Synthesis and Biological Activity of Novel Pyrazol-5-yl-benzamide Derivatives as Potential Succinate Dehydrogenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.0c08094

To promote the discovery and development of new fungicides, a series of novel pyrazol-5-yl-benzamide derivatives were designed, synthesized by hopping and inversion of amide groups of pyrazole-4-carboxamides, and evaluated for their antifungal activities. The bioassay data revealed that compound 5IIc exhibited an excellent in vitro activity against Sclerotinia sclerotiorum with an EC50 value of 0.20 mg/L, close to that of commercial fungicide Fluxapyroxad (EC50 = 0.12 mg/L) and Boscalid (EC50 = 0.11 mg/L). For Valsa mali, compound 5IIc (EC50 = 3.68 mg/L) showed a significantly higher activity than Fluxapyroxad (EC50 = 12.67 mg/L) and Boscalid (EC50 = 14.83 mg/L). In addition, in vivo experiments proved that compound 5IIc has an excellent protective fungicidal activity with an inhibitory rate of 97.1% against S. sclerotiorum at 50 mg/L, while the positive control Fluxapyroxad showed a 98.6% inhibitory effect. The molecular docking simulation revealed that compound 5IIc interact with TRP173, SER39, and ARG43 of succinate dehydrogenase (SDH) through a hydrogen bond and p-πinteraction, which could explain the probable mechanism of the action between compound 5IIc and target protein. Also, the SDH enzymatic inhibition assay was carried out to further validate its mode of action. These results demonstrate that compound 5IIc could be a promising fungicide candidate and provide a valuable reference for further investigation.

Full text of DOI:10.1021/acs.jafc.0c08094

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Synthesis and Biological Activity of Novel Pyrazol-5-yl-benzamide
Derivatives as Potential Succinate Dehydrogenase Inhibitors
Wei Wang, Jianhua Wang, Furan Wu, Huan Zhou, Dan Xu,* and Gong Xu*
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ABSTRACT: To promote the discovery and development of new fungicides, a series of novel pyrazol-5-yl-benzamide derivatives
were designed, synthesized by hopping and inversion of amide groups of pyrazole-4-carboxamides, and evaluated for their antifungal
activities. The bioassay data revealed that compound 5IIc exhibited an excellent in vitro activity against Sclerotinia sclerotiorum with
an EC50 value of 0.20 mg/L, close to that of commercial fungicide Fluxapyroxad (EC50 = 0.12 mg/L) and Boscalid (EC50 = 0.11
mg/L). For Valsa mali, compound 5IIc (EC50 = 3.68 mg/L) showed a significantly higher activity than Fluxapyroxad (EC50 = 12.67
mg/L) and Boscalid (EC50 = 14.83 mg/L). In addition, in vivo experiments proved that compound 5IIc has an excellent protective
fungicidal activity with an inhibitory rate of 97.1% against S. sclerotiorum at 50 mg/L, while the positive control Fluxapyroxad
showed a 98.6% inhibitory effect. The molecular docking simulation revealed that compound 5IIc interact with TRP173, SER39,
and ARG43 of succinate dehydrogenase (SDH) through a hydrogen bond and p−π interaction, which could explain the probable
mechanism of the action between compound 5IIc and target protein. Also, the SDH enzymatic inhibition assay was carried out to
further validate its mode of action. These results demonstrate that compound 5IIc could be a promising fungicide candidate and
provide a valuable reference for further investigation.
KEYWORDS: succinate dehydrogenase inhibitors, pyrazolamide, antifungal activity, structure−activity relationships, molecular docking
Among the marketed SDH inhibitors, pyrazole-4-carbox-
amides occupies a great proportion.15 For example, Fluxapyr-
oxad is one of the widely used pyrazole-4-carboxamide
fungicides with broad-spectrum antifungal activity,16 high
efficiency, and low toxicity.17 As shown in Figure 1, all of
these pyrazole-4-carboxamides SDH inhibitors structurally
contain an amide function and a difluoromethyl or
trifluoromethyl group as a pharmacophoric motif.
