Synthesis and in vitro antimicrobial activity of benzo[b][1,4]thiazin-3(4H) -ones via smiles rearrangement

Article abstract of DOI:10.1007/s00044-009-9288-3

New benzo[b][1,4]thiazin-3(4H)-one derivatives (compounds 12a-p) were synthesized via Smiles rearrangement and assayed in vitro for their antimicrobial activity against Gram-positive, Gram-negative bacteria and fungi. The antimicrobial activity of the benzo[b][1,4]thiazin-3(4H)-ones showed, on the whole, potent toward all tested Gram-positive and Gram-negative microorganism (minimal inhibitory concentration ranging from 16 to 64 lg/ml), whereas weak effectiveness was exhibited against fungi. Data obtained suggested that 12g, 12i, and 12o exerted the best antibacterial activity against Gram-positive bacteria and compound 12b demonstrated the best inhibition of Gram-negative bacteria. These observations provide some predictions to design further antimicrobial active compounds prior to their synthesis following with molecular modeling studies. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-009-9288-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:93–100
DOI 10.1007/s00044-009-9288-3
ORIGINAL RESEARCH
Synthesis and in vitro antimicrobial activity
of benzo[b][1,4]thiazin-3(4H)-ones via smiles rearrangement
•
Hao Yang Liang Fang Zhu-Bo Li
•
•
•
Fang-Kui Ren Li-Ying Wang Xiao Tian
•
•
•
Dong-Soo Shin Hua Zuo
Received: 26 June 2009 / Accepted: 8 December 2009 / Published online: 29 December 2009
Ó Springer Science+Business Media, LLC 2010
Abstract New benzo[b][1,4]thiazin-3(4H)-one deriva-
tives (compounds 12a–p) were synthesized via Smiles
rearrangement and assayed in vitro for their antimicrobial
activity against Gram-positive, Gram-negative bacteria and
fungi. The antimicrobial activity of the benzo[b][1,4]thia-
zin-3(4H)-ones showed, on the whole, potent toward all
tested Gram-positive and Gram-negative microorganism
(minimal inhibitory concentration ranging from 16 to
64 lg/ml), whereas weak effectiveness was exhibited
against fungi. Data obtained suggested that 12g, 12i, and
12o exerted the best antibacterial activity against Gram-
positive bacteria and compound 12b demonstrated the best
inhibition of Gram-negative bacteria. These observations
provide some predictions to design further antimicrobial
active compounds prior to their synthesis following with
molecular modeling studies.
Introduction
Benzo[b][1,4]thiazin-3(4H)-one derivatives represent one
of the most active class of compounds, which are endowed
with a variety of biological activities. They are frequently
found in biologically active compounds across a number of
different therapeutic fields, such as antihypertensive (Kaj-
ino et al., 1991), antidiabetic (Matsui et al., 1994), anti-
cataract (Kawashima et al., 1994), antiarrhythmic (Fujita
et al., 1990), vasodilator (Qar et al., 1988), etc. (Fig. 1).
They also exhibit other different pharmacological
activities: bacteriostatic (Guarda et al., 2003), antimicro-
bial (Guarda et al., 2001; Pitzurra et al., 1999; Fringuelli
et al., 1998), Na?/H? exchange system inhibitor
(Yamamoto and Watanabe, 1998), calcium antagonist
(Iwao et al., 1986; Rainer and Ulrich, 1986; Ulrich and
Rainer, 1989a, b). They are also useful as prophylactic
drugs and/or therapeutic drugs in hyperlipemia, hypergly-
cemia, obesity, diseases attributable to sugar tolerance
insufficiency, hypertension, osteoporosis, cachexia, and
complications of diabetes such as retinopathy, nephrosis,
neuropathy, cataract, coronary artery disease, and arterio-
sclerosis (Okami and Kawashima, 1997). Moreover,
4H-benzo[1,4]thiazin-3(4H)-ones are used as herbicide
(Wuemachu and Oshio, 1982).
