Discovery of Novel N-Isoxazolinylphenyltriazinones as Promising Protoporphyrinogen IX Oxidase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.9b04844

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the discovery and systematic SAR-based structure optimization of novel N-isoxazolinylphenyltriazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the new synthetic compounds displayed strong PPO inhibitory activity with K_i values in the nanomolar range. Some compounds exhibited excellent and broad-spectrum weed control at the rate of 9.375-37.5 g ai/ha by postemergence application and showed improved monocotyledonous weed control compared to saflufenacil. Most promisingly, ethyl 3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylate, 5a, with a K_i value of 4.9 nM, displayed over 2- A nd 6-fold higher potency than saflufenacil (K_i = 10 nM) and trifludimoxazin (K_i = 31 nM), respectively. Moreover, 5a showed excellent and broad-spectrum weed control against 32 kinds of weeds at 37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by postemergence application, indicating that 5a could be a potential herbicide candidate for weed control in paddy fields.

Full text of DOI:10.1021/acs.jafc.9b04844

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Agricultural and Environmental Chemistry
Discovery of Novel N-Isoxazolinylphenyltriazinones as
Promising Protoporphyrinogen IX Oxidase Inhibitors
Da-Wei Wang, Ruibo Zhang, Shuyi Yu, Lu Liang, Ismail
Ismail, Yong-Hong Li, Han Xu, Xin Wen, and Zhen Xi
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04844 • Publication Date (Web): 21 Oct 2019
Downloaded from pubs.acs.org on October 23, 2019
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Discovery of Novel N-Isoxazolinylphenyltriazinones as Promising Protoporphyrinogen IX
Oxidase Inhibitors
Da-Wei Wang,# Rui-Bo Zhang,# Shu-Yi Yu, Lu Liang, Ismail Ismail, Yong-Hong Li, Han Xu, Xin
Wen, and Zhen Xi*
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.
*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: Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide
discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists.
Here, we describe the discovery and systematic SAR-based structure optimization of novel
N-isoxazolinyl-phenyl-triazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in
vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the
new synthetic compounds displayed strong PPO inhibitory activity with Ki values in the
nanomolar range. Some compounds exhibited excellent and broad-spectrum weeds control at the
rate of 9.375-37.5 g ai/ha by postemergence application, and showed improved monocotyledonous
weeds
control
compared
to
saflufenacil.
Most
promisingly,
ethyl
3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,
5-dihydroisoxazole-5-carboxylate 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold
higher potency than saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively.
Moreover, 5a showed excellent and broad-spectrum weeds control against 32 kinds of weeds at
37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by post-emergence
application, indicated that 5a could be a potential herbicide candidate for weed control in paddy
fields.
30
31
Keywords: molecular design, herbicide, isoxazoline, protoporphyrinogen IX oxidase, triazinone,
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INTRODUCTION
Weed management has been a concern throughout the world’s history. Though significant
improvement has been made in the technologies to control weeds, there is still a high demand for
effective methods of control to help increase agricultural productivity. Weeds compete with crops
for nutrients, water, and light which, in turn, reduces the quality and yields of crops. Using
herbicides is one of the most effective ways to manage weeds and preserve crop quality and yields.
Discovering new herbicides with excellent and broad-spectrum of weed control (inclusive resistant
weeds), and good crop selectivity is a key objective for weeds management in crop fields.1
Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) inhibitory herbicides are among the most widely
used herbicides in the crop fields around the world. These herbicides can inhibit the PPO in
planta, which is the penultimate enzyme in the biosynthesis of chlorophyll. In normal conditions,
plants can catalyze protoporphyrinogen IX to protoporphyrin IX by PPO in chloroplasts and
mitochondria.2, 3 However, when treated by PPO herbicides, the activity of PPO in chloroplasts is
inhibited, resulting in the accumulation of protoporphyrinogen IX in chloroplasts. This, in turn,
causes the protoporphyrinogen IX leaks into the cytoplasm. In the cytoplasm, protoporphyrinogen
IX can undergo an auto-oxidation process to protoporphyrin IX. In the presence of light and O2,
protoporphyrin IX can generate reactive oxygen species (ROS), which can cause lipid
peroxidation and cell death.4-8 PPO herbicides have many advantageous characteristics, such as
low mammalian toxicity, benign environmental effects, broad herbicidal spectrum (including
dicotyledon and monocotyledon weeds as well as some resistant biotypes), and a low application
rate.9
The usage of PPO herbicides to decimate weeds in crop fields can be traced back to the
1960s.9 With more than a half-century of developments, many different types of PPO inhibitors
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have been discovered. They are mainly divided into diphenyl ethers, phenylpyrazoles, oxadiazoles,
thiadiazoles, triazolinones, pyrimidinediones and N-phenyl-phthalimides.10 Among them, diphenyl
ethers are considered relatively old types of herbicides, while pyrimidinediones are relatively new
types of herbicides, discovered at the beginning of the 21st century.11, 12 For example, butafenacil
(Figure 1) was commercialized by Syngenta in 2001, and it was mainly used as a non-selective
herbicide for pre- and post-emergence weed control at 75-150 g ai/ha. Saflufenacil (Figure 1) was
commercialized by BASF in 2009; it was deemed as the most successful herbicides in the past 20
years. Saflufenacil can effectively control almost all important broad-leaf biotypes by pre- or
post-emergent application at 18-125 g ai/ha. Tiafenacil (Figure 1) was commercialized by
FarmHannong Co., Ltd. in 2018, as a non-selective herbicide which mainly used for
post-emergence broadleaf and grass weeds control and pre-plant burndown at 25-250 g ai/ha.13
Trifludimoxazin (Figure 1) belongs to triazinone herbicides and is expected to be commercialized
by BASF in 2020. It is the first PPO herbicides containing novel triazinone moiety. Besides
bearing the attractive properties of PPO herbicides, trifludimoxazin can also suppress the
tough-to-control PPO-resistant weed populations, such as ragweed and pigweed species.14 In fact,
the increased market share of PPO herbicides can attribute to the widespread resistant weeds of
glyphosate, triazine, and AHAS herbicides.15 However, the extended exposure of some PPO
herbicides may lead to the development of herbicide-resistant biotypes. Therefore, discovering of
new PPO inhibitors with unique structure and improved performance is still in demand.
