Design, Synthesis, and Herbicidal Activity of N-Benzyl-5-cyclopropyl-isoxazole-4-carboxamides

Article abstract of DOI:10.1021/acs.jafc.0c03582

Based on the structures of isoxaflutole (IFT) and N-isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentenamide, a series of N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides was designed by connecting their pharmacophores (i.e., a multitarget drug design strategy). A total of 27 N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides were prepared from 5-cyclopropylisoxazole-4-carboxylic acid and substituted benzylamines, and their structures were confirmed by NMR and MS. Laboratory bioassays indicated that I-26 showed 100% inhibition against Portulaca oleracea and Abutilon theophrasti at a concentration of 10 mg/L, better than the positive control butachlor (50% inhibition for both weeds). A strong growth inhibition was observed, but a typical bleaching phenomenon of IFT could not be observed in the Petri dish assay. I-05 displayed excellent postemergence herbicidal activity against Echinochloa crusgalli and A. theophrasti at a rate of 150 g/ha, and bleaching symptoms were observed in the leaves of treated weeds. The bleaching effect of Chlamydomonas reinhardtii treated by I-05 could be reversed by adding homogentisate. Enzymatic bioassays indicated that I-05 could not inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, but II-05, an isoxazole ring-opening product of I-05, could inhibit HPPD activity with an EC50 value of 1.05 μM, similar to that of mesotrione (with an EC50 value of 1.35 μM). Detailed discussion about observed herbicidal symptoms is provided in the Results and Discussion section. This investigation provided a proof-of-concept foundation that a multitarget drug design strategy could be applied in agrochemical research.

Full text of DOI:10.1021/acs.jafc.0c03582

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Article
Design, Synthesis, and Herbicidal Activity of N‑Benzyl-5-
cyclopropyl-isoxazole-4-carboxamides
Xin-Lin Sun, Zhen-Meng Ji, Shao-Peng Wei, and Zhi-Qin Ji*
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ABSTRACT: Based on the structures of isoxaflutole (IFT) and N-isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentenamide, a series of
N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides was designed by connecting their pharmacophores (i.e., a multitarget drug design
strategy). A total of 27 N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides were prepared from 5-cyclopropylisoxazole-4-carboxylic
acid and substituted benzylamines, and their structures were confirmed by NMR and MS. Laboratory bioassays indicated that I-26
showed 100% inhibition against Portulaca oleracea and Abutilon theophrasti at a concentration of 10 mg/L, better than the positive
control butachlor (50% inhibition for both weeds). A strong growth inhibition was observed, but a typical bleaching phenomenon of
IFT could not be observed in the Petri dish assay. I-05 displayed excellent postemergence herbicidal activity against Echinochloa
crusgalli and A. theophrasti at a rate of 150 g/ha, and bleaching symptoms were observed in the leaves of treated weeds. The
bleaching effect of Chlamydomonas reinhardtii treated by I-05 could be reversed by adding homogentisate. Enzymatic bioassays
indicated that I-05 could not inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, but II-05, an isoxazole ring-opening
product of I-05, could inhibit HPPD activity with an EC50 value of 1.05 μM, similar to that of mesotrione (with an EC50 value of
1.35 μM). Detailed discussion about observed herbicidal symptoms is provided in the Results and Discussion section. This
investigation provided a proof-of-concept foundation that a multitarget drug design strategy could be applied in agrochemical
research.
KEYWORDS: 4-hydroxyphenylpyruvate dioxygenase, herbicidal activity, isoxazole, benzylamine
approaches, merging pharmacophores with and without a
linker is a well-recognized method.15,16 This method requires
the similarity of chemical structures and bioactivities of the two
lead compounds. IFT does not inhibit HPPD in plants. Its
activity comes from the product of a ring-opening conversion
(i.e., from isoxazole to diketonitrile or DKN). This ring-
opening conversion is a rapid nonenzymatic hydrolyzation and
occurs both in the plant and in soil.17 The chelating 1,3-
diketone of DKN is responsible for the binding to the active
site of HPPD. Therefore, the isoxazole-4-carbonyl moiety of
IFT is considered as its pharmacophore. Another lead
herbicide adopted for this design is N-isobutyl-N-(4-chloro-
benzyl)-4-chloro-2-pentenamide, which is a light-dependent
amide herbicide reported by Kumai Chemical Co. Ltd. It can
effectively control barnyard grass, throughout inhibiting cell
division.18 According to its structure, carbonyl benzylamine is
considered to be more important than the flexible side chain.
