Synthesis and Herbicidal Activity of Pyrido[2,3-d]pyrimidine-2,4-dione-Benzoxazinone Hybrids as Protoporphyrinogen Oxidase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.7b01990

To search for new protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors with improved bioactivity, a series of novel pyrido[2,3-d]pyrimidine-2,4-dione-benzoxazinone hybrids, 9-13, were designed and synthesized. Several compounds with improved tobacco PP

Full text of DOI:10.1021/acs.jafc.7b01990

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Article
Synthesis and herbicidal activity of pyrido[2,3-d]pyrimidine-2,4-dione
-benzoxazinone hybrids as novel protoporphyrinogen oxidase inhibitors
Da-Wei Wang, Qian Li, Kai Wen, Ismail Ismail, Dan-dan
Liu, Cong-wei Niu, Xin Wen, Guang-Fu Yang, and Zhen Xi
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 15 Jun 2017
Downloaded from http://pubs.acs.org on June 15, 2017
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Synthesis and Herbicidal Activity of Pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone
Hybrids as Protoporphyrinogen Oxidase Inhibitors
DaꢀWei Wang,† Qian Li, † Kai Wen, † Ismail Ismail,† DanꢀDan Liu, † CongꢀWei Niu, † Xin
Wen,† GuangꢀFu Yang,*‡ and Zhen Xi*†
†State Key Laboratory of ElementoꢀOrganic Chemistry, and Department of Chemical Biology,
College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering,
Nankai University, Tianjin 300071, P. R. China.
‡Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan 430079, P. R. China
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*corresponding author:
Eꢀmail: gfyang@mail.ccnu.edu.cn (G.ꢀF. Yang),
Tel: +86ꢀ27ꢀ67867800, Fax: +86ꢀ27ꢀ67867141.
Eꢀmail: zhenxi@nankai.edu.cn (Z. Xi)
Tel: +86 022ꢀ23504782, Fax: +86 022ꢀ23504782.
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ABSTRACT: To search for new protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitors with
improved bioactivity, a series of novel pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone
hybrids, 9ꢀ13, were designed and synthesized. Several compounds with improved tobacco PPO
(mtPPO)ꢀinhibiting and promising herbicidal activities were found. Among them, the most
potent compound
3ꢀ(7ꢀfluoroꢀ3ꢀoxoꢀ4ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]
oxazinꢀ6ꢀyl)ꢀ1ꢀmethylyrido[2,3ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione,11q, with a Ki value of 0.0074
ꢀM, showed six times more activity than flumioxazin (Ki = 0.046 ꢀM) against mtPPO.
Compound 11q displayed a strong and broad spectrum of weed control at 37.5ꢀ150 g of active
ingredient (ai)/ha by both postꢀ and preꢀemergence application, which were comparable to that
of flumioxazin. 11q was safe to maize, soybean, peanut and cotton at 150 g ai/ha, and selective
to rice and wheat at 75 g ai/ha by preꢀemergence application, indicating potential applicability
in these fields.
KEYWORDS: pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione, benzoxazinone, protoporphyrinogen
oxidase, herbicide, structure−activity relationship
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Introduction
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During the growing season for agricultural crops, weeds compete with crops for water,
nutrients, light and space. If weeds are left uncontrolled, the crop yield losses can be as
substantial as 50%.1, 2 Since the middle of the 20th century, synthetic organic herbicides have
played a significant role in the control of weeds. However, the overuse of some herbicides has
resulted in significant development of weed resistance and huge negative environmental
effects.3 Therefore, new compounds, covering a broad spectrum of weed control and
environmentally friendly, are globally demanded. The protoporphyrinogen oxidase (PPO, EC
1.3.3.4) inhibition compounds can provide these solutions. PPO is the penultimate enzyme in
the biosynthesis of chlorophyll and heme, belongs to a large family of flavin adenine
dinucleotide (FAD)ꢀcontaining enzymes, and catalyzes the conversion of protoporphyrinogen
IX to protoporphyrin IX.4ꢀ7 Inhibition of PPO in plants can lead to toxic accumulation of
protoporphyrin IX in cytoplasm. When exposed to light, protoporphyrin IX will react with
oxygen and generate many reactive oxygen species, destroy the cell membranes and cause the
rapid burn symptoms in plants (necrosis within one day). PPOꢀinhibiting herbicides have many
attractive properties, such as broadꢀspectrum weed control (including resistant biotypes),
benign environmental characteristics, low toxicity and low application rate.8ꢀ10
The first PPO herbicide nitrofen was introduced to the market by Dow AgroSciences in
1963. Since then, many diverse chemistries have been discovered, from which about 30
compounds were finally commercialized, which can be further divided into the following types:
phenylpyrazoles, triazolinones, Nꢀphenylꢀphthalimides, oxazolidinediones, diphenyl ethers,
oxadiazoles, pyrimidinediones and thiadiazoles. Unlike acetohydroxyacid synthase (AHAS)ꢀ
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and acetylꢀcoenzyme A carboxylase (ACCase)ꢀinhibiting herbicides, although PPO herbicides
have been used for more than six decades, there has still been no significant development of
resistant weeds.11 One possible reason is that, the structural bases of these herbicides mimic two
or three tetrapyrrole rings of the substrate protoporphyrinogen IX.12, 13 Interestingly, due to
