Design, Herbicidal Activity, and QSAR Analysis of Cycloalka[ d]quinazoline-2,4-dione-Benzoxazinones as Protoporphyrinogen IX Oxidase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b02996

In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compo

Full text of DOI:10.1021/acs.jafc.9b02996

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Agricultural and Environmental Chemistry
Design, Herbicidal Activity, and QSAR Analysis of
Cycloalka[d]quinazoline-2,4-dione-Benzoxazinones
as Protoporphyrinogen IX Oxidase Inhibitors
Da-Wei Wang, ruibo zhang, Ismail Ismail, Zhiyuan Xue, Lu Liang, Shuyi Yu, Xin Wen, and Zhen Xi
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02996 • Publication Date (Web): 29 Jul 2019
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Design, Herbicidal Activity, and QSAR Analysis of
Cycloalka[d]quinazoline-2,4-dione−Benzoxazinones as Protoporphyrinogen IX
Oxidase Inhibitors
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Da-Wei Wang,# Rui-Bo Zhang,# Ismail Ismail, Zhi-Yuan Xue, Lu Liang, Shu-Yi Yu,
Xin Wen, and Zhen Xi*
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State Key Laboratory of Elemento-Organic Chemistry, and Department of Chemical
Biology, National Pesticide Engineering Research Center, Collaborative Innovation
Center of Chemical Science and Engineering, College of Chemistry, Nankai
University, Tianjin 300071, P. R. China.
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*corresponding author: E-mail: zhenxi@nankai.edu.cn (Z. Xi), Tel: +86
022-23504782. Fax:
+86 022-23504782.
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#D.-W. W. and R.-B. Z. contributed equally to this work.
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Abstract:
In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4)
inhibitors, we designed and synthesized series of novel herbicidal
a
cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized
compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO
(NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80%
control of all three tested broadleaf weeds at 37.5-150 g ai/ha by post-emergent application,
majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha.
Promisingly, 17i (Ki = 6.7 nM) showed 6 and 4 times more potent than flumioxazin (Ki = 46 nM),
and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent and
broad-spectrum herbicidal activity even as low as 37.5 g ai/ha, and safe for wheat at 150 g ai/ha
by post-emergent application, indicating the great potential for 17i development as a herbicide for
weed control in wheat fields.
KEYWORDS: benzoxazinone; cycloalka[d]quinazoline-2,4-dione, protoporphyrinogen IX
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oxidase; herbicide; weed control
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Introduction
Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) is one of the most important targets for
herbicide discovery, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX.1-3
In planta, protoporphyrin IX is the substrate for the biosynthesis of chlorophyll, which is a key
pigment for photosynthesis. Inhibition of PPO can lead to the toxic accumulation of
protoporphyrin IX in the cytoplasm, upon exposing to light it generates reactive oxygen species,
which, in turn, result in cell death and plants bleaching.4-7 Therefore, PPO herbicides are also
called peroxidizing herbicides. The research of PPO herbicides was initiated in the 1960s, peaked
around the 1990s and decreased soon due to the development of genetically modified
glyphosate-resistant crops, such as glyphosate-resistant soybeans.8 This weed populations
resistance has resulted because of the overuse of some herbicides. PPO-inhibiting herbicides can
effectively control some triazine-, AHAS- and glyphosate-resistant weed biotypes.9 Most
interestingly, even share the same mode of action, some newly developed PPO inhibitors can also
suppress the PPO herbicides-resistance Amaranthus biotypes.10 As a consequence, in recent
years, the discovery of new PPO inhibitors has again been a very active research area for the
agrochemical industry.11
To date, there are thousands of PPO inhibitors have been reported in the literature, about 30
of them currently used as herbicides to decimate weeds in fields.12,13 Depending on the structural
features, many PPO herbicides have hydrophobic side chains to increase the foliar absorption and
translocation in plants, some of them even shared the similar core skeleton (Figure 1). For
example, saflufenacil, butafenacil and tiafenacil all belong to pyrimidinedione type inhibitors, the
different hydrophobic warheads in their structures, which, not only make them easier absorb by
foliage and transfer in whole plants, but also can improve the binding affinity of them with plants
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PPOs.14,
According to the PPO herbicides classification methodologies, flumioxazin
(N-phenyl-phthalimides), thidiazimin (thiadiazoles), and trifludimoxazin (triazinones) belong to
different types of herbicides.12 However, they all bearing the benzoxazinone skeleton as the core
motif, and the benzoxazinone systems are connected with heterocyclic moieties via the C-N
bond. Flumioxazin is one of the most widely used PPO herbicides, showing excellent
broad-spectrum of herbicidal activity including these AHAS- and glyphosate-resistant
Amaranthus weeds.15 Thidiazimin is an effective contact herbicide for dicotyledonous weeds
control in winter cereals. Trifludimoxazin is the first triazinone-containing PPO herbicide with a
novel mechanism of action.10 It shows quick burn-down action against the leaves of weeds with
one day. What makes trifludimoxazin unique is that it can effectively suppress some
PPO-resistant biotypes such as Amaranthus spp. and Ambrosia spp.
The reported studies show that it is an effective approach to obtain a new compound with
improved bioactivities by placing the heterocyclic moieties at the 6-position of benzoxazinone
16-18
ring.
