Design, Synthesis, DFT study and antifungal activity of pyrazolecarboxamide derivatives

Article abstract of DOI:10.3390/molecules21010068

A series of novel pyrazole amide derivatives were designed and synthesized by multi-step reactions from phenylhydrazine and ethyl 3-oxobutanoate as starting materials, and their structures were characterized by NMR, MS and elemental analysis. The antifungal activity of the title compounds was determined. The results indicated that some of title compounds exhibited moderate antifungal activity. Furthermore, DFT calculations were used to study the structure-activity relationships (SAR).

Full text of DOI:10.3390/molecules21010068

molecules
Article
Design, Synthesis, DFT Study and Antifungal
Activity of Pyrazolecarboxamide Derivatives
Jin-Xia Mu 1,*,†, Yan-Xia Shi 2,†, Ming-Yan Yang 3, Zhao-Hui Sun 3, Xing-Hai Liu 3,*, Bao-Ju Li 2,
and Na-Bo Sun 4
*
Received: 19 November 2015 ; Accepted: 5 January 2016 ; Published: 8 January 2016
Academic Editors: Shufeng Zhou and Wei-Zhu Zhong
1
Department of Environmental Engineering, China Jiliang University, Hangzhou 310018, China
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China;
2
shiyanxia@caas.cn
3
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100014, China;
yangmingyanzjut@163.com (M.-Y.Y.); sunzhaohuizjut@163.com (Z.-H.S.)
College of Biology and Environmental Engineering, Zhejiang Shuren University, Hangzhou 310015, China;
nabosun@gmail.com
4
*
Correspondence: mujinxiajl@sina.com (J.-X.M.); xhliu@zjut.edu.cn (X.-H.L.); libaoju@caas.cn (B.-J.L.);
Tel./Fax: +86-571-8832-0832 (X.-H.L.)
†
These authors contributed equally to this work.
Abstract: A series of novel pyrazole amide derivatives were designed and synthesized by multi-step
reactions from phenylhydrazine and ethyl 3-oxobutanoate as starting materials, and their structures
were characterized by NMR, MS and elemental analysis. The antifungal activity of the title compounds
was determined. The results indicated that some of title compounds exhibited moderate antifungal
activity. Furthermore, DFT calculations were used to study the structure-activity relationships (SAR).
Keywords: pyrazole; DFT; synthesis; antifungal activity; SAR
1. Introduction
Heterocyclic structures are important key features in natural products or synthetic medicines and
pesticides because of their high-efficiency, low toxicity and diversity of possible substituents [
This has become a hot research topic in the medicine and pesticides field. Pyrazole is an important kind
of heterocyclic nitrogen compound [ ], which derivatives exhibit a wide range of biological activities,
such as antifungal [ ], insecticidal [10], herbicidal [11], anticancer [12], anti-inflammatory [13] and so
1–5].
6–8
9
on. So far, many pyrazole derivatives such as the insecticides tebufenpyrad and chlorantraniliprole,
the fungicides penthiopyrad and pyraclostrobin, and the medicine antipyrine, etc., have been
successfully developed by different companies. On the other hand, heterocycles with amide groups are
reported as a class of compounds displaying extensive biological activities, such as anti-biofilm [14],
herbicidal [15,16], anticancer [17], antifungal [18], antiproliferative [19], plant growth regulation [20]
and so on. They represent an important class of natural and synthetic products and extremely versatile
building blocks for the manufacture of bioactive compounds in pharmaceutical drug design and the
agrochemical industry. In recent years, many succinate-dehydrogenase-inhibitor (SDHI) fungicides
had been introduced into the market for effective treatment of fruit and vegetable crops, such as
sedaxane, penflufen and benzovindiflupyr (Figure 1). Penflufen is one of the members in this new class
of fungicides for the treatment of a wide range of diseases. From Figure 1, the structure of penflufen
contains a phenyl ring, an amide group and a 1,3-dimethyl-5-fluoropyrazole moiety.
