Green synthesis of isoxazoline derivatives using microwave irradiation and their antifungal activity

Article abstract of DOI:10.14233/ajchem.2016.19900

Microwave irradiation method was used for synthesis of isoxazolines. Claisen Schmidt reaction of different aromatic aldehydes with acetanilide gave acrylamides (1a-1h) which on further reaction with hydroxylamine hydrochloride (in the presence of sodium hydroxide) afforded isoxazolines (2a-2h). Physical data of all the synthesized compounds were recorded. Isoxazolines were characterized by their IR and ¹H NMR spectra. All the synthesized isoxazolines were screened for their antifungal activity against fungi namely Drechslera maydis and Rhizoctonia solani isolated from maize. Isoxazolines having chloro substitution on benzene ring found to be most effective followed by fluoro and nitro substituted compounds. None of the compound was registered as effective as Bavistin.

Full text of DOI:10.14233/ajchem.2016.19900

Asian Journal of Chemistry; Vol. 28, No. 10 (2016), 2169-2172
A
SIAN
J
OURNAL OF HEMISTRY
C
http://dx.doi.org/10.14233/ajchem.2016.19900
Green Synthesis of Isoxazoline Derivatives Using
Microwave Irradiation and Their Antifungal Activity
1,*
2
1
2
AKHIL GOYAL , SUNITA SHARMA , JYOTI GABA and HARLEEN KAUR
1Department of Chemistry, Punjab Agricultural University, Ludhiana-141 004, India
2Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana-141 004, India
*Corresponding author: E-mail: jimmygoyal51@gmail.com
Received: 9 February 2016;
Accepted: 16 May 2016;
Published online: 30 June 2016;
AJC-17961
Microwave irradiation method was used for synthesis of isoxazolines. Claisen Schmidt reaction of different aromatic aldehydes with
acetanilide gave acrylamides (1a-1h) which on further reaction with hydroxylamine hydrochloride (in the presence of sodium hydroxide)
afforded isoxazolines (2a-2h). Physical data of all the synthesized compounds were recorded. Isoxazolines were characterized by their IR
and 1H NMR spectra. All the synthesized isoxazolines were screened for their antifungal activity against fungi namely Drechslera maydis
and Rhizoctonia solani isolated from maize. Isoxazolines having chloro substitution on benzene ring found to be most effective followed
by fluoro and nitro substituted compounds. None of the compound was registered as effective as Bavistin.
Keywords: Acrylamides, Antifungal activity, Isoxazolines, ED50.
of furoxan, which is a nitric oxide donor. Isoxazoline derivatives
controlled B. cineria on cucumbers [1], antiviral properties
against herpes type 2 viruses [2] and also possess anti-influenza
virus activity [3]. Penicillin derivatives containing isoxazole ring
were found to be antibacterial [4].
INTRODUCTION
Isoxazole is an azole (I) containing oxygen and nitrogen
atoms at 1, 2 positions, its partially saturated analogs are
isoxazolines (II-IV) and completely saturated analog of
isoxazoline (V).
Isoxazoline derivatives possess antidiabetic, diuretic,
analgesic, anthelimintic and hypolipaemic activity. There are
many interesting biological activities exhibited by these
compounds. Isoxazoline derivatives are used as corrosion
inhibitors for fuels and lubricants [5]. These derivatives also
show a good potency in animal models of thrombosis [6].
Microwave heating has emerged as a powerful technique
to promote a variety of chemical reactions [7-11]. Microwave
reactions under solvent-free conditions were attractive in
offering reduced pollution and offer low cost technique together
with simplicity in processing and handling [12-14]. Microwave-
assisted synthesis includes virtually all types of chemical
reactions such as additions, cycloadditions, substitutions, elimi-
nations, fragmentations, etc. Recently reported studies on the
microwave irradiation for the synthesis of heterocyclic
compounds revealed that it is safe rapid, economic and conve-
nient, ecofriendly method. It can be termed as e-chemistry
because it is easy, effective, economical and eco-friendly and
is believed to be a step towards green chemistry. Keeping in
view the above facts, the present study was carried out to
synthesize isoxazoline derivatives using microwave and their
evaluation for antifungal activity.
