Synthesis, antifungal activity and qsar of novel pyrazole amides as succinate dehydrogenase inhibitors

Article abstract of DOI:10.3987/COM-17-13826

We design and synthesize a series of novel pyrazole amides based on the commercialized fungicides and our previous work. The antifungal activity was tested in vitro by mycelial growth inhibition assay. The results show that all the compounds are of antifungal activities against the tested fungi at different levels. Among them, N-(2-(7-bromo-5-chloro-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vk) exhibited higher antifungal activity than boscalid against two fungi. Molecular docking study revealed that the carbonyl oxygen atom of Vk forms two hydrogen bonds toward the hydroxyl hydrogens of TYR58 and TRP173.

Full text of DOI:10.3987/COM-17-13826

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HETEROCYCLES, Vol. 96, No. 1, 2018
HETEROCYCLES, Vol. 96, No. 1, 2018, pp. 74 - 85. © 2018 The Japan Institute of Heterocyclic Chemistry
Received, 23rd October, 2017, Accepted, 27th November, 2017, Published online, 5th December, 2017
DOI: 10.3987/COM-17-13826
SYNTHESIS, ANTIFUNGAL ACTIVITY AND QSAR OF NOVEL
PYRAZOLE
INHIBITORS
AMIDES
AS
SUCCINATE
DEHYDROGENASE
Shijie Du,1 Zhonghao Li,2 Zaimin Tian,2 and Lu Xu1*
1College of Material and Chemical Engineering, Tongren University, Tongren,
2
554300, China. College of animal Science and Technology, Hebei North
University, Zhangjiakou 075131, China. E-mail: dsj5216@163.com (Shijie Du);
lxchemo@163.com (Lu Xu)
Abstract – We design and synthesize a series of novel pyrazole amides based on
the commercialized fungicides and our previous work. The antifungal activity was
tested in vitro by mycelial growth inhibition assay. The results show that all the
compounds are of antifungal activities against the tested fungi at different levels.
Among
them,
N-(2-(7-bromo-5-chloro-1H-indazol-1-yl)phenyl)-
3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vk) exhibited higher
antifungal activity than boscalid against two fungi. Molecular docking study
revealed that the carbonyl oxygen atom of Vk forms two hydrogen bonds toward
the hydroxyl hydrogens of TYR58 and TRP173.
INTRODUCTION
Succinate dehydrogenase inhibitors (SDHIs) are a class of fungicides with good inhibition effect of spore
germination, germ tube elongation and other negative effects on fungal growth. As the bactericidal
mechanism of SDHIs was illustrated in detail, pyrazole amide fungicides are becoming much more
popular. In recent years, there are many new products being developed, including boscalid (BASF, 2004),
penthiopyrad (Mitsui Corporation, 2010), isopyrazam (Syngenta, 2010), sedaxane (Syngenta, 2011),
bixafen (Bayer, 2011) penflufen (Bayer, 2012) and fluopyram (BASF, 2012). Boscalid (2-chloro-
N-(4'-chloro-[1,1'-biphenyl]-2-yl)nicotinamide) is an efficient fungicide with good inhibition of spore
germination, germ tube elongation and other effects on fungal growth. Some studies have shown that the
resistance observed in certain species towards SDHIs is due to mutations in the succinate dehydrogenase
genes. These results imply that the resistance mutations affecting specific SDHIs could be controlled by
structurally different SDHIs.1 Therefore, the introduction of further structurally diversity to SDHIs

HETEROCYCLES, Vol. 96, No. 1, 2018
75
represents a potential strategy for delaying the onset of resistance.
In our previous work, we designed and synthesized a series of amide compounds based on introducing N
atom to replace the C atom of the amine moiety of amide.2 Bioassays have shown that some target
molecules exhibited good antifungal activities against Rhizoctonia solani, Botrytis cinerea and Pythium
aphanidermatum. By comparing the structure and activity, we found that amides with indazole groups
have high antibacterial activity. In this paper, we introduce a series of substituted indazole groups into the
target molecule (Figure 1). Some have shown much better activities compared to the previous
compounds.
Figure 1. Design strategy of target compound
RESULTS AND DISCUSSION
The synthetic route to the target compounds is shown in Scheme 1. The substituted indazole ring was
constructed by o-hydroxybenzaldehyde and hydrazine hydrate. The chlorine atom in 1-chloro-
2-nitrobenzene was replaced by an amino group via an aromatic nucleophilic substitution reaction, giving
the compound III. Then the nitro group in compound III was reduced with hydrazine hydrate to provide
the key intermediates IV, which were subsequently acylated to produce the target amides V.
