Discovery of Novel Pyrazole-Quinazoline-2,4-dione Hybrids as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.0c00051

4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been identified as one of the most significant targets in herbicide discovery for resistant weed control. In a continuing effort to discover potent novel HPPD inhibitors, we adopted a ring-expansion strategy to design a series of novel pyrazole-quinazoline-2,4-dione hybrids based on the previously discovered pyrazole-isoindoline-1,3-dione scaffold. One compound, 3-(2-chlorophenyl)-6-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1,5-dimethylquinazoline-2,4(1H,3H)-dione (9bj), displayed excellent potency against AtHPPD, with an IC₅₀ value of 84 nM, which is approximately 16-fold more potent than pyrasulfotole (IC₅₀ = 1359 nM) and 2.7-fold more potent than mesotrione (IC₅₀ = 226 nM). Furthermore, the co-crystal structure of the AtHPPD-9bj complex (PDB ID 6LGT) was determined at a resolution of 1.75 ?. Similar to the existing HPPD inhibitors, compound 9bj formed a bidentate chelating interaction with the metal ion and a π-πstacking interaction with Phe381 and Phe424. In contrast, o-chlorophenyl at the N3 position of quinazoline-2,4-dione with a double conformation was surrounded by hydrophobic residues (Met335, Leu368, Leu427, Phe424, Phe392, and Phe381). Remarkably, the greenhouse assay indicated that most compounds displayed excellent herbicidal activity (complete inhibition) against at least one of the tested weeds at the application rate of 150 g of active ingredient (ai)/ha. Most promisingly, compounds 9aj and 9bi not only exhibited prominent weed control effects with a broad spectrum but also showed very good crop safety to cotton, peanuts, and corn at the dose of 150 g of ai/ha.

Full text of DOI:10.1021/acs.jafc.0c00051

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Agricultural and Environmental Chemistry
Discovery of Novel Pyrazole-Quinazoline-2,4-dione Hybrids
as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors
Bo He, Feng-Xu Wu, Liang-Kun Yu, Lei Wu, Qiong Chen, Ge-
Fei Hao, Wen-Chao Yang, Hong-Yan Lin, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00051 • Publication Date (Web): 14 Apr 2020
Downloaded from pubs.acs.org on April 15, 2020
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Discovery of Novel Pyrazole−Quinazoline-2,4-dione Hybrids as 4-Hydroxyphenylpyruvate
Dioxygenase Inhibitors
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Bo He,† Feng-Xu Wu,† Liang-Kun Yu,† Lei Wu,† Qiong Chen,† Ge-Fei Hao,† Wen-Chao Yang,†
Hong-Yan Lin,†, * Guang-Fu Yang†, #, *
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†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health, College of Chemistry,
Central China Normal University, Wuhan 430079, P.R. China
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#Collaborative Innovation Center of Chemical Science and Engineering, Nankai University,
Tianjin 300071, P.R. China
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AUTHOR INFORMATION
Corresponding Authors:
*Tel: +86-27-67867800. Fax: +86-27-67867141. E-mail: gfyang@mail.ccnu.edu.cn
*Hong-Yan Lin, E-mail: 913975503@qq.com
ORCID:
Hong-Yan Lin: 0000-0002-5627-3708
Ge-Fei Hao: 0000-0003-4090-8411
Guang-Fu Yang: 0000-0003-4384-2593
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ABSTRACT:
4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC 1.13.11.27) has been identified as one of
the most significant targets in herbicide discovery for resistant weed control. In a continuing
effort to discover potent novel HPPD inhibitors, we adopted a ring−expansion strategy to
design a series of novel pyrazole−quinazoline-2,4-dione hybrids based on the previously
discovered pyrazole−isoindoline-1,3-dione scaffold. One compound, 3-(2-chloro-
phenyl)-6-(5-hydroxy-1,3-dimethyl-1H-pyrazole-4-carbonyl)-1,5-dimethylquinazoline-2,4(1H,
3H)-dione (9bj), displayed excellent potency against AtHPPD with an IC50 value of 84 nM,
which is approximately 16-fold more potent than pyrasulfotole (IC50 = 1359 nM) and 2.7-fold
more potent than mesotrione (IC50 = 226 nM). Furthermore, the co-crystal structure of the
AtHPPD−9bj complex (PDB ID: 6LGT) was determined at a resolution of 1.75 Å. Similar to
the existing HPPD inhibitors, 9bj formed a bidentate chelating interaction with the metal ion
and a π-π stacking interaction with Phe381 and Phe424. In contrast, the o-chlorophenyl at the
N3 position of quinazoline-2,4-dione with a double conformation was surrounded by
hydrophobic residues (Met335, Leu368, Leu427, Phe424, Phe392, and Phe381). Remarkably,
the greenhouse assay indicated that most compounds displayed excellent herbicidal activity
(complete inhibition) against at least one of the tested weeds at the application rate of 150 g
ai/ha. Most promisingly, compounds 9aj and 9bi not only exhibited prominent weed control
effects with a broad spectrum, but also showed very good crop safety to cotton, peanuts and
corn at the dose of 150 g ai/ha.
