Synthesis, characterization and biological evaluation of some new isoxazoline derivatives

Article abstract of DOI:10.14233/ajchem.2013.12899

Isoxazoline represents a unique class of nitrogen- and oxygen-containing five-membered heterocycles, a class of compounds of great importance in biological chemistry. Isoxazoline is considered as one of the most potent antimicrobial compound. Isoxazoline

Full text of DOI:10.14233/ajchem.2013.12899

Asian Journal of Chemistry; Vol. 25, No. 2 (2013), 789-792
http://dx.doi.org/10.14233/ajchem.2013.12899
Synthesis, Characterization and Biological Evaluation of Some New Isoxazoline Derivatives
*
DINESH K. JAIN , NEERAJ GOYAL and UPENDRA BHADORIYA
College of Pharmacy, IPS Academy, Indore-452 012, India
*Corresponding author: E-mail: dk57@rediffmail.com
(Received: 20 October 2011;
Accepted: 20 August 2012)
AJC-11979
Isoxazoline represents a unique class of nitrogen- and oxygen-containing five-membered heterocycles, a class of compounds of great
importance in biological chemistry. Isoxazoline is considered as one of the most potent antimicrobial compound. Isoxazoline derivatives
have been found to possess appreciable antitubercular activity against Mycobacterium tuberculosis. The object of antitubercular drug
discovery has become a subject of greater challenge due to increasing incidences of tubercular drug resistance. The study aimed to
synthesize potent antitubercular isoxazline derivatives. Different substituted isoxazoline derivatives have been synthesized by cyclization
of substituted chalcone derivatives in presence of hydroxylamine hydrochloride. Synthesized derivatives were characterized by melting
1
point, TLC, FT-IR, H NMR and MS spectroscopy. Synthesized conjugates were evaluated for in vitro antitubercular activity against
M. tuberculosis. Comparable antitubercular activity was obtained as compared to isoniazide.
Key Words: Isoxazoline, Antitubercular activity, Chalcone, Hydroxylamine hydrochloride.
synthized via cyclization of substituted chalcone intermediate
in presence of hydroxylamine hydrochloride3,4.
The purpose of this study was to develop new isoxazoline
derivatives as potent antituberculer agents against M. tuber-
culosis.
INTRODUCTION
Tuberculosis is caused by Mycobacterium tuberculosis, a
deadly obligate bacterial pathogen. The major concerns for
current tuberculosis treatment are its latency, co-infection with
HIV, poor patient compliance and drug resistance issues caused
by the emergence of multidrug resistant tuberculosis (MDR-
TB) and the recent advent of extensively drug resistant tuber-
culosis (XDR-TB). Streptomycin was introduced in 1944 for
the treatment of tuberculosis which was followed by first line
tuberculosis drugs such as isoniazid, rifampicin, ethambutol
and pyrizinamide and later by second line of drugs1. Multidrug
regimen was found to be effective for the treatment of tuber-
culosis (isoniazid + rifampicin + pyrizinamide + ethambutol).
MDR is observed in all front line and second line of drugs.
Resistance toward isoniazid has arisen due to mutation of katG
and inhA, resistance toward rifampicin due to mutation of
rpoB-gene, resistance toward pyrizinamide due to mutation
of pncA gene, etc. Hence, there is a need to develop potent
and fast acting antituberculosis drugs with new modes of action
to overcome the cross-resistance with current drugs and low
toxicity profiles that can be tolerated for long treatment periods
required for tuberculosis chemotherapy2. In an effort to develop
novel antituberculosis therapeutics, we reported two series of
isoxazoline compounds through modification of the 3,5-di-
substituted isoxazoline core with potent inhibitory activity
against M. tuberculosis. The isoxazoline derivatives were
EXPERIMENTAL
Melting points were determined by thieles tube (Table-1)
1
and were uncorrected. H NMR spectra were recorded on
BrukerAvance II 400 NMR spectrometer. FT-IR spectra were
recorded on Perkin Elmer 1600 and Mass spectra were
recorded on Jeol SX-102 mass spectrometer.
Synthesis of substituted isoxazoline derivatives
Procedure for synthesis of synthesis of substituted chalcone
derivative:
Claisen-Schmidt condensation: A solution of sodium
hydroxide (40 %) in water and rectified spirit was placed in a
flask provided with a mechanical stirrer. The flask was
immersed in a bath of crushed ice. Substituted acetophenone
(0.006 M) was poured with stirring, then substituted benzal-
dehyde (0.006 M) was added. The temperature of the mixture
was kept at ca. 25 ºC (the limits being 15- 30 ºC) and was
stirred vigorously until the mixture was so thick that stirring
was no longer effective (4-6 h). The stirrer was removed and
the reaction mixture was left in refrigerator overnight. The
product was filtered with suction on a buchner funnel, washed