Cl
R
N
OH
N
NO2
H2NNH2•xHCl, EtOH
NO2
N
CHO
R
N
R
Cs2CO3
HMTA
CuI
H
(II)
(III)
(I)
DMF
O
F2HC
O
Cl
F2HC
N
R
N
N
H
N
7
N
NH2-NH2•H2O
NH2
N
N
6
N
N
TEA
CH2Cl2
Pd-C
MeOH
R
5
3
4
(V)
(IV)
Scheme 1. The synthetic routes of the title compounds

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HETEROCYCLES, Vol. 96, No. 1, 2018
The EC50 of the in vitro fungistatic activity of compounds Va–Vs and boscalid against fungi Rhizoctonia
solan and Colletotrichum orbiculare are listed in Table 1. All of the synthetic compounds exhibited
antifungal effects on the two fungi. Most compounds of the series showed strong antifungal activity with
EC50 values <100 μg·mL–1. Among them, the EC50 of compounds Vk and Vd against Colletotrichum
orbiculare were all below 10 μg·mL–1, the inhibition rates of compounds Vf, Ve and Vk against
Colletotrichum orbiculare were all below 20 μg·mL–1. The common structural feature of the outstanding
compounds is the R in the indazole with a bromine and a chlorine substitution, suggesting that the moiety
at this site plays a positive role in the mechanism of their antifungal action. Removing the 5-chlorine of
the indazole did not increase any activity against Rhizoctonia solan significantly, but was found to be
favorable to increase the activity against Colletotrichum orbiculare. With the R replaced with
5,7-di-tert-butyl, 6-diethylamino, 5-nitro and 6-benzyloxy, it was more unfavorable for the activity.
According to the different substituents, the activity decreased according to the following order: bromine >
chlorine > fluorine > aliphatic hydrocarbon group > nitro > benzyl. Careful comparison of the activity of
compounds Vk㸪Vl and Vf showed the following order: 5-substituent > 6-substituent > 7-substituent.
Small substitutent compounds were better than bulky substitutent in same position, e.g., Va > Vo > Vj.
The activities of compounds Ve and Vk exhibited stronger in vitro antifungal activity than boscalid
against Colletotrichum orbiculare. The EC50 value indicated that Vk had stronger in vitro antifungal
activity than boscalid to these two fungi. The EC50 values of the compound against Rhizoctonia solan and
Colletotrichum orbiculare were 2.38 μg·mL–1 and 6.51 μg·mL–1, respectively. Therefore, it is more
reasonable that we mainly synthesized these compounds on Table 1, which were more likely to be
developed as potential fungicides.
Table 1. Structures and EC50 values against R. solani and C. orbiculare
Structure
R
EC50 (μg·mL–1)
R. solani C. orbiculare
Compound
7-methyl
7-ethyl
7-vinyl
7-bromo
7-fluoro
7-chloro
5-fluoro
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
Vi
68.25
78.52
52.31
21.63
5.27
65.32
89.51
41.52
236.51
125.60
6.51
26.51
37.22
39.84
5.32
36.40
24.55
65.26
78.33
186.31
104.64
2.38
5,7-dimethyl
5,7-di-tert-butyl
7-allyl
5-chloro-7-bromo
6- chloro
Vj
Vk
Vl
15.63
196.37
26.31
241.25
Vm
6-diethylamino

HETEROCYCLES, Vol. 96, No. 1, 2018
77
Vn
Vo
Vp
Vq
Vr
6-benzyloxy
6-ethyl
389.55
85.15
325.61
75.42
41.80
102.72
4.85
>400
102.56
245.1
85.98
65.82
126.35
8.66
5-nitro
7-ethoxy
6-methyl
5-methoxy
Vs
Boscalid
In an effort to elucidate the possible antifungal mechanism of the fungicidal activities induced by these
compounds, molecular docking of compounds boscalid and Vk to the binding site of succinate
dehydrogenase (SDH, pdb code: 2FBW3) pdb was performed. The crystal structure of SDH was obtained
from Protein Data Bank for docking studies. The molecular docking operations in our studies were
performed by the Discovery Studio 3.5 Client to investigate the interactions between 2FBW and two
molecules. During the whole docking process, drug molecules were flexible, while the protein molecule
was kept rigid. 3D and 2D schematic diagrams clearly explained the possible optimal combination
between the ligands and receptor protein (Figure 2).
Figure 2. Docking model of boscalid and compounds Vk

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HETEROCYCLES, Vol. 96, No. 1, 2018
Boscalid and Vk were embedded well in the same protein groove established by extraction of carboxin
from the original protein 2FBW. Both were bound to the active site Qp of SDH and two hydrogen bonds
were formed between them to the amino acid residues. Interestingly, although skeleton units are similar,
the embedded subunits are different (structures shown in brown in Figure 2; (A) connolly surface of SDH
with boscalid shown as a stick model; (B) connolly surface of SDH with Vk shown as a stick model; (C)
interaction of boscalid and amino acid residues near the ligands (2D diagram); (D) interaction of Vk and
amino acid residues near the ligands (2D diagram). Two hydrogen bonds were formed between carboxyl
oxygen of boscalid and amino acid residues (the amino hydrogen of TRP173 and the hydroxyl hydrogen
of TYR58). Three d-d conjugations are simultaneously formed between boscalid to ARG 43 and TRP32.