KEYWORDS:
4-hydroxyphenylpyruvate
dioxygenase,
quinazoline-2,4-dione,
structure−activity relationship, herbicidal activity
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INTRODUCTION
It is well known that crops compete with weeds for water, nutrients, light, and space
during their whole growth process, so herbicides will continue to play an irreplaceable role in
modern agricultural production.1,
4-Hydroxyphenylpyruvate dioxygenase (HPPD, EC
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1.13.11.27) is a non-heam iron-dependent oxygenase that exists in most aerobic organisms.3, 4
In most aerobic forms of life, it catalyzes the second reaction in the catabolism of tyrosine
which is the conversion of p-hydroxyphenylpyruvic acid (HPPA) into homogentisic acid
(HGA).5, 6 In plants, HGA can be further transformed into two isoprenoids, plastoquinone and
tocopherol, which are both essential cofactors in photosynthesis.7-12 The inhibition of HPPD
results in the blockage of natural tyrosine physiological metabolism, which in turn results in the
obstruction of photosynthesis in plants and plant death with bleaching symptoms.13-17
The first-generation pyrazole-type HPPD inhibitors, such as pyrazolinate, pyrazoxyfen and
benzofenap (Figure 1), were mainly used to control weeds in the rice fields with application
rates up to 4 kg ai/ha.18, 19 After HPPD was identified as the action target of these herbicides,
structure-based discovery of novel HPPD inhibitors has become a hot area for pesticide
chemists worldwide and has produced second-generation pyrazole-type HPPD inhibitors with
significantly reduced application rates, including topramezone, pyrasulfotole, and
tolpyralate.20-23 However, these products are mainly used for the weed control of corn and
cereal fields, and new HPPD-inhibiting herbicides for other crops are of great urgency due to
the rapid development of weed resistance.24
Previously, we reported that the pyrazole-isoindoline-1,3-dione hybrid is a promising
scaffold for developing HPPD inhibitors.25 From the co-crystal structure of the
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inhibitor−AtHPPD complex (PDB ID: 6JX9, Figure 2), we could clearly observe that, apart
from the bidentate chelating interaction with the metal ion, the π−π stacking between the
isoindoline-1,3-dione moiety and the surrounding aromatic residues (Phe381 and Phe424)
contributed significantly to the binding. It has been reported that improving the π−π interaction
between inhibitors and their surrounding residues is an effective way to design new inhibitors
with improved potency.26, 27 Therefore, we designed a quinazoline-2,4-dione scaffold (Figure 2)
by inserting a nitrogen atom between the benzene ring and the carbonyl of
isoindoline-1,3-dione via a ring−expansion strategy. We expected that the six−membered
heterocyclic ring would improve the π−π interaction. Computational simulation results
indicated that the quinazoline-2,4-dione moiety was surrounded by hydrophobic residues
(Met335, Leu427, and Leu368). The binding free energy of the designed new molecule (9aa,
Figure 2) is -8.349 kcal/mol, which is significantly better than that of the corresponding
pyrazole−isoindoline-1,3-dione derivative (s1, ΔHS1 = -6.322 kcal/mol). Herein, we report the
design, syntheses, inhibitors effect against AtHPPD, structure−activity relationships, and
herbicidal activity. In addition, we resolved the co-crystal structure of AtHPPD in complex with
the representative title compound at a resolution of 1.75 Å.
MATERIALS AND METHODS
Chemicals and Instruments. During the experiment, all the solvents were either
chemically pure or analytically pure. The purity of commercially available reaction materials
was maintained above 95%. The reactions were monitored by thin-layer chromatography (TLC)
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silica gel glass plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, China). H
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and C NMR spectra were obtained on a nuclear magnetic resonance 600/400 spectrometer
(Varian Inc., Palo Alto, CA) in CDCl3 or DMSO-d6 with tetramethylsilane (TMS).