Keywords Antimicrobial activity Á
Benzo[b][1,4]thiazin-3(4H)-one Á Smiles rearrangement Á
Synthesis
H. Yang
College of Horticulture and Landscape Architecture, Southwest
University, 400715 Chongqing, China
Owing to this, and given our recent finding of a new
class of benzo[1,4]oxazin-3(4H)-ones and benzo[1,4]thia-
zin-3(4H)-ones (Zuo et al., 2008a, b, c), we decided to
extend our research to classes of analogs. Our analog-based
L. Fang
Department of Oncology, The Ninth People’s Hospital
of Chongqing, 400700 Chongqing, China
Z.-B. Li Á F.-K. Ren Á L.-Y. Wang Á X. Tian Á H. Zuo (&)
College of Pharmaceutical Sciences, Southwest University,
400715 Chongqing, China
design encompasses the synthesis of
a series of
benzo[1,4]thiazin-3(4H)-one derivatives (Scheme 1), to be
tested for their antimicrobial properties. Starting from the
knowledge of the potential of benzo[1,4]thiazin-3(4H)-one
ring system, which is a core structure in various synthetic
pharmaceuticals displaying a broad spectrum of biological
e-mail: zuohuaangel@gmail.com; zuohua@swu.edu.cn
D.-S. Shin
Department of Chemistry, Changwon National University,
641-773 Changwon, South Korea
123

94
Med Chem Res (2011) 20:93–100
Fig. 1 Some biologically active
benzo[b][1,4]thiazin-3(4H)-one
compounds
activity as mentioned above, and on the basis of the
assumption that different substitutions on benzene ring and
nitrogen atom, we replaced the benzo[1,4]thiazin-3(4H)-
one ring with analogous groups. All the compounds were
synthesized by three-step reaction via Smiles rearrange-
ment, different from the existing methods, and they were
tested in vitro antimicrobial properties against Gram-posi-
tive, Gram-negative bacteria and fungi and exhibited
moderate to excellent activity.
(Scheme 1), by which the intermediates were separated and
characterized, and the proposed Smiles rearrangement
mechanism was verified by the evidence presented in this
study.
Starting from 2-chlorobenzenethiol (10) and N-substi-
tuted-2-chloro acetamide (9), which was obtained by
reacting amine (8), chloroacetyl chloride and K2CO3 in a
molar ratio of 1:1.2:1.5 in anhydrous acetonitrile, the
S-alkylated product N-substituted-2-(2-chlorophenyl-
thio)acetamide (11) was formed in short time, usually 1 h,
with fairly good yield for all the cases. The corresponding
compound 11 thus obtained was treated with cesium car-
bonate in anhydrous DMF under reflux condition, gave the
expected benzo[b][1,4]thiazin-3(4H)-ones (12), followed
by the separation with column chromatography. The
mechanism of Smiles rearrangement involved in the for-
mation of product 12 has already been discussed (Zuo
et al., 2008b). The structure of intermediate 11 was
Materials and methods
Synthesis
The compounds described in this article were synthesized
by one-pot reaction under microwave irradiation protocol
reported earlier by us (Zuo et al., 2008b). We herein used
three-step reaction procedure under conventional condition
1
confirmed by melting point, IR, H NMR, 13C NMR, and
Scheme 1 Design and
synthesis of the target
compounds 12a–p. Reagents
and condition: (a): K2CO3,
MeCN, ice bath, 90–95%; (b):
K2CO3, anhydrous MeCN,
reflux, 82–96%; (c) Cs2CO3,
anhydrous DMF, reflux,
52–91%
123

Med Chem Res (2011) 20:93–100
95
GC-MS. The broad peak in 1H NMR of N-substituted-2-(2-
chloro phenylthio)acetamide (11) disappeared in proton
spectrum of 12, which indicated the cyclization of 11. The
further evidence was confirmed by the disappearance of
N–H stretching in IR spectrum of product 11. The spectral
data of 12a–p were in accordance with the reported data by
one-pot protocol (Zuo et al., 2008b).
127.8, 127.9, 130.4, 133.7, 136.9, 165.8; MS (EI) m/z: 317
(M?, 1%), 282 (100), 202 (21), 192 (46), 83 (20), 55 (15),
41 (7).
2-(2,3-Dichlorophenylthio)-N-((tetrahydrofuran-2-
yl)methyl)acetamide (11d)
1
An off-white solid, mp 120–121°C; H NMR (400 MHz,
General procedure for the synthesis of N-substituted-2-
(2-chlorophenylthio) acetamide (11)
CDCl3) d 1.30–1.37 (m, 1H), 1.65–1.74 (m, 1H), 1.75–1.89
(m, 2H), 3.22–3.28 (m, 1H), 3.45–3.50 (m, 1H), 3.65–3.35
(m, 4H), 3.84–3.90 (m, 1H), 6.97 (s, 1H), 7.10 (dd,
J = 8.0, 1.6 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.30 (dd,
J = 8.0, 1.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 25.8,
28.3, 36.3, 43.1, 68.2, 77.4, 124.9, 127.7, 127.8, 130.4,
133.7, 137.0, 167.2; MS (EI) m/z: 319 (M?, 2%), 284 (48),
236 (11), 214 (8), 191 (15), 142 (26), 84 (59), 71 (100), 43
(26).
The mixture of 2-chlorobenzenethiol (10) (1.0 mmol), N-
substituted-2-chloro acetamide (9) (1.1 mmol), K2CO3
(1.1 mmol) in CH3CN (20 ml) was refluxed. After com-
pletion of the reaction (monitored by TLC), the solution
was cooled; solvent was evaporated under reduced pres-
sure. The residue was poured into water and pH adjusted to
6–7 and extracted with ethyl acetate, washed with brine
and dried over anhydrous MgSO4. Removal of solvent
afforded the corresponding N-substituted-2-(2-chlorophe-
nylthio)acetamide (11).
