Antimycobacterial activity: Synthesis of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole analogues

Article abstract of DOI:10.3109/14756366.2010.529805

In this study, a series of novel 3-(substituted phenyl)-6,7-dimethoxy-3a,4- dihydro-3H-indeno[1,2-c]isoxazole analogues were synthesized and evaluated for antimycobacterial activity against Mycobacterium tuberculosis (MTB) H ₃₇Rv and isoniazid resistant M. tuberculosis (INHR-MTB). All the newly synthesized compounds were showing moderate to high inhibitory activities. The compound 6,7-dimethoxy-3-(4-chloro phenyl)-4H-indeno[1,2-c]isoxazole (4b) was found to be the most promising compound, active against MTB H₃₇Rv and INHR-MTB with minimum inhibitory concentrations of 0.22 and 0.34 μM.

Full text of DOI:10.3109/14756366.2010.529805

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(4): 598–602
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.529805
SHORT COMMUNICATION
Antimycobacterial activity: synthesis of novel 3-(substituted
phenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]
isoxazole analogues
Mohamed Ashraf Ali1,2, Rusli Ismail1, Tan Soo Choon1, Suresh Pandian2, and
Mohamed Zaheen Hassan Ansari2
1Pharmacogenetic and Novel erapeutic, Institute for Research in Molecular Medicine, Universiti of Sains Malysia,
Pennag-11800, Malaysia, and 2New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy
College, Alwar, Rajasthan 301030, India
Abstract
Inthisstudy,aseriesofnovel3-(substitutedphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazoleanalogues
were synthesized and evaluated for antimycobacterial activity against Mycobacterium tuberculosis (MTB) H37Rv and
isoniazid resistant M. tuberculosis (INHR-MTB). All the newly synthesized compounds were showing moderate to high
inhibitory activities. The compound 6,7-dimethoxy-3-(4-chloro phenyl)-4H-indeno[1,2-c]isoxazole (4b) was found to
be the most promising compound, active against MTB H37Rv and INHR-MTB with minimum inhibitory concentrations
of 0.22 and 0.34 μM.
Keywords: Isoxazole, antimycobacterial agents, isoantimycobacterial agents, tuberculosis
Introduction
developing active disease and three million people die
from TB. Currently, the available first-line anti-TB agents
such as rifampcin, ethambutol, streptomycin, and pyrazi-
namide are highly effective and are generally well toler-
ated. Problems in the chemotherapy of TB arise when any
patients develop resistance to any of these drugs. is is
due to the fact that the second-line drugs such as p-amin-
osalicylic acid, amikacin, cycloserine, capreomycin, and
ethionamide are less effective and more toxic.3 e global
mortality rate for TB is very high and the development of
new kinds of TB such as Multi-drug-resistant tuberculosis
(MDR-TB) and Extensively drug-resistant tuberculosis
(XDR-TB) alarm for the discovery of new drugs to reduce
the potential hazards caused by the fatal disease.
In the past decade, most heterocyclic systems have been
used as a source to discover new compounds with varied
biological potentials. Especially, nitrogen-containing het-
erocyclic systems such as novel pyrazolines, substituted
oxadiazole, and substituted triazole moieties play a vital
role in discovering novel candidates having antimicrobial
Many human illnesses are especially caused by infec-
tions with microbes such as virus, bacteria, or fungi.
Among various illnesses to human beings, certain viral,
tubercular, bacterial, and fungal infections are more
common and dangerous. is is because of their ten-
dency to develop new strains under any circumstances
for developing resistance with the available drugs. is
condition has paved the way for scientists to make the
efforts to work on several molecules for coming out with
novel entities to combat the illnesses.
Tuberculosis (TB) is making a worldwide resurgence.
