An efficient synthesis and biological study of substituted 8-chloro-5-methoxy/8-chloro-4H-1,4-benzothiazines, their sulphones and ribofuranosides

Article abstract of DOI:10.1007/s12039-013-0363-4

8-Chloro-5-methoxy/8-chloro-4H-1,4-benzothiazines were synthesized by condensation followed by oxidative cyclisation of 2-amino-6-chloro-3-methoxy/2- amino-3-chlorobenzenethiol with β-diketones/β-ketoesters in the presence of dimethyl sulphoxide. By treating 4H-1,4-benzothiazines with 30% hydrogen peroxide in glacial acetic acid, 4H-1,4-benzothiazine-1,1-dioxides (sulphones) were synthesized. The 4H-1,4-benzothiazines have also been used as a base to prepare ribofuranosides by the reaction with β-D-ribofuranose-1- acetate-2,3,5-tribenzoate. All the synthesized compounds have been characterized by spectral and elemental analysis and have been examined for antimicrobial and anthelmintic activity.

Full text of DOI:10.1007/s12039-013-0363-4

c
J. Chem. Sci. Vol. 125, No. 1, January 2013, pp. 85–93. ꢀ Indian Academy of Sciences.
An efficient synthesis and biological study of substituted
8-chloro-5-methoxy/8-chloro-4H-1,4-benzothiazines, their sulphones
and ribofuranosides
NISHIDHA KHANDELWAL∗, ABHILASHA, NAVEEN GAUTAM and D C GAUTAM
Department of Chemistry, University of Rajasthan, Jawahar LaL Nehru Marg, Jaipur 302 004, India
e-mail: nishidha.khandelwal@gmail.com; abhilashasuraj@gmail.com; ngautam70@yahoo.co.in;
drdcgautam@yahoo.co.in
MS received 29 December 2011; revised 29 May 2012; accepted 20 July 2012
Abstract. 8-Chloro-5-methoxy/8-chloro-4H-1,4-benzothiazines were synthesized by condensation followed
by oxidative cyclisation of 2-amino-6-chloro-3-methoxy/2-amino-3-chlorobenzenethiol with β–diketones/β–
ketoesters in the presence of dimethyl sulphoxide. By treating 4H-1,4-benzothiazines with 30% hydrogen
peroxide in glacial acetic acid, 4H-1,4-benzothiazine-1,1-dioxides (sulphones) were synthesized. The 4H-1,4-
benzothiazines have also been used as a base to prepare ribofuranosides by the reaction with β–D-ribofuranose-
1-acetate-2,3,5-tribenzoate. All the synthesized compounds have been characterized by spectral and elemental
analysis and have been examined for antimicrobial and anthelmintic activity.
Keywords. 4H-1,4-benzothiazines; sulphones; ribofuranosides; antimicrobial activity; anthelmintic activity.
1. Introduction
2. Materials and methods
Six-membered heterocyclic compounds have occu- 2.1 Experimental
pied a prominent place among various classes of
All the melting points were determined in an open cap-
organic compounds for their diverse biological activi-
ties. Among a wide variety of heterocycles that have
been explored developing pharmaceutically impor-
tant molecules, 4H-1,4-benzothiazines,1–8 their sul-
phones and rifuranosides have played an important
role in medicinal chemistry. Benzothiazines possess
a fold along nitrogen–sulphur axis which is one of
the structural specificities responsible for pharmaco-
logical activities.9–15 It has stimulated our interest
to convert benzothiazines to sulphones to understand
oxidation behaviour of 4H-1,4-benzothiazines and to
investigate changes in infrared and nuclear magnetic
resonance spectra caused by the conversion of sulphide
linkage to sulphones. 4H-1,4-benzothiazines serve as
heterocyclic base for the formation of ribofuranosides
on treatment with sugar (β-D-ribofuranosyl-1-acetate-
2,3,5-tribenzoate). All these compounds are extensively
used as sedatives, anthelmintic, antiulcer, anticancer,
antibacterial, antifungal, etc. A slight change in the sub-
stitution pattern in the benzothiazine nucleus cause dis-
tinguishable difference in their biological activities. The
newly synthesized compounds have been screened for
antimicrobial and anthelmintic activity.
illary tubes and are uncorrected. 1H NMR and 13C NMR
spectra (by broad band proton decoupling technique)
were recorded on JEOL AL-300 spectrometer at fre-
quencies of (300.40 MHz and 75.45 MHz) respectively,
in dimethylsulphoxide-d6 using TMS (tetramethyl
silane) as an internal standard. IR spectra were recorded
in KBr on SHIMADZU 8400 S FTIR spectrophotome-
ter. Mass spectra were recorded on WATERS (micro-
mass MS technologies) Q-T by electron spray ioni-
zation. 19F NMR spectra were recorded in CDCl3
using CF3COOH as standard compound. Purity of
synthesized compounds was checked by TLC (thin-
layer chromatography) using silica gel ‘G’ as adsorbent,
visualizing these by UV light or in an iodine chamber.
