Discovery of New 2-[(4,6-Dimethoxy-1,3,5-triazin-2-yl)oxy]-6-(substituted phenoxy)benzoic Acids as Flexible Inhibitors of Arabidopsis thaliana Acetohydroxyacid Synthase and Its P197L Mutant

Article abstract of DOI:10.1021/acs.jafc.7b05198

In the search for new antiresistance acetohydroxyacid synthase (AHAS, EC 2.2.1.6) inhibitors to combat weed resistance associated with AHAS mutations, a series of 2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)oxy]-6-(substituted phenoxy)benzoic acids 11-38 were de

Full text of DOI:10.1021/acs.jafc.7b05198

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Article
Discovery of New 2-[(4,6-Dimethoxy-1,3,5-triazin-2-yl)oxy]-6-
(substituted phenoxy)benzoic Acids as Flexible Inhibitors of
Arabidopsis thaliana Acetohydroxyacid Synthase and Its P197L Mutant
Ren-Yu Qu, Jing-Fang Yang, DEVENDAR PONNAM, Wei-Ming Kang, Yu-
Chao Liu, Qiong Chen, Cong-Wei Niu, Zhen Xi, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05198 • Publication Date (Web): 30 Nov 2017
Downloaded from http://pubs.acs.org on December 1, 2017
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Page 1 of 35
Journal of Agricultural and Food Chemistry
1
Discovery of New 2-[(4,6-Dimethoxy-1,3,5-triazin-2-yl)oxy]-6-(substituted
phenoxy)benzoic Acids as Flexible Inhibitors of Arabidopsis thaliana
Acetohydroxyacid Synthase and Its P197L Mutant
2
3
4
RenꢀYu Qu,† JingꢀFang Yang,† Ponnam Devendar,† WeiꢀMing Kang,† YuꢀChao Liu,† Qiong
Chen,† CongꢀWei Niu, ‡ Zhen Xi, *,‡,# and GuangꢀFu Yang*,†,#
5
6
†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University(CCNU), Wuhan 430079, P. R. China
‡State Key Laboratory of ElementoꢀOrganic Chemistry, Nankai University (NKU), Tianjin
300071, P. R. China
7
8
9
1 0
11
#Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, P.R.
China.
1 2
1 3
14
1 5
AUTHOR INFORMATION
1 6
*Corresponding Authors:
17
Tel: +86ꢀ27ꢀ67867800, Fax: +86ꢀ27ꢀ67867141, Eꢀmail: gfyang@mail.ccnu.edu.cn.
1 8
Tel: +86 022ꢀ23504782, Fax: +86 022ꢀ23504782, Eꢀmail: zhenxi@nankai.edu.cn.
1 9
ORCID:
20
GuangꢀFu Yang: 0000ꢀ0003ꢀ4384ꢀ2593
21
Zhen Xi: 0000ꢀ0002ꢀ3332ꢀ5413
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ABSTRACT: In the search for new antiꢀresistance acetohydroxyacid synthase (AHAS, EC
23
2.2.1.6) inhibitors to combat weed resistance associated with AHAS mutations, a series of
24
2ꢀ[(4,6ꢀdimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)oxy]ꢀ6ꢀ(substituted phenoxy)benzoic acids 11–38 were
25
designed and synthesized via the strategy of conformational flexibility analysis. Compounds
26
21, 22, 26, 33, 36 and 38 with high potency against both wildꢀtype AtAHAS and its P197L
27
mutant were identified as promising candidates with low resistance factors (RF, defined as
28
the ratio between the ki values towards P197L mutant and wild type AHAS) ranging from
29
0.73 to 6.32. Especially, compound 22 (RF = 0.73) was further identified as the most potent
30
antiꢀresistance AHAS inhibitor due to its significantly reduced resistance level compared with
31
tribenuronꢀmethyl (RF = 2650) and bispyribac (RF = 4.57). Furthermore, compounds 26, 33,
32
36 and 38 also displayed promising herbicidal activities against sensitive and resistant
33
(P197L) Descurainia sophia at the dosage of 75–150 g of active ingredient (ai)/ha. Notably,
34
compounds 33 and 38 still maintained over 60% herbicidal activity toward the resistant weed
35
even at much lower dosages (37.5 g·ai/ha). Therefore, the designed scaffold has the great
36
potential to discover new candidate compounds for the control of weed resistance associated
37
with AHAS mutation.
38
39
KEYWORDS: AHAS inhibitors, structureꢀactivity relationship, antiꢀresistance property,
4 0
P197L mutant, herbicidal activity, molecular docking
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INTRODUCTION
4 2
4 3
Acetohydroxyacid synthase (AHAS, EC 2.2.1.6), an essential enzyme in the
branchedꢀchain amino acids (BCAAs) biosynthesis, mainly catalyzes the synthesis of
44
4 5
2ꢀacetoꢀ2ꢀhydroxybutyrate and 2ꢀacetolactate via condensation of a single molecule of
2ꢀketobutyrate with a single molecule of pyruvate (the pathway of isoleucine biosynthesis) or
4 6
two molecules of pyruvate (the pathways of leucine and valine biosynthesis), respectively. It
47
has been considered to be an attractive target for both drug and agrochemical discovery due to
4 8
its special biological functions and gene conservation throughout diverse species (plants,
4 9
bacteria, fungus, algae) 1ꢀ3
.