Molecules 2016, 21, 68; doi:10.3390/molecules21010068
www.mdpi.com/journal/molecules

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Figure 1. The commercial pyrazole amide fungicides.
In our previous work, some amide derivatives exhibited excellent herbicidal [21] and fungicidal
activity [22]. In line with our continued efforts to synthesize bioactive lead compounds for crop
protection [23–29], the title amide compounds had modified N-substituted pyrazole pharmacophore
scaffolds. It is reported that the halogens exhibit similar biological effects. Meanwhile, the aromatic
ring also held diverse functions. In order to discover highly active pyrazole amide compounds, the
commercial amide fungicide penflufen was selected as a lead compound, and the 1-methyl and
5-fluoro groups on the pyrazole ring were replaced by a phenyl ring and chloro group, respectively.
Our original strategy is depicted in Scheme 1. It is possible that pyrazole amide derivatives possess
antifungal activities.
Scheme 1. Design strategy of the title compounds.
2. Results and Discussion
2.1. Synthesis and Spectra
In the present paper, a series of pyrazole amide analogues were designed by replacing
methyl group and fluorine of penflufen with phenyl and chlorine. First, the pyrazole ring was
synthesized from phenylhydrazine and ethyl acetoacetate as starting materials using a classical
Knorr reaction, then, using the universal Vilsmeier-Haack reaction, apyrazole with an aldehyde
group was obtained in excellent yield. The COOH group can be prepared easily through oxidation
with KMnO4. The target compounds were prepared according our previous work [30]. When
5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl chloride was reacted with a substituted amine,
organic base Et3N was used instead of the inorganic base K2CO3, while the temperature must be
maintained at 0–5 ˝C, as higher temperatures decreased the yield of product. The imide or lactam
product will be obtained.
All the compounds were identified and characterized by 1H-NMR, 13C-NMR, MS and elemental
analysis. In the 1H-NMR spectra of target compounds, all the -NH proton signals can be found around

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7.59–10.88 ppm. The appearance of a signal around at 2.5 ppm is assigned to the methyl of the pyrazole
´
ring. Meanwhile, most of the title compounds exhibited the M´H peak in the ESI-MS results.
2.2. Antifungal Activities
The in vivo fungicidal results of the title compounds against Pythium ultimum Trow, Phytophthora
infestans (Mont.) De Bary, Corynespora cassiicola, Botrytis cinerea and Rhizoctonia solani are listed in
Table 1. Zhongshengmycin, dimethomorph, fludioxonil, chlorothalonil and validamycin were used
as controls. From Table 1, some of the pyrazole compounds such as compounds 5a (77.78%), 5d
(55.56%), 5e (66.67%), 5h (66.67%), 5i (44.44%) and 5l (77.78%) exhibited good control efficacy against
Pythium ultimum Trow at a concentration of 100 µg/mL. These compounds show better activity against
Pythium ultimum Trow than that of the control. Some of them on the other hand showed low activity
(below 40%) against Pythium ultimum Trow, and some of them can’t inhibit Pythium ultimum Trow. For
example, compounds 5b
(´11.11%), 5f
(
´55.56%) and 5k
(´88.89%) had no inhibitory activity against
Pythium ultimum Trow. On the contrary, these compounds increased the fungal growth. The control
zhongshengmycin also can’t inhibit the fungus Pythium ultimum Trow. Among the new compounds,
compounds 5d (75.33%) and 5h (75.89%) exhibited excellent control efficacy against Corynespora
cassiicola, which was better than that of control chlorothalonil (45.9%), while compounds 5a (44.49%),
5c (48.41%) and 5g (46.17%) displayed the same control efficacy as chlorothalonil (45.9%). None of
the title compounds exhibited any inhibition effect against Botrytis cinerea and Phytophthora infestans
(Mont.) De Bary, except for compound 5h, which displayed weak inhibition (21.38%) of Botrytis cinerea.
For the fungus Rhizoctonia solani, most of them had no inhibitory activity, although compound 5a
showed a control efficacy of 61.11%, which was similar to that of the most active control fungicide
validamycin (62.5%).