High-resolution mass spectrometry (HRMS) of the target compounds were performed on an
Agilent 6224 TOF LC/MS (Agilent Technologies, Santa Clara, CA). the melting points of the
compounds were measured on a melting point apparatus model (Büchi model B-545, Flawil,
Switzerland), and the temperature was uncorrected.
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Molecular Simulation. The crystal structure of AtHPPD (PDB ID: 1TFZ and 1TG5) was
downloaded from the Protein Data Bank. The purified HPPD was obtained by expression of the
recombinant Arabidopsis enzyme in E. coli.28 The sequences of 1TFZ and 1TG5 contained 21
truncated amino acid residues at the N-terminus in order to express the recombinant
Arabidopsis enzyme in E. coli. The co-crystal structures of 1TFZ and 1TG5 were all homo
dimeric form and their active pockets are highly conserved. The main difference between them
was the bound inhibitors. So, we believe whatever we use 1TFZ or 1TG5 will result in the same
docking results. Herein, 1TFZ was selected at random for docking study. During molecular
docking, the number of amino acid residues was increased by 21 to make it consistent with the
wild-type. First, we used SYBYL 7.3 (Tripos Inc., St. Louis, MO) to construct and optimize the
tested compounds and used AutoDock Tools (ADT) to prepare the protein and ligand structures.
Generally, the AtHPPD-DAS645 co-crystal structure (PDB ID: 1TG5) was used as a reference,
and the Fe2+ center was regarded as the crucial activity site.29-33 Applying the GOLD version
3.0 docking program, all docking runs were carried out using the default settings of the
program on a population size of 100 individuals with a selection pressure of 1.1. We chose the
best combination mode with high docking scores to analysis the binding mode of the inhibitor
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in the active pocket.
AtHPPD Enzymatic Assay and Crystallization of complex AtHPPD−9bj. We adopted
the same method as we reported previously to express and purify recombinant AtHPPD
(Supporting Information S2).34-36 Using a classical coupled enzyme assay, the half-maximal
inhibitory concentration (IC50) values were determined, and the values are shown in Table s2
(Supporting Information S10).3, 37, 38 We selected compound 9bj with excellent enzymatic
inhibitory activity as a representative to incubate with AtHPPD and successfully obtained the
co-crystal structure (Supporting Information S3).
Herbicidal Activity Test. The broadleaf weeds Amaranthus retroflexu (AR), Eclipta
prostrata (EP), Abutilon juncea (AJ), Amaranthus tricolor (AT), and Chenopodium album (CA)
and gramineous weeds Echinochloa crusgalli (EC), Setaria faberii (SF), and Digitaria
sanguinalis (DS) were chosen as the target weeds to evaluate the postemergence herbicide
activity of compounds 9aa−9bj in a greenhouse.36, 39 We choose pyrasulfotole and mesotrione
(Supporting Information S1) as the control, and use the same method that we reported early to
test the herbicidal activity (Supporting Information S6)36. After 25 days of treatment, the results
of herbicidal activity were evaluated visually with three repetitions per treatment, and the
results are shown in Table s2 and Table 1.
Crop Safety Test. Seven major crops, maize, soybeans, cotton, wheat, rice, canola, and
peanuts were chosen as representatives to evaluate the crop selectivity of the target compounds
under greenhouse conditions.2, 34 Using self−formulated mixed soil, the crops were planted in
flowerpots (12 cm in diameter) and grown in the greenhouse at 20−25°C. Postemergence crop
safety experiments were carried out at the dose of 150 g ai/ha for the tested compounds when
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the crops reached the three-leaf stage. Crop selectivity was evaluated 25 days after treatment
with title compounds 9aj, 9bi and 9bj in triplicate, and the results are shown in Table 2.
RESULTS AND DISCUSSION
Chemistry. As shown in Figure 3, the designed pyrazole−quinazoline-2,4-dione hybrids
9aa−9bh were synthesized in eight continuous reaction steps. First, the oxidation of
5-methyl-2-nitrobenzoic acid 1 was performed with aqueous KMnO4 under alkaline conditions,
followed by esterification of the carboxyl groups and reduction of the nitro groups to yield
dimethyl 4-aminoisophthalate 4.34, 35 Intermediate 4 was refluxed with (un)substituted (R2)
phenyl isocyanates in pyridine to obtain N3-aryl-quinazoline-2,4-diones 5aa−5ba. Under
alkaline conditions, compounds 5aa−5ba were treated with various alkyl iodides (R1-I) to
prepare quinazoline-2,4-dione derivatives 6aa−6bh. Then, the ester group was hydrolyzed
under acidic conditions to obtain the corresponding key intermediate acids 7aa−7bh.