some weed biotypes developing resistance to glyphosate and other herbicides, the market
shares of PPO herbicides began to increase recently.14 Flumioxazin can effectively suppress
some glyphosateꢀ, AHASꢀ and triazineꢀresistant weed populations such as Amaranthus
rudis Sauer and Amaranthus palmeri.15ꢀ17 Saflufenacil has been the most successful herbicide
discovered in the past 20 years. By preꢀemergence application, saflufenacil can be used for
broadleaf weed control in numerous crops, such as cereals, corn, and soybean. It can effectively
control more than 90 kinds of dicotyledonous weeds as well as these triazinesꢀ, AHASꢀ and
glyphosateꢀresistant Amaranthus biotypes.18
Generally, the structures of PPO inhibitors consist of three parts: a heterocyclic moiety, a
benzene ring and a hydrophobic chain (Figure 1). The heterocyclic moiety attaches to the
benzene ring via a CꢀN or CꢀC bond, the hydrophobic chain is placed at the 4ꢀ or 5ꢀposition of
the benzene ring. In most cases, when the 4,5ꢀ or 5,6 ꢀposition of the benzene ring is fused with
a fiveꢀ or sixꢀmembered ring, compounds also show very good herbicidal activities. Examples
of this are the N‑benzothiazolyl compounds10, benzoxazinones,19 and benzoxazoles20 Among
these benzeneꢀfused ring heterocyclic inhibitors, three compounds, flumioxazin, thidiazimin
and trifludimoxazin, with benzoxazinones were commercialized (Figure 1).21 Furthermore,
researches have proved that modification of the heterocyclic moieties of the PPO inhibitors is
an effective strategy for discovering new compounds with improved bioactivities.22ꢀ24
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Quinazolineꢀ2,4ꢀdione is an important heterocyclic scaffold, exiting in many natural products
with various biological activities.25 Previously, we have found that triketoneꢀcontaining
quinazolineꢀ2,4ꢀdiones displayed excellent herbicidal activity, and the quinazolineꢀ2,4ꢀdione
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ring can form
πꢀπ stacking interactions with the Phe360 and Phe403 of Arabidopsis thaliana
4ꢀhydroxyphenylpyruvate dioxygenase (AtHPPD).26ꢀ28 It was reported that changing the CH
group in the benzene ring to N atom can significantly increase the
the target molecule.29 Based on our molecular simulation studies, the heterocyclic moieties of
PPO inhibitors can form
stacking interactions with Phe392 of tobacco PPO (mtPPO).2, 5
πꢀπ stacking possibility of
πꢀ
π
,
Therefore, we envisioned that pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione might be a promising
heterocyclic moiety to integrate with benzoxazinone. For this purpose a series of
pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone hybrids were designed. Herein, we report
the synthesis, herbicidal activity, mtPPO inhibition activity, and structure−activity relationships
(SAR) of these compounds.
MATERIALS AND METHODS
Chemicals and instruments. All chemical reagents, such as ethyl bromoacetate,
5ꢀhalogenꢀ2ꢀnitrophenols, caesium carbonate, and 4ꢀchloromethylanisole were purchased from
Innochem, Science & Technology Co., Ltd. (Beijing, China). Organic solvents, such as
dimethylformamide (DMF), petroleum ether (b.p. 60ꢀ90 °C), acetic acid, toluene, and methanol
were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). The solvents
were dried and redistilled according to the standard methods before use. 1H NMR and 13C NMR
spectra were recorded on a VARIAN MercuryꢀPlus 400 spectrometer (Bruker Corp.,
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Switzerland) in CDCl3 or DMSOꢀd6 with trimethyl chlorosilane as the internal reference. High
resolution mass spectra (HRMS) were obtained on a 6520 QꢀTOF LC/MS (Agilent, Santa Clara,
CA). Melting points of the synthesized compounds were taken on a WRSꢀ1B melting point
apparatus (Shenguang Instrument Co., Ltd. Shanghai, China) and are uncorrected.
The detailed synthetic routes, physical and spectrum data (NMR, HRMS) of compounds 3ꢀ13
are shown in the Supporting Information.
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mtPPO Inhibitory Experiments. The expression and purification of mtPPO were used the
same methods as previously reported 2,8,10,30
The enzyme product protoporphyrin IX had a
.
maximum excitation and a maximum emission wavelengths at 410 nm and 630 nm,
respectively, and the formation of the product can be monitored by using a fluorescence
detector with the maximum excitation at 410 nm and maximum emission wavelength at 630 nm.
The mtPPO reaction rate was assayed by monitoring the speed of formation of the substrate to
the product on a 96ꢀwell plate using the fluorescence detector. 8
Sigma Plot software 10.0 (SPSS, Chicago, IL) was used to calculate the kinetic parameters.
50% of the total inhibition (IC50) value of inhibitors was determined by measuring mtPPO
activity over a range of inhibitor concentrations at a fixed substrate concentration. IC50 values
were calculated by fitting v versus [I] data to a single binding site model (eq 1).
maxꢀmin
y = min +
(1)
logIC ꢀ x
50
1+10
where y is the percentage of the maximal rate, max and min being the y values at which the
curve levels off, x is the logarithm of inhibitor concentration. Calculated inhibition constant of
the enzyme reaction (Ki ) value can be determined by applying the following relationship
among Ki, Km, and IC50 at any saturating substrate concentration (eq 2).