Table 1. The antifungal activity of the title compounds in vivo at 100 ppm (%).
Pythium
ultimum
Phytophthora
infestans
Corynespora
cassiicola
Botrytis
cinerea
Rhizoctonia
solani
No.
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
77.78
´11.11
11.11
55.56
66.67
´55.56
22.22
66.67
44.44
22.22
´88.89
77.78
0.0
´0.80
44.49
21.21
48.41
75.33
23.46
34.39
46.17
75.89
6.92
´9.97
´17.69
´10.94
´52.42
´29.27
´11.90
´45.67
21.38
´13.35
´32.16
´55.31
´36.98
61.11
35.00
31.67
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
5.92
5.36
0.04
´0.80
´0.80
1.44
´0.80
6.76
´0.80
´0.80
´0.80
32.71
´0.93
´0.93
Zhongshengmycin
Dimethomorph
Chlorothalonil
Fludioxonil
Validamycin
97.8
45.9
86.98
62.5
Note: Pythium ultimum Trow for tomato, Phytophthora infestans(Mont.) De Bary for tomato, Corynespora cassiicola
for cucumber, Botrytis cinerea for cucumber and Rhizoctonia solani for cucumber; All the data were determined
three times.
2.3. DFT Calculation and SAR
The total molecular energy and frontier orbital energy levels of compound 5h and penflufen are
listed in Table 2. Energy gap between HOMO and LUMO was calculated by B3LYP.
According to the frontier molecular orbital theory, the HOMO and LUMO are the most important
factors that affect the bioactivity [31,32]. HOMO has the priority to provide electrons, while the LUMO

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can accept electrons first. Thus a study of the frontier orbital energy can provide useful information
about the biological mechanism of action. We selected the compound 5h with the best antifungal
activity among the title compounds and the commercial drug penflufen as models to compare their
frontier molecular orbital. Taking the DFT results for example, the geometry of the framework of the
compound 5h is hardly influenced by the introduction of either the phenyl ring or the pyrazole ring
(Figure 2). The HOMO of the title compound is mainly located on the 3-Me phenyl ring, pyrazole ring
and amide bond, while, the LUMO of the title compound is located on the pyrazole ring, 3-Me phenyl
ring, phenyl ring, chlorine atom and amide bond. On the other hand, the HOMO of the penflufen
is mainly located on the phenyl ring, pyrazole ring, fluorine atom, methyl group and amide bond,
while the LUMO of the penflufen is located on the pyrazole ring, phenyl ring and amide bond. From
Figure 2, the electron transition ocurrs from the 3-methylbenzene ring, amide bond and pyrazole ring
to the N-phenyl ring in compound 5h, while the energy gap between the HOMO and LUMO is 0.16616
Hartree. On the contrary, the electron transition from the phenyl ring, pyrazole ring, fluorine atom,
methyl group and amide bond to the phenyl ring, pyrazole ring and amide bond in the compound
Penflufen, while the energy gap between the HOMO and LUMO is 0.17984 Hartree. The differences
between the two compounds are the electron transition orientation and energy gap. The fact that the
title compound has strong affinity suggests the importance of the frontier molecular orbital in the
π-π stacking or hydrophobic interactions. This also implies that substituted of phenyl ring had an
important impact on the antifungal activity. Furthermore, the MO combination provided meaningful
clues as to the structural features of this new family of fungicides that will be helpful in the design of
more potent compounds in the future: first the methyl group in the 3-position of pyrazole ring had no
impact on the antifungal activity; second for the halo group in the 5-position, a higher group negativity
is better; and third, the amide bond is necessary.
Table 2. Total energy and frontier orbital energy.
DFT
5 h
Penflufen
total/Hartree b
´1394.96053044
´0.21495
´1039.42133553
´0.21213
E
E
HOMO/Hartree
LUMO/Hartree
E
´0.04879
´0.03229
∆E a/Hartree
0.16616
0.17984
a
b
∆E = ELUMO´EHOMO
;
1 Hartree = 4.35974417 ˆ 10´18 J = 27.2113845 ev.
Figure 2. Frontier molecular orbitals of compound 5h and penflufen.

