
6.64 Hz, 2H, CH2 of C2H5 at C3), 1.52 (t, 3J HH = 6.36 Hz, 3H, CH3 of C2H5
3
at C2), 1.30 (t, J HH = 6.28 Hz, 3H, CH3 of C2H5 at C3) ppm; 13C NMR
(75.45 MHz, DMSO-d6): δ 114.8 (C-2), 141.6 (C-3), 116.4 (C-5), 126.2 (C-6),
125.3 (C-7), 129.7 (C-8), 145.9 (C-4a), 119.7 (C-8a), 197.5 (C of COC2H5 at
C2), 31.2 (CH2 of C2H5 at C2), 6.8 (CH3 of C2H5 at C2), 24.7 (CH2 of C2H5
at C3), 7.9 (CH3 of C2H5 at C3) ppm; MS (FAB), m/z (%): 267.5 (M+, 49),
210.5 (70), 209.5 (34), 154.5 (52), 57 (100); Anal. Calcd. for C13H14NOSCl
(267.5): C, 58.31, H, 5.23, N, 5.23. Found: C, 58.40, H, 5.25, N, 5.20%.
Methyl-7-chloro-3-ethyl-4H-1,4-benzothiazine-2-carboxylate (5d)
m.p.: 135◦C; yield: 48%; IR (KBr): v 3390 (N–H), 1700 (C O), 1275
and 1072 (C–O–C), 2980 and 2855 (–CH3 str.), 1460 and 1370 (–CH3 def.),
1
1088 (C–S), 830 (C–Cl) cm−1; H NMR (300.40 MHz, DMSO-d6): δ 9.06
(s, 1H, N–H), 8.26–7.20 (m, 3H, Ar–H), 3.94 (s, 3H, OCH3 at C2), 2.69
3
3
(q, J HH = 6.72 Hz, 2H, CH2 of C2H5 at C3), 1.34 (t, J HH = 6.41 Hz, 3H,
CH3 of C2H5 at C3) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ 109.1 (C-2),
141.3 (C-3), 115.9 (C-5), 127.1 (C-6), 123.8 (C-7), 130.7 (C-8), 144.2 (C-
4a), 120.6 (C-8a), 165.2 (C of COOCH3 at C2), 50.5 (OCH3 at C2), 25.6
(CH2 of C2H5 at C3), 8.2 (CH3 of C2H5 at C3) ppm; MS (FAB), m/z (%):
269.5 (M+, 47), 210.5 (59), 209.5 (37), 154.5 (66), 59 (100); Anal. Calcd. for
C12H12NO2SCl (269.5): C, 53.43, H, 4.45, N, 5.19. Found: C, 53.26, H, 4.41, N,
5.18%.
General Procedure for the Synthesis of Substituted
4H-1,4-benzothiazine-1,1-dioxides (sulfones) 6a–d
Substituted 4H-1,4-benzothiazines 5a–d (0.01 mol), glacial CH3COOH
(20 mL) and 30% H2O2 (5 mL) were added together in a 50 mL R.B.
flask and the mixture was refluxed for 15–20 minutes at 50–60◦C. Heating

Benzothiazines, Their Sulfones, and Ribofuranosides
89
was stopped and another lot of H2O2 (5 mL) was added. The reaction
mixture was further refluxed for 4 hours. The contents were then poured
into a beaker containing crushed ice and the solid separated out was filtered,
washed with water and crystallized from ethanol.
