Design, synthesis, antimicrobial, anti-inflammatory, and analgesic activity of novel dihydrobenzo furo[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones

Article abstract of DOI:10.1007/s00044-013-0598-0

Novel series of dihydro benzofuro[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones 6 have been synthesized from 3,5-dimethyl-4-nitroisoxazole 1. Compound 1 on treatment with salicyl aldehydes afforded the corresponding nitrostyrylisoxazoles 3, which upon reaction

Full text of DOI:10.1007/s00044-013-0598-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0598-0
ORIGINAL RESEARCH
Design, synthesis, antimicrobial, anti-inflammatory, and analgesic
activity of novel dihydrobenzo furo[3,2-e]isoxazolo[4,5-b]
azepin-5(5aH)-ones
•
E. Rajanarendar K. Govardhan Reddy S. Rama Krishna
•
•
•
B. Shireesha Y. N. Reddy M. V. Rajam
•
Received: 21 December 2012 / Accepted: 24 April 2013
Ó Springer Science+Business Media New York 2013
Abstract Novel series of dihydro benzofuro[3,2-e]isoxaz-
olo[4,5-b]azepin-5(5aH)-ones 6 have been synthesized from
3,5-dimethyl-4-nitroisoxazole 1. Compound 1 on treatment
with salicyl aldehydes afforded the corresponding nitros-
tyrylisoxazoles 3, which upon reaction with ethyl bromo
acetate followed by cyclization with triethylamine furnished
ethyl2,3-dihydro-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]
benzofuran-2-carboxylates 5. Reductive cyclization of
compounds 5 was effected with SnCl2–MeOH to give the title
compounds 6. Compounds 4–6 were characterized by IR, 1H
NMR, 13C NMR and Mass spectral data. The title compounds
6a–g were evaluated for their antimicrobial, anti-inflamma-
tory, LOX-5 inhibitory, and analgesic activity. Compounds
6b and 6c exhibited significant antimicrobial activity, potent
anti-inflammatory and analgesic activities as that of standard
drugs.
Introduction
2,3-Disubstituted dihydrobenzofuran moiety is ubiquitous in
nature and display remarkably diverse biological properties
such as antimicrobial (Pauletti et al., 2000), antimitotic
(Pieters et al., 1999), antiangiogenic (Apers et al., 2003), and
HIV integrase inhibition activity [Abd-Elazem et al., 2002).
Remarkable examples of this class are (?)-conocarpan (1)
which exhibits insecticidal, antifungal, and antitrypanoso-
mal activity (De Campos et al., 2005), as well as anti-tumor
neo-lignane (2R, 3S)-30,4-di-o-methylcedrusin (2) (Pieters
et al., 1999). Recently benzofuran-fused azepinone system
was identified as potent and selective protein kinase D (PKD)
inhibitor (LaValle et al., 2010).
Azepine derivatives such as isoxazolo[4,5-c] azepine (3)
are found to contain many pharmacological applications
(Krogsgaard-Larsen et al., 1988), whereas isoxazolo[4,5-b]
azepine (4) and isoxazolo[5,4-b] azepine (5) (Fig. 1) are
active against Gram-positive and Gram-negative bacteria
and exhibit cytotoxicity (Rao et al., 1981a, b). Inspired by
the biological activity of 2,3-dihydrobenzofuran and isox-
azolo azepines, we embarked on the synthesis of title
compounds having isoxazolo azepine and 2,3-dihydro
benzofuran moieties embedded in a fused molecular
framework to improve specificity and efficacy of these
scaffolds against microorganisms, and these compounds
promise to offer fascinating scaffolds of fundamental
interest to both synthetic and medicinal chemists. As a part
of our ongoing research in development of new biologically
active fused isoxazole derivatives (Rajanarendar et al.,
2012a, b, c, d) from readily available starting materials, we
envisaged a novel modular synthesis leading to dihydro-
benzo furo[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-one deriv-
atives. Herein, we report the discovery of furo[3,2-e]
isoxazolo[4,5-b]azepin-5(5aH)-ones from 3,5-dimethyl-4-
Keywords Dihydrobenzofuro[3,2-e]isoxazolo[4,5-b]
azepin-5(5aH)-ones ꢀ Reductive cyclization ꢀ
Antimicrobial activity ꢀ Anti-inflammatory activity ꢀ
Analgesic activity
E. Rajanarendar (&) ꢀ K. Govardhan Reddy ꢀ S. Rama Krishna
Department of Chemistry, Kakatiya University,
Warangal 506 009, Andhra Pradesh, India
e-mail: rajanarendareligeti@gmail.com
B. Shireesha ꢀ Y. N. Reddy
Department of Pharmacology and Toxicology, Kakatiya
University, Warangal 506 009, Andhra Pradesh, India
M. V. Rajam
Department of Genetics, University of Delhi, South Campus,
New Delhi 110 02, India
123

Med Chem Res
Fig. 1 Biologically active
dihydrobenzofurans and
isoxazolo azepines
HO
OH
O
O
OMe
MeO
OMe
Conocarpan
(1)
(2R, 3S)-3,4'-di-o-methylcedrusin
(2)
2
R
Ar'
N
OR'
N
H3C
H3C
3
R
N
N
N
N
O
O
Ar
R
H
O
4
isoxazolo[4,5-c]azepine
(3)
isoxazolo[4,5-b]azepine
(4)
isoxazolo[5,4-b]azepine
(5)
nitroisoxazole synthon and their antimicrobial, anti-
inflammatory, and analgesic activities.
CH=CH, J = 12 Hz), 6.78 (d, 1H, CH=CH, J = 12 Hz),
7.10–7.68 (m, 3H, ArH), 8.60 (s, 1H, OH, D2O
exchangeable). 13CNMR (75 MHz, CDCl3) d ppm: 12.32,
41.53, 102.49, 113.29, 115.48, 120.29, 123.64, 126.81,
128.68, 132.89, 149.58, 158.03, 159.11. ESI-MS (70 eV)
m/z: 261 [M?H]?. Anal. Calcd for C13H12N2O4: C, 60.00;
H, 4.65; N, 10.76. Found: C, 60.04; H, 4.69; N, 10.79 %.
Experimental
Synthesis
General procedure for the synthesis of (E)-2-(2-(3-methyl-
4-nitroisoxazole-5-yl)vinyl)phenols (3a–g)
4-Methoxy-2-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)
phenol (3c) Yield 85 %, m.p. 152–154 °C. IR (KBr)
1
cm-1: 3448. H NMR (300 MHz, CDCl3) d ppm: 2.25 (s,
A mixture of 3,5-dimethyl-4-nitroisoxazole 1 (1 mmol)
and 2-hydroxy benzaldehyde (1 mmol) in ethanol (10 mL)
were heated at 70 °C in the presence of piperidine
(0.1 mmol) for 2 h. After completion of reaction (moni-
tored by TLC), the reaction mixture was cooled, and the
solid separated was filtered and recrystallized from ethanol
to afford the pure product.