Several factors may be responsible for the increase in the
infection rate such as infection with human immunode-
ficiency virus, change in economic and social circum-
stances, and decline in TB control programs.1 In addition,
outbreaks of multidrug resistant TB have been identified.2
When the AIDS pandemic began, one-third of the world
population was infected with Mycobacterium tubercu-
losis (MTB). Each year, eight to 10 million people are
Address for Correspondence: Mohamed Ashraf Ali, New Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy
College, Alwar, Rajasthan 301030, India. Tel.: +91-99-11-128001; Fax: +91-11-26059666. E-mail: asraf80med@rediffmail.com
(Received 30 March 2010; revised 01 October 2010; accepted 04 October 2010)
598

Antimycobacterial activity of isoxazole derivative 599
potentials.4–9 Recently, we reported that the azole deriva-
tives possess anti-TB activity.10–12 Also, substituted azole
derivatives have been evaluated for their antiproliferative
activity against human lung cancer A549.13
on to crushed ice (20g). e product obtained was
filtered, washed with water, and purified from ethanol by
recrystallization.
From the above facts, it was evident that the microbial
infections making worldwide resurgence and the whole
scientific community have to pave their useful hands in
exploring novel entities with good therapeutic response
without allowing the microbes to develop their resis-
tance. In view of these findings and in continuation of
our previous work on the synthesis of isoxazole deriva-
tives, we report the synthesis and evaluation of antimy-
cobacterial activity against MTB and isoniazid resistant
M. tuberculosis (INHR-MTB).
3-(4-Methoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4a)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–4.0
(1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.3 (6H, m,
aromatic);13C-NMR(75 MHz,CDCl3):28.4,35.1,56.1,56.1,
56.1, 91.3, 104.0, 110.4, 112.5, 120.3, 131.3, 131.9, 147.1,
149.9, 147.2, 147.8, 148.4, 152.2, 157.2; EI–MS (m/z): 326
(M+1); C19H19NO4; calculated: C = 70.14, H = 5.89, N = 4.30;
found: C = 70.12, H = 5.87, N = 4.32%.
3-(4-Chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
Materials
indeno[1,2-c]isoxazole (4b)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N), 786 (C–Cl); H-
1
e entire chemicals were supplied by Merck Limited,
Worli, Mumbai, Maharastra, India and S. D. Fine
Chemicals, Mumbai, Maharastra, India. Melting points
were determined by open-tube capillary method and
are uncorrected. Purity of the compounds was checked
on thin layer chromatography (TLC) plates (silica gel
G) in the solvent system toluene–ethyl formate–formic
acid (5:4:1) and benzene–methanol (8:2), the spots were
located under iodine vapors or UV light. Infra-red (IR)
spectrums were obtained on a Perkin–Elmer 1720 Fourier
transform infrared spectroscopy (FT-IR) (KBr pellets).
Proton nuclear magnetic resonance spectroscopy (1H-
NMR) spectra were recorded on a Bruker AC 300 MHz
spectrometer using Tetramethylsilane (TMS) as internal
standard in Dimethyl sulfoxide (DMSO)/CDCl3. e
mass spectra under Electron impact-Mass spectroscopy
(EI-MS) were recorded at 70 eV ionizing voltage with a
VG ProSpec instrument and are presented as m/z.
NMR (DMSO-d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s,
OCH3), 3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH),
6.6–7.4 (6H, m, aromatic); 13C-NMR (75 MHz, CDCl3):
28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 112.0, 127.9 128.7, 128.7,
128.9, 128.9,130.6, 131.0, 147.2, 147.8, 152.2, 157.2; EI–MS
(m/z): 330 (M+); C18H16ClNO3; calculated: C = 65.56,
H = 4.89, N = 4.25; found: C = 65.58, H = 4.87, N = 4.26%.
3-(4-Dimethyl aminophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4c)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 2.82 (6H, s, NCH3), 3.0–3.2 (2H, m, CH2), 3.82 (6H,
s, OCH3), 3.97–4.0 (1H, m, CH), 5.17 (d, J=6.4 Hz, 1H, CH),
6.6–7.0 (6H, m, aromatic); 13C-NMR (75MHz, CDCl3): 28.4,
35.1, 41.3, 41.3, 56.1, 56.1, 91.3, 104.0, 112.0, 113.0, 113.0,
127.2, 128.0,127.2, 131.2, 147.2, 147.8, 148.4,152.2, 157.2;
EI–MS (m/z): 339 (M+1); C20H22N2O3; calculated: C=70.99,
H=6.55, N=8.28; found: C=70.98, H=6.53, N=8.26%.
