Design, Synthesis, and Herbicidal Activity of Pyrimidine-Biphenyl Hybrids as Novel Acetohydroxyacid Synthase Inhibitors

Article abstract of DOI:10.1021/acs.jafc.8b00665

The issue of weed resistance to acetohydroxyacid synthase (EC 2.2.1.6, AHAS) inhibitors has become one of the largest obstacles for the application of this class of herbicides. In a continuing effort to discover novel AHAS inhibitors to overcome weed resistance, a series of pyrimidine-biphenyl hybrids (4aa-bb and 5aa-ah) were designed and synthesized via a scaffold hopping strategy. Among these derivatives, compounds 4aa (K_i = 0.09 μM) and 4bb (K_i = 0.02 μM) displayed higher inhibitory activities against Arabidopsis thaliana AHAS than those of the controls bispyribac (K_i = 0.54 μM) and flumetsulam (K_i = 0.38 μM). Remarkably, compounds 4aa, 4bb, 5ah, and 5ag exhibited excellent postemergence herbicidal activity and a broad spectrum of weed control at application rates of 37.5-150 g of active ingredient (ai)/ha. Furthermore, 4aa and 4bb showed higher herbicidal activity against AHAS inhibitor-resistant Descurainia sophia, Ammannia arenaria, and the corresponding sensitive weeds than that of bispyribac at 0.94-0.235 g ai/ha. Therefore, the pyrimidine-biphenyl motif and lead compounds 4aa and 4bb have great potential for the discovery of novel AHAS inhibitors to combat AHAS-inhibiting herbicide-resistant weeds.

Full text of DOI:10.1021/acs.jafc.8b00665

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Agricultural and Environmental Chemistry
Design, Synthesis, and Herbicidal Activity of Pyrimidine
-Biphenyl Hybrids as Novel Acetohydroxyacid Synthase Inhibitors
Ke-Jian Li, Ren-Yu Qu, Yu-Chao Liu, Jing-Fang Yang, ponnam
devendar, Qiong Chen, Cong-Wei Niu, Zhen Xi, and Guang-Fu Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00665 • Publication Date (Web): 05 Apr 2018
Downloaded from http://pubs.acs.org on April 5, 2018
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Design, Synthesis, and Herbicidal Activity of Pyrimidine−Biphenyl Hybrids
as Novel Acetohydroxyacid Synthase Inhibitors
3
KeꢀJian Li,† RenꢀYu Qu,† YuꢀChao Liu,† JingꢀFang Yang,† Ponnam Devendar,† Qiong
Chen,† CongꢀWei Niu,‡ Zhen Xi,‡,#* and GuangꢀFu Yang†,#*
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†Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of
Chemistry, Central China Normal University (CCNU), Wuhan 430079, PR China
‡State Key Laboratory of ElementoꢀOrganic Chemistry, Nankai University (NKU), Tianjin
300071, PR China
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#Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 30071, PR
China
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*Corresponding Authors:
Tel: +86ꢀ27ꢀ67867800, Fax: +86ꢀ27ꢀ67867141, Eꢀmail: gfyang@mail.ccnu.edu.cn,
zhenxi@nankai.edu.cn.
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ABSTRACT: The issue of weed resistance to acetohydroxyacid synthase (EC 2.2.1.6,
AHAS) inhibitors has become one of the largest obstacles for the application of this class of
herbicides. In a continuing effort to discover novel AHAS inhibitors to overcome weed
resistance, a series of pyrimidine−biphenyl hybrids (4aa−bb and 5aa−ah) were designed
and synthesized via a scaffold hopping strategy. Among these derivatives, compounds 4aa
(Ki = 0.09 ꢁM) and 4bb (Ki = 0.02 ꢁM) displayed higher inhibitory activities against
Arabidopsis thaliana AHAS than those of the controls bispyribac (Ki = 0.54 ꢁM) and
flumetsulam (Ki = 0.38 ꢁM). Remarkably, compounds 4aa, 4bb, 5ah, and 5ag exhibited
excellent postꢀemergence herbicidal activity and a broad spectrum of weed control at
application rates of 37.5−150 g of active ingredient (ai)/ha. Furthermore, 4aa and 4bb
showed higher herbicidal activity against AHAS inhibitorꢀresistant Descurainia sophia,
Ammannia arenaria and the corresponding sensitive weeds than that of bispyribac at
0.94−0.235 g ai/ha. Therefore, the pyrimidine−biphenyl motif and lead compounds 4aa and
4bb have great potential for the discovery of novel AHAS inhibitors to combat
AHASꢀinhibiting herbicideꢀresistant weeds.