Therefore, this moiety was assumed as a pharmacophore for
this lead herbicide.
INTRODUCTION
■
4-Hydroxyphenylpyruvate dioxygenase (HPPD) is an impor-
tant enzyme of the biosynthetic pathway to plastoquinone in
plants, and inhibition of plastoquinone biosynthesis through
HPPD blockade results in herbicidal activity with unique
bleaching symptoms.1 In 1997, HPPD was identified as the
target site of triketones.2 Great progress has been made in the
discovery of HPPD inhibitors in recent years, and triketones,
isoxazoles, and pyrazoles have been widely used in the
management of weeds.3 Because of their high activity, lack of
resistance, and low mammalian toxicity, the discovery of novel
HPPD inhibitors attracts much attention in the herbicide
industry nowadays.4−7
Isoxaflutole (IFT) is a pre-emerging HPPD herbicide for the
control of broadleaf and grass weeds in maize and sugarcane.8
Although IFT is a highly effective herbicide, the weed
spectrum and crop selectivity of IFT are not good enough in
practical applications. To overcome these shortcomings, IFT is
usually applied by mixing with flufenacet, calonifen, or atrazine
to improve its herbicidal activity against grassy weeds, and
cyprosulfamide is used as a safener in the formulation.9−11 It is
necessary to develop novel chemistry to overcome such
shortcomings.
The objective of the present study was to adopt a multitarget
drug design strategy by merging the pharmacophore of IFT
Received: June 6, 2020
Revised: October 13, 2020
Accepted: November 10, 2020
The multitarget drug design is a novel strategy in the
medicinal field, and a variety of successful examples have been
reported.12−14 However, this strategy has seldom been
reported in agrochemical research. Different approaches
could be realized for multitarget drug designs. Among these
https://dx.doi.org/10.1021/acs.jafc.0c03582
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Figure 1. Design strategy of N-benzyl-5-cyclopropylisoxazole-4-carboxamides.
Figure 2. Synthesis of compounds I-01−I-27. Reagents and conditions: (a) 60 °C, 20 h; (b) H2NOH−HCl, H2O, MeOH, 90 min, 60 °C; (c)
concentrated HCl, AcOH, 4 h, reflux; (d) oxalyl chloride, CH2Cl2, 30 min, room temperature; and (e) substituted benzylamines, CH2Cl2, 30 min,
0 °C.
1,1-dimethoxy-N,N-dimethylmethanamine (1) (24 g, 0.2 mol) and
methyl 3-cyclopropyl-3-oxo-propanoate (2) (28 g, 0.2 mol) were
mixed and heated for 20 h at 60 °C. The obtained yellow oil was first
dissolved in methanol (200 mL) and water (100 mL), and then,
hydroxylamine hydrochloride (14 g, 0.2 mol) was added. The solvents
were evaporated under vacuum after the mixture was heated for 90
min at 60 °C. The residue was dissolved in the mixture of acetic acid
(100 mL) and concentrated HCl (100 mL) and refluxed for 4 h. The
reaction mixture was diluted with water (500 mL) and extracted with
ethyl acetate (200 mL × 3). The organic layer was combined and
washed with brine and then dried with anhydrous sodium sulfate.
Ethyl acetate was evaporated under vacuum. The residue was
subjected to a silica gel column and eluted with the mixture of
ethyl acetate and petroleum ether at the ratio of 1:3 (v/v) to afford 3.
General Procedures for Preparation of 5-Cyclopropylisox-
azole-4-carbonyl Chloride (4). Oxalyl chloride (38.1 g, 0.3 mol) in
50 mL of anhydrous dichloromethane was added dropwise to the
solution of 3 (15.3 g, 0.1 mol) in 150 mL of anhydrous
dichloromethane. The mixture was reacted for 30 min at room
temperature. After completion of the reaction based on TLC
monitoring, dichloromethane was evaporated under vacuum to afford
4.
