Synthesis, Biological Evaluation, and 3D-QSAR Studies of N-(Substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1 H-pyrazole-4-carboxamide Derivatives as Potential Succinate Dehydrogenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.0c05702

A series of new fungicides that can inhibit the succinate dehydrogenase (SDH) was classified and named as SDH inhibitors by the Fungicide Resistance Action Committee in 2009. To develop more potential SDH inhibitors, we designed and synthesized a novel se

Full text of DOI:10.1021/acs.jafc.0c05702

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Article
Synthesis, Biological Evaluation, and 3D-QSAR Studies of
N‑(Substituted pyridine-4-yl)-1-(substituted phenyl)-5-
trifluoromethyl‑1H‑pyrazole-4-carboxamide Derivatives as Potential
Succinate Dehydrogenase Inhibitors
Zhibing Wu,∥ Hyung-Yeon Park,∥ Dewen Xie, Jingxin Yang, Shuaitao Hou, Nasir Shahzad,
Chan Kyung Kim,* and Song Yang*
Cite This: J. Agric. Food Chem. 2021, 69, 1214−1223
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ABSTRACT: A series of new fungicides that can inhibit the succinate dehydrogenase (SDH) was classified and named as SDH
inhibitors by the Fungicide Resistance Action Committee in 2009. To develop more potential SDH inhibitors, we designed and
synthesized a novel series of N-(substituted pyridine-4-yl)-1-(substituted phenyl)-5-trifluoromethyl-1H-pyrazole-4-carboxamide
derivatives, 4a−4i, namely, 5a−5h, 6a−6h, and 7a−7j. The bioassay results demonstrated that some title compounds exhibited
excellent antifungal activity against four tested phytopathogenic fungi (Gibberella zea, Fusarium oxysporum, Cytospora mandshurica,
and Phytophthora infestans). The EC50 values were 1.8 μg/mL for 7a against G. zeae, 1.5 and 3.6 μg/mL for 7c against F. oxysporum
and C. mandshurica, respectively, and 6.8 μg/mL for 7f against P. infestans. The SDH enzymatic activity testing revealed that the IC50
values of 4c, 5f, 7f, and penthiopyrad were 12.5, 135.3, 6.9, and 223.9 μg/mL, respectively. The molecular docking results of this
series of title compounds with SDH model demonstrated that the compounds could completely locate inside of the pocket, the body
fragment formed H bonds, and the phenyl ring showed a π−π interaction with Arg59, suggesting that these novel 5-trifluoromethyl-
pyrazole-4-carboxamide derivatives might target SDH. These results could provide a benchmark for understanding the antifungal
activity against the phytopathogenic fungus P. infestans and prompt us to discover more potent SDH inhibitors.
KEYWORDS: 5-trifluoromethyl-1H-pyrazole-4-carboxamide derivatives, fungicidal activity, succinate dehydrogenase, 3D-QSAR,
ligand docking
The target of SDHIs is the SDH complex in the respiratory
chain.1 SDH is composed of four distinct subunits, including
two hydrophilic subunits SDH A and SDH B (iron−sulfur
subunits) in the peripheral domain and two hydrophobic
membrane-spanning subunits, SDH C (Cytochrome bL) and
SDH D (Cytochrome bS).15,16 Mechanism research has
indicated that all the SDHIs could inhibit fungal respiration
by binding to the ubiquinone-binding site (UQ-site) of the
mitochondrial SDH complex II in the electron transport chain,
and this site is a functional part of the tricarboxylic acid
cycle.3,4,17,18 The UQ-site is formed by residues from subunits
SDH B, C, and D near the [3Fe−4S] cluster and heme b and is
highly conserved in bacteria and eukaryotes.5,19−22
INTRODUCTION
■
A series of novel fungicides that can inhibit succinate
dehydrogenase (SDH) was classified and named as SDH
inhibitors (SDHIs) by the Fungicide Resistance Action
Committee in 2009.1 From 1969 to 2020, 22 SDHI fungicides
have been commercialized (Figure 1). Among these com-
pounds, carboxin, a narrow-spectrum fungicide, was first
marketed in 1969 and has special activity against basidiomy-
cetes but limited activity toward other plant pathogenic
fungi.2−8 Amide bonds are the core feature of SDHIs, and
considering the introduction of structurally diverse benzene
rings and heterocycles on both sides of the amide bond,
compounds exhibit good broad spectrum activity; boscalid,
which was introduced in 2003, was the first compound with a
truly broad spectrum activity.9,10 SDHI fungicides have been
growing rapidly since 2009, and 13 commercialized products
have been released,11−14 including 10 products with a pyrazole
carboxamide structure. The structural analysis of these 10
SDHIs demonstrated that all the pyrazole rings only have a
CH3 substituent at the 1-position, six of them have CHF2 or
CF3 substituents at the 3-position, and four have two different
substituents at the 3- and 5-positions of the pyrazole ring.