3H, isoxazole–CH3), 3.68 (s, 3H, Ar OCH3), 6.69 (d, 1H,
CH=CH, J = 12 Hz), 6.81 (d, 1H, CH=CH, J = 12 Hz),
7.10–7.68 (m, 3H, ArH), 8.64 (s, 1H, OH, D2O
exchangeable). 13CNMR (75 MHz, CDCl3) d ppm: 12.31,
60.97, 101.83, 113.52, 115.39, 120.41, 123.69, 126.79,
128.59, 132.93, 149.61, 158.09, 159.28. ESI-MS (70 eV)
m/z: 277 [M?H]?. Anal. Calcd for C13H12N2O5: C, 56.52;
H, 4.38; N, 10.14. Found: C, 56.49; H, 4.41; N, 10.10 %.
Spectral data of each compound are given below
2-((E)-2-(3-Methyl-4-nitroisoxazol-5-yl)vinyl)phenol (3a)
1
4-Chloro-2-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)
Yield 79 %, m.p. 160–162 °C. IR (KBr) cm-1: 3450. H
phenol (3d)
Yield 87 %, m.p. 168–170 °C. IR (KBr)
1
NMR (300 MHz, CDCl3) d ppm: 2.30 (s, 3H, isoxazole–
CH3), 6.62 (d, 1H, CH=CH, J = 12 Hz), 6.71 (d, 1H,
CH=CH, J = 12 Hz), 7.14–7.58 (m, 4H, ArH), 8.52 (s,
1H, OH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 12.39, 102.53, 113.29, 115.28, 120.38, 123.54,
126.74, 128.75, 132.81, 149.65, 157.89, 159.89. ESI-MS
(70 eV) m/z: 247 [M?H]?. Anal. Calcd for C12H10N2O4:
C, 58.54; H, 4.09; N, 11.38. Found: C, 58.48; H, 4.08; N,
11.41 %.
cm-1: 3457. H NMR (300 MHz, CDCl3) d ppm: 2.28 (s,
3H, isoxazole–CH3), 6.67 (d, 1H, CH=CH, J = 12 Hz),
6.73 (d, 1H, CH=CH, J = 12 Hz), 7.08–7.68 (m, 3H,
ArH), 8.73 (s, 1H, OH, D2O exchangeable). 13CNMR
(75 MHz, CDCl3) d ppm: 12.29, 102.38, 113.53, 115.42,
120.32, 123.69, 126.92, 128.73, 132.69, 149.72, 158.19,
159.28. ESI-MS (70 eV) m/z: 281 [M?H]?. Anal. Calcd
for C12H9ClN2O4: C, 51.35; H, 3.23; N, 9.98. Found: C,
51.39; H, 3.19; Cl, 12.58; N, 9.95 %.
4-Methyl-2-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)phenol
Yield 82 %, m.p. 157–159 °C. IR (KBr) cm-1
3445. H NMR (300 MHz, CDCl3) d ppm: 2.28 (s, 3H,
isoxazole–CH3), 2.82 (s, 3H, Ar–CH3), 6.64 (d, 1H,
2,4-Dichloro-6-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)
phenol (3e)
(3b)
:
Yield 81 %, m.p. 163–165 °C. IR (KBr)
1
1
cm-1: 3437. H NMR (300 MHz, CDCl3) d ppm: 2.25 (s,
3H, isoxazole–CH3), 6.62 (d, 1H, CH=CH, J = 12 Hz),
123

Med Chem Res
6.73 (d, 1H, CH=CH, J = 12 Hz), 7.09–7.98 (m, 2H,
ArH), 8.71 (s, 1H, OH, D2O exchangeable). 13CNMR
(75 MHz, CDCl3) d ppm: 12.32, 101.83, 113.74, 114.98,
121.09, 123.75, 127.13, 129.03, 132.58, 150.11, 158.32,
159.35. ESI-MS (70 eV) m/z: 315 [M?H]?. Anal. Calcd
for C12H8Cl2N2O4: C, 45.74; H, 2.56; N, 8.89. Found: C,
45.71; H, 2.60; N, 8.92 %.
(75 MHz, CDCl3) d ppm: 12.29, 14.23, 60.82, 64.69,
99.78, 114.23, 120.75, 122.07, 123.21, 126.51, 128.87,
134.08, 149.76, 157.39, 157.93, 170.78. ESI-MS (70 eV)
m/z: 333 [M?H]?. Anal. Calcd for C16H16N2O6: C, 57.83;
H, 4.85; N, 8.43. Found: C, 57.88; H, 4.81; N, 8.40 %.
(E)-Ethyl 2-[4-methyl-2-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4b) Yield 73 %, m.p. 167–169 °C.
1
4-Bromo-2-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)phenol
(3f) Yield 83 %, m.p. 170–172 °C. IR (KBr) cm-1: 3440.
1H NMR (300 MHz, CDCl3) d ppm: 2.24 (s, 3H, isoxaz-
ole–CH3), 6.70 (d, 1H, CH=CH, J = 12 Hz), 6.79 (d, 1H,
CH=CH, J = 12 Hz), 7.11–7.69 (m, 3H, ArH), 8.81 (s,
1H, OH, D2O exchangeable). 13CNMR (75 MHz, CDCl3) d
ppm: 12.35, 102.23, 113.69, 115.23, 120.97, 123.69,
127.29, 128.75, 133.08, 150.24, 159.32, 160.09. ESI-MS
(70 eV) m/z: 325 [M?H]?. Anal. Calcd for C12H9BrN2O4:
C, 44.33; H, 2.79; N, 8.62. Found: C, 44.29; H, 2.82; N,
8.59 %.
IR (KBr) cm-1: 1763, 1525, 1335. H NMR (300 MHz,
CDCl3) d ppm: 0.90 (t, 3H, OCH2CH3), 2.27 (s, 3H, isox-
azole–CH3), 2.47 (s, 3H, Ar–CH3), 4.21 (q, 2H, OCH2CH3),
5.18 (s, 2H, OCH2CO), 6.60 (d, 1H, CH=CH, J = 12 Hz),
6.75 (d, 1H, CH=CH, J = 12 Hz), 7.00–7.62 (m, 3H, ArH).
13CNMR (75 MHz, CDCl3) d ppm: 12.34, 14.31, 42.23,
61.07, 64.73, 100.20, 114.37, 120.81, 122.19, 122.98, 127.06,
128.91, 134.15, 150.18, 157.43, 158.11, 169.79. ESI-MS
(70 eV) m/z: 347 [M?H]?. Anal. Calcd for C17H18N2O6: C,
58.96; H, 5.24; N, 8.09. Found: C, 58.93; H, 5.29; N, 8.14 %.
(E)-Ethyl 2-[4-methoxy-2-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4c) Yield 77 %, m.p. 162–164 °C.
2,4-Dibromo-6-((E)-2-(3-methyl-4-nitroisoxazol-5-yl)vinyl)
phenol (3g) Yield 88 %, m.p. 173–175 °C. IR (KBr)
1
IR (KBr) cm-1: 1757, 1521, 1325. H NMR (300 MHz,
1
cm-1: 3451. H NMR (300 MHz, CDCl3) d ppm: 2.27 (s,
CDCl3) d ppm: 0.91 (t, 3H, OCH2CH3), 2.30 (s, 3H, isox-
azole–CH3), 3.69 (s, 3H, OCH3), 4.27 (q, 2H, OCH2CH3),
5.28 (s, 2H, OCH2CO), 6.67 (d, 1H, CH=CH, J = 12 Hz),
6.71 (d, 1H, CH=CH, J = 12 Hz), 7.09–7.57 (m, 3H, ArH).