2-Trifluoroacetyl-3-trifluoromethyl-7-methoxy-4H-1,4-
benzothiazine-1,1-dioxide (6a)
m.p.: 290◦C; yield: 56%; IR (KBr): v 3330 (N–H), 1640 (C O), 1071
(C–S), 1165 and 1142 (SO2 sym), 560 and 540 (SO2 bend), 1345, 1280 and
1
1248 (SO2 asym) cm−1; H NMR (300.40 MHz, DMSO-d6): δ 10.97 (s, 1H,
N–H), 7.69–6.74 (m, 3H, Ar–H), 3.78 (s, 3H, CH3 of OCH3 at C7) ppm;
13C NMR (75.45 MHz, DMSO-d6): δ 100.5 (C-2), 158.7 (C-3), 115.9 (C-5),
118.1 (C-6), 151.9 (C-7), 111.3 (C-8), 138.1 (C-4a), 125.2 (C-8a), 196.8 (C of
COCF3 at C2), 128.4 (CF3 at C2), 114.2 (CF3 at C3), 56.1 (OCH3 at C7) ppm;
MS (FAB), m/z (%): 375 (M+, 51), 359 (43), 331 (22), 278 (64), 262 (29),
167 (49), 97 (100); Anal. Calcd. for C12H7NO4SF6 (375): C, 38.40, H, 1.86,
N, 3.73. Found: C, 38.52, H, 1.84, N, 3.76%.
2-Trifluoroacetyl-7-methoxy-3-methyl-4H-1,4-benzothiazine-1,
1-dioxide (6b)
m.p.: 310◦C; yield: 62%; IR (KBr): v 3325 (N–H), 1620 (C O), 1062
(C–S), 1160 and 1148 (SO2 sym.), 555 and 515 (SO2 bend), 1340, 1282,
1
and 1245 (SO2 asym) cm−1; H NMR (300.40 MHz, DMSO-d6): δ 8.65 (s,
1H, N–H), 7.46–6.39 (m, 3H, Ar–H), 3.74 (s, 3H, CH3 of OCH3 at C7), 2.32
(s, 3H, CH3 at C3) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ 100.2 (C-2),
156.3 (C-3), 116.5 (C-5), 119.9 (C-6), 152.1 (C-7), 111.8 (C-8), 137.0 (C-4a),
127.2 (C-8a), 196.1 (C of COCF3 at C2), 128.1 (CF3 at C2), 15.9 (CH3 at C3),
55.8 (OCH3 at C7) ppm; MS (FAB), m/z (%): 321 (M+, 48), 305 (33), 277
(28), 224 (61), 208 (39), 167 (53), 97 (100); Anal. Calcd. for C12H10NO4SF3
(321): C, 44.85, H, 3.11, N, 4.36. Found: C, 44.93, H, 3.09, N, 4.32%.
7-Chloro-3-ethyl-2-propionyl-4H-1,4-benzothiazine-1,
1-dioxide (6c)
m.p.: 178◦C; yield: 58%; IR (KBr): v 3370 (N–H), 1658 (C O), 1080
(C–S), 1177, 1155 (SO2 sym), 568, 540 (SO2 bend), 1350, 1290, 1260
1
(SO2 asym) cm−1; H NMR (300.40 MHz, DMSO-d6): δ 8.99 (s, 1H, N–H),
8.01–6.90 (m, 3H, Ar–H), 2.90 (q, 3J HH = 6.57 Hz, 2H, CH2 of C2H5 at C2),
2.65 (q, 3JHH = 6.75 Hz, 2H, CH2 of C2H5 at C3), 1.53 (t, 3J HH = 6.48 Hz, 3H,
CH3 of C2H5 at C2), 1.34 (t, 3J HH = 6.32 Hz, 3H, CH3 of C2H5 at C3) ppm;
13C NMR (75.45 MHz, DMSO-d6): δ 98.7 (C-2), 159.2 (C-3), 118.1 (C-5),
136.5 (C-6), 122.9 (C-7), 130.1 (C-8), 144.6 (C-4a), 126.3 (C-8a), 197.6 (C
of COC2H5 at C2), 30.9 (CH2 of C2H5 at C2), 8.4 (CH3 of C2H5 at C2), 23.7

90
K. Goyal et al.
(CH2 of C2H5 at C3), 9.2 (CH3 of C2H5 at C3) ppm; MS (FAB), m/z (%):
299.5 (M+, 44), 242.5 (62), 241.5 (36), 186.5 (50), 57 (100); Anal. Calcd. for
C13H14NO3SCl (299.5): C, 52.32, H, 4.67, N, 4.67. Found: C, 52.01, H, 4.64,
N, 4.69%.
