Synthesis and biological evaluation of novel 2-(substituted isoxazol-4-yl)-5-arylamino-1,3,4-oxadiazoles

Article abstract of DOI:10.1007/s11164-013-1062-y

A series of 2-(5-methyl-3-(4-chloro/trifluoromethylphenyl)isoxazol-4-yl)-5- arylamino-1,3,4-oxadiazoles were synthesized from 4-chloro/trifluoromethyl benzaldehyde, ethyl acetoacetate, hydroxylamine hydrochloride, hydrazine hydrate, and aryl isocyanate by

Full text of DOI:10.1007/s11164-013-1062-y

Res Chem Intermed
DOI 10.1007/s11164-013-1062-y
Synthesis and biological evaluation of novel
2-(substituted isoxazol-4-yl)-5-arylamino-1,3,4-
oxadiazoles
•
Feng Liu Meiyan Wang Xinhuan Teng
•
•
•
Peizhi Zhang Lin Jiang
Received: 19 November 2012 / Accepted: 15 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A series of 2-(5-methyl-3-(4-chloro/trifluoromethylphenyl)isoxazol-4-
yl)-5-arylamino-1,3,4-oxadiazoles were synthesized from 4-chloro/trifluoromethyl
benzaldehyde, ethyl acetoacetate, hydroxylamine hydrochloride, hydrazine hydrate,
and aryl isocyanate by multi-step reactions. The structures of the target compounds
were elucidated by IR, 1H NMR, MS, and elemental analysis. All these compounds
were tested for in vitro antifungal activities against Botrytis cinerea and Rhizoctonia
cerealis by the mycelium growth rate method, and the results indicated that some
compounds displayed high antifungal activity against Botrytis cinerea.
Keywords 1,3,4-Oxadiazole ꢀ Isoxazole ꢀ Synthesis ꢀ Antifungal activity
Introduction
Oxadiazole derivatives are an important class of heterocyclic compounds due to
their remarkable biological and pharmacological properties, such as anti-inflam-
matory [1], anticancer [2], antiviral [3], antibacterial [4], antifungal [5], insecticidal
[6], and herbicidal [7] activities. For example, metoxadiazone, oxadiazon, and
oxadiargyl have been developed as agrochemicals. Therefore, the synthesis and
biological activity of oxadiazole derivatives are a research focus. On the other hand,
many isoxazole compounds display antiviral [8], antifungal [9], insecticidal [10],
and herbicidal [11] activities, and more than ten pesticides and drugs have been
developed, e.g., clomazone, sulfamethoxazole and oxacillin. The isoxazole
F. Liu ꢀ X. Teng
College of Plant Protection, Shandong Agricultural University, Tai’an 271018, China
M. Wang ꢀ P. Zhang ꢀ L. Jiang (&)
College of Chemistry and Material Science, Shandong Agricultural University,
Tai’an 271018, China
e-mail: jiangl@sdau.edu.cn
123