Subsequently, using 2-chloro-1-methylpyridin-1-ium iodide (CMPI) as the condensing agent,
Et3N and 1,3-dimethyl-1H-pyrazol-5-ol in one pot resulted in intermediate enol esters 8aa−8bh.
After simple processing, the final title compounds 9aa−9bh were obtained by the Fries
Rearrangement.
In another synthesis scheme, we chose 2-methyl-6-nitrobenzoic acid as the starting
material to synthesize the designed compounds 9bi−9bj (Figure 4). First, we used the common
reaction conditions of esterification, nitro reduction and aromatic ring bromination to obtain the
intermediate (IV). Then, the same methods for synthesizing intermediates 5aa−5ba and
6aa−6bh were used to prepare the crucial intermediate (VI). In contrast, we used palladium
catalysis and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (xantphos) as the ligand under
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a CO atmosphere to synthesize title compounds (9bi−9bj) from the aryl bromides (VI) directly.
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The chemical structures of all the synthesized target compounds were confirmed by H NMR
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and C NMR spectroscopy and HRMS (Supporting Information S7−S9). Since the hydrogen
on the hydroxyl group is particularly active, it is very easy to exchange with D from the
solvents DMSO-d6 or CDCl3 and difficult to detect by NMR. Therefore, we do not detect the
hydrogen of the hydroxyl group. Furthermore, a transparent crystal of compound 9bi was
obtained directly from CDCl3 and confirmed by single-crystal X-ray diffraction (Figure 5). The
crystallographic data have been submitted to the Cambridge Crystallographic Data Centre
(CCDC ID: 1970427).
AtHPPD Inhibition and Structure−Activity Relationships (SAR). During the
investigation of the binding mode of 9aa (Figure 2), the N3 position was surrounded by
hydrophobic amino acid residues (Leu427 and Leu368), and a similar observation was found at
the N1 position (Met335). The results indicated that a hydrophobic substituent at the N1
position or N3 position would be beneficial for inhibitory activity in vitro. To verify the
molecular simulation results, we first introduced a hydrophobic aromatic ring at the N3 position
and a methyl at the N1 position to synthesize compound 9aa. The half-maximal inhibitory
concentration (IC50) of compound 9aa against AtHPPD reached 0.514 μM, which was two
times better than that of the control agent pyrasulfotole (IC50 = 1.359 μM) but worse than that
of mesotrione (IC50 = 0.226 μM). Furthermore, we added the hydrophobic methyl group to the
N3 benzene ring to synthesize compound 9ab (R1 = CH3, R2 = 2-CH3). The generated
compound 9ab (IC50 = 0.428 μM) showed better inhibitory activity against AtHPPD than parent
9aa. The in vitro activity was consistent with the experimental results of molecular docking.
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Hence, we tried to optimize the substituents around the N1 position (R1) and N3 phenyl ring
(R2) of quinazoline-2,4-dione.
Based on the molecular skeleton of compound 9ab, compounds 9ac−9ai were obtained by
introducing a hydrophobic alkyl chain into the N1 position (R1), and their AtHPPD inhibition
parameters are listed in Table s2 For compounds with ethyl (9ac) or n-propyl (9ad), the
AtHPPD-inhibiting activities were further improved in comparison with compound 9ab.
Remarkably, attaching an unsaturated alkyl chain to the N1 position in the corresponding
compounds 9ag (R1 = allyl) and 9ah (R1 = propargyl) also showed better inhibitory potency
compared with compound 9ab. However, when we introduced the n-butyl (9ae), iso-butyl (9af)
or benzyl (9ai) groups to the N1 position, the inhibitory activity of these compounds gradually
decreased as the spatial distribution of the substituents increased.