Although many structural diverse PPO herbicides have been discovered, the general
structure-activity relationships (SARs) of the aryl heterocycles are often conflicting.16 Studies
have shown that for inhibitors with heterocyclic moiety attached to a phenyl ring, in most cases,
substituting electron-withdrawing lipophilic groups at 2-position of phenyl ring, are favorable to
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activity, with fluorine atom gives superior result; placing substituents at 3-position can decrease
activity; installing electron-withdrawing lipophilic groups at 4-position are favorable to activity,
with chlorine atom shows the best biological activity; a variety of functional groups (including
five- or six-membered ring systems) are acceptable for activity at 5-position, the substituents at
this position have a great impact on crop selectivity and overall herbicidal activity.17 Isoxazoline is
an oxygen-nitrogen containing five-membered heterocyclic ring has attracted considerable
attention of agricultural and medicinal chemists due to its derivatives possessing various biological
important activities, such as herbicidal, insecticidal, fungicidal, anticancer, and anti-inflammatory
properties.18-22 On the basis of the above investigations, we inferred that integrating the
isoxazoline moiety into the N-phenyltriazinone skeleton may expedite the discovery of novel
herbicides. In continuation of our search for potent PPO inhibitors, herein, a series of
N-isoxazolinylphenyltriazinones with isoxazoline group at the 5-position of phenyl ring were
designed (Figure 2). The detailed synthetic methods, herbicidal activity, Nicotiana tabacum
mitochondrial PPO (NtPPO) inhibitory activity and quantitative structure-activity relationship
(QSAR) of N-isoxazolinylphenyltriazinones 5-9 are described.
MATERIALS AND METHODS
Synthesis of N-isoxazolinylphenyltriazinones 5-9.
Scheme 1 and 2 describe the detailed synthetic routes for compounds 5-9. For detailed synthetic
methods, NMR and HRMS characterization data of 5-9, see the Supporting Information.
Herbicide Activity Assay. In the first round of experiment, four representative weeds:
Abutilon juncea (ABUJU), Amaranthus tricolor (AMATR), Echinochloa crus-galli (ECHCG) and
Digitaria sanguinalis (DIGSA) were used to evaluate the post-emergence herbicidal activity of
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compounds 5-9, and the concentrations used to evaluate the herbicidal activity of 5-9 varied from
9.375 g ai/ha to 150 g ai/ha. The testing methods were similar to our previously reported
procedures.10, 23-27 In order to evaluate the post-emergence herbicidal spectrum of compound 5a,
another 28 kinds of weeds: Cassia tora (CASTO); Leptochloa panicea (LEPPA); Aeschynomene
indica (AESIN); Vicia gigantean (VICGI); Setaria faberii (SETFA); Sorghum halepense
(SORHA); Pharbitis nil (PHANI); Xanthium sibiricum (XANSI); Bidens pilosa (BIDPI);
Clinopodium chinense (CLICH); Eclipta prostrate (ECLPR); Commelina benghalensis (COMBE);
Phytolacca acinosa (PHYAC); Beckmannia syzigachne (BECSY); Orychophragmus violaceus
(ORYVI); Brassica juncea (BRAJU); Capsella bursa (CAPBU); Stellaria media (STEME);
Veronica didyma (VERDI); Chenopodium serotinum (CHESE); Trifolium repens (TRIRE); Rumex
acetosa (RUMAC); Descurainia sophia (DESSO); Polypogon fugax (POLFU); Poa annua
(POAAN); Eleusine indica (ELEIN); Isodon serra (ISOSE); Boehmeria nivea (BOENI) were
tested at the concentration of 18.75-75 g ai/ha, saflufenacil was used as a positive control.
Twenty-five days after treating by compounds, the herbicidal activities (% inhibition) were
evaluated. For each compound at each tested concentration, three replications were used. The
results are shown in Tables 1-4, S1 and S2, and Figure 3.
Crop Selectivity Test. Six conventional crops: rice, wheat, maize, peanut, soybean, and
cotton were selected for the greenhouse post-emergence crop selectivity assay.10, 12, 25 Flowerpots
(diameter = 9 cm) were filled with mixed soil (Vvegetable garden soil:Vseedling substrate = 1:2) to 3/4 of their
depth, at least 20 tested crop seeds were sowed in pots and then the seeds were covered with a thin
layer of mixed soil. When crops grew to 3-4 leaf stage, they were treated by the tested compounds
at the concentration of 150 g ai/ha. After twenty-five days post-treatment, the results (Table 5) of
crop safety were evaluated, with three replications per test.