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KEYWORDS:
acetohydroxyacid
synthase,
herbicide,
weed
resistance,
pyrimidine−biphenyl, structureꢀbased design
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INTRODUCTION
With the emergence of various field problems, weed management is still a challenge for
researchers, and continuous innovation is essential to maintain the effectiveness of weed
management.1ꢀ3 Among the herbicides classified by the Herbicide Resistance Action
Committee (HRAC), acetohydroxyacid synthase (AHAS)ꢀinhibiting herbicides have
extensive applications for controlling many weed species, and their discovery has been a
popular research topic in the herbicide field. AHAS, an important enzyme in the biosynthesis
of branchedꢀchain amino acids (BCAAs), catalyzes the conversion of 2ꢀketobutyrate and
pyruvate to 2ꢀacetoꢀ2ꢀhydroxybutyrate and the conversion of two molecules of pyruvate to
2ꢀacetolactate. In plants, BCAAs (isoleucine, leucine and valine) can promote protein
synthesis and have an influence on normal plant growth. AHAS inhibitors exert their
biological activities by interrupting the above mentioned biosynthetic pathways.4ꢀ7
AHASꢀinhibiting herbicides possess a range of advantages, such as excellent crop selectivity,
low application rate, benign environmental effects, and low toxicity for mammals.8 However,
the longꢀterm overuse of AHASꢀinhibiting herbicides has resulted in the development of
serious weed resistance in agricultural applications, and the reports of weeds that are resistant
to these herbicides have been gradually increasing in the past decade globally.9ꢀ15 For example,
AHASꢀinhibiting herbicideꢀresistant populations of Descurainia sophia, Lindernia micrantha,
Ammannia arenaria, and Monochoria korsakowii, among others, have been found around the
world over the past few years.16ꢀ18 Thus, there is a high demand to develop new
AHASꢀinhibiting herbicides with higher potency and better antiꢀresistance properties to
control resistant weeds.
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To date, extensive studies have been conducted to develop new inhibitors targeting
AHAS; however, reports of AHAS inhibitors effectively controlling both AHAS inhibiting
herbicideꢀsensitive weeds and AHAS herbicideꢀresistant weeds are quite rare.19ꢀ24 Hence, it is
necessary to design a novel AHAS inhibitor that not only displays a broader spectrum of
weed control but also overcomes the weed resistance to a certain degree. In the past decade,
our group has engaged in the design and development of novel AHAS inhibitors.25ꢀ28 We
discovered that conformational flexibility in AHAS inhibitors could improve their
antiꢀresistance properties to overcome weed resistance against commercial AHASꢀinhibiting
herbicides.29ꢀ31 Recently, we reported
a
series of conformationally flexible
triazolopyrimidineꢀsalicylate derivatives with high potency toward Arabidopsis thaliana
(At)AHAS; unfortunately, most of the inhibitors did not exhibit potent herbicidal activity
against either AHASꢀinhibiting herbicideꢀresistant weeds or the corresponding sensitive
weeds.29 Thus, it is essential to perform extensive structural modifications of the
triazolopyrimidineꢀsalicylate motif to discover more promising AHAS inhibitors.
In this context, scaffold hopping is an effective drug design methodology that has been
widely applied to the discovery of potential agrochemicals. It is beneficial to find a novel
structure that has an improved inhibitory activity and to identify the pharmacological
properties of known activities.32ꢀ35 Interestingly, pyrimidine is an important chemical motif
and structural unit of natural products, and its various derivatives are responsible for a broad
spectrum of biological activities.36, 37 Moreover, there are numerous examples of pyrimidine
moieties in the substructure of commercial herbicides (including some AHAS inhibitors),
fungicides, and insecticides.38, 39 Therefore, we considered the pyrimidine ring is a promising
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replacement for the triazolopyrimidine ring of the triazolopyrimidine–salicylate motif via
scaffold hopping and then designed a series of novel pyrimidine–biphenyl hybrids (4 and 5)
connected by a flexible oxygen linker (Figure 1). Furthermore, the results of computational
simulations revealed that the triazolopyrimidine ring (represented by A) and the pyrimidine
ring (represented by 4aa) forming similar π–π stacking interactions with amino acid residue
W574 in the active pocket of AtAHAS. Herein, we report the synthesis, AtAHAS inhibition,
herbicidal activity, antiꢀresistance properties and structure–activity relationships (SAR) of
pyrimidine−biphenyl hybrids.
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MATERIALS AND METHODS
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Chemicals, Instruments and Procedures. All reagents, catalysts, and extraꢀdry solvents
were purchased from commercial suppliers (SigmaꢀAldrich, TCI, Merck, J&K, and Aladdin
Chemicals). All reactions were monitored using thinꢀlayer chromatography (TLC) run on
silica gel glass plates (Qingdao Broadchem Industrial, Qingdao, China). Melting points were
recorded on a model Bꢀ545 melting point apparatus (Büchi, Flawil, Switzerland), and the
values were uncorrected. 1H NMR and 13C NMR spectra were recorded using a MercuryꢀPlus
600 or 400 spectrometer (Varian Inc., Palo Alto, CA) with temperature control at 21−23 °C
using DMSOꢀd6 or CDCl3 as the solvent and tetramethylsilane (TMS) as the internal
reference. In the spectra, the chemical shifts (δ) were given in parts per million (ppm). Mass
spectrometry (MS) data were obtained with a DSQ II GCꢀMS (Thermo Fisher, Austin, TX)
instrument with an electrospray ionization (ESI) source. Highꢀresolution mass spectra (HRMS)
were determined with a model 6224 timeꢀofꢀflight liquid chromatograph–mass spectrometer
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equipped with a 250 mm × 4.6 mm i.d., 5 ꢁm, Eclipse XDBꢀC18 column (Agilent
Technologies, Santa Clara, CA).