N-Benzyl-2-(2,4-dichlorophenylthio)acetamide (11e)
A white solid, mp 126–127°C; 1H NMR (400 MHz,
CDCl3) 3.70 (s, 2H), 4.41 (d, J = 6.0 Hz, 2H), 6.99 (br,
1H), 7.09–7.37 (m, 8H); 13C NMR (100 MHz, CDCl3) d
36.4, 43.8, 127.6, 127.7, 128.0, 128.7, 128.8, 129.7, 132.5,
132.8, 133.7, 137.5, 167.0; MS (EI) m/z: 325 (M?, 5%),
290 (38), 191 (20), 148 (100), 107 (23), 91 (65), 77 (8), 65
(9), 45 (8).
N-Benzyl-2-(2,3-dichlorophenylthio)acetamide (11a)
A white solid, mp 154.5–155.5°C; IR (KBr) m/cm-1: 3436,
3089, 3035, 2924, 1650, 1587, 1561, 1495, 1453, 1436,
1
1401, 1334, 1263, 1219, 1201, 1017, 770, 739, 696; H
NMR (400 MHz, CDCl3) d 3.72 (s, 2H), 4.41 (d,
J = 6.0 Hz, 2H), 7.02 (br, 1H), 7.06–7.32 (m, 8H); 13C
NMR (100 MHz, CDCl3) d 36.4, 43.8, 125.0, 127.5, 127.6,
127.8, 127.9, 128.7, 130.4, 133.8, 136.7, 137.5, 167.0; MS
(EI) m/z: 325 (M?, 1%), 290 (62), 191 (22), 148 (100), 142
(10), 107 (25), 91 (80), 77 (9), 65 (10), 45 (11).
2-(2,4-Dichlorophenylthio)-N-hexylacetamide (11f)
A white solid, mp 87–88.5°C; 1H NMR (400 MHz, CDCl3)
d 0.85 (t, J = 7.2 Hz, 3H), 1.14–1.25 (m, 6H), 1.37–1.44
(m, 2H), 3.22 (dd, J = 13.2, 7.2 Hz, 2H), 3.66 (s, 2H), 6.70
(br, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.4,
2.0 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 14.0, 22.5, 26.4, 29.3, 31.4, 36.3,
39.9, 128.0, 128.3, 130.0, 132.6, 132.8, 133.4, 166.7; MS
(EI) m/z: 319 (M?, 4%), 284 (100), 192 (53), 142 (13), 43
(16).
2-(2,3-Dichlorophenylthio)-N-hexylacetamide (11b)
A white solid, mp 86–87°C; 1H NMR (400 MHz, CDCl3) d
0.84 (t, J = 6.8 Hz, 3H), 1.13–1.24 (m, 6H), 1.36–1.43 (m,
2H), 3.22 (m, 2H), 3.68 (s, 2H), 6.71 (br, 1H), 7.06 (dd,
J = 8.0, 1.2 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.30 (dd,
J = 8.0, 1.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 14.0,
22.5, 26.4, 29.3, 31.4, 36.3, 39.9, 124.6, 127.7, 127.8,
127.9, 133.8, 136.9, 166.7; MS (EI) m/z: 319 (M?, 1%),
284 (100), 192 (47), 142 (11), 85(8), 43 (17).
N-Cyclohexyl-2-(2,4-dichlorophenylthio)acetamide (11g)
A white solid, mp 150–151°C; 1H NMR (400 MHz,
CDCl3) d 1.01–1.18 (m, 3H), 1.25–1.38 (m, 2H), 1.56–1.67
(m, 3H), 1.76–1.80 (m, 2H), 3.63 (s, 2H), 3.63–3.78 (m,
1H), 6.57 (d, J = 6.8 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H),
7.22 (dd, J = 8.4, 2.4 Hz, 1H), 7.41 (d, J = 2.4 Hz, 1H);
13C NMR (100 MHz, CDCl3) d 24.7, 25.4, 32.8, 36.7, 48.6,
127.9, 128.7, 130.0, 132.4, 132.7, 133.7, 165.8; MS (EI) m/
z: 317 (M?, 4%), 282 (100), 202 (22), 192 (58), 161 (10),
142 (9), 83 (24), 55 (17), 41 (7).