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The structural difference is also an important issue. For example, the amide group orientation
between the two compounds is opposite (Figure 3). We therefore speculate that this is the confirmation
required between the phenyl ring and pyrazole ring when they bind to the target receptor.
Figure 3. Overlay of energy-minimized structures of 5h and penflufen.
3. Experimental Section
3.1. General Information
All the chemical reagents were analytical grade or prepared in our lab. Melting points were
measured using an X-4 apparatus (Taike, Beijing, China) and were uncorrected. 1H-NMR and 13C-NMR
spectra were recorded on an Avance 500 MHz spectrometer (Bruker, Fallanden, Switzerland) using
CDCl3 as solvent. Mass spectra were determined on a LCQ Advantage LC/mass detector instrument
(Thermo Finnigan, Silicon Valley, CA, USA). Elemental analysis data of the title compounds were
obtainned by a 240C analyzer (Perkin-Elmer, Waltham, MA, USA).
3.2. Synthesis
3-Methyl-1-phenyl-1H-pyrazol-5(4H)-one (
1). Ethyl acetoacetate (13.0 g, 100 mmol) was added to a
solution of phenylhydrazine (10.8 g, 100 mmol) in ethanol (20 mL), then the mixture was refluxed
for 4 h, then the ethanol was removed under reduced pressure to give compound
(14.3 g, yield 82.6%,m.p.: 125–126 C).
1
as a yellow solid
˝
5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde (2) [33]. Phosphorus oxychloride (250 mmol) was
˝
added dropwise into N,N-dimethylformamide (100 mmol) at 0–5 C. After the mixture was stirred for
30 min, 3-methyl-1-phenyl-1H-pyrazol-5(4H) one ( , 5.22 g, 30 mmol) was added portionwise. Then
then mixture was heated to 120 C for another 1 h. The reaction mixture was poured slowly into
1
˝
crushed ice, and the precipi˝tated solid was filtered and dried, to give
2 as a light yellow solid (5.61 g,
yield: 85.0%, m.p.: 136–137 C).
5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxylic acid (3) [34]. 5-Chloro-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde ( , 5.5 g, 25 mmol) and potassium permanganate (4.74g, 30 mmol) were
2
added to water (50 mL) and refluxed under microwave irradiation for 0.5 h. The reaction mixture
was filtered, acidified to pH = 1 using HCl, to give 3 as a white solid that was filtered off and dried
˝
(5.6 g, yield: 95%,m.p.: 230–231 C).

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5-Chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl chloride (
4) [35]. To 5-chloro-3-methyl-1-phenyl-
1H-pyrazole-4-carboxylic acid (3, 7.50 mmol) thionyl chloride (30 mmol) was added and the mixture
was refluxed for 2 h. After the reaction is completed, the excess of thionyl chloride were evaporated to
give 4 as a yellow liquid that was used without further purification.
General Procedure for the Preparation of Pyrazole Amide Compounds 5a–m
To a solution of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbonyl chloride (4, 7 mmol) and
˝
Et3N (7.5 mmol) in THF (10 mL), a substituted aniline (7.50 mmol) was added dropwise under 0–5 C
for 1 h. Then the mixture was vigorously stirred at ambient temperature for 8 h, then evaporated under
reduced pressure, and subsequently the mixture was exacted with EtOAc. The organic layer was dried
over MgSO4 and evaporated. The residue was purified by chromatography on a silica gel column
˝
using petroleum ether (60–90 C) and ethyl acetate as the eluents to afford the title compounds. All the
compounds were synthesized according to this procedure (Scheme 2). All the data can be found in
Supplementary Materials (see Figures S1–S33 for more details).
Scheme 2. The synthetic route of title compounds. Reagents and Condition: i. EtOH, reflux, 4 h, 83%;
ii. POCl3/DMF, 0–5 ˝C to 120 ˝C, 1.5 h, 85%; iii. a. KMnO4 H2O, MW, 0.5 h; b. HCl, 95%; iv. SOCl2,
reflux, 2 h; v. RNH2, THF, Et3N, r.t., 8 h.