F. Liu et al.
derivatives have drawn much attention because of their wide applications in
pesticide and medicinal chemistry.
In view of the facts mentioned above, we here introduced a substituted isoxazole
moiety to the oxadiazole ring, synthesized a series of novel 2-(5-methyl-3-(4-
chlorophenyl)isoxazol-4-yl)-5-arylamino-1,3,4-oxadiazoles (4a–d) and 2-(5-methyl-
3-(4-trifluoromethylphenyl)isoxazol-4-yl)-5-arylamino-1,3,4-oxadiazoles (4e–h), and
also evaluated their in vitro antifungal activity. The synthetic route is shown in
Scheme 1.
Experimental
General
Melting points were measured on a Tech X-5 microscopic melting point apparatus
and are uncorrected. IR spectra were recorded on a Shimadzu IR-440 infrared
spectrophotometer using KBr disks. 1H NMR spectra were determined on a Bruker
AM-400 spectrometer with CDCl3 as solvent and TMS as internal standard,
Chemical shifts are reported in d (parts per million) values. ESI–MS spectra were
acquired on an Agilent 1200 spectrometer. The elemental analysis was measured by
an Elementary Vario EL III analyzer.
Chemical reagents were purchased from J&K Chemical or Aladdin Reagent, and
are of analytical grade.
NH H.H
2O
l
C
N
CS
R1
R1
H
C O
H
C
N
O
H
R1
N H
O
C
C
a
N
a .
q
H
DMF
O
(
)
l
NHNH2
O
H5
OC2
O
O
O
NH2NH2 .H
CH3
2O
H
C
O
3
R1
R1
H
H
C2 5O
O
O
N
N
-
-
2 I 2 II
,
-
-
1 I 1 II
,
R2
S
H
N
O
R2
N
N
H
N
C
N
S
c
A
O
H
H
g
(
)
2
H
C
3
R1
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N
a~
3
3h
R1
N
H
1
R
l
;
R1
=
F
C
,
3
:
:
R1
R1
R1
R1
-
-
=
=
-
-
=
N
1 I 2 I
1 II 2 II
,
,
C
1
R
l R2
H
l R2
H
OC
3
O
a
a:
4
:
3b 4b
,
:
3d 4d
,
:
3f 4f
,
:
3h 4h
,
=
=
=
3
,
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C
=
C
C
,
;
,
1
1
1
R
R
:
R
g
l R2
l
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;
l R2
B
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e
c:
=
=
=
=
r
3
3
3
4
4
4
,
,
,
C
C
C
,
,
N
R2
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,
3
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,
3
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e:
=
=
=
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;
l
F
,
3
R2
a~
4
4h
=
=
= r
B
F
C
3
C
;
,
g
Scheme 1 Synthetic route for the target compounds 4a–4h
123

Synthesis and biological evaluation
Synthesis
The key intermediates, (3-(4-chlorophenyl)-5-methyl)isoxazole-4-caroxylic acid
ethyl ester (1-I) and (3-(4-trifluoromethylphenyl)-5-methyl)isoxazole-4-carboxylic
acid ethyl ester (1-II), were synthesized from 4-chloro/trifluoromethyl benzaldehyde,
hydroxylamine hydrochloride, N-chlorosuccinimide (NCS), and ethyl acetoacetate by
three steps [12]. Compounds (3-(4-chlorophenyl)-5-methyl)isoxazole-4-carbohydra-
zide (2-I) and (3-(4-trifluoromethylphenyl)-5-methyl)isoxazole-4-carbohydrazide
(2-II) were prepared by the reaction of 1-I (or 1-II) with 80 % hydrazine hydrate [13].
General procedure for the synthesis of 2-(5-methyl-3-(4-substituted
phenyl)isoxazol-4-yl)-5-arylamino-1,3,4-oxadiazoles (4)
To a mixture of the substituted isoxazole-4-carbohydrazide 2-I or 2-II (5 mmol) in
ethanol (50 mL) was added aryl isothiocyanate (5 mmol) at room temperature, and
then the reaction mixture was refluxed with stirring for 3–4 h. Upon disappearance
of the starting materials as monitored by thin layer chromatography, the reaction
was cooled. The solid was filtered and recrystallized from 95 % ethanol to afford
5-methyl-3-(4-substituted phenyl)-isoxazole-4-carbonyl thiosemicarbazide 3 in
yields of 69–90 %.
Mercuric acetate (0.64 g, 2 mmol) was added to a mixture of the thiosemicar-
bazide 3 (2 mmol) in ethanol (50 mL). The reaction mixture was refluxed for 3 h,
and then concentrated under reduced pressure [14]. The residue was dissolved in hot
N,N-dimethylformamide (DMF) and filtered. The filtrate was removed the solvent
under reduced pressure, and the obtained solid was recrystallized from ethanol/DMF
to give the target compound 4.
2-(5-Methyl-3-(4-chlorophenyl)isoxazol-4-yl)-5-phenylamino-1,3,4-oxdiazole (4a)
Yield: 68 %, m.p. 235–237 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.78 (s, 3H, CH3),
6.97–7.54 (m, 5H, Ar–H), 7.50–7.52 (d, J = 8.4 Hz, 2H, Ar–H), 7.71–7.73 (d,
J = 8.4 Hz, 2H, Ar–H), 10.38 (s, 1H, NH); IR (KBr): m (cm-1) 3,174 (N–H), 1,635
(C=N), 1,051 (C–O–C); Anal. calcd. for C18H13ClN4O2: C 61.29, H 3.72, N 15.88;
found: C 61.40, H 3.35, N 15.74; ESI–MS m/z calcd. for C18H13ClN4O2 (M?H)?
353.7, obsd. 353.3.
2-(5-Methyl-3-(4-chlorophenyl)isoxazol-4-yl)-5-(4-methoxyphenylamino)-1,3,4-
oxdiazole (4b)
Yield: 54 %, m.p. 246–247 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.77 (s, 3H, CH3),
3.75 (s, 3H, OCH3), 6.83–6.85 (d, J = 8.8 Hz, 2H, Ar–H), 7.40–7.43 (d,
J = 8.8 Hz, 2H, Ar–H), 7.52–7.54 (d, J = 8.4 Hz, 2H, Ar–H), 7.71–7.73 (d,
J = 8.4 Hz, 2H, Ar–H), 10.23 (s, 1H, NH); IR (KBr): m (cm-1) 3,174 (N–H), 1,634
(C=N), 1,051 (C–O–C); Anal. calcd. for C19H15ClN4O3: C 59.62, H 3.95, N 14.63;
found: C 59.32, H 4.00, N 14.52; ESI–MS m/z calcd. for C19H15ClN4O3 (M?H)?
383.7, obsd. 383.3.
123