790 Jain et al.
Asian J. Chem.
TABLE-1
COMPOUNDS CODE, STRUCTURE AND IUPAC NAMES OF THE SYNTHESIZED SUBSTITUTED ISOXAZOLINE DERIVATIVES
Conjugate code
Structure of conjugates
IUPAC name of conjugates
O
N
O2N
O2N
O2N
5-(4-Bromophenyl)-3-(3-nitrophenyl)-4,5-
dihydroisoxazole
NA1
Br
O
N
5-(4-Chlorophenyl)-3-(3-nitrophenyl)-4,5-
dihydroisoxazole
NA2
Cl
O
N
NA3
4-(3-(3-Nitrophenyl)-4,5-dihydroisoxazol-5-
yl)phenol
OH
O
N
O2N
N,N-Dimethyl-4-(3-(3-nitrophenyl)-4,5-
dihydroisoxazol-5-yl)benzenamine
NA4
NA5
NA6
NB1
NB2
NB3
N(CH3)2
OCH3
CH3
Br
O
N
O2N
5-(4-Methoxyphenyl)-3-(3-nitrophenyl)-4,5-
dihydroisoxazole
O
N
3-(3-Nitrophenyl)-5-p-tolyl-4,5-
dihydroisoxazole
O2N
HO
HO
HO
O
N
3-(5-(4-Bromophenyl)-4,5-dihydroisoxazol-3-
yl)phenol
O
N
3-(5-(4-Chlorophenyl)-4,5-dihydroisoxazol-3-
yl)phenol
Cl
O
N
3-(5-(4-Hydroxyphenyl)-4,5-dihydroisoxazol-
3-yl)phenol
OH

Vol. 25, No. 2 (2013)
Synthesis, Characterization and Biological Evaluation of Some New Isoxazoline Derivatives 791
O
N
HO
3-(5-(4-(Dimethylamino)phenyl)-4,5-
dihydroisoxazol-3-yl)phenol
NB4
N(CH3)2
O
N
HO
3-(5-(4-Methoxyphenyl)-4,5-dihydroisoxazol-
3-yl)phenol
NB5
OCH3
O
N
HO
NB6
3-(5-p-Tolyl-4,5-dihydroisoxazol-3-yl)phenol
CH3
with cold water until the washings were neutral to litmus and
then with ice cold ethanol. The crude product was recrystallized
from ethanol (i).
Biological evaluation
In vitro antitubercular activity of synthesized deriva-
tives: All chemicals were dissolved in DMSO (100 mg/1 mL)
and different dilutions of chemicals in each well were filled,
such as 2.5, 5.0, 7.5 and 10 mg. Control- DMSO as a control.
(--) indicate as negative (none zone).
Addition and cyclization: A mixture of chalcone and
hydroxylamine hydrochloride in ethanol was taken in a round
bottom flask. The reaction mixture was refluxed for 6 h on a
water bath and then ice cold water was added to it at room
temperature. The mixture was kept overnight in the freezer.
The precipitates were filtered, washed with distilled water and
dried. The product was recrystallized with ethanol to afford
pure crystals (ii) (Scheme-I).
Kirby-Bauer disk diffusion method for antibacterial
stability test:The standard Kirby-Bauer disk diffusion method
was used to determine the antimicrobial chemicals. The M.
tuberculosis strain were inoculated in nutrient broth and incu-
bated at 37 ºC for overnight. After overnight incubation, the
bacterial culture was swapped in solidified Muller hinton agar
plate and made wells of 6 mm diameter were punched in the
plate. Then add different concentration of chemicals was
dispensed in separate wells such as 2.5, 5.0, 7.5 and 10 mg
and add 100 µL of DMSO to centre well as control. The plates
were incubated at 37 ºC for 24 h.After incubation, the diameter
of zone of inhibition around the wells were measured and
recorded.
O
O
C
R
H
CH
3
R
m-Substituted acetophenone
Substituted benzaldehyde
25 ºC Ethanol/NaOH
Composition of Muller Hinton Agar: Ingredients g/L.
Beef extract - 2.0 g, starch - 1.5 g, acid hydrolysate of casein
- 17.5 g, Agar - 17.0 g, final pH - 7.3 0.1 at 25 ºC.
Composition of nutrient broth: Ingredients g/L, peptic
digest of animal tissue - 5 g, sodium chloride - 5 g, Beef extract
- 1.5 g, yeast extract - 1.5 g, final pH (at 25 ºC) - 7.4 0.2.
O
C
R
R
RESULTS AND DISCUSSION
Chalcone (i)
Substituted isoxazoline derivatives were synthesized using
substituted acetophenones and substituted benzaldehydes via
cyclization of substituted chalcone. 12 such compounds were
synthesized. They were evaluated for their in vitro antituber-
cular activity against M. tuberculosis. They showed improved
antitubercular activity over the parent drug. The results are
mentioned in Table-2.
.
Ethanol NH2OH HCl
O
N
R
Structure of all the synthesized conjugates have been
established on the basis of their consistent IR, 1H NMR and
mass spectral data. The synthesized conjugates showed the
presence of amide and ester functional group along with the
presence of aromatic and aliphatic ring which was also evident
in the 1H NMR spectra of the synthesized conjugates (Table-3).
R
Isoxazoline (ii)
(R = NO2 or OH)
Scheme-I: Synthesis of 3-(3-substituted phenyl)-5-phenyl-4,5-dihydro-
isoxazole substituted isoxazoline