Synthesis of Compound 2 (Figure 2). A catalytic amount of Na2WO4·2H2O was added to
a stirred mixture of compound 1 (0.10 mol) and 100 mL of acetic acid at 25 °C. Next, 30%
H2O2 (0.11 mol) solution was added slowly at −10 °C with continued vigorous stirring. The
system temperature was maintained at 70 °C for an additional 5 h. After cooling to 0 °C, the
reaction was quenched with 200 mL of aqueous Na2SO3 solution. The resulting solid was
collected by filtration, washed with cold water (3×30 mL), and dried in a vacuum desiccator
at 60 °C to afford compound 2 as an offꢀwhite solid (19.4 g, yield 89%):1H NMR (600 MHz,
DMSOꢀd6) δ 6.57 (s, 1H), 4.00 (s, 3H), 3.40 (s, 6H). EIMS: m/z = 218.11 (M+).
General Procedure for the Synthesis of 4aa−bb (Figure 2). The key intermediate 6ꢀaryl
salicylic acid derivatives (3) were synthesized according to our previous reports.40ꢀ42 In a 100
mL flask, the derivatives 3 (2.0 mmol) were dissolved in 20 mL of toluene. Then, anhydrous
K2CO3 (8.0 mmol) was slowly added to the reaction mixture with stirring at room temperature.
After 1 h, compound 2 (2.1 mmol) was added to the reaction mixture, the whole system was
put into a preheated oil bath (120 °C), and the reaction progress was monitored by TLC until
the reaction was complete. After cooling to room temperature, the reaction was quenched with
aqueous HCl (0.5 N, 30 mL) and extracted with EtOAc (3×15 mL). The combined organic
layers were dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was
purified via flash column chromatography (nꢀhexane/acetone = 20:8) to provide target
compounds 4aa−bb (yields 32−85%).
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General Procedure for the Synthesis of 5aa−ah (Figure 2). In a 25 mL flask, compound
4aa or 4bb (1.0 mmol) and Cs2CO3 (1.1 mmol) were dissolved in 5 mL of
N,Nꢀdimethylformamide (DMF). After stirring at room temperature for 10 min, the
corresponding alkyl halide (1.50 mmol) was added to the reaction mixture. The temperature
of the reaction solution was increased to 50 °C and maintained for 2 h. After completion of
the reaction as monitored by TLC, the reaction mixture was quenched with 20 mL of water,
and the mixture was extracted with EtOAc (3×5 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified via
flash column chromatography (nꢀhexane/EtOAc = 20:4) to afford title compounds 5aa−af
(yields 80−94%).
General Procedure for the Synthesis of 5ag and 5ah (Figure 2). In a singleꢀneck
roundꢀbottomed flask, compound 4aa or 4bb (1.0 mmol) was dissolved in 10 mL of
anhydrous THF. After cooling to 0 °C in an ice bath, NaOH (1.0 mmol) was added to the
reaction system. As the reaction proceeded, a white solid was precipitated from the reaction
mixture. It was collected by filtration and washed with THF (3×5 mL) to afford compound
5ag (yield 73%) or compound 5ah (yield 65%).
Xꢀray Diffraction. A colorless crystal of compound 4bb was obtained directly from
acetone/nꢀhexane. Its Xꢀray singleꢀcrystal diffraction data were collected on a SMART APEX
DUO CCD area detector diffractometer (Bruker AXS, Madison, WI) at 296 K using Mo Kα
radiation (λ = 0.71073 Å). All the nonꢀhydrogen atoms of compound 4bb were refined with
anisotropic displacement parameters. The hydrogen atoms were placed and observed in
geometrically idealized positions. The integration of the diffraction profiles and the methods
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of structural analysis were carried out using SAINT Plus software (Bruker AXS, Madison, WI)
and SHELXS97 program (University of Gottingen, Gottingen, Germany), respectively. The
crystallographic data for crystal 4bb were deposited with the Cambridge Crystallographic
Data Centre (CCDC) with deposition number 1558576.43
AtAHAS Inhibitory Activity Assay. The expression and purification of AtAHAS
were performed using the same methods as those described in previous reports.25ꢀ31 The
inhibition of AtAHAS was measured by kinetic parameters (Ki value) between the enzyme
and inhibitors. The nonꢀlinear leastꢀsquares and simplex methods were used to calculate the
kinetic parameters by error minimization. The Ki values were calculated by fitting the data
to the following equation:
νi = ν∞+(ν0−ν∞)/(1+[I]/Kiapp
)
where [I] and Kiapp represent the concentration of the prepared inhibitors and apparent
inhibition constant (the inhibitor concentration giving 50% inhibition), respectively. ν0 and
νi are the reaction rate in the absence or presence of the inhibitor, respectively. If the initial
analysis indicated that the residual activity (ν∞) at a saturating inhibitor concentration is not
significantly larger than zero, the data were reanalyzed with ν∞ = 0.