2.2 General procedure of synthesis of substituted
4H-1,4-benzothiazines (3a–d)
To 2-amino-5-chloro-2-methoxy/3-chlorobenzenethiol
(0.01 mol) (1a–b), a stirred suspension of the appropri-
ate β-diketones/β-ketoesters (2a–d) in DMSO (5 mL)
was added and the resulting mixture was refluxed for
2–3 h. The reaction mixture was concentrated, cooled
∗For correspondence
85

86
Nishidha Khandelwal et al.
down to room temperature and filtered. The product −OCH3 at C-2), 2.42 (q, 2H, CH2 of C2H5 at C-3),
was washed with petroleum ether and crystallized from 1.45 (t, 3H, CH3 of C2H5 at C-3), 3.72 (s, 3H, −OCH3
methanol.
at C-5); 13C NMR (75.45 MHz, CDCl3, δ ppm from
TMS): 108.1 (C–2), 164.9 (C=O at C-2), 50.3 (CH3
at C-2), 141.6 (C-3), 24.0 (CH2 of C2H5 at C-3), 8.7
(CH3 of C2H5 at C-3), 55.9 (OCH3 at C-5), 147.3 (C-
5), 113.2 (C-6), 120.1 (C-7), 127.4 (C-8); MS (FAB)
10 kV, m/z (rel. int.): 299 [M]+, 301 [M+2]+, 298 (28),
270 (73), 264 (31), 240 (65), 59 (100); ‘Anal. Calcd for
C13H14NO3SCl: C, 52.08; H, 4.67; N, 4.67, Found: C,
52.23; H, 4.71; N, 4.75’.
2.2a 8-Chloro-2-trifluoroacetyl-3-trifluoromethyl-5-
methoxy-4H-1,4-benzothiazine (3a): Brown solid;
m.p.: 65◦C; yield: 56%, IR (KBr): v 3350 (N–H),
1595 (>C=O), 2965 and 2875 (−OCH3), 1285 and
1020 (C–O–C), 1335 and 1100 cm−1 (-CF3) and
780 cm−1 (C–Cl); 1H NMR spectral data (300.40 MHz,
Me2SO-d6, δ ppm from TMS): δ 8.93 (s, 1H, N–H),
8.10–6.39 (m, 2H, Ar-H), 3.87 (s, 3H, −OCH3); 13C
NMR (75.45 MHz, CDCl3, δ ppm from TMS): δ 116.2
(C–2), 196.5 (C=O at C-2), 128.8 (CF3 at C-2), 136.3
(C-3), 114.9 (CF3 at C-3), 147.0 (C-5), 56.0 (OCH3 at
C-5), 113.1 (C-6), 120.2 (C-7), 127.6 (C-8); 19F NMR
(282.65 MHz, CDCl3): δ –61.93 (s, 3F, CF3); MS
(FAB) 10 kV, m/z (rel. int.): 377 [M]+, 379 [M+2]+,
279 (41), 376 (27), 97 (100), 281 (56), 248 (36); ‘Anal.
Calcd for C12H6NO2F6SCl: C, 38.14; H, 1.59; N, 3.71,
Found: C, 38.46; H,1.60; N, 3.73’.
2.2d
5-Chloro-3-ethyl-2-propionyl-4H-1,4-benzo-
thiazine (3d): Brown solid; m.p.: 70◦C; yield: 62%, IR
(KBr): v 3290 (N–H), 1585 (>C=O), 2980 (CH3), and
720 cm−1 (C–Cl); 1H NMR spectral data (300.40 MHz,
Me2SO-d6, δ ppm from TMS): δ 8.44 (s, 1H, N-H),
8.32–6.16 (m, 3H, Ar-H), 3.17 (q, 2H, CH2 of C2H5
at C-2), 1.81 (t, 3H, CH3 of OC2H5 at C-2), 2.51 (q,
2H, CH2 of C2H5 at C-3), 1.31 (t, 3H, CH3 of C2H5
at C-3); 13C NMR (75.45 MHz, CDCl3, δ ppm from
TMS): 115.5 (C–2), 197.6 (C=O at C-2), 30.5 (CH2 of
C2H5 at C-2), 7.1 (CH3 of C2H5 at C-2), 141.1 (C-3),
2.2b
Ethyl-8-chloro-5-methoxy-3-propyl-4H-1,4- 24.2 (CH2 of C2H5 at C-3), 8.6 (CH3 of C2H5 at C-3),
benzothiazine-2-carboxylate (3b): Red solid; m.p.: 120.8 (C-5), 126.6 (C-6), 120.3 (C-7), 128.0 (C-8); MS
90◦C; yield: 58%, IR (KBr): v 3310 (N–H), 1590 (FAB) 10 kV, m/z (rel. int.): 267 [M]+, 269 [M+2]+,
(>C=O), 2955 and 2830 (−OCH3), 1280 and 1030 266 (22), 238 (44), 210 (58), 232 (76), 57 (100); ‘Anal.