Unlike the previous case, the carboxyl oxygen of Vk formed a hydrogen bond with the amino hydrogen
of TRP173. Another hydrogen bond was formed between bromine atom of the compound Vk and the
hydroxyl hydrogen of TYR58. The CDOCKER score (the scoring function value of docking program) of
boscalid and compound Vk is 78.05 and 86.33, respectively. The docking result appears to be quite
harmonious, which may be due to the sensitivity of the protein cavity to the size of the ligand. Resistant
fungal genotype analysis verified that most of those key residues involved in forming the binding cavity
were related to resistance formation.4 Thus, a stable complex between compound Vk and SDH was
formed based on these interactions. The results of this molecular docking could support the postulation
that our active compounds may act on the same enzyme target where SDH inhibitor acts, confirming the
molecular design of the reported class of antifungal agents.
Nineteen novel pyrazole amides had been synthesized. Two plant pathogenic fungi were used to measure
antifungal activities of the compounds. Compounds Vk exhibited broad-spectrum and potent antifungal
activity. By comparing the activities and structures of the compounds Vk with former synthesized lead
compounds, it could be found that the indazole ring with bromine atoms introduced to indazole ring play
a significant role in improving the activities of the molecules. Molecular docking studies further
explained the strong inhibitory activity of Vk compared to boscalid of the same series and enabled
understanding of the various interactions between the ligands and SDH active sites in detail. Further
studies on structural derivation and biometrics are in progress in our laboratory.
EXPERIMENTAL
1
13
The H NMR and C NMR spectra were recorded on a Bruker AV-500 spectrometer using TMS as the
internal standard. All melting points were obtained on an X-4 binocular microscope melting point
apparatus. The HR-MS spectra were acquired on an Agilent 6520 Accurate-Mass apparatus with an ESI
source. Solvents and reagents were obtained from commercial suppliers and were used without further
purification.

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79
Synthesis of Compounds IIa: To a solution of 2-hydroxy-3-methylbenzaldehyde (5 g, 36.7 mmol) in
EtOH (30 mL) was added hydrazine hydrochloride (in excess) in a 250 mL flask. The solution was
refluxed for 3 h and the solvent was distilled under reduced pressure.5 Purification of the residue by
column chromatography (EtOAc:PE=10:1) on silica gel was performed to give 3.5 g of
7-methyl-1H-indazole, yellow crystals (73% yield). mp 122-124 °C. 1H NMR (500 MHz, CDCl3) δ 11.50
(s, 1H, NH), 8.43 (d, J = 1.4 Hz, 1H), 7.82 (dt, J = 7.5, 1.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.13 (dd, J =
7.5, 1.4 Hz, 1H), 2.50 (s, 3H-CH3).
Synthesis of Compounds IIIa: In a 250 mL flask, 1-chloro-2-nitrobenzene (3.5 g, 22.3 mmol),
4-methyl-1H-indazole (3.0 g, 23 mmol), cesium carbonate (7.4 g, 23 mmol), HMTA (0.1 g) and CuI (0.1
g) were dissolved in DMF (100 mL). The mixture was heated to reflux for 26 h. After the complete
disappearance of the substrates, the reaction was stopped and the mixture was cooled to room temperature.
The reaction mixture was passed through a plug of celite and the filtrate was slowly added to the same
volume of water, extracted three times with EtOAc and the organic phase was combined.6 The combined
organic layer was dried over anhydrous magnesium sulfate and evaporated under reduced pressure to give
a crude product. After the organic layer was concentrated, the residue was purified by column
chromatography (EtOAc:PE=8:1) on silica gel to give 4-methyl-1-(2-nitrophenyl)-1H-indazole (3.8 g,
yellow crystals. yield 68%); mp 172-174 °C. 1H NMR (500 MHz, CDCl3) δ 8.64 (d, J = 1.4 Hz, 1H), 8.51
(dd, J = 7.5, 1.4 Hz, 1H), 8.29 (td, J = 7.4, 1.4 Hz, 1H), 8.21 (dd, J = 7.5, 1.5 Hz, 1H), 7.98 – 7.86 (m,
1H), 7.82 (dt, J = 7.5, 1.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.27 (dd, J = 7.5, 1.5 Hz, 1H), 2.55 (s,
3H-CH3).