Molecular Simulation and Comparative Molecular Field Analysis. The crystal
structure of AtAHAS (PDB ID: 5K2O) was downloaded from the Protein Data Bank.6
Computational modeling studies of representative compounds were performed using
AutoDock 4.2 software, and the parameters of the docking model were programmed to the
recommended default values. A total of 265 runs clustered by 2 Å of rootꢀmeanꢀsquare
deviation (RMSD) criteria were launched for each compound. The final binding modes with
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reference to the reported coꢀcrystal structure between bispyribac and AtAHAS were selected
by the docking score. PyMOL v1.3 software was used to visualize and analyze the docking
results. The binding free energy for each complex was estimated using the molecular
mechanics Poisson−Boltzmann surface area (MMꢀPBSA) method.44
On the basis of the best combining conformation, the threeꢀdimensional structures of
4aa−4bb were generated with the default setting of SYBYL 6.9 (Tripos Inc., St. Louis,
MO), and the molecules were subjected to energy minimization at a gradient of 1.0 kcal/mol
with a delta energy change of 0.05 cal/mol. We calculated the CoMFA descriptors and
electrostatic and steric field energies on the condition of the SYBYL default parameters,
which included a grid point spacing of 2.0 Å, a minimum σ (column filtering) of 2.0
kcal/mol, an energy cut off of 30.0 kcal/mol, and a sp3 carbon probe atom with a +1 charge.
Greenhouse Herbicidal Assay. The herbicidal activities were evaluated in the Zhejiang
Research Institute of Chemical Industry (Hangzhou, China). All the target compounds
(4aa−bb and 5aa−ah) were dissolved in 100% DMF and then diluted with Tweenꢀ80
(concentration: 100 g/L). The resulting solutions were diluted with water to the appropriate
concentrations before use. The postꢀemergence herbicidal activity of the compounds was
evaluated against three dicotyledonous weeds, Brassica juncea (B.j.), Chenopodium
serotinum (C.s.), and Rumex acetosa (R.a.), and three graminaceous weeds, Alopecurus
aequalis (A.a.), Polypogon fugax (P.f.), and Poa annua (P.a.). In addition, Abutilon
theophrasti (A.t.), Amaranthus retroflexus (A.r.), Digitaria sanguinalis (D.s.), Eclipta
prostrata (E.p.), and Echinochloa crusgalli (E.c.) were selected to evaluate the herbicidal
spectrum of representative compounds. In a greenhouse, the prepared soil was filled to 3/4 of
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the height of the flowerpots (with an inner diameter of approximately 7.5 cm). Approximately
20 seeds of each tested weed were sown and grown at temperatures alternating from
15−30 °C. When these weeds had grown to approximately the threeꢀleaf stage, they were
sprayed with a stock solution containing the inhibitors at the required concentrations (g ai/ha).
Moreover, mixtures of DMF and Tweenꢀ80 were selected as solvent control groups. After 20
days of treatment with the inhibitors, the growth inhibition rates were calculated (three
duplicates per experiment) according to previously reported methods.45
Descurainia sophia (sensitive D. Sophia and resistant D. Sophia mediated by AHAS
mutation) and Ammannia arenaria (sensitive A. arenaria and bensulfuron methylꢀresistant A.
arenaria) were selected to test the antiꢀresistance properties of the synthesized compounds.17
Similar to the above preparation, the seeds of D. sophia and A. arenaria were treated with
0.05% gibberellic acid (GA3), and then dormancy was broken after 24 h. In a phytotron, the
seeds were planted in flowerpots, and the growth temperature was maintained at 15−25 °C
(day and night). Upon reaching the fourꢀleaf stage, the seedlings were moved to a greenhouse
and treated with the selected inhibitors. Furthermore, the postꢀemergence herbicidal activity
of the compounds was visually evaluated after 35 days of treatment, and each experiment was
carried out with three replications.
RESULTS AND DISCUSSION
Chemistry. As shown in Figure 2, target compounds 4aa−bb and 5aa−ah were
prepared by using 4,6ꢀdimethoxyꢀ2ꢀ(methylthio)pyrimidine, 1, as a starting material. The
oxidation of methyl sulfide, 1, using 30% H2O2/cat. Na2WO4·2H2O in AcOH afforded
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methyl sulfone, 2, in good yield. The key intermediate 6ꢀarylsalicylates, 3, were synthesized
via two different synthetic routes (Figures 3A and B) as reported previously.40ꢀ42 When R1
in compound 3 was a hydrogen or halogen (H, F, Cl), the synthesis occurred in three steps,
as shown in method A. When R2 was a methoxyl or methyl group (OMe, Me), the synthesis
occurred in five steps, as outlined in method B. In the subsequent step, methyl sulfone, 2,
was treated with various substituted 6ꢀarylsalicylates, 3, in the presence of K2CO3, resulting
in target compounds 4aa−bb (yields 32−85%) via a nucleophilic substitution reaction. To
synthesize target compounds 5aa−af, compounds 4aa and 4bb were treated with the
corresponding alkyl halides in the presence of Cs2CO3 in DMF. In addition, compound 4aa
or 4bb reacted with NaOH in THF, yielding the corresponding sodium salts 5ag and 5ah,
respectively. The chemical structures of all the synthesized target compounds were
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identified by H NMR, 13C NMR and HRMS analyses. Furthermore, the structure of
compound 4aa was confirmed by singleꢀcrystal Xꢀray diffraction (Figure 4).