Methyl-7-chloro-3-ethyl-4H-1,4-benzothiazine-2-carboxylate-1,
1-dioxide (6d)
m.p.: 208◦C; yield: 35%; IR (KBr): v: 3400 (N–H), 1720 (C O), 1100
(C–S), 1190, 1165 (SO2 sym), 585, 560 (SO2 bend), 1380, 1300, 1270
1
(SO2 asym) cm−1; H NMR (300.40 MHz, DMSO-d6): δ 9.24 (s, 1H, N–H),
8.37–7.22 (m, 3H, Ar–H), 3.96 (s, 3H, OCH3 at C2), 2.75 (q, 3J HH = 6.74 Hz,
3
2H, CH2 of C2H5 at C3), 1.39 (t, J HH = 6.49 Hz, 3H, CH3 of C2H5 at C3)
ppm; 13C NMR (75.45 MHz, DMSO-d6): δ 93.2 (C-2), 158.7 (C-3), 116.8 (C-
5), 131.9 (C-6), 124.8 (C-7), 127.5 (C-8), 144.6 (C-4a), 125.4 (C-8a), 165.2
(C of COOCH3 at C2), 51.3 (OCH3 at C2), 25.2 (CH2 of C2H5 at C3), 9.1
(CH3 of C2H5 at C3) ppm; MS (FAB), m/z (%): 301.5 (M+, 41), 242.5 (61),
241.5 (28), 186.5 (73), 59 (100); Anal. Calcd. for C12H12NO4SCl (301.5): C,
47.76, H, 3.98, N, 4.64. Found: C, 47.92, H, 3.99, N, 4.67%.
General Procedure for the Synthesis of Substituted
N-(2ꢀ,3ꢀ,5ꢀ-tri-O-benzoyl-β-D-ribofuranosyl) Benzothiazines (7a–b)
To the solution of (5a–b) (0.002 mol) in 5 mL C6H5CH3, β-D-
ribofuranose-1-acetate-2,3,5-tribenzoate (0.002 mol) was added and the con-
tents were refluxed under vacuum with stirring on an oil bath at 155–160◦C
for 15 minutes. The vacuum was broken and reaction mixture was protected
from moisture by fitting a guard tube. Stirring was further continued for
10 hours and a vacuum was applied for 10 minutes after every 1 hour. The
viscous mass obtained was dissolved in MeOH, boiled for 10 minutes and
cooled to room temperature. The reaction mixture was filtered and the
filtrate was evaporated to dryness. The viscous residue thus obtained was
dissolved in Et2O, filtered, concentrated and kept in refrigerator overnight
to afford crystalline ribofuranosides.
