Design, Synthesis and Antifungal Evaluation of N-Substituted-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide Derivatives

Article abstract of DOI:10.1002/jhet.3400

A series of 1-(3-chloropyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives which have di-substituents on nitrogen were designed and synthesized. Bioassay results showed that all the synthetic compounds exhibited lower antifungal activi

Full text of DOI:10.1002/jhet.3400

Month 2018
Design, Synthesis and Antifungal Evaluation of N-Substituted-1-(3-
chloropyridin-2-yl)-N-(pyridin-4-yl)-5-(trifluoromethyl)-1H-pyrazole-4-
carboxamide Derivatives
a
Zhibing Wu,
Guangqian Yang,b Xin Zhao,b Jiangchun Wu,b and Shixi Wua
*
aState Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green
Pesticide and Agricultural Bioengineering, Ministry of Education, Center for R&D of Fine Chemicals of Guizhou
University, Guiyang 550025, China
bSchool of Chemistry and Chemical Engineering, Guizhou University, Guiyang 550025, China
*E-mail: wzb1171@163.com
Received August 10, 2018
DOI 10.1002/jhet.3400
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
A series of 1-(3-chloropyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide derivatives which
have di-substituents on nitrogen were designed and synthesized. Bioassay results showed that all the syn-
thetic compounds exhibited lower antifungal activities against Gibberella zeae, Cytospora mandshurica,
and Fusarium oxysporum than T3 (14.7, 21.1, and 32.7 μg/mL), but some of them exhibited better activities
against Botrytis cinerea, Phytophthora infestans, and Sclerotinia sclerotiorum than T3 (>200, >200, and
>200 μg/mL); the EC50 values of 7d and 7c against B. cinerea were 94.9 and 56.2 μg/mL, respectively.
The EC50 values of 7a, 7d, and 7c against S. sclerotiorum were 73.5, 78.7, and 68.5 μg/mL, respectively.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
chloropyridin-2-yl)-5-(trifluoromethyl)-1H-pyrazole-4-
carboxamide derivatives which have di-substituents on
nitrogen were designed and synthesized (Fig. 1). Bioassay
results showed that all the title compounds exhibited lower
antifungal activities against G. zeae, C. mandshurica, and
F. oxysporum, but some of them exhibited better activities
against Botrytis cinerea, Phytophthora infestans, and
Sclerotinia sclerotiorum than T3.
Pyrazole carboxamide compounds have been used as
fungicides in agro-chemistry for years and exhibited
good activity against many important diseases, such as
leaf spots, mildews, molds, and rusts [1–6]. As reported,
most pyrazole carboxamide fungicides inhibit fungal
respiration by binding to the ubiquinone-binding site
(Q-site) of the mitochondrial succinate dehydrogenase
complex II in the electron transport chain, which is a
functional part of the tricarboxylic acid cycle [7–9]. The
structural analysis revealed that amide linker (CONH),
which is between pyazole ring and amino part, is very
important for the activity. Most substituents groups
which replaced amide linker did not keep the activity
except thioamide (CSNH) [10].
RESULTS AND DISCUSSION
Chemistry. Title compounds 7a–7e were synthesized
in existing literature [13–15] as shown in Scheme 1.
Intermediates 2a–2e were synthesized by pyridin-4-amine
and corresponding aldehydes under nitrogen and
hydrogenated by NaBH4. Intermediate 4 was synthesized
In our previous work [11,12], a series of N-pyridinyl-
5-(trifluoromethyl)-pyrazole-4-carboxamide
were designed and synthesized. Among of them,
compound 1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (T3)
exhibited good activities against plant pathogenic fungi
Gibberella zeae, Cytospora mandshurica, and Fusarium
oxysporum, and the EC50 values were 14.7, 21.1, and
32.7 μg/mL, respectively. For further study on the role of
amide bond played in N-pyridinyl-5-trifluoromethyl
pyrazole-4-carboxamide derivatives, a series of 1-(3-
derivatives
by cyclization reaction from
1
with 3-chloro-2-
hydrazinylpyridine (3) in the ethanol. Intermediate 5 was
obtained from 4 through hydrolysis in the presence of a
weak-base lithium hydroxide. Intermediate 5 was then
refluxed in SOCl2 to obtain pyrazole acyl chloride (6).