N-Cyclohexyl-2-(2,3-dichlorophenylthio)acetamide (11c)
A white solid, mp 148–149°C; 1H NMR (400 MHz,
CDCl3) d 1.00–1.16 (m, 3H), 1.26–1.33 (m, 2H), 1.55–1.62
(m, 3H), 1.75–1.78 (m, 2H), 3.66 (s, 2H), 3.69–3.79 (m,
1H), 6.56 (br, 1H), 7.07 (dd, J = 8.0, 1.2 Hz, 1H), 7.15 (t,
J = 8.0 Hz, 1H), 7.31 (dd, J = 8.0, 1.2 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 24.6, 25.3, 31.6, 36.5, 48.6, 124.9,
123

96
Med Chem Res (2011) 20:93–100
2-(2,4-Dichlorophenylthio)-N-((tetrahydrofuran-2-
yl)methyl)acetamide (11h)
3.86–3.93 (m, 1H), 6.98 (br, 1H), 7.12 (dd, J = 8.4,
2.4 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.30 (d,
J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 25.9,
28.4, 36.1, 43.2, 68.2, 77.4, 127.2, 127.3, 130.6, 131.1,
133.5, 136.1, 166.9; MS (EI) m/z: 319 (M?, 1%), 284
(147), 236 (9), 191 (15), 142 (24), 84 (58), 71 (100), 43
(29).
A white solid, mp 101–103°C; 1H NMR (400 MHz,
CDCl3) d 1.34–1.41 (m, 1H), 1.69–1.77 (m, 1H), 1.79–1.92
(m, 2H), 3.19–3.26 (m, 1H), 3.46–3.52 (m, 1H), 3.62–3.76
(m, 4H), 3.85–3.91 (m, 1H), 6.96 (br, 1H), 7.17–7.23 (m,
2H), 7.40 (d, J = 2.0 Hz, 1H); 13C NMR (100 MHz,
CDCl3) d 25.8, 28.4, 36.4, 43.2, 68.2, 77.3, 127.8, 128.7,
129.6, 132.6, 132.8, 133.7, 167.2; MS (EI) m/z: 319 (M?,
5%), 284 (20), 236 (10), 202 (13), 191 (15), 142 (25), 84
(57), 71 (100), 43 (26).
N-Benzyl-2-(2,6-dichlorophenylthio)acetamide (11m)
A white solid, mp 154–155°C; 1H NMR (400 MHz,
CDCl3) d 3.73 (s, 2H), 4.40 (d, J = 2.0 Hz, 2H), 7.17–7.35
(m, 9H); 13C NMR (100 MHz, CDCl3) d 38.6, 44.0, 127.5,
127.6, 128.7, 129.1, 130.7, 131.3, 137.6, 140.7, 167.4; MS
(EI) m/z: 290 ([M-Cl]?, 83%), 191 (22), 148 (100), 107
(17), 91 (76), 77 (9), 65 (10), 45 (10).
N-Benzyl-2-(2,5-dichlorophenylthio)acetamide (11i)
1
An off-white solid, mp 132–134°C; H NMR (400 MHz,
CDCl3) d 3.71 (s, 2H), 4.44 (d, J = 6.0 Hz, 2H), 7.00 (br,
1H), 7.11–7.30 (m, 8H); 13C NMR (100 MHz, CDCl3) d
36.1, 43.9, 127.2, 127.4, 127.5, 127.6, 128.8, 130.7, 131.0,
133.6, 135.9, 137.5, 166.6; MS (EI) m/z: 325 (M?, 1%),
290 (85), 191 (22), 148 (100), 107 (27), 91 (80), 77 (10), 65
(11), 45 (9).
2-(2,6-Dichlorophenylthio)-N-hexylacetamide (11n)
A white solid, mp 79–80°C; 1H NMR (400 MHz, CDCl3) d
0.88 (t, J = 6.8 Hz, 3H), 1.23–1.31 (m, 6H), 1.45–1.52 (m,
2H), 3.22 (dd, J = 13.2, 7.2 Hz, 2H), 3.70 (s, 2H), 7.11 (br,
1H), 7.22 (dd, J = 8.4, 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz,
2H); 13C NMR (100 MHz, CDCl3) d 14.1, 22.6, 26.6, 29.3,
31.5, 38.7, 40.0, 129.1, 130.6, 131.5, 140.6, 167.3; MS (EI)
m/z: 284 ([M-Cl]?, 100%), 192 (44), 159 (11), 142 (13), 43
(13).
2-(2,5-Dichlorophenylthio)-N-hexylacetamide (11j)
A white solid, mp 87–88.5°C; 1H NMR (400 MHz, CDCl3)
d 0.85 (t, J = 7.2 Hz, 3H), 1.61–1.24 (m, 6H), 1.39–1.47
(m, 2H), 3.25 (dd, J = 13.2, 6.8 Hz, 2H), 3.67 (s, 2H), 6.70
(br, 1H), 7.12 (dd, J = 8.4, 2.4 Hz, 1H), 7.17 (d,
J = 2.4 Hz, 1H), 7.29 (t, J = 8.4 Hz, 1H); 13C NMR
(100 MHz, CDCl3) d 14.1, 22.5, 26.5, 29.3, 31.4, 36.2,
40.0, 127.1, 127.3, 130.7, 130.9, 133.7, 136.0, 166.4; MS
(EI) m/z: 319(M?, 1%), 284 (100), 192 (33), 142 (7), 43
(9).