Synthesis of Compounds IVa: 50 mL EtOH and palladized charcoal (0.1 g, 5%) was added to a 100 mL
three-necked flask equipped with a dropping funnel containing 4-methyl-1-(2-nitrophenyl)-1H-indazole
(3 g, 11.8 mmol), and then 80% hydrazine hydrate (10 mL) from a dropping funnel was added and kept
for 30 min.7 The reaction was then stirred for 8 h and then cooled. The solid was filtered off and the
filtrate was evaporated under vacuum to afford the corresponding crude amide product. The residue was
purified
by
column
chromatography
(EtOAc:PE=4:1)
on
silica
gel
to
give
1
2-(4-methyl-1H-indazol-1-yl)aniline (2.1 g, white crystals. yield 82%); mp 121-123 °C. H NMR (500
MHz, CDCl3) δ 8.61 (d, J = 1.4 Hz, 1H), 7.80 (dt, J = 7.5, 1.5 Hz, 1H), 7.73 (dd, J = 7.5, 1.5 Hz, 1H),
7.47 – 7.29 (m, 3H), 7.24 (dd, J = 7.5, 1.5 Hz, 1H), 7.02 (dd, J = 7.5, 1.4 Hz, 1H), 5.46 (s, 2H, -NH2),
2.49 (s, 3H, -CH3).
Synthesis of Compounds Va: A solution of 2-(4-methyl-1H-indazol-1-yl)aniline (0.5 g, 2.2 mmol), 25
mL CH2Cl2 and 0.5 mL triethylamine was added to a 50 mL flask and cooled to 0 °C in an ice bath. After
10 min, 0.84 g 3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carbonyl chloride was added. The mixture

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was stirred for 30 min, and then was concentrated under reduced pressure to give a crude product. The
pure 2-chloro-N-(2-(piperidin-1-yl)phenyl)nicotinamide (Va) was obtained by column chromatography
(EtOAc:PE=10:1) purification. Compounds Vb–Vs were synthesized according to this way.
3-(Difluoromethyl)-1-methyl-N-(2-(7-methyl-1H-indazol-1-yl)phenyl)-1H-pyrazole-4-carboxamide
(Va). white powder. yield 82%; mp 128-130 °C. 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H, NH), 8.91 (d,
J = 1.5 Hz, 1H), 8.71 (s, 1H, Pyrazole-H), 8.09 – 8.02 (m, 1H), 8.02 – 7.95 (m, 1H), 7.82 (dt, J = 7.5, 1.5
Hz, 1H), 7.79 – 7.67 (m, 2H), 7.48 – 7.42 (m, 1H), 7.30 (dd, J = 7.5, 1.5 Hz, 1H), 7.10 (t, J = 54 Hz, 1H,
-CF2H), 3.81 (s, 3H), 2.50 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 162.11, 158.95, 138.05, 137.50, 134.84,
134.40, 134.02, 130.20, 130.19, 126.66, 125.93, 124.29, 123.80, 122.59, 122.36, 117.86, 108.60, 107.12,
40.56, 18.04. HRMS (ESI), m/z calcd for C20H17F2N5O (M+H)+ 382.1435, found 382.1343.
3-(Difluoromethyl)-N-(2-(7-ethyl-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (Vb).
white crystals. yield 75%; mp 143-146 °C. 1H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H, NH), 8.87 (d, J =
1.4 Hz, 1H), 8.69 (s, 1H, Pyrazole-H), 8.08 – 8.03 (m, 1H), 8.01 – 7.96 (m, 1H), 7.82 (dt, J = 7.5, 1.4 Hz,
1H), 7.77 – 7.69 (m, 2H), 7.48 (t, J = 7.5 Hz, 1H), 7.32 (dd, J = 7.5, 1.4 Hz, 1H), 7.19 (d, J = 54 Hz, 1H,
13
-CF2H), 3.81 (s, 3H), 2.71 (q, J = 6.6 Hz, 2H), 1.18 (t, J = 6.6 Hz, 3H). C NMR (125 MHz, CDCl3) δ
162.09, 158.94, 138.03, 137.51, 135.68, 134.83, 134.01 130.34, 129.07, 128.82, 126.66, 125.93, 123.45,
122.59, 122.36, 119.38, 108.60, 107.12, 40.56, 28.09, 13.41. HRMS (ESI), m/z calcd for C21H19F2N5O
(M+H)+ 396.1591, found 396.1594.
3-(Difluoromethyl)-1-methyl-N-(2-(7-vinyl-1H-indazol-1-yl)phenyl)-1H-pyrazole-4-carboxamide (Vc).
white powder. yield 71%; mp 175-177 °C. 1H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H, NH), 8.93 (d, J =
1.5 Hz, 1H), 8.73 (s, 1H, Pyrazole-H), 8.10 – 8.03 (m, 1H), 8.02 – 7.96 (m, 1H), 7.84 – 7.77 (m, 1H),
7.76 – 7.70 (m, 2H), 7.56 (dd, J =7.5, 1.5 Hz, 1H), 7.50 – 7.41 (m, 1H), 7.33 (d, J = 54.6 Hz, 1H, -CF2H),
13
7.18 – 7.00 (m, 1H), 5.89 (dd, J = 15.0, 7.2 Hz, 1H), 5.44 (dd, J = 15.0, 7.2 Hz, 1H), 3.78 (s, 3H). C
NMR (125 MHz, CDCl3) δ 162.08, 158.92, 138.05, 137.51, 135.66, 134.84, 134.02, 133.43, 126.66,
126.61, 126.34, 125.98, 125.93, 123.04, 122.59, 122.36, 116.55, 115.05, 108.60, 107.12, 40.56. HRMS
(ESI), m/z calcd for C21H17F2N5O (M+H)+ 394.1435, found 394.1436.