General Procedures for Preparation of N-Benzyl-5-cyclo-
propylisoxazole-4-carboxamides (I-01−I-27). The target com-
pounds, I-01−I-27, were synthesized by following a reported
procedure.20 To a solution of substituted benzylamines (1 mmol)
and 4 (1 mmol) in 20 mL of anhydrous dichloromethane, pyridine (3
mmol) in 5 mL of anhydrous dichloromethane was added dropwise at
0 °C. The mixture was reacted at 0 °C for 30 min. After completion of
the reaction based on TLC monitoring, the solution was washed with
water (30 mL) and saturated sodium chloride solution (30 mL)
successively. The organic layer was dried over anhydrous sodium
sulfate. After the solvent was removed under vacuum, the residue was
(i.e., isoxazole-4-carbonyl) and the pharmacophore of N-
isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentenamide (i.e., car-
bonyl benzylamine). Because of sharing a common carbonyl
group of two pharmacophores, merging without a linker was
adopted, forming novel carboxamides (Figure 1). A total of 27
N-benzyl-5-cyclopropylisoxazole-4-carboxamides were de-
signed and synthesized, and their structures were confirmed
by NMR and MS. Their herbicidal activities were evaluated by
Petri dish assays and pot experiments in a glasshouse. Active
compounds (e.g., I-05, I-24, and I-26) were discovered, and
typical bleaching phenomenon of HPPD inhibition could be
observed with algae and postemergence pot assays for some
compounds (e.g., I-05 and I-24). These active compounds
could be used as herbicidal leads for further optimization.
MATERIALS AND METHODS
■
Reagents and Procedures. All chemical reagents were
commercially available and used without further purification.
Precoated silica gel plates (Si60 F254, Merck Chemical Co. Ltd)
were used to monitor the progress of reactions. Purification of target
compounds was performed on silica gel column chromatography
(200−300 mesh, Qingdao Marine Chemical Co. Ltd, China). 1H and
13C NMR spectra were recorded on a Bruker AVANCE III-500 NMR
spectrometer, and the residual solvent signals were used as reference.
Mass spectral analysis was carried out on a Finnegan LCQ Advantage
MAX LC/MS spectrometer equipped with an ESI source. The
melting points were conducted on a WRS-3 apparatus and
uncorrected.
General Procedures for Preparation of 5-Cyclopropylisox-
azole-4-carboxylic Acid (3). The key intermediate 5-cyclo-
propylisoxazole-4-carboxylic acid (3) was prepared by following a
procedure described in a patent with minor modification (Figure 2).19
B
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purified on a silica gel column by using ethyl acetate/petroleum ether
(1:5) as an eluent to afford I-01−I-27.
General Procedures for Preparation of N-Benzyl-2-cyano-3-
cyclopropyl-3-oxopropanamides (II-03, II-04, II-24, and II-25).
The DKNs of I-03, I-04, I-24, and I-25 were synthesized following a
procedure in literature (Figure 3).21 To a solution of I-03, I-04, I-24,
column chromatography eluted by ethyl acetate/petroleum ether
(1:4).
Petri Dish Assay. Seeds of monocotyledon weeds, such as
Echinochloa crusgalli and Digitaria sanguinalis, and dicotyledon weeds
such as Amaranthus retroflexus, Portulaca oleracea, and Abutilon
theophrasti were collected from the campus of Northwest A&F
University in 2017. Herbicidal activity of the title compounds against
these target weeds was first evaluated by Petri dish assay as described
previously.22 A total of 10 germinating seeds were placed in Petri
dishes (90 mm diameter) containing two layers of filter paper
impregnated with 5 mL of the solutions of tested compounds at 100
and 10 mg/L, respectively. Water was used as a blank control; IFT
and butachlor were used as positive controls. Then, the Petri dishes
were placed in a light incubator at 25 °C with a light intensity of 3000
lux. The growth inhibition rates of roots and stems were observed
after 5 days. Each treatment was repeated three times, and average
inhibition of stems and roots are listed in Tables 1 and 2.
Figure 3. Preparation of II-03, II-04, II-24, and II-25. Reagents and
conditions: (a) Et3N, CH2Cl2, room temperature.