In particular, AHAS has a great importance in the field of herbicide
50
research, and it is a target of five classes of herbicides, including triazolopyrimidine
51
sulfonamides
(TPs),
sulfonylureas
(SUs),
pyrimidinylꢀ(thio)benzoates
(PTBs),
52
sulfonylaminoꢀcarbonylꢀtriazolinones (SCTs) and imidazolinones (IMIs) (Figure 1).4ꢀ7
53
54
Unfortunately, AHASꢀinhibiting herbicides are facing serious weed resistance due to their
longꢀterm overuse in agricultural applications and possession of only a single ligandꢀbinding
55
site in AHAS.8ꢀ10 Weed resistance to AHAS inhibitors is mainly caused by singleꢀpoint
56
mutations in the region of the binding site, causing a reduction in the binding affinity between
57
58
the AHAS and its inhibitors.9ꢀ12 According to a recent survey, the biotypes of 144 weed species
have evolved different degrees of resistance to AHAS herbicides globally, and this number is
59
increasing year after year.13ꢀ15 In all the surveyed 144 resistant weed species associated with
60
AHAS mutations, the mutation at Proꢀ197 is the most common cause of resistance to AHAS
61
inhibitors (there are about 70 resistant weeds caused by mutation at Proꢀ197),16, 17 and 8 types
62
of mutations (Ala, Thr, Ser, Arg, Gln, Leu, His, Ile substitutions) are involved in Proꢀ197.8, 18ꢀ21
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There are currently several commercialized AHAS herbicides which show similar inhibitory
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activities against wildꢀtype AHAS as well as its P197 mutants,22 for example, imazaquin,
imazapyr, pyrithiobac and bispyribac have low degrees of resistance against Proꢀ197ꢀSer or
66
Proꢀ197ꢀHis mutants. More importantly, weed resistance caused by the Proꢀ197ꢀLeu mutation
67
has been extensively reported around the world, and this mutant has caused serious
68
crossꢀresistance to most types of AHAS inhibitors. For example, the resistance levels for most
69
sulfonylureas and other AHAS herbicides (in IMIs, TPs and PTBs) have been increased up to
7 0
more than 100ꢀfold and 10ꢀfold respectively, as a result of Proꢀ197ꢀLeu mutation.23ꢀ27 However,
71
reports of novel antiꢀresistance herbicides active against Proꢀ197ꢀLeu mutation in AHAS are
7 2
quite rare. Therefore, the development of a new antiꢀresistance AHAS inhibitor with a strong in
7 3
vitro and in vivo activity against P197L mutant is highly desirable. In the current study, we have
74
sought to design and synthesize novel AHAS inhibitors with such activities.
7 5
There are several solutions to combat mutationꢀlinked drug resistance. For example, drug
7 6
combinations, multiple targeting design, targeting highly conserved residues, targeting protein
backbone and conformational flexibility drug design. In our previous work, we discovered that
77
7 8
the ligand conformation of sulfonylurea and triazolopyrimidine sulfamide AHAS inhibitors did
7 9
not undergo any significant changes in wildꢀtype AtAHAS and its mutants because of their rigid
80
bridges (SO2NHCONH and SO2NH). As a result, these inhibitors no longer interact with the key
81
amino acids or they cause steric hindrance with mutants, and that is the resistance mechanism
82
for these inhibitors.28ꢀ31 Therefore, developing AHAS inhibitor with conformational flexibility
83
should be an effective method to overcome resistance caused by mutation. On the basis of this
84
strategy, we have recently reported the syntheses, AtAHAS inhibitory and antiꢀresistance
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activities of 2ꢀaroxylꢀ1,2,4ꢀtriazolopyrimidines,32, 33 and 2ꢀbenzoyloxyꢀ6ꢀpyrimidinyl salicylic
86
acids,34 which have flexible ether bridges. In particular, diaryl ether has received a great deal of
attention in the area of agrochemistry.35, 36 For instance, cyhalofopꢀbutyl (ACCase inhibitor) and
87
88
fomesafen (PPO inhibitor) have been used as popular commercial herbicides to eradicate weeds
89
in the past decade. Additionally, the other commercial fungicides containing diaryl ether, such
90
as quinoxyfen, famoxadone, tolfenpyrad and metominostrobin, could effectively control plant
91
pathogens.37, 38
92
In our continuing endeavor to seek new antiꢀresistance AHAS inhibitors, we design a
93
scaffold by inserting a flexible ether linker in between triazinyl and phenyl ring of
sulfonylureas (Figure 2). Some commercial examples of sulfonylureas include chlorsulfuron,
94
95
metsulfuronꢀmethyl, tribenuronꢀmethyl, chlorimuronꢀethyl and iodosulfuronꢀmethyl, which are
96
not effective against weeds with the Proꢀ197ꢀLeu mutation in AHAS.39, 40 Also, the binding
97
model shows that the designed scaffold having an orthoꢀ carboxylic acid group potentially can
98
accommodate additional hydrogen bonding interactions with the binding site (R377, K256, etc.).
99
Further, we introduced phenoxy groups on the designed scaffold (metaꢀposition) to increase the
1 00
conformational flexibility, and these final structures may lead to the discovery of antiꢀresistance
1 01
AHAS inhibitors (Figure 2). Herein, we synthesized the designed compounds 11–38, measured
1 02
the inhibitory effects (against both wildꢀtype AtAHAS and P197L mutant), antiꢀresistance
1 03
properties, and herbicidal activities (against sensitive and resistant Descurainia sophia). We
1 04
proposed the binding models of the designed compounds and performed molecular docking,
1 05
dynamics simulations and Molecular Mechanics PoissonꢀBoltzmann Surface Area (MMꢀPBSA)
1 06
calculations.
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MATERIALS AND METHODS
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Chemicals and Instruments. All purchased reagents were used as receives, if not specified
1 09
otherwise. Organic solvents were also directly used without further purification, such as
11 0
dimethylformamide (DMF), acetic acid (AcOH), and methanol (MeOH). Using
111
11 2
tetramethylsilane (TMS) as the internal reference, 1H NMR and 13C NMR spectra were recorded
in CDCl3 or DMSOꢀd6 as solvent on a MercuryꢀPlus 400 or 600 MHz spectrometer (Varian,
11 3
Palo Alto, CA). A model Bꢀ545 melting point apparatus (Büchi, Flawil, Switzerland) was used
114
to obtain melting points without calibration. Highꢀresolution mass spectra (HRMS) data was
11 5
determined with 6224 TOF LC/MS (Agilent Technologies, Santa Clara, CA) instrument. The
11 6
column used was a 250 mm × 4.6 mm i.d., 5 ꢁm, Eclipse XDBꢀC18 (Agilent Technologies,
117
Santa Clara, CA). The flow rate, injection volume and column temperature were set to 0.2
11 8
mL/min, 2.0 ꢁL and 25 °C, respectively. Mass spectrometry (MS) was conducted on a DSQII
11 9
GCꢀMS (Thermo Fisher, Austin, TX) instrument with an electrospray ionization (ESI) source.
1 20
The heat temperature was from 50 to 300 °C (heating rate of 20 °C /min) and scan ranges of
1 21
m/z 100–500 for MS. The GC column used was a 30 m × 0.25 mm i.d., 0.25 ꢁm (film
1 22
thickness), Zebron ZBꢀ35 HT Inferno (Phenomenex, Torrance, CA).