AHAS Inhibitory Activity, SAR and CoMFA. From the results of the molecular
modeling of compound 4aa with AtAHAS (PDB entry 5K2O),6 we found that it had two
evident interactions in the active pocket of AtAHAS (Figure 1). The pyrimidine ring of
compound 4aa formed a π−π interaction with W574, and the benzoic acid part showed a
hydrogen bonding interaction with residues S653, R377, and K256. The analyzed results
indicated that the introduction of various substituents into the core skeleton
(pyrimidine−biphenyl hybrid) at the R1 and R2 positions could increase the interactions with
the enzyme. We then synthesized 4ab−bb and systemically investigated the kinetic constants
(Ki values) of all the compounds against AtAHAS. The commercial AHASꢀinhibiting
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herbicides bispyribac and flumetsulam were selected as the positive controls. As shown in
Table 1, most of the synthesized compounds displayed strong inhibitory activities against
AtAHAS, and some of them showed even better potencies than the commercial controls. In
particular, among the compounds tested, compounds 4aa (Ki = 0.09 ꢁM) and 4bb (Ki = 0.02
ꢁM) displayed the highest enzyme inhibitory activities; comparatively, the Ki values of
bispyribac and flumetsulam were 0.54 ꢁM and 0.38 ꢁM, respectively. Moreover, compounds
4am (Ki = 0.18 ꢁM), 4al (Ki = 0.25 ꢁM), 4au (Ki = 0.20 ꢁM), and 4av (Ki = 0.41 ꢁM) also
showed excellent inhibitory activities that were comparable to that of flumetsulam.
On the basis of the observed Ki values of these compounds against AtAHAS, the major
SARs can be revealed. The different aromatic groups at R1 have a significant impact on
activity of the compounds. The results indicated that fiveꢀmembered furanylꢀsubstituted
compounds 4ai (Ki = 0.66 ꢁM) and 4aj (Ki = 0.51 ꢁM), and isoxazolylꢀsubstituted compound
4al (Ki = 3.78 ꢁM) displayed enhanced activity compared to (un)substituted sixꢀmembered
heterocyclic (pyridyl or pyrimidinyl)ꢀsubstituted compounds 4ab−ah (Ki range of
4.52−705.00 ꢁM). Furthermore, the compound with a bulky naphthyl group at R1 (4al, Ki =
0.25 ꢁM) showed better potency than those of the heterocyclicꢀsubstituted compounds
4ab−4aj and a similar Ki value to that of the controls bispyribac (Ki = 0.54 ꢁM) and
flumetsulam (Ki = 0.38 ꢁM). When an unsubstituted phenyl group was introduced at R1, the
resulting compound (4aa) displayed excellent activity (Ki = 0.09 ꢁM) compared to
compounds 4ab−al substituted with heterocyclic or naphthyl groups at the same position. We
further studied the influence of introducing substituted phenyl groups at R1 on inhibitory
activity. As shown in Table 1, the inhibition constants of these compounds (4am−aw)
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appeared in the micromolar to nanomolar range, and some of the compounds showed higher
potency than that of the controls. Furthermore, we found that the positions of substituents
(orthoꢀ, metaꢀ and paraꢀpositions) on the phenyl group significantly affected the AtAHAS
inhibitory activities of the resulting compounds. In most cases, when the phenyl group was
monohalogenated, the paraꢀsubstituted compound had higher inhibitory activity than that of
the metaꢀ and orthoꢀsubstituted compounds. For example, the fluoroꢀ and chloroꢀsubstituted
compounds showed the following trend in activity: 4ꢀF (4ar) > 2ꢀF (4ap) ≈ 3ꢀF (4aq); 4ꢀCl
(4au) >2ꢀCl (4as) > 3ꢀCl (4at). When different halogens were introduced at the paraꢀposition
of the phenyl group, the chloroꢀsubstituted compound demonstrated higher activity than the
bromoꢀsubstituted compound and much higher activity than the fluoroꢀsubstituted compound,
i.e., 4ꢀCl (4au) > 4ꢀBr (4av) > 4ꢀF (4ar). The nature of the substituent at the phenyl group
also affected the activity of the compound. For example, when we introduced an
electronꢀdonating group, the resulting compound exhibited more potent AtAHAS inhibitory
activity than that of compounds with an electronꢀwithdrawing group, e.g., 4ꢀMe (4am) >
4ꢀNO2 (4ao) and 4ꢀOMe (4an) > 4ꢀNO2 (4ao). From the above results, the SAR of the
paraꢀsubstituted phenyl compounds can be summarized as follows: 4ꢀMe (4am) > 4ꢀCl
(4au) > 4ꢀBr (4av) > 4ꢀF (4ar) > 4ꢀOMe (4an) > 4ꢀNO2 (4ao).
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In addition, we kept R1 as a phenyl group while simultaneously introducing different
substituents at the R2 position, which had a significant effect on the AtAHAS inhibitory
activity of the resulting compound. For instance, when we replaced the methyl group at R2 in
compound 4aa with a fluoro (4az), chloro (4ba) or methoxy (4ax) group, we found that these
changes were detrimental to the activity of the compound compared with parent 4aa.