Pre- and Postemergence Herbicidal Activity. Pre- and
postemergence herbicidal activities of the title compounds against
E. crusgalli and A. theophrasti were evaluated according to a procedure
reported previously.23 Each test compound was first dissolved in
DMSO to prepare a concentration of 100 g/L solution. Each solution
was then diluted with water containing 0.1% Tween 80 to the desired
concentrations before applications. The soil used was a mixed soil
(33.3% garden soil and 66.7% seedling substrate). Plastic pots with an
inner diameter of 7.5 cm were filled with the soil to three-fourths of
or I-25 (1 mmol) in dichloromethane (10 mL), triethylamine (3
mmol) was added, and the mixture was stirred for 12 h at room
temperature. After completion of the reaction based on TLC
monitoring, dichloromethane was evaporated under vacuum. The
residue was diluted with cold water and acidified with 1 M dilute
hydrochloric acid and then extracted with ethyl acetate (40 mL). The
organic layer was washed with brine and dried with anhydrous sodium
sulfate. II-03, II-04, II-24, and II-25 were obtained by using silica gel
Table 1. Herbicidal Activity of Title Compounds in Petri Dish Tests (100 mg/L)
inhibition rate (%)
a
a
a
a
a
EC
DS
AR
PO
AT
no.
R1
root
stem
root
stem
root
stem
root
stem
root
stem
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
I-27
II-03
II-25
isoxaflutole
butachlor
H
50
50
50
50
60
30
30
30
50
60
50
80
80
60
60
100
100
100
80
30
40
40
30
50
20
20
30
40
50
50
80
90
60
60
100
100
100
70
80
70
70
100
100
100
100
60
60
70
60
70
60
50
60
20
50
30
70
30
50
50
30
40
20
50
80
80
80
60
60
70
80
40
40
80
70
60
50
50
50
80
30
40
100
100
100
100
100
100
100
100
100
70
100
100
100
100
100
100
100
100
100
80
90
90
80
90
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
3,4-diOCH3
3,5-diOCH3
4-CH(CH3)2
4-C(CH3)3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
3-Br
2,4-diF
2,4-diCl
2,6-diF
2,6-diCl
3,4-diCl
3-Cl-4F
3-CF3
4-CF3
2-OH
3-CH3
3-CF3
100
100
100
100
100
100
100
100
60
100
100
100
100
100
100
100
100
60
50
50
60
100
100
100
100
100
100
50
100
100
100
100
100
100
100
100
60
100
100
100
100
100
100
100
100
60
50
50
50
40
70
70
60
60
70
70
100
100
100
40
100
100
100
80
60
60
40
90
100
100
100
40
60
100
40
100
100
100
50
100
100
100
80
60
60
40
90
100
100
100
40
100
100
100
100
90
80
80
80
100
100
100
20
80
100
70
100
100
100
40
100
100
100
100
90
70
80
80
100
100
100
20
100
100
100
80
100
100
100
100
80
100
100
100
80
100
100
100
100
70
80
70
60
100
90
100
90
40
50
50
100
100
100
100
70
50
100
30
100
100
100
100
80
100
100
60
100
100
100
100
80
100
100
100
30
100
100
100
100
100
100
50
50
80
100
100
100
100
100
b
b
b
b
b
10
40
60
50
50
100
100
100
100
80
80
80
80
90
90
a
Abbreviations: EC for Echinochloa crusgalli; DS for Digitaria sanguinalis; AR for Amaranthus retroflexus; PO for Portulaca oleracea; and AT for
b
Abutilon theophrasti. Exhibit bleaching symptoms.
C
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Table 2. Herbicidal Activity of Title Compounds in Petri Dish Tests (10 mg/L)
inhibition rate (%)
a
a
a
a
a
EC
DS
AR
PO
AT
no.