Methods
6,7-Dimethoxy-3-phenyl-3a,4-dihydro-3H-indeno[1,2-c]
isoxazole (4d)
Chemistry
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–4.0
(1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.6 (7H, m,
aromatic);13C-NMR(75 MHz,CDCl3):28.4,35.1,56.1,56.1,
91.3, 104.0, 112.0, 126.0, 128.0, 128.1, 128.8, 128.8,130.5,
131.2, 147.2, 147.8, 152.2, 157.2; EI–MS (m/z): 296 (M+1);
C18H17NO3; calculated: C = 73.20, H = 5.80, N = 4.74; found:
C = 73.22, H = 5.78, N = 4.76%.
General method for the preparation of 2-[(E)-1-(substituted
aryl)methylidene]-5,6-dimethoxy-1-indanone (3a–3o)
5,6-Dimethoxy-1-indanone (1.92 g, 0.01 mol) and appro-
priate aldehyde (1.02 g, 0.01 mol) were dissolved in
ethanol and sodium hydroxide (30%, 5 ml) with 10 ml of
petroleum ether. e reaction mixture was stirred at room
temperature for 4 h. e resulting solution was allowed to
stand overnight then poured into ice-cold water followed
by neutralization with HCl. e solid separated was
filtered, dried, and purified from ethanol.5,6
3-(3,4-Dimethoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4e)
General method for the preparation of 3-(3-substituted aryl)-6,7-
dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazole (4a–4o)
To 2-[(E)-1-(substituted phenyl)methylidene]-5,6-dime-
thoxy-1-indanone (3a–3o) (0.001mol) in 15ml of glacial
acetic acid, 0.002mol hydroxylamine hydrochloride was
added and the reaction mixture was refluxed for 15h and
cooled. Excess of solvent was removed under reduced
pressure and the reaction mixture was cooled and poured
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82, 3.85 (12H, s, OCH3),
3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.0
(5H, m, aromatic); 13C-NMR (75 MHz, CDCl3): 28.4, 35.1,
56.1, 56.1, 56.1, 56.1, 91.3, 104.0, 110.4, 112.5, 120.3,
131.3, 131.9, 147.1, 149.9, 147.2, 147.8, 148.4, 152.2, 157.2;
EI–MS (m/z): 356 (M+1); C20H21NO5; calculated: C = 65.59,
H = 5.96, N = 3.94; found: C = 67.57, H = 5.97, N = 3.96%.
© 2011 Informa UK, Ltd.

600 M. A. Ali et al.
(1H, m, CH), 5.17 (d, J=6.4 Hz, 1H, CH), 6.6–7.4 (2H, m,
aromatic), 7.2–7.8 (3H, m, furyl); 13C-NMR (75MHz,
CDCl3): 28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 110.2, 110.2,
112.0, 131.2, 141.5, 147.2, 147.8, 152.2, 153.5, 157.2; EI–MS
(m/z):286 (M+1); C16H15NO4; calculated: C=67.36, H=5.30,
N=4.91; found: C=67.35, H=5.28, N=4.90%.
3-(3,4,5-Trimethoxyphenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4f)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82, 3.86 (15H, s, OCH3),
3.97–4.0 (1H, m, CH), 5.17 (d, J=6.4 Hz, 1H, CH), 6.6–7.0
(4H, m, aromatic); EI–MS (m/z): 386 (M+1); 13C-NMR
(75MHz, CDCl3): 28.4, 35.1, 56.1, 56.1,56.1, 56.1, 56.1, 91.3,
104.0, 110.4, 112.5, 120.3, 131.3, 131.9, 147.1, 149.9, 147.2,
147.8, 148.4, 152.2, 157.2; C21H23NO6; calculated: C=65.46,
H=6.01, N=3.59; found: C=65.42, H=6.00, N=3.61%.