However, none of them have substituents solely at the 5-
position of the pyrazole ring.
On the basis of the active skeleton of 5-trifluoromethyl-4-
pyrazole carboxamide, which was first found by us,23,24 a series
of novel N-(substituted-pyridine-4-yl)-1-(substituted phenyl)-
5-trifluoromethyl-1H-pyrazole-4-carboxamide derivatives was
Received: September 4, 2020
Revised: December 11, 2020
Accepted: January 15, 2021
Published: January 22, 2021
https://dx.doi.org/10.1021/acs.jafc.0c05702
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Figure 1. Commercialised SDHI fungicides from 1969 to 2020.
Figure 2. Design of title compounds.
with tetramethylsilane (TMS) as the internal standard and CDCl3 or
DMSO-d6 as the solvents. High-resolution mass spectra (HRMS) data
were obtained on a Thermo Scientific Q Exactive (Thermo Scientific,
USA). Mass spectra studies were conducted by LC/MS (LC, 1100;
MS, MSD Trap VL, Agilent Technologies, USA). The single crystal
structure of the title compound was tested on a diffractometer
(SMART-1000, Bruker Corporation, Germany). The SDH enzymatic
activity data were recorded with a microplate reader (Cytation 5,
BioTek Instruments, USA). Melting points were measured with XT-4
binocular microscope melting point apparatus (uncorrected).
Commercialised SDHIs carboxin and penthiopyrad were bought
from J&K Scientific Ltd. (Beijing, China). All reagents and solvents
were of analytical grade.
designed and synthesized (Figures 2 and 3). The in vitro
antifungal activity results revealed that some title compounds
Fungi. Six plant pathogenic fungi (Gibberella zea, Fusarium
oxysporum, Cytospora mandshurica, Thanatephorus cucumeris, Phytoph-
́
thora infestans, and Botrytis cinerea) were used for antifungal testing.
All fungi were kindly provided by the College of Agriculture, Nanjing
Agricultural University, Nanjing, China. These fungi were grown on
potato dextrose agar (PDA) plates at 25 1 °C and maintained at 4
°C.
Figure 3. Synthetic route of the title compounds 4a−4i, 5a−5h, 6a−
6h, and 7a−7j. Reagents and conditions: (i) CH(OEt)3, Ac2O; (ii)
substituted phenylhydrazine, EtOH; (iii) LiOH, THF/H2O; (iv)
SOCl2; and (v) substituted pyridine amine, NaH, anhydrous THF.
Synthesis.23−27 General Procedure for the Synthesis of 1a−1h.
A mixture of ethyl trifluoroacetoacetate (0.1 mol), triethyl
orthoformate (0.2 mol), and acetic anhydride (0.3 mol) was stirred
at 130 °C for 4 h. The reaction mixture was concentrated in vacuo.
The residue was dissolved in ethanol (100 mL), and phenylhydrazine
(0.1 mol) was added slowly to the solution, reacting at 100 °C for 5 h.
The solvent was then removed under reduced pressure. The residue
was dissolved in ethyl acetate, and the organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The
crude product was purified by column chromatography (PE/EA =
20/1−50/1) to obtained 1a as a yellow oil, yield 72%. 1H NMR (500
MHz, DMSO-d6): δ 8.31 (s, 1H, pyrazole H), 7.63−7.54 (m, 5H,
benzene H), 4.32 (q, J = 7.2 Hz, 2H, CH2), 1.31 (t, J = 7.2 Hz, 3H,
CH3). MS (ESI): m/z 285 [M + H]+. The physical and spectral data
of 1b−1h are provided in the Supporting Information.
exhibited excellent antifungal activity against four kinds of
pathogenic fungi. SDH enzymatic bioassay indicated that the
title compounds have better enzyme inhibition effects than
commercial products penthiopyrad and carboxin. Furthermore,
molecular docking results revealed the binding modes of the 5-
trifluoromethyl-1H-pyrazole-4-carboxamide derivatives, thus
providing key information for the design of SDHI fungicides.
MATERIALS AND METHODS
■
Instruments and Chemicals. 1H NMR, 13C NMR, and 19F
NMR spectra were taken on a JEOL-500 (JEOL CO., Ltd., Japan) or
a Bruker 400 NMR spectrometer (Bruker Corporation, Germany)
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General Procedure for the Synthesis of 2a−2h. To a solution of
1a (0.03 mol) in THF (30 mL) was added lithium hydroxide (0.12
mmol), and the mixture was reacted at 80 °C for 1 h. The solution
was concentrated in vacuo. Hydrochloric acid (2 M) was used to
adjust the pH value to approximately 4, and some solid precipitated
and was then filtrated. The filtrate was washed with water and dried to
On the basis of the in vitro antifungal activity results, the median
effective concentrations (EC50 values) of the highly active compounds
were further determined according to the method described above. A
series of activity screening concentrations of the title compounds and
positive controls consisting of 200, 100, 50, 25, 12.5, 6.25, or 3.125
μg/mL was prepared. EC50 values were calculated with SPSS software
20.27,28 The regression equations of the title compounds are provided
in Tables S2 and S3 in the Supporting Information.
