Novel trifluoromethylpyrazole acyl urea derivatives: Synthesis, crystal structure, fungicidal activity and docking study

Article abstract of DOI:10.1016/j.molstruc.2018.06.050

Thirteen novel trifluoromethylpyrazole acyl urea derivatives were designed and synthesized. In vivo fungicidal activities of these compounds were tested against Fusarium oxysporum, Corynespora mazei, Botrytis cinerea and Pseudomonas syringae respectively, particularly compounds exhibited significant control effective at 100 mg/L. More importantly, some compounds showed the good control effective at 10 mg/L. Furthermore, docking was established to study the structure-activity relationship of the title compounds. It is possible that trifluoromethylpyrazole acyl urea derivatives, which possess good control effective against Fusarium oxysporum, Corynespora mazei and Botrytis cinerea, may become novel lead compounds for the development of fungicides with further structure modification.

Full text of DOI:10.1016/j.molstruc.2018.06.050

Accepted Manuscript
Novel trifluoromethylpyrazole acyl urea derivatives: Synthesis, crystal structure,
fungicidal activity and docking study
Han Wang, Zhi-Wen Zhai, Yan-Xia Shi, Cheng-Xia Tan, Jian-Quan Weng, Liang Han,
Bao-Ju Li, Xing-Hai Liu
PII:
S0022-2860(18)30751-8
10.1016/j.molstruc.2018.06.050
MOLSTR 25337
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 April 2018
Revised Date: 11 June 2018
Accepted Date: 11 June 2018
Please cite this article as: H. Wang, Z.-W. Zhai, Y.-X. Shi, C.-X. Tan, J.-Q. Weng, L. Han, B.-J. Li, X.-H.
Liu, Novel trifluoromethylpyrazole acyl urea derivatives: Synthesis, crystal structure, fungicidal activity
and docking study, Journal of Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.06.050.
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ACCEPTED MANUSCRIPT
Novel Trifluoromethylpyrazole Acyl Urea Derivatives: Synthesis,
Crystal Structure, Fungicidal Activity and Docking Study
Han Wanga, Zhi-Wen Zhaia, Yan-Xia Shic*, Cheng-Xia Tana, Jian-Quan Wenga, Liang Hana, Bao-Ju
Lic*, Xing-Hai Liua,b*
a College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China
b Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China
c Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100014, China
Abstract Thirteen novel trifluoromethylpyrazole acyl urea derivatives were designed and synthesized. In vivo fungicidal activities of these
compounds were tested against Fusarium oxysporum, Corynespora mazei, Botrytis cinerea and Pseudomonas syringae respectively,
particularly compounds exhibited significant control effective at 100 mg/L. More importantly, some compounds showed the good control
effective at 10 mg/L. Furthermore, docking was established to study the structure-activity relationship of the title compounds. It is possible
that trifluoromethylpyrazole acyl urea derivatives, which possess good control effective against Fusarium oxysporum, Corynespora mazei
and Botrytis cinerea, may become novel lead compounds for the development of fungicides with further structure modification.
Keywords: trifluoromethylpyrazole • urea • fungicidal activity • docking • synthesis
Introduction
Pesticide is an effective way to protect crops and increase yields in agriculture1, within which fungicides play a very important role in
modern agriculture. But the fungicide-resistance problem has appeared in recent years2. It is urgent to develop novel active compounds with
lower doses, environmental friendly and high effective in order to overcome the resistance of fungal3. In the past half century, since the
found of the first fungicide carboxin target succinate dehydrogenase (SDH), many SDHI fungicides had been introduced into the market for
effective treatment of fruit and vegetable crops4,5, such as furametpyr, penthiopyrad, isopyrazam, sedaxane, bixafen, fluxapyroxad,
pydiflumetofen, benzovindiflupyr and so on (Figure 1). Among them, many of these fungicides contain pyrazole ring with F, CHF2 or CF3
moiety. Also carboxamide is a main key group for SDHI fungicides6,7. Recent years, the carbon chain was introduced between the
carboxamide and aromatic ring in most of the commercial SDHI fungicides.
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ACCEPTED MANUSCRIPT
F2HC
F
O
Cl
O
N
O
N
N
F
Cl
Cl
O
N
H
HN
O
Cl
O
O
F
N
CF3
S
F
pydiflumetofen
2016
fluopimomide
2015
isofetamid
2015
N
F2HC
N
O
H3C
H3C
CH3
N
O
N
NH
CF3
NH
NH
CHF2
F
H
N
N
O
S
N
N
N
Cl
O
Cl
benzovindiflupyr
2013
penflufen
2012
sedaxane
2011
penthiopyrad
2009
Figure 1 The representative SDH inhibitors
In our previous work, to find high active fungicides with efficacy, lower toxicity, broad spectrum and environmental friendly, we have been
devoted to synthesizing heterocyclic derivatives and researching their pesticidal activity8-29, especially pyrazole ring with antifungal6 and
nematocidal activity30-37. The title trifluoromethylpyrazole derivatives had been designed by introducing urea group between the
trifluoromethylpyrazole ring and benzene ring in order to find high active fungicidal compounds (Figure 2). Surprisingly, all these
compounds possessed good fungicidal activity, especially against Botrytis cinerea.
F2HC
O
N
Cl
N
pydiflumetofen
2016
N
O
Cl
O
R
Cl
O
NH
NH
F3C
O
O
isofetamid
2015
N
HN
N
S
O
H3C
H3C
CH3
CF3
penthiopyrad
2009
H
N
N
S
N
O
Figure 2 Design strategy of the title compounds.
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In this study, we reported for the firstly trifluoromethylpyrazole acyl urea derivatives and their in vivo fungicidal activities. We also
discussed the relationship between structure and fungicidal activity preliminarily.
Results and Discussion
Synthesis and Spectrum
The synthetic route of key intermediate 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride is outlined in Scheme 1. The synthetic
route of title compounds is outlined in Scheme 2 and Scheme 3. Tifluoromethyl-1H-pyrazole is an important skeleton in many pesticides.
Many reference reported the synthetic methods of 3-(tifluoromethyl)-1-methyl-1H-pyrazole- 4-carboxylic acid, such as α,β-unsaturated
ketones, 1,3-diketones, β-keto esters, perfluoro acetylenes. Among them, the α,β-unsaturated ketone method was applied, which cyclized
from the starting material 2,2,2-tifluoroethyl 2-(ethoxymethyl)-4,4-difluoro-3-oxobutanoate and methylhydrazine under base condition. In
this step, the reaction temperature is below 60 oC. While the temperature increase, the yield decrease. Then the key intermediate 1-methyl-3-
(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride was obtained by hydrolysis and acyl chlorination. Finally, the acyl isocyanate reacted
with substituted aniline at room temperature. The target compounds were given easily with less by-products under this mild condition.
F3C
COOH
O
O
F3C
COOEt
O
O
CH3NHNH2
HC(OEt)3
NaOH,HCl
F3C
O
N
F3C
O
N
(CH3CO)2O
N
3
N
OEt
1
2
O
R
NH
O
F3C
CONCO
F3C
COCl
F3C
CONH2
F3C
NH
NH3H2O
RNH2
(COCl)2
SOCl2
N
N
N
N
6
N
N
4
N
N
5
7a~7m
Scheme 1 The synthetic route of title compounds
All the compounds were identified and characterized by FTIR, 1H NMR and HRMS. The infrared spectrum of acyl urea derivatives 7
showed absorption bands around at 3200 and 3100 cm−1 for N-H stretching. The characteristic stretching vibrations ν (C=O) appears at 1700
1
and 1690 cm−1 respectively. In the H NMR spectra of target compounds, all the -NH proton signals of the title compounds can be found
around 9~13 ppm. The appearance of signals at ~8.0 ppm are assigned to CH of pyrazole ring. The signal of methyl group of pyrazole is
assigned at about 4.0 ppm. Meanwhile, most of the title compounds exhibited the M+H+ peak in the ESI-MS results.
Crystal
The structure of compound 7d was further confirmed by single crystal X-ray diffraction analysis (Figure 3). The crystallographic data of
compound 7d and selected bond lengths (Å) and bond angles (°) for compound 7d are given in Table 1 and Table 2 respectively. In the
molecular structure of title compound, the pyrazole ring nearly paralleled with the benzene ring with the angle of 4.2 º. The title compound
has an extensive two dimension polymer of hydrogen bonding involving the atom O. Also it exist intermolecular hydrogen bond in the
molecule (N4-H4—O1)(see Supplementary material). The average bond lengths and bond angles of the phenyl ring38, the pyrazole ring39,
acyl urea group40 are normal.
Figure 3 The crystal structure of high active compound 7d
Table 1 Crystal Structure and Data Refinement Parameters
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Compound
7d
Empirical Formula
Color/shape
C13H10F4N4O2
colorless / rectangle
Formula Weight
Crystal System / Space Group
a / Å
330.25
Monoclinic, P2(1)/c
4.6181(11)
b / Å
18.857(4)
c / Å
15.180(4)
90
α / °
β / °
97.672(8)
γ / °
90
V / Å3
1310.1(5)
Z
4
D calc (g/cm3)
ꢀ (mm-1)
1.674
0.154
Crystal size (mm)
F(000)
0.2 × 0.18 × 0.12
672
Temp (K)
113(2)
Theta range for collection
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness of fit on F2
Final R indices [I > 2σ(I)]
R indices (all data)
Largest difference peak/hole
3.46-27.53°
16507
2977
2977 / 2 / 215
1.020
R1 = 0.0313, wR2 = 0.0782
R1 = 0.0424, wR2 = 0.0834
0.309 and -0.273
Table 2. Selected Bond lengths (Å), Selected Bond angles (°) for the Compound 7d.
Bond
Dist.
Angle
(°)
F(1)-C(1)
F(2)-C(1)
F(3)-C(1)
1.3406(16) C(2)-N(1)-N(2)
1.3432(15) C(4)-N(2)-N(1)
1.3363(15) C(4)-N(2)-C(5)
104.70(10)
112.61(10)
128.30(11)
118.95(10)
127.50(11)
121.16(11)
125.56(12)
117.57(11)
O(1)-C(6) 1.2247(15) N(1)-N(2)-C(5)
O(2)-C(7) 1.2333(15) C(6)-N(3)-C(7)
N(2)-C(4) 1.3448(16) O(1)-C(6)-C(3)
N(3)-C(7) 1.4044(16) O(2)-C(7)-N(4)
N(4)-C(7) 1.3412(16) C(9)-C(8)-C(13)
N(4)-C(8) 1.4106(15) F(4)-C(13)-C(12) 119.51(11)
C(1)-C(2) 1.4976(18) C(12)-C(13)-C(8) 123.45(12)
C(2)-C(3) 1.4162(17) N(4)-C(7)-N(3)
115.69(11)
Greenhouse in vivo fungicidal activity and SAR
The in vivo fungicidal activity of compounds 7a~7m against Fusarium oxysporum, Corynespora mazei and Botrytis cinerea in greenhouse at
different concentration are listed in Table 3. Fluxapyroxad was used as control. Meanwhile, all of these tested compounds were found safe
for the cucumber plants. As an initial evaluation, all of the title compounds were tested at a concentration of 100 ꢀg/mL against Fusarium
oxysporum, Corynespora mazei and Botrytis cinerea. As shown in Table 3, most of the title compounds were found to exhibit good control
effect against Botrytis cinerea. For example, acyl urea compound 7a (88.55±2.26), 7c (82.42±2.32), 7d (96.13±3.16), 7i (94.37±3.11), 7k
(86.18±2.85) displayed >80% control effect against Botrytis cinerea respectively, which is better than that of control. All of the title
compounds possessed good control effect (>50%) against Botrytis cinerea except compound 7f, 7g, 7h. It can be seen that substitution on the
benzene ring can greatly influence the fungicidal activity against Botrytis cinerea. When the substitution is 2,6-2F, 2,5-2F or 2,3,4-3F in urea
4

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7, there is an apparent decrease of fungicidal activity. For Corynespora mazei, acyl urea compound 7a, 7d, 7l, 7m possessed efficacy rates
70.62±3.05, 71.10±2.85, 70.01±2.30, 77.97±2.04, 83.07±2.63, and 74.29±2.33% against Corynespora mazei, respectively. All of the three
compounds were more effective than that of control. For this fungi, when compounds with an electron-donating group in the benzene ring
will affect higher fungicidal activities than those of others. However, the large electron-donating group in the benzene ring of title
compounds is favorable. Compounds 7a and 7m displayed moderate efficacy rates against Corynespora mazei, while all of them were less
effective than all of the contrasts. It was found that compounds with multi substituted in the benzene ring have a higher level of fungicidal
activity than others. Unfortunately, most of the compounds had low fungicidal activities against Fusarium oxysporum.
CK(Water) against B. cinerea The control efficacy of 7d against B. cinerea at 100 mg/L
CK(Water) against C. mazei The control efficacy of 7m against C. mazei at 100 mg/L
Figure 4 The control efficacy against B. cinerea and C. mazei of compound 7d and 7m
On the basis of the preliminary in vivo fungicidal results, the title compounds were selected for further bioassay(>50% inhibitory) at lower
dose (50 ꢀg/mL and 10 ꢀg/mL) for fungicidal activity. The subsequent results in Table 4 and Table 5 showed that the most of tested
compounds had good fungicidal activity against Botrytis cinerea with around 80% control efficacy at 10 ꢀg/mL. For example, acyl urea
compound 7d (87.35±2.35, shown in Figure 4), 7j (83.35±2.36), 7k (93.91±3.11), 7l (86.12±3.01) displayed >80% control efficacy against
Botrytis cinerea at 10 mg/L respectively, which is same as that of control. As the concentration decreased, but the fungicidal activity of these
compounds (7d, 7k and 7l) was slightly affected. For the fungal Fusarium oxysporum, however, it was found that the inhibitory activities
decreased very quickly with the decreasing of the treatment concentration. All of them held no inhibitory at these two concentrations, even
some of them can increase the fungal growth.
Table 3 The fungicidal activity of title compounds against three fungus at 100 mg/L
No
R
Fusarium oxysporum Botrytis cinerea Corynespora mazei
7a
7b
7c
7d
7e
7f
3,5-2Me
2-MeO
2,4,6-3M e
2-F
5.19±0.29
12.99±1.02
7.79±0.85
16.88±0.13
0
88.55±2.26
60.97±2.77
82.42±2.32
96.13±3.16
60.30±2.47
46.76±2.62
35.32±2.11
39.88±2.62
94.37±3.11
75.83±2.85
86.18±2.85
62.61±2.22
70.62±3.05
45.22±0.88
12.78±0.46
71.10±2.85
40.02±1.24
48.08±2.24
36.65±2.09
17.99±1.47
54.16±2.31
54.40±2.15
11.32±0.22
70.01±2.30
3,4-2F
2,6-2F
2,5-2F
2,3,4-3F
3-F
10.39±0.91
10.39±0.22
18.18±0.71
16.88±0.62
11.69±0.47
-1.30±0.02
7.79±0.62
7g
7h
7i
7j
2-CF3
2-Cl
7k
7l
4-Et
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7m
Control Fluxapyroxad 38.33±0.15
CK water
2,6-2Et
27.27±1.12
52.43±2.06
83.87±0.23
0
77.97±2.04
88.33±1.05
0
0
Table 4 The fungicidal activity of some compounds against Botrytis cinerea and Corynespora mazei at 50 mg/L
No
R
Botrytis cinerea Corynespora mazei
7a
7b
7c
7d
7e
7i
3,5-2Me
2-MeO
2,4,6-3M e
2-F
52.50±2.85
84.35±3.44
81.73±2.55
62.05±2.24
25.22±1.95
76.98±2.05
87.80±3.01
87.44±2.65
69.88±2.85
43.29±1.74
43.69±1.02
-
-
65.49±2.65
-
3,4-2F
3-F
38.52±2.04
41.86±2.06
-
7j
2-CF3
2-Cl
7k
7l
4-Et
72.46±2.00
64.81±1.85
74.06±1.01
0
7m
2,6-2Et
Control Fluxapyroxad 83.87±0.23
CK water
0
Table 5 The fungicidal activity of some compounds against Botrytis cinerea and Corynespora mazei at 10 mg/L
No
R
Botrytis cinerea Corynespora mazei
7a
7b
7c
7d
7i
3,5-2Me
2-MeO
2,4,6-3M e
2-F
65.61±3.01
70.06±3.01
50.31±2.06
87.35±2.35
68.25±3.11
83.35±2.36
93.91±3.11
86.12±3.01
-
64.81±1.63
-
-
79.28±3.03
3-F
-
7j
2-CF3
2-Cl
-
7k
7l
-
4-Et
62.05±1.14
81.64±2.39
64.56±1.02
0
7m
2,6-2Et
Control Fluxapyroxad 64.16±0.25
CK water
0
Docking Study
In order to study the action mode of high active compound and the target, the binding modes between SDH (PDB:2FBW) and the active
compound 7d were selected as exemplified in the case of representative compound by using the Discovery studio. The compound 7d can
tightly occupy binding site of SDH, the docking results is shown in Figure 5. From the docking results, the compound 7d held two hydrogen
bonds. The O atom and F atom of NHCO group in pyrazole ring of 7d can form tightly interaction with Tyr58 and Trp173 in hinge domain
of SDH via two hydrogen bonds, respectively. The results indicated that the key active group is trifluoromethyl group and acyl amide group.
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Figure 5 The docking mode of compound 7d and the SDH
Conclusions
In summary, a series of novel pyrazole acyl urea derivatives were designed and synthesized, and their fungicidal activity against four species
of fungals was evaluated. The title compound 7d possessed high in vivo activity against B. cinerea with > 80 % control efficacy at 10 mg/L.
Furthermore, the combination of DOCK provided meaningful clues as to the structural features of these new family herbicides that will be
helpful in the design of more potent compounds in the future.
Experimental Section
General
1
Melting points were determined by an X-4 apparatus and uncorrected. H NMR spectra were measured on a Bruker AV-400 or 500 MHz
instrument using TMS as an internal standard and CDCl3 or DMSO-d6 as the solvent. FT IR spectra were measured on a NICOLET-670
instrument using KBr. HRMS was determined on a JOEL AccuTOF (JMS-T 100LC) instrument equipped with electrospray ionization (ESI)
ion source.
The raw materials of trifluoroacetoacetate ethyl-(4,4,4-trifluoro)-3-oxobutyrate, triethyl orthoformate and methyl hydrazine were purchased
from Nanjing Duodian Co. Ltd., acetic anhydride, ethyl acetate, potassium hydroxide, SOCl2, aqueous ammonia, 1,2-dichloroethane, oxalyl
chloride and other routine solvents were purchased by Shanghai Chemical Reagent Co. Ltd. and used without any purification. The course of
the reactions was monitored by TLC; analytical TLC was performed on silica gel GF254
.
Experimental
Ethyl 2-(ethoxymethylene)-4,4,4-trifluoro-3-oxobutanoate (1).
In a 500 mL four-necked flask, trifluoroacetoacetate ethyl-(4,4,4-trifluoro)-3-oxobutyrate (18400 mg, 100 mmol), triethyl orthoformate
(44400 mg, 300 mmol) and acetic anhydride (61200 mg, 600 mmol) were added and heated at 120 °C for 6 hours. Then, the solvent was
removed and the product was then distilled under vacuum over a column to give a colorless liquid, yield 90%.
ethyl 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate (2).
To a solution of methyl hydrazine (1900 mg, 41 mmol) in ethyl acetate, was added 2-ethoxy methylene 4,4,4-trifluoroacetoethylacetate
(9700 mg, 41 mmol) dropwisely. The mixture was stirred under 5 °C for 1h, then further stirred at reflux temperature for 3 hours. After the
reaction is completed, the mixture was cooled and concentrated. The residue was recrystallized with yield 69%, m. p. 55~56 °C. 1H NMR
( CDCl3, 500 MHz ), δ: 1.35 ( t, 3H, J=7.32 Hz, CH3 ), 3.97 ( s, 3H, CH3 ), 4.31( m, 2H, CH2 ), 7.92 ( s, 1H, CH ).
1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid (3)
ethyl 1-Methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylate 2 (5600 mg, 25 mmol) and potassium hydroxide (4740 mg, 30 mmol) were
added to water (50 mL). The mixture was stirred for overnight, then acidified to pH = 1 using HCl, to give 3 as a white solid that was filtered
off and dried with yield 59%, m.p. 195~196 °C，1H NMR ( 500 MHz, DMSO-d6 ), δ: 12.80 ( s, 1H, COOH ), 8.44 ( s, 1H, CH ), 3.93 ( s, 1H,
CH3 ).
1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride (4)
To 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid 3 (1500 mg, 7.5 mmol) and SOCl2 (3500 mg, 30 mmol) was refluxed for 4 h.
After the reaction is completed, the excess of SOCl2 was evaporated to give 4 as a yellow liquid that was used without further purification.
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1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (5)
The resulting 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl chloride was dissolved in 50 mL of dry THF and added to a mixture of
aqueous ammonia (25%, 2720 mg, 40 mmol) at 0 °C. After stirring overnight, a large amount of solid formed and was filtered, which was
further purified by a silica gel column eluted with petroleum ether and ethyl acetate to give the 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-
carboxamide, white solid; yield 91%; m.p. 148~149 °C. 1H NMR ( 500 MHz, DMSO-d6 ), δ: 3.99 ( s, 1H, CH3 ), 8.41 ( s, 1H, CH ), 7.65 ( s,
2H, NH ).
1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl isocyanate (6)
1-Methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide (1930 mg, 10 mmol) and anhydrous 1,2-dichloroethane(8 mL) were added into a
three-neck flask and cooled to 0-5 °C with ice water. Oxalyl chloride(2520 mg, 20 mmol) was added slowly to this mixture. The reaction
mixture was stirred for 1 h at room temperature, then kept at 50-55 °C for about 3 h, further heated to 75 °C and refluxed for 6 h. Unreacted
oxalyl chloride was evaporated under reduced pressure to leave, the 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl isocyanate as an
oily solution, which was reserved for subsequent reaction.
General Synthetic Procedure for urea 7a~7n
A 10 mL round bottom was charged with 1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carbonyl isocyanate 6 (219 mg, 1 mmol) with various
substituted aniline (1 mmol) in CH2Cl2 (4 mL). The mixture was stirred at room temperature and the correspondence product was given. The
target compounds were filtered and crude solids were recrystallized from ethanol to give the title compounds 7a~7n.
N-((3,5-dimethylphenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7a), White solid, yield 59.7%, m.p.
209~210°C; FTIR(ν, cm-1): 3242 (N-H), 3131 (N-H), 1701 (C=O), 1692 (C=O), 1614 (Ar), 1569 (Ph,C=C), 1541 (C-N), 1509 (Ph, C=C),
1475 (Ph, C=C), 1298 (C-N), 1229 (C-N), 1000-1200 (C-F), 771 (Ar-H); 1HNMR (CDCl3, 400MHz), δ: 2.26(s, 6H, 2CH3), 3.98(s, 3H, CH3),
6.75(s, 1H, Ph), 7.20(s, 2H, Ph), 8.74(s, 1H, CH), 10.53(s, 1H, NH), 10.94(s, 1H, NH). ESI-HRMS for C15H15F3N4O2 m/z: Calculated,
341.1220, Found, 341.1218 [M+1]+.
N-((2-methoxyphenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7b), White solid, yield 72.6% ， m.p.
246~247°C; FTIR(ν, cm-1): 3243 (N-H), 3134 (N-H), 1705 (C=O), 1688 (C=O), 1604 (Ar), 1565 (Ph, C=C), 1541 (C-N), 1492 (Ph, C=C),
1466 (Ph, C=C), 1301 (C-N), 1247 (C-N), 1000-1200 (C-F), 761 (Ar-H), 745 (Ph, C-H). 1HNMR (CDCl3, 400MHz), δ: 3.89(s, 3H, O-CH3),
3.98(s, 3H, CH3), 6.94-6.97(m, 1H, Ph), 7.08-7.10(m, 2H, Ph), 8.17(d, J=7.5Hz, 1H, Ph), 8.73(s, 1H, CH), 10.88(s, 1H, NH), 10.97(s, 1H,
NH). ESI-HRMS for C14H13F3N4O3 m/z: Calculated, 343.1013, Found, 343.1016 [M+1]+.
N-(mesitylcarbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7c), White solid, yield 54.8%, m.p. 222~223°C; FTIR(ν,
cm-1): 3273 (N-H), 3127 (N-H), 1697 (C=O), 1625 (Ar), 1607 (Ph, C=C), 1540 (C-N), 1503 (Ph, C=C), 1478 (Ph, C=C), 1299 (C-N), 1249
(C-N), 1000-1200 (C-F), 777 (Ar-H), 759, 679 (Ph, C-H). 1HNMR (CDCl3, 400MHz), δ: 3.35(s, 3H, CH3), 3.98(s, 3H, CH3), 6.91(s, 2H, Ph),
8,74(s, 1H, CH), 9.72(s, 1H, NH), 10.88(s, 1H, NH). ESI-HRMS for C16H17F3N4O2 m/z: Calculated, 355.1376 , Found, 355.1377 [M+1]+.
N-((2-fluorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7d), White solid, yield 76.2%, m.p. 227~228°C;
FTIR(ν, cm-1): 3243 (N-H), 3160 (N-H), 1701 (C=O), 1684 (C=O), 1621 (Ar), 1599 (Ph, C=C), 1543 (C-N), 1515 (Ph, C=C), 1458 (Ph,
C=C), 1301 (C-N), 1247 (C-N), 1000-1200 (C-F), 772 (Ar-H), 759 (Ph, C-H). 1HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H, CH3), 7.113-7.17(m,
1H, Ph), 7.22(t, J=7.5Hz, 1H, Ph), 7.31-7.34(m, 1H, Ph), 8.14-8.18(m, 1H, Ph), 8.75(s, 1H, CH), 10.86(s, 1H, NH), 11.15(s, 1H, NH). ESI-
HRMS for C13H10F4N4O2 m/z: Calculated, 331.0813, Found, 331.0812[M+1]+.
N-((3,4-difluorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7e), White solid, yield 69.2%, m.p.
192~193°C; FTIR(ν, cm-1): 3207 (N-H), 3142 (N-H), 1704 (C=O), 1681 (C=O), 1615 (Ar), 1582 (Ph, C=C), 1542 (C-N), 1521 (Ph, C=C),
1
1481 (Ph, C=C), 1300 (C-N), 1269 (C-N), 1000-1200 (C-F), 771 (Ar-H), 839, 762, (Ph,C-H). HNMR (CDCl3, 400MHz), δ: 3.98(s, 3H,
CH3), 7,39-7,44(m, 2H, Ph), 7.78-7.83(m, 1H, Ph), 8.74(s, 1H, CH), 10.59(s, 1H, NH), 11.04(s, 1H, NH). ESI-HRMS for C13H9F5N4O2 m/z:
Calculated, 349.0718, Found, 349.0715[M+1]+.
N-((2,6-difluorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7f), White solid, yield 91.2%, m.p.
211~212°C; FTIR(ν, cm-1): 3245 (N-H), 3127 (N-H), 1704(C=O), 1687 (C=O), 1606 (Ar), 1587 (Ph, C=C), 1568 (Ph, C=C), 1540 (C-N),
1472 (Ph, C=C), 1298 (C-N), 1246 (C-N), 1000-1200 (C-F), 763 (Ar-H), 786, 713 (Ph,C-H). 1HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H, CH3),
7.17-7.21(m, 2H, Ph), 7.36-7.42(m, 1H, Ph), 8.75(s, 1H, CH), 9.93(s, 1H, NH), 11.14(s, 1H, NH). ESI-HRMS for C13H9F5N4O2 m/z:
Calculated, 349.0718 , Found,349.0721 [M+1]+.
N-((2,5-difluorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7g), White solid, yield 97.5%, m.p.
230~231°C; FTIR(ν, cm-1): 3243 (N-H), 3158 (N-H), 1704(C=O), 1697 (C=O), 1629 (Ar), 1587 (Ph, C=C), 1568 (Ph, C=C), 1541 (C-N),
1
1473 (Ph, C=C), 1300 (C-N), 1242 (C-N), 1000-1200 (C-F), 772 (Ar-H), 761, 731, (Ph, C-H). HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H,
CH3), 6.97-7.01(m, 1H, Ph), 7.37-7.42(m, 1H, Ph), 8.01-8.05(m, 1H, Ph), 8.76(s, 1H, CH), 11.02(s, 1H, NH), 11.26(s, 1H, NH). ESI-HRMS
for C13H9F5N4O2 m/z: Calculated, 349.0718 , Found, 349.0717 [M+1]+.