(C–O–C), and 750 cm−1 (C–Cl); 1H NMR spectral data Calcd for C13H14NOSCl: C, 58.31; H, 5.23; N, 5.23,
(300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.73 Found: C, 53.58; H, 5.26; N, 5.28’.
(s, 1H, N-H), 8.02–6.34 (m, 2H, Ar-H), 3.86 (s, 3H,
−OCH3), 2.51 (t, 2H, CH2 (terminal) of C3H7 at C-3),
2.3 General procedure of synthesis of
4H-1,4-benzothiazine sulphones (4a–d)
1.60 (sextet, 2H, CH2 (centre) of C3H7 at C-3), 1.20 (t,
3H, CH3 of C3H7 at C-3), 4.95 (q, 2H, CH2 of OC2H5
at C-2), 1.78 (t, 3H, CH3 of OC2H5 at C-2); 13C NMR
A mixture of substituted 4H-1,4-benzothiazines (3a–d)
(0.01 mol), glacial acetic acid (20 mL) and 30% hydro-
gen peroxide (5 mL) was refluxed for 15 min. Heat-
(75.45 MHz, CDCl3, δ ppm from TMS): δ 105.8 (C-2),
165.0 (C=O at C-2), 59.2 (CH2 of OC2H5 at C-2),
13.4 (CH3 of OC2H5 at C-2), 143.5 (C-3), 33.8 (CH2
ing was stopped and another lot of hydrogen perox-
(terminal) of C3H7 at C-3), 17.5 (CH2 (middle) of C3H7
ide (5 mL) was added. The reaction mixture was again
at C-3), 14.4 (CH3 (terminal) of C3H7 at C-3), 146.1
refluxed for further 4 h. The contents were poured into
(C-5), 56.4 (OCH3 at C-5), 112.1 (C-6), 119.2 (C-7),
a beaker containing crushed ice. Residue obtained was
126.2 (C-8); MS (FAB) 10 kV, m/z (rel. int.): 327 [M]+,
filtered and washed with water and recrystallized from
329 [M+2]+, 326 (48), 253 (36), 248 (43), 73 (100),
ethanol.
221 (63); ‘Anal. Calcd for C15H18NO3ClS : C, 54.96;
H, 5.49; N, 4.27, Found: C, 55.27; H, 5.51; N, 4.34’.
2.3a 8-Chloro-2-trifluoroacetyl-3-trifluoromethyl-5-
methoxy-4H-1,4-benzothiazine-1,1-dioxide
(4a):
2.2c
Methyl-8-chloro-3-ethyl-5-methoxy-4H-1,4- Brown solid; m.p.: 280◦C; yield: 48%, IR (KBr): v
benzothiazine-2-carboxylate (3c): Red solid; m.p.: 3380 (N–H), 1645 (C=O), 2950 and 2838 (−OCH3),
80◦C; yield: 63%, IR (KBr): v 3300 (N–H), 1580 1250 and 1030 (C–O–C), 1375, 1120 (-CF3), 790 (C-
1
(>C=O), 2960 and 2846 (−OCH3), 1252 and 1035 Cl), 1200 and 1140 (SO2 sym), 1080 cm−1 (C–S); H
(C–O–C) and 740 cm−1 (C–Cl); 1H NMR spectral data NMR spectral data (300.40 MHz, Me2SO-d6, δ ppm
(300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.68 from TMS): δ 9.11 (s, 1H, N–H), 8.25–6.28 (m, 2H,
(s, 1H, N–H), 8.45–6.42 (m, 2H, Ar-H), 4.01 (s, 3H, Ar-H), 3.91 (s, 3H, −OCH3); 13C NMR (75.45 MHz,

Synthesis and biological study of substituted 4H-1,4-benzothiazines
87
CDCl3, δ ppm from TMS): δ 100 (C–2), 195.6 (C=O 2.3d
5-Chloro-3-ethyl-2-propionyl-4H-1,4-benzo-
at C-2), 129.1 (CF3 at C-2), 154.3 (C-3), 115.6 (CF3 thiazine-1,1-dioxide (4d): Black solid; m.p.: 286◦C;
at C-3), 146.5 (C-5), 55.2 (OCH3 at C-5), 121.3 (C- yield: 52%, IR (KBr): v 3310 (N–H), 1645 (>C=O),
6), 119.2 (C-7), 124.6 (C-8); 19F NMR (282.65 MHz, 2985 (CH3), and 740 cm−1 (C–Cl); H NMR spectral
1
CDCl3): δ −62.11 (s, 3F, CF3); MS (FAB) 10 kV, m/z data (300.40 MHz, Me2SO-d6, δ ppm from TMS): δ
(rel. int.): 409 [M]+, 411 [M+2]+, 311 (54), 340 (38), 8.77 (s, 1H, N-H), 8.51–6.84 (m, 3H, Ar-H), 3.11 (q,
313 (68), 97 (100); ‘Anal. Calcd for C12H6NO4F6SCl: 2H, CH2 of C2H5 at C-2), 1.31 (t, 3H, CH3 of OC2H5
C, 35.16; H, 1.46; N, 3.41, Found: C, 35.48; H,1.50; at C-2), 2.65 (q, 2H, CH2 of C2H5 at C-3), 1.43 (t, 3H,
N, 3.47’.
