Synthesis and antifungal activity of n-(substituted pyridinyl)- 1-methyl(phenyl)-3-(trifluoromethyl)-1h-pyrazole-4- carboxamide derivatives

Article abstract of DOI:10.3390/molecules171214205

A series of N-(substituted pyridinyl)-1-methyl(phenyl)-3-trifluoromethyl- 1Hpyrazole- 4-carboxamide derivatives were synthesized. All target compounds were characterized by spectral data (1H-NMR, 13C-NMR, IR, MS) and elemental analysis and were bioassayed in vitro against three kinds of phytopathogenic fungi (Gibberella zeae, Fusarium oxysporum, Cytospora mandshurica). The results showed that some of the synthesized N-(substituted pyridinyl)-1-methyl-3- trifluoromethyl-1H-pyrazole-4-carboxamides exhibited moderate antifungal activities, among which compounds 6a, 6b and 6c displayed more than 50% inhibition activities against G. zeae at 100 μg/mL, which was better than that of the commercial fungicides carboxin and boscalid.

Full text of DOI:10.3390/molecules171214205

Molecules 2012, 17, 14205-14218; doi:10.3390/molecules171214205
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antifungal Activity of N-(Substituted pyridinyl)-
1-methyl(phenyl)-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide Derivatives
Zhibing Wu, Deyu Hu, Jiqing Kuang, Hua Cai, Shixi Wu and Wei Xue *
State Key Laboratory Breeding Base of Green Pesticides and Agricultural Bioengineering,
Key Laboratory of Green Pesticides and Agricultural Bioengineering, Ministry of Education,
Guizhou University, Guiyang 550025, China; E-Mails: wzb1171@163.com (Z.W.);
fcc.dyhu@gzu.edu.cn (D.H.); kuangjiqing@yeah.net (J.K.); woshicaihua@126.com (H.C.);
gzushixiwu@163.com (S.W.)
* Author to whom correspondence should be addressed; E-Mail: shouldww@126.com;
Tel./Fax: +86-851-8292-090.
Received: 18 October 2012; in revised form: 26 November 2012 / Accepted: 27 November 2012 /
Published: 30 November 2012
Abstract: A series of N-(substituted pyridinyl)-1-methyl(phenyl)-3-trifluoromethyl-1H-
pyrazole-4-carboxamide derivatives were synthesized. All target compounds were
characterized by spectral data (1H-NMR, 13C-NMR, IR, MS) and elemental analysis and
were bioassayed in vitro against three kinds of phytopathogenic fungi (Gibberella zeae,
Fusarium oxysporum, Cytospora mandshurica). The results showed that some of the
synthesized N-(substituted pyridinyl)-1-methyl-3-trifluoromethyl-1H-pyrazole-4-carboxamides
exhibited moderate antifungal activities, among which compounds 6a, 6b and 6c displayed
more than 50% inhibition activities against G. zeae at 100 µg/mL, which was better than
that of the commercial fungicides carboxin and boscalid.
Keywords: pyrazolecarboxamide derivatives; synthesis; antifungal activity; fungi
1. Introduction
The mitochondria are membrane enclosed organelles found in most eukaryotic cells [1], which are
usually described as “cellular power plants” because they generate most of the cell’s supply of
adenosine triphosphate (ATP), used as a source of chemical energy [2], and they also play an

Molecules 2012, 17
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important role in plant protection against many phytopathogenic fungi. Carboxanilides are traditional
active compounds which have been applied in agro-chemistry [3–5], and different kinds of commercial
products based on the carboxanilide structure have been developed in recent years. Early carboxanilide
fungicides such as carboxin have special activity against basidiomycetes, but limited activity towards
other plant pathogens [6,7]. The mode of action for these commercial fungicides revealed that they can
specifically bind to the ubiquinone-binding site (Q-site) of the mitochondrial complex II, interrupting
electron transport in the mitochondrial respiratory chain, so the fungi can’t produce vital energy to
form ATP [8–11].
The results of structure-activity relationships (SAR) research revealed that highly active novel
carboxanilide derivatives containing two heterocyclic rings and alkyl substitution at the amino heterocyclic
ring such as furametpyr and penthiopyrad (Figure 1) show an expanded antifungal spectrum [5,12].
Both of these commercial carboxamide fungicides have remarkable activity, not only against basidiomycetes,
but also the ascomycetes (such as gray mold, powdery mildew, and apple scab) [7,12,13].
Figure 1. The commercial carboxamide fungicides.
Penthiopyrad is one novel carboxamide fungicide, whose mode of fungicidal action indicates that it
inhibits succinate dehydrogenase and this results in the inhibition of the citric acid cycle and
mitochondrial electron transport pathways. The pyridine moiety is a traditional group with broad
activity which is often used in the molecular design of agrochemicals. Based on the molecular
structure of penthiopyrad, a new series of pyrazole carboxamide compounds containing an active
3-trifluoromethyl-1H-pyrazole-4-carboxamide skeleton have been designed by introducing active
pyridine rings into the amino part of pyrazole carboxamide. In this work 12 novel N-(substituted
pyridinyl)-1-methyl-3-trifluoromethylpyrazole carboxamide analogues and 11 novel N-(substituted
pyridinyl)-1-phenyl-3-trifluoromethylpyrazole carboxamide analogues were synthesized (Scheme 1). The
antifungal activity against Gibberella zeae (G. zeae), Fusarium oxysporium (F. oxysporium),
Cytospora mandshurica (C. mandshurica) were then evaluated.
2. Results and Discussion
2.1. Chemistry
The synthetic protocols of the target compounds are depicted in Scheme 1. Intermediate 2 was
prepared by reacting compound 1 with hydrazine hydrate [14]. The intermediates 3 and 7 were
synthesized from ethyl 3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (2) with different methods.
Ethyl 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (3) can be prepared via substitution