R1
root
stem
root
stem
root
stem
root
stem
root
stem
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
I-11
I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
I-27
II-3
H
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
30
20
20
20
0
0
0
0
0
30
20
20
20
0
0
0
0
0
50
30
30
40
20
20
20
20
30
0
40
30
20
40
20
20
20
20
20
0
50
30
0
30
20
0
50
20
0
20
20
0
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
3,4-diOCH3
3,5-diOCH3
4-CH(CH3)2
4-C(CH3)3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
3-Br
2,4-diF
2,4-diCl
2,6-diF
2,6-diCl
3,4-diCl
3-Cl-4F
3-CF3
4-CF3
2-OH
3-CH3
3-CF3
0
0
10
20
0
10
20
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
20
30
30
0
40
20
30
20
20
0
20
20
30
0
40
20
30
10
20
0
20
20
30
0
20
40
40
20
0
20
20
30
0
30
30
40
20
0
50
50
70
0
50
80
60
50
30
0
40
50
70
0
50
80
50
50
30
0
60
70
100
30
70
90
60
50
40
20
20
30
80
80
100
0
60
70
100
30
70
90
60
30
30
20
20
40
80
80
100
0
60
50
60
50
50
60
60
30
0
50
50
50
50
60
60
60
30
0
0
0
0
0
0
0
0
0
0
0
0
0
30
70
70
70
0
20
60
70
70
0
30
60
60
40
0
30
60
60
40
0
20
70
70
90
0
20
80
50
60
20
70
70
90
0
30
70
80
100
20
10
80
30
50
30
70
80
100
20
10
80
0
0
0
0
20
70
30
80
40
50
30
80
II-25
isoxaflutole
butachlor
70
40
90
70
60
30
90
60
b
b
b
b
b
20
10
20
30
20
80
90
60
50
50
a
Abbreviations: EC for Echinochloa crusgalli; DS for Digitaria sanguinalis; AR for Amaranthus retroflexus; PO for Portulaca oleracea; and AT for
b
Abutilon theophrasti. Exhibit bleaching symptoms.
their heights. Around 20 seeds of the tested weeds were sown in each
pot and covered with soil to a thickness of 0.2 cm. The pots were
maintained at temperatures from 15 to 30 °C in a glasshouse. For pre-
emergence treatments, the diluted test solutions (150 g ai/ha) were
sprayed on the surface of soil 24 h after the seeds were sown. For
postemergence treatments, the weeds were treated with the solutions
of tested compounds (150 g ai/ha) at the three-leaf stage. The
seedlings treated with the diluted solution of DMSO and Tween 80
were used as the control groups. Each treatment was performed in
four replicates. IFT was used as a positive control. The herbicidal
activity was evaluated visually at 15 days after treatment.
Effect of Exogenous Homogentisate on Chlamydomonas
reinhardtii. C. reinhardtii was grown autotrophically in the liquid
medium as literature described.24 C. reinhardtii cells in the late
exponential phase were harvested and transferred to glass tubes (with
plastic cover) at a density of 5.0 × 105 cell/mL in a volume of 5 mL
culture medium. For the exposures, I-05 (200 and 100 μM), I-05 +
homogentisate (HGA) (200 + 50 and 100 + 50 μM), and HGA (50
μM) were added to the medium. A chemical-free treatment was used
as a blank control. The algae were cultivated in an illumination
incubator with a PAR intensity of 3000 lux at 25 °C. The symptoms
were observed at 96 h after exposure.
were evaluated by using a HPLC method according to the procedures
described in literature.25 Mesotrione was used as a positive control.
Each treatment was carried out with three replicates. The content of
HGA in each assay mixture was measured by HPLC on a Hypersil C18
column (5 μm, 4.6 mm × 250 mm) by using methanol/0.1%
trifluoroacetic acid (80 + 20, v/v) as a mobile phase. The IC50 values
of tested compounds were calculated based on the average of three
replicates.
Molecular Modeling. The 3D structures of II-05 and II-24 were
constructed by using ChemBiodraw Ultra 10.0. All compounds were
then opened in Discovery studio 4.0, and energy minimization was
carried out by CHARMM force field using the ligand partial charge
method consistent force field.26 Minimization was carried out until an
energy gradient of 0.01 was reached. The CDOCKER was used for
docking of all compounds. A representative AtHPPD cocrystallized
with NTBC (PDB ID: 1TFZ) was taken from the PDB data bank.
The water molecules were deleted and hydrogen atoms were added.
Final protein was refined with CHARMM in DS 4.0 at physiological
pH. To validate the docking reliability, the cocrystallized ligand
(DAS869) was first redocked to the binding site of HPPD.
Consequently, II-05 and II-24 were docked into the same active
site, and 20 conformations of each compound were obtained through
CDOCKER.27 The conformations with the lowest energy were
selected as the most probable binding conformation. PYMOL was
used to analyze the binding modes.