13C NMR (75 MHz, CDCl3) d ppm: 12.39, 14.42, 61.23,
61.31, 65.03, 101.31, 114.43, 121.09, 122.29, 123.07, 127.12,
129.09, 134.23, 150.21, 156.98, 158.23, 170.11.ESI-MS
(70 eV) m/z: 363 [M?H]?. Anal. Calcd for C17H18N2O7: C,
56.35; H, 5.01; N, 7.73. Found: C, 56.30; H, 5.07; N, 7.67 %.
3H, isoxazole–CH3), 6.68 (d, 1H, CH=CH, J = 12 Hz),
6.81 (d, 1H, CH=CH, J = 12 Hz), 7.13–7.75 (m, 3H,
ArH), 8.69 (s, 1H, OH, D2O exchangeable). 13CNMR
(75 MHz, CDCl3) d ppm: 12.37, 101.79, 113.73, 114.98,
121.24, 123.76, 127.35, 129.17, 132.79, 150.16, 159.52,
160.62. ESI-MS (70 eV) m/z: 403 [M?H]?. Anal. Calcd
for C12H8Br2N2O4: C, 35.67; H, 2.00; N, 6.93. Found: C,
35.71; H, 2.04; N, 6.89 %.
General procedure for the synthesis of ethyl
2-[2-[(E)-2-(3-methyl-4-nitroisoxazole-5-yl)vinyl]
phenoxy]acetates (4a–g)
(E)-Ethyl 2-[2-chloro-6-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4d) Yield 77 %, m.p. 148–150 °C.
1
IR (KBr) cm-1: 1775, 1535, 1310. H NMR (300 MHz,
CDCl3) d ppm: 0.93 (t, 3H, OCH2CH3), 2.29 (s, 3H, isox-
azole–CH3), 4.12 (q, 2H, OCH2CH3), 5.25 (s, 2H,
OCH2CO), 6.58 (d, 1H, CH=CH, J = 12 Hz), 6.61 (d, 1H,
CH=CH, J = 12 Hz), 7.00–7.62 (m, 3H, ArH). 13C NMR
(75 MHz, CDCl3) d ppm: 12.42, 14.51, 60.98, 102.09,
113.89, 121.11, 122.31, 122.97, 127.29, 128.87, 134.38,
149.76, 150.53, 156.99, 158.32, 169.79.EI-MS (70 eV) m/z:
367 [M?H]?. Anal. Calcd for C16H15ClN2O6: C, 52.40; H,
4.12; N, 7.64. Found: C, 52.40; H, 4.12; N, 7.64 %.
To a solution of 3 (1 mmol), in dry acetone (10 mL), ethyl
bromoacetate (1 mmol) and anhydrous K2CO3 (2 mmol)
was added, and the contents were refluxed for 2 h. After
the completion of the reaction as indicated by TLC the
reaction mixture is filtered and the acetone is evaporated.
The crude product is purified by passing through silica gel
column by eluting with ethyl acetate:hexane (25:75).
Spectral data of each compound are given below
(E)-Ethyl 2-[2-[2-(3-methyl-4-nitro-5-isoxazolyl)vinyl]
phenoxy]acetate (4a) Yield 73 %, m.p. 137–139 °C. IR
(KBr) cm-1: 1760, 1540, 1326. 1H NMR (300 MHz,
CDCl3) d ppm: 0.87 (t, 3H, OCH2CH3), 2.32 (s, 3H, is-
oxazole–CH3), 4.10 (q, 2H, OCH2CH3), 5.21 (s, 2H,
OCH2CO), 6.62 (d, 1H, CH=CH, J = 12 Hz), 6.71 (d, 1H,
CH=CH, J = 12 Hz), 7.14–7.58 (m, 4H, ArH). .13CNMR
(E)-Ethyl 2-[2,4-dichloro-6-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4e) Yield 74 %, m.p. 141–143 °C.
1
IR (KBr) cm-1: 1765, 1540, 1315. H NMR (300 MHz,
CDCl3) d ppm: 0.86 (t, 3H, OCH2CH3), 2.31 (s, 3H, is-
oxazole–CH3), 4.00 (q, 2H, OCH2CH3), 5.20 (s, 2H,
OCH2CO), 6.66 (d, 1H, CH=CH, J = 12 Hz), 6.74 (d, 1H,
CH=CH, J = 12 Hz), 7.45 (s, 1H, ArH), 7.57 (2, 1H,
123

Med Chem Res
ArH). 13C NMR (75 MHz, CDCl3) d ppm: 12.45, 14.39,
61.07, 102.21, 114.11, 121.39, 122.42, 123.01, 127.37,
129.11, 134.43, 151.03, 157.11, 158.48, 170.09.ESI-MS
(70 eV) m/z: 401 [M?H]?. Anal. Calcd for C16H14Cl2
N2O6: C, 47.90; H, 3.52; N, 6.98. Found: C, 47.86; H, 3.48;
N, 7.02 %.
4H, ArH). 13C NMR (75 MHz, CDCl3) d ppm: 12.42,
14.29, 26.73, 41.28, 60.79, 81.59, 100.73, 113.98, 120.23,
125.78, 126.11, 130.78, 149.69, 158.23, 158.35, 172.24.
EI-MS (70 eV) m/z: 333 [M?H]?. Anal. Calcd for
C16H16N2O6: C, 57.83; H, 4.85; N, 8.43. Found: C, 57.89;
H, 4.88; N, 8.38 %.
(E)-Ethyl 2-[4-bromo-2-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4f) Yield 71 %, m.p. 145–147 °C.
Ethyl-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-2,3-dihy-
drobenzofuran-2-carboxylate (5b) Yield 70 %, m.p.
1
1
IR (KBr) cm-1: 1761, 1532, 1311. H NMR (300 MHz,
191–193 °C. IR (KBr) cm-1: 1751, 1532, 1321. H NMR
CDCl3) d ppm: 0.91 (t, 3H, OCH2CH3), 2.33 (s, 3H, isox-
azole–CH3), 4.21 (q, 2H, OCH2CH3), 5.28 (s, 2H,
OCH2CO), 6.59 (d, 1H, CH=CH, J = 12 Hz), 6.68 (d, 1H,
CH=CH, J = 12 Hz), 7.11–7.69 (m, 3H, ArH). 13C NMR
(75 MHz, CDCl3) d ppm: 12.32, 14.46, 59.78, 65.12, 99.51,
113.97, 121.35, 121.94, 123.62, 127.01, 129.11, 133.72,
149.51, 158.16, 158.19, 170.38. ESI-MS (70 eV) m/z: 411
[M?H]?. Anal. Calcd for C16H15BrN2O6: C, 46.73; H, 3.68;
N, 6.81. Found: C, 46.79; H, 3.70; N, 6.77 %.