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IC50
Ki=
(2)
⁄
S Km + 1
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Molecular Simulation and CoMFA study. The crystal structure of mtPPO was
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downloaded from the RSCB protein data bank (PDB ID: 1SEZ). The 3D structure of
compounds 9ꢀ13 were constructed and optimized using SYBYL 6.9 (Tripos, Inc.,St. Louis,
MO).31 The Gasteiger–Huckel method was used to calculate the charges of all compounds, and
the compounds were then optimized with the power method for 5000 steps until a delta energy
change lower than 0.005 kcal/mol was reached. The optimized structures were used for docking
and subsequent 3DꢀQSAR studies. The protein and ligand were prepared using the default
settings of AutoDockTools4.32 AutoDock4.2 was used to calculate the interaction of the two
molecules, and a total of 258 runs were used for each molecule. The best binding modes were
selected by the docking score as well as by comparison with the coꢀcrystal ligand in the mtPPO.
The SYBYL default parameters were used to calculate the CoMFA steric, descriptors and
electrostatic field energies. The parameters used were as follows: an sp3 carbon probe atom
with +1 charge, 2.0 Å grid points spacing, an energy cutoff of 30.0 kcal/mol and a minimum σ
(column filting) of 2.0 kcal/mol.10
Herbicidal activity. To evaluate the postꢀemergence activity of compounds 9ꢀ13, six
representative broadleaf weeds, Abutilon juncea (AJ), Amaranthus retroflexus (AR), Brassica
juncea (BJ), Chenopodium serotinum (CS), Eclipta prostrata (EP), and Rumex acetosella (RA);
six representative monocotyledon weeds, Digitaria sanguinalis (DS), Polypogon fugax (PF),
Echinochloa crusgalli (EC), Poa annua (PA), Setaria faberii (SF), and Alopecurus aequalis
(AA) were tested according to the method as in previously published papers.26,33,34 To evaluate
preꢀemergence herbicidal activity of compound 11q in this present study, we selected four
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broadleaf weeds, Amaranthus retroflexus (AR), Medicago sativa (MS), Eclipta prostrata (EP),
Ipomoea nil (IN), and two monocotyledon weeds, Chloris virgata (CV), Setaria italic (SI). One
of the most widely used PPO herbicide, flumioxazin was used as a control. Before testing,
compounds were dissolved in DMF and formulated with Tweenꢀ80 as an emulsification reagent
to a concentration of 100 g/L. Clay soil, pH 6.5, CEC 12.1 mol/kg, 37.3% clay particles, and
1.6% organic matter were used in the experiment. The active ingredient (g ai/ha) was calculated
by the total amount of active ingredients in the formulation divided by the surface area of the
pot. In the experiment, plastic pots with a diameter of 9 centimeters were filled with clay soil to
a depth of 8 centimeters. About 20 of the tested weed seeds were sown in the pots at the depth
of 1 to 3 centimeters and grown at 15ꢀ30 °C in the greenhouse. During the experiment, the air
humidity was kept at about 50%. The diluted formulation solutions were applied for both preꢀ
and postꢀemergence application. For postꢀemergence herbicidal activity evaluation,
monocotyledon weeds were tested at the oneꢀleaf stage, and broadleaf weeds were tested at the
twoꢀleaf stage. Untreated seedlings used the solvent (DMF+Tweenꢀ80) as the solvent control
group. For preꢀemergence herbicidal activity evaluation, after sowing the seeds in the plastic
pots, they were directly treated with tested compounds at the rate of 37.5, 75 and 150 g ai/ha,
with DMF+Tweenꢀ80ꢀtreated groups as solvent control. At 30 d postꢀtreatment, the results of
herbicidal activity were evaluated visually (Tables 1 and 3), with three replications per
treatment.
Crop safety. To evaluate crop selectivity of the synthesized compounds, six representative
crops were tested in the greenhouse experiments: soybean, peanut, cotton, maize, wheat, and
rice. The crops were planted in flowerpots (12 cm diameter) and grown at room temperature in
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the test soil. Postꢀemergence crop safety experiments were conducted at the rate of 75 and 150
g ai/ha when the crops had reached the fourꢀleaf stage. Preꢀemergence crop experiments were
also conducted at 75 and 150 g ai/ha after the crops seeds were sown in the flowerpots. The
crop selectivity of tested compounds were evaluated after 30 days of treatment by inhibitors,
the data are shown in Table 4, with three replications per experiment.
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RESULTS AND DISCUSSION
Synthesis. As shown in Figure 3, all the pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone
hybrids, 9ꢀ13, can be obtained in linear multistep reactions. The key intermediates
6ꢀaminoꢀ7ꢀhalogenꢀ4ꢀ(4ꢀmethoxybenzyl)ꢀ2Hꢀbenzo[b][1,4]oxazinꢀ3(4H)ꢀone, 7, (halogen = F,
Cl, or Br) was smoothly synthesized in five steps by using 5ꢀhalogenꢀ2ꢀnitrophenol 1 as the
starting materials. After reacting with ethyl bromoacetate in DMF as solvent and K2CO3 as base,
ethyl 2ꢀ(5ꢀhalogenꢀ2ꢀnitrophenoxy)acetate 3 were obtained in excellent yields (92ꢀ95%).