Target compounds 7a–7e were obtained by the reaction
of 6 with secondary amines 2a–2e with yields ranging
from 43–58% in anhydrous tetrahydrofuran by NaH.
All title compounds were identified by H-NMR, 13C-
NMR, MS, and IR spectra. Elemental analyses were also
1
© 2018 Wiley Periodicals, Inc.

Z. Wu, G. Yang, X. Zhao, J. Wu, and S. Wu
Vol 000
F. oxysporum reduced obviously at 100 μg/mL. Even so,
the antifungal activities against B. cinerea, P. infestans,
and S. sclerotiorum were enhanced effectively, such as
7b against S. sclerotiorum, 7c against P. infestans, and 7d
and 7e against B. cinereal and S. sclerotiorum. The EC50
values of 7b against S. sclerotiorum and 7c against
P. infestans were 73.5 and 41.5 μg/mL, respectively. And
the EC50 values of 7d and 7e against B. cinereal and
S. sclerotiorum were 94.9, 78.7, and 56.2, 68.5 μg/mL,
respectively, better than T3 (>200, >200, and
>200 μg/mL).
Analysis of preliminary structure–activity relationships
of title compounds 7a–7e showed that antifungal activity
against G. zeae, C. mandshurica, and F. oxysporum
obviously reduced when hydrogen of amide bond was
substituted by alkyl and pyridinyl (Table 2). The title
compounds exhibited better activity against B. cinerea
and S. sclerotiorum than T3 when isoamyl and benzyl
were introduced on -NH- position of amide bond, such as
7d and 7e, the steric effect group which introduced in the
-NH- bond may enhanced the activity against B. cinerea
and S. sclerotiorum but reduced the activity against
G. zeae, C. mandshurica, and F. oxysporum.
Figure 1. Design of the title compounds. [Color figure can be viewed at
wileyonlinelibrary.com]
consistent with the corresponding calculated values. In 1H-
NMR, -NH- proton of the secondary amine 2a–2e showed
up at δ 3.62–5.73, and the pyrazole ring proton showed up
at δ 7.31–7.90 for title compounds. In the IR spectra of the
compounds, the stretching vibration absorption peaks of
C=O appeared at 1655–1705 cmÀ1
.
Bioactivities. Preliminary antifungal activity of the title
compounds against six kinds of plant phytogenic fungi
(G. zeae, C. mandshurica, F. oxysporum, B. cinerea,
P. infestans, and S. sclerotiorum) is shown in Table 1,
and previous active compound T3 was used as the
control. For title compounds 7a, 7b, 7d, and 7e, the
antifungal activities against G. zeae, C. mandshurica, and
Scheme 1. Synthetic route of the target compounds.
Table 1
Inhibition effect of title compounds against six plant phytopathogenic fungi at 100 μg/mL.
Inhibition (%)
No.
G. zeae
C. mandshurica
F. oxysporum
B. cinerea
P. infestans
S. sclerotiorum
7a
7b
7c
7d
7e
T3
12.7 1.6
12.0 1.5
85.7 5.3
15.3 1.7
32.2 1.4
88.0 0.6
14.5 1.8
11.1 1.8
67.6 3.4
6.5 1.9
12.0 1.6
77.5 0.8
24.2 3.0
16.1 1.7
66.5 2.8
20.0 1.6
18.2 1.8
80.0 0.9
11.5 1.2
23.0 1.2
9.7 1.9
53.0 1.7
70.3 2.9
12.0 1.2
14.2 2.9
16.1 2.8
78.7 0.9
22.3 1.2
19.6 1.5
12.6 3.0
28.7 2.5
63.2 1.4
20.2 2.7
62.6 1.4
59.1 2.2
12.6 3.0
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis and Antifungal Evaluation of N-Substituted-1-(3-chloropyridin-2-
yl)-N-(pyridin-4-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide Derivatives
Table 2
The EC50 values of compounds 7a–7e against six kinds of pathogenic fungi.
EC50 (μg/mL)
Compound no.