F. Liu et al.
2-(5-Methyl-3-(4-chlorophenyl)isoxazol-4-yl)-5-(4-chlorophenylamino)-1,3,4-
oxdiazole (4c)
Yield: 87 %, m.p. 236–238 °C; 1HNMR (DMSO-d6, 400 Hz): d 2.77 (s, 3H, CH3),
7.27–7.29 (d, J = 8.8 Hz, 2H, Ar–H), 7.52–7.55 (m, 4H, Ar–H), 7.71–7.73 (d,
J = 8.8 Hz, 2H, Ar–H), 10.58 (s, 1H, NH); IR (KBr): m (cm-1) 3,170 (N–H), 1,632
(C=N), 1,050 (C–O–C); Anal. calcd. for C18H12Cl2N4O2: C 55.83, H 3.12, N 14.46;
found: C 55.60, H 3.28, N 14.85; ESI–MS m/z calcd. for C18H12Cl2N4O2 (M?H)?
388.2, obsd. 388.4.
2-(5-Methyl-3-(4-chlorophenyl)isoxazol-4-yl)-5-(4-bromophenylamino)-1,3,4-
oxdiazole (4d)
Yield: 76 %, m.p. 229–231 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.78 (s, 3H, CH3),
7.39–7.42 (d, J = 8.8 Hz, 2H, Ar–H), 7.48–7.50 (d, J = 8.8 Hz, 2H, Ar–H),
7.52–7.54 (d, J = 8.4 Hz, 2H, Ar–H), 7.71–7.73 (d, J = 8.4 Hz, 2H, Ar–H), 10.59
(s, 1H, NH); IR (KBr): m (cm-1) 3,174 (N–H), 1,632 (C=N), 1,050 (C–O–C); Anal.
calcd. for C18H12BrClN4O2: C 50.08, H 2.80, N 12.97; found: C 50.45, H 2.59, N
12.68; ESI–MS m/z calcd. for C18H12BrClN4O2 (M?H)? 432.6, obsd. 432.9.
2-(5-Methyl-3-(4-trifluoromethylphenyl)isoxazol-4-yl)-5-phenylamino-1,3,4-
oxdiazole (4e)
Yield: 79 %, m.p. 237–239 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.80 (s, 3H, CH3),
6.97–7.52 (m, 5H, Ar–H), 7.82–7.84 (d, J = 8.4 Hz, 2H, Ar–H), 7.94–7.96 (d,
J = 8.4 Hz, 2H, Ar–H), 10.45 (s, 1H, NH); IR (KBr): m (cm-1) 3,178 (N–H), 1,634
(C=N), 1,051 (C–O–C); Anal. calcd. for C19H13F3N4O2: C 59.08, H 3.39, N 14.50;
found: C 59.55, H 3.03, N 14.99; ESI–MS m/z calcd. for C19H13F3N4O2 (M?H)?
387.2, obsd. 387.3.
2-(5-Methyl-3-(4-trifluoromethylphenyl)isoxazol-4-yl)-5-(4-methoxyphenylamino)-
1,3,4-oxdiazole (4f)
Yield: 91 %, m.p. 258–260 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.79 (s, 3H, CH3),
3.75 (s, 3H, OCH3), 6.82–6.84 (d, J = 8.8 Hz, 2H, Ar–H), 7.40–7.43 (d,
J = 8.8 Hz, 2H, Ar–H), 7.81–7.83 (d, J = 8.4 Hz, 2H, Ar–H), 7.93–7.95 (d,
J = 8.4 Hz, 2H, Ar–H), 10.20 (s, H, NH); IR (KBr): m (cm-1) 3,177 (N–H), 1,629
(C=N), 1,050 (C–O–C); Anal. calcd. for C20H15F3N4O3: C 57.70, H 3.63, N 13.45;
found: C 57.25, H 3.35, N 13.51; ESI–MS m/z calcd. for C20H15F3N4O3 (M?H)?
417.3, obsd. 417.4.
2-(5-Methyl-3-(4-trifluoromethylphenyl)isoxazol-4-yl)-5-(4-chlorophenylamino)-
1,3,4-oxdiazole (4g)
Yield: 80 %, m.p. 241–243 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.80 (s, 3H, CH3),
7.25–7.27 (d, J = 8.8 Hz, 2H, Ar–H), 7.53–7.55 (d, J = 8.8 Hz, 2H, Ar–H),
123

