N-[2-[(3-Chlorophenyl)amino]-phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide: Synthesis, Crystal Structure, Molecular Docking and Biological Activities

Article abstract of DOI:10.1002/cbdv.201900118

In continuation of our previous research on the development of novel pyrazole-4-carboxamide with potential antifungal activity, compound SCU2028, namely N-[2-[(3-chlorophenyl)amino]phenyl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was synthesized by new method, structurally characterized by IR, HR-ESI-MS, ¹H- and ¹³C-NMR spectra and further identified by single-crystal X-ray diffraction. In pot tests, compound SCU2028 showed good in vivo antifungal activity against Rhizoctonia solani (R. solani) and IC₅₀ value of it was 7.48 mg L^?1. In field trials, control efficacy of compound SCU2028 at 200 g.a.i. ha^?1 was 42.30 % on the 7^th day after the first spraying and 68.10 % on the 14^th day after the second spraying, only slightly lower than that of thifluzamide (57.20 % and 71.40 %, respectively). Further in vitro inhibitory activity showed inhibitory ability of compound SCU2028 was 45-fold higher than that of bixafen and molecular docking of compound SCU2028 to SDH predicted its binding orientation in the active site of the target protein SDH. These results suggested that compound SCU2028 was a potential fungicide for control of rice sheath blight.

Full text of DOI:10.1002/cbdv.201900118

Title: N-(2-(3-Chlorophenylamino)phenyl)-3-(difluoromethyl)-1-
methyl-1H-pyrazole-4-carboxamide: Synthesis, crystal structure,
molecular docking and biological activities
Authors: Aigui Zhang, Yihua Yang, Ying Yue, Ke Tao, Taiping Hou,
and Hong Jin
This manuscript has been accepted after peer review and appears as an
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900118
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900118

10.1002/cbdv.201900118
Chemistry & Biodiversity
Chem. Biodiversity
N-(2-(3-Chlorophenylamino)phenyl)-3-(difluoromethyl)-1-methyl-1H-
pyrazole-4-carboxamide: Synthesis, crystal structure, molecular
docking and biological activities
Aigui Zhang, Yihua Yang, Ying Yue, Ke Tao, Taiping Hou* and Hong Jin*
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of
Life Sciences, Sichuan University, Chengdu 610065, Sichuan, P. R. China
e-mail:jinhong@scu.edu.cn; houtplab@scu.edu.cn
ABSTRACT: In continuation of our previous research on the development of novel pyrazole-
4-carboxamide with potential antifungal activity, compound SCU2028, namely N-(2-(3-
chlorophenylamino)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide, was
1
13
synthesized by new method, structurally characterized by IR, ESI-HRMS, H and C NMR
spectra and further identified by single-crystal X-ray diffraction. In pot tests compound
SCU2028 showed good in vivo antifungal activity against Rhizoctonia solani (R. solani) and
IC50 value of it was 7.48 mg L-1. And in field trials control efficacy of compound SCU2028 at
200 g. a.i ha-1 was 42.30 % on the 7th day after the first spraying and 68.10 % on the 14th day
after the second spraying, only slightly lower than that of thifluzamide (57.20 % and 71.40 %,
respectively). Further in vitro inhibitory activity showed inhibitory ability of compound
SCU2028 was 45-fold higher than that of bixafen and molecular docking of compound
SCU2028 to SDH predicted its binding orientation in the active site of the target protein SDH.
These results suggested that compound SCU2028 was a potential fungicide for control of rice
sheath blight.
Keywords: pyrazole-4-carboxamide • crystal structure • molecular docking • biological
activities • succinate dehydrogenase.