1 23
1 24
Synthetic Chemistry. The synthesis of the target compounds 11–38 is illustrated in
Figure 3. The synthesis included seven steps from the commercially available 3ꢀflouroanisole,
1 25
compound 1, as the starting material. The regioselective formylation of 1 with
1 26
N,NꢀDimethylformamide
(DMF)
using
nꢀBuLi/TMEDA/DIPA
(in
situ
1 27
deprotonation/metalation) afforded the aldehyde 2. The aldehyde 2 when treated with diverse
1 28
substituted phenols 3aa–bb using K2CO3 in DMF gave 2ꢀmethoxyꢀ6ꢀ(substituted
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phenoxy)benzaldehydes 4aa–bb. The subsequent oxidation of the formyl group of the
1 30
molecules 4aa–bb were achieved by using 30% H2O2 and 50% aq. KOH in MeOH followed
by acidification, which resulted 2ꢀmethoxyꢀ6ꢀsubstitutedphenoxybenzoic acids 5aa–bb. The
1 31
1 32
acids 5aa–bb were subjected to the demethylation reaction by treating with BBr3 which
1 33
yielded hydroxy acids 6aa–bb. In the next step, the acid groups of 6aa–bb were protected
1 34
with benzyl bromide 7 using KHCO3 in DMF leading to the corresponding benzyl esters
1 35
8aa–bb.
The
crude
compounds
8aa–bb
were
then
treated
with
1 36
2ꢀchloroꢀ4,6ꢀdimethoxyꢀ1,3,5ꢀtriazine 9 using K2CO3 in DMF, yielding compounds 10aa–bb.
1 37
In the final step, the ester hydrolysis of crude compounds 10aa–bb with H2 and Pd/C in
1 38
CH3OH/AcOH generated the desired target compounds 11–38. The chemical structures of the
1 39
1
synthesized target compounds were established by H NMR, 13C NMR and HRMS data.
14 0
Further, the single crystal structure of compound 22 was confirmed by Xꢀray diffraction
141
(Figure 4).
14 2
X-ray Diffraction. Compound 22 was crystallized from a mixture of nꢀhexane and
14 3
chloroform to give colorless crystals suitable for single crystal Xꢀray analysis. Crystals 22 (0.20
144
mm × 0.15 mm × 0.15 mm) were mounted in fluoropolyether oil. On a Bruker SMART APEX
14 5
DUO CCD area detector diffractometer (Bruker AXS, Madison, WI), cell dimensions and
14 6
intensities were measured at 296 K with graphiteꢀmonochromated Mo Kα radiation λ = 0.71073
147
Å; θmax =26.00; 15699 measured reflections; 4131 independent reflections Rint= 0.0231. Data
14 8
integration and data reduction were carried out with the SAINT Plus program. The intensity
14 9
data were corrected for Lorentz, polarization and absorption effects (Tmax = 0.9848; Tmin
1 50
=0.9799). The structure was solved by direct method using SHELXS97 and refined with
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SHELXL970. Based on F2 using the weight of 1/[σ2(F0 ) + (0.0788P)2 + 0.3909P], fullꢀmatrix
2
1 52
leastꢀsquares refinement gave final values of R1 = 0.0388, ωR2 = 0.1157, and GOF(F) = 1.044
1 53
for 303 variables, 303 parameters, and 4131 contributing reflections. Maximum/minimum
1 54
residual electron density = 0.177 per ꢀ0.213 e.Åꢀ3, and maximum shift/error = 0.000. Hydrogens
1 55
were either found or placed in calculated positions and isotropically refined using a riding
1 56
model. The nonꢀhydrogen atoms were refined anisotropically. We deposited the
1 57
crystallographic data for compound 22 with the Cambridge Crystallographic Data Centre
1 58
(CCDC) with deposition no. 1558575.41
1 59
AtAHAS Inhibitory Experiments. As described in our previous studies,33, 34 the
1 60
wildꢀtype AtAHAS and P197L mutant were expressed from the plasmids pET28a
(+)ꢀAtAHAS and pET28a (+)ꢀAtAHASꢀPro197Leu, respectively. In addition, the in vitro
1 61
1 62
inhibitory activity (Ki value) of the synthesized compounds against wildꢀtype AtAHAS and
1 63
P197L mutant were studied using the method described previously.42ꢀ44 The results are
1 64
summarized in Table 1.
1 65
1 66
Computational Modeling and Calculation of Binding Free Energies. The crystal
structure of wildꢀtype AtAHAS (PDB ID:5K2O) was taken from the Protein Data Bank.7 The
1 67
P197L mutant was introduced to the wildꢀtype ligandꢀprotein complexes by using the
1 68
biopolymer mutation modeling tool in SYBYL v2.0. The conformations of the mutant
1 69
complexes were further minimized by using the Powell method of 3000 steps for residues in
17 0
3Å around the mutational site. The AutoDock v4.2 software was used to dock representative
171
inhibitors into AtAHAS which clustered by 2 Å of RootꢀMeanꢀSquare Deviation (RMSD).
17 2
Criteria and molecule modeling was visualized by PyMOL v1.3. On the basis of the result of
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previous 1~2 ns equilibrating Molecular Dynamics (MD) simulation of commercial
174
17 5
compounds and the reported crystal structure of bispyribac, we chose the final conformations
of target compounds to calculate binding free energy via MolecularꢀMechanics
17 6
PoissonꢀBoltzmann Surface Area (MMꢀPBSA) method.45
177
Herbicidal Activity. Sensitive and resistant (Proꢀ197ꢀLeu in AHAS) Descurainia
17 8
sophia (D. sophia) were selected as representative weeds for screening the compounds, and
17 9
the herbicidal activities were evaluated as described previously.33, 34 Before testing, all of the
1 80
synthesized compounds were dissolved in DMF and diluted with Tweenꢀ80 (100 mg/mL).
1 81
The solutions were further diluted with water to the tested concentrations. In the greenhouse,
1 82
the seeds of D. sophia were incubated with 0.05% GA3 solution for 1 day, and then prepared
1 83
seeds were planted at flowerpots (inner diameter of 7.5 cm) and its growth at temperatures
1 84
kept at 15 °C (night) and 25 °C (day). When D. sophia had grown to the fourꢀleaf stage, they
1 85
were treated by the inhibitorꢀcontaining solutions at the required concentrations (150, 75 or
1 86
37.5 g ai/ha). Herbicidal activity was evaluated visually after 35 days via postꢀemergence
1 87
treatment, with three replicates per treatment. The results of herbicidal activity of all the
1 88
compounds are described in Table 2.