N-(2-(7-Bromo-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vd).
1
white powder. yield 69%; mp 162-164 °C. H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H, NH), 8.92 (d, J =
1.6 Hz, 1H), 8.71 (s, 1H), 8.10 – 8.02 (m, 1H), 8.02 – 7.95 (m, 1H), 7.86 (dt, J = 7.5, 1.5 Hz, 1H), 7.81 –
7.69 (m, 2H), 7.68 – 7.61 (m, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.11 (d, J = 56 Hz, 1H, -CF2H), 3.81 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 162.13, 158.97, 138.06, 137.53, 135.44, 134.84, 134.02, 130.55, 130.07,
126.66, 126.11, 125.93, 122.59, 122.36, 121.97, 113.62, 108.60, 107.12, 40.56. HRMS (ESI), m/z calcd
for C19H14BrF2N5O (M+H)+ 447.0329, found 447.0333.

HETEROCYCLES, Vol. 96, No. 1, 2018
81
3-(Difluoromethyl)-N-(2-(7-fluoro-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (Ve).
1
white powder. yield 86%; mp 153-155 °C. H NMR (500 MHz, CDCl3) δ 9.30 (s, 1H, NH), 9.11 (d, J =
1.6 Hz, 1H), 8.75 (s, 1H), 8.13 – 8.06 (m, 1H), 8.04 – 7.98 (m, 1H), 7.89 (dt, J = 7.5, 1.5 Hz, 1H), 7.85 –
7.73 (m, 2H), 7.73 – 7.68 (m, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.16 (d, J = 56 Hz, 1H, -CF2H), 3.86 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 162.11, 158.99, 143.95, 138.05, 137.50, 134.84, 134.02, 129.78, 127.42,
126.66, 125.93, 125.86, 122.59, 122.36, 118.48, 115.58, 108.64, 107.16, 40.58. HRMS (ESI), m/z calcd
for C19H14F3N5O (M+H)+ 386.1184, found 386.1185.
N-(2-(7-Chloro-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vf).
white crystals. yield 77%; mp 127-129 °C. 1H NMR (500 MHz, CDCl3) δ 9.25 (s, 1H, NH), 8.93 (d, J =
1.6 Hz, 1H), 8.71 (s, 1H), 8.12 – 7.94 (m, 2H), 7.86 – 7.77 (m, 1H), 7.72 (dd, J = 7.5, 1.6 Hz, 2H), 7.49
13
(dd, J = 7.5, 1.6 Hz, 2H), 7.45 – 7.40 (m, 1H), 7.16 (d, J = 54 Hz, 1H, -CF2H), 3.85 (s, 3H). C NMR
(125 MHz, CDCl3) δ 162.16, 158.55, 138.05, 137.37, 134.79, 134.08, 132.66, 129.24, 128.79, 126.66,
125.93, 124.85, 122.59, 122.36, 121.94, 120.14, 108.60, 107.12, 40.87. HRMS (ESI), m/z calcd for
C19H14F3N5O (M+H)+ 403.0825, found 403.0824.
3-(Difluoromethyl)-N-(2-(5-fluoro-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (Vg).
light yellow crystals. yield 81%; mp 180-188 °C. 1H NMR (500 MHz, CDCl3) δ 9.33 (s, 1H, NH), 8.93 (d,
J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.30 (dd, J = 7.5, 1.5 Hz, 1H), 8.11 – 7.93 (m, 2H), 7.84 – 7.67 (m, 2H),
7.60 (dt, J = 7.5, 1.5 Hz, 1H), 7.27 (dd, J = 7.5, 1.5 Hz, 1H), 7.10 (d, J = 56 Hz, 1H, -CF2H), 3.81 (s, 3H).
13C NMR (125 MHz, CDCl3) δ 161.04, 160.82, 158.95, 137.88, 136.69, 136.53, 134.81, 133.28, 131.01,
126.09, 125.90, 122.76, 122.60, 117.08, 114.16, 109.14, 107.57, 106.45, 40.32. HRMS (ESI), m/z calcd
for C19H14F3N5O (M+H)+ 386.1184, found 386.1185.
3-(Difluoromethyl)-N-(2-(5,7-dimethyl-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide
(Vh). white crystals. yield 73%; mp 156-158 °C. 1H NMR (500 MHz, CDCl3) δ 9.45 (s, 1H, NH), 8.89 (d,
J = 1.5 Hz, 1H), 8.69 (s, 1H), 8.09 – 7.92 (m, 2H), 7.78 – 7.64 (m, 2H), 7.57 (t, J = 1.5 Hz, 1H), 7.31 (d, J
13
= 54 Hz, 1H, -CF2H), 7.15 (d, J = 1.5 Hz, 1H), 3.80 (s, 3H), 2.49 (s, 3H), 2.30 (s, 3H). C NMR (125
MHz, CDCl3) δ 161.70, 157.05, 139.11, 137.95, 134.84, 134.02, 132.28, 132.03, 131.05, 130.54, 126.66,
125.93, 125.23, 122.59, 122.36, 120.61, 108.60, 107.12, 40.56, 21.83, 18.73. HRMS (ESI), m/z calcd for
C21H19F2N5O (M+H)+ 396.1591, found 396.1589.