SDH Enzyme Assay. The SDH enzyme activities of 4c, 5f, 7f, and
penthiopyrad were determined by using a succinate dehydrogenase
assay kit (Solarbio, BC0955) and assessed as reported previously.29 P.
infestans was grown in potato dextrose (PD) medium for 4 days and
then treated with 4c, 5f, 7f, or penthiopyrad at different
concentrations (200, 100, 50, 25, 12.5, 6.25, or 3.125 μg/mL). The
SDH enzymatic activity was measured after 48 h of treatment with the
selected compounds, and the absorbance value was measured at 600
nm by using a microplate reader. The inhibitions values were
calculated by GraphPad Prism 6.0. The EC50 values of the title
compounds were further determined according to the method
described in the Bioassays section. Differences between the groups
were compared by one-way analysis of variance (ANOVA; Duncan’s
multiple range test) with P < 0.05. Statistical tests were performed
using the SPSS software package (version 20).
Homology Modeling. The target protein SDH consists of four
subunits (SDH A, SDH B, SDH C, and SDH D), and the UQ-site is
formed by the residues from the B, C, and D subunits of SDH.17−22
Therefore, the B, C, and D subunits of P. infestans SDH (PiSDH)
were built on the basis of the available sequence data from the NCBI
database: strain B-XP_002901751.1, strain C-XP_002900462.1, and
strain D-XP_002896752.1. To identify a suitable parent standard for
docking, the FUGUE sequence-structure homology recognition
program was used to identify protein candidate hits.30 Alignments
for the highest-scoring hits produced by FUGUE were formatted with
JOY (Mizuguchi) and analyzed visually to highlight the conservation
of the structurally important residues.30,31 Profile−profile matching
between the target sequence and the HOMTRAD database generated
initial hits for homology recognition and alignment.31 The model was
constructed with ORCHESTRA on the basis of the result of
FUGUE.32 The model structure was validated by Protable, and visual
inspection was performed using 3D graphics software.33 The initial
model was energy-minimized by the conjugate gradient method until
the energy gradient norm converged to 0.01 kcal/mol.
1
obtained 2a as a yellow solid, yield 92%, mp 191−192 °C. H NMR
(500 MHz, DMSO-d6): δ 13.38 (s, 1H, COOH), 8.25 (s, 1H,
pyrazole H), 7.62−7.53 (m, 5H, benzene H). MS (ESI) m/z: 279 [M
+ Na]+. The physical and spectral data of 2b−2h are provided in the
Supporting Information.
General Procedure for the Synthesis of 3a−3h. Intermediates
3a−3h were prepared with a previously reported procedure using
SOCl2 as a solvent. The solvent was removed under a vacuum after
the reaction was finished, and the crude product was directly used for
the next reaction.
General Procedure for the Synthesis of 4a−4i, 5a−5h, 6a−6h,
and 7a−7j. 4-Aminopyridine (1.0 mmol), 3a (1.1 mmol), NaH (2.0
mmol), and anhydrous THF (5 mL) were added into a 25 mL three-
neck round-bottom flask and stirred at room temperature for 4 h. The
THF was removed under reduced pressure. The residue was dissolved
in ethyl acetate (25 mL). Then, the organic layer was washed by
brine, dried over anhydrous Na2SO4, and filtered. The solvent was
removed under a vacuum. The residue was further purified by column
chromatography on a silica gel to obtain 4c as a white solid, yield
1
65%, mp 108−109 °C. H NMR (400 MHz, CDCl3): δ 8.47 (d, J =
8.0 Hz, 2H, pyridine H), 8.01 (s, 1H, pyrazole H), 7.63 (d, J = 8.0 Hz,
2H, pyridine H), 7.51−7.49 (m, 3H, benzene H and NH), 7.39 (d, J
= 8.0 Hz, 2H, benzene H). 13C NMR (101 MHz, CDCl3): δ 160.18,
160.16, 150.51, 150.49, 145.54, 150.49, 145.54, 145.50, 139.59,
138.91, 130.33, 129.48, 125.91, 120.71, 120.49, 120.48, 118.02,
114.27, 14.36. 19F NMR (376 MHz, CDCl3): δ −55.56. HRMS: calcd
for C16H11F3N4O, [M − H]− 331.08012, found 331.08090. The
physical and spectral data of 4a−4i, 5a−5h, 6a−6h, and 7a−7j are
provided in the Supporting Information.