CH3 of C2H5 at C-3); 13C NMR (75.45 MHz, CDCl3, δ
ppm from TMS): 99.1 (C–2), 197.8 (C=O of OC2H5 at
C-2), 30.3 (CH2 of C2H5 at C-2), 7.6 (CH3 of C2H5 at
C-2), 158.1 (C-3), 23.9 (CH2 of C2H5 at C-3), 9.1 (CH3
of C2H5 at C-3), 121.5 (C-5), 135.0 (C-6), 120.8 (C-7),
125.5 (C-8); MS (FAB) 10 kV, m/z (rel. int.): 299 [M]+,
301 [M+2]+, 298 (28), 270 (46), 242 (55), 264 (70),
57 (100); ‘Anal. Calcd for C13H14NO3SCl: C, 52.08; H,
4.67; N, 4.67, Found: C, 52.23; H, 4.70; N,4.76’.
2.3b
Ethyl-8-chloro-5-methoxy-3-propyl-4H-1,4-
benzothiazine-2-carboxylate-1,1-dioxide (4b): Red-
dish yellow solid; m.p.: 293◦C; yield: 50%, IR (KBr): v
3340 (N–H), 2973 and 2870 (−OCH3), 1630 (>C=O),
1180, 1150 (SO2 sym), 1085 (C–S) and 735 cm−1 (C–
Cl); 1H NMR spectral data (300.40 MHz, Me2SO-d6, δ
ppm from TMS): δ 8.97 (s, 1H, N-H), 8.07–6.44 (m,
2H, Ar-H), 4.43 (s, 3H, −OCH3), 2.23 (t, 2H, CH2
(terminal) of C3H7 at C-3), 1.45 (sextet, 2H, CH2 (mid-
dle) of C3H7 at C-3), 1.65 (t, 3H, CH3 of C3H7 at C-3),
4.22 (q, 2H, CH2 of OC2H5 at C-2), 1.89 (t, 3H, CH3
of OC2H5 at C-2); 13C NMR (75.45 MHz, CDCl3, δ
ppm from TMS): δ 99 (C-2), 197.5 (C=O at C-2), 30.2
(CH2 of OC2H5 at C-2), 7.4 (CH3 of OC2H5 at C-2),
158 (C-3), 33.8 (CH2 (terminal) of C3H7 at C-3), 18.0
(CH2 (middle) of C3H7 at C-3), 14.1 (CH3 (terminal) of
C3H7 at C-3), 147.1 (C-5), 56.4 (OCH3 at C-5), 121.1
(C-6), 120.9 (C-7), 124.9 (C-8); MS (FAB) 10 kV, m/z
(rel. int.) : 359 [M]+, 361 [M+2]+, 358 (20), 286 (39),
316 (28), 73 (100); ‘Anal. Calcd for C15H18NO5ClS:
C, 50.06; H, 5.00; N, 3.89, Found: C, 50.31; H, 5.06;
N, 3.93’.
2.4 General procedure for the synthesis of substituted
N-(2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyl-β-D-ribofuranosyl)
benzothiazines (5a, b)
To the solution of 4H-1,4-benzothiazine (3a, b)
(0.002 mol) in 5 mL toluene, β-D-ribofuranose-1-
acetate-2,3,5-tribenzoate (0.002 mol) was added and the
contents were refluxed under vacuum with stirring on
an oil bath at 155–160◦C for 15 min. The vacuum was
removed and reaction mixture was protected from mois-
ture by fitting a guard tube. Stirring was further contin-
ued for 10 h and a vacuum was applied for 10 min after
every 1 h. The viscous mass obtained was dissolved in
methanol, boiled for 10 min and cooled to room temper-
ature. The reaction mixture was filtered and the filtrate
was evaporated to dryness. The viscous residue thus
obtained was dissolved in ether, filtered, concentrated
and kept in refrigerator overnight to afford crystalline
ribofuranosides.