Compounds 4 were synthesized in 81ꢀ86% yields, by using 3 and iron powder in acetic acid
under the reflux. Initially, we tried to do nitration of 4 in 98% H2SO4, but unfortunately, some
byproducts formed immediately while adding nitric acid to the reaction mixture, even though
by the temperature was kept as low as ꢀ15 °C. The synthesis of compounds 5 were finally
accomplished by using 80% H2SO4 as solvent. Then 5 was reacted with protecting group
4ꢀchloromethylanisole
in
DMF
with
Cs2CO3
as
base,
7ꢀhalogenꢀ4ꢀ(4ꢀmethoxybenzyl)ꢀ6ꢀnitroꢀ2Hꢀbenzo[1,4]oxazinꢀ3(4H)ꢀone, 6, were obtained in
78ꢀ84% yields. The intermediates 7 were synthesized by using iron powder and NH4Cl as
reducing reagents in 90% ethanol under reflux.
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After three consecutive steps of reactions, 3ꢀ(7ꢀhalogenꢀ4ꢀ(4ꢀmethoxybenzyl)ꢀ3ꢀoxoꢀ3,4ꢀ
dihydroꢀ2Hꢀbenzo[b][1,4]oxazinꢀ6ꢀyl)pyrido[2,3ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione, 8, were
prepared in good yields.26 Subsequently, compounds 8 reacted with methyl iodide in DMF with
Cs2CO3 as base to give hybrids 9 in the yields of 87ꢀ92%. The 4ꢀmethoxybenzyl group of 9 can
be easily removed by using CF3SO3H and CF3CO2H in dichloromethane to give compounds 10.
Finally, 10 reacted with various nucleophilic reagents R1I or R1Br; compounds 11ꢀ13 were
prepared in satisfactory yields.
The structure of all the pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone hybrids 9ꢀ13
were characterized by 1H and 13C NMR, and HRMS spectrometric data.
PPO
inhibition
and
SAR.
As
we
envisaged,
pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone hybrids might be a promising scaffold for
potent PPO inhibitors. In our previous work, we have found that the optimum substituent at the
Nꢀ1 position of quinazolineꢀ2,4ꢀdione was a –CH3 group.26, 28 In this work we also placed the –
CH3 group at the Nꢀ1 position of pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione. Before structural
optimization, we investigated the binding modes of a lead compound 10a with mtPPO. As
shown in Figure 2, the pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione moiety of 10a can form a
πꢀπ
stacking interaction with the Phe392 residue. The distance between benzene ring of Phe392 and
pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione ring is 3.5 Å, indicating the strong interactions of two
aromatic rings. The benzoxazinone ring is sandwiched between aliphatic Leu372 and Leu356.
Furthermore, the carbonyl group of benzoxazinone can form a 2.6 Å, strong hydrogen bond
with the Arg98. On the basis of the above analysis, we decided to synthesize 10a (Figure 3) and
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evaluate its mtPPOꢀinhibiting activity. Though the Ki value of 10a (Ki = 0.26 ꢀM) was not as
potent as flumioxazin (Ki = 0.046 ꢀM) (Table 1), the promising results also suggested that its
worth further optimization. Because the NH group of 10a faced to the hydrophobic residues of
the open binding pocket, therefore, introducing some nonpolar groups at this position might
improve the inhibitory potency of this series of compounds.35 Bearing this in mind, compounds
11 with different hydrophobic groups at the Nꢀ1 position of benzoxazinone were synthesized.
As shown in Table 1, introducing a –CH3 group at Nꢀ1 position of benzoxazinone,
compound 11a (Ki = 0.048 ꢀM) showed five times more potency than the parent lead
compound 10a, nearly comparative to that of flumioxazin. When introducing ꢀCH2CH3, 11b,
and ꢀCH2CH2CH3, 11c, groups at this position, the mtPPOꢀinhibiting activity of these
compounds were further improved. However, when introducing ꢀCH2CH2CH2CH3, 11d, and
ꢀCH2CH(CH3)2, 11e, groups, compared with 11b the activities did not improve much. Based on
the above results, the second round optimization was mainly based on the ethyl group of 11b
and the nꢀpropyl group of 11c. When the terminal hydrogen atom of ꢀCH2CH3, 11b, or
ꢀCH2CH2CH3, 11c, was replaced by other hydrophobic groups, it was found that
electronꢀwithdrawing groups were detrimental to activity (11b >11f, 11c > 11j), and
substituting electronꢀdonating groups was favorable for activity (11i > 11b). Furthermore, the
fluoroꢀsubstituted analogues displayed higher activity than chloroꢀsubstituted analogues
(11f >11h, 11g >11k), and the two fluorine atom substituents in 11g showed better activity than
that of singlyꢀsubstituted 11f. As for allylꢀsubstituted compounds (11lꢀ11o), substituting two
terminal hydrogen atoms of the allyl group with two methyl groups was found to be detrimental
towards activity, while changing the hydrogen atom on the 2ꢀposition of the allyl group to a –
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CH3 group led to a 5ꢀfold activity decrease. If the allyl group was changed to a –CH2CN group,
the resulting compound 11p underwent a more than a 31ꢀfold activity decrease compared with
that of 11I. The possible reason for this is that, –CH2CN is a hydrophilic group, which causes
the repulsion among the hydrophobic residues of mtPPO and 11p.