Crystal Structure Determination. A single crystal of title
compound 4c was grown from EtOH. A sample of size 0.20 × 0.18 ×
0.10 mm3 was selected for the crystallographic study. The diffraction
measurement was performed at a temperature of 113 K using graphite
monochromated Mo Kα radiation (λ = 0.71073 Å) and an Enraf-
Nonius CAD-4 four-circle diffractometer. The accurate cell
parameters and orientation matrix were obtained by the least-squares
refinement of the setting angles of 1472 reflections at the h range of
2.12 < θ < 27.87. The systematic absences and intensity symmetries
indicated the orthorhombic Pbcn space group. Corrections for LP
factors were applied. The structure was solved by direct methods and
refined by full-matrix least-squares techniques on F2 with anisotropic
thermal parameters for all nonhydrogen atoms. The calculations were
performed with the SHELXL-97 program.
Bioassays. The fungicidal activities of 4a−4i, 5a−5h, 6a−6h, and
7a−7j were tested in vitro against six plant pathogenic fungi (G. zeae,
F. oxysporum, C. mandshurica, T. cucumeris, P. infestans, and B. cinerea)
using a mycelial growth inhibition method.24 The preliminary activity
screening concentration of the title compounds was 100 μg/mL. The
mycelia dishes of fungi that were used to for testing were cut from the
PDA medium, cultivated at 25 1 °C and approximately 4 mm in
diameter, were inoculated in the middle of a PDA plate with a germ-
free inoculation needle, and then were incubated for 4−5 days at the
same temperature. DMSO (1%) in sterile distilled water served as a
blank control, whereas commercialized SDHI fungicides carboxin and
penthiopyrad served as the positive controls. Each treatment
condition consisted of three replicates. When the mycelia of the
blank control grew to 6 cm, the diameter of the mycelia treated with
the title compounds was recorded. Inhibitory effects on these fungi
were calculated by the formula I (%) = [(C − T)/(C − 0.4)] × 100,
where C represents the diameter of fungal growth of the blank control,
T represents the diameter of the fungi with treated compound, and I
represents the inhibition rate. Standard deviation (SD) values were
calculated on the basis of the inhibition data of three repetitions for
each test compound.
Molecular Docking. The constructed homology model of PiSDH
was used for the docking study. The binding site was identified by
SITEID.34 Twenty title compounds were selected as the docking
ligands, and the structures were drawn using the sketch module of the
SYBYL package and minimized using the Tripos force field with the
Gasteiger-Huckel charge until the RMS gradient was less than 0.05.
̈
The molecular docking studies were performed on the inhibitor−
SDH interactions using SYBYL packages to examine the binding
energies of the synthesized SDH inhibitor candidates.35−37 Docking
of the inhibitors was carried out using the Run-Multiple ligand option
of Surflex-Dock.3 The docking score, which estimates the free energy
of binding (ΔG) for the protein−ligand complex, was calculated using
̈
a modified Bohm scoring function, which includes entropic, hydrogen
bonding, ionic, aromatic, and lipophilic terms.
RESULTS AND DISCUSSION
■
Chemistry. The synthetic route is shown in Figure 3. Ethyl
4,4,4-trifluoro-3-oxobutanoate and triethyl orthoformate were
used as the starting materials to synthesis a transition
intermediate that reacted with substituted-phenyl hydrazines
to obtain the key intermediates 1a−1h. Compounds 2a−2h
were obtained from 1a−1h through hydrolysis in the presence
of lithium hydroxide and then refluxed in SOCl2 to obtain 3a−
3h, which were reacted with different pyridine amines to
obtain the title compounds 4a−4i, 5a−5h, 6a−6h, and 7a−7j.
1
The key synthetic intermediates were characterized by H
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nuclear magnetic resonance (NMR) and ESI-MS, and the title
1
compounds were characterized by H NMR, 13C NMR, 19F
NMR, and HRMS.
X-ray Diffraction. To confirm the trifluoromethyl func-
tional group at the 5-position of the pyrazole ring, we
determined the crystal structure of title compound 4c. The
skeleton of the new compound 4c contains a pyrazole ring, a
pyridine ring, and an amide bond connected to C(8) and
C(12). The benzene ring and trifluoromethyl group are
directly attached to the N(1) and C(9), respectively, which are
part of the pyrazole ring (Figure 4). The C(9)N(1) bond
length (1.362 Å) was slightly longer than that of a typical C
N bond (1.34 Å), indicating a significant double bond
Figure 4. Single crystal structure of 4c.