Introduction
Plant diseases caused by fungal pathogens led to detrimental agricultural crop losses.[1]
Among the plant diseases rice sheath blight is one of the most important diseases worldwide
and can cause heavy losses in rice yield.[2-4] At present agrochemicals are still the better
choice for controlling rice sheath blight and in China the succinate dehydrogenase inhibitor
(SDHI) thifluzamide had been registered for rice sheath blight and exhibited excellent
efficacy in controlling this disease.[5] Besides it in SDHI fungicides pyrazole carboxamides
were also a class of important SDHIs and widely used, especially CF2H substituted pyrazole
1
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10.1002/cbdv.201900118
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carboxamides such as fluxapyroxad, bixafen, sedaxane, isopyrazam and isoflucypram (Figure
1).[6] However, the extensively application of a single chemical for a long time might increase
the risk of resistance development of certain fungi.[7] Therefore, development and application
of a new fungicide is one of the most important strategies.[8]
Small molecules containing the diarylamine moieties displayed excellent agricultural
activities such as antifungal, insecticidal and herbicidal activities.[9-11] In previous studies we
discovered a novel fenfuram-diarylamine hybrid by replacing the phenyl group in fenfuram
with the diarylamine moieties, which had exhibited the better antifungal activities than
fenfuram.[12] Inspired by this result, bixafen was choose as a lead compound, the diarylamine
moieties were introduced to replace the biphenyl group and pyrazole carboxamide with
diarylamine-modified scaffold were synthesized. Among them, compound SCU2028, namely
N-(2-(3-chlorophenylamino)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-
carboxamide showed good in vitro antifungal activities against Rhizoctonia solani (R. solani)
and IC50 value of it reached 0.015 mg L-1 (Figure 1).[13]
Figure 1. Structures of the commercial pyrazole-4-carboxamide fungicides and compound
SCU2028.
Thus, as our continuous work on studying compound SCU2028 in depth, in paper
compound SCU2028 was synthesized by new synthesis method and its structure was further
identified by single-crystal X-ray diffraction. In addition, to further evaluate its biological
activities, its in vivo antifungal activities in pot tests and field trials and in vitro inhibitory
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activity against SDH were evaluated. Furthermore, molecular docking study predicted the
binding pose of it to SDH.
Results and Discussion
Chemistry
Scheme 1 detailed the synthesis and chemical structure of compound SCU2028. Initially,
Intermediate 3 was obtained by condensation reaction and then transformed into the
corresponding compound 4 through reduction reaction. Then the key pyrazole acid 5 directly
reacted with compound 4 under EDCI and DMAP immediately to afford compound
SCU2028.[14] And this method was different from previous method.[13] At last, the structure of
compound SCU2028 was confirmed by1H NMR, 13C NMR, IR and ESI-HRMS spectroscopic
data.
Scheme 1. Synthetic route of compound SCU2028.
X-ray crystallography
The crystallographic data and refinement information of compound SCU2028 is summarized
in Table 1. CCDC 1870000 contains the supplementary crystallographic data for this paper.
These
data
can
be
obtained
free
of
charge
via
http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033).
Table 1. Crystal data for compound SCU2028.
Parameter
SCU2028
C18H15ClF2N4O
376.79
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
293.01(10)
triclinic
P-1
3
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a/Å
7.0253(4)
b/Å
9.0047(4)
c/Å
13.6853(7)
α/°
89.605(4)
β/°
82.635(4)
γ/°
88.091(4)
Volume/Å3
Z
858.12(8)
2
ρcalcg/cm3
μ/mm-1
1.458
2.298
F (000)
388.0
Crystal size/mm3
Radiation
0.65 × 0.45 × 0.25
CuKα (λ = 1.54184)
9.828 to 145.098
-8 ≤ h ≤ 8, -9 ≤ k ≤ 11, -16 ≤ l ≤ 16
2Θ range for data collection/°
Index ranges
Reflections collected
9409
Independent reflections
3329 [Rint = 0.0256, Rsigma = 0.0223]
Data/restraints/parameters
Goodness-of-fit on F2
3329/0/236
1.049
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
R1 = 0.0534, wR2 = 0.1467
R1 = 0.0568, wR2 = 0.1509
0.36/-0.55
The molecular structure of compound SCU2028 was illustrated in Figure 2. Crystal data
and the selected bond lengths and angles of SCU2028 were listed in Tables 1 and 2.