(300 MHz, CDCl3) d ppm: 0.97 (t, 3H, OCH2CH3), 2.32 (s,
3H, isoxazole–CH3), 2.65 (s, 3H, ArCH3), 3.39 (dd, 1H,
J = 4.5, 18 Hz, CH2), 3.66 (dd, 1H, J = 5.5, 18 Hz, CH2),
4.06 (m, 1H, H-3), 4.31 (q, 2H, OCH2CH3), 5.42 (m, 1H,
H-2), 7.13–7.67 (m, 3H, ArH). 13C NMR (75 MHz,
CDCl3) d ppm: 12.48, 14.31, 41.39, 27.09, 40.89, 61.09,
81.63, 100.81, 114.02, 120.53, 126.09, 131.11, 150.07,
158.35, 158.44, 172.38. ESI-MS (70 eV) m/z: 347
[M?H]?. Anal. Calcd for C17H18N2O6: C, 39.21; H, 2.88;
N, 5.72. Found: C, 39.25; H, 2.84; N, 5.76 %.
(E)-Ethyl 2-[2,4-dibromo-6-[2-(3-methyl-4-nitro-5-isoxazolyl)
vinyl]phenoxy]acetate (4g) Yield 75 %, m.p. 153–155 °C.
Ethyl-5-methoxy-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-
2,3-dihydrobenzofuran-2-carboxylate (5c) Yield 70 %,
1
IR (KBr) cm-1: 1755, 1530, 1315. H NMR (300 MHz,
1
CDCl3) d ppm: 0.95 (t, 3H, OCH2CH3), 2.30 (s, 3H, is-
oxazole–CH3), 4.22 (q, 2H, OCH2CH3), 5.11 (s, 2H,
OCH2CO), 6.61 (d, 1H, CH=CH, J = 12 Hz), 6.70 (d, 1H,
CH=CH, J = 12 Hz), 7.39 (s, 1H, ArH), 7.48 (s, 1H, ArH).
13C NMR (75 MHz, CDCl3) d ppm: 12.49, 14.47, 61.29,
102.21, 113.97, 12.53, 121.98, 122.79, 127.48, 129.18,
133.97, 151.03, 157.11, 158.49, 170.09.ESI-MS (70 eV) m/
z: 489 [M?H]?. Anal. Calcd for C16H14Br2N2O6: C, 39.21;
H, 2.88; N, 5.72. Found: C, 39.24; H, 2.82; N, 5.67 %.
m.p. 191–193 °C. IR (KBr) cm-1: 1752, 1537, 1333. H
NMR (300 MHz, CDCl3) d ppm: 0.90 (t, 3H, OCH2CH3),
2.28 (s, 3H, isoxazole–CH3), 3.40 (dd, 1H, J = 4.5, 18 Hz,
CH2), 3.64 (dd, 1H, J = 5.5, 18 Hz, CH2), 3.68 (s, 3H,
OCH3), 4.03 (m, 1H, H-3), 4.25 (q, 2H, OCH2CH3), 5.44
(m, 1H, H-2), 7.00–7.64 (m, 3H, ArH). 13C NMR
(75 MHz, CDCl3) d ppm: 12.40, 15.09, 26.98, 40.91,
60.78, 61.23, 82.09, 101.1, 114.23, 121.13, 125.97, 126.29,
131.28, 149.78, 158.43, 158.71, 173.08. ESI-MS (70 eV)
m/z: 363 [M?H]?. Anal. Calcd for C17H18N2O7: C, 56.35;
H, 5.01; N, 7.73. Found: C, 56.41; H, 4.98; N, 7.76 %.
General procedure for the preparation of ethyl
2,3-dihydro-3-[(3-methyl-4-nitroisoxazole-5-
yl)methyl]benzofuran-2-carboxylates (5a–g)
Ethyl-7-chloro-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-
2,3-dihydrobenzofuran-2-carboxylate (5d) Yield 74 %,
1
To a solution of 4 (1 mmol) in ethanol, Et3N (0.5 mmol)
was added and the contents are heated at 100 °C for 8 h.
After completion of the reaction as indicated by TLC,
ethanol is evaporated and the crude product is purified by
passing through a column of silica gel with ethylace-
tate:hexane (20:80) as elute.
m.p. 178–180 °C. IR (KBr) cm-1: 1755, 1528, 1310. H
NMR (300 MHz, CDCl3) d ppm: 0.90 (t, 3H, OCH2CH3),
2.26 (s, 3H, isoxazole–CH3), 3.42 (dd, 1H, J = 4.5, 18 Hz,
CH2), 3.67 (dd, 1H, J = 5.5, 18 Hz, CH2), 4.10 (m, 1H,
H-3), 4.26 (q, 2H, OCH2CH3), 5.40 (m, 1H, H-2),
7.11–7.73 (m, 3H, ArH). 13C NMR (75 MHz, CDCl3) d
ppm: 12.43, 16.21, 27.03, 41.12, 60.83, 100.79, 114.38,
121.73, 126.15, 131.42, 150.04, 158.59, 158.83, 173.08.
ESI-MS (70 eV) m/z: 367 [M?H]?. Anal. Calcd for
C16H15ClN2O6: C, 52.40; H, 4.12; N, 7.64. Found: C,
52.45; H, 4.18; N, 7.60 %.
Spectral data of each compound are given below
Ethyl-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-2,3-dihy-
drobenzofuran-2-carboxylate (5a) Yield 70 %, m.p.
1
170–172 °C. IR (KBr) cm-1: 1746, 1520, 1315. H NMR
(300 MHz, CDCl3) d ppm: 0.98 (t, 3H, OCH2CH3), 2.32 (s,
3H, isoxazole–CH3), 3.48 (dd, 1H, J = 4.5, 18 Hz, CH2),
3.65 (dd, 1H, J = 5.5, 18 Hz, CH2), 4.03 (m, 1H, H-3),
4.27 (q, 2H, OCH2CH3), 5.48 (m, 1H, H-2), 7.02–7.69 (m,
Ethyl-5,7-dichloro-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-
2,3-dihydrobenzofuran-2-carboxylate (5e) Yield 73 %,
1
m.p. 181–183 °C. IR (KBr) cm-1: 1742, 1521, 1322. H
123

Med Chem Res
NMR (300 MHz, CDCl3) d ppm: 0.96 (t, 3H, OCH2CH3),
2.30 (s, 3H, isoxazole–CH3), 3.42 (dd, 1H, J = 4.5, 18 Hz,
CH2), 3.64 (dd, 1H, J = 5.5, 18 Hz, CH2), 4.00 (m, 1H,
H-3), 4.29 (q, 2H, OCH2CH3), 5.44 (m, 1H, H-2), 7.31 (s,
1H, ArH), 7.43 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3)
d ppm: 12.49, 15.78, 27.19, 41.37, 61.12, 82.31, 101.03,
144.53, 121.95, 126.53, 127.08, 131.59, 149.78, 158.35,
159.13, 172.81. ESI-MS (70 eV) m/z: 401 [M?H]?. Anal.
Calcd for C16H14Cl2N2O6: C, 47.90; H, 3.52; N, 6.98.