Introducing a propargyl group on the Nꢀ1 position significantly increased the mtPPO
inhibitory activity. For example, the activity of compound 11q (Ki = 0.0074 ꢀM) was 35 times
more potent than lead compound 10a, and 6ꢀfold more potent than flumioxazin. It was also
detrimental to activity when the terminal hydrogen atom of the propargyl group was changed to
other groups (11q>11r and 11s). In addition, if a bigger hydrophobic group such as
ꢀCH2C6H4(4ꢀOCH3) was introduced at R1, compound 9a also displayed good mtPPO inhibitory
activity. As we have observed previously,8ꢀ10 the fluoroꢀsubstituted compounds 11 always
demonstrated higher activity than the chloroꢀsubstituted 12 and bromoꢀsubstituted 13, while the
R1 groups stayed the same. Furthermore, the SAR observed in the fluoroꢀsubstituted
compounds 11 can also apply to chloroꢀsubstituted 12 and bromoꢀsubstituted 13.
CoMFA analysis. Quantitative structureꢀactivity relationship (QSAR) is an important
descriptor widely utilized in understanding chemicalꢀbiological interactions in herbicide
research.36 To understand the relationship between chemical structures and mtPPO inhibition
activities, we performed CoMFA analysis of the synthesized compounds 9ꢀ13. 28 compounds
were selected as the training set (Figure 4A), the experimental and calculated mtPPOꢀinhibiting
activities are shown in Table 2. The conventional coefficient r2 of the CoMFA model is 0.949,
the crossꢀvalidated q2 value is 0.632, and the predicted nonꢀcrossꢀvalidated r2 value is 0.814.
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The steric isocontour diagram of CoMFA model is shown in Figure 4B, the yellow regions
indicate that a bulk group would be detrimental to the mtPPO inhibition activity. For example,
the fluoroꢀcontaining compound 11q (X = F, R1 = ꢀCH2C≡CH, Ki = 0.0074 ꢀM) exhibited
higher mtPPO inhibition activity than the chloroꢀsubstituted 12f (X = Cl, R1 = ꢀCH2C≡CH, Ki =
0.085 ꢀM) and bromoꢀsubstituted 13f (X = Br, R1 = ꢀCH2C≡CH, Ki = 0.099 ꢀM). In contrast,
placing a bulk group at the green area was advantageous to activity. For the electrostatic
contour map (Figure 4C), the red polyhedra means that, increasing electronꢀnegative groups at
this site is favorable, whereas substituting electronꢀpositive groups on the blue contour region
will increase activity. As shown in Figure 4C, the red contour mainly locates on the Nꢀ1
position of benzoxazinone ring, which is consistent with our experimental results, in which 11q
(R1 = ꢀCH2C≡CH, Ki = 0.0074 ꢀM) displayed higher mtPPOꢀinhibiting activity than 11l (R1 =
ꢀCH2CH=CH2, Ki = 0.016 ꢀM) and 10a (R1 = H, Ki = 0.26 ꢀM). The blue contour is located
around the terminal alkyl groups, which supports our experimental data, in which placing
electronꢀwithdrawing groups on the terminal hydrogen atom of ꢀCH2CH3, 11b, or ꢀCH2CH2CH3,
11c, was detrimental to activity.
Herbicidal activity and SAR. To investigate the herbicidal activity of the synthesized
compounds 9ꢀ13, six broadleaf weeds (E. prostrata, R. acetosella, A. retroflexus, C. serotinum,
A. juncea, B. juncea,) and six monocotyledon weeds (D. sanguinalis, P. fugax, E.crusgalli, P.
annua, S. faberii, A. aequalis) were selected as representatives for the postemergence herbicidal
activity evaluation. The commercial PPO herbicide flumioxazin was selected as a control. In
most cases, the herbicidal activities of these compounds were consistent with their in vitro
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mtPPOꢀinhibiting activities (Table 1). For example, when R1 groups remained the same, the
fluoroꢀsubstituted compounds always exhibited higher herbicidal activity than the chloroꢀ and
bromoꢀsubstituted analogues. After treatment with the tested compounds, most of the sensitive
weeds showed quick burn symptoms within one or two days, followed by bleaching and death,
indicating these compounds are PPO inhibitors. As with most of the PPO herbicides, this series
of compounds also displayed strong inhibition against tested broadleaf weeds.37 As shown in
Table 1, most of the fluoroꢀcontaining derivatives 11 exhibited over 80% strong inhibition
against six test broadleaf weeds. Even at a rate as low as 37.5 g ai/ha, compound 11q still
displayed more than 80% inhibition against the test weeds, which were comparable to that of
flumioxazin.