Synthesis and biological evaluation
7.80–7.82 (d, J = 8.4 Hz, 2H, Ar–H), 7.93–7.95 (d, J = 8.4 Hz, 2H, Ar–H), 10.56
(s, H, NH); IR (KBr): m (cm-1) 3,170 (N–H), 1,627 (C=N), 1,049 (C–O–C); Anal.
calcd. for C19H12ClF3N4O2: C 54.24, H 2.88, N 13.31; found: C 54.75, H 2.99, N
13.07; ESI–MS m/z calcd. for C19H12ClF3N4O2 (M?H)? 421.7, obsd. 421.3.
2-(5-Methyl-3-(4-trifluoromethylphenyl)isoxazol-4-yl)-5-(4-bromophenylamino)-
1,3,4-oxdiazole (4h)
Yield: 89 %, m.p. 240–241 °C; 1H NMR (DMSO-d6, 400 Hz): d 2.80 (s, 3H, CH3),
7.26–7.28 (d, J = 8.8 Hz, 2H, Ar–H), 7.49–7.52 (d, J = 8.8 Hz, 2H, Ar–H),
7.84–7.86 (d, J = 8.4 Hz, 2H, Ar–H), 7.92–7.94 (d, J = 8.8 Hz, 2H, Ar–H), 10.58
(s, 1H, NH); IR (KBr): m (cm-1) 3,160 (N–H), 1,628 (C=N), 1,050 (C–O–C); Anal.
calcd. for C19H12BrF3N4O2: C 49.05, H 2.60, N 12.04; found: C 49.73, H 2.25, N
12.41; ESI–MS m/z calcd. for C19H12BrF3N4O2 (M?H)? 466.2, obsd. 466.0.
Bioassays of antifungal activity
The in vitro fungicidal activities of the target compounds 4a–h against Botrytis
cinerea and Rhizoctonia cerealis were evaluated using the mycelium growth rate
method [15, 16]. The culture media, with known concentrations (10, 50, 100, and
200 lg/mL) of the test compounds, were obtained by mixing the water suspension
(1 mL) of 4 with potato dextrose agar (PDA, 9 mL) at 50 °C. The medium was
poured onto 9-cm Petri dishes. When the medium solidified, it was inoculated onto a
5-mm-diameter agar disc which was grown with mycelium and cultivated for
3 days. The discs were placed in the center of the dish and in a light incubator at
25 1 °C for 48–96 h, and then assessed by measurement of the colony diameter.
A commercial fungicide carbendazim was used as the positive control, and sterile
water was used as the blank. Three replications were used per treatment. The
inhibition rate was expressed as the mean of values obtained in three independent
experiments. Effective concentrations (EC50) that inhibited mycelium growth by
50 % were obtained using log-probit analysis (Table 1). The inhibition rate was
calculated according to the formula:
Inhibition rate (%Þ ¼ ½ðD0 ꢁ D1Þ= D0ꢂ ꢃ 100
where D0 is the expansion diameter of mycelia in the blank test, and D1 is the
expansion diameter of mycelia in the presence of tested compounds.
Results and discussion
Spectra
The signals of the CH3 protons connected to the isoxazole ring were observed in the
1H NMR spectra of target compounds at d 2.77–2.80 ppm, and singlet signals at d
10.20–10.59 ppm were assigned to NH proton. ArH protons appeared at d
6.82–7.95 ppm. Moreover, owing to the strong electron-withdrawing effect of CF3,
123