Found: C, 47.84; H, 3.48; N, 7.01 %.
under vacuum and purified by recrystallization from etha-
nol to give pure product 6.
3-Methyl-10b,11-dihydro-4H-benzofuro[3,2-e]isoxazolo
[4,5-b]azepin-5(5aH)-one (6a) Yield 73 %, m.p.
198–200 °C. IR (KBr) cm-1: 3200, 1710. 1H NMR
(300 MHz, CDCl3) d ppm: 2.22 (s, 3H, isoxazole–CH3),
3.50 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.66 (dd, 1H,
J = 6.6, 18 Hz, 11-CH2), 4.05 (m, 1H, H-10b), 5.48 (d,
1H, J = 4 Hz, H-5a), 7.01–7.83 (m, 4H, ArH), 8.32 (bs,
1H, NH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 12.51, 26.21, 40.79, 85.63, 114.21, 115.76, 119.58,
126.78, 126.91, 150.08, 156.54, 160.21, 174.79. ESI-MS
(70 eV) m/z: 257 [M?H]?. Anal. Calcd for C14H12N2O3:
C, 65.62; H, 4.72; N, 10.93. Found: C, 65.66; H, 4.78; N,
10.89 %.
Ethyl-7-bromo-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-
2,3-dihydrobenzofuran-2-carboxylate (5f) Yield 70 %,
1
m.p. 175–177 °C. IR (KBr) cm-1: 1740, 1525, 1315. H
NMR (300 MHz, CDCl3) d ppm: 0.91 (t, 3H, OCH2CH3),
2.30 (s, 3H, isoxazole–CH3), 3.39 (dd, 1H, J = 4.5, 18 Hz,
CH2), 3.64 (dd, 1H, J = 5.5, 18 Hz, CH2), 4.05 (m, 1H,
H-3), 4.30 (q, 2H, OCH2CH3), 5.45 (m, 1H, H-2),
7.11–7.70 (m, 3H, ArH). 13C NMR (75 MHz, CDCl3) d
ppm: 12.39, 27.83, 42.11, 60.79, 82.48, 101.28, 114.75,
122.16, 127.13, 132.09, 150.11, 159.11, 159.27, 173.08.
ESI-MS (70 eV) m/z: 412 [M?H]?. Anal. Calcd for
C16H15BrN2O6: C, 46.73; H, 3.68; N, 6.81. Found: C,
46.77; H, 3.62; N, 6.87 %.
3,9-Dimethyl-10b,11-dihydro-4H-benzofuro[3,2-e]isoxaz-
olo[4,5-b]azepin-5(5aH)-one (6b) Yield 73 %, m.p.
222–225 °C. IR (KBr) cm-1: 3210, 1725. 1H NMR
(300 MHz, CDCl3) d ppm: 2.25 (s, 3H, isoxazole–CH3),
2.48 (s, 3H, ArCH3), 3.52 (dd, 1H, J = 4.6, 18 Hz,
11-CH2), 3.64 (dd, 1H, J = 6.6, 18 Hz, 11-CH2), 4.13 (m,
1H, H-10b), 5.52 (d, 1H, J = 4 Hz, H-5a), 7.10–7.64 (m,
3H, ArH), 8.11 (bs, 1H, NH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3) d ppm: 12.48, 25.79, 41.13, 41.79,
86.08, 113.79, 115.53, 120.12, 127.29, 127.58, 131.96,
149.79, 156.63, 160.48, 175.04. ESI-MS (70 eV) m/z: 271
[M?H]?. Anal. Calcd for C15H14N2O3: C, 66.66; H, 5.22;
N, 10.36. Found: C, 66.70; H, 5.28; N, 10.33 %.
Ethyl-5,7-dibromo-3-[(3-methyl-4-nitro-5-isoxazolyl)methyl]-
2,3-dihydrobenzofuran-2-carboxylate (5g) Yield 77 %,
1
m.p. 184–186 °C. IR (KBr) cm-1: 1745, 1535, 1320. H
NMR (300 MHz, CDCl3) d ppm: 0.93 (t, 3H, OCH2CH3),
2.26 (s, 3H, isoxazole–CH3), 3.43 (dd, 1H, J = 4.5, 18 Hz,
CH2), 3.65 (dd, 1H, J = 5.5, 18 Hz, CH2), 4.04 (m, 1H,
H-3), 4.30 (q, 2H, OCH2CH3), 5.41 (m, 1H, H-2), 7.33 (s,
1H, ArH), 7.47 (s, 1H, ArH). 13C NMR (75 MHz, CDCl3)
d ppm: 12.45, 28.11, 61.27, 82.42, 100.98, 114.79, 122.09,
127.12, 127.29, 131.75, 150.09, 158.73, 160.15, 172.57.
ESI-MS (70 eV) m/z: 491 [M?H]?. Anal. Calcd for
C16H14Br2N2O6: C, 39.21; H, 2.88; N, 5.72. Found: C,
39.17; H, 2.93; N, 5.68 %.
9-Methoxy-3-methyl-10b,11-dihydro-4H-benzofuro[3,2-e]
isoxazolo[4,5-b]azepin-5(5aH)-one (6c) Yield 75 %,
1
m.p. 219–221 °C. IR (KBr) cm-1: 3218, 1725. H NMR
(300 MHz, CDCl3) d ppm: 2.20 (s, 3H, isoxazole–CH3),
3.54 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.58 (s, 3H,
OCH3), 3.63 (dd, 1H, J = 6.6, 18 Hz, 11-CH2), 4.00 (m,
1H, H-10b), 5.40 (d, 1H, J = 4 Hz, H-5a), 7.13–7.83 (m,
3H, ArH), 8.33 (bs, 1H, NH, D2O exchangeable). 13C NMR
(75 MHz, CDCl3) d ppm: 12.53, 26.13, 41.78, 62.21,
85.97, 114.09, 115.74, 119.78, 127.81, 128.11, 131.23,
149.78, 156.63, 160.49, 175.04. ESI-MS (70 eV) m/z: 287
[M?H]?. Anal. Calcd for C15H14N2O4: C, 62.93; H, 4.93;
N, 9.79. Found: C, 62.89; H, 4.90; N, 9.74 %.
General procedure for the synthesis of 3-methyl-10b,11-
dihydro-4H-benzofuro[3,2-e] isoxazole[4,5-b]azepin-
5(5aH)-ones (6a–g)
Spectral data of each compound are given below
Compound 5 (1 mmol) and SnCl2ꢀ2H2O (3 mmol) were
dissolved in 20 mL of methanol and refluxed for 4 h. After
completion of the reaction (monitored by TLC), solvent
was removed in vacuum. The solid mass was decomposed
with cold water and the reaction solution was carefully
adjusted to pH 8 with 10 % NaHCO3 solution and then
extracted with ethyl acetate (2 9 20 mL). The combined
organic layers were dried over Na2SO4 and evaporated
9-Chloro-3-methyl-10b,11-dihydro-4H-benzofuro[3,2-e]
isoxazolo[4,5-b]azepin-5(5aH)-one (6d) Yield 77 %,
1
m.p. 211–213 °C. IR (KBr) cm-1: 3215, 1723. H NMR
(300 MHz, CDCl3) d ppm: 2.28 (s, 3H, isoxazole–CH3),
3.48 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.64 (dd, 1H,
J = 6.6, 18 Hz, 11-CH2), 4.00 (m, 1H, H-10b), 5.42 (d,
1H, J = 4 Hz, H-5a), 7.00–7.81 (m, 3H, ArH), 8.21 (bs,
123

Med Chem Res
1H, NH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 12.41, 26.73, 42.11, 86.24, 113.89, 116.31, 120.12,
128.09, 128.29, 131.83, 150.12, 157.13, 161.11, 174.79.