Roy K. and Paul S. have found that the electrostatic potential surface of the
heterocyclic moieties of benzoxazinone derivatives is crucial to bioactivities.17 Compounds with
the electrostatic positively charged surface at this position were found to showed better activity,
whereas, inhibitors with negatively charged surface were proved detrimental to PPO-inhibiting
activity. To date, PPO inhibitors are designed by mimicking part of the enzyme substrate
protoporphyrinogen IX.12 We have found that the protoporphyrinogen IX bound to the active
site of Nicotiana tabacum PPO (NtPPO) with two pyrrole rings buried in the bottom of the
pocket, and two carboxyl groups orientated to the product channel.19 The catalytic pocket of
PPO is mainly formed by a number of hydrophobic residues, such as Leu334, Phe392, Leu372,
and Leu356, indicating that compounds with hydrophobic groups may be advantageous to
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PPO-inhibiting activity. Based on the above-mentioned studies, we thought that integrated
hydrophobic and heterocyclic moieties and benzoxazinone system into a molecular architecture
would promote the discovery of novel PPO inhibitors with improved activity. Cycloalkanes and
pyrimidine-2,4(1H,3H)-dione are biologically important functional scaffolds exist in many
natural products, agrochemicals, and pharmaceuticals.20-22 Previously, we have reported a series
of pyridopyrimidine-2,4-dione−benzoxazinones, the results showed that the fluorine atom was
the optimal substituent at the 7-position of the benzoxazinone ring.23 As a continuation of our
program to search for novel PPO inhibitors, herein,
a
novel series of
cycloalka[d]quinazoline-2,4-dione-benzoxazinones possessing a fluorine atom at the 7-position
of benzoxazinone system were designed and synthesized (Figure 2). The NtPPO inhibitory
activity, structure-activity relationships (SAR), herbicidal activity, and crop selectivity of the
newly synthesized compounds were systematically explored.
MATERIALS AND METHODS
Preparation of cycloalka[d]quinazoline-2,4-dione-benzoxazinones 8, 9, 17, and 19. The
synthetic routes for 8, 9, 17, and 19 are shown in Scheme 1-3, the detailed synthetic methods,
13
characterization data (1H and C NMR, HRMS, melting point) of the compounds are shown in
the Supporting Information.
X-ray Diffraction. The single crystals of compound 17a were obtained by slow evaporating
from chloroform solution. The supplementary crystallographic data for 17a had been deposited in
the Cambridge Crystallographic Data Centre (CCDC, http://www.ccdc.cam.ac.uk/), the
deposition number is 1911322. The crystal structure of 17a is shown in Figure 3.
PPO Inhibitory Experiments. The expression and purification of Nicotiana tabacum
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mitochondrial PPO2 (NtPPO) were performed as described previously.23-25 The enzyme substrate
protoporphyrinogen IX was synthesized by reduction of protoporphyrin IX with freshly prepared
sodium amalgam. Due to the chemical nature of protoporphyrin IX, it has a maximum excitation
at 410 nm and a maximum emission of 630 nm. In the kinetic inhibition assays, we used a
fluorescence detector to monitor the formation of the protoporphyrin IX by setting the emission
wavelengths to 631 nm and excitation wavelengths to 410. Inhibitors were dissolved in dimethyl
sulfoxide (DMSO) as stock solution, and diluted to the different concentration ranges from 0.05
M to 50 mM just before using. The total volume of reaction solution was 200 L, which
consists of 0-40 g PPO, 5 M flavin adenine dinucleotide (FAD), 5 mM DTT, 1 mM EDTA, 0.2
M imidazole, 0.1 M potassium phosphate buffer (pH 7.4), and 0.03% Tween 80 (v/v). To initiate
the PPO reaction, 0-6.5 M protoporphyrinogen IX was added to the assay solution.
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The half maximal inhibitory concentration (IC50) value of inhibitors was calculated by fitting
v versus [I] data to a single binding site model (eq 1). The kinetic parameters were calculated by
Sigma Plot software 10.0 (SPSS, Chicago, IL).
max - min
(1)
y = min +
1 + 10logIC
- x
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in this equation, y is the percentage of the maximal rate, min and max being the y values at which
the curve levels off, x is the logarithm of inhibitor concentration. The inhibition constant of the
enzymatic reaction (Ki) was calculated by using the following relationship among IC50, Ki, and
Km (1.52 μM) at any saturated substrate concentration (eq 2). The results of Ki values of inhibitors
are shown in Table 1.
IC50
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122
Ki =
(2)
S
Km + 1
Molecular Modeling and 3D-QSAR Analysis. The structure of NtPPO (PDB ID: 1SEZ) was
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downloaded and prepared by standard methods using Autodock Tools Package before docking.26
The 3D structures of cycloalka[d]quinazolinedione−benzoxazinones were constructed by
SYBYL-X 6.9 (Tripos, Inc., St. Louis, MO) based on the crystal structure of 17a, and
subsequently optimized with the conjugate-gradient and steepest-descent algorithm to a
convergence criterion of 0.005 kcal/mol. Docking calculations of two molecules were performed
on AutoDock4.2 by using a genetic algorithm, for each ligand the docking runs were set to 500.
After calculation, the results were clustered, and the best binding modes were selected by the
docking energy as well as by comparison with the co-crystal ligand.27 The binding free energies
of 8i and 9i with NtPPO were calculated by molecular mechanics-Possion-Boltzmann surface
area (MM-PBSA) method, and the results are shown in Table S1. Open3DAlign was used to
align the 36 synthesized molecules, the best align mode was selected both referred to the docking
modes and the crystal structure of 17a, then the aligned compounds were transferred to
Open3DQSAR to build the QSAR model.28 A grid box with 5.0 Å margin and 0.5 Å step size
was set around the compounds. The compounds number for training and test sets were 28 and 8,
respectively. Electrostatic potential and steric factors of molecular interaction fields was then
calculated, other parameters used for the calculation were the same as described by Tosco et al.29
The experimental and predicted activity values are depicted in Table 2, and PyMOL was used to
visualize the QSAR results.