2.3c
Methyl-8-chloro-3-ethyl-5-methoxy-4H-1,4-
benzothiazine-2-carboxylate-1,1-dioxide (4c): Brown
solid; m.p.: 275◦C; yield: 46%, IR (KBr): v 3360
(N–H), 1610 (>C=O), 2965 and 2865 (−OCH3) and
760 cm−1 (C–Cl); 1H NMR spectral data (300.40 MHz,
Me2SO-d6, δ ppm from TMS): δ 8.91 (s, 1H, N–H),
8.65–6.72 (m, 2H, Ar-H), 3.96 (s, 3H, −OCH3 at C-2),
2.58 (q, 2H, CH2 of C2H5 at C-3), 1.59 (t, 3H, CH3 of
C2H5 at C-3), 3.82 (s, 3H, −OCH3 at C-5); 13C NMR
(75.45 MHz, CDCl3, δ ppm from TMS): 92 (C–2),
165.2 (C=O of OCH3 at C-2), 50.7 (CH3 at C-2),
159.1 (C-3), 23.6 (CH2 of C2H5 at C-3), 9.1 (CH3 of
C2H5 at C-3), 56.3 (OCH3 at C-5), 147.5 (C-5), 121.2
(C-6), 121.0 (C-7), 124.9 (C-8); MS (FAB) 10 kV,
m/z (rel. int.): 331 [M]+, 333 [M+2]+, 330 (25), 302
(71), 296 (36), 272 (61), 59 (100); ‘Anal. Calcd for
C13H14NO5SCl: C, 47.05; H, 4.22; N, 4.22, Found: C,
47.29; H, 4.26; N, 4.33’.
2.4a N-(2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyl-β-D-ribofuranosyl)-8-
chloro-2-trifluoroacetyl-3-trifluoromethyl-5-methoxy-
4H-1,4-benzothiazine (5a): Red solid; m.p.: 86◦C;
yield: 78%, IR (KBr): v 1700 (>C=O), 1170 (C–O–
C), 1355 and 1170 cm−1 (-CF3) and 795 cm−1 (C–Cl);
1H NMR spectral data (300.40 MHz, Me2SO-d6, δ ppm
from TMS): δ 8.56–7.54 (m, 2H, Ar-H), 3.80 (s, 3H,
–OCH3); 13C NMR (75.45 MHz, CDCl3, δ ppm from
TMS): δ 116.4 (C–2), 196.9 (C=O at C-2), 129.0 (CF3
at C-2), 136.7 (C-3), 114.5 (CF3 at C-3), 145.6 (C-5),
56.3 (OCH3 at C-5), 113.5 (C-6), 118.8 (C-7), 128.1
(C-8), 80.2 (C-1ꢁ), 74.1 (C-2ꢁ), 70.6 (C-3ꢁ), 69.5 (C-
4ꢁ); 19F NMR (282.65 MHz, CDCl3): δ –62.35 (s, 3F,
CF3); MS (FAB) 10 kV, m/z (rel. int.): 821 [M]+, 823

88
Nishidha Khandelwal et al.
[M+2]+, 723 (49), 820 (34), 97 (100), 725 (65), 692 disc were observed. Each testing is done in triplicate.
(53); ‘Anal. Calcd for C38H26NO9F6SCl: C, 55.51; H, Ciprofloxacin at conc. 50 μgmL−1 was used as standard
3.16; N, 1.71 Found: C, 55.76; H,3.19; N, 1.81’.
drug for antibacterial activity. Results were interpreted
in terms of diameter (mm) of zone of inhibition. The %
activity index for the compound was calculated by the
formula as under:
2.4b N-(2ꢁ,3ꢁ,5ꢁ-tri-O-benzoyl-β-D-ribofuranosyl)-
ethyl-8-chloro-5-methoxy-3-propyl-4H-1,4-benzo-
thiazine-2-carboxylate (5b): Red solid; m.p.: 79◦C;
yield: 85%, IR (KBr): v 1680 (>C=O), 1160 (C–
% Activity Index
Zone of inhibition by test compound (diameter)
=
Zone of inhibition by standard (diameter)
1
O–C), and 810 cm−1 (C–Cl); H NMR spectral data
×100.
(300.40 MHz, Me2SO-d6, δ ppm from TMS): δ 8.32–
7.13 (m, 2H, Ar-H), 3.78 (s, 3H, –OCH3), 1.95 (t, 2H,
CH2 (terminal) of C3H7 at C-3), 1.36 (sextet, 2H, CH2
(centre) of C3H7 at C-3), 0.94 (t, 3H, CH3 of C3H7 at
C-3), 4.20 (q, 2H, CH2 of OC2H5 at C-2), 1.30 (t, 3H,
CH3 of OC2H5 at C-2); 13C NMR (75.45 MHz, CDCl3,
δ ppm from TMS): δ 106.0 (C-2), 165.2 (C=O at C-2),
58.9 (CH2 of OC2H5 at C-2), 13.8 (CH3 of OC2H5 at
C-2), 144.0 (C-3), 31.8 (CH2 (terminal) of C3H7 at
C-3), 18.1 (CH2 (middle) of C3H7 at C-3), 13.8 (CH3
(terminal) of C3H7 at C-3), 144.5 (C-5), 56.1 (OCH3
at C-5), 113.1 (C-6), 118.8 (C-7), 127.6 (C-8), 81.4
(C-1ꢁ), 75.5 (C-2ꢁ), 72.2 (C-3ꢁ), 70.3 (C-4ꢁ); MS (FAB)
10 kV, m/z (rel. int.): 771 [M]+, 773 [M+2]+, 770 (41),
697 (30), 692 (54), 73 (100), 665 (60); ‘Anal. Calcd for
C41H38NO10SCl : C, 63.77; H, 4.93; N, 1.81, Founds:
C, 63.98; H, 4.98; N, 1.95’.