5-Chloro-N-(2,4-difluorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5a). Yellow solid, m.p.
˝
138–139 C, yield 94%, 1H-NMR
δ
: 2.62 (s, 3H, CH3), 6.89–6.94 (m, 2H, Ph-H), 7.49–7.51 (m, 1H, Ph-H),
7.53–7.54 (m, 4H, Ph-H), 8.21 (s, 1H, NH), 8.38–8.44 (m, 1H, Ph-H); 13C-NMR
δ: 14.66, 103.57, 111.19,
111.36, 112.79, 122.86, 122.93, 125.54, 126.12, 129.20, 129.27, 137.31, 152.34, 159.78; ESI-MS: 346.04
[M ´ H]´, 348.01 [M
´
H + 2]+, 347.97 [M + H]+, 349.97 [M + H + 2]+; Elemental anal. calculated for
C17H12ClF2N3O (%): C, 58.72; H, 3.48; N, 12.08; found: C, 58.90; H, 3.17; N, 12.00.
5-Chloro-3-methyl-N-(4-nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxamide (5b). Yellow solid, m.p.
˝
159–161 C, yield 98%, 1H-NMR
δ: 2.62 (s, 3H, CH3), 7.51–7.55 (m, 5H, Ph-H), 7.83–7.85 (m, 2H, Ph-H),
8.25–8.27 (d, J = 8.0 Hz, 2H, Ph-H), 8.38 (s, 1H, NH); ESI-MS: 355.04 [M
356.97 [M + H]+, 358.98 [M + H + 2]+; Elemental anal. calculated for C17H13ClN4O3 (%): C, 57.23; H,
3.67; N, 15.70; found: C, 57.44; H, 3.57; N, 15.65.
´
H]´, 356.97 [M ´ H + 2]+
,

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5-Chloro-N-(2,6-dichlorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5c). Yellow solid, m.p.
151–153 ˝C, yield 99%, 1H-NMR
δ: 2.61 (s, 3H, CH3), 7.20–7.24 (m, 1H, Ph-H), 7.42–7.43
(d,J = 4.0 Hz, 2H, Ph-H), 7.48–7.55 (m, 5H, Ph-H), 7.76 (s, 1H, NH); ESI-MS: 379.91 [M + H]+, 381.92
[M + H + 2]+; Elemental anal. For C17H12Cl3N3O (%), calculated: C, 53.64; H, 3.18; N, 11.04; found: C,
53.56; H, 3.17; N, 11.32.
˝
5-Chloro-3-methyl-1-phenyl-N-(o-tolyl)-1H-pyrazole-4-carboxamide (5d). Yellow solid, m.p. 132–133 C
,
1
yield 38.4%, H-NMR
δ
: 2.36 (s, 3H, CH3), 2.62 (s, 3H, CH3), 7.09–7.13 (m, 1H, Ph-H), 7.22–7.24
(d, J = 8.0 Hz, 2H, Ph-H), 7.49–7.54 (m, 5H, Ph-H), 7.85 (s, 1H, NH), 8.05–8.07 (m, 1H, Ph-H); 13C-NMR
δ
: 14.65, 18.13, 113.34, 122.87, 125.08, 125.53, 126.81, 128.61, 129.06, 129.20, 130.50, 135.66, 137.40, 152.40,
´
159.90; ESI-MS: 324.04 [M
anal. calculated for C18H16ClN3O (%): C, 66.36; H, 4.95; N, 12.90; found: C, 66.43; H, 4.89; N, 13.02.
´
H] , 326.11 [M
´
H + 2]+, 326.01 [M + H]+, 327.99 [M + H + 2]+; Elemental
5-Chloro-3-methyl-N-(4-methyl-2-nitrophenyl)-1-phenyl-1H-pyrazole-4-carboxamide (5e). Yellow solid, m.p.