Herbicidal activity. The post-emergence herbicidal activity of compounds 8, 9, 17, and 19
against three dicot weeds, Eclipta prostrata (E.p.), Abutilon juncea (A.j.), and Amaranthus
retroflexus (A.r.); and three monocotyledonous weeds, Setaria faberii (S.f.), Digitaria sanguinalis
(D.s.), and Echinochloa crusgalli (E.c.) were evaluated by using the similar reported
procedures.30-35 To further evaluate the herbicidal spectrum bioactivities of compounds 8i, 9i and
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17i, another thirteen weeds: Ipomoea nil (I.n.), Bidens pilosa (B.p.), Commelina benghalensis
(C.b.), Cyperus rotundus (C.r.), Amaranthus spinosus (A.s.), Chenopodium album (C.a.),
Phytolacca Americana (P.a.), Bidens tripartite (B.t.), linopodium chinense (L.c.), Nicandra
physaloides (N.p.), Cassia obtusifolia (C.o.), Solanum nigrum (S.n.), and Vicia gigantean (V.g.)
were tested at the concentration of 37.5-150 g ai/ha, using flumioxazin as a control. The
inhibition percentages were evaluated at 25 days after treating by compounds, for each treatment,
three replications were performed. The herbicidal activity results are shown in Tables 1, 3 and S2.
Crop Selectivity. Six representative crops: wheat, maize, rice, soybean, peanut, and cotton were
used in the crop safety experiment.23, 36 In this test, we evaluated the post-emergence crop safety
of all the compounds that showed more than 80% control against at least three of the E. prostrata,
A. juncea, A. retroflexus, S. faberii, D. sanguinalis, and E. crusgalli at 37.5 g ai/ha. Flowerpots
with 12 cm diameter were filled with mixed soil (Vseedling substrate:Vvegetable garden soil = 2:1) to 9 cm
depth. Crop seeds were planted and grown at 25 °C (day) and 15 °C (night) in the greenhouse.
The application rate for the tested compounds was 150 g ai/ha. The results of crop selectivity
(Tables 4 and S3) were evaluated after 25 days of treatment, with three replications per test.
RESULTS AND DISCUSSION
Chemistry. Three similar synthetic routes were designed to synthesize 8, 9, 17, and 19, which
were dependent on the number of n, and the substituents of R1 and R2. When n = 1 or 2, R1 and
R2 are hydrogen atoms, the detailed synthetic routes for 8 and 9 are depicted in Scheme 1; when n
= 2, R1 and R2 are fluorine atoms, the synthetic routes for 17 are outlined in Scheme 2; when n =
3, R1 and R2 are hydrogen atoms, the synthetic routes for 19 are shown in Scheme 3. By using the
similar synthetic procedures we have reported, compounds 8 and 9 can be synthesized in 9 to 11
steps.23 In this work, we found that compounds 8a and 9a could be synthesized by a four-step
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consecutive reaction. Firstly, 7 reacted with triphosgene in refluxing toluene afforded the
corresponding
isocyanate,
which
was
directly
reacted
with
ethyl
2-aminocyclopent-1-ene-1-carboxylate or ethyl 2-aminocyclohex-1-ene-1-carboxylate to give the
corresponding urea, and got precipitated from the solution upon cooling, without further
purification the urea then underwent intramolecular ring closure reactions in CH3ONa-CH3OH
solution and methylation reaction in DMF with K2CO3 as a base to afford 8a and 9a. 8b and 9b
were synthesized by deprotecting the 4-methoxybenzyl groups of 8a and 9a in
trifluoromethanesulfonic acid and trifluoroacetic acid. 8b and 9b reacted with various
electrophilic reagents R3I or R3Br in DMF with K2CO3 as base to give 8c-k and 9c-l in yields of
58-88%.
Initial attempts to synthesize 13 by using similar methods as 4 were proved problematic.
Ethyl 2,2-difluoro-2-(5-fluoro-2-nitrophenoxy)acetate can be smoothly prepared by reacting of
5-fluoro-2-nitrophenol 1 with ethyl 2-bromo-2,2-difluoroacetate 11 in DMF with K2CO3 as a
base in 87% yield. We have tried various methods to reduce the nitro group of ethyl
2,2-difluoro-2-(5-fluoro-2- nitrophenoxy)acetate by using H2/Pd(C), Fe/acetic acid, or Fe/NH4Cl,
and then followed by a ring-closure reaction to obtain 13. However, the reaction seemed to be
unsatisfactory. The reason may be that ethyl 2-(2-amino-5-fluorophenoxy)-2,2-difluoroacetate
with two fluorine atoms on -position of the ether bond significantly reduce the nucleophilic
potency of the amino group.
So, another alternative synthetic route shown in Scheme 2 was designed to synthesize 13.
The required intermediate 13 can be synthesized in two steps by reacting of 10 with 11 in
tetrahydrofuran with NaH as a base, and then followed by an intramolecular ring closure reaction
using Cs2CO3 as a base in DMF. Subsequently, 14 was achieved by reacting of 13 with HNO3 in
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concentrated H2SO4 in 93% yield. While synthesizing 15 by using a similar method as 6, the
reaction could not proceed due to the weak nucleophilic ability of NH group of 14 made it hard to
react with 4-methoxybenzyl chloride. 4-methoxybenzyl bromide is more active than
4-methoxybenzyl chloride, therefore, 15 was prepared by reacting 4-methoxybenzyl bromide in
DMF with Cs2CO3 as a base in a yield of 86%. The fluorine-substituted compounds 17 were
obtained in the same fashion as 8 and 9 in yields of 58-86%. Compound 19 was prepared in a
yield of 61% with 6-amino-7-fluoro-4-(prop-2-yn-1-yl)-2H-benzo[b][1,4]oxazin-3(4H)-one 18 as
the starting material, the synthetic methods were the same as 8a,9a, and 17a.
PPO Inhibitory Activity and SAR. According to the structural feature of the designed
cycloalka[d]quinazoline-2,4-dione-benzoxazinones, we intended to optimize it in three ways: one
was to explore the size of cycloalkanes ring effect on PPO inhibitory activity; another was to
study the substituents change at the 2-positions of benzoxazinone ring (defined as 2-position)
effect on activity; the third was to search for the optimum substituents at R3. Previously, we have
reported that for compounds containing of pyrimidine-2,4(1H,3H)-dione skeleton in their
structures, placing a methyl group at the N-1-position of pyrimidinedione ring would most
favorable to herbicidal activity.23 Therefore, for the cycloalka[d]quinazoline-2,4-dione
derivatives, we also kept its N-1 position as a methyl group.
Initially, we intended to explore compounds with cyclopropane or cyclobutane ring as the
hydrophobic groups that fused to pyrimidine-2,4(1H,3H)-dione, however, through various
synthetic methods were tried, we still could not obtain the needed compounds (data not shown).
Compounds containing a cyclopentane ring could be easily synthesized (Scheme 1, compounds
8). One lead compound, 8a, was obtained in a yield of 65%, and it showed good PPO inhibition
activity with a Ki value of 0.21 M (Table 1). Inspired by this, we made further optimization of
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8a by substituting various groups at the N-4-position of benzoxazinone ring (defined as
N-4-position). It was found that placing a hydrogen atom (8b, Ki = 0.34 M) at this position was
detrimental to activity, whereas, placing hydrophobic and medium-chain fatty groups (2-4 carbon
atoms) were found favorable to activity. Interesting, compound 8i with a propargyl group was
found to display better PPO inhibition activity comparatively with other substituents at the same
position.
Cyclohexane is not only bigger than cyclopentane in size, but also shows more
hydrophobicity than cyclopentane. Because introducing the electrostatic positively charged
surface at the 6-position of benzoxazinone system is advantageous to activity.17 Therefore, we
envisaged that cyclopentane ring might be a more suitable cycloalkane ring to fuse with
pyrimidinedione. The results indicated that in almost all cases, compounds 9 showed higher
PPO-inhibiting activity than compounds 8, which were consistent with our hypothesis. Inspired
by this, we decided to change the cyclohexane ring to a seven-membered cycloheptane ring, in
order to make further improvement of PPO inhibitory activity. As we have found that the
propargyl group was a suitable substituent at R3 for cyclopentane- and cyclohexane-fused
derivatives, 8 and 9, respectively. Therefore, compound 19 was synthesized. Disappointedly, 19
did not show improved PPO-inhibiting activity than 9i and 8i (9i, Ki = 0.0067 M > 8i, Ki =
0.014 M > 19, Ki = 0.072 M). In addition, it should be noted that efforts to synthesize more
membered ring that seven to fuse with pyrimidinedione was proved unsuccessful. Considering
the above PPO inhibitory results, we can conclude that the cyclohexane ring was the optimum
cycloalkane ring to fuse with pyrimidinedione in the present study.
Having identified the most suitable cycloalkane ring, we then transferred to optimize the
substituents at the 2-position. In our previous work, we have demonstrated that introduced methyl
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group(s) at this position was detrimental to PPO inhibitory activity, due to the steric clashing
between the methyl group(s) with the surrounding residues.25 These results also suggested that
the low substituent tolerance at the 2-position, substituents introduced at this site should be
smaller than the methyl group. It is reported that fluorine atom is smaller than methyl group in
size, introducing fluorine atom to a compound can increase its hydrophobicity and thermal
stability, which in turn can improve its bioactivity.37-39 Therefore, we introduced two fluorine
atoms at the 2-positions. As shown in Table 1, in some cases substituting hydrogen atoms with
fluorine atoms were advantageous to the PPO inhibitory activity. For example, the activity of 17h
(R1 = R2 = F, Ki = 0.0098 M) showed a slightly higher active than its parent compound 9h (R1 =
R2 = H, Ki = 0.011 M), the propargyl-containing 17i (Ki = 0.0067 M) was also showed higher
PPO-inhibiting activity than 9i (Ki = 0.0078 M). The promising results demonstrated that
introducing the fluorine atoms at 2-positions could improve the binding affinity of inhibitors with
PPO.
Though, there was some variation in the SAR of R3 among compounds 8, 9 and 17 (Table
1). However, the general trends of substituents changes affected on PPO inhibitory activity still
shared some similarity. For examples, substituting the propargyl group (8i, 9i, 17i) at R3 was
found most favorable to activity; compounds (8h, 9h, 17h) with allyl groups were also found
exhibited very strong PPO-inhibiting activity; placing medium-chain fatty or ester groups at this
site was a benefit to activity as well. It should be noted that sometimes even introducing the same
group at R3, the PPO-inhibiting activities of compounds with different motifs still had some
variations. In order to understand this molecular basis, we conducted molecular modeling
analyses of four representative compounds 8i, 9i, 17i, and 19 with PPO. As shown in Figure 4,
there were mainly three similar interactions of 8i, 9i and 17i with surrounding key residues: first,
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was the - interaction between Phe392 and the pyrimidinedione moieties of the three
compounds; second, was the 2.7 Å strong hydrogen bonded interaction between Arg98 and the
carbonyl group of benzoxazinone system; and the third, was the sandwich like hydrophobic
interactions among benzoxazinone moiety, Leu372, and Leu356. In addition, we found that the
binding modes of 8i and 9i with NtPPO were almost identical, however, 9i (Ki = 0.0078 M)
showed higher activity than 8i (Ki = 0.014 M). To explain this, we calculated the binding free
energies of 8i and 9i with NtPPO by using MM-PBSA method. The results showed that the total
gas-phase binding energies of 8i (-76.70 kcal/mol) and 9i (-76.76 kcal/mol) were almost same
(Table S1). However, the higher solvation penalty (8i = 39.99 kcal/mol, 9i = 37.70 kcal/mol) and
enthalpy change (8i = 9.61 kcal/mol, 9i = 9.41 kcal/mol) made a lower binding affinity of 8i
(-27.10 kcal/mol) when compared with 9i (-29.65 kcal/mol), indicating that introduction of
cyclohexa[d]pyrimidinedione system can reduce the solvation penalty and entropy change and
thereby favorable to activity. The fluorine atom of 17i at 2-position can form a favorable 2.3 Å
hydrogen bonding with Gly354 (Figure 4C), indicating that introduced the two fluorine atoms at
the 2-position of 9i could improve its PPO inhibitory activity. Cycloheptane ring is sterically
bulky than cyclopentane ring. Hence, as the docking results suggest the binding mode of 19 with
PPO was different with that of 8i, 9i, and 17i, due to the steric repulsive effects between the
cycloheptane group of 19 and the surrounding residues. Interestingly, though significantly
different binding modes were observed in 19, it still showed acceptable PPO-inhibiting activity
(Ki = 0.072 M). The retained activity of it may attributed to the two hydrogen bonded
interaction with Arg98 (3.0 Å) and FAD (2.7 Å).
QSAR Analysis. To elucidate the substitution effect on PPO inhibitory activity of the
synthesized
compounds,
we
studied
the
QSAR
of
the
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cycloalka[d]quinazoline-2,4-dione-benzoxazinones, the results of experimental and predicted
activity values are listed in Table 2. The conventional coefficient (r2), cross-validated coefficient
(q2) and noncross-validated coefficient (r2pred) of the QSAR model are 0.95, 0.66, and 0.83,
respectively (Figure 5, Table S4). To better elucidate the QSAR results, compound 17i, with best
PPO-inhibiting activity was selected as a representative and placed in the center of the model
(Figure 6). According to the steric contribution contour maps, the green contours mainly located
near the N-4-position of 17i, indicating that placing bulky groups at this position was favorable to
activity (Figure 6B). For examples, compounds 8c-g, 9c-g, and 17c-g with hydrophobic and
medium-chain fatty groups at the N-4-position showed higher activity than those with hydrogen
atoms substituted compounds 8b, 9b, and 17b, respectively; most of the compounds 9 displayed
improved activity than their corresponding compounds 8. On the contrary, there is almost no
yellow polyhedron around 17i, indicating that placing sterically small groups was not acceptable
for activity. For example, substituting a hydrogen atom at R3 was found detrimental to activity.
As shown in Figure 6C, there is almost no red polyhedron around 17i, indicating that introducing
electronegative groups to cycloalka[d]quinazolinedione-benzoxazinones have an unfavorable
contribution to activity. The blue polyhedrons are situated around the N-4-position of 17i as well,
meaning that compounds with electron-positive groups at this position would show high activity.
This is consistent with the case of compound 9l and 17l, in which, the electronegative nitrogen
atom is at the terminal of R3 showed reduced activity. In contrast, 9i and 17i both have
electron-positive groups at the terminal of R3 showed significantly improved activity.
Herbicidal Activity. In the greenhouse tests, we observed that the leaves of sensitive plants
became droopy several hours after treating by the tested compounds, followed by bleaching and
withering, this process is generally within three days under sunlight. As shown in Table 1, the
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herbicidal activities of compounds 8, 9, 17, and 19 correlated well with their PPO-inhibiting
activities. In most cases, compounds displayed strong PPO inhibition activity showed higher
herbicidal activity as well. Almost all the synthesized compounds displayed nearly 100%
inhibition against three broad–leaved weeds (A. juncea, A. retroflexus, and E. prostrata) at the
rate of 150 g ai/ha. Nine compounds, 8h, 8i, 8k, 9h, 9i, 17h, 17i, 17m and 19, exhibited at least 4
of the six tested weeds at 150 g ai/ha. It is worth mentioning that, 8i, 9i, 17i and 19 even
displayed comparable herbicidal activity to that of flumioxazin and trifludimoxazin against the
tested weeds at the 150 g ai/ha.
Due to the excellent herbicidal activity of the synthesized compounds, we performed three
rounds of herbicidal evaluation against these compounds. In the first round screening, 37
compounds were tested at the dosage of 150 g ai/ha, a total of 33 compounds (8b, 8d-i, 8k, 9b-l,
17 and 19) showed more than 80% inhibition against at least half of the six tested weeds, most of
them exhibited 100% control against the tested broad-leaf weeds (Table 1). Then the 33
compounds were tested at the relative lower concentrations: 75 and 37.5 g ai/ha. The results
(Tables 1 and S2) showed that, 29 compounds (8d-i, 9b-j, 9l, 17b-m and 19) exhibited over 80%
control against more than three of the six weeds at the concentration of 75 g ai/ha, and 20
compounds (8d-g, 8i, 9c-e, 9h, 9i, and 17b-j, 17m) showed more than 80% inhibition against at
least three of the tested weeds at 37.5 g ai/ha. Furthermore, 6 compounds 8i, 9h, 9i, 17b 17h, and
17i still exhibited 100% inhibition against three tested broadleaf species at the rate as low as 37.5
g ai/ha. Most promising, 17i even displayed more than 80% control against the six tested weeds
at 37.5 g ai/ha, which were comparable to that of flumioxazin. In most cases, substituting 2
fluorine atoms at the 2-position of compounds can improve their herbicidal activity to some
extent. For example, most of compounds 17 showed higher and broader herbicidal actives than
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their corresponding mother compounds 9. We inferred the possible reason is that introducing two
fluorine atoms at the 2-positions can increase the lipophilicity of compounds, which will make
better absorption of compounds by plant foliage.
Based on the results of the second round screening, three compounds (8i, 9i and 17i) with
excellent and broad-spectrum of weeds control were further evaluated against another 13 kinds of
weeds (I. nil., B. pilosa, C. benghalensis, C. rotundus, A. spinosus, C. album, P. Americana, B.
tripartite, l. chinense, N. physaloides, C. obtusifolia, S. nigrum, and V. gigantean) at the rate of
37.5-150 g ai/ha. A shown in Table 3, all of the three compounds showed more than 80%
inhibition against the 13 test weeds at 150 g ai/ha and over 80% control against 12 of 13 test
weeds at 75 g ai/ha. At the rate of 37.5 g ai/ha, 8i showed at least 80% control against 6 kinds of
weeds; 9i displayed more than 80% inhibition against 9 kinds of weeds; 17i exhibited over 80%
inhibition against 12 kinds of weeds, which were comparable to that of flumioxazin, and more
potent than that of 9i and 8i (17i > 9i > 8i).
Crop selectivity. 20 compounds (8d-g, 8i, 9c-e, 9h, 9i, and 17b-j, 17m) with strong and
broad-spectrum of weeds control at 37.5-150 g ai/ha were selected for post-emergent crop safety
studies, the results are shown in Tables S3 and 4. Most of the tested compounds displayed no
phytotoxicity toward at least one kind of the tested crops at the rate of 150 g ai/ha. For examples,
8d, 8f, 9c, and 9d exhibited high safety for maize; wheat exhibited relative to high tolerance to
8f, 8i, 9d, 9i, and 17i, suggesting the great potential for using these compounds in maize or wheat
fields. To our surprise that maize, wheat, and rice showed extremely high tolerance to 17b-h, 17j,
and 17m, whereas, flumioxazin and trifludimoxazin were not safe for the six tested crops at the
same condition. The promising results indicated that 17b-h, 17j, and 17m had great potential to
develop as herbicides application in maize, wheat and rice fields for broad-leaf weeds control.
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In summary, we have presented the design, synthesis, herbicidal activity and QSAR studies of
cycloalka[d]quinazoline-2,4-dione−benzoxazinones as novel PPO inhibitors. The bioassay results
indicated that, most of the synthesized compounds exhibited strong PPO inhibition activity; 20 of
the 37 synthesized compounds displayed over 80% inhibition against A. juncea, A. retroflexus,
and E. prostrata at the rate of 37.5-150 g ai/ha by post-emergence application, most of them
exhibited high selectivity toward at least one kind of crop at 150 g ai/ha. Compounds 8i and 9i
not only exhibited strong and broad-spectrum weeds control comparable to that of flumioxazin at
75-150 g ai/ha, but also showed highly safe to wheat at 150 g ai/ha. Most promisingly, 17i (Ki =
0.0067 M) displayed excellent and wide-spectrum herbicidal activity even as low as 37.5 g
ai/ha, and relatively safe for wheat at 150 g ai/ha by post-emergent application, indicating the
great possibility of 17i to be a herbicide for weed control in wheat fields. Furthermore, a highly
predictive QSAR model was built to understand the activity of the newly synthesized compounds.
The results showed that the highest activity is obtained for inhibitors with
cyclohexa[d]pyrimidine-2,4-dione moiety at the 6-position of benzoxazinone ring, hydrophobic
and medium-sized groups at the N-4-position. The molecular docking analyses revealed that
placing fluorine atoms at the 2-position was advantageous to activity by forming the fluorine
atom mediated hydrogen binding with the surrounding residues. Our present study not only
provides a series of potential candidates for herbicide discovery but also be useful for the design
and development of novel benzoxazinone-containing compounds.
Supporting Information
The detailed synthetic routes, 1H and 13C NMR and HRMS spectra of compounds 8, 9, 17 and 19;
calculated binding free energies of 8i and 9i with NtPPO (Table S1), post-emergence herbicidal
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activity of compounds 8b, 8d-g, 9b-g, 9j-l, 17a-g and 17j-l at 37.5-75 g ai/ha (Table S2);
post-emergence crop selectivity of compounds 8d-g, 9c-e, 9h, 17b-h, 17j, and 17m at 150 g ai/ha
(Table S3); and statistical data of 3D-QSAR model (Table S4 ) are shown in the Supporting
Information.
Acknowledgment
This research was funded in part by the National Key Research and Development Program of
China (No. 2017YFD0200501), the National Natural Science Foundation of China (No.
21702111, 21672118, 21332004), the Tianjin Natural Science Foundation (No.
16JCYBJC20200), and the China Postdoctoral Science Foundation Funded Project (No.
2016M591384).
Notes
The authors declare no competing financial interest.
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Figure and Scheme Captions:
Figure 1. Chemical structures of some commercial PPO herbicides and the synthesized
compounds 8, 9, 17, and 19.
Figure 2. Design protocol of novel cycloalka[d]quinazolinedione-benzoxazinones.
Figure 3. Crystal structure of compound 17a.
Figure 4. Simulated binding modes of 8i, 9i, 17i, and 19 with NtPPO. The key residues around
the active site are shown in blue sticks, (A) Binding mode of 8i with NtPPO; (B) Binding mode
of 9i with NtPPO; (C) Binding mode of 17i with NtPPO; (D) Binding mode of 19 with NtPPO.
Figure 5. Correlation of experimental and predicted pKi values.
Figure 6. (A) Alignment of 28 training set compounds. (B) Contour maps of steric contribution,
compound 17i is located in the center of the filed. (C) Contour maps of electrostatic contribution,
compound 17i is located in the center of the filed.
Scheme 1. Synthesis of compounds 8 and 9. Reagents and conditions: (a) K2CO3, DMF, r.t.; (b)
Fe, acetic acid, reflux; (c) HNO3, H2SO4, 0 C-r.t.; (d) 4-methoxybenzyl chloride (PMBCl),
K2CO3, DMF, r.t.; (e) Fe, NH4Cl, C2H5OH (90%), reflux; (f) CO(OCCl3)2, Et3N, toluene, 0 C to
reflux;
(g)
ethyl
2-aminocyclopent-1-ene-1-carboxylate
or
ethyl
2-aminocyclohex-1-ene-1-carboxylate, toluene, reflux; (h) NaOCH3, CH3OH, r.t.; (i) CH3I,
K2CO3, DMF, r.t.; (j) CF3SO3H, CF3CO2H, CH2Cl2, r.t.; (k) R3Br or R3I, K2CO3, DMF, r.t..
Scheme 2. Synthesis of compounds 17. Reagents and conditions: (a) NaH, THF, -15 C - r.t.; (b)
Cs2CO3, DMF, 75 C; (c) HNO3, H2SO4, -10 C-r.t.; (d) 4-methoxybenzyl bromide (PMBBr),
Cs2CO3, DMF, r.t.; (e) Fe, NH4Cl, C2H5OH (90%), reflux; (f) CO(OCCl3)2, Et3N, toluene, 0 C to
reflux; (g) ethyl 2-aminocyclohex-1-ene-1-carboxylate, toluene, reflux; (h) NaOCH3, CH3OH,
r.t.; (i) CH3I, K2CO3, DMF, r.t.; (j) CF3SO3H, CF3CO2H, CH2Cl2, r.t.; (k) R3Br or R3I, K2CO3,
DMF, r.t..
Scheme 3. Synthesis of compound 19. Reagents and conditions: (a) CO(OCCl3)2, Et3N, toluene,
0 C to r.t.; (b) toluene, reflux; (c) NaOCH3, CH3OH, r.t.; (d) CH3I, K2CO3, DMF, r.t..
531
532
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533
534
535
536
Tables:
Table 1. Post-emergence herbicidal activity and NtPPO inhibitory activity of compounds 8, 9, 17,
and 19.
% inhibition
A.j.a A.r. E.p. D.s. E.c. S.f.
dosage
g ai/ha
150
150
150
150
150
150
150
150
75
compds
n
R1 R2
R3
Ki/Mb
8a
8b
8c
8d
8e
8f
1
1
1
1
1
1
1
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2C6H4(4-OCH3)
H
30
80
80
50
30
80
80
0
0
0
0
0
0
0
0
0
0
0
0.21
0.34
100
100
CH3
0
0
0.27
CH2CH3
100 100 100
100 100 100
100 100 90
100 100 100
0
0
0.098
0.069
0.12
CH2CH2CH3
CH2CH2CH2CH3
CH2CH(CH3)2
CH2CH=CH2
0
0
0
0
8g
8h
0
0
0.17
100 100 100 80
0
80
30
0
0.020
100
100
80 100 30
40 80
30
0
37.5
150
75
0
8i
1
H
H
CH2C≡CH
100 100 100 90 100 100 0.014
100 100 100 50
100 100 100 30
85
80
0
70
30
0
37.5
150
150
75
8j
1
1
H
H
H
H
CH2C≡CSi(CH3)3
100 100 70
0
0
0
0
0
0
2.50
8k
CH2CO2CH2CH3
80
60
50
70
80
50
30
70
80
80
70
70
0
80
60
0
0.048
30
0
37.5
150
150
150
150
150
150
150
150
75
9a
9b
9c
9d
9e
9f
2
2
2
2
2
2
2
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH2C6H4(4-OCH3)
H
0
0
0.20
0.21
100 100 100
0
0
CH3
100 100 100 45
100 100 100
100 100 100 50
50
0
45
0
0.16
CH2CH3
0
0.098
0.044
0.046
0.10
CH2CH2CH3
CH2CH2CH2CH3
CH2CH(CH3)2
CH2CH=CH2
50
0
50
0
100 100 100
100 100 100
0
0
9g
9h
0
0
100 100 100 80
80
80
0.011
100 100 100 30 30 30
37.5 100 100 100
0
0
0
9i
2
H
H
CH2C≡CH
150
100 100 100 100 100 100 0.0078
75
100 100 100 80 90 80
37.5 100 100 100 60 70 60
9j
2
2
2
2
2
2
2
2
2
H
H
H
F
F
F
F
F
F
H
H
H
F
F
F
F
F
F
CH2C≡CSi(CH3)3
CH2CO2CH2CH3
CH2CN
150
150
150
150
150
150
150