6,7-Dimethoxy-3-(2-thienyl)-3a,4-dihydro-3H-indeno[1,2-c]
isoxazole (4l)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3),
3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.0
(2H, m, aromatic), 7.2–7.5 (3H, m, thiophenyl); 13C-NMR
(75 MHz, CDCl3): 28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 112.0,
124.9, 125.1, 126.4, 131.2, 142.9, 147.2, 147.8, 152.2, 157.2;
EI–MS (m/z):302 (M+1); C16H15NO3S; calculated: C = 63.77,
H = 5.02, N = 4.65; found: C = 63.75, H = 5.00, N = 4.66%.
3-(4-Flurophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4g)
1
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N), 776 (C–F); H-
NMR (DMSO-d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s,
OCH3), 3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH),
6.6–7.2 (6H, m, aromatic); 13C-NMR (75 MHz, CDCl3):
28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 112.0, 115.6, 115.6,
127.9, 127.9, 131.2, 134.1, 147.2, 147.8, 152.2, 157.2, 160.2;
EI–MS (m/z): 314 (M+1); C18H16FNO3; calculated: C = 69.00,
H = 5.15, N = 4.47; found: C = 69.02, H = 5.13, N = 4.46%.
3-(4-Bromophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4m)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–
4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.2 (6H,
m, aromatic); 13C-NMR (75 MHz, CDCl3): 28.4, 35.1, 56.1,
56.1, 91.3, 104.0, 112.0, 120.4, 128.2, 128.2, 131.2, 131.7,
137.5, 147.2, 147.8, 152.2, 157.2, 160.2; EI–MS (m/z):
374 (M+1); C18H16BrNO3; calculated: C = 57.77, H = 4.31,
N = 3.74; found: C = 57.74, H = 4.29, N = 3.76%.
3-(2-Chlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4h)
1
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N), 786 (C–Cl); H-
NMR (DMSO-d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s,
OCH3), 3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH),
6.6–7.4 (6H, m, aromatic); 13C-NMR (75 MHz, CDCl3):
28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 112.0, 126.9,127.4 128.9,
133.4, 128.9, 135.2, 131.0, 147.2, 147.8, 152.2, 157.2; EI–MS
(m/z): 330 (M+1); C18H16ClNO3; calculated: C = 65.56,
H = 4.89, N = 4.25; found: C = 65.58, H = 4.87, N = 4.26%.
4-(6,7-Dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazol-3-
yl)-2-benzonitrile (4n)
1
IR:(KBr) cm−1: 3042 (CH), 1320 (C–N); H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–4.0
(1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.2 (6H, m,
aromatic);13C-NMR(75 MHz,CDCl3):28.4,35.1,56.1,56.1,
91.3, 104.0, 109.9, 112.0, 118.6, 127.3, 127.3, 131.2, 132.3,
132.3, 142.8, 147.2, 147.8, 152.2, 157.2; EI–MS (m/z): 321
(M+1); C19H16N2O3; calculated: C = 71.24, H = 5.03, N = 8.74;
found: C = 71.22, H = 5.01, N = 8.72%.
3-(2,6-Dichlorophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4i)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N), 786 (C–Cl); H-
1
NMR (DMSO-d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s,
OCH3), 3.97–4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH),
6.6–7.5 (5H, m, aromatic); 13C-NMR (75 MHz, CDCl3):
28.4, 35.1, 56.1, 56.1, 91.3, 104.0, 112.0, 127.8,128.9,130.0,
130.0,133.8, 134.8, 131.2, 147.2, 147.8, 152.2, 157.2; EI–MS
(m/z): 364 (M+1); C18H15Cl2NO3; calculated: C = 59.36,
H = 4.15, N = 3.85; found: C = 59.34, H = 4.13, N = 3.83%.