N-(2-(5,7-Di-tert-butyl-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxam-
1
ide (Vi). yellow crystals. yield 78%; mp 161-163 °C. H NMR (500 MHz, CDCl3) δ 9.33 (s, 1H, NH),
8.95 (d, J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.14 – 8.02 (m, 1H), 8.02 – 7.96 (m, 1H), 7.81 – 7.65 (m, 3H), 7.51
13
(d, J = 1.6 Hz, 1H), 7.26 (t, J = 56 Hz, 1H, -CF2H), 3.81 (s, 3H), 1.35 (s, 9H), 1.32 (s, 9H). C NMR
(125 MHz, CDCl3) δ 162.11, 158.95, 150.47, 141.67, 140.04, 138.05, 137.95, 134.84, 134.73, 134.02,
126.66, 125.87, 125.54, 122.59, 122.36, 119.30, 108.60, 107.12, 40.56, 38.64, 35.51, 31.36, 31.15.

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HRMS (ESI), m/z calcd for C27H31F2N5O (M+H)+ 480.2530, found 480.2529.
N-(2-(7-Allyl-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vj).
white crystals. yield 71%; mp 147-148 °C. 1H NMR (500 MHz, CDCl3) δ 9.26 (s, 1H, NH), 8.93 (d, J =
1.4 Hz, 1H), 8.71 (s, 1H), 8.10 – 8.02 (m, 1H), 8.02 – 7.96 (m, 1H), 7.92 (dt, J = 7.3, 1.5 Hz, 1H), 7.82 –
7.66 (m, 2H), 7.54 (dd, J = 7.5, 1.4 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.25 (t, J = 57.2 Hz, 1H, -CF2H),
5.92 (ddt, J = 16.2, 7.2, 6.1 Hz, 1H), 5.06 (d, 1H, J = 6.1 Hz), 4.82 (d, 1H, J = 16.2 Hz), 3.81 (s, 3H), 3.33
(d, J = 7.2 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 163.15, 157.55, 137.12, 137.58, 136.82, 135.36,
134.84, 134.02, 128.54, 127.69, 126.66, 126.38, 125.93, 122.59, 122.36, 121.34, 119.07, 115.06, 108.60,
107.12, 40.56, 38.75. HRMS (ESI), m/z calcd for C22H19F2N5O (M+H)+ 408.1591, found 408.1591.
N-(2-(7-Bromo-5-chloro-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxa-
1
mide (Vk). white crystals. yield 69%; mp 164-166 °C. H NMR (500 MHz, CDCl3) δ 9.15 (s, 1H, NH),
8.96 (d, J = 1.5 Hz, 1H), 8.71 (s, 1H), 8.11 – 8.02 (m, 1H), 8.02 – 7.96 (m, 1H), 7.84 (t, J = 1.6 Hz, 1H),
7.76 – 7.70 (m, 2H), 7.69 (d, J = 1.6 Hz, 1H), 7.25 (t, J = 56 Hz, 1H, -CF2H), 3.81 (s, 3H). 13C NMR (125
MHz, CDCl3) δ 163.45, 157.58, 138.05, 136.82, 134.79, 134.34, 134.02, 132.01, 131.48, 131.26, 126.66,
125.93, 122.59, 122.36, 122.28, 114.68, 108.62, 107.19, 45.86. HRMS (ESI), m/z calcd for
C19H13BrClF2N5O (M+H)+ 480.9940, found 480.9942.
N-(2-(6-Chloro-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Vl).
yellow crystals. yield 74%; mp 151-153 °C. 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H, NH), 8.90 (d, J =
1.6 Hz, 1H), 8.71 (s, 1H), 8.09 – 8.02 (m, 1H), 8.02 – 7.97 (m, 1H), 7.96 (d, J = 1.5 Hz, 1H), 7.86 (dd, J =
7.5, 1.6 Hz, 1H), 7.79 – 7.67 (m, 2H), 7.34 (dd, J = 7.5, 1.6 Hz, 1H), 7.10 (d, J = 55.4 Hz, 1H, -CF2H),
3.81 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 161.79, 159.25, 138.77, 137.65, 136.17, 134.81, 133.28,
130.09, 128.25, 126.09, 125.90, 123.79, 122.76, 122.60, 122.03, 112.28, 108.60, 107.12, 40.56. HRMS
(ESI), m/z calcd for C19H14ClF2N5O (M+H)+ 403.0825, found 403.0823.