Simultaneously, keeping the N1 position as a methyl group, a single substituent was
introduced to the N3 phenyl ring as R2. The in-vitro activity results of compounds (9aj−9at) are
shown in Table s2. Generally, a substituent at the para position of the N3 phenyl ring could
improve the inhibitory effect better than a substituent in the ortho position; i.e., 4-CH2CH3 (9ao,
IC50 = 0.177 μM) > 2-CH2CH3 (9aj, IC50 = 0.401 μM) and 4-OCF3 (9as, IC50 = 0.235 μM) >
2-OCF3 (9am, IC50 = 0.284 μM). In particular, at the para position, the inhibitory activity was
enhanced with increasing steric hindrance of the substituent. The order of the inhibition activity
was 4-OPh (9at, IC50 = 0.130 μM) > 4-CH2CH3 (9ao, IC50 = 0.177 μM) > 4-CH3 (9an, IC50 =
0.361 μM). In addition, regardless of whether the substituent was in the ortho or para position
of the N3 phenyl ring, the inhibitory activity against AtHPPD increased with enhancement of
the electron−withdrawing ability of the substituent; i.e., 2-OCF3 > 2-OCH3 (9al) > 2-CH3 (9ab),
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and 4-CF3 (9aq) > 4-OCF3 (9as) > 4-OCH3 (9ar) > 4-CH3 (9an). A special case was the
compound containing a strong electron−withdrawing group, 4-NO2 (9ap, IC50 = 0.139 μM),
which displayed lower inhibitory activity than 9aq (4-CF3, IC50 = 0.075 μM).
In recent years, other specific nonbonded contacts have been reported, e.g., halogen bonds,
CH-π, and cation-π interactions.40-43 The halogen bond (XB), an emerging noncovalent
intermolecular interaction analogous to the hydrogen bond (HB), has made significant
contributions to the molecular recognition in protein-ligand interactions.44 Thus, keeping N1
position as a methyl group, we synthesized monohalogenated derivatives on the N3 benzene
ring (R2: F, Cl, Br) and investigated their bioactivity in vitro (9au−9bc). At the ortho or meta
position of the N3 phenyl ring, introducing a chloro substituent was helpful to increase the
inhibitory activity compared to fluoro or bromo substituents. However, the enzyme inhibition
activity was discrepant for the same substituent at the different positions of the N3 phenyl ring.
The trend of those compounds was 2-F > 3-F > 4-F, 3-Cl > 2-Cl > 4-Cl and 4-Br > 2-Br > 3-Br.
Therefore, there may be a certain interaction among fluorine, chlorine or bromine and the
enzyme active pocket that has not yet been demonstrated.
In addition, we evaluated the inhibitory activities of these compounds with multiple
substitutions (di, tri) on the N3 phenyl ring (9bd−9bh, Table s2). The compound with the
diethyl substitution (9be) was better than that the compound with the dimethyl substitution
(9bd). Moreover, an electron−withdrawing group at the para position of the N3 phenyl ring
also resulted in increasing inhibitory activity (R2: 2-Cl-4-NO2 > 2,4-di-Cl). When we added a
tri-methyl group (9bf) to the phenyl ring, the inhibitory activity was better than that of the
dimethyl derivative (9bd), but it was similar to that of the monomethyl derivative (9ab)
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(2,4,6-tri-CH3 ≈ 2-CH3 > 2,6-di-CH3). In addition, keeping the R2 group as a single methyl or
chlorine on the ortho position of the N3 phenyl ring, we synthesized compounds 9bi−9bj with a
methyl at the 5 position of quinazoline-2,4-dione. The in vitro test results confirmed that
compounds 9bi and 9bj were better than compounds 9ab and 9ax, respectively. In particular,
compound 9bj showed high AtHPPD inhibitory activity with an IC50 value of 84 nM, which
was approximately 2.7 times higher than that of mesotrione (IC50 = 226 nM). Furthermore, we
carried out molecular docking. When compounds 9ab and 9bi maintain a similar binding
conformation (Figure 6A and 6B ), we observed that the binding model of 9bi was in a
suboptimal conformation according to its docking score ranking and binding free energy, which
was only -3.113 kcal/mol. However, after optimizing the conformation (Figure 6C), the very
significant change was that the orientation of the N3 benzene ring was deflected. Moreover, the
binding free energy of 9bi was reduced to -9.859 kcal/mol, which was better than 9ab (ΔH =
-5.861 kcal/mol). Thus, all the results suggested that a methyl group at the 5-position of the
quinazoline-2, 4-dione fragment would be essential for improving biological activity.