In a pesticide or a pharmaceutical drug development
process, the design strategies by scaffold hopping18,19 and
bioisosterism20 have been extensively used for lead compound
optimization, which can provide compounds with novel
skeletons, improved potency, reduced toxicity, enhanced
physicochemical properties, more favorable absorption,
metabolism, distribution, and excretion (ADME) profiles.21−24
Other novel techniques, such as high-performance computa-
tion, structure biology, and artificial intelligence, have also
been widely utilized.25−27 For example, some high potent SDH
inhibitors were discovered by Yang and Zhu using the
pharmacophore-linked fragment virtual screening (PFVS)
method.7,28−30 In this work, to develop novel pyrazolamide
SDH inhibitors, pyrazole-4-carboxamides were used as lead
compounds, and we attempted to transfer the amide groups
INTRODUCTION
■
Plant pathogenic fungi may cause crop yield losses,1,2 which
not only causes huge economic losses3 but also threatens
global food security.4 Chemical fungicide protection is one of
the most economical and effective measures to control fungal
diseases at present.5 Succinate dehydrogenase (SDH) inhib-
itors, which can inhibit the growth of pathogens through
disrupting the mitochondrial tricarboxylic acid cycle and the
respiration chain,6,7 have been widely used for crop protection
against plant diseases due to their broad-spectrum and
excellent antifungal activities.8,9 Carboxin, the first commercial
SDH inhibitor, was invented by Uniroyal in 1969.10 To date,
there are 24 SDH inhibitors in the market, which are divided
into 11 categories, including oxathilin carboxamides (Carbox-
in, 1969), furan carboxamides (Fenfuram, 1974), phenyl-
benzamides (Flutolanil, 1986), thiazole-carboxamides (Thi-
fluzamide, 1997), pyridinyl-ethyl benzamides (Fluopyram,
1997), pyridine carboxamides (Boscalid, 2003), N-methoxy-
(penylethyl)-pyrazole-carboxamides (Pydiflumetofen, 2016),
pyrazine carboxamides (Pyrzziflumid, 2018), phenyl-oxo-
ethyl-thiophene amides (Isofetamid, 2005), N-cyclopropyl-
N-benzyl-pyrazole-carboxamides (Isoflucypram, 2020), and
pyrazole-4-carboxamides (Fluxapyroxad, 2012)11 (Flubene-
teram, 2020).12 Nevertheless, with the repeated use and
misuse of SDH inhibitors, fungal resistance has become an
increasingly serious problem. Also, due to a lack of effective
alternative control options, plant pathogenic fungi are still
challenging to control.13 Therefore, it is an urgent and unmet
need to develop novel SDH inhibitors with unique skeletons
and improved fungicidal activity.14
Received: December 25, 2020
Revised: March 25, 2021
Accepted: May 5, 2021
Published: May 14, 2021
https://doi.org/10.1021/acs.jafc.0c08094
J. Agric. Food Chem. 2021, 69, 5746−5754
© 2021 American Chemical Society
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Figure 1. Representative chemical structures of pyrazole-containing SDHIs.
Figure 2. Design strategy for the target compounds.
from the 4th position to the 5th position of the pyrazole by
scaffold hopping and inverse the amide groups based on the
concept of bioisosterism. A series of novel N-pyrazol-5-yl-
benzamide derivatives were designed and synthesized by a
simple synthetic method (Figure 2). Structurally, pyrazol-5-
amine and benzoic acid were spliced through an amide bond,
and therefore the two types of SDHI phenylbenzamides and
pyrazole-4-carboxamides were well combined. All of the target
compounds were identified by 1H NMR, 13C NMR, and high-
resolution mass spectrometry (HRMS). The in vitro fungicidal
activities of these compounds were evaluated against five
common plant pathogenic fungi, and in vivo fungicidal
activities of compounds 5Ic and 5IIc with high antifungal
activities were tested against Sclerotinia sclerotiorum. Further-
more, molecular docking and SDH enzymatic inhibition of
compound 5IIc were evaluated to validate its mode of action.
MATERIALS AND METHODS
■
Instruments and Chemicals. All chemicals (reagent grade) were
purchased from commercial sources (Energy, Shanghai, China). All of
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Figure 3. General synthesis of compounds 5. Reagents and conditions: (a) NaH, THF, 80 °C; (b) EtOH, 80 °C; and (c) Et3N, DCM, 0 °C.
1
the H and 13C NMR spectra were measured on a Bruker AV 300,
400, 500, or 600 spectrometer or an Agilent DD2 600 Hz
spectrometer with CDCl3 or DMSO-d6 as the solvent and
tetramethylsilane as the internal standard. Chemical shifts were
reported in ppm (δ). Electrospray ionization mass spectrometry (ESI-
MS) spectra were carried out on a Mariner System 5304 mass
spectrometer. Melting points (mp) were recorded on a WRS-1B
melting point apparatus (Jingsong, Shanghai, China) and were
uncorrected. The crystal structure was recorded on a Bruker APEX-II
CCD diffraction meter. The microscopic morphology of the fungal
hyphae was observed using a scanning electron microscope (Hitachi,
S-3400 N, Tokyo, Japan). Molecular docking studies were performed
with Discovery Studio 4.0.
room temperature for 3 h. Water was added and the resulting mixture
was extracted with dichloromethane (3 × 5 mL). The combined
organic layers were washed with brine (3 × 5 mL), dried over
anhydrous MgSO4, and concentrated in vacuo. The crude product
was purified by silica gel flash chromatography (petroleum ether/ethyl
acetate = 3:1) to afford compounds 5Ia−5IIq. The physical data of
5Ia−5IIq in detail are provided in the Supporting Information.