N-(2ꢀ,3ꢀ,5ꢀ-tri-O-benzoyl-β-D-ribofuranosyl)-2-Trifluoroacetyl-
3-trifluoromethyl-7-methoxy-4H-1,4-benzothiazine (7a)
m.p.: 54◦C; yield: 446%; IR (KBr): v 1632 (C O), 1165 (C–O–C), 1350
and 1160 (–CF3) cm−1; 1H NMR (300.40 MHz, DMSO-d6): δ 7.62–6.70 (m,
3
3H, Ar–H), 3.75 (s, 3H, CH3 of OCH3 at C7, 5.59 (d, J HH = 6.9 Hz, 1H,
1ꢁ-H), 5.21 (dd, 3J HH = 6.5, 6.5 Hz, 1H, 2ꢁ-H), 4.99 (dd, 3J HH = 7.0, 8.0 Hz,
3
1H, 3ꢁ-H), 5.04 (m, 1H, 4ꢁ-H), 4.51 (d, J HH = 7.4 Hz, 2H, CH2 of sugar),
8.02 (m, 1H, 2ꢁꢁ-H or 6ꢁꢁ-H of Bz), 7.49 (m, 1H, 3ꢁꢁ-H or 5ꢁꢁ-H of Bz), 7.62
(m, 1H, 4ꢁꢁ-H of Bz) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ 116.7 (C-2),

Benzothiazines, Their Sulfones, and Ribofuranosides
91
138.1 (C-3), 112.2 (C-5), 110.8 (C-6), 148.4 (C-7), 115.3 (C-8), 137.6 (C-4a),
116.9 (C-8a), 196.9 (C of COCF3 at C2), 128.8 (CF3 at C2), 113.4 (CF3 at
C3), 56.0 (OCH3 at C7), 84.5 (C-1ꢁ), 73.7 (C-2ꢁ), 71.6 (C-3ꢁ), 68.9 (C-4ꢁ) ppm;
MS (FAB), m/z (%): 787 (M+, 55), 690 (61), 97 (100); Anal. Calcd. for
C38H27NO9SF6 (787): C, 57.94, H, 3.43, N, 1.77. Found: C, 58.12, H, 3.46,
N, 1.79%.
N-(2ꢀ,3ꢀ,5ꢀ-tri-O-benzoyl-β-D-ribofuranosyl)-2-Trifluoroacetyl-7-
methoxy-3-methyl-4H-1,4-benzothiazine (7b)
m.p.: 73◦C; yield: 62%; IR (KBr): v 1610 (C O), 1150 (C–O–C), 1340
and 1155 (–CF3) cm−1; 1H NMR (300.40 MHz, DMSO-d6): δ 7.31–6.36 (m,
3H, Ar–H), 3.72 (s, 3H, CH3 of OCH3 at C7), 2.31 (s, 3H, CH3 at C3), 5.64 (d,
3J HH = 7.2 Hz, 1H, 1ꢁ-H), 5.12 (dd, 3J HH = 7.7, 7.8 Hz, 1H, 2ꢁ-H), 4.97 (dd,
3J HH = 6.8, 6.3 Hz, 1H, 3ꢁ-H), 5.06 (m, 1H, 4ꢁ-H), 4.61 (d, 3J HH = 8.1 Hz, 2H,
CH2 of sugar), 7.99 (m, 1H, 2ꢁꢁ-H or 6ꢁꢁ-H of Bz), 7.43 (m, 1H, 3ꢁꢁ-H or 5ꢁꢁ-H
of Bz), 7.64 (m, 1H, 4ꢁꢁ-H of Bz) ppm; 13C NMR (75.45 MHz, DMSO-d6): δ
116.4 (C-2), 136.2 (C-3), 112.9 (C-5), 111.7 (C-6), 149.8 (C-7), 115.9 (C-8),
137.1 (C-4a), 116.8 (C-8a), 196.5 (C of COCF3 at C2), 128.6 (CF3 at C2), 15.1
(CH3 at C3), 56.2 (OCH3 at C7), 83.9 (C-1ꢁ), 74.2 (C-2ꢁ), 72.1 (C-3ꢁ), 68.4
(C-4ꢁ) ppm; MS (FAB), m/z (%): 733 (M+, 53), 636 (66), 97 (100); Anal.
Calcd. for C38H30NO9SF3 (733): C, 62.21, H, 4.09, N, 1.91. Found: C, 62.32,
H, 4.03, N, 1.88%.
ANTIOXIDANT ACTIVITY
All the synthesized compounds were screened for their antioxidant ac-
tivity by DPPH radical scavenging assay (Table 2 and Figure 1) and ABTS
radical cation decolorization assay (Table 3 and Figure 2). In antioxidant
activity assays (both DPPH and ABTS+ assays), ascorbic acid was used as a
positive control.