N-Cyclohexyl-2-(2,6-dichlorophenylthio)acetamide (11o)
A white solid, mp 189–191°C; 1H NMR (400 MHz,
CDCl3) d 1.10–1.21 (m, 3H), 1.27–1.38 (m, 2H), 1.59–1.64
(m, 1H), 1.68–1.73 (m, 2H), 1.82–1.86 (m, 2H), 3.64–3.73
(m, 1H), 3.69 (s, 2H), 6.97 (br, 1H), 7.22 (dd, J = 8.4,
7.6 Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H); 13C NMR
(100 MHz, CDCl3) d 24.9, 25.5, 32.8, 38.7, 48.9, 129.0,
130.6, 131.6, 140.6, 166.4; MS (EI) m/z: 282 ([M-Cl]?,
100%), 202 (21), 192 (45), 83 (18), 55 (17).
N-Cyclohexyl-2-(2,5-dichlorophenylthio)acetamide (11k)
A white solid, mp 141–141.5°C; 1H NMR (400 MHz,
CDCl3) d 1.03–1.18 (m, 3H), 1.28–1.39 (m, 2H), 1.59–1.67
(m, 3H), 1.79–1.83 (m, 2H), 3.65 (s, 2H), 3.72–3.83 (m,
1H), 6.53 (d, J = 10.4 Hz, 1H), 7.13 (dd, J = 8.4, 2.4 Hz,
1H), 7.19 (d, J = 2.4 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H);
13C NMR (100 MHz, CDCl3) d 24.7, 25.4, 32.7, 36.5, 48.6,
127.4, 127.6, 130.7, 131.2, 133.6, 136.0, 165.5; MS (EI) m/
z: 317 (M?, 1%), 282 (100), 202 (18), 192 (43), 142 (7), 83
(19), 55 (14), 32 (10).
2-(2,6-Dichlorophenylthio)-N-((tetrahydrofuran-2-
yl)methyl)acetamide (11p)
A white solid, mp 125–126°C; 1H NMR (400 MHz,
CDCl3) d 1.46–1.54 (m, 1H), 1.85–1.98 (m, 3H),
3.14–3.21 (m, 1H), 3.47–3.53 (m, 1H), 3.69 (s, 2H),
3.72–3.78 (m, 1H), 3.84–3.89 (m, 1H), 3.92–3.99 (m,
1H), 7.20–7.41 (m, 4H); 13C NMR (100 MHz, CDCl3) d
25.8, 28.6, 38.7, 43.6, 68.1, 77.4, 129.0, 130.6, 131.6,
140.9, 167.8; MS (EI) m/z: 319 (M?, 1%), 284 (88), 249
(7), 236 (30), 214 (20), 191 (23), 155 (10), 142 (35), 84
(63), 71 (100), 43 (34).
2-(2,5-Dichlorophenylthio)-N-((tetrahydrofuran-2-
yl)methyl)acetamide (11l)
A white solid, mp 76–78°C; 1H NMR (400 MHz, CDCl3) d
1.37–1.44 (m, 1H), 1.72–1.82 (m, 1H), 1.84–1.95 (m, 2H),
3.25–3.31 (m, 1H), 3.47–3.53 (m, 1H), 3.67–3.71 (m, 4H),
123

Med Chem Res (2011) 20:93–100
97
General procedure for the synthesis
ATCC 74212). The minimal inhibitory concentrations (MIC,
lg/ml) were defined as the lowest concentrations of com-
pound that completely inhibited the growth of each strain.
Ampicillin, streptomycin, ceftazidime, and clotrimazole
were used as reference antibacterial and antifungal sub-
stances, respectively. Test compounds were dissolved in
distilled dimethylsulfoxide and then were plated in 96-well
microplates. Each tested compound (1024 lg/ml in
DMSO) was transferred to each microplate well, to be
diluted in 100 ll of broth and 100 ll of inocula (Broth
Agar Medium for all Gram-positive and Gram-negative
bacteria and Sabouraud Liquid Medium for fungi) to obtain
final concentrations at 512, 256, 128, 64, 32, 16, 8, 4, 2,
1 lg/ml, respectively. Dimethylsulfoxide never exceeded
1% v/v. The amount of inocula was 5.00 9 105 bacteria/ml
and 1.00 9 104 fungi/ml. After incubation at 37°C for 24 h
(bacteria) and at 30°C for 48 h (fungi), the last tube
remaining clear with no growth of microorganism was
recorded to represent MIC. The MICs were read after
incubation at 37°C for 24 h (bacteria) and at 30°C for 48 h
(fungi). Growth controls consisting of media and media
with 1% v/v dimethylsulfoxide were employed.
of benzo[b][1,4]thiazin-3(4H)-ones (12)
The reaction mixture of appropriate N-substituted-2-(2-
chlorophenylthio)acetamide (11) (1.0 mmol) and Cs2CO3
(1.2 mmol) was refluxed in dry DMF (10 ml). After the
completion of the reaction (monitored by TLC), the DMF
was removed under vacuum. Water (20 ml) was added to
the residue. The aqueous solution was extracted by ethyl
acetate (4 9 30 ml), and the combined organic layers were
dried over anhydrous MgSO4. The solvent was removed
under vacuum to obtain the crude product which was
purified by column chromatography (silica gel) to afford
the desired pure compound.