Crystal Structure Determination. Crystal structure determi-
nation of compound 5Ic was carried out on a Bruker APEX-II CCD
equipped with graphite monochromated MoKa (l 1/4 0.71073 Å)
radiation (Figure 4). The structure was solved by direct methods and
Fungi. The plant pathogenic fungi (S. sclerotiorum, Physalospora
piricola, Valsa mali, FusaHum graminearum sehw., and Botrytis cinerea
pers) were provided by the College of Plant Protection, Northwest
A&F University (Yangling, China).
Synthetic Procedures. The synthetic routes of the target
compounds 5Ia−5IIp are outlined in Figure 3.31,32
General Synthesis Procedure for Intermediate 2. To a 50 mL
round-bottomed flask, acetonitrile (10 mmol), substituted ethyl
acetate 1 (10 mmol), NaH (10 mmol), and tetrahydrofuran (THF)
(30 mL) were added. The reaction flask was put into a preheated oil
bath (80 °C) and monitored by TLC (thin layer chromatography)
until the reaction was completed. Then, the reaction was cooled to
room temperature, hydrochloric acid (10 mL, 12 mol/L) was added,
and the resulting mixture was extracted with ethyl acetate (3 × 20
mL). The organic layers were combined and dried over anhydrous
Na2SO4. The solvent was removed under reduced pressure to afford
the residue, which was purified by flash column chromatography to
give the intermediate 2 in a 81−92% yield.
General Synthesis Procedure for Intermediate 3. Intermedi-
ate 2 (10 mmol) was added to a solution of methylhydrazine sulfate in
ethanol (15 mL). The mixture was heated at 80 °C for 4 h. The
reaction was cooled to room temperature and concentrated in vacuo.
The crude product was purified by silica gel flash chromatography
(petroleum ether/ethyl acetate = 5:1) to afford the intermediate 3.
1-Methyl-3-(trifluoromethyl)-1H-pyrazol-5-amine (Intermediate
3a). White solid; yield: 95%; mp: 96.3−97.0 °C; 1H NMR (500 MHz,
chloroform-d) δ 5.76 (s, 1H, Ar−H), 3.67 (s, 3H, N−CH3), 3.58 (s,
2H, NH2); 13C NMR (126 MHz, chloroform-d) δ 145.5, 140.7 (q, J =
37.8 Hz), 121.3 (q, J = 268.3 Hz), 89.5, 34.8; 19F NMR (376 MHz,
dimethyl sulfoxide (DMSO)-d6) δ −61.1; HRMS (ESI) m/z: [M +
H]+ calculated for C5H7F3N3: 166.0587, found 166.0588.
Figure 4. X-ray crystal structure of compound 5Ic (CCDC number:
2005916, displacement ellipsoids are drawn at the 50% probability
level).
refined on F2 by full-matrix least-squares methods using SHELXTL.
All nonhydrogen atoms were refined with anisotropic thermal
parameters. All hydrogen atoms with the exception of those on
nitrogen atoms were geometrically fixed and refined using a riding
model. X-ray diffraction data and the refinement are shown in the
Supporting Information.
Biological Assay. In Vitro Fungicidal Activities. All synthesized
compounds were screened for their in vitro antifungal activities
against S. sclerotiorum (Lib.) de bary, P. piricola, V. mali, F.
graminearum sehw., and Botrytis cinereal pers at 50 mg/L for the
preliminary screening according to a mycelial growth inhibition
method.33,34
In Vivo Fungicidal Activities against S. sclerotiorum. The Brassica
napus L. leaves of rape were collected from the Key Laboratory of
Botanical Pesticide R&D of Northwest A&F University. For
protective activity assay, healthy leaves of B. napus L. were sprayed
with the target compounds (50 mg/L), respectively, and then
cultivated at 25 °C for 24 h before inoculation with S. sclerotiorum.
For curative activity assay, healthy leaves of B. napus L. were sprayed
with the target compounds (50 mg/L), respectively, and then
cultivated at 25 °C for 24 h after inoculation with S. sclerotiorum.