Crystal Structure of AtHPPD−9bj Complex (PDB ID: 6LGT). Using the method we
reported earlier, we successfully obtained the co-crystal structure of AtHPPD−9bj.15, 25, 45 The
structure of the AtHPPD−9bj complex was solved by molecular replacement, and the resolution
of the complex was refined to 1.75 Å (Supporting Information S4 and S5). As shown in Figure
7A, the interaction between the inhibitor and protein in the co-crystal structure of AtHPPD−9bj
was similar to that of the reported commercial HPPD inhibitor, which contained a bidentate
chelating interaction with the metal ion, and a π-π stacking interaction with Phe381 and Phe424.
In contrast, from the omit map of 9bj (Figure 7B), due to the free rotation of the C−N bond, the
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o-chlorophenyl at the N3 position showed double conformations. In addition, two water
molecules are observed near the inhibitor in Figure 7C. The distance from water (a) to water (b)
is 2.8 Å, and water (a) made a potential hydrogen bond with the nitrogen of Asn282. Water (b)
is 3.4 Å away from the carbonyl group of the inhibitor and made other potential hydrogen
bonds with the nitrogen atoms of Gln293 and Gln307, which are fully conserved in all known
HPPD proteins. To gain insight into the differences in binding modes between 9bj and the
commercial herbicide in the active pocket, we superimposed this complex structure onto the
complex structure of AtHPPD-mesotrione (Figure 7D). In general, in addition to common
interactions, the nitro group of mesotrione and the methyl group on the 5-position of
quinazoline-2,4-dione maintain almost the same orientation. The biggest difference was that the
terminal o-chlorophenyl fragment was near the entrance of the active pocket and was
surrounded by hydrophobic amino acids (such as Met335, Leu368, Leu427, Phe424, Phe392,
and Phe381; Supporting Information S4). Thus, the introduction of hydrophobic substituents at
the N3 position was beneficial for improving the biological activity of the inhibitor.
Herbicidal Activity. We tested the postemergence herbicidal activities of all synthetic
compounds at a dose of 150 g ai/ha. The weeds produced the same unique bleaching symptoms
after dealing with our synthetic compounds and controls. The herbicidal activity results are
shown in Table s2. For the N1 position, methyl−substituted compounds 9aa and 9ab showed
more than 50% of the control effect for all the tested weeds. The other compounds with
unsaturated alkyl chains also displayed more than 50% inhibition for most of the tested weeds.
However, when the alkyl chain had more than three carbon atoms, as in compounds 9ae, 9af
and 9ai, there was obvious herbicidal activity under the same test conditions. Thus, at the N1
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position, the methyl group would be the best for improving the herbicide activity.
On the N3 phenyl ring, some compounds (9aj−9at) with mono substitutions displayed a
complete control effect for one of the tested weeds (Abutilon juncea or Amaranthus retroflexus)
and showed a better control effect on Setaria faberii (SF) than the control. In particular, the
compound 9aj (2-CH2CH3) can completely control the three types of broadleaf weeds (AJ, AR
and EP), maintaining a control effect of more than 70% for the tested grass weeds. In addition,
the compounds with mono substitutions at the ortho position of the N3 phenyl ring displayed
better weed control effects than substitutions at the para position. For example, 2-CH3 > 4-CH3,
2-OCH3 > 4-OCH3 and 2-OCF3 > 4-OCF3. In addition, compounds that contained strong
electron-withdrawing groups or phenoxy groups at the para position of the N3 phenyl ring had
better inhibitory activity against AtHPPD, but they exhibited poor herbicidal activities.
Moreover, for compounds with monohalogenated substitution, a similar trend was observed,
and the herbicidal activity of the ortho-substituted compounds led to an obvious increase in
potency when compared to the para- or meta-substituted derivatives. For example, compounds
9au (2-F), 9ax (2-Cl) and 9ba (2-Br), had more than 50% control effects on the tested weeds.
The control potential of these compounds displayed a better control effect on Setaria faberii
(SF) compared with the control. Furthermore, compounds with multiple substitutions on the N3
phenyl ring (9bd−9bh) did not improve herbicidal activity. More importantly, compounds 9bi
and 9bj with a methyl at the 5-position of quinazoline-2,4-dione displayed excellent control
efficiency for all the tested weeds and was significantly improved compared to their
corresponding parent compounds 9ab and 9ax, respectively. Thus, substituting the 5-position of
the quinazoline-2, 4-dione fragment would be essential for improving herbicide activity.