3.1b Antifungal activity: Antifungal activity of syn-
thesized compounds was tested on following fun-
gal strains: A. niger, C. albican, using disc diffusion
method. In the disc-diffusion method, disc impregnated
with compounds dissolved in solvent DMF at con-
centrations 25, 50 and 100 μgmL−1 were spread over
microorganism culture in nutrient agar medium. The
plates were incubated at 25◦C for 48 h for fungal strains.
After incubation, the growth inhibiting zones around
the disc were observed. Growth inhibiting zone indi-
cates that the compounds inhibit growth of microorgan-
ism. Each experiment is done in triplicate. Griseofulvin
at concentration 50 μgmL−1 was used as standard drug
for antifungal activity. Results were interpreted in terms
of diameter (mm) of zone of inhibition. The percentage
inhibition was calculated by the following equation
3. Biological activity
% Inhibition = (C − T) 100/C,
3.1 In vitro antibacterial and antifungal activity
where C and T are the diameters of the fungal colony in
the control and the test plates, respectively.
3.1a Antibacterial activity: Antibacterial activity
was tested against S. aureus, B. subtilis (Gram positive)
and E. coli, Pseudomonas aeruginosa (Gram negative)
microorganisms using paper disc diffusion method
3.2 Minimal inhibitory concentrations
using nutrient agar medium. The paper disc diffusion Minimum inhibitory concentrations (MICs) are defined
method of assay of drug potency is based on the mea- as the lowest concentration of antimicrobials that will
surement of the zone of microbial growth inhibition inhibit the visible growth of a microorganism after
surroundings discs containing various concentrations overnight incubation at 37◦C. Determination of the
of test compounds, which are placed on the surface of MIC is a semiquantitative test, which gives an approxi-
a solid nutrient previously inoculated with the culture mate idea of the least concentration of an antimicro-
of suitable microorganism. Inhibition produced by the bial (test) solution needed to parent microbial growth.
test drug is compared with that produced by known The MIC was determined by the liquid dilution method.
concentration of reference standard.
Two Gram positive (Staphylococcus aureus and Bacil-
In this method, paper disc impregnated with com- lus subtilis) and two Gram negative (Escherichia coli
pounds dissolved in solvent DMF at concentrations and Pseudomonas aeruginosa) were used as quality
25, 50 and 100 μgmL−1. Then the disc impregnated control strains. Candida albicans and Aspergilius niger
with the solution was placed on the surface of the were tested for the antifungal activities of the com-
media inoculated with the bacterial strain. The plates pounds. Ciprofloxacin and Griseofulvin were used as
were incubated at 35◦C for 24 h for bacterial cultures. standard antibacterial and antifungal agents. The stock
After incubation, the zones of inhibition around the solutions of test compounds with 1 to 20 μgmL−1

Synthesis and biological study of substituted 4H-1,4-benzothiazines
89
R1
NH2
R1
R1
R2
O
C
O
OH
H
NH2
SH
NH2
R3-C=CH-C-R4
S
S
S
R4
2a-d
R2
R2
2
O
C
S
N
H
R3
R4
R1
R2
R2
2
N
H
R3
1a-b
R1
DMSO
BzO
OCOCH3
O
O
R2
R2
O
C
1
S
2
Bz
O
C
R4
1
3
8
8
S
R4
2
7
6
where Bz = Benzoyl group
Toluene under vacuum, 155-160°C
7
5
4
6
N
3
R3
4
R1
N
R3
5
H
BzO
O
R1
4´
3´
1´
3a-d
2´
OBz
BzO
30% H2O2
glacial acetic acid
5a-b
R2
O
O
O
8
S
1
C
R4
2
7
6
3
4
N
H
R3
5
R1
4a-d
Compound R1
R2
R3
R4
Compounds
3a-d
Yield range
56-63%
46-52%
3a, 4a,5a
3b,4b,5b
3c, 4c
OCH3
Cl
Cl
Cl
H
CF3
CF3
4a-d
5a-b
OCH3
OCH3
Cl
C3H7
C2H5
C2H5
OC2H5
OCH3
C2H5
78-85%
3d, 4d
Scheme 1. Synthesis of substituted 4H-1,4-benzothiazines.