Effect of 5IIc on the Mycelial Morphology of S. sclerotiorum. S.
sclerotiorum grew in Fries medium for 3 days. Fresh fungus dishes (5
mm in diameter) were made from the edge of the colonies. The
mycelia were inoculated on PDA medium plates containing no
compound (negative control) and 5IIc with a concentration of 1 mg/
L. Then, the mycelia were cultured at 25 °C for 2 days. The mycelial
3-(Difluoromethyl)-1-methyl-1H-pyrazol-5-amine (Intermediate
1
3b). Yellow solid; yield: 31.6%; mp: 29.9−32.6 °C; H NMR (500
MHz, DMSO-d6) δ 6.66 (t, J = 55.1 Hz, 1H, CHF2), 5.47 (s, 1H, Ar−
H), 5.39 (s, 2H, NH2), 3.54 (s, 3H, CH3); 13C NMR (126 MHz,
DMSO-d6) δ 148.6, 144.2 (t, J = 28.1 Hz), 112.6 (t, J = 230.7 Hz),
85.3, 34.9; 19F NMR (376 MHz, DMSO-d6) δ −110.3; HRMS (ESI)
m/z: [M + H]+ calculated for C5H8F2N3: 148.0681, found 148.0681.
General Synthetic Procedure for the Target Compounds
5Ia−5IIq. Intermediate 3 (1 mmol) and compound 4 (1 mmol) were
dissolved in dichloromethane (3 mL), and triethylamine (0.55 mL)
was added into the solution at 0 °C. Then, the mixture was stirred at
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a
Table 1. In Vitro Fungicidal Activities (Inhibition Rate %) of All Compounds at 50 mg/L
b
c
d
e
f
compd.
5Ia
5Ib
5Ic
5Id
5Ie
5If
5Ig
5Ih
5Ii
5Ij
5Ik
5Il
5Im
5In
5Io
R1
R2
S. s.
P. p.
V. m.
F. g.
B. c.
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
H
13.33 1.6
45.71 2.8
100
55.24 1.3
22.86 1.4
87.14 3.2
71.33 2.1
<5.0
55.91 1.0
22.81 0.2
58.06 1.8
24.73 1.9
25.81 2.1
40.43 1.0
29.35 1.9
24.73 1.1
30.11 1.0
39.78 2.4
24.73 3.3
39.78 1.0
38.71 1.8
12.90 1.9
35.48 1.9
25.81 1.8
61.2 0.7
39.78 1.9
78.71 1.9
35.48 1.8
12.90 1.9
31.20 1.0
25.81 1.9
17.20 3.8
43.24 2.1
34.41 1.9
17.20 1.1
33.33 1.0
24.73 2.1
35.21 4.3
48.38 3.3
38.48 1.3
45.16 2.3
59.39 0.6
55.00 1.4
61.67 3.6
77.50 2.5
52.12 0.9
45.00 1.4
72.50 3.3
65.15 3.0
52.50 1.4
62.50 0.1
12.50 3.3
46.67 1.9
61.66 2.2
53.33 3.6
50.00 2.4
61.67 1.9
53.33 0.4
70.00 0.8
70.83 1.4
95.00 0.4
74.16 1.7
65.00 1.4
80.20 0.5
78.33 0.7
51.67 0.8
77.40 1.4
65.00 2.8
32.55 3.8
61.21 1.5
56.67 0.8
44.16 2.2
88.83 0.8
76.28 2.2
65.83 1.5
78.67 0.7
29.43 4.1
23.69 0.9
55.48 2.4
32.87 2.1
22.96 4.3
53.76 2.4
27.12 1.0
<5.0
55.48 2.4
13.91 3.5
18.27 1.6
54.83 1.1
33.73 2.3
15.45 1.9
58.49 1.1
36.95 0.9
22.83 1.9
33.72 1.5
43.06 1.3
29.31 1.6
42.58 1.4
51.22 1.1
44.40 1.9
22.83 1.8
32.62 1.7
33.66 2.6
<5.0
31.25 1.7
26.12 2.1
43.48 0.9
50.03 1.6
41.76 1.2
37.86 2.5
17.78 3.6
27.78 1.2
65.55 1.1
36.67 1.9
21.11 0.9
62.56 1.1
46.67 1.9
20.10 2.0
58.89 1.1
40.00 0.3
16.77 2.6
53.33 3.3
47.78 1.1
11.12 3.0
36.67 0.4
22.35 1.5
32.22 1.1
23.33 1.8
89.33 1.9
40.00 2.0
51.11 1.1
71.28 0.2
62.22 2.1
34.44 1.1
55.55 0.7
40.00 0.2
13.47 3.5
51.14 1.8
32.22 2.2
34.44 1.1
77.78 0.4
53.33 2.0
86.91 1.1
82.67 0.7
2-CH3
3-CH3
4-CH3
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
2-OCH3
3-OCH3
4-OCH3
2-CF3
3-CF3
4-CF3
H
2-CH3
3-CH3
4-CH3
2-Cl
77.14 2.1
47.14 0.8
23.16 3.7
65.71 1.1
37.14 3.3
48.10 1.6
83.33 2.2
77.10 0.8
21.42 1.2
25.41 1.3
100
63.33 2.2
26.10 0.7
98.67 2.1
85.71 2.8
37.14 1.4
94.76 0.8
47.62 2.2
40.00 1.3
88.57 1.4
47.14 6.5
61.00 2.8
96.19 0.8
90.95 0.7
100
5Ip
5IIa
5IIb
5IIc
5IId
5IIe
5IIf
5IIg
5IIh
5IIi
5IIj
5IIk
5IIl
5IIm
5IIn
5IIo
5IIp
Boscalid
Fluxapyroxad
3-Cl
4-Cl
2-F
3-F
4-F
2-OCH3
3-OCH3
4-OCH3
2-CF3
3-CF3
4-CF3
100
38.33 1.0
a
b
c
d
e
The data are the mean values of triplicate experiments. S. s. Sclerotinia sclerotiorum. P. p. Physalospora piricola. V. m. Valsa mali. F. g. FusaHum
f
graminearum sehw. B. c. Botrytis cinerea pers.