Table 1. Structures and Inhibition Rates of Title Compounds 4a−4i, 5a−5h, 6a−6h, and 7a−7j at 100 μg/mL on Pathogenic
a
Fungi
inhibition rate (%)
compound number
R1
R2
G. zeae
F. oxysporum
C. mandshurica
T. cucumeris
P. infestans
B. cinerea
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
6g
6h
7a
7b
7c
7d
7e
7f
7g
H
H
H
H
H
H
H
H
H
4-Cl
4-Cl
4-Cl
4-Cl
4-CH3
4-CH3
4-CH3
4-CH3
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-CH3
3-CH3
3-CH3
2-Cl
2-Cl
2-Cl
2-Cl
2-F
2-F
2-CH3
2-CH3
2-CH3
2-CH3
2-pyridinyl
3-pyridinyl
4-pyridinyl
66.9 2.7
38.1 2.1
84.1 1.7
64.9 2.9
65.6 1.8
69.6 1.5
85.1 2.3
54.6 1.5
34.5 2.7
37.3 1.8
79.0 2.1
34.6 2.3
19.3 1.1
44.1 2.2
36.4 2.5
9.7 1.4
34.4 1.7
20.5 3.9
93.2 2.8
77.5 2.4
77.8 2.9
75.9 3.2
100
63.8 1.4
47.7 1.2
26.6 2.2
72.9 2.5
25.1 1.9
16.3 1.1
34.3 2.2
27.8 3.5
9.4 1.7
0
50.8 1.6
37.2 1.8
56.2 1.9
78.2 3.1
0
61.0 3.0
87.7 2.8
0
28.7 1.7
28.9 1.0
94.3 3.5
76.8 2.6
79.4 3.2
76.4 2.5
100
69.5 2.1
63.1 3.4
22.2 1.4
81.4 2.2
35.2 2.1
13.5 1.2
25.9 1.6
26.3 2.1
15.7 1.5
−
50.8 1.1
31.4 1.4
24.1 1.9
19.2 1.9
61.6 2.4
56.9 1.3
57.8 2.0
25.9 1.3
24.1 1.1
92.9 3.2
72.4 3.3
29.2 1.8
−
35.6 2.2
42.0 2.0
9.6 1.5
7.4 2.9
56.7 2.3
61.5 2.9
77.2 3.8
49.0 2.4
22.8 3.9
49.7 2.3
52.6 2.2
10.4 3.9
51.6 2.2
72.1 3.5
30.4 1.8
31.7 3.9
31.7 2.5
90.8 2.9
36.5 1.9
73.4 2.4
29.2 1.8
51.5 3.5
88.3 4.3
100
22.8 1.0
24.0 0.9
100
57.9 1.7
70.9 1.8
68.5 2.4
100
−
57.1 1.5
28.9 1.1
24.3 1.5
−
3-CH3-4-pyridinyl
3-Cl-4-pyridinyl
3-Br-4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
2-Br-4-pyridinyl
4-pyridinyl
3-Cl-4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
3-Cl-4-pyridinyl
4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
4-pyridinyl
3-CH3-4-pyridinyl
3-Cl-4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
4-pyridinyl
3-Cl-4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
4-pyridinyl
2-CH3-4-pyridinyl
4-pyridinyl
3-Cl-4-pyridinyl
2-CH3-4-pyridinyl
2-Cl-4-pyridinyl
−
41.6 1.1
−
−
77.5 2.6
75.4 4.0
59.8 3.0
−
45.3 2.9
72.5 3.5
72.8 3.9
59.8 3.5
69.9 4.4
68.8 2.4
61.2 3.5
70.3 3.5
54.8 2.8
73.9 3.4
76.1 2.6
66.6 3.5
45.7 2.2
59.8 2.7
74.3 2.8
61.8 3.7
−
59.1 1.3
30.8 2.9
66.1 3.7
54.2 3.1
27.8 1.8
−
35.9 1.8
34.5 3.3
9.2 1.5
7.0 2.9
51.6 2.2
41.5 2.2
65.0 2.6
67.6 2.9
15.5 3.9
44.9 2.4.
46.1 3.2
0
5.2 2.0
60.2 1.8
62.9 2.4
56.0 2.0
26.9 2.6
15.0 1.8
62.0 2.6
62.9 2.7
57.3 4.2
98.4 3.2
66.8 2.2
88.7 2.7
33.7 2.3
81.2 3.7
88.6 2.7
90.6 3.6
73.2 2.7
85.3 3.1
50.9 2.2
67.1 2.7
56.7 2.3
54.5 1.4
25.5 2.2
59.4 2.3
66.9 3.5
−
64.2 2.5
71.0 2.9
100
100
100
87.7 2.6
88.9 3.3
28.0 1.6
94.6 2.4
91.0 2.7
94.1 2.3
91.8 3.2
95.4 3.3
66.7 4.0
28.4 1.6
60.0 3.7
76.0 2.7
81.7 2.2
−
90.7 3.1
90.1 3.4
100
86.5 2.1
80.8 3.4
−
76.5 3.7
77.1 1.9
32.7 4.4
92.3 2.5
95.4 3.2
94.4 3.8
71.2 3.2
84.3 1.4
74.8 2.9
38.1 1.7
21.2 1.9
75.6 2.7
45.7 2.4
79.7 1.4
63.4 2.3
74.4 3.0
100
7h
7i
7j
carboxin
penthiopyrad
−
76.0 3.6
100
a
Values are mean SD of three replicates. “−” not tested.