G. zeae
C. mandshurica
F. oxysporum
B. cinerea
P. infestans
S. sclerotiorum
7a
7b
7c
7d
7e
T3
>200
>200
45.7 1.9
>200
163.5 3.4
14.7 1.4
>200
>200
73.5 1.6
>200
>200
21.1 2.7
>200
>200
69.2 1.3
>200
>200
32.7 3.1
>200
>200
>200
94.9 3.2
56.2 2.2
>200
>200
>200
41.5 3.1
>200
>200
>200
>200
73.5 1.6
>200
78.7 1.7
68.5 1.1
>200
CONCLUSIONS
slowly, and the mixture was stirred at room temperature
for 12 h. Afterward, the mixture was added to ice water
(30 mL) and extracted by ethyl acetate (20 mL); the
organic layer was dried with anhydrous sodium sulfate
and filtered; the solvent was then removed under vacuum.
The crude residue was further purified by flash column
chromatography (DCM/MeOH = 30/1) on a silica gel to
obtain the secondary amine 2a–2e.
N-propylpyridin-4-amine (2a): colorless oil, yield 59%.
1H-NMR (500 MHz, DMSO-d6) δ 7.95 (d, J = 6.3 Hz,
2H, pyridine H), 6.41 (d, J = 6.3 Hz, 2H, pyridine H),
4.12 (s, 1H, NH), 2.95 (t, J = 9.8 Hz, 2H, CH2), 1.58–
1.41 (m, 2H, CH2), 0.88 (t, J = 6.5 Hz, 3H, CH3). 13C-
NMR (125 MHz, DMSO-d6) δ 154.17, 149.85, 107.50,
43.94, 22.18, 12.05.
N-butylpyridin-4-amine (2b): colorless oil, yield 41%.
1H-NMR (500 MHz, DMSO-d6) δ 7.95 (d, J = 5.1 Hz,
1H, pyridine H), 6.41 (d, J = 5.1 Hz, 1H, pyridine H),
3.70 (s, 1H, NH), 2.98 (t, J = 9.3 Hz, 2H, CH2), 1.51–
1.42 (m, 2H, CH2), 1.39–1.24 (m, 2H, CH2), 0.87
(t, J = 6.8 Hz, 3H, CH3). 13C-NMR (125 MHz,
DMSO-d6) δ 154.18, 149.77, 107.47, 41.82, 31.02,
20.23, 14.27.
A series of N-substituted-1-(3-chloropyridin-2-yl)-N-
(pyridin-4-yl)-5-(trifluoromethyl)-1H-pyrazole-4-
carboxamide derivatives were designed and synthesized.
Bioassay results showed that when hydrogen of amide
bond was substituted by alkyl or benzyl, all the synthetic
compounds exhibited lower antifungal activities against
G. zeae, C. mandshurica, and F. oxysporum than T3
(14.7, 21.1, and 32.7 μg/mL), but some of them exhibited
better activities against B. cinerea, P. infestans, and
S. sclerotiorum than T3 (>200, >200, and 200 μg/mL).
The EC50 values of 7d and 7c against B. cinerea were
94.9 and 56.2 μg/mL, respectively. The EC50 values of
7a, 7d, and 7c against S. sclerotiorum were 73.5, 78.7,
and 68.5 μg/mL, respectively.
EXPERIMENTAL
Instruments. Melting points of the compounds were
determined on an XT-4 binocular microscope (Beijing
Tech Instrument Co., China) and were not corrected.
1H-NMR and 13C-NMR spectra were recorded with a
JEOL ECX 500 NMR spectrometer operated at 500
and 125 MHz, respectively, using DMSO-d6 or CD3Cl
as solvent and tetramethylsilane as an internal
standard. Chemical shifts were reported in parts per
million (ppm) down field from tetramethylsilane with
the solvent resonance as internal standard. Coupling
constants (J) were reported in Hz and referred to
apparent peak multiplications. Mass spectral studies
were conducted on an Agilent 5973 mass spectrometer.
Elemental analysis was performed using Vario-III
CHN analyzer.
N-isobutylpyridin-4-amine (2c): colorless oil, yield
50%. 1H-NMR (500 MHz, DMSO-d6)
δ
7.95
(d, J = 5.7 Hz, 2H, pyridine H), 6.44 (d, J = 5.7 Hz, 2H,
pyridine H), 3.62 (s, 1H, NH), 2.82 (t, J = 6.3 Hz, 2H,
CH2), 1.80–1.70 (m, 1H, CH), 0.87 (d, J = 6.7 Hz, 6H,
CH3). 13C-NMR (125 MHz, DMSO-d6) δ 154.5, 149.4,
107.5, 49.88, 27.9, 20.8.