As to the fluoroꢀsubstituted compounds 9a, 10a and 11, modifying the substituent on the
Nꢀ1 position of benzoxazinone ring had a big impact on the herbicidal activity. Compound 10a
with a hydrogen atom at this position, showed almost no herbicidal activity. However, when
changed to a methyl group, 11a displayed total inhibition against A. retroflexus. There were
also some variations in herbicidal activity when different hydrophobic alkyl, allyl and
propargyl groups were introduced. Substituting an ethyl group in this position can further
improve the herbicidal potency; 11b displayed 100% inhibition against E. prostrata, A. juncea,
and A. retroflexus at 150 g ai/ha, whereas a ꢀCH2CH2CH3 group in this position produces a
broader spectrum of weed control (11c > 11b). Prolonging the carbon chain of 11c was found
detrimental to herbicidal activity (11c > 11d). For the alkyl halideꢀsubstituted analogues,
compounds with a fluorine atom on the carbon terminal displayed more improved herbicidal
activity than those with a chlorine atom (11f > 11h, 11j > 11k). It seemed that, placing an
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electronꢀdonating group on the 2ꢀpostion of allyl group was favorable for potency (11n > 11l >
11m). Furthermore, placing a substituent on the 1ꢀposition of the propargyl group was found
detrimental to herbicidal activity (11q > 11s >11r).
It is known that most of the PPO herbicides demonstrate good herbicidal activity when
applied both postꢀemergence and preꢀemergence. Compound 11q had an excellent broad
spectrum of postꢀemergence herbicidal activity, and was selected for further preꢀemergence
herbicidal activity evaluation. Six representative weeds were tested: A. retroflexus, M. sativa, E.
prostrata, I. nil, C. Virgata and S. italic, the results are shown in Table 3. At the rate of 150 g
ai/ha, 11q displayed 100% inhibition against A. retroflexus, I. nil, C. virgata and S. italic and
over 90% inhibition against M. sativa and E. prostrata, which is as potent as flumioxazin. Even
at a dosage as low as 37.5 g ai/ha, 11q still exhibited strong inhibition against six of the seven
test weeds (> 80% inhibition).
Crop selectivity of 11q. During the herbicide discovery process, crop safety is one of the
issues. To evaluate whether 11q is safe to a target crop or not, we tested its postꢀ and
preꢀemergence crop selectivity against six representative crops: maize, wheat, rice, soybean,
cotton and peanut. As shown in Table 4, both 11q and flumioxazin were nonꢀselective
(inhibition >10%) for six crops at 75 and 150 g of active ingredient (ai)/ha in postꢀemergence
application. The possible reason for the limited postꢀemergence crop safety of 11q is that, when
11q was absorbed by crops, it inhibited the PPO activity in crops and generated a lot of reactive
oxygen species in a relatively short time, causing rapid disruption of the membrane. On the
basis of the above analysis, we evaluated the preꢀemergence crop selectivity of 11q. Maize,
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soybean, peanut and cotton displayed high tolerance to 11q at 150 g ai/ha in preꢀemergence
application. Furthermore, rice and wheat exhibited good tolerance to 11q at 75 g ai/ha; however,
flumioxazin was nonꢀselective to rice and wheat at the same rate. These promising results
suggest that 11q has potential for development as a preꢀemergence herbicide for weed control
in maize, soybean, peanut and cotton fields.
In summary, in order to obtain new compounds that can form more favorable
πꢀπ
interactions with Phe392 of mtPPO, 37 new pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone
hybrids, 9ꢀ13, were designed and synthesized. Several compounds with improved
mtPPOꢀinhibiting and good herbicidal activities were found. Among them, the most potent
compound
3ꢀ(7ꢀfluoroꢀ3ꢀoxoꢀ4ꢀ(propꢀ2ꢀynꢀ1ꢀyl)ꢀ3,4ꢀdihydroꢀ2Hꢀbenzo[b][1,4]oxazinꢀ6ꢀyl)ꢀ
1ꢀmethylpyrido[2,3ꢀd]pyrimidineꢀ2,4(1H,3H)ꢀdione, 11q, with a Ki value of 0.0074 ꢀM, which
demonstrated sixꢀfold more activity than flumioxazin (Ki = 0.046 ꢀM) against mtPPO.
Compound 11q displayed a strong and broad spectrum of weed control at 37.5 g ai/ha by both
postꢀ and preꢀemergence application, which was comparable to that of flumioxazin.
Furthermore, 11q was safe to maize, soybean, peanut and cotton in preꢀemergence application
at 150 g ai/ha, and selective to rice and wheat at 75 g ai/ha, indicating great applicability in
these fields. Our work points out that pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinones may
be worth further optimization to attain more applicability, while the structural modifications
and field trials of 11q may be efficient for the development of a new platform for research in
the field of herbicides
Supporting Information
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Supporting Information includes the detailed synthetic routes, physical and spectrum data
(NMR, HRMS) of compounds 3ꢀ13, and the preꢀemergence herbicidal activity of compounds
9ꢀ13. These materials are available free of charge via internet at http://pubs.acs.org.
Acknowledgment
This research was supported in part by the National Natural science Foundation of China (No.
21332004, 21172122).
Notes
348
349
350
The authors declare no competing financial interest.