N
O
HN
O HN
O
HN
O
N
O
I
II
III
IV
V
Isoxazole nucleus is a structural moiety found in natural
products such as ibotenic acid and in synthetic compounds
with vital medicinal value. Isoxazoles have illustrious history
e.g., their chemistry is associated with Ludwig Claisen, who
first recognized the cyclic structure of 3-methyl-5-phenyl-
isoxazole in 1888 and was shown to possess typical properties
of an aromatic system under certain reaction conditions
particularly in basic media, it is highly labile.A very significant
contribution to the development of isoxazole chemistry came
between 1930-1946 from Quilico’s studies on the synthesis
of ring system from nitrile oxides and unsaturated compounds.
Isoxazoles also form the basis for a number of drugs,
including the COX-2 inhibitor valdecoxib (bextra), a derivative

2170 Goyal et al.
Asian J. Chem.
(C-O str.), 1053 (C-Cl str.), 745 (N-H bending). 1H NMR (400
MHz, CDCl3, δ, ppm) 3.77-3.83 (dd, 1H, CH2, J = 17.3, 6.3
Hz), 4.03-4.07 (dd, 1H, CH2, J = 16.5, 7.4 Hz), 5.04-5.09 (dd,
1H, CH, J = 16.6, 6.2 Hz)., 7.68-7.8.24 (m, 9H, Ar-H), 9.92
(s, 1H, NH).
5-(4-Fluorophenyl)-N-phenyl-4,5-dihydro isoxazol-3-
amine (2d): Yield 66 %, m.p.: 94-95 °C. IR (KBr, νmax, cm-1):
3349 (N-H str.), 3050 (Ar C-H str.), 1594 (C=N str.), 1239 (C-
O str.), 1050 (C-F str.), 768 (N-H bending).1H NMR (400 MHz,
CDCl3, δ, ppm) 3.77-3.83 (dd, 1H, CH2, J = 17.3, 6.3 Hz),
4.13-4.20 (dd, 1H, CH2, J = 17.5, 7.4 Hz), 5.14-5.19 (dd, 1H,
CH, J = 16.6, 6.2 Hz), 7.49-8.29 (m, 9H, Ar-H), 9.72 (s, 1H,
NH).
EXPERIMENTAL
All the recorded melting points were determined in open
capillaries and are uncorrected. Structures of compounds were
confirmed by routine spectrometric analysis. IR and 1H NMR
spectra were scanned at SophisticatedAnalytical Instrumentation
Facility (SAIF), Central Instrument Laboratory (CIL), Panjab
University, Chandigarh. The IR spectra of compounds were
recorded using KBr discs on Perkin-Elmer FTIR spectrophoto-
meter. 1H NMR spectra were recorded on a Bruker Avance II
400 MHz instrument using TMS as an internal standard. The
chemical shifts were expressed in δ (ppm) values.
General procedure forsynthesis of isoxazolines (2a-2h):
Equimolar amounts of already prepared acrylamides [15] (1a-
1h) (0.005 mol), hydroxylamine hydrochloride (0.005 mol)
in ethanol (30 mL) were taken in an Erlenmeyer flask of 100
mL. 2-3 drops of NaOH solution (30 %) was added. The reaction
mixture was mixed thoroughly and irradiated at 720 W with
intermitted irradiation for 30 s. The progress of reaction was
checked with TLC. On completion of reaction resultant mass
was poured into ice cold water with stirring. The solid obtained
was filtered, washed and recrystallized from ethanol. Physical
data of all the synthesized compounds is given in Table-1.
2-[3-(Phenylamino)-4,5-dihydro isoxazol-5-yl]phenol
(2e): Yield 71 %. IR (KBr, νmax, cm-1): 3399 (broad N-H, O-H
str.), 1594 (C=N str.), 1393 (C-N str.), 1268 (C-O str.), 759
(N-H bending). 1H NMR (400 MHz, D2O, δ, ppm) 2.58-2.63
(dd, 1H, CH2, J = 18.3, 4.3 Hz), 3.31-3.39 (dd, 1H, CH2, J =
19.7, 12.6 Hz), 5.15-5.20 (dd, 1H, CH, J = 11.5, 4.4 Hz), 7.61-
8.15 (m, 9H, Ar-H), 10.24 (s, 1H, NH), 10.42 (s, 1H, O-H).