N-isopentylpyridin-4-amine (2d): colorless oil, yield
63%. 1H-NMR (500 MHz, DMSO-d6)
δ
7.95
(d, J = 4.5 Hz, 2H, pyridine H), 6.41 (d, J = 4.5 Hz, 2H,
pyridine H), 3.60 (s, 1H, NH), 3.0 (dd, J = 12.0, 5.6 Hz,
2H, CH2), 1.70–1.53 (m, 2H, CH2), 1.43–1.30 (m, 1H,
CH), 0.86 (dd, J = 6.6, 2.2 Hz, 6H, CH3). 13C-NMR
(125 MHz, DMSO-d6) δ 154.1, 149.8, 107.5, 39.9, 37.8,
25.8, 22.9.
N-benzylpyridin-4-amine (2e): colorless oil, yield 79%.
1H-NMR (500 MHz, DMSO-d6) δ 7.96 (d, J = 3.6 Hz,
2H, pyridine H), 7.48–6.93 (m, 5H, benzene H), 6.46
General procedure for the preparation of intermediate 2a–
2e.
Pyridin-4-amine (1 g, 10.6 mmo1), corresponding
aldehyde (10.6 mmo1), and ethanol (15 mL) were added
into a 25-mL three-round bottom flask. The reaction
mixture was refluxed for 6 h under nitrogen. Then
NaBH4 (482.4 mg,12.8 mmol) was added at ice-bath
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Z. Wu, G. Yang, X. Zhao, J. Wu, and S. Wu
Vol 000
(d, J = 3.6 Hz, 2H, pyridine H), 5.73 (s, 1H, NH), 4.28 (s,
2H, CH2). 13C-NMR (125 MHz, DMSO-d6) δ 154.0,
149.9, 139.7, 128.9, 127.7, 127.4, 108.0, 45.8.
151.2, 149.8, 147.7, 147.1, 139.8, 139.6, 129.5, 126.8,
121.9, 120.1, 119.9, 49.5, 29.9, 20.1, 13.8. IR (KBr
cmÀ1): νmax 3398, 3065, 2948, 2913, 2872, 1655, 1584,
1559, 1481, 1438, 1420, 1394, 1381, 1290, 1234, 1064,
1050, 986, 694 cmÀ1. MS (ESI): m/z 424 [M + H]+.
Anal. Calcd for C19H17ClF3N5O: C, 53.85; H, 4.04; N,
16.52; Found: C, 54.14; H, 4.36; N, 16.71.
General procedure for the preparation of intermediate 3.
2, 3-Dichloropyridine (20.0 g, 135.1 μmol) and 80%
hydrazine hydrate (67.7 g, 1.4 mmol) were added into a
250-mL three-round bottom flask. The reaction mixture
was refluxed for 3 h, cooled to room temperature, and
filtered; the residue was washed by ethanol, dried, and
obtained 3, white solid, yield 93.0%, m.p. 161–162°C. 1H-
NMR (500 MHz, DMSO-d6) δ 8.04 (d, J = 5.1 Hz, 1H,
pyridine H), 7.61 (s, 1H, NH), 7.57 (d, J = 7.6 Hz,
pyridine H), 6.61 (dd, J = 5.1 Hz, J = 7.6 Hz, 1H, pyridine
H), 4.21 (s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6) δ:
156.3, 146.2, 136.7, 114.1, 113.7.
N-Isobutyl-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (7c).