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Figure captions:
Figure 1. Structures of Saflufenacil, commercial benzoxazinoneꢀcontaining PPO herbicides
and the synthesized hybrid compounds 10a and 11.
471
472
473
474
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477
478
479
480
481
482
483
484
485
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487
Figure 2. Simulated binding mode of lead pyrido[2,3ꢀd]pyrimidineꢀ2,4ꢀdione−benzoxazinone
hybrid compound 10a with mtPPO and the designed compounds 11ꢀ13. 10a is shown with
magenta bonds, and the key residues around it are shown with green bonds.
Figure 3. Synthesis of compounds 11ꢀ13. Reagents and conditions: (a) K2CO3, DMF, 50 °C; (b)
Fe, acetic acid, reflux; (c) HNO3, H2SO4, 0ꢀroom temp.; (d) 4ꢀchloromethylanisole, Cs2CO3,
DMF, room temp.; (e) Fe, NH4Cl, C2H5OH, reflux; (f) CO(COCl3)2, Et3N, 1,4ꢀdioxane, 0 °C to
room temp.; (g) methyl 2ꢀaminonicotinate, toluene, reflux; (h) NaOCH3, CH3OH, room temp.;
(i) CH3I, Cs2CO3, DMF, room temp.; (j) CF3SO3H, CF2CO2H, dichloromethane, room temp.; (k)
R1I or R1Br, Cs2CO3, DMF, room temp..
Figure 4. (A) Alignment of 28 training set molecules. (B) Steric contour map of CoMFA
model, 11q is shown with grey bonds. The yellow contour indicates that, placing a steric bulky
group at this site is detrimental to activity, whereas the green contour represents that, increasing
the size of a substituent is favorable to activity. (C) Electrostatic contour map of CoMFA model,
11q is shown in grey stick. The red polyhedra means that, increasing electronꢀnegative charge
at this site is favorable; on the contrary, substituting electronꢀpositive charge on the blue
contour region will increase activity.
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Table 1. Chemical Structure, Postꢀemergence Herbicidal Activity and mtPPO Inhibitory
Activities of Compounds 9ꢀ13.
dose
Cmpds
X
R1
AJ/BJa AR/CS EP/RA DS/PF EC/PA SF/AA Ki (ꢂM)b
(g ai/ha)
150
150
150
75
c
10a
11a
11b
F
F
F
H
−
−
−
++
++++
+
−
−
−
−
−
−
0.26
0.048
0.03
CH3
−
++++
++++
++++
++++
++++
++++
++++
++
CH2CH3
++++
+
−
−
−
−
−
−
37.5
150
75
−
−
−
−
−
11c
F
CH2CH2CH3
++++
−
+++
+
+++
+
++
−
++++
+
0.016
37.5
150
150
150
75
−
−
−
−
+
11d
11e
11f
F
F
F
CH2CH2CH2CH3
CH2CH(CH3)2
CH2CH2F
++++
++++
++++
+++
−
++
−
−
−
−
0.022
0.051
0.038
+++
−
−
−
++++
++++
++++
++++
++++
++++
++++
++++
+++
++++
+++
++
+++
+
−
−
−
−
−
−
37.5
150
75
−
−
−
11g
11h
11i
F
F
F
F
F
F
CH2CHF2
CH2CH2Cl
++++
++
−
−
−
0.010
0.051
0.018
0.032
0.040
0.016
−
−
−
37.5
150
75
−
+
−
−
−
++++
+++
+
+++
+
−
−
−
−
−
−
37.5
150
75
−
−
−
−
CH2CH2OCH3
CH2CH2CH2F
CH2CH2CH2Cl
CH2CH=CH2
++++
+++
−
++++
++++
++++
++++
+++
++++
+++
+
−
−
−
−
−
−
37.5
150
75
−
−
−
11j
11k
11l
++++
+++
++
+++
+
−
−
−
−
−
−
37.5
150
75
+++
−
−
−
−
++++
+++
+
++++
++++
+++
+++
+
−
−
−
−
−
−
37.5
150
75
+
−
−
−
++++
++
++++
++++
++++
−
+++
+
−
−
−
−
−
−
37.5
150
150
75
+
−
−
−
−
11m
11n
F
F
CH2CBr=CH2
++++
++++
+++
++
−
−
−
−
0.014
0.084
CH2C(CH3)=CH2
++++
++++
++++
++++
++++
++++
+++
+++
+
−
−
−
−
−
−
37.5
150
150
75
−
−
−
−
11o
11p
F
F
CH2CH=C(CH3)2
CH2CN
++++
++++
++
−
−
−
−
0.027
0.50
+++
+
−
−
−
−
−
−
37.5
150
+
−
−
−
−
11q
F
CH2C
≡CH
++++
++++
++++
++++
++++
++++
0.0074
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Page 25 of 33
Journal of Agricultural and Food Chemistry
75
37.5
150
150
75
++++
++++
++++
++
++++
++++
++++
+++
+++
−
++++
++++
++++
+++
11r
11s
F
F
CH2C≡CCH3
+
−
−
−
0.027
0.053
CH2C≡CSi(CH3)3
++++
++++
++++
++++
−
++++