4-(6,7-Dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]isoxazol-3-
yl)-2-methoxyphenol (4o)
1
IR: (KBr) cm−1: 3042 (CH),1320 (C–N); H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (9H, s, OCH3), 3.97–4.0
(1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 5.6 (1H, s, OH),
6.6–7.3 (5H, m, aromatic); 13C-NMR (75 MHz, CDCl3):
28.4, 35.1, 56.1, 56.1, 56.1, 91.3, 104.0, 109.9, 110.8, 112.0,
120.7, 132.1, 142.8, 146.0, 147.6, 147.2, 147.8, 152.2, 157.2;
EI–MS (m/z):342 (M+1); C19H19NO5; calculated: C = 66.85,
H = 5.61, N = 4.10; found: C = 66.83, H = 5.59, N = 4.08%.
3-(4-Nitrophenyl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazole (4j)
1
IR:(KBr) cm−1:3042 (CH), 1320 (C–N); H-NMR (DMSO-
d6) ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–
4.0 (1H, m, CH), 5.17 (d, J= 6.4 Hz, 1H, CH), 6.6–7.7 (6H,
m, aromatic); 13C-NMR (75 MHz, CDCl3): 28.4, 35.1, 56.1,
56.1, 91.3, 104.0, 112.0, 124.0, 124.0, 129.0, 129.0, 131.2,
144.6, 145.2,147.2, 147.8, 152.2, 157.2; EI–MS (m/z): 341
(M+1); C18H16N2O5; calculated: C = 63.53, H = 4.74, N = 8.23;
found: C = 63.51, H = 4.76, N = 8.26%.
Results and discussion
3-(Substituted
aryl)-6,7-dimethoxy-3a,4-dihydro-3H-
indeno[1,2-c]isoxazoleanalogues(4a–o)describedinthis
study are shown in Table 1 and a reaction sequence for the
preparation is outlined in Scheme 1. In the initial step, 5,6-
dimethoxy-2-[(E)-1-arylmethylidene]-1-indanone was
3-(2-Furyl)-6,7-dimethoxy-3a,4-dihydro-3H-indeno[1,2-c]
isoxazole (4k)
IR: (KBr) cm−1: 3042 (CH), 1320 (C–N); 1H-NMR (DMSO-
d6)ppm: 3.0–3.2 (2H, m, CH2), 3.82 (6H, s, OCH3), 3.97–4.0
Journal of Enzyme Inhibition and Medicinal Chemistry

Antimycobacterial activity of isoxazole derivative 601
Table 1. Physical constants and antimycobacterial activity of the synthesized compounds.
N
O
O
O
H3C
H3C
R
4a-o
MIC (µM)
Comp.
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
R
Yield (%)
84
MP (°C)
144
162
143
141
128
103
176
145
164
184
198
168
154
164
192
–
MTBa
>6.25
0.22
MTBb
>6.25
0.34
Cytotoxicity
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
>62.5
4-Methoxy phenyl-
4-Chloro phenyl-
4-Dimethylamino phenyl-
Phenyl-
60
82
4.94
24.78
>6.25
>6.25
>6.25
2.10
70
>6.25
>6.25
>6.25
1.22
3,4-Dimethoxy phenyl-
3,4,5-Trimethoxy phenyl-
4-Fluoro phenyl-
2-Chloro phenyl-
2,6-Dichloro phenyl-
3-Nitro phenyl-
Furyl-
62
75
82
65
0.78
1.72
67
1.98
3.96
72
3.46
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
11.37
80
4.59
iophenyl
62
5.68
4m
4n
4o
INH
4-Bromo phenyl-
4-Cyano phenyl-
4-Hydroxy, 3-methoxy phenyl-
–
71
>6.25
>6.25
>6.25
0.73
66
72
–
aMycobacterium tuberculosis H37Rv.
bINH resistant Mycobacterium tuberculosis.