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Due to the excellent level of control weed activity, compounds 9aj, 9bi, and 9bj were
chosen for further testing at lower doses (Table 1). When the dosage was lowered from 120 g
ai/ha to 30 g ai/ha, the herbicidal activities of compounds 9aj and 9bj also decreased. However,
the control weed potency of compound 9bi was maintained at more than 70%, except for
Abutilon juncea (AJ). In our work, the in vitro activity of some compounds was better than that
of the control, but they did not show obvious control effects on the tested weeds. As we know,
in plants, the process of absorption, conduction, and metabolism is very important for
herbicides to exert their effects.46, 47 These factors were very likely the cause of the differences
in activity observed in vitro and in vivo.
Crop Selectivity. The crop safety of compounds 9aj, 9bi and 9bj was evaluated at the
dose of 150 g ai/ha (Table 2). The results indicated that compound 9aj showed no injury to
cotton or peanuts. However, obvious injury symptoms appeared in cotton after using
mesotrione in the greenhouse. Additionally, compound 9bi displayed good tolerance to corn.
Compound 9bj had an excellent herbicidal effect, but the phytotoxicity was also obvious to the
seven crops under the tested conditions. Thus, these three compounds have potential application
prospects in the future.
In summary, using a ring−expansion strategy, a series of novel quinazoline−2,4-dione
derivatives were designed and synthesized. The two synthetic routes were chosen to prepare
title compounds (9aa−9bj). Of the two routes, one was catalysed by palladium under a CO
atmosphere to prepare the designed compounds 9bi−9bj. From the SAR analysis, at the N1
position (R1), a methyl would be the best group for improving herbicide activity. For the N3
position, on one hand, an electron-withdrawing group on the N3 phenyl ring was beneficial for
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improving enzyme inhibition activity, but it was not good for enhancing herbicidal activity. On
the other hand, the pata-substituted derivatives were better for in vitro inhibition activity than
the ortho-substituted derivatives. However, the ortho-substituted derivatives displayed better
herbicidal activity. In addition, the co-crystal structure of AtHPPD−9bj was obtained at a
resolution of 1.75 Å and confirmed the mechanism of action at the atomic level. Furthermore,
under greenhouse conditions, many compounds displayed excellent postemergence herbicidal
activities against one of the tested weeds at the dose of 150 g ai/ha. In particular, compound 9aj
was selective for cotton and peanuts, which was promising for development as a selective
postemergence HPPD-target herbicide. Compound 9bi provided an excellent weed control
effect and showed better safety to corn at the dose of 150 g ai/ha. In addition, compound 9bj
has superior herbicidal activity, but its crop safety needs further improvement. Thus,
pyrazole−quinazoline-2,4-dione derivatives will be very promising for the development of
novel HPPD inhibitors.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website; S1.
The structure of commercial HPPD herbicides; S2. Protein expression and purification; S3.
Crystallization and structure determination of complex AtHPPD−9bj (PDB ID: 6LGT); S4. The
overall co-crystal structure of AtHPPD-9bj (PDB ID: 6LGT); S5. Data collection and
refinement statistics of the crystal structure of AtHPPD−9bj (PDB ID: 6LGT); S6. The testing
method of herbicide activity; S7. Synthetic method and physical data of compounds 9aa−9bh;
S8. Synthetic method and physical data of compounds 9bi−9bj; S9. Spectra of representative
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compounds 9aa−9bj; and S10. Chemical Structures and Bioactivity of Compounds 9aa−9bj.
Acknowledgments
This work was supported by the National Key Research and Development Program of China
(2018YFD0200100) and the National Natural Science Foundation of China (Nos. 21837001,
21672079 and 21772058).
Notes
The authors declare no competing financial interest.
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Figure captions:
Figure 1. Chemical structures of some commercial pyrazole HPPD herbicides.
Figure 2. The design strategy of compounds 9aa−9bj. The binding free energy of s1, △Hs1 =
-6.322 kcal/mol. The binding free energy of 9ab, △H9aa = -8.349 kcal/mol.
Figure 3. Synthetic route of the title compounds 9aa−9bh. Reagents and conditions: (a)
KMnO4, KOH, 80°C; (b) CH3OH, H2SO4, reflux; (c) H2, 10% Pd/C, rt; (d) (un)substituted
penylisocyanates, pyridine, 100°C; (e) alkyl iodide , Cs2CO3, DMF, rt; (f) HOAc, H2SO4, H2O,
100°C;
(g)
2-chloro-1-methylpyridin-1-ium
iodide
(CMPI),
Et3N,
DCM,
1,3-dimethyl-1H-pyrazol-5-ol, rt; (h) acetone cyanohydrin, Et3N, acetonitrile, 25°C.