SCU2028 was composed of a pyrazole ring moiety, two phenyl moieties, a chlorine atom, a
CF2 methyl group, a methyl group and an amide group. For CF2 methyl group, bond length of
C5-F2 is 1.35 Å and the bond angle of corresponding F2-C5-C4 is 109.82°. In pyrazole ring
moiety, bond length of N2-C4 is 1.33 Å, shorter than that of C2-C3 (1.38 Å). In the amide
group, bond length of the C6-O1 is 1.22 Å, and bond length of the C6-N3 is 1.36 Å. The bond
angle of C3-C6-N3 is 115.53°, while the bond angle of C6-N3-C7 is 124.97°. From the
molecular structure, it can be seen that the bond angle of C12-N4-C13 is 127.78° and the
bond length of the C17-Cl1 is 1.75 Å. The dihedral angle between the two benzene rings is
4
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41.55°, which indicates that the two rings are not coplanar in the molecular structure.
Table 2. Selected bond lengths (Å) and angles (°) for compound SCU2028.
Bond Lengths/angles
C5- F2
Calculated Values
1.35 Å (2)
Bond Lengths/angles
C2-C3
Calculated Values
1.38 Å (3)
C6-O1
1.22 Å (2)
C6-N3
1.36 Å (2)
N2-C4
1.33 Å (2)
C17-Cl1
1.75 Å (2)
C6-N3-C7
C3-C6 -N3
124.97°(16)
115.53°(16)
C12-N4-C13
F2-C5-C4
127.78°(17)
109.82°(17)
Figure 2. Molecular structure of compound SCU2028.
Crystal data for compound SCU2028: C18H15ClF2N4O (M =376.79 g/mol): triclinic, space
group P-1 (no. 2), a = 7.0253 (4) Å, b = 9.0047(4) Å, c = 13.6853 (7) Å, α = 89.605 (4)°, β =
82.635 (4)°, γ = 88.091 (4)°, V = 858.12 (8) Å3, Z = 2, T = 293.01 (10) K, μ(CuKα) = 2.298
mm-1, Dcalc = 1.458 g/cm3, 9409 reflections measured (9.828° ≤ 2Θ ≤ 145.098°), 3329
unique (Rint = 0.0256, Rsigma = 0.0223) which were used in all calculations. The final R1
was 0.0534 (I > 2σ(I)) and wR2 was 0.1509 (all data).
Biological activities
Pot tests
To further assess biological activities of compound SCU2028, the in vivo antifungal activities
in pot tests and field trials and inhibitory activity against SDH were evaluated. Firstly the in
vivo antifungal activities of compound SCU2028 against R. solani in pot tests were evaluated
in a greenhouse environment according to the previously reported procedure and thifluzamide
was selected as positive control in Table 3.[15] As shown in Table 3, compound SCU2028
displayed promising antifungal activity (90 %) against R. solani at a dosage of 80 mg L-1.
5
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Even at a dosage of 20 mg L-1, it still showed antifungal activity of 80 %. At a lower dosage
of 10 mg L-1, it displayed 60 % antifungal activity against R. solani. When its concentration
was reduced to 5 mg L-1, the effect was still 35 %. Although IC50 value of compound
SCU2028 was 7.48 mg L-1 and higher than that of thifluzamide (IC50 = 3.12 mg L-1), the
novel fungicide SCU2028 with diarylamine scaffold had potentially antifungal activity
against R. solani. So, the in vivo antifungal activities of compound SCU2028 in field trials
were carried out in the next trials.[15-17]
Table 3. In vivo antifungal activities against R. solani of compound SCU2028 in pot tests.
Compd
Dosage (mg L-1)
Antifungal activities (%)
IC50 (mg L-1)
SCU2028
80
40
20
10
5
90
85
80
60
35
15
100
100
95
85
80
55
15
7.48
2.5
thifluzamide 80
3.12
40
20
10
5
2.5
1.25
Field trials
The in vivo antifungal activities of compound SCU2028 against rice sheath blight in field
trials were evaluated in according to the previously reported procedure and thifluzamide was
selected as positive control in Table 4.[15-16] As shown in Table 4, applications of compounds
SCU2028 and thifluzamide at 200 g.ai ha-1 recorded protection effects with 42.30 % and
57.20 % on the 7th day after the first spraying. Obviously, the effect of thifluzamide was
slightly better than that of compound SCU2028 at 200 g.ai ha-1 after the first spraying.