Spectral data of the selected product (12a): A light-
yellow powder; mp 96–97°C; IR (KBr) m/cm-1: 3070,
3026, 3001, 2955, 2916, 1662, 1575, 1562, 1494, 1450,
1375, 1312, 1275, 1148, 883, 776, 733, 698; 1H NMR
(400 MHz, CDCl3) d: 3.53 (s, 2H), 5.20 (s, 2H), 6.92 (dd,
J = 8.0, 1.2 Hz, 1H), 7.00–7.09 (m, 2H), 7.17–7.33 (m,
5H); 13C NMR (100 MHz, CDCl3) d: 31.1, 48.8, 116.6,
124.6, 126.3, 127.1, 127.4, 128.9, 129.1, 132.6, 136.3,
140.8, 164.9; MS (EI) m/z: 289 (M?, 69%), 246 (7), 170
(28), 134 (7), 108 (7), 91 (100), 65 (12).
The minimal bactericidal concentrations (MBC, lg/ml)
were measured by subculturing 100 ll of each sample
remaining clear in tubes containing 1 ml of fresh medium.
The tubes were then incubated at 37°C for 24 h. All the
experiments were performed in duplicate and repeated three
times.
Antimicrobial susceptibility test
Test compounds
Test compounds were 12a–p synthesized according to the
above procedure.
Results and discussion
The benzo[b][1,4]thiazin-3(4H)-ones (12) were assayed in
vitro for their antimicrobial activity against a panel of
selected Gram-positive, Gram-negative bacteria and fungi
in Table 1, in comparison with those of the reference drugs
ampicillin, streptomycin, ceftazidime, and clotrimazole,
and the MICs that inhibited the growth of the tested
microorganisms were detected.
Strains
Gram-positive bacteria were Staphylococcus aureus ATCC
25923; Bacillus subtilis ATCC 6633; Micrococcus luteus
ATCC 4698, and Gram-negative bacteria were Escherichia
coli ATCC 25922; Proteus vulgaris ATCC 6896; Pseu-
domonas aeruginosa ATCC 27853. Fungi were Candida
albicans ATCC 76615 and Saccharomyces cerevisiae
ATCC 74212.
All the compounds displayed good inhibition of the
growth of Gram-positive and Gram-negative bacteria,
including Staphylococcus aureus, Bacillus subtilis,
Micrococcus luteus, Escherichia coli, Proteus vulgaris,
and Pseudomonas aeruginosa. Most compounds demon-
strated MIC values in the range of 16–64 lg/ml, and the
MIC values are represented in Table 1 and Fig. 2. All of
the novel benzo[b][1,4]thiazin-3(4H)-ones exhibited
activity lower than those of the reference drugs ampicillin,
streptomycin, ceftazidime, and clotrimazole.
MIC tests
The antimicrobial activity was assayed in vitro by the two-
fold broth dilution technique (Jorgensen and Sahm, 1995)
against Gram-positive bacteria (Staphylococcus aureus
ATCC 25923, Bacillus subtilis ATCC 6633, and Micro-
coccus luteus ATCC 4698), Gram-negative bacteria (Esch-
erichia coli ATCC 25922, Proteus vulgaris ATCC 6896, and
Pseudomonas aeruginosa ATCC 27853), fungi (Candida
albicans ATCC 76615 and Saccharomyces cerevisiae
In order to evaluate the influence of N-substitution on the
antimicrobial activity, different amines including benzyl-
amine, n-hexylamine, cyclohexylamine, and tetrahydrofuranyl
amine were conducted for the reaction and then for activity
123

98
Med Chem Res (2011) 20:93–100
Table 1 Antimicrobial activity of compounds 12a–p expressed as
MIC (lg/ml)
Table 1 continued
Microorganismsa
Gram-positive
SA BS ML EC PV PA CA SC
12n 32 32 32 32 32 64
Microorganismsa
Product
Gram-negative Fungib
Product
Gram-positive Gram-negative Fungib
SA BS ML EC PV PA CA SC
–
–
Cl
N
S
O
12a 64 64 32 64 64
32 128 64
N
S
O
O
12o 16 32 16 32 32 32 64 128
Cl
Cl
Cl
Cl
N
O
12b 32 16 32 32 16
32 64 64
S
N
O
12p 32 32 32 32 32 64 64 128
S
N
S
O
12c 16 32 32 32 32
64 128 64
Ampicillin
Streptomycin
Ceftazidime
Clotrimazole
a
2
2
4
1
32
2
16 16 16 128
–
–
–
2
–
–
–
4
N
O
8
4
4
4
4
2
S
Cl
Cl
16
128
O
12d 32 16 32 64 64 128 128 128
512 512 512 512 512 512
N
O
Gram-positive bacteria: SA, Staphylococcus aureus ATCC 25923;
BS, Bacillus subtilis ATCC 6633; ML, Micrococcus luteus ATCC