150
150
100 100 90
100 100 100
100 100 100
100 100 80
0
0
0
0
0
0
0.26
0.037
0.11
9k
0
0
9l
0
0
17a
17b
17c
17d
17e
17f
CH2C6H4(4-OCH3)
H
0
0
0.20
100 100 100 50
100 100 100 30
50
30
0
50
30
0
0.48
CH3
0.14
CH2CH3
100 100 100
0
0.11
CH2CH2CH3
CH2CH2CH2CH3
100 100 100 60
100 100 100 60
60
60
60
60
0.058
0.021
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17g
17h
2
2
F
F
F
F
CH2CH(CH3)2
CH2CH=CH2
150
150
75
100 100 90
0
0
0
0.084
100 100 100 50
100 100 100 40
100 100 100 30
80
70
30
70 0.0098
60
30
37.5
150
75
17i
2
F
F
CH2C≡CH
100 100 100 100 90 100 0.0067
100 100 100 90
100 100 100 80
85
80
0
90
80
0
37.5
150
150
150
150
75
17j
17k
17l
2
2
2
2
F
F
F
F
F
F
F
F
CH2C≡CSi(CH3)3
CH2CO2Et
100 100 90
100 100 85
100 100 100
0
0
0
0.17
0.014
0.20
0
0
CH2CN
0
0
17m
CH2CH2OCH3
100 100 100 80
80
80
0.12
100 100 85 50 50 50
37.5 100 100 80 30 30 30
19
3
F
F
CH2C≡CH
150
100 100 100 100 100 100 0.072
75
100
100
90
40
80
50
50
0
50
0
50
0
37.5
150
75
flumioxazin
trifludimoxazin
100 100 100 100 100 95
0.046
100 100 100 95
100 100 100 90
95
80
80
70
37.5
150
75
100 100 100 100 100 100 0.031
100 100 100 100 100 100
100 100 100 95 100 100
37.5
537
538
aAbbreviations: A.j., Abutilon juncea; A.r., Amaranthus retroflexus; E.P., Eclipta prostrata; D.s.,
Digitaria sanguinalis; E.C., Echinochloa crusgalli; S.f., Setaria faberii.
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Table 2. Experimental and Calculated pKi Values of Compounds 8, 9, 17, and 19.
pKia
pKia
compds
compds
exptl calcd
6.68 6.61
6.77 6.54
6.57 6.57
7.01 6.80
7.16 7.11
6.92 6.95
6.77 6.82
7.70 7.45
7.85 7.94
7.32 7.15
6.70 6.75
7.00 6.90
6.80 6.91
7.01 7.16
7.36 7.43
7.34 7.25
7.00 7.00
7.96 7.93
exptl calcd
8.11 8.27
6.77 6.91
7.43 7.37
7.43 7.23
6.68 6.67
6.32 6.35
8a
8bb
8c
9i
9j
9k
9l
17a
17b
8db
8e
8f
8g
8hb
8i
8k
9a
9bb
9cb
9d
9e
17cb 6.86 6.54
17d
17e
6.96 6.84
7.24 7.18
17fb 6.70 6.89
17g
17h
17i
17j
17k
17l
7.08 7.18
8.01 7.83
8.17 7.98
6.82 6.73
7.85 7.99
6.70 6.92
9f
9g
9h
17m 6.92 7.01
19b
8.17 8.13
a
b
540
541
pKi = −log Ki, test set compunds.
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542
Table 3. Herbicidal spectrum of compounds 8i, 9i, and 17i (post-emergence).
% inhibition
I.n. B.p. C.b. C.r. A.s. C.a. P.a. B.t. L.c. N.p. C.o. S.n. V.g.
dosage
compds
a
g ai/ha
8i
150
75
95 100 100 95
90 100 100 90
85 100 95
80 100 75
75 100 70
90 100 95
70 100 85
95 100 100 95 100 100
90
90
95 100 90 100 80
60 100 80 100 50
37.5
150
75
75 100 70
70
9i
17i
100 100 100 90
95 100 100 80
95 100 100 100 100 100
90 100 100 95 100 100
37.5
150
75
85 100 95
60
50
60
80
80
90 100 80 100 70
100 100 100 80 100 100 100 100 100 100 100 100 100
95 100 100 75
90 100 95 70
90 100 95
80 100 80
90 100 100 95 100 90
90 90 100 80 100 85
37.5
150
75
flumioxazin
100 100 100 90 100 100 100 100 100 100 100 100 100
100 100 100 85 100 100 100 100 100 100 100 100 100
100 100 100 80 100 100 100 80 100 100 100 100 100
37.5
543
544
545
546
547
aAbbreviations: I.n., Ipomoea nil; B.p., Bidens pilosa; C.b., Commelina benghalensis; C.r.,
Cyperus rotundus; A.s., Amaranthus spinosus; C.a., Chenopodium album; P.a.; Phytolacca
Americana; B.t., Bidens tripartite; L.c., linopodium chinense; N.p., Nicandra physaloides; C.o.,
Cassia obtusifolia; S.n., Solanum nigrum; V.g., Vicia gigantean.
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Table 4. Crop Selectivity of compounds 8i, 9i, and 17i (post-emergence, 150 g ai/ha).
% injury
compds
wheat
10
maize
15
rice
40
50
50
80
90
peanut
40
cotton soybean
8i
9i
17i
70
70
80
80
10
20
70
100
15
30
60
80
50
50
80
100
70
80
flumioxazin
80
80
trifludimoxazin
100
100
549
550
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