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a
Table 2. EC50 Values of Some Title Compounds against Four Kinds of Pathogenic Fungi
EC50(μg/mL)
compound number
G. zeae
F. oxysporum
C. mandshurica
P. infestans
4c
4d
4e
4f
4g
5b
5f
5g
19.1 1.5
57.9 2.1
82.6 1.5
83.5 2.3
9.6 1.8
39.4 2.3
138.0 2.7
>200
24.3 2.4
87.7 2.7
39.8 2.2
43.8 2.9
8.4 1.3
62.6 1.4
>200
33.7 4.1
85.8 3.2
30.2 1.1
44.0 1.2
15.9 3.5
42.6 2.7
>200
7.9 1.1
94.6 2.6
70.1 1.8
82.6 1.7
7.2 1.1
90.7 3.5
>200
>200
>200
>200
6b
6d
6f
6g
7a
7b
7c
7e
7f
7g
7h
7i
69.1 3.5
>200
>200
>200
126.5 2.7
77.2 2.2
132.6 3.4
121.7 3.5
8.9 1.3
39.2 2.4
16.9 1.4
13.6 2.4
6.8 1.7
11.8 1.6
38.5 2.8
22.6 1.5
117.1 3.2
>200
46.8 1.6
68.1 2.3
23.0 1.4
9.5 2.6
23.8 1.3
1.5 0.9
10.2 2.7
11.7 1.3
11.0 1.9
26.3 1.4
3.7 1.8
120.3 2.3
69.6 2.4
51.1 2.5
85.7 2.5
48.5 2.7
8.7 1.4
37.2 2.7
3.6 1.3
9.1 1.4
12.7 1.9
10.1 2.7
33.2 1.7
11.6 1.2
−
65.1 1.9
79.0 2.0
1.8 0.2
62.6 2.5
8.0 1.6
7.5 1.1
18.9 2.1
2.9 1.1
61.0 1.9
16.7 1.1
38.0 2.0
71.9 1.7
carboxin
penthiopyrad
74.4 3.1
a
Values are mean SD of three replicates. “−” not tested.
and 113.97°, respectively. The supplementary data for 4c have
been deposited in the Cambridge Crystallographic Data
Centre (http://www.ccdc.cam.ac.uk/conts/retrieving.html)
under deposition number 880862. The crystallographic data
of title compound 4c are provided in Table S1 in the
Supporting Information.
Antifungal Activity. The preliminary antifungal activity
results of the title compounds against six plant phytogenic
fungi (G. zeae, C. mandshurica, F. oxysporum, B. cinerea, P.
infestans, and S. sclerotiorum) at 100 μg/mL are shown in Table
1. As indicated in Table 1, some title compounds exhibited
excellent antifungal activity against G. zeae, C. mandshurica, F.
oxysporum, and P. infestans at 100 μg/mL. Among theses
compounds, 4g and 7a exhibited 100% inhibition against F.
Table 3. IC50 values of 4c, 5f and 7f against P. infestans
SDH
a
compound number
IC50 (μg/mL)
4c
5f
7f
12.5 1.9 c
135.3 3.5 b
3.8 1.7 c
penthiopyrad
223.9 20.9 a
a
Different lower case letters (a, b, and c) in a column indicate
significant differences between mean values evaluated by Duncan’s
multiple range test (P < 0.05).
character. The N(2)N(1)C(6), C(15)N(4)C(14)
and C(8)C(11)N(3) bond angles were 117.45°, 116.46°,
Figure 5. (a) PiSDH structure obtained from homology modeling, and (b) the binding pocket depicted by the electrostatic potential map. The
electronegative region is represented in blue, while the electropositive region is represented in red.
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Figure 6. Docking poses of 20 ligands in the binding pockets of P. infestans.
μg/mL, respectively, which were obviously superior to those of
carboxin (38.0 μg/mL) and penthiopyrad (71.9 μg/mL). The
EC50 values of 4g, 7a, 7c, and 7i against F. oxysporum were 8.4,
9.5, 1.5, and 3.7 μg/mL, respectively, which were much better
than those of carboxin (120.3 μg/mL) and penthiopyrad (69.6
μg/mL). The EC50 values of 7a, 7c, and 7e against C.
mandshurica were 8.7, 3.6, and 9.1 μg/mL, respectively, which
were superior to that of penthiopyrad (74.4 μg/mL). The EC50
values of 4c, 4g, 7a, 7c, and 7f against P. infestans were 7.9, 7.2,
8.9, and 6.8 μg/mL, respectively, which were much better than
that of carboxin (117.1 μg/mL). Preliminary structure−activity
relationship (SAR) analysis revealed that the title compounds
exhibited good antifungal activity when 4-pyridinyl was
introduced to the amine moiety of the pyrazole carboxamide
structure, and a phenyl group was introduced at the 1-position
Figure 7. Ligand was divided into three fragments, namely, head
(phenyl), body (5-trifluoromethyl pyrazole-4-carboxamide), and tail
(pyridine).
oxysporum, 4g, 7a, and 7g exhibited 100% inhibition against C.
mandshurica, and 4c, 4g, and 7a exhibited 100% inhibition
against P. infestans. As shown in Table 2, the EC50 values of 4g,
7a, 7c, 7e, and 7g against G. zeae were 9.6, 1.8, 8.0, 7.5, and 2.9
Figure 8. Linear relationship between the docking score and EC50 value that was studied for this series of title compounds.