Molecules 2012, 17
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reaction of 2 with iodomethane [15], and ethyl 1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxylate (7) can be synthesized by a Suzuki coupling reaction of 2 with phenylboronic acid [16].
Scheme 1. Synthetic route to compounds 6a–l and 10a–k.
Then intermediates 3 and 7 reacted with lithium hydroxide in THF to afford pyrazole acids 4 and 8,
respectively. Subsequently 4 and 8 were refluxed in SOCl2 to give pyrazole acid chlorides 5 and 9.
Compounds 6a–l and 10a–k were then obtained by reaction of 5 or 9 with different substituted
pyridylamines, respectively [17,18]. The 1H-NMR, 13C-NMR, IR and MS data and elemental analysis
for the synthesized new compounds were consistent with assigned structures.
2.2. Antifungal Activity
As indicated at Table 1, most of the synthesized compounds possessed antifungal activity to a
certain extent, and some of the synthesized compounds exhibited moderately antifungal activity, such
as 6a, 6b and 6c that displayed more than 50% inhibition activity against G. zeae at 100 µg/mL, which
were better than that of the commercial fungicides carboxin and boscalid. The inhibition rates of
compound 6a against G. zeae, F. oxysporum and C. mandshurica were 73.2%, 53.5%, 48.7% respectively,
better than that of the other compounds.

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Table 1. Inhibition effect of compound 6a–l and 10a–k against phytopathogenic fungi at
100 µg/mL.
Inhibition rate a (%)
Compound R1
R2
G. zeae
F. oxysporum C. mandshurica
6a
6b
CH3
CH3
51.6 ± 1.3
39.1 ± 1.4
53.5 ± 1.5
35.6 ± 2.2
48.7 ± 2.0
73.2 ± 1.7
60.1 ± 1.0
6c
CH3
CH3
5.9 ± 1.2
9.1 ± 1.6
6d
34.7 ± 0.9
22.27 ± 1.3
20.8 ± 1.5
6e
6f
CH3
CH3
40.9 ± 1.7
26.3 ± 1.0
19.5 ± 0.8
11.8 ± 1.5
9.0 ± 1.9
2.9 ± 1.7
6g
6h
6i
CH3
CH3
CH3
25.2 ± 8.2
30.4 ± 1.8
40.4 ± 1.1
6.9 ± 1.1
22.5 ± 0.9
14.9 ± 0.9
1.74 ± 1.6
13.6 ± 1.5
4.9 ± 2.2
6j
CH3
CH3
39.3 ± 1.5
26.0 ± 1.4
8.3 ± 0.8
0
6k
30.1 ± 1.1
13.6 ± 1.8
6l
CH3
Ph
25.2 ± 1.8
11.3 ± 1.0
16.2 ± 1.9
10.9 ± 1.8
7.9 ± 2.1
0
6.4 ± 1.3
10a
10b
0
0
Ph
10c
10d
10f
Ph
Ph
Ph
10.7 ± 1.2
35.2 ± 1.6
22.6 ± 1.3
5.7 ± 1.2
23.9 ± 2.1
27.2 ± 2.9
0
17.0 ± 1.2
8.4 ± 1.0
10g
Ph
6.7 ± 1.1
11.5 ± 3.6
6.1 ± 0.8

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Table 1. Cont.
Inhibition rate a (%)
F. oxysporum C. mandshurica
Compound R1
R2
G. zeae
10h
Ph
9.8 ± 0.9
4.23 ± 1.7
0
10i
10j
10k
Ph
Ph
Ph
6.1 ± 1.2
16.5 ± 1.2
6.4 ± 1.5
10.6 ± 3.8
14.2 ± 1.0
20.5 ± 0.8
0
10.1 ± 0.9
0
Carboxin
Boscalid
56.3 ± 2.4
35.9 ± 3.5
23.6 ± 9.4
4.3 ± 1.3
--
3.4 ± 1.5
a Average of three replicates. “--” Not tested.
2.3. Toxicity of Target Compound 6b on Three Kinds of Pathogenic Fungi
The EC50 values of compound 6b against three plant pathogens (G. zeae, F. oxysporum and
C. mandshurica) were 81.3 µg/mL, 97.8 µg/mL, 176.5 µg/mL, respectively (Table 2). The preliminary
structure-activity relationship analysis indicated that the antifungal activity of the synthesized
compounds was obviously decreased when the methyl group at N-1 of the pyrazole ring was replaced
by a phenyl group.
Table 2. Toxicity of compound 6b on three kinds of pathogenic fungi a.
Compounds
Fungi
G. zeae
Toxic regression equation EC50 (μg/mL)
R
y = 2.8606x − 0.4639
y = 1.3314x + 2.2993
y = 0.7853x + 3.1312
76.3 ± 1.3
97.8 ± 2.7
132.5 ± 2.1
0.949
0.983
0.967
6b
F. oxysporum
C. mandshurica
a Average of three replicates.
3. Experimental
3.1. Chemistry
All the melting points were determined with a XT-4 binocular microscope melting point apparatus
1
(Beijing Tech Instrument Co., Beijing, China), while the thermometer was uncorrected. H-NMR and
13C-NMR were recorded with a JEOL-ECX500 NMR spectrometer operating at 500 MHz and 125
MHz respectively, and tetramethylsilane (TMS) was used as the internal standard. Mass spectral
studies were conducted on an Agilent 5973 organic mass spectrometer. Elemental analysis was
performed on an Elementar Vario-III CHN analyzer (Hanau, Germany). Analytical thin-layer
chromatography (TLC) was performed on silica gel GF254. Column chromatographic purification was
carried out using silica gel, the spots were visualized with ultraviolet (UV) light. All reagents were of
analytical reagent grade or chemically pure. All solvents were dried, deoxygenated, and redistilled
before use.