cell wall structures of S. sclerotiorum at the top of each treated colonies
were selected and observed under an S-3400 N scanning electron
microscope (SEM) (Hitachi, Ltd., Tokyo, Japan).
characterization data are provided in the Supporting
Information.
Fungicidal Activities and SAR Discussion. The
fungicidal activities of all synthesized compounds were
evaluated against five fungi including S. sclerotiorum, P. piricola,
V. mali, F. graminearum sehw., and B. cinereal, where
Fluxapyroxad and Boscalid were used as positive controls.
The results are presented in Table 1, and the EC50 values of
the selected compounds are shown in Table 2.
As shown in Table 1, most of the compounds showed
antifungal activity against five kinds of phytopathogenic fungi
at 50 mg/L, and some of them displayed good fungicidal
activity against specific fungi. Among them, nine target
compounds displayed good antifungal activities against S.
sclerotiorum (inhibition rate > 80%) at 50 mg/L. Compounds
5If, 5Io, 5IIf, 5IIi, 5IIl, 5IIo, and 5IIp showed fungicidal
activities of 87.14, 83.33, 98.67, 94.76, 88.57, 96.19, and
90.95%, respectively. In addition, compounds 5Ic and 5IIc
showed excellent fungicidal activity toward S. sclerotiorum with
a 100% inhibition rate, which was same as those of
Fluxapyroxad and Boscalid (100%). As for P. piricola, the
two compounds 5IIa and 5IIc have higher antifungal activities
than Fluxapyroxad (59.36%). For V. mali, three target
Molecular Docking. The crystal structures of succinate dehydro-
genase (PDB entry: 2FBW) were retrieved from the Protein Data
Bank. The molecular docking procedure was performed using the
CDOCKER protocol for receptor−ligand interaction section of DS
4.0 (Discovery Studio 4.0, Accelrys, Inc., San Diego, CA).35
SDH Enzyme Assay In Vitro. The SDH enzyme activity of 5IIc and
Fluxapyroxad were determined using a succinate dehydrogenase
assay kit (Mlbio, ml076573). S. sclerotiorum was grown in the
sterilized Fries medium for 3 days and then treated with different
concentrations of 5IIc and Fluxapyroxad, separately. The SDH
enzyme activity was measured after 24 h of treatment with the
selected compounds, and the absorbance value was recorded at 600
nm using a microplate reader. Compounds were tested at five
different concentrations with three replicates, and the IC50 value was
calculated by SPSS 24.0.
RESULTS AND DISCUSSION
■
Synthesis of Target Compounds. A series of novel
pyrazol benzamide derivatives were synthesized, and the
general pathway is outlined in Figure 3. The chemical
structures of all synthesized compounds were confirmed by
1H NMR, 13C NMR, and HRMS (ESI) studies, and
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Table 2. Fungicidal Activities with EC50 against S. sclerotiorum and V. mail of the Selected Compounds
a
fungi
compd.