concentrations were prepared with aqueous methanol. at 4 μgmL−1 concentrations. Suspensions of samples
were prepared by triturating synthesized compounds
(200 mg) with Tween 80 (0.5%) and distilled water and
the resulting mixtures were stirred using a mechani-
cal stirrer for 30 min. The suspensions were diluted to
contain 0.4% w/v of the test samples. Suspension of
reference drug mebendazole was prepared with same
concentration in a similar way. Three sets of five earth-
worms of almost similar sizes (3 inch in length) were
placed in petri plates of 4 inch diameter containing
50 mL of suspension of test sample and reference drug
at room temperature. Another set of five earthworms
was kept as control in 50 ml suspension of distilled
water and Tween 80 (0.5%). The paralyzing and death
times were noted and their mean was calculated for trip-
licate sets. The death time was ascertained by placing
Inoculums of the overnight culture were prepared. In
a series of tubes, 1 mL each of stock solutions of test
compound with different concentration are taken and
0.4 mL of the inoculums was added to each tube. Fur-
ther 4.0 mL of sterile water was added to each of the test
tubes. These test tubes were incubated for 22–24 h and
observed for the presence of turbidity. The absorbance
of the suspension of the inoculums was observed with
spectrophotometer at 555 nm. The end result of the
test was the minimum concentration of antimicrobial
(test) solutions, which gave clear solution, i.e., no visual
growth.
3.3 Anthelmintic activity
Anthelmintic studies were carried out against Eudrilus the earthworms in warm water (50◦C), which stimulate
species of earthworms by Garg and Atal method the movement, if the worm was alive.

90
Nishidha Khandelwal et al.
Figure 1. % Activity index of E. coli and B. subtilis.
Figure 2. % Activity index of P. aeruginosa and S. aureus.
Figure 3. % Inhibition of C. albicans and A. niger.

Synthesis and biological study of substituted 4H-1,4-benzothiazines
91
4. Results and discussion
compounds were also screened for their antioxidant
and antimicrobial activities (scheme 1).
Substituted 2-amino-6-chloro-3-methoxy/2-amino-3-
A series of novel heterocyclic compounds (3a–d)
chlorobenzenethiol (1a–b) and the appropriate and (4a–d) were synthesized. All the compounds tested
β-diketones/β-ketoesters (2a–d) were refluxed in against for their in vitro antibacterial activity against
dimethyl sulphoxide which results in oxidative the four strains of bacteria (two Gram−ve E.coli, Pseu-
cyclization. This reaction proceeds with the forma- domonas aeruginosa and two Gram+ve Bacillus sub-
tion of an intermediate enaminoketone. The bis-(2- tilis, Staphylococcus aureus) and antifungal activity
aminophenyl)disulphides were obtained which then against the two strains of fungi (C. albicans and A.
cyclizes to 4H-1,4-benzothiazine (3a–d) by the scis- niger). The results have been summarized in figures 1,
sion of sulphur–sulphur bond, due to high reactivity of 2 and 3. The results show that all the compounds act as
α-position of enaminoketone system towards nucleo- antimicrobial agent against selected bacteria and fungi.
philic attack. On refluxing the compound (3a–d), with The antibacterial and antifungal activities increase with
30% hydrogen peroxide in glacial acetic acid, their increase concentration of test compounds. The mini-
corresponding sulphones (4a–d) were obtained. Com- mum inhibitory concentration (MIC) of the compounds
pounds (3a–b) were treated with β-D-ribofuranose-1- varies from 9.25 to 17.30 μgmL−1 (tables 1 and 2).
acetate-2,3,5-tribenzoate in toluene stirred in vacuum Among compounds of the obtained series, 3a, 4a, 4c
in an oil both at 155–160◦C for 10 h to form their and 5a showed good activity and remaining showed
ribofuranosides (5a–b). The structures proposed for the moderate activity against bacteria E. coli. Compounds
synthesized compounds were elucidated by spectro- 4a showed good and remaining moderate activity
scopic data and elemental analysis. All the synthesized against bacteria B. subtilis. For bacteria P. aeruginosa,
Table 1. Minimum inhibitory concentrations (μgmL−1) of synthesized compounds.
Bacterial minimal inhibitory concentrations (μgmL−1
)
Compound No.
E. coli
B. subtilis
P. aeruginosa
S. aureus
3a
3b
3c
3d
4a
4b
4c
4d
10.10 0.22
13.20 0.26
13.00 0.17
14.15 0.35
09.36 0.28
11.30 0.20
11.05 0.29
12.50 0.32
09.78 0.19
12.22 0.31
04.10 0.10
12.00 0.10
16.10 0.15
15.80 0.27
16.70 0.20
11.10 0.19
15.50 0.22
15.15 0.26
16.30 0.16
11.82 0.29
15.72 0.17
04.90 0.13
10.15 0.27
11.40 0.23
11.15 0.31
12.16 0.19
09.25 0.24
11.09 0.18
10.80 0.35
12.45 0.28
09.67 0.34
11.16 0.25
03.85 0.15
11.30 0.09
17.30 0.32
16.40 0.33
17.26 0.17
10.52 0.19
16.70 0.27
15.94 0.33
16.90 0.25
11.00 0.24
17.12 0.21
04.90 0.11
5a
5b
Ciprofloxacin
Table 2. MIC (μgmL−1) of synthesized compounds.