Molecules 2012, 17
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3.2. Ethyl 3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (2)
A mixture of ethyl 3,3,3-trifluoro-2-oxo-butanoate (30.0 g, 0.16 mol), triethyl orthoformate (75.7 g,
0.33 mol) and acetic anhydride (49.0 g, 0.49 mol) was stirred at 120 °C for 12 h. The reaction mixture
was concentrated in vacuo. The residue was dissolved in methanol (150 mL), hydrazine hydrate (12.2 g,
0.24 mol) was added, and reacted at 80 °C for 3 h. The reaction mixture was concentrated in vacuo.
The residue was extracted with ethyl acetate, washed with water and brine, dried over anhydrous
magnesium sulfate and concentrated in vacuo. The residue was recrystallized from ethyl acetate-hexane
to give compound 2 as a light yellow solid (20.4 g, yield 60.0%), m.p. 141~142 °C. 1H-NMR (DMSO-d6):
14.13 (s, 1H, pyrazole NH), 8.59 (s, 1H, pyrazole H), 4.24 (q, J = 7.2 Hz, 2H, CH2), 1.27 (t, J = 7.2 Hz,
3H, CH3).
3.3. Ethyl 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (3)
Ethyl 3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (2, 5.0 g, 21.2 mmol) and NaH (1.3 g, 42.4 mmol)
were dissolved in DMF (30 mL), CH3I (3.3 g, 23.3 mmol) in DMF (10 mL) was added slowly into the
solution while the temperature was kept under 0 °C, and reacted for 20 min, then the resulting mixture
was monitored by TLC and reacted at ambient temperature for about 20 h. After completion of the
reaction, water was added, and the reaction mixture was subsequently extracted with ethyl acetate, the
organic layer were washed with brine, dried over anhydrous magnesium sulfate and concentrated
in vacuo. The residue was recrystallized from ethyl acetate-petroleum to give compound 3 as a white
crystalline solid (8.2 g, yield 73.8%), m.p. 58~60 °C; 1H-NMR (DMSO-d6) δ: 8.55(s, 1H, pyrazole H),
4.24 (q, J = 7.2 Hz, 2H, OCH2CH3), 3.95 (s, 3H, N-CH3), 1.27 (t, J = 7.2 Hz, 3H, OCH2CH3); MS
(ESI) m/z: 223 [M+H]+.
3.4. Ethyl 1-Phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (7)
Ethyl 3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (2, 8.0 g, 38.4 mmol) was dissolved in DMF
(80 mL), then phenylboronic acid (9.4 g, 76.8 mmol), cupric acetate (10.5 g, 57.7 mmol) and pyridine
(6.1 g, 76.9 mmol) were added to the solution, the resulting mixture was monitored by TLC and
reacted at ambient temperature for about 24 h, then filtrated with kieselguhr, and extracted with ethyl
acetate, the organic layer were washed with NaHCO3, dried over anhydrous magnesium sulfate and
concentrated in vacuo. The residue was recrystallized from ethanol to give compound 7 as a white
1
crystalline solid, yield 91.0%, m.p. 89~90 °C; H-NMR (DMSO-d6): 9.33 (s, 1H, pyrazole H), 7.95
(d, J = 8.6 Hz, 2H, benzene H), 7.58 (t, J = 8.0 Hz, 2H, benzene H), 7.48 (t, J = 7.5 Hz, 1H, benzene H),
4.31 (q, J = 7.2 Hz, 2H, CH2), 1.32 (t, J = 7.2 Hz, 3H, CH3); MS (ESI): m/z 285 [M+H]+.
3.5. General Synthetic Procedure for Intermediates 4 and 8
To a solution of compound 3 (4.0 g, 18.0 mmol) in THF (20 mL), lithium hydroxide (1.72 g, 0.72 mol)
were added, reacted at 70 °C for about 2 h. The solution was concentrated in vacuo, the pH was
adjusted to 5 with hydrochloric acid, the reaction mixture was filtered, and the filtrate was washed with
water to give 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (4), as a white crystalline