1 89
1 90
RESULTS AND DISCUSSION
1 91
Enzyme Inhibition Activities (against P197L Mutant and Wild-Type AtAHAS) and
1 92
Structure-Activity Relationship. According to the previously mentioned methods, the
1 93
inhibition constants (Ki values) of compounds 11–38 were investigated against wildꢀtype
1 94
AtAHAS and P197L mutant. The commercial AHAS herbicide tribenuronꢀmethyl and
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bispyribac were chosen as the positive controls.
1 96
1 97
Among the 28 newly synthesized compounds (Table 1), most of them exhibited moderate
to excellent activities against P197L mutant. In particular, compounds 21 (3,4ꢀbenzo) and 22
1 98
(4ꢀph) were identified as the most potent inhibitors against P197L mutant with Ki values of 3.17
1 99
ꢁM and 2.71 ꢁM respectively. In order to optimize compound 11 (R1 = H), we initially focused
200
on introducing a single substituent (R1) on the phenoxy ring, which could affect the enzyme
201
inhibitory activity. In most cases, the different mono substitutions at the paraꢀ or metaꢀ position
202
of the phenoxy ring gave rise to compounds with higher inhibitory activities compared with the
203
compounds modified at the orthoꢀ position. Taking the fluorine substituent as an example, the
204
activity of compound 14 (2ꢀF) was reduced to 108.00 ꢁM (Ki, P197L value), when compared with
205
compounds 16 (4ꢀF, Ki, P197L = 21.40 ꢁM) and 15 (3ꢀF, Ki, P197L= 60.50 ꢁM). (The same
206
phenomenon applies to chlorine substituent). Additionally compared with the single substituted
207
derivatives 12–20 (F, Cl, Me substitution), 31 with 2,4ꢀdiꢀCl (Ki, P197L= 7.58 ꢁM) substitutions
208
at the R1 position showed increased inhibitory activity against P197L mutant, which implied
209
introducing dual substituted groups on the phenoxy group might enhance the inhibitory activity.
21 0
Unfortunately, when –Cl at different positions or other halogens (such as –F, ꢀBr) substituents
211
were introduced, the inhibitory activity did not improved and, even in some cases decreased,
21 2
for instance compounds 23 (2,3ꢀdiꢀF, Ki, P197L= 228.00 ꢁM), 24 (2,4ꢀ diꢀF, Ki, P197L= 84.30 ꢁM),
21 3
30 (2,3ꢀdiꢀCl, Ki, P197L= 75.70 ꢁM), 32 (2,5ꢀdiꢀCl, Ki, P197L= 55.30 ꢁM) and 37 (3ꢀBrꢀ4ꢀF, Ki,
214
P197L= 145.00 ꢁM). However, upon introducing both a methyl group and a halogen atom on the
21 5
phenoxy ring, the inhibitory activity improved as compared with the compounds with a single
21 6
substitution (12–20). For example, the inhibitory activities of compounds 26 (2ꢀFꢀ3ꢀCH3, Ki,
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P197L= 3.35 ꢁM), 33 (2ꢀClꢀ4ꢀCH3, Ki, P197L= 3.70 ꢁM), and 38 (3ꢀCH3ꢀ4ꢀBr, Ki, P197L= 3.34 ꢁM)
21 8
reached a micromolar level, which were more than 55ꢀfold higher than the commercial control
21 9
tribenuronꢀmethyl (Ki, P197L=212.00 ꢁM).
220
At the same time, larger sterically hindered structures also had a favorable effect on
221
increasing the inhibitory activity. For example, compounds 21 (3,4ꢀbenzo, Ki, P197L= 3.17 ꢁM)
222
and 22 (4ꢀPh, Ki, P197L= 2.71 ꢁM) showed higher activity than 11 (Ki, P197L= 52.80 ꢁM) and
223
almost similar activity to bispyribac (Ki, P197L= 2.47 ꢁM). We then pursued the experiments with
molecular modeling to analyze the binding mode of the representative compound 21 with the
224
225
P197L mutant. As shown in Figure 5A, compound 21 had a conservative πꢀπ stacking
226
interaction with W574 and its carboxyl group formed strong hydrogen bonds with many
227
residues in the P197L mutant AHAS, such as G120, K256, R377 and S653. Although all the
228
compounds had similar binding pattern with compound 21, the van der Waals (VDW)
229
interactions and binding free energy were all remarkably enhanced in presence of steric
230
hindrance on the structures (compounds 21 and 22) (Table S1). We conclude that, hybrids of
231
triazineꢀsalicylic acids (phenoxyꢀsubstituted) connecting through ether bridges could therefore
232
be used as a new scaffold for discovering novel AHAS inhibitors against P197L mutant.
233
234
Interestingly, most of the compounds exhibited good enzyme inhibiting activities against
wildꢀtype AtAHAS as well. As shown in Table 1, compound 11 (R1= H) exhibited good activity
235
(Ki, WT = 4.20 ꢁM), and the introduction of substituents at R1 improved the inhibitory activity.
236
For example, compound 26 (2ꢀFꢀ3ꢀCH3) showed excellent inhibitory activity (Ki, WT = 0.53 ꢁM),
237
equal to the commercial control bispyribac (Ki,
= 0.54 ꢁM). The structureꢀactivity
WT
238
relationship (SAR) can be summarized as follows: in general, compared with the substitution at
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the paraꢀ and orthoꢀ position of the substrates, substitution of groups at metaꢀposition increased
24 0
the enzyme inhibitory activities against wildꢀtype AtAHAS. For example, the inhibitory activity
of compound 15 (mꢀF, Ki, WT = 2.01 ꢁM) was 6ꢀfold higher than 16 (pꢀF, Ki, WT = 11.90 ꢁM), and
241
24 2
13ꢀfold higher than 14 (oꢀF, Ki, WT = 27.40 ꢁM). Compounds with ꢀCl, ꢀMe substituents
24 3
followed the same trend: mꢀCl > pꢀCl > oꢀCl (18 > 19 > 17); mꢀCH3 > pꢀCH3 (13 > 12) (Table
244
1).