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Figure 9. Docking poses of ligands (a) 4c (mode A) and (b) 7h (mode B) in the binding pocket of P. infestans.
of the pyrazole ring, such as in 4c. However, compounds 7a,
7e, and 7g exhibited excellent antifungal activity against four
plant phytogenic fungi, because CH3, Cl, or F was introduced
to the adjacent position of the phenyl group in these
compounds. Considering that CH3 was introduced to the
adjacent position of the pyridinyl of the amine, compounds 4g,
7c, 7f, and 7g exhibited excellent antifungal activity against the
four plant phytogenic fungi.
Enzymatic Inhibition Activity of SDH. Compounds 4c,
5f, and 7f were selected and evaluated for SDH enzymatic
inhibition determination for target site validation. As shown in
Table 3, 4c and 7f exhibited good SDH inhibition with IC50
values of 12.5 and 6.9 μg/mL, respectively, but 5f exhibited
low SDH inhibition with an IC50 value of 135.3 μg/mL. The
IC50 value of penthiopyrad was 223.9 μg/mL.
SDH Model Analysis. The three-dimensional structure of
the PiSDH protein (PDB: 1ZOY, ID% = 49.9) obtained from
the homology modeling studies is depicted in Figure 5a. The
binding site depicted in Figure 5b shows that the pocket size
was 866.4 Å3. Moreover, the binding pocket consisted of more
than nine residues from subunits B and C and only a few
residues from subunit D, and the pocket was located in a
similar position compared with the available sequence data
(see the details in Table S4 in the Supporting Information).
Molecular Modeling Study. To elucidate the mechanism
of potential SDH inhibitors and explain the SAR in detail, we
performed docking studies. As shown in Figure 5b, the
electrostatic potential maps can be easily classified in the
PiSDH model; the top part of the binding pocket (site 1)
belongs to the electronegative region, whereas the bottom part
(site 2) belongs to the electropositive region. The docking
results for 20 ligands are depicted in Figure 6. The head
fragment is oriented toward the negatively charged region (site
1), whereas the tail fragment is oriented toward the positively
charged region (site 2) to match the characteristics of the
binding pocket (Figure 7). A plot of the docking score versus
EC50 value is shown in Figure 8, and it shows a linear
relationship between the computed and experimental results
with a regression coefficient (R2) of 0.72. The detailed analysis
of Figure 6 demonstrated two distinct docking poses (Figures
S11− S15) for all docking structures (docking scores are
summarized in Table S5 in the Supporting Information). The
docking modes for 4c (mode A) and 7h (mode B) are shown
in Figure 9. Among the 20 ligands, 15 ligands (4c, 4d, 4e, 4f,
4g, 5b, 5f, 5g, 6f, 7a, 7b, 7c, 7e, 7f, and 7g) were mode A
binders while the five other ligands (6b, 6d, 6g, 7h, and 7i)
were mode B binders, in which the pyrazole ring was rotated
by 180°.
In mode A, the polar groups (CF3, CO, and N2
pyrazole) in the body fragment formed H bonds and the
phenyl ring showed π−π interactions with Arg59. Ligand 4c
formed five H bonds (2.05−2.68 Å) with Ser55, Arg59,
Tyr134, and Trp197 and a π−π interaction between the phenyl
ring and Arg59; the distance between the center of the phenyl
ring and the central carbon connecting the three nitrogen
atoms of Arg59 was 3.84 Å (Figure 9a). By introducing
substituents into the head and tail fragments, the steric
repulsion and electronegativity could induce a subtle change in
the docking pose and the score. For instance, when a
substituent was introduced into the 3-pyridyl position (4d,
4e, and 4f), the ligand moved upward to alleviate steric
repulsion with the site 2 pocket, thus decreasing the docking
score and increasing the EC50 value. In other words, the
antifungal activity of the compounds was significantly reduced.
The docking scores of these ligands were partly compensated
by the electronegativity and increased according to the order
Cl > Br > C. Considering that the 2-CH3 substituent in the
pyridine ring (4g) experienced less repulsion with the binding
pocket, its docking score was slightly smaller than that of 4c,
thus improving the antifungal activity of 4g. When two
substituents were introduced into the phenyl and pyridine
rings, two interactions could operate together. (1) The 3-
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phenyl substituents in 6b, 6d, 6f, and 6g experienced the
second strongest repulsion with the site 1 pocket. (2) The 2-
and 3-pyridinyl substituents in 6b, 6d, and 6g also have
repulsive interactions with the site 2 pocket. Considering the
insufficient space to accommodate these ligands in the binding
pocket, the body fragment had to rotate to form another
favorable pose in mode B. As shown in Figure 9b, the CF3
group of 7h formed H bonds with Ser55 (2.49 Å) and N2
pyrazole formed H bonds with Arg59 (2.88 Å) and Tyr134
(2.51 Å). This configuration reduced the antifungal activity of
these title compounds.