Furthermore, the control effects would respectively go up to 68.10 % and 71.40 % on the 14th
day after the second spraying for compound SCU2028 and thifluzamide. When the treatment
concentration of compounds decreased to 100 g.ai ha-1, compound SCU2028 displayed a
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slightly lower efficacy after the two sprayings, 39.50 % and 62.10 % respectively. At the
same time control efficacy of thifluzamide also slightly dropped at 100 g.ai ha-1. Obviously,
although the effect of thifluzamide was slightly better than that of compound SCU2028, the
difference between the control effects of the two compounds was weak at 200 g.ai ha-1. This
suggested that compound SCU2028 could be a potential antifungal candidate.
Table 4. In vivo control efficacy of compound SCU2028 against rice sheath blight in field
trials.
treatment
CTa
7th after the first spraying
14th after the second spraying
(g. ai
ha-1)
Disease
index
Control
effects
(%)
Disease index
Control effects (%)
200
100
50
1.24
1.30
1.46
0.92
0.94
1.18
2.15
42.30
39.50
32.10
57.20
56.10
45.10
--
0.96
1.14
1.26
0.86
0.92
1.01
3.01
68.10
62.10
58.10
71.40
69.40
66.40
--
SCU2028
thifluzamide 200
100
50
control
0
a: concentration
SDH inhibition
In order to investigate whether the SDH is a potential target enzyme of compound SCU2028
or not, the fungal SDH inhibition assay was performed.[12] Compounds SCU2028 and bixafen
(a commercial SDH inhibitor) were selected to test in vitro inhibitory activity against SDH
from mitochondria of R. solani. As demonstrated in Table 5, compound SCU2028 (IC50 =
0.0392 mg L-1) showed higher inhibitory activity against SDH than bixafen (IC50 = 1.766 mg
L-1), which indicated that the inhibitory ability of compound SCU2028 was 45-fold higher
than that of bixafen. It proved that the SDH is one of the important action targets of
compound SCU2028.
Table 5. In vitro inhibitory activity of compound SCU2028 against SDH.
Compd
IC50 (mg L-1)*
0.0392 ± 0.020
SCU2028
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bixafen
1.766 ± 0.105
*Data are means ± SE, n=3
Molecular docking studies
In addition, in order to theoretically illuminate the mechanism of action of compound
SCU2028 against SDH, molecular docking of compound SCU2028 into the binding site of
the SDH was performed according to previous method.[18-20] The theoretical binding mode
between compound SCU2028 and SDH was shown in Figure 3. Compound SCU2028 fit in
the gap composed of subunit B, C and D of SDH. The phenyl group linking amide bond in the
middle of compound SCU2028 was occupied the hydrophobic pocket composed of the
residues B/Pro-202, B/Ile-251, C/Ile-77 and C/Trp-73, while the 4-methylpyrazole scaffold of
compound SCU2028 located at another hydrophobic pocket, surrounded by the residues
B/Trp-205, B/Trp-206, C/Phe-64 and C/Trp-73, forming a stable hydrophobic binding.
Detailed analysis showed that a π-π stacking interaction was observed between the phenyl
group linking amide bond of compound SCU2028 and sidechain of the residue C/Trp-73. In
addition, 4-methylpyrazole group of compound SCU2028 formed CH-π interaction with the
residue C/Trp-73. Moreover, the 3-chlorophenyl group of SCU2028 formed cation-π
interaction C/Arg-80 (Figure 3). All these interactions helped compound SCU2028 anchor in
the binding site of SDH. The above molecular simulations gave us rational explanation of the
interactions between compound SCU2028 and SDH, which also provided valuable
information for further discovery of the SDHIs.
Figure 3. The putative binding mode between SCU2028 and SDH.
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Conclusions
In summary, compound SCU2028 was synthesized by new method and evaluated as a new
fungicide candidate. Its structure was confirmed by using different spectroscopic techniques
and its configuration was determined via its single crystal X-ray analysis. By pot tests, it
showed good promising in vivo antifungal activity against R. solani with IC50 value of
7.48 mg L-1. Moreover, the field trials showed that the control effect of compound SCU2028
was only slightly lower than that of thifluzamide against rice sheath blight at 200 g.ai ha-1.