24 5
24 6
Furthermore, the number and position of substitutions on the phenoxy group also could
affect their inhibitory activity against wildꢀtype AtAHAS. More specifically, introducing both a
247
mꢀmethyl group and an oꢀfluoro atom at R1, compound 26 (2ꢀFꢀ3ꢀCH3, Ki, WT = 0.53 ꢁM),
24 8
produced the best inhibitory activity. From the binding models, the binding of compound 26
24 9
(Figure 5C) in wildꢀtype AtAHAS active channel was very similar to that of compound 11
250
(Figure 5B). But the introduction of oꢀF atom (compound 26) could form a strong hydrogen
251
bond with S654 (3.5 Å) and the introduction of the mꢀMe increased the hydrophobic interaction,
252
which resulted in an elevation of electrostatic energy and VDW energy. Finally, compound 26
253
exhibited a higher binding free energy (
ꢀ
Gcal, WT = −25.60 kcal/mol) against wildꢀtype
254
AtAHAS as compared with the unsubstituted compound 11 ( Gcal, WT = −20.81 kcal/mol)
ꢀ
255
(Table 1).
256
The Molecular Basis of Anti-resistance Mechanism. To illuminate the relationship
257
between the structure and resistance at molecular level, we carried out molecular simulation
258
for representative molecules and binding free energy calculations for the synthesized
259
derivatives. As shown in Table 1, the ranges of the calculated binding free energies (ꢀ Gcal)
260
were from −13.98 to −25.60 kcal/mol and −12.01 to −21.74 kcal/mol for wildꢀtype AtAHAS
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261
and P197L mutant respectively. More importantly, the ꢀꢀ G (
ꢀ
GP197L− ꢀ GWT) indicated that
262
263
the difference between the binding free energy for P197L mutant and wildꢀtype AtAHAS, and
it can be used as a criterion to determine resistance caused by P197L mutation. The calculated
264
ꢀꢀ Gcal ranged from 7.26 to −0.90 kcal/mol, whereas the experimental ꢀꢀ Gexp ranged from
265
7.83 to −0.77 kcal/mol. It is worth mentioning that the relative order of the calculated
266
ꢁꢁ Gcal of these analogues corresponded with that of actual ꢀꢀ Gexp qualitatively
ꢁꢁGexp = ꢀ RT Ln Ki, P197L/ Ki, WT ). A linear correlation between the calculated ꢀꢀ Gcal and
267
(
268
experimental ꢁꢁ Gexp was acquired with a correlation coefficient of R2 = 0.92 (Figure 6),
269
which confirmed the reliability and rationality of the antiꢀresistance theoretical models built
27 0
in this work. In addition, in order to quantitate the degree of resistance of inhibitors caused by
271
P197L mutation, we defined the resistance factor (RF) as the ratio between the Ki values of
27 2
the mutant to that of wildꢀtype enzyme. This ratio was calculated for evaluating the effects of
27 3
the compounds 11
–
38 on resistance (RF
= Ki, P197L/ Ki, WT). It turned out the larger the value
274
of RF, the lower the drug resistance or it could be deduced that the smaller the value of
27 5
27 6
277
ꢀꢀ
G
cal, the higher drug resistance.
As shown in Table 1, the Ki values of compound 11 (unsubstituted) against P197L
mutant and wildꢀtype AtAHAS were determined to be 52.80 M and 4.20 M respectively.
Comparing these values indicated that the resistance factor (RF) value was 12.57, which was
ꢁ
ꢁ
27 8
27 9
far smaller than the positive control, tribenuronꢀmethyl with RF = 2650. The different
280
substituent groups (R1) on phenoxy ring of compound 11 could greatly affect the resistance
281
282
factor (RF). In comparison with the parent compound 11, the RF values of compounds with
3ꢀF, 2ꢀCl or 3ꢀCl substitutions (compounds 15
,
17 or 18) were not conducive to the reduction
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283
of the resistance factor (RF values were increased by over 2ꢀfold). Taking compound 15 as an
284
example, although it had a good inhibitory activity (
K
i, WT = 2.01
M toward the P197L mutant.
From energy component analysis (Table S1 and Table S2), we found that the
ele) and VDW energy ( vdw) of compound 15 showed a higher
value for wildꢀtype AtAHAS compared with the P197L mutant, which resulted in enthalpy
ꢁM) against wildꢀtype
285
AtAHAS, its Ki value was 60.50 ꢁ
286
287
electrostatic energy (
ꢀ
E
ꢀ
E
288
289
contribution ( ꢀ
H
) to having a smaller value for wildꢀtype AtAHAS. Hence, the binding free
energy of compound 15 Gcal = −22.39 kcal/mol) against wildꢀtype AtAHAS was lower
than the same against P197L mutant (ꢀ Gcal = −15.30 kcal/mol) and its ꢀꢀ cal value was
7.09 kcal/mol, which was consistent with the experimental value ( ꢀꢀ Gexp = 8.44 kcal/mol
290
291
292
293
(
ꢀ
G
;
RF = 30.10). Furthermore, the compounds bearing 4ꢀCH3, 4ꢀF, 3.4ꢀbenzoꢀ and 4ꢀCl, 2ꢀFꢀ
294
substitutions (compounds 12
,
16, 21 and 29) had slightly lower binding affinity against the
295
P197L mutant (
ꢀ Gcal = −17.54, −16.56, −21.74 and −17.73 kcal/mol respectively) than
296
against wildꢀtype AtAHAS ( ꢀ Gcal = −18.94, −19.00, −23.38 and −19.58 kcal/mol
297
298
respectively). These numbers were also in good agreement with the experimental values (RF
= 1.31, 1.80, 1.77 and 1.20 respectively). These results indicated that these inhibitors had low
299
resistance level. It is fair to conclude that the compounds which had a lower binding free
300
energy against the P197L mutant than the same against wildꢀtype AtAHAS ( ꢀꢀ Gcal < 0
301
kcal/mol), could be considered as potential antiꢀresistance inhibitors. In line with this, when
302
the R1 group on the phenoxy group changed from ꢀH to 4ꢀPh, 2,3ꢀCl, the compounds showed
303
less resistance, for instance in compounds 22 (RF = 0.73) and 30 (RF = 0.81). However, in
304
consideration of their inhibitory activity, compound 22 appears to have a better antiꢀresistance
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305
AHAS inhibitory activity simply because it had the lowest resistance factor and displayed
306
excellent inhibitory activities against both the P197L mutant and wildꢀtype AtAHAS.