A detailed analysis of the docking results for this series of
title compounds revealed that, when a substituent (CH3, F or
Cl) was introduced into the 2-phenyl position, compounds
such as 7a, 7e, and 7g exhibited enhanced antifungal activity
against P. infestans. Moreover, when CH3 was introduced into
the 2-pyridinyl position, compounds 4g, 7c, 7f, and 7i
exhibited better antifungal activity against P. infestans. These
results are consistent with the result of the preliminary SAR
analysis. Therefore, this molecular docking study could provide
a benchmark for understanding the antifungal activity against
the phytopathogenic fungus P. infestans and prompt us to
develop more potent SDH inhibitors.
A series of novel N-(susbstituted pyridine-4-yl)-1-(substi-
tuted phenyl)-5-trifluoromethyl-1H-pyrazole-4-carboxamide
derivatives 4a−4i, 5a−5h, 6a−6h, and 7a−7j was designed
and synthesized to discover the potential SDH inhibitors. The
bioassay results showed that some title compounds exhibited
excellent antifungal activity against four phytopathogenic fungi
(G. zeae, F. oxysporum, C. mandshurica, and P. infestans). The
EC50 values were 1.8 μg/mL for 7a against G. zeae, 1.5 and 3.6
μg/mL for 7c against F. oxysporium and C. mandshurica,
respectively, and 6.8 μg/mL for 7f against P. infestans. SDH
enzymatic activity testing revealed that the IC50 values of 4c,
5f, 7f, and penthiopyrad were 12.5, 135.3, 6.9, and 223.9 μg/
mL, respectively. The molecular docking of this series of title
compounds with the SDH model demonstrated that the body
fragment formed H bonds, and the phenyl ring showed π−π
interactions with Arg59, suggesting that these novel 5-
trifluoromethyl-pyrazole-4-carboxamide derivatives might tar-
get SDH. A detailed analysis of the docking results revealed
that, when a substituent (CH3, F, or Cl) and CH3 were
introduced into the 2-phenyl and 2-pyridinyl positions,
respectively, the antifungal activity of the title compounds
was notably improved. However, when a substituent was
introduced into the 3-phenyl and 3-pyridinyl positions, the title
compounds exhibited reduced antifungal activity. These results
could provide a benchmark for understanding the antifungal
activity against the phytopathogenic fungus P. infestans and
prompt us to develop more potent SDH inhibitors.
AUTHOR INFORMATION
Corresponding Authors
■
Chan Kyung Kim − Department of Chemistry and Chemical
Engineering, Center for Design and Applications of Molecular
Catalysts, Inha University, Incheon 22212, Korea;
orcid.org/0000-0002-4716-5553; Phone: +82-32-860-
7684; Email: kckyung@inha.ac.kr; Fax: +82-32-867-5604
Song Yang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals of Guizhou
University, Guiyang 550025, China; orcid.org/0000-
0003-1301-3030; Phone: +86-851-88292171;
Email: jhzx.msm@gmail.com; Fax: +86-851-88292170
Authors
Zhibing Wu − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals of Guizhou
University, Guiyang 550025, China; orcid.org/0000-
0002-2724-9725
Hyung-Yeon Park − Department of Chemistry and Chemical
Engineering, Center for Design and Applications of Molecular
Catalysts, Inha University, Incheon 22212, Korea
Dewen Xie − College of Pharmacy, Guizhou University,
Guiyang 550025, China
Jingxin Yang − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals of Guizhou
University, Guiyang 550025, China
Shuaitao Hou − State Key Laboratory Breeding Base of Green
Pesticide and Agricultural Bioengineering, Key Laboratory of
Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals of Guizhou
University, Guiyang 550025, China
Nasir Shahzad − Department of Chemistry and Chemical
Engineering, Center for Design and Applications of Molecular
Catalysts, Inha University, Incheon 22212, Korea
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c05702
Author Contributions
∥Z.W. and H.-Y.P. should be considered joint first authors.
Funding
This work was supported by the National Natural Science
Foundation of China (Nos. 21662008 and 21877021), the Key
Technologies R&D Program (No. 2014BAD23B01), and the
Science and Technology Project of Guizhou province (No.
[2017]5788).
ASSOCIATED CONTENT
Notes
■
The authors declare no competing financial interest.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c05702.
ACKNOWLEDGMENTS
C.K.K. would like to thank Inha University for support of this
work.
■
1
Discussions of H NMR, 13C NMR, 19F NMR, HRMS,
and MS data and molecular docking analysis, tables of
crystallographic data, regression equations, character-
istics of SDH binding pockets of P. infestans, and
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