Molecules 2012, 17
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1
solid (3.1 g, yield 89.2%), m.p. 133–134 °C; H-NMR (DMSO-d6) δ: 12.90 (s, 1H, COOH), 8.46
(s, 1H, pyrazole H), 3.93 (s, 3H, N-CH3); MS (ESI) m/z: 195 [M+H]+.
Intermediate 8 (1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid) can be prepared by the
1
same method as 4. White solid, yield 97.6%, m.p. 223~224 °C; H-NMR (DMSO-d6): 9.23 (s, 1H,
pyrazole H), 7.93 (d, J = 8.6 Hz, 2H, benzene H), 7.57 (t, J = 7.8 Hz, 2H, benzene H), 7.47 (t, J = 7.2 Hz,
1H, benzene H); MS (ESI): m/z 257 [M+H]+.
3.6. General Synthetic Procedure for Intermediates 5 and 9
1-Methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride (5) and 1-phenyl-3-(trifluoromethyl)-
1H-pyrazole-4-carbonyl chloride (9) were prepared by refluxing 4 and 8 in thionyl chloride for
8 h, respectively.
3.7. Preparation of the Target Compounds 6 and 10
To a solution of 2-amino-5-(trifluoromethyl) pyridine (163 mg, 1 mmol) and 80% sodium hydride
(45 mg, 1.5 mmol) in anhydrous THF (10 mL), compound 5 (214 mg, 1 mmol) was added slowly,
while the temperature was controlled under 10 °C, then reacted at ambient temperature for 10 h. The
solvent was distilled off, and the residue was dissolved in ethyl acetate, washed with water and brine,
dried over by anhydrous sodium sulfate. After the solvent was removed under reduced pressure, the
residue was purified by column chromatography on silica gel (ethyl acetate/petroleum ether 1:15) to afford
the target product, N-(5-(trifluoromethyl)-pyridin-2-yl)-1-methyl-3-(trifluoro-methyl)-1H-pyrazole-4-
carboxamide (6a). The following compounds 6b–l and 10a–k was prepared from the corresponding
starting materials in a similar manner to that described for 6a. The physical and spectral data for
compounds 6b–l and 10a–k are listed below.
1-Methyl-3-(trifluoromethyl)-N-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrazole-4-carboxamide (6a): White
solid, yield 86.4%, m.p. 128~130 °C. 1H-NMR (DMSO-d6) δ: 11.19 (s, 1H, NH), 8.76 (s, 1H, pyrazole H),
8.73 (1H, pyridine H), 8.34 (d, J = 8.6 Hz, 1H, pyridine H), 8.20 (d, J = 8.6 Hz, 1H, pyridine H), 3.97
(s, 3H, N-CH3); 13C-NMR (DMSO-d6): δ 160.03, 155.60, 145.82, 136.42, 136.07, 123.3, 122.40,
121.25, 120.27, 115.68, 114.21, 40.51; IR (KBr): ν 3305.9, 3121.8, 1681.9, 1591.1, 1542.3, 1525.6,
1051.2, 848.6, 752.5 cm−1; MS(ESI): m/z 339 [M+H]+; Anal. Calc. for C12H8F6N4O: C, 42.62; H, 2.38;
N, 16.57. Found: C, 42.22; H, 2.05; N, 16.11.
1-Methyl-N-(5-bromopyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6b): White solid,
1
yield 74.2%, m.p. 170~173 °C. H-NMR (DMSO-d6) δ: 10.91 (s, 1H, NH), 8.68 (s, 1H, pyrazole H),
8.49 (s, 1H, pyridine H), 8.12 (d, J = 9.15 Hz, 1H, pyridine H), 8.03 (d, J = 8.6 Hz, 1H, pyridine H),
3.94 (s, 3H, N-CH3); 13C-NMR (DMSO-d6): δ 159.67, 151.45, 149.03, 141.18, 135.81, 122.44, 120.30,
116.39, 115.94, 114.38, 40.48; IR (KBr): ν 3429.4, 3128.5, 3261.6, 1674.2, 1543.0, 1521.1, 1496.7,
1053.1, 835.1 cm−1; MS(ESI) m/z: 349 [M+H]+; Anal. Calc. for C11H8BrF3N4O: C, 37.84; H, 2.31; N,
16.05. Found: C, 37.38; H, 1.99; N, 15.75.

Molecules 2012, 17
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1-Methyl-N-(2-chloropyridin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6c): White solid,
1
yield 45.1%, m.p. 129~131 °C. H-NMR (DMSO-d6) δ:10.00 (s, 1H, NH), 8.59 (s, 1H, pyrazole H),
8.29 (1H, pyridine H), 8.07 (s, 1H, pyridine H), 7.49 (1H, pyridine H), 3.97 (s, 3H, N-CH3); 13C-NMR
(DMSO-d6): δ 159.43, 147.06, 145.98, 136.80, 135.43, 132.02, 124.01, 122.40, 120.27, 115.79, 40.51;
IR (KBr): ν 3284.7, 3121.1, 1660.7, 1657.7, 1548.8, 1541.1, 1521.8, 1456.2, 1496.7, 1078.21, 1051.1,
800 cm−1; MS(ESI): m/z 305 [M+H]+; Anal. Calc. for C11H8ClF3N4O: C, 43.37; H, 2.65; N, 18.39.
Found: C, 43.11; H, 2.34; N, 17.93.
1-Methyl-N-(6-methylpyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6d): Light yellow
solid, yield 66.3%, m.p. 133~136 °C. 1H-NMR (DMSO-d6) δ: 10.61(s, 1H, NH), 8.66 (s, 1H, pyrazole
H), 8.19 (m, 1H, pyridine H), 8.02 (d, J = 8.0 Hz, 1H, pyridine H), 7.63 (d, J = 8.0 Hz, 1H, pyridine
13
H), 3.96 (s, 3H, N-CH3); 2.26 (s, 3H, CH3); C-NMR (DMSO-d6): δ 159.41, 150.27, 148.19, 139.05,
135.52, 129.21, 122.51, 120.36, 116.33, 114.32, 40.49, 17.80; IR (KBr): ν 3365.7, 3130.4, 1678.0,
1591.2, 1541.1, 1533.4, 1330.8, 1282.6, 869.9 cm−1; MS(ESI): m/z 285 [M+H]+; Anal. Calc. for
C12H11F3N4O: C, 50.71; H, 3.90; N, 19.71. Found: C, 50.53; H, 3.65; N, 19.48.
1-Methyl-N-(pyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6e): White solid, yield
1
75.6%, m.p. 159~162 °C. H-NMR (DMSO-d6) δ: 10.70 (s, 1H, NH), 8.68 (s, H, pyrazole H), 8.36
(d, J = 3.5 Hz, 1H, pyridine H), 8.13 (d, J = 8.6 Hz, 1H, pyridine H), 7.81 (t, J = 7.7 Hz, 1H, pyridine H),
7.15 (t, J= 6.3 Hz, 1H, pyridine H), 3.96 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 159.62, 152.46,
148.47, 140.13, 138.73, 135.65, 120.29, 116.25, 115.81, 114.78, 40.54; IR(KBr): ν 3312.1, 3226.9,
1683.8, 1579.7, 1541.1, 1527.6, 1506.4, 1055.0, 823.6, 790.8, 754.1 cm−1; MS(ESI): m/z 271 [M+H]+;
Anal. Calc. for C11H9F3N4O: C, 48.89; H, 3.36; N, 20.73. Found: C, 48.44; H, 3.08; N, 20.22.
1-Methyl-N-(pyridin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6f): White solid, yield
1
59.7%, m.p. 205~208 °C. H-NMR (DMSO-d6) δ:10.46 (s, 1H, NH), 8.57(s, H, pyrazole H), 8.46
(d, J = 1.7 Hz, 1H, pyridine H), 8.45 (d, J = 1.2 Hz, 1H, pyridine H), 7.66 (d, J = 1.2 Hz, 1H, pyridine H),
7.65 (d, J = 1.7 Hz, 1H, pyridine H), 3.99 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 159.94, 150.93,
146.11, 139.96, 135.37, 122.39, 116.31, 114.12, 40.53; IR (KBr): ν 3044.1, 3226.9, 1697.3, 1583.5,
1570.0, 1541.1, 1003.8, 835.1, 775.3 cm−1; MS(ESI): m/z 271 [M+H]+; Anal. Calc. for C11H9F3N4O:
C, 48.89; H, 3.36; N, 20.73. Found: C, 48.56; H, 3.01; N, 20.39.
1-Methyl-N-(4-methylpyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6g): Light yellow
solid, yield 47.8%, m.p. 138~141 °C, 1H-NMR (DMSO-d6) δ: 10.61 (s, 1H, NH), 8.41 (s, 1H, pyrazole
H), 8.19 (s, 1H, pyridine H), 7.71 (d, J = 5.2 Hz, 1H, pyridine H), 7.17 (d, J = 5.2 Hz, 1H, pyridine H),
3.89 (s, 3H, N-CH3); 2.30 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 160.04, 152.82, 150.68, 140.73,
136.64, 124.80, 119.95, 116.56, 115.15, 113.91, 40.49, 21.12; IR (KBr): ν 3363.8, 3112.5, 1670.3,
1575.8, 1541.1, 1506.4, 1456.2, 1055.0, 873.7, 825.5, 752.4 cm−1; MS(ESI): m/z 285 [M+H]+; Anal.
Calc. for C12H11F3N4O: C, 50.71; H, 3.90; N, 19.71. Found: C, 50.27; H, 3.66; N, 19.54.
1-Methyl-N-(5-fluoropyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6h): Yellow solid,
1
yield 82.4%, m.p. 135~137 °C. H-NMR (DMSO-d6) δ: 10.83 (s, 1H, NH), 8.65 (s, H, pyrazole H),
13
8.37(d, 1H, pyridine H), 8.16 (1H, pyridine H), 7.76 (1H, pyridine H), 3.96 (s, 3H, CH3); C-NMR