N-(2-(6-(Diethylamino)-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxam-
1
ide (Vm). white crystals. yield 77%; mp 174-176 °C. H NMR (500 MHz, CDCl3) δ 9.35 (s, 1H, NH),
8.83 (d, J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.09 – 8.02 (m, 1H), 8.02 – 7.96 (m, 1H), 7.79 – 7.67 (m, 3H), 7.37
(d, J = 56 Hz, 1H, -CF2H), 7.24 (d, J = 1.5 Hz, 1H), 6.69 (dd, J = 7.5, 1.5 Hz, 1H), 3.81 (s, 3H), 3.40 (q, J
13
= 7.2 Hz, 4H), 1.12 (t, J = 7.2 Hz, 6H). C NMR (125 MHz, CDCl3) δ 162.13, 158.82, 148.37, 145.10,
138.05, 136.17, 134.81, 133.28, 126.09, 125.90, 124.44, 122.86, 122.76, 122.60, 113.87, 108.60, 107.12,
96.99, 46.85, 41.78, 12.99. HRMS (ESI), m/z calcd for C23H24F2N6O (M+H)+ 439.2013, found 439.2015.
N-(2-(6-(Benzyloxy)-1H-indazol-1-yl)phenyl)-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide
(Vn). white powder. yield 73%; mp 138-140 °C. 1H NMR (500 MHz, CDCl3) δ 9.40 (s, 1H, NH), 8.88 (d,
J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.09 – 8.03 (m, 1H), 8.02 – 7.96 (m, 1H), 7.95 – 7.88 (m, 1H), 7.80 – 7.67
(m, 2H), 7.54 (d, J = 1.6 Hz, 1H), 7.51 – 7.45 (m, 2H), 7.44 – 7.29 (m, 3H), 7.14 (d, J = 56 Hz, 1H,

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13
-CF2H), 6.96 (dd, J = 7.5, 1.6 Hz, 1H), 5.14 (s, 2H), 3.81 (s, 3H). C NMR (125 MHz, CDCl3) δ 162.21,
159.05, 157.93, 139.24, 138.05, 137.56, 136.17, 134.81, 133.28, 129.01, 128.19, 128.16, 126.09, 125.90,
124.13, 122.76, 122.60, 119.91, 112.03, 108.60, 107.12, 97.21, 70.96, 40.56. HRMS (ESI), m/z calcd for
C26H21F2N5O2 (M+H)+ 474.1697, found 474.1699.
3-(Difluoromethyl)-N-(2-(6-ethyl-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide (Vo).
1
white powder. yield 75%; mp 133-135 °C. H NMR (500 MHz, CDCl3) δ 9.19 (s, 1H, NH), 8.88 (d, J =
1.5 Hz, 1H), 8.71 (s, 1H), 8.10 – 8.02 (m, 1H), 8.02 – 7.97 (m, 1H), 7.96 – 7.89 (m, 1H), 7.84 (d, J = 1.6
Hz, 1H), 7.79 – 7.65 (m, 2H), 7.23 (d, J = 1.5 Hz, 1H), 7.15 (d, J = 55.8 Hz, 1H, -CF2H), 3.81 (s, 3H),
2.72 (q, J = 6.2 Hz, 2H), 1.18 (t, J = 6.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 164.51, 162.37, 142.08,
138.05, 136.17, 135.42, 134.81, 133.28, 126.09, 125.90, 123.12, 122.76, 122.60, 122.60, 120.81, 108.60,
108.46, 107.12, 40.56, 28.44, 13.19. HRMS (ESI), m/z calcd for C21H19F2N6O (M+H)+ 396.1591, found
396.1591.
3-(Difluoromethyl)-1-methyl-N-(2-(5-nitro-1H-indazol-1-yl)phenyl)-1H-pyrazole-4-carboxamide (Vp).
white powder. yield 84%; mp 159-161 °C. 1H NMR (500 MHz, CDCl3) δ 9.36 (s, 1H, NH), 9.01 (d, J =
1.6 Hz, 1H), 8.79 (t, J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.58 (d, J = 7.5 Hz, 1H), 8.30 (dd, J = 7.5, 1.6 Hz, 1H),
8.12 – 8.03 (m, 1H), 8.03 – 7.95 (m, 1H), 7.80 – 7.65 (m, 2H), 7.25 (t, J = 56 Hz, 1H, -CF2H), 3.66 (s,
3H). 13C NMR (125 MHz, CDCl3) δ 163.51, 160.15, 144.01, 142.09, 138.05, 136.69, 134.81, 133.28,
126.09, 125.90, 122.76, 122.60, 121.82, 121.14, 117.58, 113.96, 108.60, 107.12, 42.51. HRMS (ESI), m/z
calcd for C19H14F2N6O3 (M+H)+ 413.1129, found 413.1128.