DPPH RADICAL SCAVENGING ASSAY
Radical scavenging activity of synthesized compounds against stable
DPPH radical was determined spectrophotometrically by the method de-
scribed by Blois.[8] DPPH is stable free radical by virtue of delocalization of
electron over the whole molecule. For hydrogen atom transfer, antioxidants
quench the free radicals by donating hydrogen, while for single electron
transfer, antioxidants transfer one electron to the radical.
Different concentrations (10, 50, 100, 200, 400, 600, 800, and
1000 μg/mL) of each compound MeOH were dissolved in DMSO and
taken in test tubes in triplicates. Then 5 mL of 0.1 mM EtOH solution
of DPPH was added to each of the test tubes and were shaken vigorously.

92

Benzothiazines, Their Sulfones, and Ribofuranosides
93
DPPH activity (at different concentrations) of 4H-1,4-benzothiazines (5a-d)
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
Concentration (µg/mL)
5a
5b 5c 5d
Ascorbic acid
DPPH activity (at different concentrations) of 4H-1,4-benzothiazine's sulfones (6a-d)
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
Concentration (µg/mL)
6a
6b 6c 6d
Ascorbic acid
DPPH activity (at different concentrations) of 4H-1,4-benzothiazine's ribofuranosides (7a-b)
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
Concentration (µg/mL)
7a 7b Ascorbic acid
FIGURE 1 Free radical scavenging of synthesized compounds by dpph assay. Each value represented
the mean sd of three replicates (n = 3) (Color figure available online).
They were then allowed to stand at 37◦C for 20 minutes. MeOH was used for
base line corrections in absorbance (OD) of sample measured at 517 nm.
DPPH is a purple-colored stable free radical, when reduced it becomes the
yellow-colored diphenylpicrylhydrazine. The percentage of inhibition was

94
K. Goyal et al.
activity (at different time intervals) of 4H-1,4-benzothiazines (5a-d)
ABTS
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Initial
0 min
1 min
2 min
4 min
6 min
Time (min)
5a
5b
5c
5d
Ascorbic acid
activity (at different time intervals) of 4H-1,4-benzothiazine's sulfones (6a-d)
0.8
ABTS
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Initial
0 min
1 min
2 min
4 min
6 min
Time (min)
6a
6b
6c
6d
Ascorbic acid
activity (at different time intervals) of 4H-1,4-benzothiazine's ribofuranosides (7a-b)
0.8
ABTS
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1
2
3
4
5
6
Time (min)
7a
7b Ascorbic acid
FIGURE 2 The effect of time on the suppression of absorbance of abts by synthesized compounds.
After addition of 1 mL of diluted abts solution (at 734 nm = 0.700 0.020) to 10 μL of the compound
the absorbance reading was taken at 30◦C exactly 1 minute, after initial mixing and every minute upto
6 minutes. All determinations were repeated three times (Color figure available online).

Benzothiazines, Their Sulfones, and Ribofuranosides
95
TABLE 3 Antioxidant activity of synthesized compounds (ABTS+ Assay)
ABTS+ activity at different intervals (min)
Compd. no.
Initial
0 minutes
1 minutes
2 minutes
4 minutes
6 minutes
5a
5b
5c
5d
6a
6b
6c
6d
0.708
0.705
0.712
0.720
0.711
0.708
0.719
0.727
0.709
0.707
0.717
0.688
0.680
0.696
0.704
0.692
0.687
0.698
0.713
0.690
0.683
0.340
0.394
0.342
0.461
0.503
0.452
0.381
0.530
0.581
0.416
0.359
0.049
0.198
0.126
0.238
0.432
0.276
0.201
0.318
0.525
0.227
0.163
0.039
0.155
0.085
0.208
0.422
0.253
0.191
0.311
0.513
0.178
0.114
0.03
0.134
0.078
0.197
0.420
0.248
0.185
0.310
0.513
0.172
0.113
0.028
7a
7b
Ascorbic acid
calculated using the following formula.