Molecules 2012, 17
14213
(DMSO-d6): δ 160.07, 151.35, 147.66, 140.10, 137.88, 125.26, 120.23, 119.17, 118.52, 114.73, 40.50;
IR (KBr): ν 3.454.5, 3136.2, 1670.3, 1541.1, 1521.8, 1489.0, 1392.6, 1055.0, 840.1, 771.2 cm−1;
MS(ESI): m/z 289 [M+H]+; Anal. Calc. for C11H8F4N4O: C, 45.84; H, 2.80; N, 19.44. Found: C, 45.56;
H, 2.63; N, 19.14.
1-Methyl-N-(2-chloropyridin-3-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6i): Light yellow
solid, yield 33.3%, m.p. 107~109 °C. 1H-NMR (DMSO-d6) δ: 10.25 (s, 1H, NH), 8.48 (s, 1H, pyrazole H),
8.34 (d, J = 7.5 Hz,1H, pyridine H), 8.03 (d, J = 8.0 Hz, 1H, pyridine ), 7.59 (q, J = 4.0 Hz, 1H,
pyridine H), 3.89 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 163.57, 150.55, 149.23, 140.76, 136.72,
133.98, 125.19, 121.98, 119.84, 115.68, 40.51; IR (KBr): ν 3415.5, 3127.1, 1691.5, 1585.4, 1541.1,
1514.1, 1490.9, 1037.7, 815.8, 763.8, 732.9 cm−1; MS(ESI): m/z 305 [M+H]+; Anal. Calc. for
C11H8ClF3N4O: C, 43.37; H, 2.65; N, 18.39. Found: C, 42.88; H, 2.14; N, 18.25.
1-Methyl-N-(5-methylpyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6j): Yellow solid,
1
yield 43.9%, m.p. 135~138 °C. H-NMR (DMSO-d6) δ: 10.34 (s, 1H, NH), 8.80 (s, 1H, pyrazole H),
8.53 (s, 1H, pyridine H), 8.29 (d, J = 7.2 Hz, 1H, pyridine H), 8.11 (d, J = 7.2 Hz, 1H, pyridine H),
3.99 (s, 3H, N-CH3); 2.26 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 159.55, 151.23, 145.11, 141.89,
139.82, 135.11, 127.36, 124.18, 116.48, 115.94, 40.50, 24.37; IR (KBr): ν 3545.1, 3105.3, 1653.0,
1589.3, 1506.4, 1496.7, 1473.6, 1039.6, 891.1, 808.7, 789.9. cm−1; MS(ESI): m/z 285 [M+H]+; Anal.
Calc. for C12H11F3N4O: C, 50.71; H, 3.90; N, 19.71. Found: C, 50.43; H, 3.77; N, 19.62.
1-Methyl-N-(5-chloropyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6k): White solid,
1
yield 30.1%, m.p. 138~140 °C, H-NMR (DMSO-d6) δ: 10.14 (s, 1H, NH), 8.41 (s, 1H, pyrazole H),
8.15 (d, J = 9.2 Hz, 1H, pyridine H), 8.10 (s, 1H, pyridine H), 7.94 (d, J = 7.5 Hz, 1H, pyridine H),
4.03 (s, 3H, NCH3); 13C-NMR (DMSO-d6): δ 160.28, 150.99, 146.92, 139.30, 138.52, 126.18, 121.25,
119.95, 119.10, 115.84, 40.52; IR (KBr): ν 3282.8, 2964.5, 1701.2, 1575.8, 1525.6, 1456.2, 1292.8,
1155.3, 1006.8, 837.1, 736.8 cm−1; MS(ESI): m/z 305 [M+H]+; Anal. Calc. for C11H8ClF3N4O: C,
43.37; H, 2.65; N, 18.39. Found: C, 43.02; H, 2.18; N, 18.12.
1-Methyl-N-(2-bromopyridin-4-yl)-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (6l): White solid,
1
yield 56.4%, m.p. 174~176 °C. H-NMR (DMSO-d6) δ: 10.67 (s, 1H, NH), 8.57 (s, 1H, pyrazole H),
8.29 (d, J = 5.8 Hz, 1H, pyridine H), 7.84 (s, 1H, pyridine H), 7.59 (d, J = 5.2 Hz, 1H, pyridine H),
13
3.99 (s, 3H, CH3); C-NMR (DMSO-d6): δ 160.05, 151.48, 150.95, 148.69, 135.55, 122.32, 120.17,
115.85, 113.53, 113.40, 40.50; IR (KBr): ν 3330.5, 3115.0, 1699.2, 1587.4, 1541.1, 1489.0, 1049.2,
842.1, 772.1 cm−1; MS(ESI): m/z 349 [M+H]+; Anal. Calc. for C11H8BrF3N4O: C, 37.84; H, 2.31; N,
16.05. Found: C, 37.39; H, 2.11; N, 15.87.
N-(Pyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10a): White solid, yield
1
80.5%, m.p. 244~245 °C. H-NMR (DMSO-d6) δ: 10.63 (s, 1H, NH), 9.35 (s, 1H, pyrazole H), 8.50
(d, J = 6.3 Hz, 2H, pyridine H), 7.89 (d, J = 8.1 Hz, 2H, benzene H), 7.69 (d, J = 6.3 Hz, 2H, pyridine H),
13
7.64 (t, J = 8.1 Hz, 2H, benzene H), 7.51 (t, J = 7.5 Hz, 1H, benzene H); C-NMR (DMSO-d6): δ
159.72, 151.04, 145.94, 141.81, 138.76, 136.44, 132.74, 130.54, 129.04, 120.10, 117.99, 114.14; IR
(KBr): ν 3419.7, 3290.5, 2978.0, 1716.6, 1683.8, 1624.0, 1543.0, 1508.3, 1400.2, 1284.5 cm−1;