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Surprisingly, if we introduced a hydrogen atom in place of methyl group, the activity of the
resultant compound (4bb, Ki = 0.02 ꢁM) was 4.5 times greater than that of compound 4aa (Ki
= 0.09 ꢁM). To understand the structural basis, we performed molecular modeling on
representative compounds 4aa, 4ax, 4az, and 4bb (R2 = Me, OMe, F, H, respectively). As
depicted in Figures 5A−D, the compounds 4ax, 4az, and 4bb had a similar docking mode to
that of compound 4aa, which was discussed earlier (Figure 1). However, the Ki values of
these four compounds showed statistically significant differences. Considering their
molecular structures, the main difference was in the R2 group, indicating that this position
might have a significant effect on the binding between the ligand and AtAHAS. To explain
this finding, we further conducted binding free energy calculation and compared the binding
conformations among the different compounds. The results (Table 2) revealed that the van der
Waals (VDW) interaction of compound 4aa was enhanced compared with that of 4bb
(
∆∆ EVDW = ∆EVDW,4aa− ∆EVDW,4bb = −1.81 kcal/mol). However, the number of rotatable
bonds in 4aa (R2 = Me) decreased compared with that of 4bb (R2 = H) because of the
increased steric hindrance in the active pocket, which caused an obvious increase in entropy
compensation (−T∆∆ S = 4.08 kcal/mol). By integrating the energy, compound 4bb showed a
better binding affinity with AtAHAS than that of compound 4aa (∆∆ GCal
= ∆ GCal,4aa −
∆
GCal,4bb = 1.68 kcal/mol). When analyzing the binding modes of compounds 4ax and 4bb
(Figure 5B, D), we observed that the binding conformation of 4ax was offset upward
compared with that of 4bb because the OMe group of 4ax displayed steric repulsion with
residue S168. This repulsion resulted in weakened hydrogen bonding between the ligand and
amino acid residues S653 and K256. From the perspective of the binding free energy (Table
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2), the increased distance caused a dramatic reduction in the electrostatic energy of 4ax
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relative to that of compound 4bb ( ∆∆ EELE = ∆ EELE,4ax − ∆ EELE,4bb = 16.96 kcal/mol).
Additionally, the VDW energy and entropy of compound 4ax (R2 = OMe) were increased
over those of compound 4bb (R2 = H) due to the introduction of a substituent group at R2.
Overall, the energy component analysis indicates that the binding free energy of 4az with
AtAHAS is lower than that of 4bb (∆∆ GCal
= ∆GCal,4az −∆GCal,4bb = 4.06 kcal/mol). The
binding free energy of the fluoroꢀsubstituted compound (4az) was the lowest among the four
representative compounds. A possible reason for this finding is that the fluorine atom causes
the molecule to be more electronegative, which may induce electrostatic repulsion with
negatively charged residue D376 (Figure 5C). According to the above energy component
analysis and the binding mode comparison, compounds 4aa and 4bb could be better AtAHAS
inhibitors.
To further understand the relationship between the substituents and AtAHAS inhibition,
a brief comparative molecular field analysis (CoMFA) of 27 representative compounds
4aa−bb was performed. As shown in Figure 6, a linear correlation between the
experimental and calculated AtAHAS inhibitory activities (pKi) was acquired with a
correlation coefficient (R2) of 0.933, which confirmed the reliability of the theoretical
models constructed in this work. The predicted CoMFA model of this series collected for
the training set was statistically significant with a crossꢀvalidated coefficient (q2) of 0.752, a
conventional coefficient (r2) of 0.935, and a predicted correlation coefficient (r2 pred) of
0.919. The electrostatic contour map is shown in the blue and red highlighted areas (Figure
7A). The contour plot of the steric contributions is shown in the yellow and green
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highlighted regions in Figure 7B. The yellow polyhedra show that introducing a bulky
group in this position negatively affects the AtAHAS inhibitory of these compounds. For
example, compound 4bb (R2 = H, Ki = 0.02 ꢁM) is a more potent inhibitor than 4ax (R2 =
OMe, Ki = 26.80 ꢁM) against AtAHAS, even though they have the same R1 group (R1 = Ph).
A similar phenomenon was also displayed by compounds 4aa and 4ax and compounds 4au
and 4ay. In addition, the green positions denote where a sterically bulky group would be
beneficial for in vitro AtAHAS inhibition. Above all, these rules further illuminated the
molecular mechanism of the SAR.
Herbicidal Activity and SAR. The postꢀemergence herbicidal activity of title
compounds 4aa−bb and 5aa−ah was evaluated against six kinds of representative weeds, B.j.,
C.s., R.a., A.a., P.f., and P.a., under greenhouse conditions. The AHASꢀinhibiting herbicides
flumetsulam and bispyribac were selected as positive controls, and the herbicidal activity
results are shown in Table 1. In most cases, the weed inhibition of these compounds was
consistent with their in vitro results. When applied at a rate of 150 g ai/ha, several compounds
showed greater than 60% weed control against some of the tested weeds, and 4am and 4aq
displayed 70−100% inhibition against five of the six tested weeds. Very promisingly, 4aa,
4bb, 5ag, and 5ah exhibited a weed control spectrum (inhibition >80%) that was broader than
or equivalent to that of the commercial herbicides flumetsulam and bispyribac at the same
dosage.