HPPD Enzymatic Assay. HPPD originating from etiolated dark-
grown corn seedlings was extracted and purified according to the
method described in literature.25 The inhibitory effects of HPPD by I-
05, I-24, and the active entities of I-05 and I-24 (i.e., II-05 and II-24)
D
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RESULTS AND DISCUSSION
■
Chemistry. The title compounds were designed based on
the chemical structures of IFT and N-isobutyl-N-(4-chloro-
benzyl)-4-chloro-2-pentenamide. IFT has a complex chemical
structure; therefore, a longer synthetic route leads to higher
production cost.28 The synthetic route for the title compounds
is much easier than that of IFT. 5-Cyclopropylisoxazole-4-
carbonyl acid was the only intermediate needed to be
prepared, and all substituted benzylamines used in this study
were commercially available. 5-Cyclopropylisoxazole-4-carbox-
ylic acid could be prepared from 1,1-dimethoxy-N,N-dimethyl-
methanamine and methyl 3-cyclopropyl-3-oxo-propanoate via
a one-pot three-step reaction. We observed that the yield of the
final product in the pot was strongly affected by the
concentration of HCl. Retaining excess water in the second
step resulted in low yield of 5-cyclopropylisoxazole-4-
carboxylic acid, so removal of solvents before the final reaction
was a crucial factor. To further increase the yield, we examined
the amount of raw materials and found that the optimal molar
ratio of 3-cyclopropyl-3-oxo-propanoate to 1,1-dimethoxy-
N,N-dimethyl-methanamine was 1:1.5. Under the optimal
conditions, the yield of the final product was above 80%. For
preparation of 5-cyclopropylisoxazole-4-carbonyl chloride, we
examined the effect of temperature on the yield, and found that
the yield of acyl chloride was close to 100% at room
temperature. The preparation of N-benzyl-5-cyclopropylisox-
azole-4-carboxamides was carried out under an ice-bath
condition. Because of the high reactivity of benzylamine, the
reaction proceeding at room temperature tended to produce a
bisamide. The title compounds could be readily converted to
their corresponding DKNs in dichloromethane containing
triethylamine at room temperature.
Figure 4. Symptoms of P. oleracea treated by IFT, butachlor, and I-26.
1: CK; 2, 3: IFT at 100 and 10 mg/L; 4, 5: butachlor at 100 and 10
mg/L; 6, 7: I-26 at 100 and 10 mg/L.
broadleaf weeds were stronger than that of butachlor. For
example, I-26 completely inhibited the growth of P. oleracea at
both 100 and 10 mg/L. Its poison symptoms were similar to
that of N-isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentena-
mide, one of its lead compounds, and we speculated the title
compounds also exhibited herbicidal activity through an
inhibition of cell division. However, no bleaching effect was
observed in the groups treated by the title compounds.
Because the conversion from IFT to DKN is essential for the
herbicidal activity, we speculated whether the title compounds
were not converted into the active form in vivo. To testify this
speculation, II-03 and II-25, two DKN derivatives, were
prepared from I-03 and I-25 in the laboratory, and their
herbicidal activities were evaluated by the Petri dish assay
(Tables 1 and 2). These two DKN derivatives showed similar
inhibitory effects with their corresponding precursor mole-
cules, and no bleaching effect was observed. No bleaching
phenomenon might result from poor uptake and translocation
of these compounds or their poor stability in seedlings so that
their quantity in leaves of seedlings could not accumulate
enough.
Pre- and Postemergence Herbicidal Activity. Pre- and
postemergence herbicidal activities of the newly prepared
compounds against E. crusgalli and A. theophrasti were assessed
at an application rate of 150 g/ha. Unfortunately, none of the
tested compounds showed pre-emergence herbicidal activity at
this application rate. But several compounds showed good
postemergence herbicidal activity, and the results are
illustrated as Figure 5. I-05 and I-24 exhibited strong
herbicidal activity against E. crusgalli and A. theophrasti, and
their activities were comparable to IFT. The inhibition rates of
I-12 and I-19 against monocotyledon E. crusgalli were all
above 60%; however, they only showed weak activity against A.
theophrasti. In contrast, I-23 showed good herbicidal activity
against the broadleaf weed A. theophrasti. Furthermore, these
active compounds resulted in a bleaching effect similar to that
of HPPD herbicides.