Molecules 2012, 17
14214
MS(ESI): m/z 333 [M+H]+; Anal. Calc. for C16H11F3N4O: C, 57.83; H, 3.34; N, 16.86. Found: C,
57.65; H, 3.12; N, 16.54.
N-(2-Chloropyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10b): White solid,
1
yield 16.5%, m.p. 216~218 °C. H-NMR (DMSO-d6) δ: 10.23 (s, 1H, NH), 8.69 (s, 1H, pyrazole H),
8.51 (d, J = 5.8 Hz, 1H, pyridine H), 7.76 (d, J = 5.8 Hz, 1H, pyridine H), 7.58 (d, J = 8.1 Hz, 2H,
benzene H), 7.43 (t, J = 7.6 Hz, 2H, benzene H), 7.51 (t, J = 7.5 Hz, 1H, benzene H), 7.36 (s, 1H,
pyridine H); 13C-NMR (DMSO-d6): δ 159.35, 159.21, 149.78, 145.69, 138.66, 132.23, 129.97, 129.12,
125.54, 122.14, 120.04, 116.75, 114.03, 110.28; IR (KBr): ν 3444.8, 3383.1, 1701.2, 1653.0, 1577.7,
1490.9, 1356.8, 1298.0, 765.7 cm−1; MS(ESI): m/z 367 [M+H]+; Anal. Calc. for C16H10ClF3N4O: C,
52.40; H, 2.75; N, 15.28. Found: C, 52.22; H, 2.59; N, 15.04.
N-(2-Bromopyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10c): White solid,
1
yield 26.9%, m.p. 235~236 °C. H-NMR (DMSO-d6) δ: 10.83 (s, 1H, NH), 9.35 (s, 1H, pyrazole H),
8.35 (d, J = 5.7 Hz, 1H, pyridine H), 7.89 (s, 1H, pyridine H), 7.87 (d, J = 3.4 Hz, 2H, benzene H),
7.65–7,61 (m, 3H, benzene 2H and pyridine H), 7.51 (t, J = 7.2 Hz, 1H, benzene H); 13C-NMR
(DMSO-d6): δ 159.86, 151.57, 151.12, 148.51, 143.56, 138.70, 132.98, 130.58, 129.15, 120.15,
117.56, 116.72, 113.56, 113.44; IR (KBr): ν 3365.8, 3111.1, 3066.8, 2922.1, 2850.7, 1705.0, 1579.7,
1543.0, 1494.8, 1386.8, 1298.0, 1043.4, 688.5 cm−1; MS(ESI): m/z 411 [M+H]+, 412 [M+2H]+; Anal.
Calc. for C16H10BrF3N4O: C, 46.74; H, 2.45; N, 13.63. Found: C, 46.34; H, 2.18; N, 13.47.
N-(Pyridin-2-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10d): White solid, yield
1
40.5%, m.p. 149~151 °C. H-NMR (DMSO-d6) δ: 10.85 (s, 1H, NH), 9.52 (s, 1H, pyrazole H), 8.23
(d, J = 8.0 Hz, 1H, benzene H), 7.87–7.49 (m, 9 H, pyridine and benzene H); 13C-NMR (DMSO-d6): δ
159.33, 152.36, 148.50, 138.81, 133.44, 132.97, 130.39, 128.82, 120.39, 120.35, 119.96, 118.36, 117.75,
114.64; IR (KBr): ν 3130.4, 3068.7, 2987.7, 2927.7, 1695.4, 1600.9, 1490.9, 1435.0, 1307.7 cm−1;
MS(ESI): m/z 333 [M+H]+; Anal. Calc. for C16H11F3N4O: C, 57.83; H, 3.34; N, 16.86. Found: C,
57.66; H, 3.04; N, 16.49.
N-(3-Chloropyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10e): White solid,
1
yield 74.9%, m.p. 146~147 °C. H-NMR (DMSO-d6) δ: 10.08 (s, 1H, NH), 9.41 (s, 1H, pyridine H),
8.69 (s, 1H, pyrazole H), 8.51 (d, J = 5.7 Hz, 1H, pyridine H), 7.99 (d, J = 5.7 Hz, 1H, pyridine H),
7.91 (d, J = 8.0 Hz, 2H, benzene H), 7.64 (t, J = 7.8 Hz, 2H, benzene H), 7.51 (t, J = 7.5 Hz, 1H,
benzene H); 13C-NMR (DMSO-d6): δ 159.46, 150.34, 149.26, 142.23, 138.76, 133.26, 130.49, 129.04,
123.34, 120.22, 119.04, 118.56, 117.31, 110.23; IR (KBr): ν 3387.0, 3101.5, 3034.0, 1707.0, 1581.6,
1496.7, 1406.1, 1294.2, 763.8 cm−1; MS(ESI): m/z 367 [M+H]+; Anal. Calc. for C16H10ClF3N4O: C,
52.40; H, 2.75; N, 15.28. Found: C, 51.98; H, 2.54; N, 14.96.
N-(3-Bromopyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10f): White solid,
1
yield 69.7%, m.p. 136~138 °C. H-NMR (DMSO-d6) δ: 9.98 (s, 1H, NH), 9.39 (s, 1H, pyridine H),
8.80 (s, 1H, pyrazole H), 8.53 (d, J = 5.2 Hz, 1H, pyridine H), 7.90–7.87 (m, 3H, pyridine and benzene
H), 7.63 (d, J = 8.0 Hz, 2H, benzene H), 7.64 (t, J = 7.8 Hz, 2H, benzene H), 7.51 (t, J = 6.9 Hz, 1H,
benzene H); 13C-NMR (DMSO-d6): δ 159.26, 152.92, 149.69, 143.61, 138.76, 133.09, 130.49, 129.05,