307
To explain the antiꢀresistance property, we investigated the binding modes using
308
compound 22 as a representative. As depicted in Figures 5D–F, compound 22 had a
309
conservative πꢀπ stacking interaction with W574 and its carboxyl group could form hydrogen
31 0
bonds with the residues (G120, K256, R377 and S653) of the both wildꢀtype AtAHAS and
311
P197L mutant. When the amino acid residue was mutated (P197L), a steric clash occurred
31 2
between L197 and the biphenyl structure of compound 22. The flexible bridge of the
31 3
molecule led to the ligand shifting deeply into the pocket, thereby increasing the VDW
314
interactions ( ꢀꢀ
Evdw = −2.49 kcal/mol). At the same time, this strengthened the hydrogen
31 5
bond between the ligand and G120, but it could not compensate for the weakened hydrogen
31 6
bonds between the ligand and amino acid residues R377 and K256 ( ꢀꢀ
E
ele = 1.29 kcal/mol).
On the other hand, the mutation reduced the loss of solvation energy ( ꢀꢀ pbsol = −1.83
ꢀꢀ S = 2.54 kcal/mol). Above all,
compound 22 showed better binding affinities for the P197L mutant than wildꢀtype AtAHAS
317
E
31 8
kcal/mol) and increased entropy compensation (−
T
31 9
320
(
ꢀꢀ Gcal = −0.48 kcal/mol) (Table 1). This could also justify why compound 22 could
321
potentially be an antiꢀresistance AtAHAS inhibitor (ꢀꢀ
G
exp < 0 kcal/mol, RF <1).
322
To overcome resistance associated with AHAS mutation, we needed to consider the
323
factors mentioned earlier (i.e. inhibitory activity and resistance factor). On the basis of above
324
observations, compounds 21
,
22, 26, 33 and 38 had low RF values and showed excellent
325
inhibitory activities against both wildꢀtype AtAHAS and P197L mutant. Notably, the
326
inhibitory activity of compound 22 (4ꢀPh) was improved significantly compared to
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327
unsubstituted compound 11 (20ꢀfold), tribenuronꢀmethyl (78ꢀfold) and was equivalent to
328
bispyribac (0.9ꢀfold) against P197L mutant. At the same time, the RF value of compound 22
329
was also reduced about 3630ꢀfold and 6ꢀfold in comparison to the positive controls
330
tribenuronꢀmethyl and bispyribac respectively. The energy component analysis (Table 1) also
331
showed that compound 22 had equal and low binding free energy against wildꢀtype AtAHAS
332
and P197L mutant respectively, which corresponded with the experimental results.
333
334
Herbicidal Activity against Sensitive and Resistant Descurainia sophia. To further
38, we
selected sensitive and resistant (Proꢀ197ꢀLeu mutation in AHAS) D. sophia as representative
investigate the greenhouse in vivo herbicidal activity of the synthesized compounds 11
–
335
336
weeds. We selected commercial AHAS herbicides tribenuronꢀmethyl and bispyribac as
controls.46 Some of the synthesized compounds displayed promising herbicidal activity against
337
338
both sensitive and resistant weeds. Furthermore, the growth of inhibited weeds could be
339
recovered to a certain extent when we added additional branchedꢀchain amino acids (valine,
34 0
leucine and isoleucine) to the herbicidal assays (data not shown). This indicated that these
341
compounds targeted AHAS in the tested weeds.
34 2
As shown in Table 2, most derivatives exhibited moderate to good control against sensitive
18 26 33, 36 and 38 clearly exhibited over
90% inhibition. Even at lower dosage of 37.5 g ai/ha, compounds 15 and 38 displayed more
34 3
D. sophia at the rate of 150 g ai/ha. Compounds 15
,
,
,
344
34 5
than 80% inhibition against the sensitive weeds, and these results were comparable to that of
34 6
tribenuronꢀmethyl and bispyribac. It can be noted that some compounds also demonstrated
347
good herbicidal activity against resistant D. sophia (P197L). Compounds 21, 22, 26, 31, 33 and
34 8
36 at 150 g ai/ha showed more than 60% weed control, and compound 38 displayed almost
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34 9
100% inhibition against the resistant weed, which was a stronger inhibitory effect than the
350
tribenuronꢀmethyl inhibition against the resistant weed. When the dosage was reduced to 37.5 g
351
ai/ha, tribenuronꢀmethyl showed weak herbicidal activity against the resistant D. sophia (<30%
352
inhibition). In contrast, compounds 33 and 38 as well as another control, bispyribac, exhibited
353
good inhibition (> 60%) against the resistant D. sophia. This was consistent with the
354
observation from their AHASꢀinhibiting activities in vitro against the P197L mutant (Table 1).
355
Surprisingly, although some of the synthesized compounds had good antiꢀresistance properties
356
in vitro, their herbicidal activities against the sensitive or resistant weeds were not satisfactory.
357
For example, compounds 21 and 22 showed a low RF value and excellent inhibitory activity
358
against the two enzymes (P197L mutant and wildꢀtype AtAHAS); however, their activities
359
against the resistant and sensitive weeds could not compete with that of tribenuronꢀmethyl and
360
bispyribac. The main reason for such discrepancy could be that these compounds (21 and 22
)
361
might not possess the expected absorption, distribution, metabolism, and excretion (ADME)
362
properties.
363
364
To summarize our findings, compounds 26, 33, 36 and 38 displayed good herbicidal
activity against both the sensitive and resistant D. sophia. In particular, compounds 33 and 38
365
showed better or equivalent inhibition against the resistant D. sophia compared with
366
tribenuronꢀmethyl and bispyribac at three dosages (37.5, 75 and 150 g ai/ha). Therefore it can
367
368
be inferred that those compounds not only ensured high herbicidal potency, but also showed
better antiꢀresistance properties than tribenuronꢀmethyl.
369
In conclusion, in this study a series of 28 hybrids of triazineꢀsalicylic acids (phenoxy
37 0
substituted) connected through an ether bridge were designed through conformational
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371
flexibility and scaffold hopping. These compounds were synthesized and evaluated for their
37 2
AtAHAS inhibition, antiꢀresistance property and herbicidal activity. Compounds 21
37 3
(3,4ꢀbenzo
),
22 (4ꢀPh), 26 (2ꢀFꢀ3ꢀCH3), 33 (2ꢀClꢀ4ꢀCH3), 36 (3ꢀCH3ꢀ4ꢀCl) and 38
374
(3ꢀCH3ꢀ4ꢀBr) had low resistance factor (RF ranging from 0.73 to 6.32) and showed excellent
37 5
inhibitory activities against both wildꢀtype AtAHAS and its P197L mutant. In particular,
37 6
compound 22
(
Ki, WT = 3.69
ꢁM, Ki, P197L = 2.71 ꢁM, RF=0.73) was found to be the most
377
37 8
potent antiꢀresistance AtAHAS inhibitor and its resistance level was reduced about 3630ꢀfold,
and 6ꢀfold when compared with control tribenuronꢀmethyl (RF=2650) and bispyribac
37 9
(RF=4.57), respectively. Computational simulations revealed that the conformational
380
flexibility of these compounds was the main reason for the low resistance causing by P197L
381
mutation. Further greenhouse in vivo assays showed that compounds 26
,
33, 36 and 38
382
383
displayed promising herbicidal activity against both the sensitive and resistant (P197L) D.
sophia. We believe that compounds 33 and 38 have the potential to overcome weed resistance
384
conferred by P197L mutant in AHAS because they maintained more than 60% inhibition
385
against both the sensitive and resistant D. sophia at an application rate as low as 37.5 g ai/ha.