(AB − AA)
% Inhibition =
× 100
AB
Where
AB = Absorbance of control
AA = Absorbance of test sample
The antioxidant activity of each sample was expressed in terms of IC50
(micromolar concentration required to inhibit DPPH radical formation by
50%), calculated from the inhibition curve (Figure 1).
ABTS RADICAL CATION DECOLORIZATION ASSAY
The ABTS radical cation decolorization test was also used to assess the
antioxidant activity of the synthesized compounds. The ABTS+ assay was
carried out using the improved assay of Re et al.[10] ABTS+ was generated
by oxidation of ABTS with K2S2O8. For this purpose, ABTS was dissolved in
deionized H2O at a concentration of 7 mM, and K2S2O8 was added to a con-
centration of 2.45 mM. The reaction mixture was left at room temperature
overnight (12–16 hours) in the dark before use; the ABTS+ solution then
was diluted with EtOH to an absorbance of 0.700 0.020 at 734 nm. After
addition of 1 mL of the diluted ABTS solution to 10 μL of compound and
mixing, absorbance readings were taken at 30◦C at intervals of exactly 1–6
minutes, later. All determinations were carried out in triplicate.

96
K. Goyal et al.
ANTIMICROBIAL ACTIVITY
The MICs (μg mL−1) of the chemical compounds assays were carried
out by broth microdilution method as per NCCLS-1992 manual. Two Gram-
positive (Bacillus cereus MTCC 4317 and Staphylococcus aureus MTCC 3160)
and two Gram-negative (Escherichia coli DH5 alpha MTCC 1652 and Pseu-
domonas aeruginosa MTCC 4676) bacteria were used as quality control strains.
For determining antifungal activities of the compounds, the following refer-
ence strains were tested: Aspergillus niger MTCC 282, Aspergillus flavus MTCC
2456, Fusarium oxysporum MTCC 6659 and Rhizopus stolonifer MTCC 2591.
Ampicillin sodium salt and Ketoconazole were used as standard antibac-
terial and antifungal drugs, respectively. Solutions of test compounds and
standard drugs were prepared in DMSO. Each synthesized drug was diluted
obtaining 1000 μg/mL concentration as a stock solution. In primary screen-
ing, 500, 250, and 125 μg/mL concentrations of the synthesized drugs were
taken. The active synthesized drugs found in this primary screening were
further tested in a second set of dilution against all microorganisms. The
drugs found active in primary screening were similarly diluted to obtain 100,
50, 25, 20, 15 μg/mL concentrations. The highest dilution showing at least
99% inhibition was taken as MIC. This means that the lowest concentration
of each chemical compound in the tube with no growth (i.e., no turbidity) of
inoculated bacteria/fungi was recorded as minimum inhibitory concentra-
tion of that compound. Antibacterial activities of the bacterial strains were
carried out in Luria broth (Himedia) medium and all fungi were cultivated
in Sabouraud Dextrose Agar (Himedia), at pH 6.9, with an inoculum of 108
cfu/mL by the spectrophotometric method and an aliquot of 10 μL was
added to each tube of the serial dilution and incubated on a rotary shaker at
37◦C for 24 hours at 150 rpm. At the end of incubation period, MIC values
were recorded. The MICs of tested compounds in μg/mL against certain
bacteria and fungi are shown in Table 1.
REFERENCES
1. Clercq, E.D. Target for the antiviral and antitumor activities of nucleoside nucleotide and oligonu-
cleotide analogues, Nucleos. Nucleot. Nucl. 1985, 4, 3–11.
2. Vysokov, V.I.; Charushin, V.N.; Afanasyeva, G.B.; Chupakhin, O.N. The synthesis of fluorinated 4H-
1,4-benzothiazine-2-carboxylic acid-1,1-dioxides-thionated analogues of Pefloxacin. Mendeleev Com-
mun. 1993, 3, 159–160.