Further in vitro inhibitory activity showed inhibitory ability of compound SCU2028 was 45-
fold higher than that of bixafen and molecular docking study showed the possible binding
mode of compound SCU2028 to the SDH. It was believed that compound SCU2028 could be
further utilized as a potential fungicide to control rice sheath blight. Further synthesis
optimization and toxicological studies are currently in progress.
Experimental Section
Reagents and Materials
All chemicals and reagents were purchased from commercial sources and were used as
received. All reactions were monitored by thin layer chromatography (TLC) using pre-coated
silica gel GF254 plates. Column chromatography purification was performed over silica gel
(300-400 mesh, Qingdao Marine Chemical Ltd., P.R. China). Melting points (uncorrected)
were determined on a SPSIC WRS-1B digital melting point apparatus. IR spectra were
recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer (KBr presser method). ESI-
HRMS spectra were recorded on a Bruker Daltonics ESI-BioTOF-Q High Definition Mass
Spectrometer. 1H NMR and 13C NMR spectra were collected on a Bruker AVII-400 NMR
spectrometer (Bruker Company, Germany) using DMSO-d6 as solvent. Chemical shifts (δ) are
reported in parts per million (ppm) with reference to internal TMS, and coupling constants (J)
are provided in Hertz (Hz). Single crystal X-Ray diffraction measurements were conducted on
a Bruker Smart Apex II diffractometer.
General Procedure
Synthesis
A reaction mixture containing 1-chloro-2-nitrobenzene 1 (20 mmol), 3-chlorobenzenamine 2
(30 mmol) and KF (30 mmol) was stirred at 220 °C and were monitored by TLC. After
reaction completion, the desired intermediate 3 was obtained via column chromatographic
purification. Secondly, pure compound 3 (20 mmol), reductive iron powder (60 mmol),
9
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ammonium chloride (60 mmol) and ethanol aqueous solution (75 %, 50 mL) were mixed and
refluxed for 2 h and the key intermediate 4 was obtained. At last, 3-(difluoromethyl)-1-
methyl-1H-pyrazole-4-carboxylic acid 5 (30 mmol), EDCI (30 mmol), DMAP (0.6 mmol)
and compound 4 (25 mmol) were dissolved in DCM (CH2Cl2, 50 mL). The mixture was
stirred at room temperature for 6 h and the target compound SCU2028 was gained and
purified via recrystallization. Recrystallisation from anhydrous methanol afforded a white
powder: yield, 70 %; mp, 128-129 °C; 1HNMR (400 MHz, DMSO-d6) δ 3.93 (s, 3H, N-CH3),
6.76 (dd, 1H, J = 7.6, 1.6 Hz, Ph-H), 6.82-6.86 (m, 2H, Ph-H), 7.07 (td, 1H, J = 7.7, 1.3 Hz,
Ph-H), 7.16 (t, 1H, J = 8.0 Hz, Ph-H), 7.19 (td, 1H, J = 7.6, 1.5 Hz, Ph-H), 7.31 (t, 1H, JH-F=
54.0 Hz, CHF2), 7.33 (s, 1H, Ph-H), 7.55 (d, 1H, J = 7.6 Hz, Ph-H), 7.73 (s, 1H, Ph-NH-Ph),
8.37 (s, 1H, Py-H), 9.51 (s, 1H, CO-NH); 13C NMR (100MHz, DMSO-d6) δ 39.42 (-NMe-C),
109.74 (t, JC-F = 232.9 Hz, -CF2H-), 114.07 (Ph-C), 114.94 (Ph-C), 116.06 (t, JC-F = 3.5 Hz,
Py-C), 118.42 (Ph-C), 121.45 (Ph-C), 122.76 (Ph-C), 126.08 (Ph-C), 126.49 (Ph-C), 129.61
(Ph-C), 130.58 (Ph-C), 133.15 (Py-C), 133.54 (Ph-C), 135.51 (Ph-C), 144.91 (t, JC-F = 23.1
Hz, Py-C), 146.15 (Ph-C), 160.23 (-CONH-); IR (KBr, cm-1): 1663.86 (C = O); ESI-HRMS:
m/z [M+H]+ calcd. for C18H16ClF2N4O 377.0981, found 377.0973.