4-[3-(Phenylamino)-4,5-dihydro isoxazol-5-yl]phenol
(2f): Yield 70 %, m.p.: 106-108 °C. IR (KBr, νmax, cm-1): 3440
(broad N-H, O-H str.),1592 (C=N str.), 1250 (C-O str.), 770
1
(N-H bending). H NMR (400 MHz, CDCl3, δ, ppm) 3.75-
TABLE-1
3.79 (dd, 1H, CH2, J = 16.4, 6.3 Hz), 4.03-4.08 (dd, 1H, CH2,
J = 16.7, 7.4 Hz), 5.04-5.09 (dd, 1H, CH, J = 16.6, 6.2 Hz),
7.68-8.24 (m, 9H,Ar-H), 9.92 (s, 1H, O-H),10.55 (s, 1H, NH).
5-(2-Nitrophenyl)-N-phenyl-4,5-dihydro isoxazol-3-
amine (2g): Yield 58 %, m.p.: 97-99 °C. IR (KBr, νmax, cm-1):
3340 (N-H, str.), 3070 (Ar C-H str.), 1593 (C=N str.), 1510
(N=O str.), 1255 (C-O str.), 765 (N-H bending). 1H NMR (400
MHz, CDCl3, δ, ppm) 2.77-2.82 (dd, 1H, CH2, J = 17.3, 6.3
Hz), 3.03-3.07 (dd, 1H, CH2, J = 16.9, 7.4 Hz), 5.12-5.17 (dd,
1H, CH, J = 12.6, 6.2 Hz), 7.44-8.04 (m, 9H, Ar-H), 9.52 (s,
1H, NH).
PHYSICAL PARAMETERS OF ISOXAZOLINES
Yield
(%)
m.p.
(°C)
111-113
115-116
90-91
94-95
–
106-108
97-99
116-118
Compd.
m.f.
Colour
White
Rf
2a
2b
2c
2d
2e
2f
C15H14N2O
50
68
70
66
71
70
58
63
0.61
0.59
0.58
0.56
0.54
0.55
0.54
0.65
C15H13N2OCl Off white
C15H13N2OCl Yellow
C15H13N2OF
C15H14N2O2
C15H14N2O2
C15H15N3O3
C15H13N3O3
Yellow
Brown
Light brown
Orange
Yellow
2g
2h
5-(4-Nitrophenyl)-N-phenyl-4,5-dihydro isoxazol-3-
Testing of antifungal activity: Synthesized isoxazolines
(2a-2h) were evaluated for their in vitro antifungal activity
against D. maydis and R. solani by poisoned food technique
[16]. The per cent inhibition and ED50 of synthesized compounds
against both the test fungi was determined.
amine (2h): Yield 63 %, m.p.: 116-118 °C. IR (KBr, νmax
,
cm-1): 3380 (N-H, str.), 3028 (Ar C-H stretching), 1596 (C=N
str.), 1550 (N=O str.), 1253 (C-O str.), 785 (N-H bending). 1H
NMR (400 MHz, CDCl3, δ, ppm) 3.77-3.81 (dd, 1H, CH2, J =
17.1, 6.3 Hz), 4.15-4.19 (dd, 1H, CH2, J = 16.5, 7.4 Hz), 5.04-
5.08 (dd, 1H, CH, J = 16.6, 6.2 Hz), 7.68-8.24 (m, 9H, Ar-H),
10.52 (s, 1H, NH).
N-5-Diphenyl-4,5-dihydro isoxazol-3-amine (2a):Yield
50 %, m.p.: 111-113 °C. IR (KBr, νmax, cm-1): 3360 (N-H str.),
3038 (Ar C-H str.), 1597 (C=N str.), 1263 (C-O str.), 757 (N-
1
H bending). H NMR (400 MHz, CDCl3, δ, ppm) 2.57-2.64
RESULTS AND DISCUSSION
(dd, 1H, CH2, J = 18.3, 4.3 Hz), 3.33-3.40 (dd, 1H, CH2, J =
18.5, 11.4 Hz), 4.22-4.26 (dd, 1H,CH, J = 11.6, 4.4 Hz), 7.49-
7.89 (m, 10H, Ar-H), 10.25 (s, 1H, NH).