ESI-MS (70 eV) m/z: 291 [M?H]?. Anal. Calcd for
C14H11ClN2O3: C, 57.84; H, 3.81; N, 9.64. Found: C,
57.80; H, 3.86; N, 9.62 %.
Pharmacological screening
Antibacterial assay
The ready-made nutrient broth medium (Himedia, 24 g)
was suspended in distilled water (100 mL) and heated until
it dissolved completely. The medium and test tubes were
autoclaved at a pressure of 15 lb/inc2 for 20 min. A set of
sterilized test tubes with nutrient broth medium was capped
with cotton plugs. The test compound was dissolved in
acetone and a concentration of 100 lg/mL of the test
compound was added in the first test tube, which was
serially diluted. A fixed volume of 0.5 mL of overnight
culture was added in all the test tubes which were incu-
bated at 37 °C for 24 h. After 24 h these tubes were
measured for turbidity.
7,9-Dichloro-3-methyl-10b,11-dihydro-4H-benzofuro[3,2-e]
isoxazolo[4,5-b]azepin-5(5aH)-one (6e)
Yield 72 %,
1
m.p. 218–220 °C. IR (KBr) cm-1: 3210, 1720. H NMR
(300 MHz, CDCl3) d ppm: 2.31 (s, 3H, isoxazole–CH3),
3.50 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.65 (dd, 1H,
J = 6.6, 18 Hz, 11-CH2), 4.13 (m, 1H, H-10b), 5.44 (d,
1H, J = 4 Hz, H-5a), 7.21 (s, 1H, ArH), 7.35 (s, 1H, ArH),
8.28 (bs, 1H, NH, D2O exchangeable). 13C NMR (75 MHz,
CDCl3) d ppm: 12.49, 27.19, 42.08, 86.11, 113.89, 116.31,
120.09, 128.09, 128.29, 132.19, 149.78, 157.13, 161.11,
174.79. ESI-MS (70 eV) m/z: 325 [M?H]?. Anal. Calcd
for C14H10Cl2N2O3: C, 51.72; H, 3.10; N, 8.62. Found: C,
51.68; H, 3.13; N, 8.60 %.
Antifungal assay
For the antifungal assay, the ready-made potato dextrose
agar medium (Himedia, 39 g) was suspended in distilled
water (100 mL) and heated until it dissolved completely.
The medium and glass petri dishes were autoclaved at a
pressure of 15 lb/in C2 for 20 min. The medium was
poured into sterile petri dishes under aseptic conditions in a
Laminar flow chamber. When the medium in the plates
solidified, 0.5 mL of the culture (one-week-old) of fungal
spore suspension was inoculated and uniformly spread over
the agar surface with a sterile L-shaped rod. Solutions were
prepared by dissolving test compound in acetone. After
inoculation, cups were scooped out with 6-mm sterile cork
borer and the lids of the dishes were replaced. To each cup,
different concentrations of test solution were added. Con-
trols were maintained with acetone and fluconazole. The
treated and the controls were kept at room temperature for
72–96 h. The minimum inhibitory concentration (MIC)
was recorded in lg/mL. Three to four replicates were
maintained for each treatment.
9-Bromo-3-methyl-10b,11-dihydro-4H-benzofuro[3,2-e]is-
oxazolo[4,5-b]azepin-5(5aH)-one (6f) Yield 70 %, m.p.
205–207 °C. IR (KBr) cm-1: 3216, 1720. 1H NMR
(300 MHz, CDCl3) d ppm: 2.30 (s, 3H, isoxazole–CH3),
3.53 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.61 (dd, 1H,
J = 6.6, 18 Hz, 11-CH2), 4.11 (m, 1H, H-10b), 5.39 (d,
1H, J = 4 Hz, H-5a), 7.11–7.61 (m, 3H, ArH), 8.11 (bs,
1H, NH, D2O exchangeable). 13C NMR (75 MHz, CDCl3)
d ppm: 12.44, 28.03, 41.79, 84.55, 114.23, 117.11,
120.42,127.89, 128.53, 133.03, 150.11, 157.91, 163.11,
174.79. ESI-MS (70 eV) m/z: 335 [M?H]?. Anal. Calcd
for C14H11BrN2O3: C, 50.17; H, 3.31; N, 8.36. Found: C,
50.12; H, 3.35; N, 8.31 %.
7,9-Dibromo-3-methyl-10b,11-dihydro-4H-benzofuro[3,2-
e]isoxazolo[4,5-b]azepin-5(5aH)-one (6g) Yield 76 %,
1
m.p. 215–217 °C. IR (KBr) cm-1: 3225, 1716. H NMR
(300 MHz, CDCl3) d ppm: 2.28 (s, 3H, isoxazole–CH3),
3.48 (dd, 1H, J = 4.6, 18 Hz, 11-CH2), 3.67 (dd, 1H,
J = 6.6, 18 Hz, 11-CH2), 4.00 (m, 1H, H-10b), 5.43 (d,
1H, J = 4 Hz, H-5a), 7.25 (s, 1H, ArH), 7.37 (s, 1H, ArH),
8.18 (bs, 1H, NH, D2O exchangeable). 13C NMR (75 MHz,
CDCl3) d ppm: 12.79, 28.45, 41.94, 85.38, 114.58, 117.51,
121.34,128.42, 129.31, 133.59, 149.79, 158.13, 163.42,
175.05. EI-MS (70 eV) m/z: 413 [M?H]?. Anal. Calcd for
C14H10Br2N2O3: C, 40.61; H, 2.43; N, 6.77. Found: C,
40.57; H, 2.40; N, 6.80 %.
Anti-inflammatory analysis
Anti-inflammatory activity was determined by carra-
geenan-induced paw edema method (Winter et al., 1990).
Wistar rats of either sex weighing 150–200 g were divided
into 6 groups (n = 6) and they were fasted 18 h before the
experiment with water ad libitum. Group-I received 1 %
sodium CMC (negative control), Group-II received ibu-
profen at a dose of 100 mg/kg (positive control), and
Group-III–VI were given the compounds 6a–g (100 mg/
kg). All the compounds 6a–g were given in oral route.
After 30 min, 0.1 mL of 1 % carrageenan suspension in
normal saline was injected into the subplantar region of the
Seven new derivatives for each class of compounds (4,
5, and 6) are herein reported. The structures of all newly
synthesized compounds were confirmed by analytical and
1
spectral data (IR, H NMR, and MS).