1
187–188 ˝C, yield 71.5%, H-NMR
δ: 2.42 (s, 3H, CH3), 2.61 (s, 3H, CH3), 7.48–7.50 (m, 1H, Ph-H),
7.51–7.53 (m, 5H, Ph-H), 8.04 (s, 1H, Ph-H), 8.71–8.73 (m, 1H, Ph-H), 10.81 (s, 1H, NH); ESI-MS:
368.98 [M
H]´ , 370.95 [M H + 2]+, 371.01 [M + H]+ , 373.00 [M + H + 2]+; Elemental anal.
´
´
For C18H15ClN4O3 (%), calculated: C, 58.31; H, 4.08; N, 15.11; found: C, 58.23; H, 4.17; N, 14.99.
5-Chloro-N-(4-chloro-2-nitrophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5f). Yellow solid, m.p.
˝
163–165 C, yield 96%, 1H-NMR
δ
: 2.63 (s, 3H, CH3), 6.79–6.81 (m, 1H, Ph-H), 7.51–7.53 (m, 5H, Ph-H),
´
8.25–8.26 (m, 1H, Ph-H), 8.88–8.91 (m, 1H, Ph-H), 10.88 (s, 1H, NH); ESI-MS: 389.01 [M
[M
H + 2]+, 392.91 [M H + 4]+, 391.05 [M + H]+, 393.01 [M
Elemental anal. calculated for C17H12Cl2N4O3 (%): C, 52.19; H, 3.09; N, 14.32; found: C, 52.33; H, 3.13;
N, 14.15.
´
H] , 390.97
´
´
´
H + 2]+ , 394.99 [M + H + 4]+;
5-Chloro-˝N-(4-fluorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5g). Yellow solid, m.p.
1
154–156 C, yield 62.2%, H-NMR
δ
: 2.61 (s, 3H, CH3), 7.06–7.10 (m, ´2H, Ph-H), 7.50–7.55 (m, 5H,
Ph-H), 7.59–7.61 (m, 2H, Ph-H), 7.59 (s, 1H, NH); ESI-MS: 328.06 [M
[M + H]+, 332.02 [M + H + 2]+; Elemental anal. calculated for C17H13ClFN3O (%): C, 61.92; H, 3.97; N,
12.74; found: C, 61.90; H, 4.17; N, 12.65.
´
H] , 330.10 [M
´
H + 2]+, 329.96
˝
5-Chloro-3-methyl-1-phenyl-N-(m-tolyl)-1H-pyrazole-4-carboxamide (5h). Yellow solid, m.p. 78–81 C,
yield 99%, 1H-NMR
: 2.37 (s, 3H, CH3), 2.61 (s, 3H, CH3), 6.96–6.98 (m, 1H, Ph-H), 7.23–7.26 (m, 2H,
Ph-H), 7.36–7.38 (d, J = 8.0Hz, 1H, Ph-H), 7.51–7.53 (m, 5H, Ph-H), 7.91 (s, 1H, NH); 13C-NMR
: 14.55,
δ
δ
21.49, 113.36, 117.26, 120.84, 125.41, 125.54, 128.90, 129.08, 129.24, 137.41, 137.53, 139.07, 152.18, 159.91;
´
ESI-MS: 324.08 [M
´
H] , 326.01 [M
´
H + 2]+, 326.00 [M + H]+, 328.03 [M + H + 2]+; Elemental anal.
calculated for C18H16ClN3O (%): C, 66.36; H, 4.95; N, 12.90; found: C, 66.58; H, 4.86; N, 12.13.
5-Chloro-3-methyl-1-phenyl-N-(3-(trifluoromethyl)phenyl)-1H-pyrazole-4-carboxamide (5i). Yellow solid,
m.p. 97–98 ˝C, yield 72.5%, 1H-NMR
δ: 2.61 (s, 3H, CH3), 7.39–7.41 (m, 1H, Ph-H), 7.47–7.49 (m, 1H,
Ph-H), 7.50–7.54 (m, 5H, Ph-H), 7.80–7.82 (m, 1H, Ph-H), 7.95 (s, 1H, Ph-H), 8.14 (s, 1H, NH); ESI-MS:
´
378.02 [M
´
H] , 380.00 [M
´
H + 2]+, 380.02 [M + H]+, 381.93 [M+H+2]+; Elemental anal. calculated
for C18H13ClF3N3O (%): C, 58.72; H, 3.48; N, 12.08; found: C, 58.59; H, 3.45; N, 12.12.