Isoxazolines (2a-2h) were prepared by reaction of synthe-
sized acrylamides with hydroxylamine hydrochloride using
microwave method. The reactants were irradiated for 3-4 min
to form the isoxazolines as shown in Fig. 1. Synthesis of
isoxazolines was confirmed by their characteristics peaks in
IR and 1H NMR spectra. Three double doublets in the 1H NMR
spectra confirmed the formation isoxazoline ring system. IR
spectral data provided further confirmation to the formation
of isoxazoline ring. The physical parameters (colour, yield,
melting point, Rf values) of synthesized compounds were
determined and are presented in Table-1.
5-(2-Chlorophenyl)-N-phenyl-4,5-dihydro isoxazol-3-
amine (2b): Yield 68 %, m.p.: 115-116 °C. IR (KBr, νmax
,
cm-1): 3320 (N-H str.), 3020 (Ar C-H str.), 1585 (C=N str.),
1260 (C-O str.), 1025 (C-Cl str.), 760 (N-H bending). 1H NMR
(400 MHz, CDCl3, δ, ppm) 2.57-2.63 (dd, 1H, CH2, J = 16.3,
5.3 Hz), 3.38-3.44 (dd, 1H, CH2, J = 16.3, 11.4 Hz), 5.23-
5.27 (dd, 1H, CH, J = 11.6, 4.4 Hz), 7.19-7.89 (m, 9H, Ar-H),
10.82 (s, 1H, NH).
5-(4-Chlorophenyl)-N-phenyl-4,5-dihydro isoxazol-3-
amine (2c): Yield 70 %, m.p.: 90-91 °C. IR (KBr, νmax, cm-1):
3350 (N-H str.), 3056 (Ar C-H str.), 1605 (C=N str.), 1256
Infrared: Infrared spectral data revealed the formation
of isoxazolines. N-H, C=N and C-O stretching vibrations were

Vol. 28, No. 10 (2016)
Green Synthesis of Isoxazoline Derivatives Using Microwave Irradiation and Their Antifungal Activity 2171
O
O
CHO
NH
H
R
NH
C
HN
C
NH2OH.HCl
NaOH
O
CH3
+
H
N
R
R
1a-1h
2a-2h
e
f
g
R = 2-OH
a
b
c
d
R = H
R = 2-Cl
R = 4-Cl
R = 2-F
R = 4-OH
R = 2-NO2
R = 4-NO2
h
Fig. 1. Scheme for the preparation of acrylamide and isoxazolines
TABLE-2
EFFECT OF DIFFERENT ISOXAZOLINES AT DIFFERENT
CONCENTRATIONS (µg/mL) ON THE GROWTH OF D. maydis
also observed in range of 3440-3320, 1605-1588 and 1268-
1239 cm-1, respectively. The characteristic >C=O stretching
vibration of acrylamides was absent in IR spectra of isoxazoline
which again assured the ring formation. Band for O-H stret-
ching was observed for compounds 2e and 2f having hydroxy
group on aromatic ring.
Inhibition (%)
Compd.
1000
26.2
500
250
100
6.55
50
3.27
10.56
17.31
19.67
9.83
4.91
6.55
9.83
48.56
0.003
2a
2b
2c
2d
2e
2f
2g
18.03
40.56
39.34
35.63
26.22
26.22
28.65
29.36
76.23
0.005
9.83
51.36
54.26
45.96
29.5
31.14
34.76
35.65
85.97
0.007
30.25
29.50
29.5
21.31
19.67
20.85
24.23
68.65
0.006
20.56
22.95
22.95
14.75
11.47
13.11
17.53
60.63
NS
1H NMR: The formation of isoxazolines was further con-
firmed by 1H NMR spectrum. The two hydrogens on C-3 of
isoxazoline ring (Fig. 2) were not equiva-lent. Both of these
protons were observed as double doublets in range of 2.57-
3.83 and 3.03-4.20 ppm. Proton at C-4 of isoxazoline also
appeared as double doublet in the range of 4.22-5.27 ppm.