Figure 4. Synthetic route of the title compounds 9bi−9bj. Reagents and conditions: (a) CH3OH,
H2SO4, reflux; (b) H2, 10% Pd/C, ethyl acetate, rt; (c) NBS, 1,2-dichloroethane, 0°C; (d)
substituted penylisocyanates, pyridine, 100°C; (e) iodomethane, Cs2CO3, DMF, rt; (f) Xantphos,
PdCl2, Et3N, K2CO3, CO, DMF, 100°C.
Figure 5. X-ray crystal structure of compound 9bi.
Figure 6. The molecular docking model of compounds 9ab and 9bi with AtHPPD (PDB No.
1TFZ), where green represents the inhibitor molecule and blue represents the amino acid
residue at the active pocket. (A) Binding free energy of 9ab, ΔH = -5.861 kcal/mol; (B)
Binding free energy of 9bi, non-optimal combined conformation, ΔH = -3.123 kcal/mol; and (C)
Binding free energy of 9bi, optimal combined conformation, ΔH = -9.857 kcal/mol.
Figure 7. Co-crystal structure of AtHPPD−9bj (green) and the structural comparison with
AtHPPD-mesotrione (purple). Inhibitors and the key surrounding residues of the active pocket
are highlighted as sticks. The chelation interactions and hydrogen bonds are indicated with
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531
532
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535
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540
541
542
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547
black dashed lines. (A) The binding mode of compound 9bj with AtHPPD and its interaction
with adjacent amino acid residues. (B) 2Fo-Fc map (contoured at 1.0 σ) of compound 9bj. (C)
Water molecules mediating the hydrogen bonding network in the AtHPPD−9bj active pocket.
(D) The superposition of the binding modes of 9bj and mesotrione in AtHPPD.
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First generation
H3C
H3C
O
O
O
S
O
O
Cl
O
Cl
Cl
O
O
O
O
CH3
Cl
H3C
N
N
H3C
H3C
N
N
Cl
N
N
CH3
Cl
CH3
CH3
Pyrazolynate
Second generation
Pyrazoxyfen
Benzofenap
H3C
O
O
O
O
CH3
N
O
HO
CH3
O
O O
CH3
S
CH3
O
HO
O
CH3
H3C
O
H3C
O
N
N
CH3
O
H3C
N
N
N
CH3
S
N
H3C
S
CF3
O
O O
Topramezone
Pyrasulfotole
Tolpyralate (SL573)
548
549
Figure 1
550
551
552
553
554
555
556
557
558
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O
O
N
HO
O
HO
O
5
4
6
N
3
H3C
N
N
H3C
N
N
N
7
O
2
CH3
1
CH3
8
O
9aa
s1
Ring-expansion
Strategy
Complex AtHPPD-s1 (PDB ID: 6JX9)
559
560
Figure 2
561
562
563
564
565
566
567
568
569
570
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O
O
O
O
O
O
O
a
b
c
H3C
H3C
CH3
H3C
CH3
OH
NO2
HO
OH
NO2
O
O
O
O
NO2
NH2
1
2
3
4
O
O
O
O
R2
R2
O
O
R2
H3C
d
e
g
f
H3C
O
N
HO
N
O
N
N
R1
O
N
R1
O
N
H
O
5aa 5ba
6aa 6bh
7aa 7bh



H3C
N
O
O
HO
R2
O
O
N
R2
h
N
H3C
N
N
N
O
H3C
N
R1
O
CH3
N
R1
O
8aa 8bh
9aa-9bh

571
572
Figure 3
573
574
575
576
577
578
579
580
581
582
583
584
585
586
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Page 29 of 35
Journal of Agricultural and Food Chemistry
CH3
O
CH3
O
CH3
O
CH3 O
a
b
c
CH3
CH3
Br
CH3
d
OH
O
O
O
NO2
CH3
NO2
NH2
NH2
III
II
IV
I
CH3
O
N
O
CH3 O
O
HO
e
Br
f
Br
N
R2
R2
H3C
R2
N
N
N
N
O
N
O
N
H
O
CH3
CH3
CH3
Vaa R2 = 2-CH3
Vab R2 = 2-Cl
VIaa R2 = 2-CH3
VIab R2 = 2-Cl
9bi R2 = 2-CH3
9bj R2 = 2-Cl
587
588
Figure 4
589
590
591
592
593
594
595
596
597
598
29
ACS Paragon Plus Environment