386
On the basis of the aforementioned discussion, the designed hybrid structures can be regarded
387
as new leads for the development of antiꢀresistance AHAS inhibitors to overcome weed
388
resistance.
389
390
Supporting Information
391
Supporting Information includes the energy component analysis of compounds 11–38 in P197L
392
mutant and wildꢀtype AtAHAS (Table S1 and Table S2), inhibition curves for
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393
compounds 22 and 38 against wildꢀtype AtAHAS and P197L mutant (Figure S1), the detailed
394
synthetic procedures, physical and spectrum data of compounds 11–38. This material is
395
available free of charge via the Internet at http://pubs.acs.org.
396
397
Acknowledgment
398
We thank Prof. Jie Chen at Zhejiang Research Institute of Chemical Industry for her help in
399
screening for herbicidal activity. This research was supported by the National Key R&D
4 00
Program (2017YFD0200507) and the National Natural Science Foundation of China (No.
4 01
21332004).
4 02
4 03
Notes
4 04
The authors declare no competing financial interest.
4 05
4 06
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51 6
759ꢀ770.
517
(39) Sada, Y.; Ikeda, H.; Kizawa, S., Resistance levels of sulfonylureaꢀresistant
51 8
Schoenoplectus juncoides (Roxb.) Palla with various Pro197 mutations in acetolactate
51 9
synthase
to
imazosulfuron,
bensulfuronꢀmethyl,
13, 53ꢀ61.
(40) Boigegrain, R. A.; Liautard, J. P.; Kohler, S., Targeting of the virulence factor
metsulfuronꢀmethyl
and
520
521
522
imazaquinꢀammonium. Weed Biol. Manage. 2013
,
acetohydroxyacid synthase by sulfonylureas results in inhibition of intramacrophagic
523
multiplication of Brucella suis. Antimicrob. Agents Chemother. 2005, 49, 3922ꢀ3925.
524
(41) The Cambridge Crystallographic Data Centre (CCDC). https://www.ccdc.cam.ac.uk/
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Journal of Agricultural and Food Chemistry
525
(Accessed: 06 June 2017).
526
(42) Singh, B. K.; Stidham, M. A.; Shaner, D. L., Assay of acetohydroxyacid synthase. Anal.
527
biochem. 1988
,
171, 173ꢀ179.
528
(43) Li, Y. X.; Luo, Y. P.; Xi, Z.; Niu, C. W.; He, Y. Z.; Yang, G. F., Design and syntheses
529
of novel phthalazinꢀ1(2H)ꢀone derivatives as acetohydroxyacid synthase inhibitors. J. Agric.
530
Food. Chem. 2006
,
54, 9135ꢀ9139.
531
532
(44) Chang, A. K.; Duggleby, R. G., Herbicideꢀresistant forms of Arabidopsis thaliana
acetohydroxyacid synthase: characterization of the catalytic properties and sensitivity to
533
inhibitors of four defined mutants. Biochem. J. 1998
,
333 , 765ꢀ777.
534
535
(45) Sitkoff, D.; Sharp, K. A.; Honig, B., Accurate calculation of hydration free energies
using macroscopic solvent models. J. Phys. Chem. 1994, 98, 1978ꢀ1988.
536
(46) Cui, H. L.; Zhang, C. X.; Wei, S. H.; Zhang, H. J.; Li, X. J.; Zhang, Y. Q.; Wang, G. Q.,
Acetolactate synthase gene proline (197) mutations confer tribenuronꢀmethyl resistance in
537
538
flixweed (Descurainia sophia) populations from China. Weed Sci. 2011, 59, 376ꢀ379.
539
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54 0
FIGURE CAPTIONS:
541
Figure 1. Chemical structures of commercial AHAS inhibitors.
54 2
Figure 2. Design strategy of the novel antiꢀresistance AHAS compounds.
54 3
Figure 3. Synthetic route of the title compounds 11
–
38. Reagents and Conditions: (i)
nꢀBuLi,
544
54 5
TMEDA, DIPA, DMF, ꢀ78 °C; (ii) K2CO3, DMF, 100 °C; (iii) KOH, 30% H2O2, CH3OH,
60 °C; (iv) BBr3, CH2Cl2, ꢀ78 °C; (v) KHCO3, DMF, 100 °C; (vi) K2CO3, DMF, 60 °C; (vii) H2,
54 6
Pd/C(10%), CH3OH, AcOH, RT.
547
Figure 4. Xꢀray crystal structures for compound 22
.
54 8
Figure 5. (A) The binding mode of compound 21 in P197L mutant interaction; (B and C)
54 9
Binding modes for compounds 11 and 26 interactions with wildꢀtype AtAHAS; (D and E)
550
Binding modes of compound 22 interactions with wildꢀtype AtAHAS and P197L mutant; (F)
551
552
Binding mode for compound 22 interactions with wildꢀtype AtAHAS (blue), and the P197L
mutant (green) are overlaid. [Simulated binding modes of the interaction between
553
representative compounds (11
,
21
, 22 and 26) and wildꢀtype AtAHAS or P197L mutant. The
554
555
black dashed lines represent hydrogen bonds between ligands and key residues].
Figure 6. The correlation between the difference of calculated and experimental binding free
556
energy ( ꢀꢀ G).
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Table 1. Inhibitory Activities and the Binding Free Energy of Compounds 11
WildꢀType AtAHAS and P197L Mutant.
–38 against
Ki (ꢁM)
ꢀ
Gcal (kcal/mol)
ꢀꢀ G (kcal/mol)
Com
pd.