X-ray analysis
Condition on crystal growing of compound SCU2028: SCU2028 (1.0 g) was dissolved in 60
ml anhydrous methanol, heated to 50 °C and stirred until the mixture approaching saturated
solution. Subsequently, the hot solution was filtered immediately and solvent slowly was
evaporated at room temperature and colorless crystal suitable for XRD was obtained.
Single crystal X-ray diffraction measurements were conducted on a Bruker APEX-II
diffractometer using Cu-Kα radiation (λ = 1.54184 Å) with a graphite monochromator at
293.01(10) K in scan mode. Collected data and cell refinement were reduced using Olex2[21]
and the structure was solved with the ShelXT[22] structure solution program using Direct
Methods and refined with the ShelXL[23] refinement package using Least Squares
minimisation.
Biologal activities
Pot tests for compound SCU2028 against R. solani
In vivo fungicidal activity of compound SCU2028 against R. solani was tested according to
the procedure described previously.[13] Rice plants were grown under greenhouse conditions
(Relative Humidity = 70-80 %, Temperature = 24-26 °C, Light Intensity ≥ 2000 lux) in vinyl
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planting pots. Compound SCU2028 dissolved in acetone and distilled water containing
Tween-80 (0.05 %) at the given concentration was sprayed over the plant and subsequently
cultivated for 24 h. The blank control groups and the treated rice seedlings at the third-leaf
stage were inoculated with strain R. solani, then the symptoms were examined 5 days later.
Pots were arranged as a randomized complete block with three replicates per treatment. The
inhibition percentage was expressed as the mean of values obtained in three independent
experiments. Commercial fungicide thifluzamide was used as a control.
Field trials for compound SCU2028 in controlling rice sheath blight
A rice field naturally infected by R. solani was selected to perform the field trials by using the
standard method.[17] Field trials were conducted in Chengdu, Sichuan Province, China, to
determine the potential control of rice sheath blight in summer rice in 2018. The field was
divided into 21 plots, and the size of each plot was 5 × 6 m. Each of the plots was separated
by a 50 cm interval with untreated rice plants. The concentrations used were 0, 50, 100, 200
g.ai ha-1 for compound SCU2028 and thifluzamide treatments. All field trials were arranged
in a block design and each treatment on a given test was replicated a minimum of three times
throughout the field. Foliar application of fungicides was made by using a hand sprayer after
the emergence of disease. Each treatment took up in three plots and was distributed randomly
in each field. Fungicides were sprayed upon initiation of disease on August 4, 2018 for the
first time and the second application was carried out seven days later. The relative control
effects were assessed at the 7th and 14th day of foliar application of compound SCU2028 and
thifluzamide.
The disease severity was scored by using the following scale: [15-17]
0: no symptoms;
1: lesions limited to lower 1/4 of leaf sheath area;
3: lesions present on lower 1/2 of leaf sheath area;
5: lesions present on more than 1/2 of leaf sheath area, with slight infection on lower (3rd or
4th) leaves;
7: lesions present on more than 3/4 of leaf sheath, with severe infection on upper leaves (flag
and 2nd leaf);
9: lesions reaching top of tillers, with severe infection on all leaves and some plants killed.
Disease severity and the control control efficacy were calculated as follows:
Disease severity = [Σ (The number of diseased plants in this index × Disease index)/(Total
number of plants investigated × The highest disease index)]×100 %.
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Control efficacy = [(Disease severity of control−Disease severity of treated group)/Disease
severity of control] ×100 %.