Fungal minimal inhibitory
concentrations (μgmL−1
)
Compound No.
C. albicans
A. niger
3a
3b
3c
3d
4a
4b
4c
4d
10.40 0.26
13.10 0.22
12.35 0.19
14.30 0.27
10.00 0.35
11.20 0.21
10.90 0.23
13.75 0.31
10.26 0.18
12.13 0.34
03.10 0.08
10.13 0.31
15.40 0.16
15.90 0.29
16.10 0.32
09.39 0.17
15.00 0.26
14.30 0.32
15.60 0.20
09.77 0.29
15.14 0.21
04.80 0.10
5a
5b
Griseofulvin

92
Nishidha Khandelwal et al.
Table 3. Anthelmintic activity of synthesized compound.
Time (min)
Paralyzing time
100 mgmL−1
Death time
100 mgmL−1
Compound No.
200 mgmL−1
50 mgmL−1
200 mgmL−1
50 mgmL−1
3a
3b
3c
3d
4a
4b
4c
4d
11.00 0.67
15.00 0.77
13.26 1.08
15.16 1.24
08.79 0.67
12.85 1.22
12.00 1.00
13.16 0.75
09.54 0.88
12.20 1.00
12.50 1.04
14.83 0.83
17.00 1.00
16.00 1.10
18.50 1.00
13.50 1.00
16.66 0.94
15.00 0.90
18.00 0.78
14.00 1.10
16.50 1.20
16.00 0.98
21.55 1.00
23.45 0.93
22.50 0.50
24.56 0.70
19.48 0.77
22.78 0.60
22.20 1.00
25.00 0.70
20.03 0.68
23.36 1.00
21.80 1.34
15.53 0.69
19.31 1.00
18.16 0.76
20.16 0.90
13.15 0.67
15.40 0.77
20.63 0.62
19.12 0.60
15.16 1.24
17.33 1.52
15.26 0.84
20.50 0.50
27.16 0.76
26.10 0.73
29.16 0.67
19.00 0.66
24.65 0.79
24.13 1.09
27.08 1.00
19.50 0.50
27.00 1.10
22.65 0.90
31.03 0.90
35.75 0.57
34.50 0.50
38.83 0.98
28.16 0.76
35.23 0.91
34.12 0.60
38.43 1.00
28.90 1.05
35.50 0.89
30.55 1.00
5a
5b
Mebendazole
compounds 3a, 4a, 4c and 5a showed good activity and activities against four strains of bacteria. Compounds
remaining showed moderate activity. Compounds 4a (4a–d) exhibited significant antibacterial and antifun-
and 5a showed good and remaining showed moderate gal activities as compared to their benzothiazines (3a–
activity against bacteria S. aureus.
d), respectively due to electron withdrawing effect of
Compounds 3a, 4a, 4c and 5a showed good acti- oxygen in (4a–d). In general, the presence of electron
vity against fungi C. albicans. For fungi A. niger, withdrawing group on the aromatic ring increases the
compounds 3a, 4a and 5a showed good activity and antimicrobial activities of tested compounds when com-
remaining showed moderate activity.
pared to compounds having electron donating groups.
The above screened compounds were tested for
anthelmintic activity. The results have been summa-
rized in table 3. The results showed that compounds
Acknowledgements
3a, 4a, 4c and 5a showed significant paralytic time The authors are thankful to the Department of Chem-
than standard drug Mebendazole, at given concentra- istry, University of Rajasthan, Jaipur, for providing
tions. Test compounds 5b show better death time of laboratory facilities and spectral data analysis. Authors
earthworms with that of standard drug Mebendazole. are also thankful to Rajiv Academy for Pharmacy,
The synthesized compounds in overall estimation con- Mathura for assistance in carrying out the biological
firms the better activity against Eudrilus species of activities. The University Grants Commission (UGC),
earthworms.
New Delhi is duly acknowledged for financial support
through Major Research Project and UGC Research
Award Scheme.
5. Conclusion
The present paper is focused on the synthesis of novel
heterocyclic compounds as possible antibacterial and
antifungal agents. The structures of synthesized com-
pounds are well-supported by spectral data and ele-
mental analysis. In the preparation of ribofuranosides
(5a–b), ribosylation takes place mostly by replac-
ing hydrogen atom attach to nitrogen in 4H-1,4-
benzothiazines (3a–d).
The present study elucidated that the synthesized
compounds showed good anthelmintic activity. Com-
pounds 3a, 4a, c and 5a showed significant paralytic
time and compounds 5b showed better death time of
earthworms with that of standard drug Mebendazole. In
antimicrobial activity, 4a exhibited good antibacterial
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