3. Gupta, R.R.; Saraswat, V.; Gupta, V.; rajoria, C.M., Gupta, A.; Jain, M. Synthesis of 5,6 and 5,7-
dichloro-3-methyl-4H-1,4-benzothiazines and their conversion into sulfones. J. Heterocycl. Chem. 1993,
30, 803–806.
4. Jain, M.; Alabdalla, M.A.; Gupta, R.R. Synthesis of substituted 4H-1,4-benzothiazines and their con-
version into sulfones. Heterocycl. Commun. 1995, 1, 281–288.
5. Thomas, L.; Gupta, A.; Gupta, V. Synthesis of 2-amino-5-chloro-3-(trifluoromethyl) benzenethiol and
conversion into 4H-1,4-benzothiazines and their sulfones. J. Fluorine Chem. 2003, 122, 207–213.
6. Gautam, N.; hans, D.; Gautam, D.C. Antifungal activity of some 4H-1,4-benzothiazine compound.
Orient. J. Chem. 2005, 21, 299–302.

Benzothiazines, Their Sulfones, and Ribofuranosides
97
7. Rathore, B.S.; Kumar, M. design and synthesis of 4H-1,-benzothiazines containing thiazole ring
system for use as potential biopharmaceuticals. Res. Chem. Intermed. 2006, 32, 647–651.
8. Gautam, V.; Sharma, M.; Panwar, M.; Gautam, N.; Kumar, A.; Sharma, I.K.; Gautam, D.C. Synthetic
methodology, spectral elucidation and antioxidative properties of benzothiazines and their sulfones.
Phosphorus Sulfur 2009, 184, 3090–3109.
9. Gupta, S.; Ajmera, N.; Meena, P.; Gautam, N.; Kumar, A.; Gautam, D.C. Synthesis and biological
evaluation of new 1,4-thiazine containing heterocyclic compounds. Jordan J. Chem. 2009, 4, 209–221.
10. Blois, M.S. Antioxidant determination by the use of a stable free radical. Nature 1958, 181, 1199–1200.
11. Cuendet, M.; Hostettmann, K.; Potterat, O.; Dyatmiko, W. Iridoid Glucosides with free radical scav-
enging properties from Fagraea blumei. Helv. Chim. Acta 1997, 80, 1144–1152.
12. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant activity
applying an improved ABTS radical cation decolorization assay. Free Radical Biol. Med. 1999, 26, 1231.
13. Braca, A.; Nunziatina, D.T.; Lorenzo, D.B.; Pizza, C.; Politi, M.; Morelli, I. Antioxidant principles
from Bauhinia tarapotensis. J. Nat. Prod. 2001, 64, 892–895.
14. Culbertson, T.P. Synthesis of 4H-1,4-benzothiazine-1-oxide and 1,1-dioxide analogs of quinolone
antibacterial agents. J. Heterocycl. Chem. 1991, 28, 1701–1703.
15. Dixit, Y.; Dixit, R.; Gautam, N.; Gautam, D.C. Synthesis and antimicrobial activities of novel bi-
ologically active heterocycles: 10H-phenothiazines, their ribofuranosides and sulfone derivatives.
nucleosides Nucleotodes Nucleic Acids 2009, 28, 998–1006.
16. Gautam, N.; Goyal, K.; Saini, O.P.; Kumar, A.; Gautam, D.c. Synthesis and biological activity of sub-
stituted 3-fluoro/3-trifluoromethyl 10H-phenothiazines, its ribofuranosides and sulfones. J. Fluorine
Chem. 2011, 132, 420–426.
17. Sharma, P.K.; Fogla, A.; Rathore, B.S.; Kumar, M. Synthesis and antimicrobial activity of structurally
flexible heterocycles with the 1,4-thiazine heterosystem. Res. Chem. Intermed. 2011, 37, 1103–1111.