The chemical structures of the synthesized compounds were
confirmed by NMR and MS spectrometric analysis.
Herbicidal Activity in Petri Dish Assays. As shown in
Table 1, most of the title compounds at 100 mg/L exhibited
high herbicidal activity against target weeds, especially
broadleaf weeds. The herbicidal activities of I-16−I-18 and
I-24−I-26 were stronger than other compounds, and their
inhibition rates on all tested weeds were 100%. These results
indicated that introduction of electron-withdrawing groups on
the benzene ring is more beneficial to herbicidal activity than
that of electron donating groups. I-12, I-15, and I-19−I-22
showed weaker activity than other halogenated compounds,
implying that the introduction of halogen atoms to the meta-
and para-positions of the benzene ring is better for herbicidal
activity than the ortho-position. To further assess the SAR,
herbicidal activities of the title compounds at 10 mg/L were
evaluated, and the results are listed in Table 2. I-26 was the
most potent compound, and it could effectively inhibit the
growth of A. retroflexus, P. oleracea, and A. theophrasti at 10
mg/L. The influence of the substitution position of halogen
atoms on the benzene ring could be summarized as: para- >
meta- > ortho-position. All derivatives which bear electron-
donating groups on the benzene ring including I-01−I-11 and
I-27 showed slight or even no herbicidal activity at this dose.
The symptoms of weeds treated by IFT, butachlor, and I-26
on P. oleracea are illustrated as Figure 4. As an effective HPPD
inhibitor, IFT resulted in characteristic bleaching symptoms in
the leaves but only slightly inhibited the growth of P. oleracea.
The synthesized compounds showed strong inhibition against
weeds, and the inhibitory rates of some compounds against
The structure−activity relationships of several ortho-
substituted derivatives obtained from pot experiments and
Petri dish assay had great discrepancies. The inhibitory effects
of I-02 and I-05 in the Petri dish assay were weaker than those
of many meta- or para-halogenated derivatives, whereas they
showed stronger postemergence herbicidal activity. It seems
that an introduction of electron-donating groups to the ortho-
position is helpful for postemergence herbicidal activity.
Additionally, the introduction of fluorine and chlorine to the
meta- and para-positions on the benzene ring is beneficial to
the activity.
Effect of Exogenous HGA on C. reinhardtii. HPPD
herbicides indirectly inhibit carotenoid biosynthesis, through-
out preventing the production of HGA, and this inhibition can
be reversed by a supplement of HGA. To assess whether a
E
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Figure 5. Postemergence herbicidal activity of several title compounds (150 g/ha). EC for Echinochloa crusgalli; AT for Abutilon theophrasti.
supplement of HGA on the inhibition of C. reinhardtii by I-05,
we ran the following test: C. reinhardtii grew normally with
green color upon the treatments of HGA and the chemical-free
control at 96 h after exposure to I-05 (Figure 6E,F). However,
Table 3. Inhibitory Effects of HPPD by Tested Compounds
compounds
IC50 (μM)
I-05
>10
II-05
I-24
1.05 0.16
>10
II-24
mesotrione
1.28 0.07
1.34 0.09
respectively. However, I-05 and I-24 showed no inhibitory
effect on HPPD.
Combined information such as no HPPD inhibition by I-05,
HPPD inhibition by II-05, algae bleaching, and postemergence
bleaching phenomena from I-05 treatments implies that I-05
could be transferred to II-05 in vivo (e.g., algae and plants)
slowly; therefore, I-05 and other active compounds (e.g., I-24)
possibly work as prodrugs. For IFT, the ring-opening
conversion of isoxazole is a rapid nonenzymatic hydrolysis,
happening in both plants and in soil. The rapid hydrolysis may
come from the strong effects of the electron-withdrawing
carbonyl group located between two rings, which could form a
much stable equilibrium among triketones and ketone enols.
For title compounds (e.g., I-05 and I-24), the electron-
withdrawing effects of the carbonyl in the carboxamide group
become weaker because of the stronger electronegative
nitrogen. Therefore, the isoxazole ring hydrolysis of title
compounds may become very slow or may happen by special
metabolic enzymes, which could explain why bleaching
symptoms could be observed in the algae assay and
postemergence pot assays, but not in the Petri dish assay.