4698; Gram-negative bacteria: EC, Escherichia coli ATCC 25922;
PV, Proteus vulgaris ATCC 6896; PA, Pseudomonas aeruginosa
ATCC 27853
S
c
12e 64 64 16 32 32
64
–
–
b
N
S
O
O
Fungi: CA, Candida albicans ATCC 76615; SC, Saccharomyces
cerevisiae ATCC 74212
Cl
c
Not tested. Compounds resulted inactive against the fungi
12f 32 32 64 32 64
64 128 256
32 64 128
N
S
Cl
12g 16 32 16 32 32
N
S
O
Cl
O
12h 32 64 16 32 32
64 128 128
N
S
O
Cl
Cl
12i 16 32 16 32 32
32 64 64
N
S
O
O
12j 32 32 16 32 32
64
–
–
–
Cl
N
S
12k 32 64 32 64 64 128 256
Cl
N
O
Fig. 2 Antibacterial activity of compounds 12a–p expressed as MIC
(lg/ml)
S
O
12l 64 32 32 64 64 128 64 128
Cl
N
O
test. The result showed that compounds containing N-alkyl
substitution such as n-hexyl and cyclohexyl exhibited higher
average antibacterial activity than those with either aryl or
heterocycle (Table 1). Moreover, the position of chlorine
atom at benzene ring also exerted a certain effect on the
S
12m 32 32 32 16 64
32
– 128
Cl
N
S
O
123

Med Chem Res (2011) 20:93–100
99
Conclusions
New benzo[b][1,4]thiazin-3(4H)-one derivatives (com-
pounds 12a–p), carrying 5-chloro, 6-chloro, 7-chloro, and
8-chloro on the benzene ring, or n-hexyl, cyclohexyl,
benzyl, and tetrafuryl methyl groups attached to nitrogen
atom in the molecules, were synthesized via Smiles rear-
rangement and assayed in vitro for their antimicrobial
activity against Gram-positive, Gram-negative bacteria and
fungi. The method presents an efficient protocol for syn-
thesizing benzo[b][1,4]thiazin-3(4H)-ones. The antimicro-
bial activity of the benzo[b][1,4]thiazin-3(4H)-ones
showed, on the whole, potent toward a wide spectrum of
Gram-positive and Gram-negative microorganism (MIC
ranging from 16 to 128 lg/ml), whereas weak effective-
ness was exhibited against fungi. Data obtained suggest
that compounds 12g, 12i and 12o exerted the best anti-
bacterial activity against Gram-positive bacteria, and
compound 12b demonstrated the best inhibition of Gram-
negative bacteria. The good properties of this new class of
antibacterial substances deserve further investigation for
deeper insight into structure–activity relationship and to
optimize the effectiveness of this series of molecules.
Fig. 3 Average value of antibacterial activity of compounds 12a–
p expressed as MIC (lg/ml)
activity of these compounds against Gram-positive and
Gram-negative bacteria. Compounds holding chloro at 5-C
position possessed the strongest antibacterial activity with
the lowest average MIC values, while those with chloro at 6-
C positionexhibited the weakest antibacterial activity, which
can be seen from the average MIC values of 12a–d, 12e–h,
12i–l, 12m–p at 43, 37, 45, 34 lg/ml, respectively, accord-
ing to the calculation. Thus, compounds with N-n-hexyl
(12b, 12j, 12n, average MIC values at 27, 35, 37 lg/ml,
respectively) and N-cyclohexyl (12c, 12g, 12o, average MIC
values at 35, 29, 27 lg/ml, respectively) showed higher
antimicrobial activity in respect to other substances (average
MIC values at 45–64 lg/ml), whereas, 12k with 6-chloro
exhibited much weaker activity even they held N-alkyl
moiety in the molecules (Fig. 2). It is interesting to point out
that compound 12i (with both N-benzyl and 6-chloro) also
attributed to good activity among all the compounds.
As regard to the average MIC value against all the tested
Gram-positive and tested Gram-negative bacteria, the
average MIC value of antibacterial activity against Gram-
positive was much stronger than the activity against Gram-
negative (Fig. 3).
Acknowledgments The authors acknowledge the financial support
of the grants from Natural Science Foundation Project of CQ CSTC,
2009BB5007.