3-(Difluoromethyl)-N-(2-(7-ethoxy-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide
1
(Vq). yellow crystals. yield 82%; mp 182-184 °C. H NMR (500 MHz, CDCl3) δ 9.23 (s, 1H, NH), 8.94
(d, J = 1.4 Hz, 1H), 8.71 (s, 1H), 8.13 – 8.02 (m, 1H), 7.99 (dt, J = 7.5, 1.6 Hz, 1H), 7.79 – 7.68 (m, 2H),
7.60 (dt, J = 7.5, 1.4 Hz, 1H), 7.48 (t, J = 7.5 Hz, 1H), 7.29 (dd, J = 7.5, 1.4 Hz, 1H), 7.20 (d, J = 56.3 Hz,
1H, -CF2H), 4.07 (q, J = 5.9 Hz, 2H), 3.81 (s, 3H), 1.34 (t, J = 5.9 Hz, 3H). 13C NMR (125 MHz, CDCl3)
δ 165.13, 160.05, 147.82, 138.05, 137.50, 134.84, 134.02, 130.20, 128.50, 126.66, 125.93, 124.58, 122.59,
122.36, 118.16, 112.85, 108.60, 107.12, 64.46, 40.56, 13.80. HRMS (ESI), m/z calcd for C21H19F2N5O2
(M+H)+ 412.1540, found 412.1539.
3-(Difluoromethyl)-1-methyl-N-(2-(6-methyl-1H-indazol-1-yl)phenyl)-1H-pyrazole-4-carboxamide
(Vr). white powder. yield 76%; mp 147-148 °C. 1H NMR (500 MHz, CDCl3) δ 9.11 (s, 1H, NH), 8.87 (d,
J = 1.6 Hz, 1H), 8.71 (s, 1H), 8.10 – 8.02 (m, 1H), 8.02 – 7.96 (m, 1H), 7.87 (dd, J = 7.5, 1.5 Hz, 1H),
7.79 (t, J = 1.6 Hz, 1H), 7.72 (dd, J = 7.5, 3.6 Hz, 2H), 7.37 (d, J = 56 Hz, 1H, -CF2H), 7.17 (dd, J = 7.5,
13
1.5 Hz, 1H), 3.81 (s, 3H), 2.44 (s, 3H). C NMR (125 MHz, CDCl3) δ 167.84, 161.78, 138.29, 138.65,
136.17, 135.81, 134.81, 133.28, 126.09, 125.90, 124.08, 122.79, 122.76, 122.60, 122.07, 111.68, 108.62,
107.12, 45.33, 21.23. HRMS (ESI), m/z calcd for C20H17F2N5O (M+H)+ 382.1435, found 382.1437.

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3-(Difluoromethyl)-N-(2-(5-methoxy-1H-indazol-1-yl)phenyl)-1-methyl-1H-pyrazole-4-carboxamide
(Vs). white powder. yield 69%; mp 140-142 °C. 1H NMR (500 MHz, CDCl3) δ 9.17 (s, 1H, NH), 8.90 (d,
J = 1.5 Hz, 1H), 8.71 (s, 1H), 8.32 (d, J = 7.5 Hz, 1H), 8.13 – 8.03 (m, 1H), 8.02 – 7.93 (m, 1H), 7.82 –
7.61 (m, 2H), 7.43 (t, J = 1.5 Hz, 1H), 7.33 (d, J = 56 Hz, 1H, -CF2H) , 7.06 – 6.96 (m, 1H), 4.42 (s, 3H),
3.81 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 167.05, 162.14, 159.38, 139.42, 136.69, 135.08, 134.81,
133.28, 130.49, 126.09, 125.90, 122.76, 122.60, 114.15, 110.39, 108.60, 106.05, 105.95, 56.48, 48.26.
HRMS (ESI), m/z calcd for C20H17F2N5O2 (M+H)+ 398.1384, found 398.1386.
The fungicidal activity of the target compounds was tested in vitro against the Rhizoctonia solani and
Colletotrichum orbiculare using the method of mycelia growth inhibition.8 The synthesized compounds
were dissolved in DMSO to prepare a 10 mg·mL–1 solution and further diluted to 100, 50, 25, 12.5, 6.25,
3.125 and 1.56 μg·mL–1. Each test was repeated three times. The antifungal activities were tested
according to the methods in reference.9 We used SPSS 19.0 to perform the statistical analysis.
The automated docking was performed with CDOCKER implemented in Discovery Studio 3.5. The
three-dimensional structures of picked compounds were constructed using Chem. 3D ultra 14.0 software.
Geometrical structures were energetically optimized by using MOPAC with 100 iterations and minimum
RMS gradient of 0.10. The Gasteiger–Hückel charges of ligands were assigned. All bound water and
ligands were eliminated from the protein, and the polar hydrogens and the Kollman-united charges were
added to the proteins.
ACKNOWLEDGEMENTS
This work was supported by the Guizhou Engineering Research Center (QJHKYZ[2017]024) and
Tongren University Dr. fund (trxyDH1509).
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