Molecules 2012, 17
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122.21, 120.24, 120.07, 117.37, 114.93, 110.58; IR (KBr): ν 3400.5, 3325.2, 2924.0, 1670.3, 1579.7,
1494.8, 1406.8, 1235.3, 682.8 cm−1; MS(ESI): m/z 411 [M+H]+; Anal. Calc. for C16H10BrF3N4O: C,
46.74; H, 2.45; N, 13.63. Found: C, 46.57; H, 2.33; N, 13.47.
N-(3-Methylpyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10g): White solid,
yield 65.3%, m.p. 207~208 °C. 1H-NMR (DMSO-d6) δ: 9.83 (s, 1H, NH), 9.35 (s, 1H, pyridine H), 8.
44 (s, 1H, pyrazole H), 8.38 (d, J = 5.2 Hz, 1H, pyridine H), 7.90 (d, J = 8.0 Hz, 2H, benzene H), 7.73
(d, J = 5.2 Hz, 1H, benzene H), 7.63 (t, J = 6.9 Hz, 2H, benzene H), 7.51 (d, J = 6.3 Hz, 1H, pyridine
H), 2.31 (s, 3H, CH3); 13C-NMR (DMSO-d6): δ 159.39, 152.05, 148.40, 143.70, 138.83, 132.86,
130.48, 128.97, 125.91, 120.16, 118.07, 117.78, 114.93, 111.23, 15.33; IR (KBr): ν 3244.2, 3116.9,
3030.1, 1699.2, 1581.6, 1544.9, 1490.9, 1409.9, 1300.0 cm−1; MS(ESI): m/z 347 [M+H]+; Anal. Calc.
for C17H13F3N4O: C, 58.96; H, 3.78; N, 16.18. Found: C, 58.66; H, 3.59; N, 16.01.
N-(2-Methylpyridin-4-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10h): White solid,
1
yield 60.9%, m.p. 196~197 °C. H-NMR (DMSO-d6) δ: 10.53 (s, 1H, NH), 9.34 (s, 1H, pyridine H),
8.37 (s, 1H, pyrazole H), 7.88 (d, J = 8.0 Hz, 1H, pyridine H), 7.64–7.62 (m, 3H, pyridine and benzene
13
H), 7.50 (d, J = 9.0 Hz, 2H, benzene H), 2.46 (s, 3H, CH3); C-NMR (DMSO-d6): δ 159.64, 159.33,
150.27, 146.22, 138.76, 132.67, 130.54, 129.03, 126.12, 122.34, 120.10, 118.04, 113.03, 111.58,
24.84; IR (KBr): ν 3261.6, 3115.0, 3066.8, 1683.8, 1604.7, 1588.4, 1490.9, 1409.9, 1301.9 cm−1;
MS(ESI): m/z 347 [M+H]+; Anal. Calc. for C17H13F3N4O: C, 58.96; H, 3.78; N, 16.18. Found: C,
58.67; H, 3.63; N, 15.94.
N-(Pyridin-3-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10i): White solid, yield 79.8%,
1
m.p. 196~197 °C. H-NMR (DMSO-d6) δ: 10.50 (s, 1H, NH), 9.32 (s, 1H, pyridine H), 8.84 (s, 1H,
pyrazole H), 8.33 (d, J = 3.5 Hz, 1H, pyridine H), 8.14 (d, J = 8.6 Hz, 1H, pyridine H), 7.88 (d, J = 7.5 Hz,
2H, benzene H), 7.63 (t, J = 8.0 Hz, 2H, benzene H), 7.50 (t, J = 7.5 Hz, 1H, benzene H), 7.42 q,
J = 7.5 Hz, 1H, pyridine H); 13C-NMR (DMSO-d6): δ 161.11, 159.32, 145.34, 141.85, 138.79, 135.92,
132.50, 130.55, 129.00, 127.34, 124.30, 122.28, 120.07, 118.18; IR (KBr): ν 3259.7, 3122.7, 3066.8,
1695.4, 1653.0, 1600.9, 1490.9, 1419.6, 1303.8 cm−1; MS(ESI): m/z 333 [M+H]+; Anal. Calc. for
C16H11F3N4O: C, 57.83; H, 3.34; N, 16.86. Found: C, 57.75; H, 3.16; N, 16.62.
N-(5-Chloropyridin-2-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10j): White solid,
1
yield 67.8%, m.p. 101~102 °C. H-NMR (DMSO-d6) δ: 10.17 (s, 1H, NH), 9.36 (s, 1H, pyrazole H),
8.33 (d, J = 4.6 Hz, 1H, pyridine H), 8.13 (d, J = 8.1 Hz, 1H, pyridine H), 7.90 (d, J = 8.1 Hz, 2H,
benzene H), 7.64 (t, J = 7.7 Hz, 2H, benzene H), 7.54–7.50 (m, 2H, benzene and pyrazole H); 13C-NMR
(DMSO-d6): δ 160.97, 159.36, 146.94, 138.57, 135.39, 133.14, 130.49, 130.28, 129.00, 126.14,
120.30, 117.44, 115.71, 113.84; IR (KBr): ν 3421.7, 2926.0, 1705.0, 1653.0, 1575.8, 1506.4, 1386.8,
1298.0, 765.7 cm−1; MS(ESI): m/z 367 [M+H]+; Anal. Calc. for C16H10ClF3N4O: C, 52.40; H, 2.75; N,
15.28. Found: C, 52.03; H, 2.61; N, 14.98.
N-(5-Methylpyridin-2-yl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (10k): White solid,
1
yield 64.4%, m.p. 182~183 °C. H-NMR (DMSO-d6) δ: 11.17 (s, 1H, NH), 9.36 (s, 1H, pyrazole H),
8.33 (d, J = 4.6 Hz, 1H, pyridine H), 8.14 (d, J = 8.1 Hz, 2H, benzene H), 7.90 (d, J = 8.1 Hz, 2H,