White
1
solid, yield 57%, m.p. 202–203°C. H-NMR (500 MHz,
CDCl3) δ 8.72 (d, J = 4.7 Hz, 1H, pyridine H), 8.50
(d, J = 4.6 Hz, 2H, pyridine H), 8.16 (d, J = 8.0 Hz, 1H,
pyridine H), 7.96 (s, 1H, pyrazole H), 7.54 (dd, J = 7.0,
4.6 Hz, 1H, pyridine H), 7.26 (d, J = 4.8 Hz, 2H,
pyridine H), 3.54 (d, J = 13.9 Hz, 2H, CH2), 2.27–1.80
(m, 1H, CH), 1.25 (d, J = 14.6 Hz, 6H, 2CH3). 13C-NMR
(125 MHz, CDCl3) δ 159.3, 150.7, 148.0, 147.3, 145.0,
140.5, 139.8, 137.8, 129.3, 127.0, 123.5 120.0, 114.1,
57.6, 29.8, 21.5. IR (KBr cmÀ1): νmax 3479, 2945, 2919,
2830, 1696, 1627, 1528, 1480, 1419, 1383, 1363, 1330,
1300, 1246, 1061, 1035, 999, 880751 cmÀ1. MS (ESI):
m/z 424 [M + H]+. Anal. Calcd for C19H17ClF3N5O: C,
53.85; H, 4.04; N, 16.52; Found: C, 53.45; H, 4.46; N,
16.76.
General procedure for the preparation of the target
compounds 7a–7e.
Intermediates 2 (586.4 μmol), 6
(645.0 μmol), NaH (2.4 mmol), and anhydrous
tetrahydrofuran (5 mL) were added into a 25-mL three-
round bottom flask. The reaction mixture was stirred at
room temperature for 10 h. The organic solvent was
removed under reduced pressure. Afterward, the residue
was re-dissolved by ethyl acetate (20 mL). Finally, the
organic layer was washed by saturated salt water, dried
with anhydrous sodium sulfate, and filtered; the solvent
was then removed under vacuum. The crude residue was
further purified by flash column chromatography (PE/
N-Isopentyl-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (7d).
White
1
solid, yield 49%, m.p. 132–133°C. H-NMR (500 MHz,
CDCl3) δ 8.55 (d, J = 8.2 Hz, 1H, pyridine H), 8.47
(d, J = 4.6 Hz, 2H, pyridine H), 7.90 (s, 1H, pyrazole H),
7.45 (dd, J = 7.8, 5.0 Hz, 1H, pyridine H), 7.39
(d, J = 8.1, 1H, pyridine H), 7.04 (d, J = 7.2 Hz, 2H,
pyridine H), 3.95 (m, 2H, CH2) 2.18–1.11 (m, 3H, CH2
and CH), 0.90 (d, J = 12.8 Hz, 6H, 2CH3). 13C-NMR
(125 MHz, CDCl3) δ 161.3, 151.2, 149.8, 147.7, 147.2,
139.8, 139.6, 130.6, 129.5, 126.8, 121.9, 120.1, 120.0,
119.6, 118.0, 48.3, 36.5, 26.1, 22.5. IR (KBr cmÀ1): νmax
3451, 3068, 2953, 2914, 2870, 1658, 1587, 1562, 1483,
1437, 1417, 1400, 1382, 1287, 1239, 1063, 1052, 988,
692, 596 cmÀ1. MS (ESI): m/z 438 [M + H]+. Anal.
Calcd for C20H19ClF3N5O: C, 54.86; H, 4.37; N, 16.00;
Found: C, 55.13; H, 4.78; N, 16.38.
EA = 2:1) on a silica gel to obtain the desired product 7a–7e.
N-Propyl-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
(trifluoromethyl)-1H-pyrazole-carboxamide (7a).
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1
solid, yield 43%, m.p. 122–123°C. H-NMR (500 MHz,
CDCl3) δ 8.52 (d, J = 9.2 Hz, 1H, pyridine H), 7.90
(d, J = 7.8 Hz, 2H, pyridine H), 7.45 (dd, J = 7.0,
4.6 Hz, 1H, pyridine H), 7.39 (s, 1H, pyrazole H), 7.06
(d, J = 2.8 Hz, 2H, pyridine H), 3.90 (t, J = 7.2 Hz, 2H,
CH2), 1.64 (m, 2H, CH2), 0.94 (t, J = 7.2 Hz, 3H, CH3).
13C-NMR (125 MHz, CDCl3) δ 161.4, 151.3, 149.8,
148.0, 147.12, 139.8, 139.6, 131.5, 129.5, 126.8, 121.9,
120.1, 119.9, 118.0, 51.2, 21.2, 11.2. IR (KBr cmÀ1):
νmax 3446, 3052, 29483, 2917, 2871, 1659, 1585, 1559,
1469, 1440, 1417, 1381, 1286, 1234, 1058, 1051,
968 cmÀ1. MS (ESI): m/z 410 [M + H]+. Anal. Calcd for
N-Benzyl-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
(trifluoromethyl)-1H-pyrazole-4-carboxamide (7e).