R1
R2
EC50
95% CI
regression equation
R2
S. sclerotiorum
5Ic
5If
5Ig
5Ii
5Io
5Ip
5IIc
5IIf
5IIg
5IIi
5IIo
5IIp
Boscalid
Fluxapyroxad
5Ic
5If
5Ig
5Ii
5Io
5Ip
5IIc
5IIf
5IIg
5IIi
5IIo
5IIp
Boscalid
Fluxapyroxad
CF3
CF3
CF3
CF3
CF3
CF3
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
3-CH3
3-Cl
4-Cl
1.19
3.02
21.22
5.63
7.78
9.24
0.20
2.62
7.36
2.68
4.64
5.07
0.11
0.12
8.30
6.46
10.08
13.51
9.65
21.19
3.68
4.11
6.07
6.12
5.01
8.08
14.83
12.67
0.92−1.48
1.47−4.58
17.40−26.85
3.90−7.88
5.53−10.51
7.76−10.87
0.16−0.25
2.08−3.20
5.93−9.06
1.95−3.42
3.83−5.51
4.06−6.19
0.07−1.13
0.07−0.17
5.67−11.22
0.91−9.50
7.63−12.97
8.44−24.57
5.57−14.88
13.16−47.01
2.37−4.98
2.48−5.72
3.40−8.81
4.40−8.38
3.12−6.18
6.51−9.77
8.33−30.32
9.20−20.12
y = −0.0862 + 1.1564x
y = −0.4204 + 0.8772x
y = −1.8741 + 1.4125x
y = −0.5908 + 0.7864x
y = −0.7776 + 0.8729x
y = −1.6679 + 1.7273x
y = 0.9094 + 1.3097x
y = −0.7511 + 1.3582x
y = −1.0354 + 1.1944x
y = −0.5298 + 1.2383x
y = −1.0478 + 1.5713x
y = −0.9140 + 1.2958x
y = 1.0464 + 1.0523x
y = 0.6599 + 0.7285x
y = −0.7939 + 0.8639x
y = −0.9509 + 1.1737x
y = −1.0499 + 1.0462x
y = −0.7060 + 0.6244x
y = −0.5928 + 0.6022x
y = −0.7125 + 0.5373x
y = −0.6659 + 1.1747x
y = −0.6079 + 0.9898x
y = −0.5732 + 0.7316x
y = −0.6689 + 0.8497x
y = −0.6830 + 0.9763x
y = −1.2658 + 1.4136x
y = −0.5625 + 0.4802x
y = −0.9514 + 0.8626x
0.978
0.953
0.981
0.998
0.994
0.972
0.991
0.955
0.996
0.996
0.963
0.992
0.990
0.962
0.956
0.964
0.986
0.976
0.993
0.956
0.968
0.965
0.954
0.998
0.965
0.951
0.968
0.998
3-F
3-CF3
4-CF3
3-CH3
3-Cl
4-Cl
3-F
3-CF3
4-CF3
V. mail
CF3
CF3
CF3
CF3
CF3
CF3
CHF2
CHF2
CHF2
CHF2
CHF2
CHF2
3-CH3
3-Cl
4-Cl
3-F
3-CF3
4-CF3
3-CH3
3-Cl
4-Cl
3-F
3-CF3
4-CF3
a
Confidence interval.
Table 3. In Vivo Activity of the Target Compounds against
a
S. sclerotiorum-Infected B. napus L. Leaves at 50 mg/L
protective activity
curative activity
control
control
diameter of
lesions (cm)
efficacy
diameter of
lesions (cm)
efficacy
compd.
5Ic
5IIc
Fluxapyroxad
CK
(%)
(%)
1.1 0.2
0.6 0.1
0.5 0.1
4.0 0.3
82.8
97.1
98.6
3.7 0.4
1.1 0.1
0.8 0.1
4.3 0.2
15.8
84.2
92.1
a
Data are given as the mean of three experiments.
compounds did not show high inhibition activities (less than
60%), but some of them were more active than Fluxapyroxad
(38.33%). Notably, compounds 5Ic and 5IIc exhibited a
broader fungicidal spectrum than other compounds, in which
the 3-methyl phenyl moiety seems to be an important
functional group for high activity.
To further determine the fungicidal potency and probe the
SAR of these novel derivatives, several compounds with
superior fungicidal activities were selected for further study.
Their EC50 values against S. sclerotiorum and V. mail are shown
in Table 2. In general, the compounds bearing the 3-
difluoromethyl group of a pyrazole ring showed better
fungicidal activities than that with the 3-trifluoromethyl
group. For example, 5IIc (EC50 = 0.20, 3.68 mg/L) was
more active than 5Ic (EC50 = 1.19, 8.30 mg/L) and 5IIo (EC50
= 4.64, 5.01 mg/L) was more active than 5Io (EC50 = 7.78,
9.65 mg/L) against S. sclerotiorum and V.mali, respectively.
Figure 5. In vivo activity of the target compounds against S.
sclerotiorum-infected B. napus L. leaves. (A) Protective activity and
(B) curative activity.
compounds exhibited higher activities than Fluxapyroxad
(78.67%), and compound 5IIc displayed the best fungicidal
activity of 95.00%. However, for B. cinerea, only compound
5IIc (89.33%) displayed higher activity than Fluxapyroxad
(82.67%). Unfortunately, for F. graminearum sehw., most
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Figure 6. Scanning electron micrographs of the hyphae from the colony of S. sclerotiorum. (A) Partial enlarged view of the untreated hypha, (B)
partial enlarged view of the hypha treated with 5IIc (1 mg/L), (C) overall image of the untreated hypha, and (D) overall image of the hypha
treated with 5IIc (1 mg/L).