Molecules 2012, 17
14216
benzene H), 7.64–7.52 (m, 3H, benzene and pyridine H); 13C-NMR (DMSO-d6): δ 159.32, 147.12,
145.62, 138.80, 136.49, 132.76, 131.89, 130.51, 129.02, 124.07, 120.20, 117.48, 116.13, 112.32,
14.54; IR (KBr): ν 3282.8, 2918.3, 1654.9, 1544.9, 1490.9, 1388.7, 1224.8, 1080.1 cm−1; MS(ESI):
m/z 347 [M+H]+; Anal. Calc. for C17H13F3N4O: C, 58.96; H, 3.78; N, 16.18. Found: C, 58.78; H, 3.55;
N, 16.02.
3.8. Antifungal Bioassays
The fungicidal activity of the target compounds 6a–l and 10a–k were tested in vitro against
G. zeae, F. oxysporium, and C. mandshurica, and their relative inhibitory ratio (%) was determined
using the mycelium growth rate method [19,20]. Carboxin and boscalid were used as controls. After
the mycelia grew completely, the diameters of the mycelia were measured, and the inhibition rate was
calculated according to the formula:
I = (D1 − D2)/D1 × 100%
where I is the inhibition rate, D1 is the average diameter of mycelia in the blank test, and D2 is the
average diameter of mycelia in the presence of those compounds. The inhibition ratios of those
compounds at the dose 100 μg/mL have been determined, and the experimental results are summarized
in Table 1.
Compound 6b has been tested against three pathogenic fungi (Gibberella zeae, F. oxysporum,
C. mandshurica,) at concentrations of 100, 50, 25, 12.5, 6.25, 0 μg/mL. The EC50 (effective dose for
50% inhibition μg/mL) values were estimated statistically by Probit analysis with the help of the Probit
package of the SPSS software version 11.5 using a personal computer. The average EC50 was taken
from at least three separate analyses for inhibition of growth using the basic EC50 program of SPSS
version 11.5.
4. Conclusions
In conclusion, a novel series of pyrazolecarboxamide compounds containing an active
3-trifluoromethyl-1H-pyrazole-4-carboxamide skeleton were designed and synthesized using penthiopyrad
13
as the lead structure. All target compounds were characterized by spectral data (1H-NMR, C-NMR,
IR, MS) and elemental analysis. The compounds were tested in vitro for antifungal activity against
three kinds of phytopathogenic fungi (Gibberella zeae, Fusarium oxysporum, Cytospora
mandshurica). The results showed that some of the synthesized N-(substituted pyridinyl)-1-methyl-3-
trifluoromethyl-1H-pyrazole-4-carboxamide compounds exhibited moderately antifungal activity, and
compounds 6a, 6b and 6c displayed more than 50% inhibition activities against G. zeae at 100 μg/mL,
which were better than that of the commercial fungicides carboxin and boscalid. The preliminary
structure-activity relationship analysis indicated that the antifungal activity of the synthesized
compounds was obviously decreased when a methyl at the 1-position of the pyrazole ring was
substituted by a phenyl. The structures of the target products need to be optimized to enhance their
antifungal activity. Future structural modification and biological evaluation to explore the full potential
of this novel class of antifungal molecules are under way.

Molecules 2012, 17
14217
Acknowledgments
This work was supported by Natural Science Foundation of Guizhou Province (No. 2010008) and
Science and Technology Foundation of Guizhou Province (No. 20112064).
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Sample Availability: Samples of the compounds 6a–l and 10a–k are available from the authors.
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