5-Chloro-˝N-(4-chlorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5j). Yellow solid, m.p.
1
161–162 C, yield 78.1%, H-NMR
δ
: 2.52 (s, 3H, CH3), 7.24–7.26 (m, ´2H, Ph-H), 7.43–7.45 (m, 5H,
Ph-H), 7.49–7.51 (m, 2H, Ph-H), 7.89 (s, 1H, NH); ESI-MS: 343.92 [M
[M + H]+, 347.92 [M + H + 2]+; Elemental anal. calculated for C17H13Cl2N3O (%): C, 58.98; H, 3.78; N,
12.14; found: C, 59.11; H, 3.95; N, 12.19.
´
H] , 345.89 [M
´
H + 2]+, 345.91
5-Chloro-N-(4-methoxy-2-nitrophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5k). Yellow solid,
m.p. 157–159 ˝C, yield 66%, 1H-NMR
δ: 2.61 (s, 3H, CH3), 3.88 (s, 3H, CH3), 7.28–7.29 (m, 1H, Ph-H),
7.53–7.55 (m, 5H, Ph-H), 7.70 (s, 1H, Ph-H), 8.71–8.74 (m, 1H, Ph-H), 10.69 (s, 1H, NH) ; ESI-MS: 385.07

Molecules 2016, 21, 68
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[M
´
H]´, 387.14 [M
´
H + 2]+, 386.95 [M + H]+, 389.03 [M+H+2]+; Elemental anal. calculated for
C18H15ClN4O4 (%): C, 55.89; H, 3.91; N, 14.49; found: C, 55.98; H, 4.17; N, 14.38.
5-Chloro-3-methyl-1-phenyl-N-(p-tolyl)-1H-pyrazole-4-carboxamide (5l). Yellow solid, m.p. 115–116 C,
yield 73.7%, 1H-NMR
: 2.34 (s, 3H, CH3), 2.60 (s, 3H, CH3), 7.16–7.18 (m, 2H, Ph-H), 7.47–7.49 (m,
2H, Ph-H), 7.51–7.53 (m, 5H, Ph-H), 7.90 (s, 1H, NH); ESI-MS: 324.03 [M
326.02 [M + H]+, 328.05 [M + H + 2]+; Elemental anal. calculated for C18H16ClN3O (%): C, 66.36; H,
4.95; N, 12.90; found: C, 66.48; H, 5.13; N, 12.88.
˝
δ
´
H]´, 326.02 [M
´
H + 2]+,
5-Chlo˝ro-N-(3-chlorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide 5m. Yellow solid, m.p.
81–82 C, yield 88%, 1H-NMR
δ: 2.60 (s, 3H, CH3), 7.11–7.14 (m, 1H, Ph-H), 7.28–7.30 (m, 1H, Ph-H),
7.44–7.46 (m, 1H, Ph-H), 7.52–7.53 (m, 5H, Ph-H), 7.77 (s, 1H, Ph-H), 7.99 (s, 1H, NH); ESI-MS: 343.98
[M
H]´, 345.98 [M H + 2]+, 345.96 [M + H]+, 347.93 [M + H + 2]+; Elemental anal. calculated for
´
´
C17H13Cl2N3O (%): C, 58.98; H, 3.78; N, 12.14; found: C, 59.05; H, 3.87; N, 12.33.