123

Med Chem Res
left hind paw of each rat to induce edema. The edema
volumes of the injected paw were measured with the help
of plethysmograph at the interval of 0, 1, 2, 4, and 6 h. The
difference between the paw volumes of treated animals
were compared with that of the control group and the mean
edema volume was calculated. Percentage inhibition was
calculated as per the formula, %inhibition = [Vo - Vt)/
Vo] 9 100, where Vo = volume of the paw control at time
t, Vt = volume of the paw of drug treated at time t.
Results and discussion
Synthesis
The synthesis of title compounds was accomplished by
synthetic sequence shown in Scheme 1. Knoevenagel
condensation of 3,5-dimethyl-4-nitroisoxazole (1) (pur-
chased from Sigma-Aldrich) with salicyl aldehydes (2) in
refluxing ethanol in the presence of piperidine affords ni-
trostyrylisoxazoles (3) in good yields (Martinez, 1980;
Chimichi, 1989; Malla Reddy et al., 1981). Compound 3
on treatment with ethyl bromoacetate in dry acetone in the
presence of anhydrous K2CO3 under refluxing condition
led to the formation of ethyl 2-[2-[(E)-2-(3-methyl-4-nitro-
5-isoxazolyl)vinyl]phenoxy]acetates (4). Compounds 4
underwent cyclization to afford ethyl 2,3-dihydro-3-((3-
methyl-4-nitro-5-isoxazolyl)methyl)benzofuran-2-carbox-
ylates (5) on treatment with triethyl amine in refluxing
alcohol. These compounds (5) were further converted into
3-methyl-10b,11-dihydro-4H-benzofuro[3,2-e]isoxazol-
o[4,5-b]azepin-5(5aH)-ones (6) by reductive cyclization on
treatment with SnCl2–MeOH (Scheme 1).
5-Lox inhibitory activity
The in vitro anti-inflammatory activity (5-LOX inhibition
assay) was determined using UV–Vis spectrophotometer
(Reddenna et al., 1990; Ulusu et al., 2002). The assay
mixture contained 80 mM linoleic acid and sufficient
amount of potato 5-lipoxygenase enzyme in 50 mM
phosphate buffer (pH 6.3). The reaction was initiated by
the enzyme buffer mix to substrate (linoleic acid) and the
enzyme activity was monitored as an increase in absor-
bance at 234 nm. The reaction was monitored for 10 s.
using UV Kinetic mode on Varian Cary-50-UV–Vis
spectrophotometer. In the inhibition studies, the activities
were measured by incubating various concentrations of test
substance with enzyme buffer mix for 2 min before addi-
tion of the substrate. The assay was performed in triplicate
and mean values were used for the calculation. The activity
of 5-Lipoxygenase was compared with the standard posi-
tive control LI01020. Percentage inhibition was calculated
as per the formula, % inhibition = 1 - [O.D. in sample
well)/(O.D. in control well)] 9 100.
Biological screening
Antimicrobial activity
The newly synthesized title compounds 6a–g were evalu-
ated for their in vitro antibacterial activity against Gram-
positive bacteria viz., Bacillus subtilis (MTCC 441),
Bacillus sphaericus (MTCC 511), and Staphylococcus
aureus (MTCC 96) and Gram-negative bacteria viz.,
Pseudomonas aeruginosa (MTCC 741), Klobsinella aer-
ogenes (MTCC 39), and Chromobacterium violaceum
(MTCC 2656) at 100 lg/mL concentration. The in vitro
antibacterial activity of the tested compounds was assessed
by MIC using broth dilution method (National committee
for clinical laboratory standards (NCCLS) 1982). Cipro-
floxacin was used as standard drug for comparison. The
results of antibacterial screening (Table 1) reveal that the
compounds 6a–g displayed a better activity and were more
active than the standard Ciprofloxacin. Compounds 6b and
6c carrying methyl and methoxy substituents on the ben-
zene ring showed best activity and more active than the
standard Ciprofloxacin. Compounds 6d, 6e, 6f, and 6g
carrying chloro and bromo substitutions on benzene ring
did not exhibit much activity. Compound 6a showed least
activity because it has no substituent on the benzene ring.
However, the degree of inhibition varied both with the test
compound and with the bacteria used in the present
investigation. In conclusion, compounds 6b and 6c showed
Analgesic analysis
The analgesic activity was determined by acetic acid-
induced writhing method (Syamsudin and Rahayn, 2010).
Swiss albino mice (n = 6) of either sex selected by random
sampling technique were used for the study and divided
into 6 groups. The animals were fasted 18 h before the
experiment with water ad libitum. Diclofenac (50 mg/kg)
was administered as standard drug for comparison. The test
compounds 6a–g (50 mg/kg) were administered orally
30 min after the administration of compounds. All the mice
were given 1.0 % v/v solution of acetic acid, i.p. the dose
being 1 mL/100 g of the mice and the writhings produced
in these animals were counted for 20 min. The number of
writhings produced in the treated groups was compared
with those in the control group. Percentage inhibition was
calculated as per the formula, % inhibition = [(Average
writhes in control - Average writhes in test)/Average
writhes in control] 9 100.
123

Med Chem Res
N
N
O
O
O
CH3
CH3
R'
R'
CHO
OH
H3C
NO2
R'
(ii)
(i)
NO2
NO2
+
N
O
OH
CH3
O
R
R
R
O
1
2
3
4
(iii)
H3C
NO2
N
O
R'
O
O
O
R
5
(iv)
N
O
CH3
R'
NH
O
O
R
6
Scheme 1 Synthesis of novel dihydrobenzo furo[3,2-e]isoxazolo[4,5-
b]azepin-5(5aH)-ones (6a–g). 3, 4, 5, and 6: (a) R=H, R0=H; (b) R=H,
R0=CH3; (c) R=H, R0=OCH3; (d) R=H, R0=Cl; (e) R=Cl, R0=Cl;
(f) R=H, R0=Br; (g) R=Br, R0=Br. Reagents and conditions: (i) ethanol,
piperidine, D; (ii) BrCH2COOEt, K2CO3, acetone, reflux, D; (iii) Et3N,
ethanol, 100 °C, 8 h; (iv) SnCl2ꢀ2H2O/MeOH, 90 °C, 24 h
maximum activity by inhibiting the growth of all the
bacteria under investigation compared to standard Cipro-
floxacin, hence can be exploited for the formulation of
bacteriocides after further studies.
fluconazole, hence, may be exploited for control of wilt
diseases of different crops as fungicides after further
studies.
The title compounds 6a–g were also evaluated for their
antifungal activity against Fusarium oxysporum, Verticil-
lium dahliae, Alternaria solani, Rhizoctonia solani, Col-
letotrichum capsici, and Pythium aphanidermatum in
acetone by agar cup bioassay method (Margery Linday,
1962), using fluconazole as the standard drug. The anti-
fungal activity data (Table 2) reveal that compounds 6a–
g are highly toxic toward all the fungi under investigation.