As shown in Table 1, most of the heterocyclicꢀ or naphthylꢀsubstituted (at R1)
compounds (4ab−al) did not show promising herbicidal activity. Only compounds 4ab, 4ac,
4ad, and 4af exhibited over 80% inhibition against one or two of the tested weeds (B.j./A.a.).
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The introduction of the (un)substituted phenyl group at R1 was favorable to the herbicidal
activity compared to the activity of compounds 4ab−al. Notably, compound 4aa with the
phenyl group at R1 displayed >80% inhibition against the three tested dicotyledonous weeds
(B.j./C.s./R.a.) and even had 90% control against the three graminaceous weeds
(A.a./P.f./P.a.). Furthermore, we observed a clear SAR when introducing the substituted
phenyl at R1 (4am−aw). For example, compounds featuring a phenyl ring substituted with an
electronꢀwithdrawing group (4am, 4ꢀNO2ꢀPh) displayed lower herbicidal activity than those
featuring a phenyl ring substituted with an electronꢀdonating group (4am, 4ꢀMeꢀPh; 4an,
4ꢀOMeꢀPh). When a halogenꢀsubstituted phenyl was introduced at R1, the SAR was
summarized as follows (with a few exceptions against some of the tested weeds): (i) pꢀCl
(4au) > pꢀBr (4av) > pꢀF (4ar), (ii) pꢀF (4ar) > mꢀF (4aq) > oꢀF (4ap) and (iii) pꢀCl (4au) >
mꢀCl (4at) > oꢀCl (4as).
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The introduction of substituents at the R2 position also had a significant influence on the
herbicidal activity. As shown in Table 1, compound 4bb (R2 = H) exhibited greater than 80%
control against the six tested weeds, which was comparable to that of compound 4aa (R2 = Me)
at 150 g ai/ha. However, compounds bearing fluoro, chloro and methoxy groups at the R2
position (4ax−ba) lost their herbicidal activity (except compound 4ba against B.j.) against all
the weeds at the tested application rate. These results were consistent with the AtAHAS
inhibitory activity of these compounds.
It is well known that proꢀdrugs can improve the absorption, distribution, metabolism, and
excretion (ADME) properties of a compund.46 In this work, the carboxyl group in the core
structure was regarded as a good modification site to design a proꢀdrug. We synthesized
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another set of R3ꢀsubstituted compounds (5aa−ah) based on compounds 4aa and 4bb and
examined the effect of R3 substitution on herbicidal activity. Steric factors had a significant
impact on herbicidal activity. Compound 5aa (R3 = Me) showed moderate control (30−50%)
against four different weeds (B.j./R.a./A.a./P.f.) at 150 g ai/ha. However, compounds 5ab (R3
= Et), 5ad (R3 = CH2COOMe), 5ae (R3 = CH2COOEt), and 5af (R3 = CHCH3COOMe) with
sterically larger substituents at R3 (compared to 5aa) displayed poor herbicidal activities
against all the tested weeds. Compounds 5aa (R2 = Me, R3 = Me) and 5ac (R2 = H, R3 = Me)
had dramatically decreased herbicidal activities relative to their parent compounds 4aa and
4bb, respectively. These results implied that introduction of the ester group (proꢀdrug) had a
detrimental effect on herbicidal activity. A conceivable explanation for the poor in vivo
activity of 5aa−af is that the ester group exerts a large steric hindrance effect because of its
ortho substituents, which could make the esterꢀcontaining group difficult for plants to
hydrolyze. However, carboxylic acid sodium salts 5ag and 5ah displayed equivalent
herbicidal activity to that of carboxylic acids 4aa and 4bb.
Herbicidal Spectrum and Antiꢀresistance Properties against Resistant Weeds. Based
on the herbicidal activity results, we selected four promising compounds, 4aa, 4bb, 5ag, and
5ah, to evaluate their herbicidal activity against more weed biotypes (11 kinds) at application
rates of 37.5−150 g ai/ha. As shown in Table 3, compounds 4aa, 4bb, 5ag, and 5ah displayed
70−100% control against all the tested weeds at an application rate of 150 g ai/ha. Even at an
application rate as low as 37.5 g ai/ha, these compounds still displayed excellent inhibitory
activities against all the investigated weeds. For example, compounds 4aa and 5ag had almost
100% control against A.r. and B.j.; more than 80% inhibition against E.p., E.c., A.a., and P.a.;
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and 60% control against A.t., C.s., and R.a.. These results indicated that these compounds
were nearly as potent as bispyribac. It is worth noting that compounds 4bb and 5ah not only
displayed >60% inhibition against all the tested weeds but also completely inhibited the
growth of A.t., A.r., E.p., and B.j. at the lowest application rate of 37.5 g ai/ha.
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As previously mentioned, antiꢀresistance properties are one of the main criteria in
AHASꢀinhibiting herbicide discovery because commercial AHAS inhibitors are suffering
from serious weed resistance problems. To have a better in vivo antiꢀresistance properties, the
inhibitor could display equal or high herbicidal activities against sensitive weeds and its
resistant weeds. To evaluate whether the target compounds can be potentially developed as
antiꢀresistance herbicides, we further selectively tested the herbicidal activity of representative
compounds 4aa and 4bb against AHAS herbicideꢀresistant weeds and the corresponding
sensitive weeds (D. sophia and A. arenaria) at application rates of 0.235−15.0 g ai/ha.