Aromatic protons appeared as multiplet in range of 7.19-8.29
ppm.A singlet corresponding to -NH group was obtained in the
range of 9.55-10.82 ppm. More confir-mation to the formation
of isoxazolines was provided by coupling constant values of
double doublets.
2h
Bavistin
CD (p = 0.05)
inhibition at all the test concentration than all other isoxazo-
lines.
ED50 of only two compounds were found to be less than
1000 mg/mL against D. maydisas shown in Table-3. Isoxazo-
lines (2b) and (2c) having chloro group at ortho and para
position on benzene ring showed better control as compared
to other tested compounds. ED50 of (2b) and (2c) were 945
and 863 mg/mL, respectively while all other compounds had
ED50 value more than 1000 mg/mL.
H
3
N
2
R
4
N
O
1
5
R = H, Cl, F, OH, NO2
Fig. 2. Structure of isoxazoline ring
TABLE-3
ED50 OF DIFFERENT ISOXAZOLINES AGAINST D. maydis
Antifungal activity: Isoxazolines (2a-2h) were screened
in vitro for their antifungal activity against Drechslera maydis
and Rhizoctonia solani by poisoned food technique. The results
are expressed in ED50 values i.e. the effective dose at which
50 % inhibition has occurred. All the compounds showed
moderate antifungal potential against test fungi.
Compd.
ED50 (µg/mL)
Compd.
ED50 (µg/mL)
2a
2b
2c
2d
2e
–
945
863
–
2f
2g
2h
Bavistin
–
–
–
–
60
–
–
Antifungal activity of isoxazolines against D. maydis:
The data presented in Table-2 given below exhibited that all
the isoxazolines were active against D. maydis at 50 µg/mL
with inhibition ranging from 3.27 to 19.67 % but less than
bavistin (48.56 %). Isoxazoline (2c) having chloro group at
para position of benzene ring showed maximum activity i.e.
54.26 % inhibition at 1000 µg/mL which was significantly
higher than other tested compounds. Isoxazoline having no
substitution on benzene ring exhibited lowest per cent inhibi-
tion at all the concentrations. It was followed by compounds
having hydroxyl group at ortho and para position of benzene
ring, respectively. Bavistin recorded to have highest per cent
Antifungal activity of isoxazolines against R. solani:
The data presented in Table-4 exhibited that all the compounds
except compound having hydroxyl group at ortho position
were active against R. solani at 100 µg/mL with inhibition
ranging from 1.63 to 31.14 %. Compound having chloro group
at para position of benzene ring showed maximum inhibition
percentage of 86.80 at 1000 µg/mL, which was significantly
higher than other tested compounds. Isoxazolines were found
to be moderately effective against R. solani but none of the
compound was found effective as compared to the standard
fungicide bavistin (ED50 5 mg/mL).

2172 Goyal et al.
Asian J. Chem.
line in 3-4 min. Chloro substituted isoxazolines were found to
be more effective than other compounds. Further para chloro
substituted isoxazoline was observed to have more activity
than ortho chloro substituted compound. But all the compounds
showed less antifungal activity as compared to standard
bavistin.
TABLE-4
EFFECT OF DIFFERENT ISOXAZOLINES AT DIFFERENT
CONCENTRATIONS (µg/mL) ON THE GROWTH OF R. solani
Inhibition (%)
Compd.
1000
27.86
68.85
86.8
63.93
13.27
19.83
54.09
59.01
100
500
250
13.27
34.42
45.90
40.98
7.63
100
11.63
11.47
24.59
31.14
0
1.63
19.65
21.31
100
50
0
1.63
14.75
21.31
0
2a
2b
2c
2d
2e
2f
2g
2h
Bavistin
18.19
57.37
68.85
55.73
11.63
14.91
34.42
40.98
100
ACKNOWLEDGEMENTS
The authors are thankful to Panjab University, Chandigarh,
India for providing IR and 1H NMR spectral data of synthesized
compounds.
8.27
0
22.95
29.50
100
16.39
18.03
100
0.003
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CD (p = 0.05)
0.003
0.004
0.005
NS
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Conclusion
Reaction of different acrylamides with hydroxylamine
hydrochloride under microwave irradiations yielded isoxazo-