Figure 7. Binding model of 5Ic (A), 5IIc (B), and Fluxapyroxad (C) in the active site of SDH (PDB entry: 2FBW). The H bond is displayed as a
line and π interaction is displayed as a solid yellow line.
Table 4. CDOCKER_INTERACTION_ENERGY (kcal/mol) Obtained from the Docking Study of All Synthesized
Compounds by the CDOCKER Protocol
compd.
R1
R2
CDOCKER_INTERACTION_ENERGY (kcal/mol)
5Ic
5IIc
Fluxapyroxad
CF3
CHF2
3-CH3
3-CH3
−35.8619
−37.1253
−39.1853
Meanwhile, the compounds with meso-substitution of the
phenyl ring showed better fungicidal activities than that with
para-substitution. For instance, 5If (EC50 = 3.02, 6.46 mg/L)
was more active than 5Ig (EC50 = 21.22, 10.08 mg/L) and
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Table 5. SDH Enzymatic Inhibition Activity (IC50)
a
compd.
5IIc
Fluxapyroxad
IC50 (mg/L)
95% CI
regression equation
R2
0.39
0.23
0.19−1.18
0.10−0.74
y = −1.0902 + 0.4202x
y = −0.8291 + 0.3519x
0.963
0.969
a
Confidence interval.
5IIo (EC50 = 4.64, 5.01 mg/L) was more active than 5IIp
(EC50 = 5.07, 8.08 mg/L) against S. sclerotiorum and V. mali,
respectively. Notably, the fungicidal activity of compound 5IIc
(EC50 = 0.20 mg/L) was close to those of Fluxapyroxad (EC50
= 0.12 mg/L) and Boscalid (EC50 = 0.11 mg/L) against S.
sclerotiorum. Meanwhile, 5IIc (EC50 = 3.68 mg/L) showed
significantly higher activity than Fluxapyroxad (EC50 = 12.67
mg/L) and Boscalid (EC50 = 14.83 mg/L) against V. mali.
When R1 is the difluoromethyl group and R2 is fixed at the
meso-position of the phenyl ring, the influence of R2
substituents was compared, and the results showed that the
meso-methyl group increases the inhibition activity on S.
sclerotiorum and V. mali, and the compound 5IIc (EC50 = 0.20,
3.68 mg/L) showed the best antifungal activity. An electro-
negative group (Cl or CF3) at the meso-position of the phenyl
ring could decrease the inhibition property. For the compound
5IIf (EC50 = 2.62, 4.11 mg/L) that contains a Cl substituent,
the activities are higher than that of 5IIo (EC50 = 4.64, 5.01
mg/L) with a trifluoromethyl group. The same effects were
also shown for compounds 5Ia−p, in which R1 is the
trifluoromethyl group.
Å), similar to the binding mode of Fluxapyroxad in 2FBW
(angle = 154.6°, distance = 1.91 Å). In addition, the fluorine
atom of compounds 5Ic and 5IIc formed H bonds with SER39
(5Ic, angle = 109.4°, distance = 2.40 Å and angle = 145.1°,
distance = 2.00 Å; 5IIc angle = 110.6°, distance = 2.27 Å). As
presented in Figure 7 A/B/C-2, the phenyl tail of
Fluxapyroxad formed the π−π interaction and p−π con-
jugation with TYR58 and ARG43, respectively. Also, the
pyrazole rings of 5IIc and Fluxapyroxad formed the p−π
conjugation with ARG43. In addition, the CDOCKER_IN-
TERACTION_ENERGY for docking was in the following
order: Fluxapyroxad (−39.1853 kcal/mol) > 5IIc (−37.1253
kcal/mol) > 5Ic (−35.8619 kcal/mol) (Table 4), which could
explain the observation that 5Ic (R1 = CF3) has lower activity
than 5IIc (R1 = CHF2).
SDH Enzymatic Inhibition Activity. To further prove the
possible antifungal mechanism of the novel pyrazol-5-yl-
benzamide derivatives, compound 5IIc with promising
fungicidal activity was selected and evaluated for SDH
enzymatic inhibition determination for its target site validation.
As shown in Table 5, 5IIc exhibited significant SDH inhibition
with an IC50 value of 0.39 mg/L, which is the same level of
inhibitory activity as Fluxapyroxad (IC50 = 0.23 mg/L). These
IC50 values are consistent with the order of their CDOCK-
ER_INTERACTION_ENERGY, which may be related to the
fact that 5IIc does not form the π−π interaction with TYR58.