Bispyribac, a commercially available AHASꢀinhibiting herbicide with the lowest degree of
resistance against resistant weeds at present, was used as a positive control. As shown in
Table 4, compounds 4aa and 4bb exhibited nearly 100% control against AHAS
inhibitorꢀsensitive D. sophia and A. arenaria and even 90−100% inhibition against AHAS
inhibitorꢀresistant D. sophia and A. arenaria, indicating their antiꢀresistance properties were
comparable to those of bispyribac at 15.0 g ai/ha. When the spraying dosage was reduced
further (3.75−0.94 g ai/ha), compound 4bb maintained complete inhibition against both
sensitive and resistant D. sophia, and displayed greater than 92.5% herbicidal activity against
both sensitive and resistant A. arenaria. Compound 4aa showed slightly lower herbicidal
activity (>85% inhibition against the four tested weeds) than that of compound 4bb; however,
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the commercial herbicide bispyribac only moderately inhibited sensitive and resistant weeds
at the same application rate. Most promisingly, compounds 4aa and 4bb still displayed
excellent weed control against both sensitive and resistant A. arenaria (>82.5% inhibition)
relative to bispyribac at an application rate as low as 0.235 g ai/ha, indicating that 4aa and
4bb had better antiꢀresistance properties than those of bispyribac. These promising findings
indicate that compounds 4aa and 4bb have great potential to be developed as new lead
compounds to combat weeds with resistance to commercial AHASꢀinhibiting herbicides.
Additional structural optimization and field trial experiments of compounds 4aa and 4bb are
ongoing.
Supporting Information
Detailed information about the singleꢀcrystal data of 4bb (Table S1), comparison of the
experimental pKi and calculated pKi values (Table S2), inhibition curves for
compounds 4aa and 4bb against AtAHAS (Figure S1), HRMS data for compounds 4aa and
4bb (Figure S2), binding energy calculation, physical and spectral data of target compounds
4aa−bb and 5aa−ah. This material is available free of charge via the Internet at
http://pubs.acs.org.
Acknowledgment
We thank Prof. Jie Chen and her research group at Zhejiang Research Institute of Chemical
Industry for their help with screening for herbicidal activity. This research was supported by
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the National Key R&D Program (2017YFD0200507) and the National Natural Science
Foundation of China (No. 21332004).
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Notes
The authors declare no competing financial interest.
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FIGURE CAPTIONS:
Figure 1. Design of the target compounds 4 and 5 and simulated binding modes of
representative compounds A and 4aa with AtAHAS. The key residues surrounding the
active site are shown as blue sticks, and the structure of representative compounds A and
4aa are shown in green and pink, respectively.
Figure 2. Synthetic routes of compounds 4aa–bb and 5aa–ah. Reagents and conditions: (a)
30% aqueous H2O2, Na2WO4·2H2O, AcOH, 50 °C; (b) K2CO3, anhydrous toluene, reflux; (c)
1 N HCl solution; (d) R3ꢀX (X = I or Br), Cs2CO3, DMF, RT; (e) NaOH, THF, RT.
Figure 3. Synthetic routes of key intermediates 3. Reagents and conditions: Method A: (a) I2,
Pd(OAc)2, PhI(OAc)2, DMF, 100 °C; (b) R1B(OH)2, Pd(PPh3)4, K2CO3, DME/H2O,
microwave−assisted, 110 °C; (c) BBr3, anhydrous CH2Cl2, −78 °C. Method B: (d) CO2,
KHCO3, glycerol, 100 °C; (e) acetone, SOCl2, DME; (f) Tf2O, Et3N, CH2Cl2; (g) R1B(OH)2,
Pd(PPh3)4, NaHCO3, DME/H2O, microwave−assisted, 110 °C; (h) KOH, THF/H2O, reflux; (i)
concentrated HCl.
Figure 4. Xꢀray crystal structure of compound 4bb.
Figure 5. Simulated binding mode of compounds 4aa, 4ax, 4az and 4bb with AtAHAS. The
key residues and molecules in the active site are shown as blue sticks. The hydrogen bond
distance (Å) is shown as black lines. (A) Binding mode of 4aa with AtAHAS. (B) Binding
mode of 4ax with AtAHAS. The red arrow represents the direction of its conformational
excursion compared with that of 4bb. (C) Binding mode of 4az with AtAHAS. The pink
circle covering D376 indicates the electrostatic repulsion between this residue and compound
4az. (D) Binding mode of 4bb with AtAHAS.
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Figure 6. Correlation between the experimental and calculated AtAHAS inhibitory activities
(pKi).
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Figure 7. (A) CoMFA map for electrostatic contribution. Compound 4bb is shown inside the
field. Blue contours mean the increase of positive charge, which will promote the activity; on
the contrary, the activity is enhanced if the negative charges are increased in the red contour
areas. (B) CoMFA prediction of steric contribution. Compound 4bb is shown inside the graph.
The yellow map represents the sterically disadvantageous areas where bulkier groups are
beneficial for the enzyme inhibitory activity, and the green area means the opposite. (C)
Alignments of 21 compounds of the training set.
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