Compounds 6b and 6c exhibited high antifungal activity by
inhibiting the growth of fungi to a remarkable extent, when
compared to standard drug fluconazole which may be due
to the presence of methyl and methoxy substituents on the
benzene ring. Compound 6a showed good activity. Com-
pounds 6d, 6e, 6f, and 6g are moderately active. However,
the degree of spore germination inhibition varied with the
test compound as well as with the fungi under investiga-
tion. It is noteworthy that compounds 6b and 6c showed
better activity, when compared with the standard drug
Anti-inflammatory activity
The anti-inflammatory activity of the title compounds, 6a–
g were evaluated by carrageenan-induced paw edema
method in rats (Winter et al., 1990) at a dose of 100 mg/kg
body weight using ibuprofen as a reference drug. Results
were expressed as a mean SE. The anti-inflammatory
properties were recorded at successive intervals of 0, 1, 2,
4, and 6 h and compared with that of standard ibuprofen.
The anti-inflammatory activity data (Table 3) indicated
that all the compounds 6a–g exhibited significant activity
by decreasing the paw volume that was produced by car-
rageenan. Among all the compounds tested, it is interesting
to note that the compounds 6b and 6c showed better anti-
inflammatory activity, may be due to the presence of
methyl and methoxy substituents on the benzene ring,
besides benzofuran-fused isoxazoloazepine moiety. Com-
pounds 6b and 6c exhibited maximum activity at the
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Med Chem Res
Table 1 Antibacterial activity of dihydrobenzo furo[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones (6a–g)
Compound
Minimum inhibitory concentration in lg/mL (MIC)
Gram ?ve bacteria
Gram –ve bacteria
B. subtilis
B. sphaericus
S. aureus
P. aeruginosa
K. aerogenes
C. violaceum
6a
18
8
20
10
10
18
22
12
15
22
20
9
22
8
20
7
20
6
6b
6c
10
20
18
16
18
20
8
10
21
24
16
16
25
8
10
16
18
16
18
22
6d
22
21
14
16
26
19
18
15
18
20
6e
6f
6g
Ciprofloxacin
Negative control (acetone)—No activity
Values are indicated in lg/mL
Table 2 Antifungal activity of dihydrobenzo furo[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones (6a–g)
Compound
Minimum inhibitory concentration in lg/mL (MIC)
F. oxysporum
V. dahale
A. solani
R. solani
C. capsici
P. aphanidermatum
6a
16
8
14
8
12
10
11
14
15
18
20
20
12
8
15
9
18
9
6b
6c
8
10
16
14
17
16
16
10
16
14
14
12
16
10
16
18
17
18
18
8
6d
16
14
14
16
18
15
20
17
20
22
6e
6f
6g
Flucanozole
Negative control (acetone)—No activity
interval of 6 h. The presence of electron withdrawing
chloro and bromo groups on benzene ring (6d, 6e, 6f, and
6g) did not influence the activity much.
a dose of 50 mg/Kg body weight. All the compounds 6a–
g produced significant activity when compared to standard
diclofenac. Compounds 6b and 6c showed more significant
activity compared to other test compounds (Table 5).
These compounds 6b and 6c decreased the number of
writhings produced in mice to a remarkable extent. Rest of
the compounds (6a, 6d, 6e, 6f, and 6g) are moderately
active.
5-Lox inhibitory activity
The 5-Lox inhibitory assay of compounds 6a–g were
evaluated using UV–Vis spectrophotometer (Reddenna et
a., 1990; Ulusu et al., 2002). All the compounds 6a–
g showed dose-dependant inhibition of 5-lipoxygenase
enzyme. The compounds 6b and 6c showed more signifi-
cant 5-Lox inhibitory activity compared to other com-
pounds. Rest of the compounds are moderately active. The
results are illustrated in Table 4.
Conclusion
In conclusion, we report the synthesis of novel dihydro
benzofuro[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones using
inexpensive and commercially available materials with
potential medicinal properties. The newly synthesized title
compounds 6a–g were evaluated for their antimicrobial,
anti-inflammatory, and analgesic activities. It has been
found that compounds 6a–g exhibited good antimicrobial,
anti-inflammatory, and analgesic activity compared to that
Analgesic activity
The analgesic activity of the newly synthesized compounds
6a–g were determined in vivo by acetic acid-induced
writhing method (Syamsudin and Rahayn, 2010) in mice at
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Med Chem Res
Table 3 Anti-inflammatory activity of dihydrobenzo furo[3,2-e]isoxazolo[4,5-b]azepin-5(5aH)-ones (6a–g)
Paw volume (mL of Hg)a
Groupb
0 h
1 h
2 h
4 h
6 h
6a
0.36 0.03
0.26 0.06
0.22 0.02
0.30 0.05
0.26 0.08
0.31 0.04
0.34 0.03
0.36 0.3
0.30 0.5
0.76 0.03
0.53 0.8
0.86 0.08ns
0.70 0.05ns
0.71 0.04
0.83 0.03ns
0.90 0.05ns
0.84 0.03
0.80 0.03
1.07 0.08
0.70 0.05***
0.76 0.06**
0.66 0.03**
0.58 0.02**
0.83 0.03**
0.83 0.03**
0.81 0.03*
0.82 0.04*
1.2 0.05
0.63 0.03**
0.36 0.01***
0.40 0.03***
0.55 0.05**
0.60 0.01**
0.53 0.02**
0.54 0.02**
0.96 0.03
6b
6c
0.56 0.06
0.70 0.05
0.80 0.05
0.78 0.02
0.82 0.04
0.90 0.05
0.66 0.03*
6d
6e
6f
6g
Control
Ibuprofen
0.60 0.05***
0.40 0.05***
n = 6, number of animals used in each group
ns
Nonsignificant, compared to control
*
**
***
Statistically significant compound to respective control value P \ 0. 05, P \ 0.01,
P \ 0.001
a
Values are expressed as mean SE
b
Dose levels: test compound (100 mg/kg b.wt) Ibuprofen (100 mg/kg b.wt)
Table 4 5-LOX inhibitory activity (in vitro anti-inflammatory
activity) of dihydrobenzo furo[3,2-e] isoxazolo[4,5-b]azepin-5(5aH)-
ones (6a–g)
of reference drugs. Among all the test compounds, it is
interesting to note that compounds 6b and 6c showed better
antimicrobial, anti-inflammatory, and analgesic activity.
Compounda
IC50 (lg/mL)b
Acknowledgments The authors are thankful to the Head, Depart-
ment of Chemistry, Kakatiya University, Warangal for providing the
facilities, the Director, Indian Institute of Chemical Technology,
Hyderabad for recording 1H NMR, and Mass Spectral data. Financial
assistance from the UGC SAP (phase-1)-DRS programme, New
Delhi, India, is greatly acknowledged. We are grateful to Prof. J.R.
Falck, Departments of Biochemistry and Pharmacology, University of
Texas, Southwestern Medical Center, Texas, USA for helpful dis-
cussions. One of the authors (K. Govardhan Reddy) is thankful to
UGC, New Delhi for financial assistance.
6a
19.90 1.0
13.31 0.2
14.62 0.3
28.29 1.5
20.99 1.2
25.21 0.3
24.25 0.4
4.33 0.2
6b
6c
6d
6e
6f
6g
LI0I020
a
Values are expressed as mean SEM
b
IC50 conentration corresponding to 50 % inhibition
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