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PPO Inhibition Assay. The expression and purification of Nicotiana tabacum mitochondrial
PPO2 (NtPPO) were performed as described previously.10-12, 28, 29 Protoporphyrinogen IX was
prepared by reduction of protoporphyrin IX with newly synthesized sodium amalgam.
Protoporphyrin IX has a maximum excitation at 410 nm and a maximum emission of 630 nm.
Therefore, in the kinetic inhibition assays, we used a fluorescence detector to monitor the
formation of the protoporphyrin IX by setting the emission wavelengths to 630 nm and excitation
wavelengths to 410. Compounds were dissolved in dimethyl sulfoxide (DMSO) as a stock solution
and diluted to the different concentration ranges from 0.5 nM to 50 mM just before testing. The
total volume of reaction solution was 200 L, which consists of 100 mM potassium phosphate
buffer (pH 7.4), 5 M FAD, 0-40 g PPO enzyme, 200M imidazole, 5 mM DTT, 0.03% Tween
80 (v/v) and 1 mM EDTA.
Molecular Docking and 3D-QSAR studies. The crystal structure of NtPPO (PDB ID: 1SEZ) was
taken from RCSB Protein Data Bank and prepared by AutoDockTools-1.5.6 with the standard
method before calculation studies.2, 30 Because there is a chiral center at the 5-position of
isoxazoline ring, in order to understand which isomer is favorable towards activity, we used
molecular mechanics Poisson−Boltzmann surface area (MM-PBSA) method to calculate the
binding free energy of a representative compound 5a. The results indicated that R-configuration
showed higher PPO inhibitory activity than S-configuration (Table S3). Therefore, in this study,
we mainly investigate the effect of R-configuration of 5-9 on activity. SYBYL-X 6.9 (Tripos, Inc.,
St. Louis, MO) was used to build the 3D structures of N-isoxazolinylphenyltriazinones 5-9.
AutoDock4.2 was used for docking calculations of inhibitors with NtPPO. The docking runs for
each molecule were set as 300 times, and the optimum binding modes were selected on the basis
of the docking energy. Open3DAlign and Open3DQSAR were used to build the QSAR model of
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the synthetic N-isoxazolinylphenyltriazinones.31, 32 The best binding mode of R-5a was used as a
template, and then the other compounds were superimposed to R-5a by Open3DAlign. The aligned
compounds were transferred to Open3DQSAR to calculate the QSAR model. According to the
results of Open3DQSAR, 42 compounds were finally selected to build the 3D-QSAR model, and
33 and 9 compounds were used as training and test sets, respectively. A grid box with 5.0 Å
margin and 0.5 Å step size was set around the molecules. Electrostatic potential and steric factors
of molecular interaction fields were then calculated. PyMOL 1.3 was used to visualize the QSAR
results.
Stability Study of 5a in the Enzymatic Reaction Solution. 2 L of 5a (1 mM), 33 L of the
testing buffer, 100 L of water, 60 L of NtPPO, and 5 L protoporphyrinogen IX were mixed in
the reaction tube for 10 min. Then, 200 L of CH3OH was added to the reaction solution and
stirred variously for 10 min. Next, the mixture was centrifuge at 12000 g for 10 min. The
supernatant was filtered through 0.22 m filter membrane and the solution was then analyzed by
High-performance liquid chromatography (HPLC) (Agilent 1200 Infinity Series, 5.0 m, 4.6 150
mm) at 254 nm with the follow rate of 1 mL/min. The mobile phases used were acetonitrile and
water, Vacetonitrile : Vwater = 60 : 40.
Degradation Study of 5a in Abutilon juncea. Three-leaf old Abutilon juncea in the herbicidal
assay was chosen as the test weed.33 A solution of 5a (75 M, containing 1‰ DMF and 1‰ tuwen
80) was sprayed on the surface of the leaves, then the treated weeds were moved to the greenhouse,
about 2 h post-treatment, the leaves of Abutilon juncea began to drop. Next, the leaves were cut,
and about 1 g of leaves were used in further analysis. The leaves were washed with 10 g of water
(containing 1‰ DMF and 1‰ tuwen 80) for 5 min, and then the water on the surface of leaves
was removed by filter paper. The leaves were transferred to a triturator and triturated together with
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liquid nitrogen, and then 1 mL of acetonitrile was added. After 1 h, the mixture was centrifuged
and the supernatant was used in the UPLC-HRMS (Waters Acquity®UPLC H-class, Waters Xevo
G2-XS Q-TOF) analysis.