R1
RFa
WildꢀType
P197L
Mutant
WildꢀType P197L
b
ꢀꢀ Gcal ꢀꢀ Gexp
AtAHAS
AtAHAS Mutant
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
H
4.20 ± 0.37
17.80 ± 1.60
6.45 ± 1.20
52.80 ± 7.50
23.40 ± 1.70
20.10 ± 1.90
12.57
1.31
3.12
3.94
30.10
1.80
25.63
23.59
12.42
2.55
1.77
0.73
14.43
5.73
4.59
6.32
3.33
11.40
1.20
0.81
3.58
3.84
2.33
10.26
2.82
4.79
20.89
2.37
2650
4.57
5.09
1.40
2.36
2.62
7.09
2.44
6.54
7.26
4.51
2.88
1.64
6.27
0.68
2.82
3.40
8.44
1.45
8.04
7.83
6.24
2.33
1.42
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
20.81
18.94
20.15
17.34
22.39
19.00
19.17
23.21
20.27
19.05
23.38
20.69
18.90
19.84
20.95
25.60
21.12
21.34
19.58
13.98
22.55
18.55
23.18
21.60
21.75
23.61
19.69
23.27
29.73
25.42
−15.72
−17.54
−17.79
−14.72
−15.30
−16.56
−12.63
−15.94
−15.76
−16.16
−21.74
−21.17
−12.01
−14.86
−17.48
−21.72
−17.19
−16.53
−17.73
−14.88
−20.11
−16.37
−21.62
−16.37
−19.54
−20.44
−13.21
−21.11
−12.77
−22.14
4ꢀCH3
3ꢀCH3
2ꢀF
27.40 ± 9.90 108.00 ± 11.00
3ꢀF
2.01 ± 0.46
11.90 ± 1.10
8.27 ± 9.60
1.84 ± 0.36
4.51 ± 0.37
25.70 ± 6.40
1.79 ± 0.18
3.69 ± 0.89
15.8 ± 1.90
14.70 ± 3.60
5.72 ± 1.36
0.53 ± 0.03
6.07 ± 0.79
5.37 ± 0.65
15.70 ± 2.30
92.90 ± 9.90
2.12 ± 0.35
14.40 ± 2.50
1.59 ± 0.45
4.58 ± 1.22
3.25 ± 0.56
1.34 ± 0.28
6.94 ± 0.68
1.41 ± 0.28
0.08 ± 0.01
0.54 ± 0.06
60.50 ± 9.40
21.4 ± 2.50
212.00 ± 23.00
43.40 ± 6.70
56.00 ± 7.20
65.70 ±7.50
3.17 ± 0.15
2.71 ± 0.12
228.00 ± 24.00
84.30 ± 7.00
26.30 ± 3.90
3.35 ± 0.42
20.20 ± 1.80
61.2 ± 7.20
18.90 ± 2.70
75.70 ± 7.80
7.58 ± 1.02
55.30 ± 6.50
3.70 ± 0.51
47.0 ± 3.80
9.16 ± 1.71
6.42 ± 0.80
145.00 ± 15.00
3.34 ± 0.40
212.00 ± 47.00
2.47 ± 0.41
4ꢀF
2ꢀCl
3ꢀ Cl
4ꢀ Cl
2ꢀBr
3.4ꢀbenzo
4ꢀPh
−
0.48
6.89
4.98
3.46
3.88
3.93
4.81
1.85
−
0.77
6.62
4.33
3.78
4.57
2.98
6.03
0.46
2,3ꢀdiF
2,4ꢀdiF
2,5ꢀdiF
3ꢀCH3ꢀ2ꢀF
4ꢀCH3ꢀ2ꢀF
5ꢀCH3ꢀ2ꢀF
2ꢀFꢀ4ꢀCl
2,3ꢀdiCl
2,4ꢀdiCl
2,5ꢀdiCl
2ꢀClꢀ4ꢀCH3
2ꢀBrꢀ4ꢀF
2ꢀCH3ꢀ4ꢀCl
3ꢀCH3ꢀ4ꢀCl
3ꢀBrꢀ4ꢀF
3ꢀCH3ꢀ4ꢀBr
−
0.90
2.44
2.19
1.56
5.24
2.22
3.17
6.48
2.16
16.96
3.28
−
0.51
3.16
3.34
2.09
5.77
2.57
3.88
7.53
2.14
24.33
3.76
Tribenuron-methyl
Bispyribac
aRF = Ki, P197L/ Ki, WT
;
b ꢀꢀ Gexp
=
−
RT Ln(Ki, P197L/ Ki, WT).
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Table 2. Herbicidal Activity of Compounds 11–
38 against Sensitive and Resistant D.sophiaa.
Dose
Sensitive Resistant
D.sophia D.sophiab
Dose
Sensitive Resistant
D.sophia D.sophiab
Compd.
Compd.
(g ai/ha)
(g ai/ha)
++++c
++
++
++
−
++++
+++
++
+
11
12
13
14
15
150
150
150
150
150
75
27
28
29
30
31
150
150
150
150
150
75
+++
++++
++++
+++++
++++
++++
++++
+++++
+++++
++++
+++
++
+
−
+
++
−
++++
+++
+++
+
−
−
37.5
150
150
150
75
37.5
150
150
75
+
16
17
18
32
33
++
+++
+
++++
+++
+++
+
−
+++++
+++++
+++
++
+
37.5
150
150
150
75
−
37.5
150
150
150
75
34
35
36
++++
+++
19
20
21
++++
+++
+
+
++
+++
+
+++++
+++++
+++
++++
++
+++
++
−
−
37.5
150
150
75
−
37.5
150
75
37
38
+
+++
22
+++
+++
++
−
+++++
+++++
++++
+++++
+++++
+++++
+++++
++++
++++
+++++
++++
+++
+++
++
++
−
37.5
150
150
150
150
75
37.5
150
75
−
23
24
25
26
+++
++++
++++
+++++
++++
++
Tribenuron-
methyl
−
37.5
150
75
++
++++
+++
++
+
Bispyribac
+++++
+++
+++
37.5
37.5
b
aAbbreviations: D.sophia
:
Descurainia sophia. The mutation site of resistant D.sophia
:
c
Proꢀ197ꢀLeu in AHAS. Rating scale of herbicidal activity (percentage of inhibition): +++++,
≥90%; ++++, 80−89%; +++, 60−79%; ++, 30−59%; +, 10−29%; −, <10%.
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Journal of Agricultural and Food Chemistry
Figure 1
.
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