3.3. Antifungal Activity
The antifungal activity of compounds 5a
(Mont.) De Bary, Corynespora cassiicola, Botrytis cinerea and Rhizoctonia solani was evaluated according
to reference [36 37]. A potted plant test method was adopted. Germination was induced by soaking
–5m against Pythium ultimum Trow, Phytophthora infestans
,
cucumber seeds in water for 2 h at 50 ˝C and then keeping the seeds moist for 24 h at 28 ˝C in an
incubator. When the radicles were 0.5 cm, the seeds were grown in plastic pots containing a 1:1 (v/v)
mixture of vermiculite and peat. Cucumber plants used for inoculations were at the stage of two
cotyledons, and tomato plants were five euphyllas. Tested compounds and commercial fungicides
were sprayed with a hand sprayer on the surface of the leaves and on a fine morning, at the standard
concentration of 100
(200 L). Dimethomorph, fludioxonil, chlorothalonil, validamycin, and zhongshengmycin were used as
controls. After 2 h, inoculations of Phytophthora infestans, Corynespora cassiicola and Botrytis cinerea were
carried out by spraying fungal spore suspension with 1
104 spore/mL, inoculation of Rhizoctonia
solani and Pythium ultimum were carried out by spraying mycelial suspension of 2
104 CFU/mL,
which was smashed with a T10 basic ULTRA-TURRAX® (IKA, Guangzhou, China). Each kind of
inoculum was sprayed at 300 L/plant. Each treatment was replicated four times. After inoculation,
µg/mL, each plant was sprayed with compounds and commercial fungicides
µ
ˆ
ˆ
µ
˝
˝
the plants were maintained at 18–30 C (mean temperature of 24 C and above 80% relative humidity
(RH)). The antifungal activity was evaluated when the non-treated plant (blank) fully developed
symptoms. The area of inoculated treated leaves covered by disease symptoms was assessed and
compared to that of nontreated ones to determine the average disease index. The relative control
efficacy of compounds compared to the blank assay was calculated via the following equation:
relative control efficacy p%q “ pCK´PTq{CK ˆ 100%
(1)
where CK is the average disease index during the blank assay and PT is the average disease index after
treatment during testing. All experiments were replicated three times.
3.4. Theoretical Calculations
According to the above crystal structure, a molecular unit was selected as the initial structure,
while the DFT-B3LYP/6-31G (d,p) methods in the Gaussian 03 package [38] were used to optimize
the structure of the title compound. Vibration analysis showed that the optimized structures were
in accordance with the minimum points on the potential energy surfaces, which means no virtual
frequencies, proving that the obtained optimized structures were stable. All the convergent precisions
were the system default values, and all the calculations were carried out on the DELL computer.

Molecules 2016, 21, 68
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4. Conclusions
Some interesting pyrazole amide derivatives were designed and synthesized. Their structures
were confirmed by NMR, MS and elemental analysis. The antifungal evaluation of the newly
synthesized pyrazole amide derivatives showed that among the tested compounds 5-chloro-N-(2,4-
difluorophenyl)-3-methyl-1-phenyl-1H-pyrazole-4-carboxamide (5a), 5-chloro-3-methyl-1-phenyl-
N-(o-tolyl)-1H-pyrazole-4-carboxamide (5d), 5-chloro-3- methyl-N-(4-methyl-2-nitrophenyl)-1-
phenyl-1H-pyrazole-4-carboxamide
(5e), 5-chloro-3-methyl-1-phenyl-N-(m-tolyl)-1H-pyrazole-
4-carboxamide (5h) and 5-chloro-3-methyl-1-phenyl-N-(p-tolyl)-1H-pyrazole-4-carboxamide (5l
)
showed good antifungal activity against Pythium ultimum. Interestingly, compound 5d and 5h
still exhibited good antifungal activity against Corynespora cassiicola. The best activity compound
5h was selected as a model and its frontier orbitals studied in comparison with the commercial
fungicide penflufen.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/
21/1/68/s1.
Acknowledgments: This work was supported financially by Zhejiang Provincial Science Foundation of China
(No. LY16C140007, LQ13B020003) and National Natural Science Foundation of China (No. 31401691, 21205109).
Author Contributions: Jin-Xia Mu, Yan-Xia Shi, Ming-Yan Yang, Zhao-Hui Sun carried out experimental work,
Jin-Xia Mu prepared the manuscript, Xing-Hai Liu, Bao-Ju Li designed the material and supervised the project.
Na-Bo Sun revised the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons by Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).