F. Liu et al.
Table 1 Antifungal activity for
the target compounds 4a–h
Compds
R1
R2
EC50, (lg/mL)*
B. cinerea pers
R. cerealis
4a
Cl
H
46.36 1.22
18.15 1.07
48.63 1.28
24.88 1.15
40.42 1.38
14.03 1.05
16.89 1.13
36.69 1.19
48.68 1.21
47.02 1.16
58.46 1.14
80.05 1.46
61.48 1.30
37.39 1.17
54.44 1.22
61.70 1.34
54.53 1.25
1.52 0.26
4b
Cl
OCH3
Cl
4c
Cl
4d
Cl
Br
4e
CF3
CF3
CF3
CF3
H
4f
OCH3
Cl
4g
4h
Br
*Antifungal activity was
expressed as EC50
Carbendazim
both the two doublet signals of the 4-trifluoromethylphenyl protons moved
downfield (4e–h, d 7.80–7.86 and 7.92–7.96 ppm, respectively). In their IR spectra,
the N–H stretching vibrations were observed at 3,160–3,178 cm-1 and the C=N
absorption bands of the heterocycles were near 1,630 cm-1. The bands around
1,050 cm-1 were attributed to the C–O–C absorption of the oxadiazole ring.
Antifungal activity
As shown in Table 1, the EC50 values of the target compounds ranged from 14.03 to
48.63 lg/mL against Botrytis cinerea, near to or less than that of carbendazim
(EC50 = 48.68 lg/mL), suggesting moderate to high antifungal activities. Among
them, 4b, 4f and 4g displayed high activities due to their low EC50 values of less
than 20 lg/mL. However, compared with carbendazim, their EC50 values against
Rhizoctonia cerealis were quite large (37.39–80.05 lg/mL), which means that the
target compounds displayed low activities against this fungus.
Conclusion
In summary, we have synthesized four 2-(5-methyl-3-(4-chlorophenyl)isoxazol-4-yl)-5-
arylamino-1,3,4-oxadiazolesandfour2-(5-methyl-3-(4-trifluoromethylphenyl)isoxazol-
4-yl)-5-arylamino-1,3,4-oxadiazoles by multi-step reactions, which were elucidated by
IR, 1H NMR, MS, and elemental analysis. All these compounds were tested for in vitro
antifungal activities against Botrytis cinerea and Rhizoctonia cerealis by the mycelium
growth rate method, and the results indicated that three compounds displayed high
antifungal activity against Botrytis cinerea, with EC50 values of less than 20 lg/mL.
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