RESULTS AND DISCUSSION
Chemistry. The synthetic routes for N-isoxazolinylphenyltriazinones 5-9 are illustrated in Scheme
1 and 2. Starting with 2-chloro-4-substituted-5-nitrobenzaldehyde 1, the corresponding title
compounds 5a, 6a, 7a, and 8a were prepared in a linear six-step reaction.19, 34 The aldehydes 1
reacted with hydroxylamine hydrochloride in ethanol/water solution to afford the corresponding
aldoximes 2 in excellent isolated yields (91-96%). Then the aldoximes 2 reacted with
N-chlorosuccinimide
(NCS)
in
DMF
to
provide
the
2-chloro-4-substituted-N-hydroxy-5-nitrobenzimidoyl chlorides, which subsequently underwent
cycloadditions with CH2=CR1R2 to give isoxazolines 3 in yields of 20-99%. The key intermediates
4 can be easily obtained by reducing the nitro groups of 3 with iron powder and NH4Cl in
refluxing ethanol solution (90%). Finally, 4 reacted with CO(OCCl3)2 in toluene with Et3N as a
catalyst to provide the corresponding isocyanates, without further purification, the isocyanates
then reacted with CS(NHR3)2 and followed by
a
ring-closure reaction with
N,N'-carbonyldiimidazole to give the title compounds 5a, 6a, 7a, and 8a in 39-82% yields.
The synthesis of compounds 5b-r, 6b-e, 7b-e, 8b-e and 9 started with hydrolyzing the
ethoxycarbonyl groups of 5a, 6a, 7a and 8a in mixed acids solutions (H2SO4, HOAc, and H2O) at
100 C to give the corresponding carboxylic acids 5b, 6b, 7b and 8b in good isolated yields
(67-81%). Then the carboxylic acid derivatives reacted with various R4I or R4Br in DMF with
K2CO3 as a base at room temperature to afford ester-containing compounds 5c-r, 6c-e, 7c-e and
8c-e in 55-95% yields. The amide-containing compounds 9 were prepared in a consecutive
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two-step reaction began with carboxylic acid 5b, which underwent acyl chlorination with (COCl)2
to give the acid chloride. Subsequently, the acid chloride reacted with R5NH2 in CH2Cl2 with Et3N
as a base to provide the amide derivatives 9 in 69-85% yields.
Herbicidal Activity and SAR. According to the general SARs of the aryl heterocycles we have
discussed in the introduction, compound 5a was first synthesized, and then its herbicidal activity
was evaluated against two representative dicotyledon weeds (A. juncea and A. tricolor) and two
representative monocotyledon weeds (E. crus-galli and D. sanguinalis) by both pre- and
post-emergence application. One of the most widely used PPO inhibitory herbicides saflufenacil
was selected as a positive control. As shown in Table S1, 5a exhibited complete control against 4
kinds of tested weeds by both pre- and post-emergence application at 150 g ai/ha, which were
more active than saflufenacil in pre-emergence weeds control and as potent as saflufenacil in
post-emergence weeds control. It is worth mentioning that even at the post-emergence application
rate as low as 9.375 g ai/ha, 5a still showed over 80% control against the four tested weeds,
however, at the same test conditions saflufenacil only displayed mild inhibition against the tested
monocotyledon weeds (E. crus-galli and D. sanguinalis). Inspired by the promising herbicidal
activity results of 5a, we intended to make systematically structural optimization and explore the
SARs of the designed N-isoxazolinylphenyltriazinones 5-9. Because the post-emergent herbicidal
activity of 5a was better than its pre-emergent activity, herein, in this work we mainly study the
post-emergence activity of 5-9. The results (Tables 1-4) indicated that most of the synthetic
compounds displayed excellent post-emergence herbicidal activity against the four weeds tested,
some of them showed higher monocotyledon weeds control than saflufenacil and nearly
equipotent to that of the new PPO herbicide trifludimoxazin.
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At first, we kept the X group as a fluorine atom and studied the effect of the substituents of R1,
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R2, and R3 on herbicidal activity (Table 1). It was found that when sterically bulky groups, such as
ethyl and n-propyl groups were introduced to R3, compounds would show decreased activity with
the increased size of substituents (-CH3 > -CH2CH3 > -CH2CH2CH3). Having identified methyl
group was the optimum substituent at R3 we turned to modify of R1 and R2, compounds 5a3-a5
were designed and synthesized by fixing R3 as the methyl groups (Scheme 1). Substituting the
methyl group of 5a at R1 with a hydrogen atom (5a3) could significantly reduce its herbicidal
activity against the broadleaf weeds (A. juncea and A. tricolor). Changing the ethoxycarbonyl
group of 5a to an ethyl group (5a4) were found detrimental to monocotyledon weeds (E. crus-galli
and D. sanguinalis) control. However, when the terminal -CH3 in the ethyl group of 5a4 was
changed to an acetoxy group (5a5), both the herbicidal potency and spectrum were significantly
reduced compared to its parent compound 5a. Based on the above discussion, we can conclude
that the size of substituents at R3 had a significant impact on herbicidal potency, and the herbicidal
spectrum and activity could be ‘tuned’ by modifying of the substituents at R1 and R2.
In most cases, for the N-phenyl heterocyclic types of PPO inhibitors, the SAR of 2-position of
35, 36
phenyl ring is F > Cl > Br.10,
In order to study whether the novel
N-isoxazolinylphenyltriazinones fit these rules or not, we designed and synthesized compounds
5b-f and 6-8 by keeping the R1 and R3 fixed as methyl groups, and introduced of fluorine,
hydrogen, chlorine, and bromine atoms at the X group (Table 2). It was found that when other
substituents were kept the same, fluoro-substituted compounds (5a-f) always displayed
significantly enhanced herbicidal potency than their corresponding hydrogen, chlorine, and
bromine substituted analogs 8, 6, and 7, respectively (F > H > Cl >Br). Therefore, we can
conclude that for the SAR of 2-position of the phenyl ring of the new
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N-isoxazolinylphenyltriazinones also conform to classical SARs of the N-phenyl heterocycles.
Having obtained some very useful SARs on the basis of the preliminary herbicidal activity,
namely, the introduction of methyl groups at R1 and R3 were favorable to activity, and placing
fluorine atom at the X group could significantly improve the potency of the target compounds. We
then considered varying the spatial and electronic properties of the ester group of 5a, the results of
these changes are shown in Tables 2-4.
As shown in Tables 2 and 3, substituting the ethyl group of 5a with a hydrogen atom (5b)
caused a significant drop in herbicidal potency against broadleaf weeds (A. juncea and A. tricolor),
and a slight drop in monocotyledon weeds (E. crus-galli and D. sanguinalis) control
at
9.375-37.5 g ai/ha. However, the replacement of ethyl group with methyl, n-propyl, or n-butyl
groups, as in compounds 5c, 5d, and 5e, did not affect much on herbicidal activity. A possible
reason for the decreased in the activity of 5b is that the carboxyl group at R2 would decrease the
absorption of inhibitor by leaves. Therefore, in the next round of optimization, a variety of
compounds (5g-r) with lipophilic substituents at R4 were synthesized and tested (Scheme 2 and
Table 3). The installment of one or two fluorine atom(s) on the terminal -CH3 of -CH2CH3 (5a) or
-CH2CH2CH3 (5d) caused a slight drop of herbicidal activity. For examples, compound 5g (R4 =
-CH2CH2F) showed a limited drop in A. juncea and A. tricolor control in comparison with 5a at
9.375 g ai/ha; 5h (R4 = -CH2CHF2) exhibited decreased E. crus-galli and D. sanguinalis control
than 5g; 5i (R4 = -CH2CH2CH2F) displayed diminished potency than 5d at 9.375 g ai/ha. In
addition, we have found that there was some variation in herbicidal potency when increasing the
length and size of the R4 group. While it is clear that in most cases increasing the size and length
of the R4 group could lead to an increased herbicidal effect in comparison with the non-substituted
compound 5b. For example, substituting -CH2CO2C2H5 (5j), -CH(CH3)CO2C2H5 (5k),
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-C(CH3)2CO2C2H5 (5l), and -CF2CO2C2H5 (5n) at R4 were found beneficial to herbicidal potency
against A. juncea and A. tricolor; similarly, placing a benzyl group at R4 resulted in enhanced
weeds control than 5b (5o > 5b). When a strong electron-withdrawing group (-CF3, 5p) was
introduced to the para-position of benzyl group, the herbicidal potency of 5o was impaired in large
extent (5o > 5p), while substituting weak electron-withdrawing (F, 5q) or electron-donating (-CH3,
5r) group at the para-position resulted in a slight drop in E. crus-galli and D. sanguinalis
inhibition.
To gain additional SAR of the R2, compounds 9 with amide groups at R2 were designed and
synthesized (Scheme 2 and Table 4). Unfortunately, all the amide-containing compounds proved
to significantly reduce herbicidal potency compared to their relative ester analogs, exemplified by
compounds 9a (R5 = -CH3), 9b (R5 = -CH2CH3) or 9d (R5 = -CH(CH3)2) which showed decreased
herbicidal effect in comparison with compounds 5c (R4 = -CH3), 5a (R2 = -CO2C2H5) or 5f (R4 =
-CH(CH3)2), respectively. Studies have proved that the N-isopropyl-N-methylsulfamide side chain
37, 38
of saflufenacil is crucial for the absorption and translocation of the herbicide in plants.
Therefore, we inferred that introducing the N-isopropyl-N-methylsulfamide to a compound would
improve its herbicidal performance. As envisaged, compound 9i exhibited significantly improved
herbicidal activity than other amide-containing analogs 9a-h. It should be mentioned that 9i
displayed complete control against E. crus-galli and over 90% inhibition against D. sanguinalis
even as low as 9.375 g ai/ha, which were far more potent than saflufenacil at the same condition.
Herbicidal Spectrum. In order to investigate the herbicidal spectrum of the synthetic
N-isoxazolinylphenyltriazinones, compound 5a with excellent weeds control were selected for
further herbicidal evaluation against another 29 different kinds of weeds. Saflufenacil was used as
a positive control. As shown in Figure 3 and Table S2, 5a provided more than 85% control against
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28 weeds at 75 g ai/ha and of 27 weeds at 37.5 g ai/ha. The average inhibition ratios of 5a against
28 weeds at 75 and 37.5 g ai/ha was 97.9% and 95.4%, respectively; and of saflufenacil at 75 and
37.5 g ai/ha was 97.3% and 91.6%, respectively. The overall results revealed that 5a also
displayed improved monocotyledon weeds (S. faberii, S. halepense, B. syzigachne, E. indica)
control than saflufenacil at the rate of 37.5 g ai/ha in this test.
Crop Selectivity. The post-emergence crop safety of compound 5a was tested against six
representative crops: rice, wheat, maize, peanut, soybean, and cotton. As shown in Table 5, the
entire tested crop exhibited high sensitivity to saflufenacil at 150 g ai/ha, and the phytotoxicity of
5a on wheat, maize, soybean, cotton, and peanut was similar to that of saflufenacil. At the same
test conditions, rice injury to 5a was far less than with saflufenacil. Although temporary leaf burn
symptom was observed at the first several days of rice treated by 5a, however, at the seven days
post-application rice could outgrow from the initial injury, and the growth of new leaves was
almost not affected. The promising results indicated that 5a could be developed as an herbicide for
weeds control in paddy fields.
PPO inhibitory activity. To gain additional structural information of the synthetic
N-isoxazolinylphenyltriazinones as PPO inhibitors, we tested the Ki values of 5-9 against NtPPO.
As can be seen from Tables 1-4, the majority of the synthesized compounds exhibited strong PPO
inhibitory activity with binding affinities (Ki) values generally in the single-digit nanomolar range.
For examples, compound 5a (Ki = 4.9 nM), showed two and six times higher potency than
saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively; compound 5c (Ki = 1.7
nM), about 5 and 18 times more activity than saflufenacil and trifludimoxazin, respectively.
In most cases, the in vitro activity of 5-9 was consistent with their corresponding in vivo
herbicidal activity. For instance, introducing sterically-bulky groups at R3 was found detrimental
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to PPO inhibitory activity (-CH3 > -CH2CH3 > -CH2CH2CH3); when other substituents remained
the same, the fluoro-substituted analogs (X = F) exhibited higher activity than the hydro-, chloro-
and bromo-substituted analogs (F > H > Cl > Br). However, there was some inconsistency when
comparing the PPO-inhibiting activity of the amide-containing compounds 9a-h with their
herbicidal activity. The PPO inhibitory activity of 9a-h was comparable to their corresponding
ester analogs, whereas the herbicidal potency of 9a-h decreased in large extent. This data indicated
that the introduction of the amide groups at R2 was advantageous to PPO inhibition, while the
presence of amide groups of 9a-h may affect the absorption of compounds by plants.
To elucidate the effect of substituents on PPO inhibitory activity, we performed molecular
modeling studies of four representative compounds 5a, 5a1, 5a2 and 9h with NtPPO. By
calculating the binding free energy (ΔGbind) of R-5a and S-5a with NtPPO using MM-PBSA
method, we observed that the ΔGbind for R-5a and S-5a were -29.11 and -28.59 kcal/mol,
respectively (Table S3). The result insinuated that both isomers of 5a can bind tightly to the
receptor, however, R-5a showed a slightly more potent than S-5a. So, in the subsequent molecular
modeling studies, we mainly investigated the interaction of R-configuration of inhibitors with the
receptor. As shown in Figure 4A, the binding mode of 5a was similar to other reported PPO
inhibitors which bound to the activity site of NtPPO by simulating part of the structure of the
enzyme substrate protoporphyrinogen IX.2, 4, 10 The thiotriazinone ring of 5a established a π−π
interaction with the residue of F392, the benzene ring was sandwiched between the residues of
L372 and L356, and the oxygen atom of the isoxazoline ring could form two favorable hydrogen
bonding interactions with the residue of R98 (1.3 Å and 2.6 Å). Because ethyl is a relatively bulky
group than methyl, as the docking results suggested that the introduction of ethyl groups at R3
would increase the steric clashes with surrounding residues that, in turn, precluded the
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thiotriazinone ring from binding to its original site (Figure 4B). However, the retained activity of
5a1 could attribute to the hydrogen bonding interactions of thiotriazinone ring with R98 (3.0 Å)
and ester group with FAD (3.2 Å), and a T-shape π−π interaction of benzene ring with F192.
Replacement of the ethyl groups with n-propyl groups at R3 also made a significant conformational
change of 5a2 relative to 5a1 (Figure 4C). It is clear that substituting the sterically bulky benzyl
group at R5 did not alter the binding conformation of 9h when compared with 5a. As depicted in
Figure 4D, 9h was nicely accommodated in the binding pocket. Almost all the important
interactions of 5a with the surrounding residues were retained in 9h, and the benzyl ring of 9h
stretched toward the bottom of the binding pocket and formed a π−π interaction with the
isoalloxazine ring of FAD. Therefore, we inferred that these privileged interactions were the key
driving forces for excellent inhibitory activity of 9h.
3D-QSAR Modeling. To gain additional molecular mechanism on PPO inhibitory activity, we
investigated the three-dimensional quantitative structure-activity relationships (3D-QSAR) of the
N-isoxazolinylphenyltriazinones 5-9 by using the Open3DQSAR software.39 The characteristics of
the QSAR model were as follows: conventional coefficient r2 was 0.93, standard deviation of error
of calculation (SDEC) was 0.18, non-cross-validated coefficient r2
was 0.88, cross-validated
pred
coefficient q2 was 0.74, and standard deviation of error of prediction (SDEP) was 0.20 (Table S4),
suggesting the high reliability of prediction. The calculated and predicted values (pKi) of all
compounds used in the QSAR study are reported in Table 6, the alignments of 33 set compounds
were shown in Figure S1, and the correlation plot of predicted versus calculated Ki values was
p
depicted in Figure S2. As can be seen from Figure 5A, the yellow polyhedral of the steric
contribution contour maps was located at the 2-position of the phenyl ring of 5a, indicating that
the introduction of bulky substituents at this site was detrimental to activity. For examples,
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compounds 5a (X = F, Ki = 4.9 nM) and 8a (X = H, Ki = 50 nM) showed higher activity than 6a
(X = Cl, Ki = 121 nM) and 7a (X = Br, Ki = 172 nM). Whereas only two very small green contours
were located near the 2-position of the isoxazoline ring, suggesting that the steric effect at this
position on activity was not significant. From Figure 5B we can observe that there are mainly two
red regions of electrostatic contribution. The big one was situated near the 2-position of the phenyl
ring, which meant that introducing the electronegative groups at this site are advantageous to
activity, exemplified by compounds 5 which exhibited improved activity over the corresponding
analogs 6 and 7. The relatively small red region was located near 2-position of isoxazoline ring,
however, several small blue contours also situated near the 2-position insinuated that the
electrostatic contribution to activity was not obvious.
In vitro and In vivo Degradation Study of 5a. It is reported that isoxazoline-containing
compounds are prone to decompose and/or rearrange in vivo.21 In order to understand whether the
synthesized N-isoxazolinylphenyltriazinones are pro-herbicides or if the isoxazoline functional
group is intact in the enzyme active site. A representative compound 5a was selected for further
degradation studies. As shown in Figure 6A and S3, the purity of 5a is about 99.58%, and the
retention time of 5a under the test condition is 6.96 min. When incubated in the enzymatic
reaction solution for 10 min, there was almost no hydrolysis occurred of 5a, indicating that 5a was
intact during the enzymatic assay. The in vivo degradation study suggested that after 2 hours, there
was still some 5a in the weeds (Figure 6B, 6C and S4). Although it is hard to draw a definite
conclusion about whether the isoxazoline functional group of 5a is degraded in Abutilon juncea or
not, because no specific metabolites peaks were detected; however, it is clear that until Abutilon
juncea completely wither, 5a still remained in the plants, this may because of the quick burn down
effect of 5a.
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In summary, we have discovered a novel series of N-isoxazolinylphenyltriazinones 5-8 by
using scaffold hopping strategy and systematic SAR-guided structure optimization. Many of the
synthetic compounds showed strong PPO inhibitory activity with Ki values in single-digit or
double-digit nanomolar range. Some compounds exhibited an excellent and broad spectrum of
weeds control at the rate of 9.375-37.5 g ai/ha by postemergence application, compared with
saflufenacil, the monocotyledonous weeds control which was improved in large extent. Especially,
compound 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold higher potency than
saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively. The herbicidal spectrum
experiments indicated that 5a showed excellently and broad-spectrum of weeds control against 32
kinds of tested weeds at 37.5-75 g ai/ha by post-emergent application, which was a slightly
improved than that of saflufenacil at the same conditions. Most promisingly, 5a exhibited
relatively safe to rice at 150 g ai/ha by post-emergence application, insinuated that 5a may be used
for the development of potential herbicide candidate for weeds control in paddy fields. Our present
work has demonstrated that N-isoxazolylphenyltriazinone motif can be used as a novel scaffold for
herbicide discovery.
Supporting Information
The detailed synthetic routes, 1H and 13C NMR and HRMS spectra for
N-isoxazolinylphenyltriazinones 5-9; superimposition of the structures 33 training set compounds
(Figure S1); plot of the Open3DQSAR calculated versus experimental pKi values (Figure S2);
pre-emergent herbicidal activity of 5a (Table S1); post-emergent herbicidal spectrum of 5a (Table
S2); calculated binding free energies of R-5a and S-5a with NtPPO (Table S3); statistics of
3D-QSAR model (Table S4) are shown in the Supporting Information.
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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, 21877066), and Tianjin Natural Science Foundation (No.
16JCYBJC20200).
Notes
The authors declare no competing financial interest.
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syntheses of novel N-(benzothiazol-5-yl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione and
N-(benzothiazol-5-yl)isoindoline-1,3-dione as potent protoporphyrinogen oxidase inhibitors.
J. Agric. Food Chem. 2011, 59, 6172-6179.
37. Grossmann, K.; Niggeweg, R.; Christiansen, N.; Looser, R.; Ehrhardt, T., The herbicide
saflufenacil (Kixor™) is a new inhibitor of protoporphyrinogen IX oxidase activity. Weed Sci.
2010, 58, 1-9.
38. Grossmann, K.; Hutzler, J.; Caspar, G.; Kwiatkowski, J.; Brommer, C. L., Saflufenacil
(Kixor™): biokinetic properties and mechanism of selectivity of a new protoporphyrinogen
IX oxidase inhibiting herbicide. Weed Sci. 2011, 59, 290-298.
39. Tosco, P.; Balle, T., A 3D-QSAR-driven approach to binding mode and affinity prediction. J.
Chem. Inf. Model. 2012, 52, 302-307.