White
C18H15ClF3N5O: C, 52.76; H, 3.69; N, 17.09; Found: C,
52.25; H, 4.03; N, 16.93.
N-Butyl-1-(3-chloropyridin-2-yl)-N-(pyridin-4-yl)-5-
1
solid, yield 58%, m.p. 139–140°C. H-NMR (500 MHz,
CDCl3) δ 8.57 (dd, J = 4.7, 1.3 Hz, 2H, pyridine H),
8.46–8.42 (m, 1H, pyridine H), 8.28 (dd, J = 8.2, 1.2 Hz,
1H, pyridine H), 7.90 (s, 1H, pyrazole H), 7.72
(dd, J = 8.2, 4.7 Hz, 1H pyridine H), 7.33–7.18 (m, 5H,
benzene H), 7.14 (d, J = 6.0 Hz, 2H, pyridine H), 5.15
(s, 2H, CH2). 13C-NMR (125 MHz, CDCl3) δ 161.7,
151.247, 149.1, 148.3, 147.0, 140.9, 140.9, 136.8, 129.1,
129.0, 128.7, 128.3, 128.1, 122.7, 120.4, 51.9. IR (KBr
cmÀ1): νmax 3248, 3180, 3122, 3065, 3033, 2968, 1705,
1602, 1576, 1548, 1482, 1456, 1385, 1033, 980,
(trifluoromethyl)-1H-pyrazole-4-carboxamide (7b).
White
1
solid, yield 49%, m.p. 147–148°C. H-NMR (500 MHz,
CDCl3) δ 8.55 (d, J = 4.7 Hz, 1H, pyridine H), 8.47
(d, J = 4.6 Hz, 2H, pyridine H), 7.90 (d, J = 8.0 Hz, 1H,
pyridine H), 7.44 (dd, J = 8.0 Hz, 4.7 Hz, 1H, pyridine
H), 7.31 (s, 1H, pyrazole H), 7.05 (d, J = 4.8 Hz, 2H,
pyridine H), 3.92 (dd, J = 15.1, 8.3 Hz, 2H, CH2), 2.07–
1.06 (m, 4H, CH2CH2), 0.90 (dd, J = 5.9, 7.5 Hz, 3H,
CH3). 13C-NMR (125 MHz, CDCl3) δ 161.4, 151.5,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Design, Synthesis and Antifungal Evaluation of N-Substituted-1-(3-chloropyridin-2-
yl)-N-(pyridin-4-yl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide Derivatives
697 cmÀ1. MS (ESI): m/z 458 [M + H]+. Anal. Calcd for
C22H15ClF3N5O: C, 57.71; H, 3.30; N, 15.30; Found: C,
57.69; H, 3.24; N, 15.04.
Acknowledgments. This work was financially supported by
National Natural Science Foundation of China (nos. 21462011
and 21662008) and Science and Technology Foundation of
Guizhou Province (no. 20175788).
Antifungal activities assay. The antifungal activity of
the synthetic compounds was tested in vitro against six
plant pathogenic fungi (G. zeae, C. mandshurica,
F. oxysporium, B. cinerea, P. infestans, and
S. sclerotiorum) using mycelia growth inhibition
method [16–18]. The title compounds were dissolved
in DMSO to prepare the 10 mg/mL stock solution
before mixing with potato dextrose agar (PDA 90 mL).
The compounds were tested at a concentration of
100 μg/mL. All fungi were cultivated in PDA at
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were incubated at 27
1°C for 5 days. DMSO in
sterile distilled water served as the negative control,
whereas hymexazol, carboxin, and boscalid served as
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analyzed. The in vitro inhibitory effects of the test
compounds on these fungi were calculated by formula
I (%) = [(C À T)/(C À 0.4)] × 100, where C
represents the diameter of fungal growth on untreated
PDA, T represents the diameter of fungi on treated
PDA, and I represents the inhibition rate. EC50 was
defined as the concentration for 50% of maximal effect
of mycelia growth and was calculated with SPSS 17.0
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet