Fragmenlt Recombination Design, Synthesis, and Safener Activity of Novel Ester-Substituted Pyrazole Derivatives

Article abstract of DOI:10.1021/acs.jafc.1c02221

Fenoxaprop-p-ethyl (FE), a type of acetyl-CoA carboxylase (ACCase) inhibitor, has been extensively applied to a variety of crop plants. It can cause damage to wheat (Triticum aestivum) even resulting in the death of the crop. On the prerequisite of not reducing herbicidal efficiency on target weed species, herbicide safeners selectively protect crops from herbicide injury. Based on fragment splicing, a series of novel substituted pyrazole derivatives was designed to ultimately address the phytotoxicity to wheat caused by FE. The title compounds were synthesized in a one-pot way and characterized via infrared spectroscopy, 1H nuclear magnetic resonance, 13C nuclear magnetic resonance, and high-resolution mass spectrometry. The bioactivity assay proved that the FE phytotoxicity to wheat could be reduced by most of the title compounds. The molecular docking model indicated that compound IV-21 prevented fenoxaprop acid (FA) from reaching or acting with ACCase. The absorption, distribution, metabolism, excretion, and toxicity predictions demonstrated that compound IV-21 exhibited superior pharmacokinetic properties to the commercialized safener mefenpyr-diethyl. The current work revealed that a series of newly substituted pyrazole derivatives presented strong herbicide safener activity in wheat. This may serve as a potential candidate structure to contribute to the further protection of wheat from herbicide injury.

Full text of DOI:10.1021/acs.jafc.1c02221

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Article
Fragmenlt Recombination Design, Synthesis, and Safener Activity of
Novel Ester-Substituted Pyrazole Derivatives
Ling Jia,# Shuang Gao,# Yuan-Yuan Zhang, Li-Xia Zhao, Ying Fu,* and Fei Ye*
Cite This: J. Agric. Food Chem. 2021, 69, 8366−8379
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ABSTRACT: Fenoxaprop-p-ethyl (FE), a type of acetyl-CoA carboxylase (ACCase) inhibitor, has been extensively applied to a
variety of crop plants. It can cause damage to wheat (Triticum aestivum) even resulting in the death of the crop. On the prerequisite
of not reducing herbicidal efficiency on target weed species, herbicide safeners selectively protect crops from herbicide injury. Based
on fragment splicing, a series of novel substituted pyrazole derivatives was designed to ultimately address the phytotoxicity to wheat
1
caused by FE. The title compounds were synthesized in a one-pot way and characterized via infrared spectroscopy, H nuclear
magnetic resonance, 13C nuclear magnetic resonance, and high-resolution mass spectrometry. The bioactivity assay proved that the
FE phytotoxicity to wheat could be reduced by most of the title compounds. The molecular docking model indicated that compound
IV-21 prevented fenoxaprop acid (FA) from reaching or acting with ACCase. The absorption, distribution, metabolism, excretion,
and toxicity predictions demonstrated that compound IV-21 exhibited superior pharmacokinetic properties to the commercialized
safener mefenpyr-diethyl. The current work revealed that a series of newly substituted pyrazole derivatives presented strong
herbicide safener activity in wheat. This may serve as a potential candidate structure to contribute to the further protection of wheat
from herbicide injury.
KEYWORDS: ester-substituted pyrazole derivatives, synthesis, bioassay, safener activity, molecular simulation
converted into free carboxylic acid-containing phenoxy groups,
which destroys the physiological effect of the normal growth of
weeds and gradually withers leaves. It is a highly selective stem
and leaf treatment agent.9,10 However, due to the long-term
and widespread use of FE, crops suffer varying degrees of
phytotoxicity.11−13 The application of FE results in crop
yellowing, crop growth inhibition, and even crop death in
serious cases. To reduce damage to crops, many methods have
been reported that incorporate the scientific management of
crop rotation and cultivation and the research and develop-
ment of new herbicides.14,15 In addition, the use of herbicide
safeners is also considered to be an efficient solution.16−18
Herbicide safeners, also known as herbicide detoxifiers, are
chemicals that increase plant tolerance to herbicides without
affecting weed control efficacy.19,20 The phenomenon of
herbicide safeners was first discovered in 1947, when
Hoffmann accidentally observed that after treatment with 2-
(2,4,6-trichlorophenoxy) acetic acid, no phytotoxicity occurred
when the herbicide was exposed to 2,4-dichlorophenoxyacetic
acid (2,4-D). As the phenomenon of a safener was discovered,
the first herbicide safener, 1,8-naphthalic anhydride (NA), was
created in the mid-20th century.21 Afterward, nearly 20
commercial safeners, produced by agrochemical companies
INTRODUCTION
■
In the biosynthesis process of fatty acids, acetyl-CoA
carboxylase (ACCase) exerts a significant impact on the
sustaining function of biological organisms including bacteria,
fungi, plants, humans, and animals.1−3 ACCase catalyzes the
formation of malonyl CoA, which plays an important role in
the maintenance of cell functions during the biosynthesis of
long-chain fatty acids.4 Malonyl CoA is produced in two stages,
starting with acetyl-CoA and CO2 followed by being catalyzed
by ACCase. First, the biotin carboxylase subunit of ACCase
catalyzes the ATP-dependent carboxylation of biotin. Then,
the carboxyltransferase subunit catalyzes the process of
transferring an activated group containing carboxyl groups to
its acceptor acetyl-CoA.5 Any exogenous substance inhibits the
production of malonyl CoA, thereby inhibiting the synthesis of
fatty acids, resulting in an increased permeability of the cell
membrane structure of monocotyledonous plants, damage to
the membrane structure, and ultimately death of the plants.
Therefore, ACCase is the target of a series of important
herbicides including aryloxyphenoxypropionates (AOPPs),
cyclohexanediones (CHDs), and phenylpyrazolines (DENs).6
ACCase inhibitors are highly selective, conductive in plants,
have a low toxicity and long-lasting effects, are safe for
subsequent crops, and can effectively control annual or
perennial gramineous weeds after seedlings.
Received: April 16, 2021
Revised: July 12, 2021
Accepted: July 13, 2021
Published: July 26, 2021
Fenoxaprop-p-ethyl (FE), a typical AOPP herbicide, is an
inner-absorption herbicide for postemergence treatment. It is
applied to control annual grasses and some broad-leaf weeds in
fields.7,8 FE is transmitted to the meristem and root growth
points through stem and leaf absorption and is then rapidly
https://doi.org/10.1021/acs.jafc.1c02221
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© 2021 American Chemical Society
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Scheme 1. Design of the New Fungicide Fluopyram
Scheme 2. Design of the Target Compounds
can be classified into diverse chemical families, including oxime
ether derivatives, sulfonamides, dichloroacetamides, carboxylic
acid derivatives, and phenyl pyrazoles.22
of the novel fungicide fluopyram was formed by splicing the
fragments of flutolanil and fluopicolide (Scheme 1).36
Over the past several years, some heterocyclic rings bearing
N and O atom safeners have been found with excellent
biological activities.37−40 Based on our previous research,41−45
the herbicide safener mefenpyr-diethyl was used as a precursor
to splice the biologically active ester group structure of the
herbicides fluazifop-butyl and fenoxaprop-p-ethyl with pyrazole
compounds via amide bonds. A sequence of new ester-
substituted pyrazole derivatives has been designed (Scheme 2).
The title compounds were all characterized via infrared (IR)
Studies have shown that herbicide safeners can elevate crop
detoxification and metabolism by upregulating ATP-binding
cassette (ABC) transporter proteins, glutathione S-transferases
(GSTs), glutathione (GSH), and cytochrome P450 (CYP450),
which counteract poison herbicides.23,24 Mefenpyr-diethyl is an
herbicide safener discovered by the Hoechst Company and
belongs to the chemical category of phenyl pyrazoles. It was
determined that it has a certain effect on preventing crops from
herbicide damage.25 In herbicide-tolerant wheat production
systems, the combination of FE and mefenpyr-diethyl is an
excellent method for weed control.26,27 It is worth noting that a
safener is treated as an “inert ingredient” in the USA, and there
are few studies on its environmental behavior and toxicities.
The usage of safeners is growing with the herbicide resistances.
Commercial safeners have shown a series of risks to the
environment.28−30 To reduce the risk to the environment, it is
necessary to design more safeners with a lower toxicity and
higher activity. Therefore, research on safeners with a higher
phenyl pyrazole activity is also more valuable. The research
shows that delicate modifications to the structure of mefenpyr-
diethyl will impact its biological activity, and it is determined
that retaining the pyrazole heterocyclic group and ester group
is the necessary chemical fragment for its safener activity.25,31
One of the main techniques for designing and optimizing
novel framework structures with the target biological activity is
the fragment splicing method. Many instances of fragment
splicing have been demonstrated;32−35 for example, the design
1
spectroscopy, H nuclear magnetic resonance (1H NMR), 13C
nuclear magnetic resonance (13C NMR), and high-resolution
mass spectrometry (HRMS) to characterize the accuracy of
their structures. The safener activity of each compound was
evaluated by measuring the growth index, ACCase activity,
GST activity, and CYP450 content in wheat. The possible
detoxification mechanism was determined, and novel effective
safeners were designed through molecular structure compar-
isons and molecular docking. Furthermore, the pharmacoki-
netic properties of the compounds were evaluated through
absorption, distribution, metabolism, excretion, and toxicity
(ADMET) prediction. This research might provide an
appropriate reference for the progress of more efficient
pyrazole derivative herbicide safeners in the future.
MATERIALS AND METHODS
■
Instruments and Materials. All reagents were of analytical
reagent grade, and no purification in later procedures was required
(Table S1). The infrared (IR) spectra were recorded on an ALPHA-T
(Bruker Corp., Billerica, MA, USA) infrared spectrophotometer using
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potassium bromide pellets. Nuclear magnetic resonance (NMR)
spectra were recorded on a Bruker AV400 spectrometer (Bruker
Corp., Billerica, MA, USA), using deuterated chloroform as the
solvent and tetramethylsilane (TMS) as an internal standard. The
melting points were obtained using a Beijing Taike melting point
apparatus (X-4; Beijing Taike, Beijing, China) and without
corrections. The mass spectrum (HRMS; Bruker Corp., Billerica,
MA, USA) was determined via high-resolution mass spectrometry.
The X-ray diffraction data of the target compound were gathered on a
Rigaku R-AXIS RAPID area-detector diffractometer (Rigaku Corp.,
Tokyo, Japan).
Isopropyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-5. Orange oily liquid; yield: 47%; IR (KBr) v (cm−1):
3131−2989 (C−H), 1773 (O−CO), 1547 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.53−6.52 (m, 4H, Ar−H), 5.29−5.23
(m, 1H, CH), 1.43 (d, J = 6.3 Hz, 6H, 2 × CH3); 13C NMR (100
MHz, CDCl3) δ (ppm): 148.56, 143.85, 142.35, 138.75, 125.40,
121.66, 118.98, 113.52, 111.72, 74.47, 21.52, 21.52; HRMS (ESI):
calcd for C12H12F3N2O3 ([M + H+]), 311.0621; found, 311.0614.
Isobutyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-6.
White solid; yield: 70%; m.p. 104.0−104.8 °C; IR (KBr) v (cm−1):
3107−2875 (C−H), 1748 (O−CO), 1573 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.86−6.49 (m, 6H, Ar−H), 4.24 (d, J =
6.8 Hz, 2H, CH2), 2.61 (s, 3H, CH3), 2.22−2.16 (m, 1H, CH), 1.06
(d, J = 6.7 Hz, 6H, 2CH3); 13C NMR (100 MHz, CDCl3) δ (ppm):
154.00, 150.75, 145.13, 131.97, 128.94, 128.94, 128.61, 126.41,
126.41,107.73, 73.93, 27.86, 19.11, 19.11, 14.59; HRMS (ESI): calcd
for C15H19N2O2 ([M + H+]), 259.1444; found, 259.1447.
Isobutyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-7. White
solid; yield: 66%; m.p. 90.7−91.7 °C; IR (KBr) v (cm−1): 2967−
2875 (C−H), 1767 (O−CO), 1562 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.93−6.71 (m, 11H, Ar−H), 4.09 (d, J = 6.8 Hz,
2H, CH2), 1.94−1.82 (m, 1H, CH), 0.82 (d, J = 6.7 Hz, 6H, 2 ×
CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 154.16, 150.20,
147.98, 131.71, 131.34, 129.14, 129.14, 129.06, 128.88, 128.88,
128.71, 127.99, 127.99, 126.48, 126.48, 109.19, 74.21, 27.61, 18.83,
18.83; HRMS (ESI): calcd for C20H21N2O2 ([M + H+]), 321.1606;
found, 321.1603.
Preparation of Pyrazole Intermediates II.46 1,3-Dione (15
mmol) was dissolved in an anhydrous ethanol solution (50 mL) in a
three-neck flask. Hydrazine hydrate (15 mmol) was added, and then,
the mixture was incubated at 78 °C for 2 h. The solvent was removed
under vacuum, and the crude products were purified via
recrystallization with light petroleum ether and ethyl acetate to
obtain the intermediate pyrazole II. Pyrazole intermediate II provided
yields in the range of 73−88%.
General Procedure for Synthesis of Compounds IV-1−IV-
35.47 Compound II (7 mmol) and 50 mL of tetrahydrofuran were
sequentially added to a three-neck flask. Subsequently, triethylamine
(11 mmol) and acid chloride III (11 mmol) were added to the
system. The next dropwise addition was complete, and the reaction
was conducted under reflux for 1−1.5 h at 65 °C. After the reaction
was complete (TLC monitoring), the mixture was washed three times
with distilled water, and then, the organic phase was dried with
anhydrous MgSO4 for 0.5 h. Compound IV was then obtained at a
yield rate of 33−87% after the solvent was removed under a reduced
pressure, and the crude products were purified via column
chromatography on a silica gel, eluted with ethyl acetate and
petroleum ether (1:10−1:50) or recrystallized with ethyl acetate and
light petroleum ether.
Isopropyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-1.
Light yellow oily liquid; yield: 73%; IR (KBr) v (cm−1): 3064−
2880 (C−H), 1761 (O−CO), 1572 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.91−6.47 (m, 6H, Ar−H), 5.29−5.23 (m, 1H,
CH), 2.59 (s, 3H, CH3), 1.48 (d, 6H, 2 × CH3); 13C NMR (100
MHz, CDCl3) δ (ppm): 153.90, 150.17, 145.09, 132.03, 128.88,
128.88, 128.58, 128.58, 126.46, 107.71, 72.66, 21.83, 21.83, 14.68;
HRMS (ESI): calcd for C14H17N2O2 ([M + H+]), 245.1289; found,
245.1290.
Isopropyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-2. Light
yellow oily liquid; yield: 65%; IR (KBr) v (cm−1): 3102−2877 (C−
H), 1759 (O−CO), 1562 (CN); 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.94−6.69 (m, 10H, Ar−H), 5.13−5.06 (m, 1H, CH), 1.20
(d, J = 6.3 Hz, 6H, 2 × CH3); 13C NMR (100 MHz, CDCl3) δ
(ppm): 154.01, 149.45, 147.85, 131.75, 131.55, 129.11, 129.11,
129.05, 129.05, 128.74, 128.74, 128.65, 127.83, 126.48, 126.48,
108.96, 72.77, 21.40, 21.40; HRMS (ESI): calcd for C19H19N2O2 ([M
+ H+]), 307.1446; found, 307.1447.
Isobutyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-carboxylate
IV-8. White solid; yield: 62%; m.p. 41.2−41.7 °C; IR (KBr) v (cm−1):
2992−2896 (C−H), 1775 (O−CO), 1571 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.88−7.18 (m, 6H, Ar−H), 4.31 (d, J =
6.8 Hz, 2H, CH2), 2.23−2.16 (m, 1H, CH), 1.05 (d, J = 6.7 Hz, 6H, 2
× CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 153.50, 148.29,
130.37, 129.79, 129.79, 128.90, 126.44, 126.44, 110.86, 110.83, 77.23,
75.32, 27.76, 18.90, 18.90; HRMS (ESI): calcd for C15H16F3N2O2
([M + H+]), 313.1162; found, 313.1164.
Isobutyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxy-
late IV-9. White solid; yield: 55%; m.p. 72.1−72.6 °C; IR (KBr) v
1
(cm−1): 3117−2877 (C−H), 1776 (O−CO), 1588 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.78−7.14 (m, 5H, Ar−H), 4.30
(d, J = 6.7 Hz, 2H, CH2), 2.39 (s, 3H, CH3), 2.21−2.15 (m, 1H, CH),
1.06 (d, J = 6.7 Hz, 6H, 2 × CH3); 13C NMR (100 MHz, CDCl3) δ
(ppm): 153.56, 149.28, 139.94, 129.59, 128.91, 127.55, 127.55,
126.76, 126.76, 120.55, 108.27, 75.26, 27.76, 21.42, 18.90, 18.90;
HRMS (ESI): calcd for C16H18F3N2O2 ([M + H+]), 327.1323; found,
327.1320.
Isobutyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-10. Orange oily liquid; yield: 40%; IR (KBr) v (cm−1):
3150−2880 (C−H), 1779 (O−CO), 1554 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.53−6.52 (m, 4H, Ar−H), 4.25 (d, J =
6.9 Hz, 2H, CH2), 2.17−2.10 (m, 1H, CH), 1.00 (d, J = 6.8 Hz, 6H, 2
× CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 149.26, 144.00,
144.00, 142.33, 138.96, 121.64, 111.81, 109.59, 107.36, 75.20, 27.76,
18.86, 18.86; HRMS (ESI): calcd for C13H14F3N2O3 ([M + H+]),
303.0956; found, 303.0957.
Propyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-11.
White solid; yield: 87%; m.p. 70.2−70.6 °C; IR (KBr) v (cm−1):
3088−2832 (C−H), 1732 (O−CO), 1576 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.78−6.43 (m, 6H, Ar−H), 4.35 (t, J =
6.9 Hz, 2H, CH2), 2.54 (s, 3H, CH3), 1.85−1.79 (m, 2H, CH2), 0.99
(t, J = 7.4 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm):
154.06, 150.72, 145.30, 131.93, 128.94, 128.60, 128.60, 126.46,
126.46, 107.77, 69.61, 22.03, 14.74, 10.29; HRMS (ESI): calcd for
C14H17N2O2 ([M + H+]), 245.1286; found, 245.1285.
Isopropyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-carboxy-
late IV-3. Light yellow oily liquid; yield: 58%; IR (KBr) v (cm−1):
3057−2882 (C−H), 1775 (O−CO), 1570 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.90−7.16 (m, 6H, Ar−H), 5.37−5.28
(m, 1H, CH), 1.49 (d, J = 6.3 Hz, 6H, 2 × CH3); 13C NMR (100
MHz, CDCl3) δ (ppm): 153.42, 148.45, 147.48, 130.42, 129.73,
129.73, 128.09, 126.47, 120.58, 120.58, 110.76, 74.51, 21.49, 21.49;
HRMS (ESI): calcd for C14H14F3N2O2 ([M + H+]), 321.0826; found,
321.0827.
Isopropyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxy-
late IV-4. Light yellow oily liquid; yield: 52%; IR (KBr) v (cm−1):
3123−2874 (C−H), 1772 (O−CO), 1568 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.77−7.13 (m, 5H, Ar−H), 5.35−5.29
(m, 1H, CH), 2.39 (s, 3H, CH3), 1.47 (d, J = 6.3 Hz, 6H, 2 × CH3);
13C NMR (100 MHz, CDCl3) δ (ppm): 153.48, 148.61, 147.54,
Propyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-12. White
solid; yield: 82%; m.p. 89.3−90.0 °C; IR (KBr) v (cm−1): 3027−
2832 (C−H), 1752 (O−CO), 1599 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.93−6.71 (m, 11H, Ar−H), 4.27 (d, J = 13.6 Hz,
2H, CH2), 1.67−1.62 (m, 2H, CH2), 0.85 (t, J = 7.4 Hz, 3H, CH3);
13C NMR (100 MHz, CDCl3) δ (ppm): 154.14, 150.13, 148.11,
139.86, 129.57, 129.57, 128.99, 127.61, 126.37, 126.37, 120.64,
110.70, 74.41, 21.48, 21.48; HRMS (ESI): calcd for C15H16F3N2O2
([M + H+]), 313.1162; found, 313.1164.
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131.70, 131.27, 129.12, 129.12, 129.06, 129.06, 128.86, 128.86,
128.69, 127.91, 127.91, 126.50, 109.16, 69.79, 21.74, 10.09; HRMS
(ESI): calcd for C19H19N2O2 ([M + H+]), 307.1442; found, 307.1441.
Propyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-carboxylate
IV-13. White solid; yield: 78%; m.p. 50.0−50.7 °C; IR (KBr) v
Butyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxy-
late IV-20. White solid; yield: 54%; m.p. 33.9−34.5 °C; IR (KBr) v
1
(cm−1): 3145−2930 (C−H), 1776 (O−CO), 1507 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.53−6.52 (m, 4H, Ar−H), 4.52
(t, J = 6.9 Hz, 2H, CH2), 1.88−1.81 (m, 2H, CH2), 1.52−1.43 (m,
2H, CH2), 0.99 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3) δ (ppm): 148.07, 145.88, 143.74, 143.74, 111.84, 111.84,
110.61, 109.63, 109.63, 69.48, 30.34, 18.83, 13.63; HRMS (ESI):
calcd for C13H14F3N2O3 ([M + H+]), 303.0957; found, 303.0953.
2-Chloroethyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-
21. Light yellow oily liquid; yield: 65%; IR (KBr) v (cm−1): 3109−
2890 (C−H), 1751 (O−CO), 1572 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.90−6.51 (m, 6H, Ar−H), 4.69 (t, J = 6.0 Hz, 2H,
CH2), 3.87 (t, J = 6.0 Hz, 2H, CH2), 2.61 (d, J = 1.0 Hz, 3H, CH3);
13C NMR (100 MHz, CDCl3) δ (ppm): 154.59, 150.19, 145.60,
1
(cm−1): 3041−2861 (C−H), 1758 (O−CO), 1561 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.89−7.18 (m, 6H, Ar−H), 4.48
(t, J = 6.8 Hz, 2H, CH2), 1.94−1.85 (m, 2H, CH2), 1.06 (t, J = 7.4
Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 153.57,
130.33, 129.71, 129.55, 129.06, 128.91, 128.91, 128.17, 126.47,
126.47, 110.86, 71.00, 21.65, 10.00; HRMS (ESI): calcd for
C14H14F3N2O2 ([M + H+]), 299.1003; found, 299.1002.
Propyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxylate
IV-14. White solid; yield: 70%; m.p. 42.0−42.8 °C; IR (KBr) v
1
(cm−1): 2952−2860 (C−H), 1761 (O−CO), 1605 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.78−7.14 (m, 5H, Ar−H), 4.47
(t, J = 6.8 Hz, 2H, CH2), 2.39 (s, 3H, CH3), 1.92−1.85 (m, 2H,
CH2), 1.05 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ
(ppm): 1149.24, 148.84, 145.17, 139.70, 128.94, 128.94, 128.94,
128.86, 128.86, 126.70, 108.26, 70.66, 21.70, 21.41, 9.99; HRMS
(ESI): calcd for C15H16F3N2O2 ([M + H+]), 313.1156; found,
313.1158.
131.67, 129.14, 128.65, 128.65, 126.48, 126.48, 108.15, 66.86, 40.80,
14.51; HRMS (ESI): calcd for C13H14ClN2O2 ([M + H+]), 265.0746;
found, 265.0744.
2-Chloroethyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-22.
White solid; yield: 57%; m.p. 122.9−123.3 °C; IR (KBr) v (cm−1):
3057−2952 (C−H), 1763 (O−CO), 1564 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.97−6.74 (m, 11H, Ar−H), 4.54 (t, J
= 6.0 Hz, 2H, CH2), 3.65 (t, J = 6.0 Hz, 2H, CH2); 13C NMR (100
MHz, CDCl3) δ (ppm): 154.73, 149.70, 148.47, 131.46, 130.93,
129.36, 129.14, 129.14, 129.08, 129.08, 128.78, 128.78, 128.00,
123.57, 126.57, 109.62, 67.03, 40.48; HRMS (ESI): calcd for
C18H16ClN2O2 ([M + H+]), 327.0900; found, 327.0900.
Propyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxy-
late IV-15. Light yellow oily liquid; yield: 64%; IR (KBr) v (cm−1):
3125−2862 (C−H), 1765 (O−CO), 1495 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.53−6.52 (m, 4H, Ar−H), 4.48 (t, J =
6.9 Hz, 2H, CH2), 1.93−1.81 (m, 2H, CH2), 1.04 (t, J = 7.4 Hz, 3H,
CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 148.07, 145.76,
143.76, 120.35, 117.68, 113.83, 111.86, 110.60, 109.66, 71.11, 21.79,
10.08; HRMS (ESI): calcd for C12H12F3N2O3 ([M + H+]), 289.0800;
found, 289.0799.
Butyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-16.
White solid; yield: 81%; m.p. 46.4−46.8 °C; IR (KBr) v (cm−1):
3111−2873 (C−H), 1750 (O−CO), 1573 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.85−6.49 (m, 6H, Ar−H), 4.46 (t, J =
6.9 Hz, 2H, CH2), 2.60 (s, 3H, CH3), 1.84 (m, 2H, CH2), 1.54−1.45
(m, 2H, CH2), 1.00 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3) δ (ppm): 154.04, 150.75, 145.29, 131.94, 128.94, 128.60,
128.60, 126.44, 126.44, 107.73, 67.96, 30.63, 19.06, 14.52, 13.72;
HRMS (ESI): calcd for C15H19N2O2 ([M + H+]), 259.1443; found,
259.1441.
Butyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-17. White
solid; yield: 76%; m.p. 104.3−104.5 °C; IR (KBr) v (cm−1): 3048−
2870 (C−H), 1766 (O−CO), 1562 (CN); 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.92−6.71 (m, 11H, Ar−H), 4.31 (t, J = 6.7 Hz,
2H, CH2), 1.59 (m, 2H, CH2), 1.29−1.20 (m, 2H, CH2), 0.87 (t, J =
7.4 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 154.20,
150.15, 148.10, 131.71, 131.30, 129.15, 129.15, 129.08, 129.08,
128.85, 128.85, 127.92, 127.92, 126.51, 126.51, 109.18, 68.16, 30.33,
18.87, 13.63; HRMS (ESI): calcd for C20H21N2O2 ([M + H+]),
321.1595; found, 321.1598.
(Trifluoromethyl)-1H-pyrazole-1-carboxylate IV-18. White solid;
yield: 70%; m.p. 43.2−44.1 °C; IR (KBr) v (cm−1): 3080−2865 (C−
H), 1775 (O−CO), 1572 (CN); 1H NMR (400 MHz, CDCl3) δ
(ppm): 7.53−7.17 (m, 6H, Ar−H), 4.52 (t, J = 6.8 Hz, 2H, CH2),
1.85 (m, 2H, CH2), 1.52−1.43 (m, 2H, CH2), 1.00 (t, J = 7.4 Hz, 3H,
CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 153.55, 148.24,
130.36, 129.79, 129.05, 128.89, 128.89, 128.14, 126.46, 126.46,
110.83, 69.33, 30.36, 18.87, 13.63; HRMS (ESI): calcd for
C15H16F3N2O2 ([M + H+]), 313.1160; found, 313.1158.
2-Chloroethyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-23. Light yellow oily liquid; yield: 54%; IR (KBr) v
1
(cm−1): 3125−2884 (C−H), 1776 (O−CO), 1574 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.93−7.21 (m, 6H, Ar−H), 4.75
(t, J = 5.9 Hz, 2H, CH2), 3.88 (t, J = 5.9 Hz, 2H, CH2); 13C NMR
(100 MHz, CDCl3) δ (ppm): 154.05, 147.85, 130.10, 128.96, 128.96,
126.52, 126.52, 125.72, 120.40, 117.72, 113.34, 68.05, 40.34; HRMS
(ESI): calcd for C13H11ClF3N2O2 ([M + H+]), 319.0461; found,
319.0463.
2-Chloroethyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-24. White solid; yield: 43%; m.p. 77.4−78.0 °C; IR
(KBr) v (cm−1): 3154−2923 (C−H), 1772 (O−CO), 1528 (C
1
N); H NMR (400 MHz, CDCl3) δ (ppm): 7.84−7.17 (m, 5H, Ar−
H), 4.74 (t, J = 6.0 Hz, 2H, CH2), 3.87 (t, J = 6.0 Hz, 2H, CH2), 2.40
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 154.10,
147.89, 140.19, 129.65, 129.65, 127.29, 126.42, 126.42, 120.44,
117.76, 111.28, 67.98, 40.35, 21.42; HRMS (ESI): calcd for
C14H13ClF3N2O2 ([M + H+]), 333.0618; found, 333.0607.
2-Chloroethyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-
carboxylate IV-25. Light yellow oily liquid; yield: 38%; IR (KBr) v
1
(cm−1): 3148−2966 (C−H), 1784 (O−CO), 1506 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.54−6.53 (m, 4H, Ar−H), 4.75
(t, J = 6.0 Hz, 2H, CH2), 3.87 (t, J = 6.0 Hz, 2H, CH2); 13C NMR
(100 MHz, CDCl3) δ (ppm): 147.71, 146.28, 145.51, 143.97, 120.24,
117.56, 111.92, 111.05, 110.04, 68.11, 40.26; HRMS (ESI): calcd for
C11H9ClF3N2O3 ([M + H+]), 309.0254; found, 309.0250.
3-Chloropropyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-26. White solid; yield: 63%; m.p. 37.1−37.7 °C; IR
(KBr) v (cm−1): 3061−2853 (C−H), 1748 (O−CO), 1572 (C
1
N); H NMR (400 MHz, CDCl3) δ (ppm): 7.84−6.49 (m, 6H, Ar−
H), 4.61 (t, J = 6.2 Hz, 2H, CH2), 3.74 (t, J = 6.3 Hz, 2H, CH2), 2.61
(s, 3H, CH3), 2.35−2.29 (m, 2H, CH2); 13C NMR (100 MHz,
CDCl3) δ (ppm): 154.24, 150.52, 145.51, 131.80, 129.06, 128.65,
128.65, 126.42, 126.42, 107.90, 64.63, 40.86, 31.53, 14.43; HRMS
(ESI): calcd for C14H16ClN2O2 ([M + H+]), 279.0902; found,
279.0900.
Butyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-carboxylate
IV-19. White solid; yield: 66%; m.p. 76.4−77.0 °C; IR (KBr) v
1
(cm−1): 2966−2878 (C−H), 1775 (O−CO), 1602 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.33−6.59 (m, 5H, Ar−H), 4.34
(t, J = 6.7 Hz, 2H, CH2), 2.41 (s, 3H, CH3), 1.68−1.54 (m, 2H,
CH2), 1.30−1.20 (m, 2H, CH2), 0.88 (t, J = 7.4 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3) δ (ppm): 149.22, 148.78, 145.55, 145.17,
139.68, 128.85, 128.85, 126.74, 121.79, 119.03, 108.25, 69.07, 30.26,
21.40, 18.80, 13.58; HRMS (ESI): calcd for C16H18F3N2O2 ([M +
H+]), 327.1319; found, 327.1315.
3-Chloropropyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-27.
White solid; yield: 57%; m.p. 143.7−144.7 °C; IR (KBr) v (cm−1):
3112−2850 (C−H), 1761 (O−CO), 1567 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.98−6.72 (m, 11H, Ar−H), 4.46 (t, J
= 6.0 Hz, 2H, CH2), 3.36 (t, J = 6.4 Hz, 2H, CH2), 2.09−2.03 (m,
2H, CH2); 13C NMR (100 MHz, CDCl3) δ (ppm): 154.46, 149.87,
148.07, 131.52, 131.21, 129.28, 129.28, 129.05, 129.05, 128.76,
8369
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Article
1
128.76, 128.05, 128.05, 126.51, 126.51, 109.41, 64.79, 40.71, 31.29;
HRMS (ESI): calcd for C19H18ClN2O2 ([M + H+]), 341.1057; found,
341.1051.
N); H NMR (400 MHz, CDCl3) δ (ppm): 7.49−6.86 (m, 3H, Ar−
H), 6.63−6.59 (m, 1H, CH), 6.47 (dd, J = 3.5, 1.8 Hz, 1H, Ar−H),
1.90 (d, J = 5.8 Hz, 3H CH3); 13C NMR (100 MHz, CDCl3) δ
(ppm): 147.21, 144.29, 141.85, 139.71, 121.44, 118.76, 114.39,
111.97, 107.66, 84.50, 25.04; HRMS (ESI): calcd for C18H16ClN2O2
([M + H+]), 309.0254; found, 309.0247.
X-ray Diffraction. The crystal of compound IV-17 with a size of
0.13 mm × 0.12 mm × 0.10 mm was measured at 293(2) K using
graphite-monochromated Mo Kα radiation (λ = 0.71073 Å) on a
3-Chloropropyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-28. White solid; yield: 52%; m.p. 46.8−47.5 °C; IR
(KBr) v (cm−1): 2969−2852 (C−H), 1775 (O−CO), 1471 (C
N); 1H NMR (400 MHz, CDCl3) δ (ppm): 7.87−7.19 (m, 6H, Ar−
H), 4.31 (t, J = 6.0 Hz, 2H, CH2), 3.64 (t, J = 6.3 Hz, 2H, CH2),
2.17−2.11 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ (ppm):
154.90, 153.78, 148.01, 130.18, 129.94, 128.95, 128.95, 126.47,
126.47, 117.79, 111.11, 64.67, 40.66, 31.28; HRMS (ESI): calcd for
C14H13ClF3N2O2 ([M + H+]), 333.0618; found, 333.0612.
3-Chloropropyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-
carboxylate IV-29. Light yellow oily liquid; yield: 40%; IR (KBr) v
Rigaku R-AXIS-RAPID area-detector diffractometer (Japan). θmax
=
25.24, 20,730 measured reflections and 3503 independent reflections
(Rint = 0.0292) (Figure 1). The crystalline structure was solved with
SHELXS 97 direct methods and deposited to the Cambridge
Crystallographic Data Centre under Supplementary Publication No.
CCDC 2064120.
1
(cm−1): 3144−2873 (C−H), 1775 (O−CO), 1473 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.31−6.60 (m, 5H, Ar−H), 4.49
(t, J = 6.0 Hz, 2H, CH2), 3.38 (t, J = 6.4 Hz, 2H, CH2), 2.42 (s, 3H,
CH3), 2.13−2.07 (m, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ
(ppm): 148.97, 148.83, 145.82, 145.43, 139.92, 126.62, 126.62,
126.62, 121.72, 121.72, 119.03, 65.65, 40.48, 31.16, 21.41; HRMS
(ESI): calcd for C15H15ClF3N2O2 ([M + H+]), 340.0774; found,
347.0765.
3-Chloropropyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-
carboxylate IV-30. Light yellow oily liquid; yield: 35%; IR (KBr) v
1
(cm−1): 3148−2929 (C−H), 1780 (O−CO), 1507 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.47−6.46 (m, 4H, Ar−H), 4.55
(t, J = 6.1 Hz, 2H, CH2), 3.58 (t, J = 6.2 Hz, 2H, CH2), 2.23−2.17
(m, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ (ppm): 149.01,
144.13, 142.16, 139.15, 121.56, 118.87, 113.98, 111.88, 107.41, 65.92,
40.65, 31.24; HRMS (ESI): calcd for C12H11ClF3N2O3 ([M + H+]),
323.0410; found, 323.0409.
Figure 1. X-ray crystal structure for compound IV-17.
1-Chloroethyl-3-methyl-5-phenyl-1H-pyrazole-1-carboxylate IV-
Plant Material and Growth Conditions. Wheat seeds (College
of Agriculture, Northeast Agricultural University, Harbin, China)
were soaked in warm water for 0.5 h. Next, the samples were soaked
in 0.6% carbendazim for approximately 0.5 h. The seeds were then
washed with distilled water; afterward, the seeds were soaked in the
title compounds (10 mg/kg) for 12 h. After germination at 26.5 °C
for 24 h, 180 g of soil (Northeast Agricultural University, Harbin,
China) was placed and sown in paper cups at a density of seven wheat
seeds per cup, sprayed with an appropriate amount of water, and
covered with 30 g of topsoil. The seeds were then placed in an
incubator at 26.5 °C under a 12 h light/12 h dark photoperiod. The
stems and leaves were sprayed with 200 g.a.i/hm2 FE when the
seedlings reached the two-leaf stage. Equal volumes of water were
sprayed on the control plants. After 7 days of cultivation, the injury
recovery rate (IRR) of each growth index (root length, plant height,
and root and plant fresh weights) was measured. The injury recovery
rate is calculated according to the following equation
31. White solid; yield: 54%; m.p. 132.9−133.8 °C; IR (KBr) v
1
(cm−1): 3061−2848 (C−H), 1760 (O−CO), 1573 (CN); H
NMR (400 MHz, CDCl3) δ (ppm): 7.77−7.21 (m, 5H, Ar−H),
6.67−6.63 (m, 1H, CH), 6.43 (s, 1H, Ar−H), 2.53 (s, 3H, CH3), 1.93
(d, J = 5.8 Hz, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm):
154.91, 148.46, 145.95, 131.57, 129.27, 128.69, 128.69, 126.54,
126.54, 108.50, 83.81, 25.25, 14.48; HRMS (ESI): calcd for
C15H15ClF3N2O2 ([M + H+]), 265.0744; found, 265.0741.
Chloroethyl-3,5-diphenyl-1H-pyrazole-1-carboxylate IV-32.
White solid; yield: 50%; m.p. 73.6−73.8 °C; IR (KBr) v (cm−1):
2998−2938 (C−H), 1762 (O−CO), 1562 (CN); 1H NMR
(400 MHz, CDCl3) δ (ppm): 7.93−6.75 (m, 11H, Ar−H), 6.61−6.57
(m, 1H, CH), 1.72 (d, J = 5.8 Hz, 3H, CH3); 13C NMR (100 MHz,
CDCl3) δ (ppm): 155.08, 148.63, 147.78, 131.32, 130.71, 129.46,
129.23, 129.14, 129.14, 128.78, 128.78, 128.03, 128.03, 126.61,
126.61, 109.88, 83.65, 24.83; HRMS (ESI): calcd for C18H16ClN2O2
([M + H+]), 327.0900; found, 327.0892.
1-Chloroethyl-5-phenyl-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-33. White solid; yield: 40%; m.p. 93.7−94.3 °C; IR
(KBr) v (cm−1): 3127−2941 (C−H), 1785 (O−CO), 1471 (C
N); 1H NMR (400 MHz, CDCl3) δ (ppm): 7.98−7.21 (m, 6H, Ar−
H), 6.77−6.73 (m, 1H, CH), 2.01 (d, J = 5.8 Hz, 3H, CH3); 13C
NMR (100 MHz, CDCl3) δ (ppm): 154.35, 146.03, 136.35, 130.13,
128.97, 126.57, 126.57, 120.34, 120.34, 117.66, 111.74, 84.44, 24.99;
HRMS (ESI): calcd for C18H16ClN2O2 ([M + H+]), 319.0461; found,
319.0456.
1-Chloroethyl-5-(p-tolyl)-3-(trifluoromethyl)-1H-pyrazole-1-car-
boxylate IV-34. White solid; yield: 37%; m.p. 58.9−59.8 °C; IR
(KBr) v (cm−1): 3131−2921 (C−H), 1778 (O−CO), 1476 (C
N); 1H NMR (400 MHz, CDCl3) δ (ppm): 7.29−6.62 (m, 5H, Ar−
H), 6.58−6.54 (m, 1H, CH), 2.43 (s, 3H, CH3), 1.79 (d, J = 5.8 Hz,
3H, CH3); 13C NMR (100 MHz, CDCl3) δ (ppm): 149.52, 147.08,
146.42, 140.03, 129.07, 129.07, 128.95, 126.12, 121.61, 118.93,
108.78, 84.17, 24.81, 21.43; HRMS (ESI): calcd for C18H16ClN2O2
([M + H+]), 333.0618; found, 333.0612.
Injury Recovery Rate (IRR)(%)
treated with safener and FE − treated with FE
=
× 100%
contrast − treated with FE
GST, CYP450, ACCase Extraction, and Assay In Vivo. The
Plant GST ELISA Kit, Plant CYP450 ELISA Kit, and Plant ACCase
ELISA Kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd.) were
used to determine the activities of GST and CYP450 and the content
of ACCase, respectively. All the experiments were performed at 0−4
°C. First, 0.1 g of the wheat root or leaf tissue was put into the mortar
after being washed with distilled water and dried, and a certain
amount of PBS (pH = 7.4) was added to the mortar, which was
quickly frozen, mashed to a homogenate, poured into a centrifuge
tube, and centrifuged for 20 min. The supernatant was carefully
collected and used for testing the enzymes. The activities of GST,
CYP450, and ACCase were assayed according to the manufacturer’s
instructions with three duplicates per experiment.
Statistical Analysis. Each reported value represents the mean
standard deviation (SD) of at least three replicate data points for each
measurement method. Three different batches of wheat plants were
repeatedly tested for the purpose of determining the growth indexes.
1-Chloroethyl-5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazole-1-
carboxylate IV-35. White solid; yield: 33%; m.p. 41.9−42.5 °C; IR
(KBr) v (cm−1): 3158−2964 (C−H), 1774 (O−CO), 1507 (C
8370
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Article
Scheme 3. Route for the Synthesis of the Title Compounds
SPSS 20 (IBM Corp., Armonk, NY, USA) was used for the statistical
analysis. Through Duncan’s tests, significant differences between
treatment methods were identified when p < 0.05.
membered furan ring. This phenomenon might be attributed
to the high electronegativity of the O atom in the furan ring,
the fact that the electron cloud distribution is not as uniform as
that of the benzene ring, and that the conjugation effect is
lower than that of the benzene ring. Notably, the introduction
of −CH3 on the benzene ring would reduce the yield of
compound IV. This may be caused by the addition of −CH3
on the benzene ring in the structure, which further increased
the steric hindrance and was not conducive to the reaction.
The R2 substituent on the pyrazole ring significantly affected
the yields of products. The yields of compounds IV-11 (R2 =
−CH3), IV-12 (R2 = phenyl), and IV-13 (R2 = −CF3) were
87, 82, and 78%, respectively. The yield of compounds with
−CH3 substituents at R2 was the highest. The reason for this
phenomenon may be that the electron-donating group −CH3
on the ring increased the electron cloud density, which was
conducive to the reaction. In contrast, the strong electron-
withdrawing group −CF3 substituent on the heterocyclic ring
reduces the electron cloud density, leading to a lower yield,
and the conjugation effect caused by the benzene ring led to an
increased yield. Apparently, the spatial structure of R3 has a
significant impact on the yield of the products. In general, the
yield of the linear chain structure is essentially higher than that
of the branched chain structure. The yields of compounds IV-
1, IV-6, IV-11, and IV-16 were 73, 70, 87, and 81%,
respectively.
Docking Study. The 3D structures of mefenpyr-diethyl,
compound IV-21, and FA were created in the Sketch module of
SYBYL-X 2.0 (Tripos, Inc., St. Louis, MO, USA). The Gasteiger−
Huckel charges were computed, and the molecule was further
̈
optimized. The crystalline structure of ACCase was downloaded from
the Protein Data Bank (PDB ID: 1UYR) and pretreated to repair the
main chain, the side chain, and the terminal of the protein to make the
structure complete. Molecular docking was then performed using the
CDOCKER module in Discovery Studio 3.5 (BIOVIA, Inc., San
Diego, CA, USA).48 Water and a number of other cocrystallized small
molecules were eliminated. The protein structure was obtained with
the use of the CHARMM force field. The carboxyltransferase (CT)
domain of acetyl-CoA carboxylase (ACC) is the site of action of
commercial herbicides, such as haloxyfop, diclofop, and sethoxydim.
The inhibitors are bound in the active site, i.e., at the interface of the
dimer of the CT domain. Inhibitor binding requires large conforma-
tional changes for several residues in this interface, which create a
highly conserved hydrophobic pocket that extends deeply into the
core of the dimer. The active site was defined as a subregion of 13.0 Å
from the center of the known ligand. According to the principle of
complementary shapes and properties of the receptor and the ligand, a
small molecule is placed at the active site to make the ligand and the
receptor complementary in shape and nature.49 The Top Hits
parameter was set to 100, and the remaining parameters were default
values. The native ligand was redocked to ensure the accuracy of
protein preparation. The small molecule−receptor protein complex
was evaluated using the interaction energy as an evaluation index.
The yields of compounds IV-21 to IV-35 ranged from 33 to
65% (Table S3). It could be observed that the conclusion
obtained from the yields of compounds IV-1 to IV-20 were
also applicable to the yields of compounds IV-21 to IV-35.
The yields of compounds IV-35 and IV-21 were 33 and 65%,
respectively, which were lower than those of compounds IV-5
and IV-11, which were 47 and 87%, respectively. This may be
because the addition of Cl atoms might significantly increase
the volume of the acid chloride, and the excessive steric
hindrance impeded the reaction, resulting in a decrease in the
acid chloride reaction activity. Meanwhile, it weakened its
electron-withdrawing effect, which made the acyl chloride
carbonyl carbon less positively charged. The C−Cl bond of the
acyl portion was difficult to break, which led to a decrease in
the yield. The results showed that due to the introduction of
the Cl atom on R3 into the structure, the yields of compounds
IV-11 to IV-35 were lower than those of compounds IV-1 to
IV-20. On the whole, the highest yields were obtained when R1
and R2 were replaced by the benzene ring and −CH3, with the
introduction of a benzyl or furyl on R1 and −CF3 or a phenyl
on R2 reducing the yields. The yield of the linear chain
structure on R3 is essentially higher than that of the branched
chain structure (Figure 2).
RESULTS AND DISCUSSION
■
Chemistry. The synthetic route is outlined in Scheme 3.
Key intermediates II were obtained by cyclizing 1,3-dione I
and hydrazine hydrate. It was observed that the solvent
substantially affects the product yield. The results indicate that
anhydrous ethanol is a better solvent than no solvent or
methanol. The corresponding intermediates II-1 to II-5 had
yields of 73−88% (Table S2).
The synthetic route of compound IV is depicted in Scheme
3. With tetrahydrofuran as a solvent, the acid chloride was
reacted with intermediate II and stirred for 1−1.5 h to obtain
compound IV (Table S3). The yields of the target compounds
IV-1 to IV-20 were within the range of 40−87% (Table S3).
The R1 substituent on the pyrazole ring significantly affected
the yield of the products. The yields of compounds IV-11 to
IV-13 (R1 = phenyl group), IV-14 (R1 = benzyl), and IV-15
(R1 = furyl) were 87, 82, 78, 70, and 64%, respectively. The
target compounds with the six-membered benzene ring
structure exhibited better yields than those with the five-
membered furan ring. The structure of the six-membered
benzene ring is likely to be more stable than that of the five-
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Figure 2. Structure and yield comparison of final products IV-5, IV-35, IV-11, and IV-21.
Table 1. Protective Effect of Target Compounds on the Growth Indexes of Wheat
a b c
, ,
compound
root length IRR (%)
plant height IRR (%)
root fresh weight IRR (%)
plant fresh weight IRR (%)
mefenpyr-diethyl
IV-1
IV-2
IV-3
IV-4
IV-5
IV-6
IV-7
IV-8
79.15 1.53a
60.99 0.72cde
30.49 0.34fgh
51.52 0.32e
36.80 0.17fg
9.42 0.26h
97.87 1.76abc
69.78 0.56ef
83.02 0.54cd
124.47 1.31a
103.49 0.92abc
88.16 0.72cd
60.95 0.78ef
27.34 0.29g
98.34 1.04bc
101.67 2.62abc
106.23 3.17ab
80.32 1.83cde
102.34 1.46ab
93.46 1.36bcd
98.23 1.49abc
93.22 0.81bcd
71.36 1.27de
69.78 1.95ef
72.07 0.74bc
69.23 0.61bc
54.42 0.52de
95.01 0.46a
55.82 0.31de
68.69 0.73bc
85.80 0.83ab
40.34 0.73ef
67.87 0.92bcd
61.23 0.98cde
69.87 0.86bc
41.56 0.78ef
64.32 0.91cd
27.08 0.36gh
40.67 0.61ef
51.67 0.58de
15.68 0.16h
116.15 2.01a
123.17 1.95a
65.88 0.42ef
74.69 0.97de
108.98 1.42a
7.17 0.15h
79.15 1.54a
69.57 0.78b
1.33 0.12gh
57.29 0.47cd
66.93 0.65bc
−4.15 0.19h
78.34 0.78a
14.33 0.23g
56.78 1.57cd
61.23 1.08bcd
10.43 0.37g
61.00 1.46bcd
36.17 0.52def
50.27 1.85cde
61.69 0.82bcd
55.96 1.38cd
69.34 0.93b
35.23 0.25def
65.34 0.64bc
51.44 0.36cde
71.57 0.89ab
85.43 0.61a
62.04 0.35bcd
65.16 0.62bc
78.23 0.92a
49.34 0.52de
76.34 0.73a
57.24 0.74cd
60.34 0.86bcd
49.34 0.29de
64.23 0.41bc
43.45 0.62def
54.45 0.85cde
56.43 0.45cd
36.23 0.36def
51.44 0.68cde
65.98 0.92c
57.08 0.81de
34.78 0.45fg
53.78 0.89e
21.06 0.41gh
75.26 1.68ab
56.82 2.68de
80.97 1.16a
59.96 1.03cde
40.49 0.72ef
69.05 1.65bc
43.97 1.08ef
55.09 0.51de
63.87 0.76cd
12.65 0.15h
86.80 0.36a
42.06 0.41ef
64.14 0.74cd
81.48 1.38a
−47.32 0.46i
73.56 0.53bc
40.97 0.83efg
56.97 0.59de
72.05 0.57ab
26.98 0.38gh
51.02 0.53e
34.54 0.46fg
49.56 0.70ef
40.21 0.63efg
99.43 1.03abc
57.32 0.82f
71.23 0.74de
85.94 1.86cd
00.21 0.06h
103.20 3.63abc
74.26 0.93de
99.64 2.49abc
80.97 2.14cde
96.91 1.90abc
106.42 1.97ab
92.03 0.69bcd
97.87 1.02abc
69.78 0.75ef
83.02 0.72cd
124.47 0.82a
103.49 1.31abc
88.16 0.74cd
60.95 0.65ef
27.34 0.27g
98.34 1.04bc
101.67 1.75abc
106.23 1.07ab
80.32 0.64cde
102.34 1.40ab
93.46 1.04bcd
98.23 1.77abc
93.22 1.64bcd
71.36 0.62de
69.78 0.91ef
IV-9
IV-10
IV-11
IV-12
IV-13
IV-14
IV-15
IV-16
IV-17
IV-18
IV-19
IV-20
IV-21
IV-22
IV-23
IV-24
IV-25
IV-26
IV-27
IV-28
IV-29
IV-30
IV-31
IV-32
IV-33
IV-34
IV-35
−15.43 0.31i
69.23 0.72bc
a
b
c
Data are means of three replicates. Water treated was used as contrast. Statistical analyses of the data were conducted using SPSS software, and
different lowercase letters in the table indicate significant differences (p < 0.05).
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Figure 3. Sequence of activity with different substitutions.
Figure 4. Effect of compounds on GST activity (A), ACCase content (B), and CYP450 activity (C) in vivo of wheat.
Biological Activity and Structure−Activity Relation-
ships (SARs). The safener activity of target compound IV in a
greenhouse was assessed. All new compounds were assessed
for their protective effects on wheat in vivo against FE damage
at a concentration of 200 g/hm2 (Table 1). It is exciting that
most of the compounds presented excellent effects on the
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Figure 5. Metabolic hydrolysis of FE.
recovery of the growth index, indicating that the title
compounds were successfully designed.
197.71 U/L in the shoot protein, which were lower than those
in CK (Figure 4). These results prove that the GST activity of
wheat could be decreased by FE. As shown in Figure 4,
obvious enhancements in the GST activity by 374.38, 202.10,
217.27, 322.80, and 307.61 U/L in shoots and 424.41, 421.12,
560, 610.21, and 624.49 U/L in roots were found after
treatment with mefenpyr-diethyl and compounds IV-11, IV-
16, IV-21, and IV-24, respectively. The GST activity was
obviously increased, and the induction rate was 197.71 U/L in
shoots and 388.52 U/L in roots after treatment with FE.
Apparently, the promotion of GST in the wheat root and shoot
tissues after pretreatment with FE was lower than that after
treatment with the target compounds. From the above
conclusions, it can be indicated that the four test compounds
showed excellent protective effects and raised the tolerance of
wheat to FE by enhancing the GST activity. Among these
compounds, compound IV-21 had the best effect and achieved
GST activities of 610.21 U/L in roots and 332.8 U/L in
shoots, which were even better than those of mefenpyr-diethyl.
To further explore the protection mechanism of the
compounds, the ACCase content and CYP450 activity assays
of these four compounds were determined.
ACCase Content. FE is an ACCase inhibitor, provoking
significant chlorosis and growth inhibition in wheat. The
ACCase content in vivo was measured to verify the influence of
the synthesized compound on the target enzyme of the
herbicide. Compounds IV-11, IV-16, IV-21, and IV-24, with
good safener activities, were selected to detect the ACCase
content based on the bioassay test. Mefenpyr-diethyl was used
as a control.
As shown in Figure 4, the contents of ACCase in wheat
treated with FE were 24.58 pmol/L in roots and 23.51 pmol/L
in shoots, which were lower than those in CK. The content of
ACCase was obviously decreased by FE. It was found that the
ACCase content was recovered after treatment with com-
pounds IV-11, IV-16, IV-21, and IV-24, similar to mefenpyr-
diethyl. The contents of ACCase in wheat treated with IV-11
were 25.14 pmol/L in roots and 26.57 pmol/L in shoots,
which were higher than those in wheat treated with mefenpyr-
diethyl. Among them, compound IV-21 recovered the ACCase
content to the CK level, with its content in the roots reaching
31.91 pmol/L. In general, all the selected compounds
interfered with the inhibition of ACCase via FE.
As shown in Table 1, a majority of the title compounds
promoted the recovery rate of the growth indexes. Among the
rest, compound IV-21 showed an excellent activity against FE.
The IRRs of compound IV-21 on the root length, plant height,
root fresh weight, and plant fresh weight were 86.80, 124.47,
95.01, and 85.43%, respectively, which demonstrates that
compound IV-21 is even better than mefenpyr-diethyl.
Notably, diverse biological activities were presented by
different substituents. For the same replacement in R3, the
compounds with other substitutions that had phenyl at the R1
position showed an excellent biological activity compared with
those with furyl at the R1 position. For example, the recovery
rate of the root length of compound 4(S3) was 5.5 times
higher than that of its corresponding compound IV-5.
Compounds with the electron-donating group −CH3 at R2
showed a higher safener biological activity than that of the
structure with the strong electron-withdrawing group −CF3.
For instance, the IRR of root fresh weight was just 88.16% for
compound IV-23 (R2 = −CF3), while it was 124.47% for
compound IV-21 (R2 = −CH3). Obviously, the target
compounds with diverse substitutions of the ester had a
significant impact on the biological activity. Target compound
IV with a Cl atom at R3 showed an excellent biological activity
compared with the corresponding structure with an alkyl. For
example, recoveries of the root length in the case of
compounds IV-21 and IV-29 were 86.80 and 72.05%, which
were better than those in the case of compounds IV-11 and
IV-19, with 75.26 and 63.87%, respectively. Based on the
overall biological activity, the chlorine atom may increase the
activity of the target compounds.
In summary, the SAR at R1 can be briefly summarized as
follows (Figure 3): phenyl > benzyl > furyl. The biological
activity was obtained when R2 was replaced by −CH3, which
was essentially higher than that of −CF3. The safener activity
of the linear chain structure on R3 is essentially higher than
that of the branched chain structure, and introduction of the Cl
atom increased the safener activity. The SAR results indicated
that the structure of compound IV-21 showed a greater
similarity to mefenpyr-diethyl. This indicated that the
structure−activity correlations could be used to determine
the bioactivity, as they provided valuable information about the
required substituents for biological activity.
CYP450 Activity. CYP450 is a key enzyme in the
metabolism of herbicides, and safeners work through the
induction of enzyme systems. Compounds IV-11, IV-16, IV-
21, and IV-24 were selected, and mefenpyr-diethyl was used as
a control. The CYP450 activity of wheat was measured, and
the results are shown in Figure 4.
The four test compounds increased the CYP450 activity of
wheat. Compounds IV-16 and IV-21 showed a better effect in
vivo than mefenpyr-diethyl. Compound IV-21 was the most
effective, with CYP450 activities of 170.47 and 102.85 U/L in
roots and in shoots, respectively. The effects of compound IV-
11 were weak. The results of the comprehensive effects of
GST Activity. GST, present in a plant from the early
embryonic stage to the mature stage, plays a significant role in
the process of herbicide detoxification and protects the plant
from oxidative damage.50−53 To further explore the specific
influence of compound IV on GST activity, compounds IV-11,
IV-16, IV-21, and IV-24 with a better bioactivity were singled
out. Next, their detoxification mechanisms were further
explored, and the commercialized safener mefenpyr-diethyl
was used as a control.
The results of the enzyme assay showed that the activity of
GST in wheat treated with FE was 388.52 U/L in roots and
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Table 2. Chemical Property Comparisons of FA, Mefenpyr-diethyl, and Compound IV-21
a
b
Discovery Studio 3.5 for interaction energy, MW, log p, rotatable bonds (RBs), aromatic rings (ARs), and surface area (SA). Electronegativity
was predicted using SYBYL-X 2.0 (Tripos, Inc., St. Louis, MO, USA).
Figure 6. Receptor−ligand interactions of FA (A), mefenpyr-diethyl (B), and compound IV-21 (C) with the active site of ACCase.
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Figure 7. Docking modeling of FA (A), mefenpyr-diethyl (B), and compound IV-21 (C) with ACCase.
safeners on the GST activity and ACCase content showed that
safeners induced GST activity and the ACCase content in
plants and increased the CYP450 activity in wheat.
30.07 kcal/mol, which was higher than those of mefenpyr-
diethyl and compound IV-21 (Table 2), which indicated that
the binding of FA and ACCase was stronger than that of the
other two compounds. It was thus speculated that both
compound IV-21 and mefenpyr-diethyl interacted with the
amino residues in the active site of ACCase, which hindered
herbicide insertion.
Furthermore, geometric matching models were established
(Figure 7). FA was well-embedded in the active pocket, filling
the entire active pocket and acting on multiple amino acid
sites, thereby causing the inhibition effect. Notably, neither
mefenpyr-diethyl nor compound IV-21 matched well in terms
of spatial geometry as FA did, so it could not exert a similar
weed control effect. However, mefenpyr-diethyl and com-
pound IV-21 occupied part of the active site. This prevented
FA from reaching or acting with ACCase when compound IV-
21 was applied before or with an herbicide.
Molecular Structure Comparisons. The pharmacological
activity and its interaction with the target enzyme of the
compounds are greatly affected by the physicochemical
properties. The similar property principle (SPP) refers to the
fact that molecules share similar structures, display similar
properties, and use similar binding modes when molecules
interact with the target proteins.54,55 FE is hydrolyzed into the
corresponding acid in plants; here, FA is selected as the
structure for comparison (Figure 5).49 By comparing the
physicochemical properties of compound IV-21, mefenpyr-
diethyl, and FA (Table 2), it is not difficult to see that the log
p, hydrogen-bond donors (HBDs), aromatic rings (ARs),
surface area (SA), and electronegativity of the three
compounds had a strong resemblance. In addition, more
hydrogen-bond acceptors (HBAs) were found in mefenpyr-
diethyl. The molecular weight (MW) of compound IV-21 was
relatively low, which was conducive to the metabolism of
exogenous compounds for crops. The rotatable bonds (RBs) of
compound IV-21 were lower than those of mefenpyr-diethyl,
which could allow for the dominant conformation to be
successfully locked and was conducive to the efficacy of the
drug. This finding indicated that compound IV-21 has
enormous potential to become the leading structure of new
herbicide safeners.
ADMET Prediction. Many drugs have failed to enter the
market due to their toxicity. The pharmacokinetic properties of
the active compounds are predicted in Table 3. The solubility
Table 3. ADMET Prediction of FA, Mefenpyr-diethyl, and
Compound IV-21
FA
mefenpyr-diethyl
compound IV-21
a
solubility level
2
0
2
0
2
0
b
absorption level
CYP2D6 prediction
c
false
4.071
true
true
3.951
true
false
3.664
true
Molecular Docking. Molecular docking is considered to
be a highly efficient method for predicting the interactions
between ligands and receptors.56 To further verify the
detoxification mechanism of safeners on wheat, a molecular
docking study was performed to confirm the interactions
between the active compounds and ACCase.
d
AlogP98
e
PPB# prediction
a
b
Solubility level: categorical solubility level. 2: Yes, low. Absorption
level: absorption level. 0: Good absorption. CYP2D6: cytochrome
c
P450 2D6. <0.161: False, noninhibitor; >0.161: true, inhibitor.
d
AlogP98: the logarithm of the partition coefficient between n-octanol
The crystalline structure of ACCase (PDB ID: 1UYR) was
used to carry out the molecular docking experiment. As shown
in Figure 6, FA interacted with six amino acid residues, of
which Gly 1734 formed amide−π stacked interactions with the
phenyl of FA. Moreover, FA formed π−alkyl interactions with
Val 1627, Val 1733, and Ile 1735. There were also alkyl and π−
alkyl nonpolar interactions between compound IV-21 and Ala
1627, Ile 1735, Leu 1756, and Tyr 1738. It was clear that
compound IV-21 formed an additional π−alkyl interaction
with Ile 1735 compared to mefenpyr-diethyl. Therefore, the
mechanism of compound IV-21 is similar to that of mefenpyr-
diethyl, except that compound IV-21 bonds are more stable
with ACCase. Although there are more HBAs in mefenpyr-
diethyl, HBAs hardly affect the interaction in the active site. By
comparing the interaction energy of the three compounds, it
was not difficult to see that the interaction energy of FA was
and water. <4.0: Binding is <90%; >4.0: binding is >90% and binding
is <95%. PPB: plasma protein binding ability. <−2.209: ≥90%, False;
> −2.209: ≤90%, true.
e
level, absorption level, and plasma protein binding (PPB#
prediction) of the three compounds were exactly analogical.
The PPB ability was less than 90%, indicating that mefenpyr-
diethyl, compound IV-21, and FA bear good bioavailability
and would not attach to the carrier protein. Notably, the
CYP2D6 prediction showed no inhibition of the CYP2D6
enzyme via compound IV-21, indicating that it could pass
through the first stage of metabolism smoothly, while
mefenpyr-diethyl inhibited the CYP2D6 enzyme, and metab-
olism was easily blocked. In summary, compound IV-21
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exhibited better pharmacokinetic properties than mefenpyr-
diethyl.
ACKNOWLEDGMENTS
■
This work was supported by the National Nature Science
Foundation of China (31572042 and 31801784) and the
Natural Science Foundation of Heilongjiang Province
(ZD2017002).
In conclusion, a sequence of new ester-substituted pyrazole
derivatives was designed and synthesized based on the
fragment splicing method. The bioactivity assay showed that
the safening activity was possessed by most of the target
compounds, to some extent, preventing wheat from FE injury.
Compound IV-21 showed the greatest activity against FE and
enhanced the tolerance of wheat by recovering the ACCase
content and enhancing the GST and CYP450 activities. The
molecular structure comparisons and molecular docking results
indicated that the better bioactivity of compound IV-21 came
from the occupation in the active site of ACCase. The
ADMET prediction revealed that compound IV-21 exhibited
excellent pharmacokinetic properties. This information in-
dicated that the ester-substituted pyrazole is a potent safener
skeleton to be further optimized and developed.
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ASSOCIATED CONTENT
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* Supporting Information
The Supporting Information is available free of charge at
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AUTHOR INFORMATION
■
Corresponding Authors
Ying Fu − Department of Chemistry, College of Arts and
Sciences, Northeast Agricultural University, Harbin 150030,
China; orcid.org/0000-0003-4265-6879; Phone: +86-
451-55190070; Email: fuying@neau.edu.cn
Fei Ye − Department of Chemistry, College of Arts and
Sciences, Northeast Agricultural University, Harbin 150030,
China; orcid.org/0000-0002-7731-752X; Phone: +86-
451-55191507; Email: yefei@neau.edu.cn
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2017, 73, 2227−2235.
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ACCase-Resistant goosegrass (Eleusine indica) in the United States.
Weed Sci. 2016, 64, 399−408.
Ling Jia − Department of Chemistry, College of Arts and
Sciences, Northeast Agricultural University, Harbin 150030,
China
Shuang Gao − Department of Chemistry, College of Arts and
Sciences, Northeast Agricultural University, Harbin 150030,
China; orcid.org/0000-0003-2012-8768
Yuan-Yuan Zhang − Department of Chemistry, College of Arts
and Sciences, Northeast Agricultural University, Harbin
150030, China
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Isoxadifen-Ethyl derivatives protect rice from fenoxaprop-P-ethyl-
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Li-Xia Zhao − Department of Chemistry, College of Arts and
Sciences, Northeast Agricultural University, Harbin 150030,
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.1c02221
(17) Kang, T.; Gao, S.; Zhao, L.-X.; Zhai, Y.; Ye, F.; Fu, Y. Design,
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Author Contributions
#L.J. and S.G. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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