Molecular Simulations. The interactions of II-05 and II-
24, the active entities of I-05 and I-24, with HPPD were
studied via a molecular docking method (Figure 7). As
illustrated in Figure 7, II-05 and II-24 formed bidentate
interactions with FeII, and the benzene ring could form π−π
interactions with Phe360 and Phe403. The CN group of II-05
formed a hydrogen bond with the amino group of Asn261,
which was suspected to result in an increase in herbicidal
activity. By comparing the distances of π−π interactions, we
found that the distance between the benzene ring of II-05 and
Phe360 was approximately equal to that of II-24, whereas that
between II-05 and Phe403 (3.7 Å) was smaller than that of II-
24 (4.3 Å). The increased distance might be the reason that
the herbicidal activity of I-05 was slightly stronger than that of
I-24. In the CDOCKER module of DS, the −CDOCKER
interaction energy is used to evaluate the docking results. A
Figure 6. Reversal of bleaching effects of I-05 on C. reinhardtii by
adding HGA. (A) I-05 (200 μM). (B) I-05 + HGA (200 + 50 μM).
(C) I-05 (100 μM). (D) I-05 + HGA (100 + 50 μM). (E) HGA (50
μM). (F) control.
the algae treated with 200 μM of I-05 solution were clearly
bleached (Figure 6A), and 100 μM of I-05 resulted in slight
bleaching effect on C. reinhardtii (Figure 6C). The algae
treated with I-05 + HGA (Figure 6B, 200 + 50 μM; Figure 6D,
100 + 50 μM) grew normally. The phenomena implied that
(1) the bleaching effect induced by I-05 could be reversed by
exogenous HGA; (2) I-05 showed activity through inhibition
of the formation of HGA; (3) I-05 might be transferred to its
active DKN in algae so that the typical bleaching effects could
be observed; and (4) a dose−response bleaching phenomenon
further suggested that no bleaching effects during the Petri dish
assay may come from low quantity of the title compound in
seedling leaves.
HPPD Enzymatic Assays. A difference may exist between
crop HPPD and weed HPPD (e.g., corn HPPD and Arabidopsis
thaliana HPPD or AtHPPD); this leads to selectivity of an
herbicide between crops and weeds. Our purpose was to see
whether HPPD could be one of the targets of the title
compounds (i.e., whether HPPD) and could be inhibited by
the title compounds as well as the positive control, a
commercial herbicide mesotrione (i.e., a side-by-side compar-
ison). Corn HPPD could be easily prepared in our lab, but
AtHPPD was not readily available in our lab; therefore, corn
HPPD was used for this purpose. As shown in Table 3, II-05,
II-24, and mesotrione showed inhibitory effects on HPPD
activity, and their IC50 values were 1.05, 1.28, and 1.34 μM,
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Article
Shao-Peng Wei − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, P. R. China; Shaanxi
Province Key Laboratory Research & Development on
Botanical Pesticides, Northwest A&F University, Yangling
712100, Shaanxi Province, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jafc.0c03582
Funding
This work was financially supported by the National Natural
Science Foundation of China (no. 31872011).
Figure 7. Simulated binding modes of II-05 and II-24 with AtHPPD.
The key residues in the active site are shown in blue sticks, and FeII is
shown as a cyan sphere. (A) Binding mode of II-05 with AtHPPD. II-
05 is shown in green sticks. (B) Binding mode of II-24 with AtHPPD.
II-24 is shown in orange-magenta sticks.
Notes
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jafc.0c03582.
■
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AUTHOR INFORMATION
Corresponding Author
■
Zhi-Qin Ji − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, P. R. China; Shaanxi
Province Key Laboratory Research & Development on
Botanical Pesticides, Northwest A&F University, Yangling
712100, Shaanxi Province, China; orcid.org/0000-0002-
1257-9238; Phone: +86-15309203829; Email: jizhiqin@
nwsuaf.edu.cn; Fax: +86-29-87092191
Authors
Xin-Lin Sun − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, P. R. China
Zhen-Meng Ji − College of Plant Protection, Northwest A&F
University, Yangling 712100, Shaanxi, P. R. China
G
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