8

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1-methyl-3-(trifluoromethyl)-N-((2,3,4-trifluorophenyl)carbamoyl)-1H-pyrazole-4-carboxamide(7h), White solid, yield 47.8%, m.p.
240~241°C; FTIR(ν, cm-1): 3209 (N-H), 3156 (N-H), 1707(C=O), 1683 (C=O), 1625 (Ar), 1581 (Ph, C=C), 1543 (C-N), 1517 (Ph, C=C),
1
1483 (Ph, C=C), 1301 (C-N), 1254 (C-N), 1000-1200 (C-F), 773 (Ar-H), 821, 761 (Ph, C-H). HNMR (CDCl3, 400MHz), δ: 4.00(s, 3H,
CH3), 7.34(s, 1H, Ph), 7.86(s, 1H, Ph), 8.76(s, 1H, CH), 10.77(s, 1H, NH), 11.23(s, 1H, NH). ESI-HRMS for C13H8F6N4O2 m/z: Calculated,
367.0624 , Found, 367.0620 [M+1]+.
N-((3-fluorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7i), White solid, yield 70.0%, m.p. 205~206°C,
FTIR(ν, cm-1): 3237 (N-H), 3146 (N-H), 1710 (C=O), 1684 (C=O), 1606 (Ar), 1541 (C-N), 1513 (Ph, C=C), 1481 (Ph, C=C), 1448 (Ph,
1
C=C), 1300 (C-N), 1279 (C-N), 1000-1200 (C-F), 771 (Ar-H), 884, 847, 711 (Ph, C-H). HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H, CH3),
6.93-6.96(m, 1H, Ph), 7.33-7.38(m, 2H, Ph), 7.60-7.62(m, 1H, Ph), 8.74(s, 1H, CH), 10.65(s, 1H, NH), 11.03(s, 1H, NH). ESI-HRMS for
C13H10F4N4O2 m/z: Calculated, 331.0813, Found, 331.0809 [M+1]+.
1-methyl-3-(trifluoromethyl)-N-((2-(trifluoromethyl)phenyl)carbamoyl)-1H-pyrazole-4-carboxamide(7j)，White solid, yield 90.8%，m.p.
192~193°C; FTIR(ν, cm-1): 3241 (N-H), 3137 (N-H), 1705 (C=O), 1695(C=O), 1616 (Ar), 1559 (Ph, C=C), 1541 (C-N), 1477 (Ph, C=C),
1
1456 (Ph, C=C), 1322 (C-N), 1301 (C-N), 1000-1200 (C-F), 767 (Ar-H); HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H, CH3), 7.38(t, J=7.5Hz,
1H, Ph), 7.71(t, J=7.5Hz, 1H, Ph), 7.75(d, J=8.0Hz, 1H, Ph), 8.76(s, 1H, CH), 10.95(s, 1H, NH), 11.20(s, 1H, NH). ESI-HRMS for
C14H10F6N4O2 m/z: Calculated, 381.0781, Found, 381.0780 [M+1]+.
N-((2-chlorophenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7k), White solid, yield 96.4%，m.p. 218~219°C;
FTIR(ν, cm-1): 3259 (N-H), 3127 (N-H), 1701 (C=O), 1682 (C=O), 1596 (Ph, C=C), 1540 (C-N), 1510 (Ph, C=C), 1291 (C-N), 1236 (C-N),
1000-1200 (C-F), 757 (Ar-H), 734 (Ph, C-H). 1HNMR (CDCl3, 400MHz), δ: 3.99(s, 3H, CH3), 7.15-7.18(m, 1H, Ph), 7.37-7.39(m, 1H, Ph),
7.55-7.56(m, 1H, Ph), 8.76(s, 1H, CH), 11.09(s, 1H, NH), 11.17(s, 1H, NH). ESI-HRMS for C13H10ClF3N4O2 m/z: Calculated, 347.0517,
Found, 347.0512 [M+1]+.
N-((4-ethylphenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7l), White solid, yield 72.3% ， m.p. 246°C;
FTIR(ν, cm-1): 3244 (N-H), 3127 (N-H), 1701(C=O), 1697 (C=O), 1598 (Ph, C=C), 1541 (C-N), 1514 (Ph, C=C), 1471 (Ph, C=C), 1296 (C-
N), 1235 (C-N), 1000-1200 (C-F), 826 (Ph, C-H), 765 (Ar-H). 1HNMR (CDCl3, 400MHz), δ: 1.17(t, J=7.5Hz, 3H, CH3), 2.58(m, 2H, CH2),
3.98(s, 3H, CH3), 7.18(d, J=8.5Hz, 2H, Ph), 7.48(d, J=8.5Hz, 2H, Ph), 8,74(s, 1H, CH), 10.49(s, 1H, NH), 10.93(s, 1H, NH). ESI-HRMS for
C15H15F3N4O2 m/z: Calculated, 341.1220, Found, 341.1222 [M+1]+.