O
O
O
O
H3C
H3C
O
O
CH3OH-NaOH
H3C
H3C
+
R-CHO
2a-o
R
3a-o
1
NH2OH.HCl/CH3OH
N
O
O
O
H3C
H3C
R
4a-o
Scheme 1. Protocol for synthesis.
synthesized by condensing 5,6-dimethoxy-1-indanone
with appropriate aromatic aldehyde in dilute methano-
lic sodium hydroxide solution at room temperature. e
product 5,6-dimethoxy-2-[(E)-1-phenylmethylidene]-
1-indanone obtained in the first step is treated with
hydroxylamine hydrochloride in the presence of glacial
acetic acid to get titled compounds in 62–84% yield after
recrystallization with ethanol. e purity of the com-
pounds was checked by TLC and elemental analyses.
Both analytical and spectral data (1H-NMR, IR) of all the
synthesized compounds were in full agreement with the
proposed structures. e elemental analysis results were
within 0.4% of the theoretical values.
e synthesized compounds (4a–o) were tested for
their antimycobacterial activity in vitro against MTB
and INHR-MTB by agar dilution method using double-
dilution technique similar to that recommended by the
National Committee for Clinical Laboratory Standards.14
© 2011 Informa UK, Ltd.

602 M. A. Ali et al.
e INHR-MTB clinical isolate was obtained from
Tuberculosis Research Center, Alwar, India. e MIC was
defined as the minimum concentration of compound
required to inhibit 90% of bacterial growth and MICs of
the compounds were reported in Table 1 with standard
drug INH for comparison.
and MDR. e isoxazole derivatives discovered in this
study may provide valuable therapeutic intervention for
the treatment of anti-tubercular diseases.
Acknowledgements
e authors thank Institute for Research in Molecular
Medicine, Universiti of Sains Malysia, Pennag, Malaysia
and Alwar Pharmacy College, Alwar, Rajasthan, India, for
providing research facilities.
Among the 15 compounds synthesized eight com-
pounds were found to be most active compounds with
minimum inhibitory concentration of <6 μΜ and were
more active than INH against MTB. Compounds with
electron withdrawing group substituted on the aryl ring
were showing better activity. Among the 15 newly synthe-
sized compounds, compound 6,7-dimethoxy-3-(4-chloro
phenyl)-4H-indeno[1,2-c]isoxazole (4b) was found to be
the most active agent against MTB H37Rv and INHR-MTB
with minimum inhibitory concentration of <0.30 μΜ.
When compared with INH, compound (4b) was found to
be 3.32- and 33.4-fold more active against MTB and INHR-
MTB, respectively. Following these compounds, 2-chlo-
rophenyl (4h) and 2,6-dichlorophenyl (4i) substituents
were found to be more active than INH against MTB H37Rv
and INHR-MTB with MIC of 0.78 μΜ, 1.72 μΜ, and 1.28
μΜ, 3.96 μΜ, respectively. However, the electron with-
drawing group such as 4-cholrophenyl, 2-chlorophenyl,
2,6-dichlorophenyl, thiophenyl, furyl, and 4-nitrophenyl
substituted analogues showed good to excellent inhibitory
activity against MTB H37Rv and moderate to good activ-
ity against INHR-MTB. On the other hand, the electron
donating group containing analogues, such as (OCH3)
group substituted 4-methoxy phenyl (4a), 3,4 dimethoxy
phenyl (4e), 3,4,5 trimethoxy phenyl (4f), and 3-hydroxyl,
4-methoxy substituted compound (4o), and electron with-
drawing group substituted 4-dimethylaminophenyl (4c)
and 4-flurophenyl (4g) showed moderate anti-tubercular
activity. ese reports clearly show that the substitution
of electron withdrawing group such as 4-chloro makes
remarkable improvement in antimycobacterial activity.
All the compounds were tested for cytotoxicity (IC50) in
VERO cells at concentrations of 62.5 μg/ml or 10×. After
72h of exposure, viability was assessed on the basis of cellu-
lar conversion of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl
tetrazolium bromide (MTT) into a formazan product using
the Promega Cell Titer 96 non-radioactive cell proliferation
method.15 Most of the active compounds were found to be
nontoxic up to the concentration of 62.5 μg/ml.
Declaration of interest
e authors declare no conflicts of interest.
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antimycobacterial activity against MTB and INHR-MTB.
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Journal of Enzyme Inhibition and Medicinal Chemistry