In vitro enzyme assay for compound SCU2028 against SDH
Isolation of R. solani
Fungus mitochondrial was isolated according to a previously reported method.[12] Cultures
were inoculated at 0.05 OD600 nm and grown on a reciprocal shaker (180 rpm, 25 °C) for 5
days in Sabouraud maltose broth (SMB) medium. Cells were harvested by vacuum filtration
and disrupted in liquid nitrogen using a mortar and pestle. The resultant powder was
resuspended to 10 % w/v in mitochondrial extraction buffer (10 mM KH2PO4, pH 7.2, 10 mM
KCl, 10 mM MgCl2, 0.5 M sucrose, 0.2 mM EDTA, 2 mM PMSF). The extract was clarified
by centrifugation (5000 g, 4 °C for 10 min, 2 times), and intact mitochondria were then
pelleted at 10000 g for 20 min at 4 °C and resuspended in the same buffer. Mitochondrial
suspensions were brought to a concentration of 10 mg/mL and stored at −80 °C until use.
SDH activity was found to remain stable for months.
Succinate:Ubiquinone/DCPIP activity inhibition
Mitochondrial suspensions were diluted 1/20 in extraction buffer and preactivated at 30 °C for
30 min in the presence of 10 mM succinate. Succinate:ubiquinone/DCPIP activity inhibition
measurements were performed by adding 10 μL of preactivated mitochondria to 200 μL of
assay buffer (50mM phosphate-sodium, pH 7.2, 250mM sucrose, 3mM NaN3, 10 mM
succinate) supplemented with 140 μM dichloro- phenolindophenol (DCPIP) and 1 mM 2,3-
dimethoxy-5-methyl-1,4-benzoquinone (Q0). Inhibitor concentrations ranged between 0.0050
and 15 μg/mL, with uniform 5× dilution factor steps (six inhibitor concentrations + DMSO
control). A total of 96 well plates were pre-equilibrated at reaction temperature (30 °C) for 10
min before the reactions were started by the addition of 10 μL of preactivated R. solani
mitochondrial suspension. DCPIP reduction was conducted at 30 °C and monitored at 595 nm.
Calculated absorbance slopes (OD/h) were used for half-inhibitory concentration (IC50)
calculations using GraphPad Prism 5.0 software.
Homology modeling and molecular docking
Homology modeling
The NCBI protein database (http://www.ncbi.nlm.nih.gov/protein/) was used to search the
SDH amino acid sequence of R. solani. The employed protein sequence was CUA72490.1,
CUA71217.1, CUA73421.1 and CUA73959.1 reported by Wibberg. The BLAST server
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10.1002/cbdv.201900118
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(http://blast.ncbi.nlm.nih.gov) was used to search a template for the chain. We applied SDH
from avian (PDB ID:1YQ3) as the template, and the homology of amino acid sequence was
aligned. Homology modeling of SDH from R. solani was carried out using MODELER 9.15
(http://salilab. org/modeller/).
Molecular docking
Molecular docking studies were performed to investigate the binding mode of the compound
to the SDH using Autodock vina 1.1.2.[18-19] The 3D structure of compound SCU2028 was
drawn by ChemBioDraw Ultra 14.0 and ChemBio3D Ultra 14.0 softwares. The
[20]
AutoDockTools 1.5.6 package
was employed to generate the docking input files. The
ligand was prepared for docking by merging non-polar hydrogen atoms and defining rotatable
bonds. The search grid of SDH was identified as center_x: 86.459, center_y: 65.6, and
center_z: 85.537 with dimensions size_x: 15, size_y: 15, and size_z: 15. The value of
exhaustiveness was set to 20. For Vina docking, the default parameters were used if it was not
mentioned. The best-scoring pose as judged by the Vina docking score was chosen and
visually analyzed using PyMoL 1.7.6 software (http://www.pymol.org/).
Acknowledgements
This study was supported by the National Key Research and Development Program of China
(2016YFC0502004, 2017YFD0200503) for the financial support.
Author Contribution Statement
H. Jin designed and managed the project. A. G. Zhang contributed to synthetic work. Y. Yue
and Y. H. Yang contributed to activity evaluation. K. Tao and T. P. Hou conducted the optical
experiments. Y. H. Yang worked on the molecular docking studies.
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Entry for the Graphical Illustration
Twitter Text
In continuation
of
the
development
of
pyrazolecarboxamide,
N-(2-(3-
chlorophenylamino)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide
showed good in vivo antifungal activity against Rhizoctonia solani.
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