−
−
−
+++
+++
−
−
−
37.5
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
150
75
+
++
−
−
−
9a
10b
12a
F
CH2C6H4(4ꢀOCH3)
H
−
−
−
−
−
0.028
0.74
0.32
0.34
0.26
0.12
0.38
0.085
0.68
6.88
0.75
0.17
0.6
Cl
Cl
Cl
Cl
Cl
Cl
Cl
−
−
−
−
−
−
CH3
++++
++++
−
+
−
−
−
12b
12c
CH2CH3
−
+++
−
++
−
CH2CH2CH3
CH2CH2CH2CH3
CH2CH=CH2
CH2C≡CH
++++
++++
−
−
−
−
−
12d
12e
−
−
−
−
−
−
+++
−
−
−
−
−
12f
−
+
−
−
−
9b
Cl CH2C6H4(4ꢀOCH3)
−
−
−
−
−
−
10c
Br
Br
Br
Br
Br
Br
Br
H
−
−
−
−
−
−
13a
CH3
−
−
−
−
−
−
13b
13c
CH2CH3
−
−
−
−
−
−
−
−
−
−
CH2CH2CH3
CH2CH2CH2CH3
CH2CH=CH2
−
−
13d
13e
−
−
−
−
−
−
0.44
1.45
0.099
23.39
0.046
−
−
−
−
−
−
13f
CH2C
≡CH
−
−
−
−
−
−
9c
Br CH2C6H4(4ꢀOCH3)
−
−
−
−
−
−
flumioxazin
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
++++
+++
+
37.5
aAbbreviations: AJ: Abutilon juncea; BJ: Brassica juncea; AR: Amaranthus retroflexus; CS:
Chenopodium serotinum; EP: Eclipta prostrata; RA: Rumex acetosella; DS: Digitaria
sanguinalis; PF: Polypogon fugax; EC: Echinochloa crusgalli; PA: Poa annua; SF: Setaria
b
c
faberii; AA: Alopecurus aequalis. Inhibition constant of the enzymatic reaction. Rating scale
of herbicidal activity (percentage of inhibition): ++++, ≥90%; +++, 80−89%; ++, 60−79%; +,
50−59%; −, <50%.
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Journal of Agricultural and Food Chemistry
Page 26 of 33
Table 2. Experimental and Calculated pKi Values of Compounds 9ꢀ13.
pKia
Cmpds exptl
pKia
calcd
calcd Cmpds exptl
6.5850 6.6098
7.3188 7.2697
7.5229 7.7300
7.7959 7.7518
7.6576 7.6142
7.2924 7.5178
7.4202 7.3378
8.0000 8.0433
7.2924 7.3666
7.7447 7.7318
7.4949 7.3478
7.3979 7.2704
7.7959 7.3430
7.2757 7.2980
7.5528 7.5869
6.1308 5.8226
6.4949 6.4890
6.4685 6.9622
6.5850 6.8230
6.9208 6.8579
6.4202 6.6137
7.0706 7.0744
6.1675 6.0439
5.1624 5.1598
6.1249 5.8233
6.7696 6.3294
6.2218 6.3449
6.3565 6.2214
5.8386 5.9372
7.0044 6.4379
4.6310 4.7280
10a
11a
11b
11c
11d
11e
11f
11g
11h
11i
11s
9a
10b
12a
12b
12c
12d
12e
12f
9b
10c
13a
13b
13c
13d
13e
13f
9c
11j
11k
11l
11m 7.8539 7.4127
7.0757 7.0382
7.5686 7.3788
6.301 6.5782
8.1308 7.8344
7.5686 7.6708
11n
11o
11p
11q
11r
apKi = ꢀlogKi.
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Journal of Agricultural and Food Chemistry
Table 3. Preꢀemergence Herbicidal Activity Compound 11q and Flumioxazin.
Cmpds
11q
dose (g ai/ha) ARa MS EP IN CV SI
++++b ++++ ++++ ++++ ++++ ++++
150
++++
+++
++++ +++ ++++ ++++ ++++
+++ +++ ++++ +++
75
37.5
150
75
+
++++
++++
++++
++++ ++++ ++++ ++++ ++++
++++ ++++ ++++ ++++ ++++
++++ +++ ++++ ++++ ++++
flumioxazin
37.5
aAbbreviations: AR: Amaranthus retroflexus; MS: Medicago sativa; EP: Eclipta prostrata; IN:
Ipomoea nil; CV: Chloris virgata; SI: Setaria italic. bRating scale of herbicidal activity
(percentage of inhibition): ++++, ≥90%; +++, 80−89%; ++, 60−79%; +, 50−59%; −, <50%.
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Journal of Agricultural and Food Chemistry
Page 28 of 33
Table 4. Postꢀ and Preꢀemergence Crop Selectivity of Compound 11q and Flumioxazin.
postꢀemergence
preꢀemergence
Dose
Cmpds
(g ai/ha)
maize wheat rice soybean cotton peanut maize wheat rice soybean cotton peanut
11q
150
75
30
20
50
30
40
30
80
70
40
30
75
60
80
70
80
60
70
50
85
80
0
0
0
0
20
10
30
20
30
10
70
60
5
0
0
0
5
0
5
0
0
0
0
0
flumioxazin
150
75
100
100
100
100
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Page 29 of 33
Journal of Agricultural and Food Chemistry
Figure 1.
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