N-((2,6-diethylphenyl)carbamoyl)-1-methyl-3-(trifluoromethyl)-1H-pyrazole-4-carboxamide(7m) ， White solid, yield 46.3% ， m.p.
219~220°C; FTIR(ν, cm-1): 3234 (N-H), 3124 (N-H), 1705(C=O), 1697 (C=O), 1591 (Ph, C=C), 1521 (C-N), 1299 (C-N), 1230 (C-N), 1000-
1
1200 (C-F), 777 (Ar-H), 760 (Ph, C-H). HNMR (CDCl3, 400MHz), δ: 1.13(t, J=7.5Hz, 6H, 2CH3), 2.56(m, 4H, 2CH2), 3.98(s, 3H, CH3),
7.14(d, J=7.5Hz, 2H, Ph), 7.21-7.24(m, 1H, Ph), 8,75(s, 1H, CH), 9.81(s, 1H, NH), 10.92(s, 1H, NH). ESI-HRMS for C17H19F3N4O2 m/z:
Calculated, 369.1533, Found, 369.1533 [M+1]+.
Greenhouse in vivo fungicidal evaluation
Antifungal activities of compound 7a~7m against Fusarium oxysporum, Corynespora mazei and Botrytis cinerea were determined according
to our previous work41. The potted plants cucumbers were used. The determine concentration of control fluxapyroxad and the title
compounds is 100 ꢀg/mL, 50ꢀg/mL, 10ꢀg/mL respectively. The three fungals Fusarium oxysporum, Corynespora mazei and Botrytis cinerea
were inoculated when the cucumber is at the stage of two seed leaves. The relative control efficacy of compounds compared to the blank
assay was calculated via the following equation:
relative control efficacy (%)=(CK-PT)/CK × 100%
where CK is the average disease index during the blank assay and PT is the average disease index after treatment during testing. All
experiments were replicated three times.
Crystal Structure Determination
The crystal of compound 7d with dimensions of 0.20mm × 0.18mm × 0.12 mm was mounted on a Rigaku saturn diffractometer with a
graphite-monochromated MoKα radiation (λ = 0.71073Å) by using a phi and scan modes at 113(2) K in the range of 3.46°≤θ≤27.53°. The
crystal belongs to monoclinic system with space group P2(1)/c and crystal parameters of a= 4.6181(11) Å, b = 18.857(4) Å, c = 15.180(4) Å,
α = 90°, β = 97.672(8)°, γ = 90°, V= 1310.1(5) A3，Dc= 1.674 g/cm3. The absorption coefficient ꢀ= 0.154 mm-1, and Z = 4. The structure
was solved by direct methods with SHELXS-9742 and refined by the full-matrix least squares method on F2 data using SHELXL-97. The
empirical absorption corrections were applied to all intensity data. H atom of N-H was initially located in a difference Fourier map and were
refined with the restraint Uiso(H) = 1.2Ueq(N). Other H atoms were positioned geometrically and refined using a riding model, with d(C---
H) = 0.93-0.97 Å and Uiso(H) = 1.2Ueq(C) or 1.5Ueq(Cmethyl). The final full-matrix least squares refinement gave R = 0.0313 and wR =
2
0.0782 (w = 1/[σ2(Fo ) + (0.0426P)2] where P = (Fo2 + 2Fc2)/3] S = 1.02.
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Supplementary material
CCDC 1848186 contain the supplementary crystallographic data for 7d. Copies of this information can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html/datarequest/cif, or from The Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK, Fax :( +44)-1223-336033/ Email: deposit@ccdc.cam.ac.uk. Also the NMR, FTIR and HRMS data can be found
in supplementary material.
Acknowledgements
This work was supported financially by the Opening Foundation of the Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Guizhou University, grant No. 2018GDGP0104 and Zhejiang Provincial Natural Science Foundation of China (No.
LY16C140007).
Author Contribution Statement
It is no conflict.
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Highlights
►The new trifluoromethylpyrazole acyl urea derivatives were
designed and synthesized.
► All the structures were determined by NMR, FTIR and HRMS.
The highest active compound were determined by X-ray diffraction.
► All newly synthesized urea derivatives are potent antifungal
agents compared to commercial drug Fluxapyroxad.
► Among all the synthesized urea derivatives, compound 7d
possessed high in vivo activity against B. cinerea with > 80 %
control efficacy at 10 mg/L.
► The mode of action was done using Docking method.