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527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
Schemes and Figures:
Scheme 1. Synthesis of compounds 6a-8a. Reagents and conditions: (a) C2H5OH, H2O, 0 C-r.t.;
(b) NCS, DMF, 35 C; (c) CH2=CR1R2, Et3N, CH2Cl2, 0 C；(d) Fe, NH4Cl, C2H5OH (90%),
reflux; (e) CO(OCCl3)2, Et3N, toluene, 0 C-reflux; (f) CS(NHR3)2, N,N'-carbonyldiimidazole,
Et3N, toluene, r.t.-85 C.
Scheme 2. Synthesis of compounds 5b-u, 6b-f, 7b-f, 8b-f, and 9. Reagents and conditions: (a)
H2SO4, HOAc, H2O, 100 C; (b) R4I or R4Br, K2CO3, DMF, r.t.; (c) (COCl)2, CH2Cl2, 0 C-r.t.；(d)
R5NH2, Et3N, CH2Cl2, 0 C-r.t..
Figure 1. Chemical structures, introduction companies and commercialization years of butafenacil,
saflufenacil, tiafenacil, and trifludimoxazin.
Figure 2. The design strategy of N-isoxazolinylphenyltriazinones 5-9.
Figure 3. Post-emergence herbicidal spectrum of compound 5a and saflufenacil. Abbreviations:
CASTO, Cassia tora; LEPPA, Leptochloa panicea; AESIN, Aeschynomene indica; VICGI, Vicia
gigantean; SETFA, Setaria faberii; SORHA, Sorghum halepense; PHANI, Pharbitis nil; XANSI,
Xanthium sibiricum; BIDPI, Bidens pilosa; CLICH, Clinopodium chinense; ECLPR, Eclipta
prostrate; COMBE, Commelina benghalensis; PHYAC, Phytolacca acinosa; BECSY,
Beckmannia syzigachne; ORYVI, Orychophragmus violaceus; BRAJU, Brassica juncea; CAPBU,
Capsella bursa; STEME, Stellaria media; VERDI, Veronica didyma; CHESE, Chenopodium
serotinum; TRIRE, Trifolium repens; RUMAC, Rumex acetosa; DESSO, Descurainia sophia;
POLFU, Polypogon fugax; POAAN, Poa annua; ELEIN, Eleusine indica; ISOSE, Isodon serra;
BOENI, Boehmeria nivea.
Figure 4. The molecular docking results of 5a, 5a1, 5a2 and 9h with NtPPO, the R configuration
of the four inhibitors were used in this study. The inhibitors are shown as magenta sticks and the
key residues around the active site are shown as blue sticks. (A) Binding mode of 5a with NtPPO.
(B) Binding mode of 5a1 with NtPPO. (C) Binding mode of 5a2 with NtPPO. (D) Binding mode
of 9h with NtPPO.
Figure 5. Contour maps of the 3D-QSAR model, 5a is located inside the field. (A) Steric and (B)
electrostatic contributions.
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555
556
557
558
Figure 6. Degradation analysis of 5a in the assay solution and in Abutilon juncea. (A) HPLC
analysis of 5a and 5a in the enzymatic solution; (B) UPLC analysis of the degradation of 5a in
Abutilon juncea; (C) HMRS analysis of the peak in the retention time of 6.6 min of (B).
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X
Cl
X
Cl
X
Cl
a
b
c
N
NH2OH HCl
+
N
O2N
O
O2N
CHO
O2N
OH
R2
1a
1b
1c
1d
: X = F
R1
2a
: X = F
2b
: X = Cl
2c
: X = Br
2d
: X = H
3a
: X = F
: X = Cl
: X = Br
: X = H
: X = Cl
: X = Br
: X = H
3b
3c
3d
X
Cl
X
Cl
O
R3
N
N
d
f
e
H2N
N
N
O
O
R2
R2
S
N
R3
O
R1
R1
4a
: X = F
5a
6a
7a
8a
: X = F
4b
: X = Cl
4c
: X = Br
4d
: X = H
: X = Cl
: X = Br
: X = H
Scheme 1.
X
Cl
O
N
N
N
N
O
O
O
R4
S
X
N
Cl
X
Cl
O
5c-r
: X = F
: X = Cl
: X = Br
: X = H
b
c
O
N
O
N
a
6c-e
7c-e
8c-e
N
N
N
N
N
O
O
O
OH
d
S
O
S
O
F
Cl
O
O
O
N
5a
6a
7a
8a
: X = F
5b
6b
7b
8b
: X = F
: X = Cl
: X = Br
: X = H
: X = Cl
: X = Br
: X = H
N
N
N
O
H
N
R5
S
O
9
O
Scheme 2.
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Cl
O
F
Cl
O
O
N
O
N
H
O
N
N
N
O
N
S
O
O
F3C
O
O
F3C
O
Butafenacil
(Syngenta, 2001)
Saflufenacil
(BASF, 2009)
F
F
Cl
S
F
O
O
N
O
N
F
H
N
O
N
N
N
N
O
O
O
F3C
O
S
O
Tiafenacil
(FarmHannong, 2018)
Trifludimoxazin
(BASF, 2020)
Figure 1.
F
F
O
N
O
N
F
N
N
O
S
O
Trifludimoxazin
O
F3C
X
Cl
N
O
R3
S
S
N
N
N
N
N
Scaffold Hopping
O
O
O
OCHF2
R2
N
R3
O
R1
Pyroxasulfone
O
5-9
N
O
HO
N
O
N
S
O
Topramezone
Figure 2.
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Figure 3.
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