792 Jain et al.
Asian J. Chem.
TABLE-2
PHYSICAL CONSTANTS OF SYNTHESIZED
ISOXAZOLINE DERIVATIVES
Derivatives NB2, NB3, NB4 and NB6 showed greater zone
of inhibition than Rifampicin at 10 mg conc. and were the
most potent antitubercular derivatives. Derivatives NA4 and
NB1 showed greater zone of inhibition than Rifampicin at 10
mg conc. but showed intermediate anti-tubercular activity at
other concentrations. Derivatives NA1, NA2, NA3, NA5, NA6
and NB5 showed poor antitubercular activity at all concen-
trations (Table-4).
Conjugate
code
m.f.
m.w.
m.p. (ºC)
Rf* value
NA1
NA2
NA3
NA4
NA5
NA6
NB1
NB2
NB3
NB4
NB5
NB6
C15H11N2O3Br
C15H11N2O3Cl
C15H12N2O4
C17H17N3O3
C16H14N2O4
C16H14N2O3
C15H12NO2Br
C15H12NO2Cl
C15H13NO3
347
302
284
311
298
282
318
273
255
282
269
253
110-115
135-140
90-95
0.67
0.73
0.69
0.81
0.83
0.79
0.84
0.83
0.72
0.74
0.66
0.56
100-105
120-125
80-85
TABLE-4
RESULT OF IN VITRO ACTIVITY OF SYNTHESIZED
ISOXAZOLINE DERIVATIVES
120-125
135-140
200-205
120-125
115-120
155-160
Concentration of chemicals (zone of
inhibition in mm)
C17H18N2O2
C16H15NO3
C16H15NO2
Compounds
2.5 mg
–
5.0 mg
14
14
12
11
–
7.5 mg
15
10.0 mg
17
NA1
NA2
NA3
11
–
15
16
TABLE-3
SPECTRAL DATA OF THE SYNTHESIZED DERIVATIVES
Mass spectra
15
17
NA4*
NA5
10
–
14
19
Comp.
1H NMR spectra (δ) in ppm
10
13
(molecular
ion peak)
code
NA1
NA6
–
11
13
20
17
16
14
17
13
14
NB1*
NB2*
NB3*
NB4*
NB5
12
18
15
14
12
15
15
20
347.99
303.05
285.08
312.13
299.10
283.10
319.00
275.05
256.09
283.14
270.11
254.11
7.61-8.51 (r, 4H, aromatic ring), 7.08-7.36
(s, 4H, aromatic ring), 4.5 (m, 1H, CH),
3.39-3.52 (s, 2H, CH2 in ring)
21
22
21
25
NA2
NA3
NA4
NA5
NA6
NB1
NB2
NB3
NB4
NB5
NB6
7.61-8.51 (r, 4H, aromatic ring), 7.13-7.20
(s, 4H, aromatic ring), 4.5 (m, 1H, CH),
3.39-3.52 (s, 2H, CH2 in ring)
25
27
15
17
NB6*
21
25
7.61-8.49 (r, 4H, aromatic ring), 7.77-8.02
(s, 4H, aromatic ring), 7.57(r, OH), 3.37 (s,
2H, CH2 in ring)
Standard (Rifampicin)
10 mg
18
–
Control (DMSO)
7.59-8.59 (r, 4H, aromatic ring), 6.63-7.26
(s, 4H, aromatic ring), 3.04 (s, 2H, CH2 in
ring), 2.70 (s, 6H, CH3)
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7.62-8.42 (r, 4H, aromatic ring), 6.77-7.10
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(s, 4H, aromatic ring), 5.0 (r, 2H,
OH), 3.33 (s, 2H, CH2 in ring)
6.80-7.20 (r, 4H, aromatic ring), 6.52-7.01
(s, 4H, aromatic ring), 5.0 (r, 1H, OH), 2.85
(s, 6H, CH3), 3.33 (s, 2H, CH2 in ring)
6.80-7.20 (r, 4H, aromatic ring), 6.70-7.08
(s, 4H, aromatic ring), 5.0 (r, 1H, OH), 3.73
(s, 3H, CH3), 3.33 (s, 2H, CH2 in ring)
6.80-7.20 (r, 4H, aromatic ring), 6.99-7.07
(s, 4H, aromatic ring), 5.0 (r, 1H, OH), 2.35
(s, 3H, CH3), 3.33 (s, 2H, CH2 in ring)