References
Fringuelli R, Schiaffella F, Bistoni F, Pitzurra L, Vecchiarelli A
(1998) Azole derivatives of 1,4-benzothiazine as antifungal
agents. Bioorg Med Chem 6(1):103–108
Fujita M, Ito S, Ota A, Kato N, Yamamoto K, Kawashima Y,
Yamauchi H, Iwao J (1990) Synthesis and calcium ion antag-
onistic activity of 2-[2-[(amino alkyl) oxy]-5-methoxyphenyl]-
3,4-dihydro-4-methyl-3-oxo-2H-1,4-benzothiazines. J Med Chem
33(7):1898–1905
Guarda VLM, Perrissin M, Thomasson F, Ximenes EA, Galdino SL,
Pitta IR, Luu-Duc C (2001) Synthesis and microbiological
activity of some 4-butyl-2H-benzo[1,4]thiazin-3-one derivatives.
Il Farmaco 56(9):689–693
Guarda VLM, Perrissin M, Thomasson F, Ximenes EA, Galdino SL,
Pitta IR, Luu-Duc C, Barbe J (2003) Synthesis of 4-octyl-2H-
1,4-benzothiazin-3-ones. Eur J Med Chem 38(7–8):769–773
Iwao J-I, Iso T, Oya M (1986) Platelet anti-aggregative- and calcium
antagonistic-1,4-benzothiazin-3-one derivatives, compositions,
and methods of use therefor. U.S. Patent 4584300
Concerning fungi Candida albicans and Saccharomyces
cerevisiae, most compounds were inactive against them.
As represented in Table 1, only 12b and 12i showed
moderate activity against the fungi.
Jorgensen JH, Sahm DF (ed) (1995) Manual of clinical microbiology.
ASM Press, Washington, pp 1275–1434
Among all the tested novel compounds, compounds 12g,
12i, and 12o exerted the best antibacterial activity against
Gram-positive and compound 12b demonstrated the best
inhibition of Gram-negative bacteria.
Kajino M, Mizuno K, Tawada H, Shibouta Y, Nishikawa K, Meguro
K (1991) Synthesis and biological activities of new 1,4-
benzothiazine derivatives. Chem Pharm Bull 39(11):2888–2895
Kawashima Y, Ota A, Mibu H (1994) 3-Oxo-1,4-benzothiazine
derivative. W.O. Patent 9405647
All the active compounds exhibited microbiostatic
properties having MFC values higher than the corre-
sponding MICs (data not shown).
Matsui T, Nakamura Y, Ishikawa H, Matsuura A, Kobayashi F (1994)
Pharmacological profiles of a novel aldose reductase Inhibitor,
123

100
Med Chem Res (2011) 20:93–100
SPR-210, and its effects on streptozotocin-induced diabetic rats.
Jpn J Pharmacol 64(2):115–124
Wuemachu T, Oshio H (1982) Process for preparing 1,4-benzo
thiazine derivatives. K.R. Patent 820000849
Okami Y, Kawashima Y (1997) Production of benzothiazine deriv-
ative. J.P. Patent 9012576
Yamamoto T, Watanabe I (1998) Benzo[1,4]thiazine derivatives and
drugs comprising the same. W.O. Patent 9813357
Pitzurra L, Fringuelli R, Perito S, Schiaffella F, Barluzzi R, Bistoni F,
Vecchiarelli A (1999) A new azole derivative of 1,4-benzothi-
azine increases the antifungal mechanisms of natural effector
cells. Antimicrob Agents Chemother 43:2170–2175
Zuo H, Meng L, Ghate M, Hwang K-H, Cho Y-K, Chandrasekhar S,
Reddy CR, Shin D-S (2008a) Microwave-assisted one-pot
synthesis of benzo[b][1,4]oxazin-3(4H)-ones via Smiles rear-
rangement. Tetrahedron Lett 49(23):3827–3830
Qar J, Barhanin J, Romey G, Henning R, Lerch U, Oekonomopulos R,
Urbach H, Lazdunski M (1988) A novel high affinity class of
Ca2? channel blockers. Mol Pharmacol 33(4):363–369
Rainer H, Ulrich L (1986) Benzothiazine derivatives. U.S. 4595685
Ulrich L, Rainer H (1989a) Benzothiazinone-oxides, processes for
their preparation, medicaments containing them, and their use, as
well as intermediates for their preparation. EP Patent 0303180
Ulrich, L, Rainer, H (1989b) Novel benzothiazinone derivatives,
processes for their preparation, medicaments containing them
and their use. U.S. Patent 4831028
Zuo H, Li Z-B, Ren F-K, Falck JR, Lijuan M, Ahn C, Shin D-S
(2008b) Microwave-assisted one-pot synthesis of benzo[b]
[1,4]thiazin-3(4H)-ones via Smiles rearrangement. Tetrahedron
64(41):9669–9674
Zuo H, Kam K-H, Kwon H-J, Meng L, Ahn C, Won T-J, Kim T-H,
Reddy CR, Chandrasekhar S, Shin D-S (2008c) Microwave-
assisted synthesis of 2H-benzo[b][1,4]oxazin-3(4H)-ones and
1H-Pyrido[2,3-b][1,4] oxazin-2(3H)-ones via smiles rearrange-
ment. Bull Korean Chem Soc 29(7):1379–1385
123