The results indicate that SDH is a possible target enzyme of
the synthesized compound 5IIc, and its binding mode is not
exactly the same as that of the marketed SDHI Fluxapyroxad,
which may provide some valuable clues for further optimizing
the SDHIs and solving the drug-resistance problem.
In summary, a series of novel pyrazol-5-yl-benzamide
derivatives were designed, synthesized, and evaluated for
their antifungal activities against five fungi (S. sclerotiorum, P.
piricola, V. mali, F. graminearum sehw., and B. cinereal). The
antifungal bioassay discovered a highly active compound 5IIc
with a broad spectrum of excellent in vitro fungicidal activity
and better in vivo efficacy against S. sclerotiorum. The
molecular docking for SDH target validation indicated that
compound 5IIc exhibited similar properties as that of the
SDHI positive control Fluxapyroxad. These results demon-
strate that compound 5IIc could be a promising fungicide
candidate and provide a valuable reference for further
investigation.
The SAR of the selected compounds can be summarized as
follows: (1) the compounds bearing a difluoromethyl group at
the 3 position of pyrazole showed better fungicidal activities
than those with a trifluoromethyl group and (2) the
compounds with the meso-substitution of the phenyl ring
may be essential for enhancing antifungal activity.
In Vivo Fungicidal Activities. In vivo fungicidal activity of
the target compounds against S. sclerotiorum was carried out on
B. napus L. leaves. The efficacy of the protective and curative
activity is shown in Figure 5. The compounds 5Ic and 5IIc
showed promising protective activities of 82.8% and 97.1%,
respectively. Notably, compound 5IIc showed apparent
antifungal effects comparable to Fluxapyroxad (98.6%) at a
concentration of 50 mg/L. In curative activity assay, unlike 5Ic
with low curative activity (15.8%), compounds 5IIc and
Fluxapyroxad displayed significant efficacies of 84.2 and
92.1%, respectively. Thus, remarkable differences existed
between the treated and untreated groups for disease control
experiments in the greenhouse cole leaves. In vivo fungicidal
assay exhibited the same tendency as the data acquired using
the mycelial growth inhibition assay in vitro (Table 3).
Scanning electron micrographs (SEM) of S. sclerotiorum
revealed morphological changes in the fungal cell surface. As
shown in Figure 6, the surface of S. sclerotiorum cells in the
untreated groups (A) and (C) was relatively smooth, regular,
and even the pores could be seen, whereas when treated with
5IIc (B) and (D) at 1 mg/L, there was an obvious shrinkage.
This showed that 5IIc had a certain degree of damage to the
cell wall of S. sclerotiorum.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Molecular Docking. The binding modes of compounds
5Ic, 5IIc, and Fluxapyroxad with SDH(PDB: 2FBW) are
shown in Figure 7. Both the pyrazole rings of 5Ic and 5IIc
were embedded into the binding pocket, and the oxygen of
their amide bonds formed H bonds with TRP173 (5Ic, angle =
145.1°, distance = 2.0 Å; 5IIc, angle = 128.7°, distance = 2.0
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c08094.
The X-ray crystallography data of 5Ic and physical and
spectroscopic data of compounds 5Ia−5IIp (PDF)
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AUTHOR INFORMATION
■
Corresponding Authors
Dan Xu − College of Chemistry & Pharmacy, Northwest A&F
University, Yangling 712100, Shaanxi, China; State Key
Laboratory of Crop Stress Biology for Arid Areas, Key
Laboratory of Botanical Pesticide R&D in Shaanxi Province,
Shaanxi Key Laboratory of Natural Products & Chemical
Biology, Yangling 712100, Shaanxi, China; Email: danxu@
nwafu.edu.cn
Gong Xu − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China; State Key
Laboratory of Crop Stress Biology for Arid Areas, Key
Laboratory of Botanical Pesticide R&D in Shaanxi Province,
Shaanxi Key Laboratory of Natural Products & Chemical
Biology, Yangling 712100, Shaanxi, China; orcid.org/
0000-0002-4305-4370; Email: gongxu@nwafu.edu.cn
Authors
Wei Wang − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Jianhua Wang − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Furan Wu − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
Huan Zhou − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, China
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c08094
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (21801207), the Department of Science
and Technology of Shaanxi Province (2021NY-140, 2021JQ-
157), the Program for Science & Technology Innovation Team
of Shaanxi Province (2020TD-035), and the Scientific
Research Foundation of Northwest A&F University. The
authors thank Prof. Zhi-qing Ji (College of Plant Protection,
Northwest A&F University, Yangling) for computational
molecular docking assistance. Fu-zhen Guo (College of Plant
Protection, Northwest A&F University, Yangling) is acknowl-
edged for scanning electron microscopy (SEM) analysis.
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