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Syntheses and Herbicidal Activity of Triketone-Quinoline Hybrid as Novel 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors Da-Wei Wang, Hong-Yan Lin, Run-Jie Cao, Tao Chen, Feng-Xu Wu, Ge-Fei Hao, Qiong Chen, Wen-Chao Yang, and Guang-Fu Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01530 • Publication Date (Web): 26 May 2015 Downloaded from http://pubs.acs.org on May 30, 2015

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Journal of Agricultural and Food Chemistry

1

Syntheses

and

Herbicidal

Activity

of

Triketone-Quinoline

2

4-Hydroxyphenylpyruvate Dioxygenase Inhibitors

Hybrid

as

Novel

3 4

Da-Wei Wang,† Hong-Yan Lin,† Run-Jie Cao, † Tao Chen,† Feng-Xu Wu,† Ge-Fei Hao,† Qiong

5

Chen,†* Wen-Chao Yang,† and Guang-Fu Yang†, ‡

6

†

Chemistry, Central China Normal University, Wuhan 430079, P. R. China

7 8

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College of

‡

Collaborative Innovation Center of Chemical Science and Engineering, Tianjing 30071, P.R.China

9 10 11

*corresponding authors:

12

E-mail: [email protected]

13

Tel: +86-27-67867800, Fax: +86-27-67867141.

14

1

ACS Paragon Plus Environment


15

ABSTRACT: 4-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is one of the

16

most important targets for herbicide discovery. To search for new HPPD inhibitors with novel

17

scaffolds, triketone-quinoline hybrids were designed and subsequently optimized based on the

18

structure-activity relationship (SAR) studies. Most of the synthesized compounds displayed

19

potent inhibition of Arabidopsis thaliana HPPD (AtHPPD), and some of them exhibited

20

broad-spectrum and promising herbicidal activity at the rate of 150 g ai/ha by post-emergence

21

application. Most promisingly, compound III-l, 3-hydroxy-2-(2-methoxy-7-(methylthio)

22

-quinoline-3-carbonyl)cyclohex-2-enone (Ki = 0.009 µM, AtHPPD), had broader spectrum of

23

weed control than mesotrione. Furthermore, compound III-l was much safer to maize at the rate

24

of 150 g ai/ha than mesotrione, demonstrating its great potential as herbicide for weed control in

25

maize field. Therefore, triketone-quinoline hybrid may serve as a new lead structure for novel

26

herbicides discovery.

27 28

KEYWORDS: 4-Hydroxyphenylpyruvate dioxygenase; Herbicide; quinoline; triketone;

29

rational design.

30

2


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31


INTRODUCTION

32

Modern weed management is still demanding of new herbicides that can control a wide

33

spectrum of weeds, including herbicide-resistant biotypes of these species. In addition, a new

34

herbicide should also be 'selective' to the crops and friendly to the environment.1, 2 Among the

35

known

36

HPPD)-inhibiting herbicides can offer such solutions.3, 4 HPPD is an important enzyme in the

37

metabolism of tyrosine, catalyzing the conversion of 4-hydroxyphenyl pyruvic acid (HPPA) into

38

homogentisic acid (HGA). In plants, HGA can be further transformed into tocopherols and

39

plastoquinone, both of them are crucial for the normal growth of plants. Inhibition of HPPD

40

causes a reduction in carotenoids levels, which indirectly affects photosynthesis. Then the plants

41

exposed to sunlight will be severely damaged, developing unique bleaching symptoms followed

42

by necrosis and death.5-8 HPPD-inhibiting herbicides have many advantages, such as

43

broad-spectrum weed control (including resistant to other herbicides), excellent crop selectivity,

44

benign environmental effects, low application rate and low toxicity.

herbicide

classes,

4-hydroxyphenylpyruvate

dioxygenase

(EC

1.13.11.27,

45

Up to now, there are about 13 commercial HPPD herbicides, which are mainly classified into

46

three categories: triketones, pyrazoles, and isoxazoles (diketonitrile). The minimum substructure

47

of these inhibitors is mainly based around 2-benzoylethen-1-ol or 2-heteroaroylethen-1-ol,

48

which is involved in the binding of these inhibitors to the ironII in the active site of HPPD.9, 10

49

Among the commercialized herbicides, triketone derivatives are one of the most widely studied,

50

due to the structural features of these derivatives. The structure feature of these triketones can be

51

further divided into two parts, triketone and aromatic moieties. Extensive studies have proved

52

that, modification of the aromatic part is an effective way to obtain new inhibitors with

3



53

improved potency.3, 4, 11 Additionally, many natural and synthetic triketones have aliphatic side

54

chains that demonstrate the importance of lipophilicity for activity.12, 13

55

A series of novel triketone-containing quinazoline-2,4-diones by modifying the aromatic part

56

have designed previously.14-16 Most of the synthesized inhibitors showed 'good' to 'excellent'

57

herbicidal activity. As a follow up to discover novel HPPD inhibitors with improved potency, a

58

series of compounds with new heterocyclic aromatic side chains have been prepared. Quinoline

59

is an important structural motif of many natural products, and its derivatives are important

60

pharmaceutical and agrochemical intermediates.17-20 For example, in pharmaceuticals, the

61

derivatives of quinoline exhibited extensive biological activities, including antimalarial,

62

antitumor, anti-infective, and anti-inflammatory; in agrochemicals, quinoline is a substructure of

63

many herbicides, fungicides and insecticides.21-23 Therefore, quinoline might be a promising

64

aromatic part to integrate with the triketone pharmacophore. Therefore, a series of novel

65

triketone-quinoline hybrids was designed. Herein, we will report the detail of rational design,

66

synthetic chemistry, inhibitory activity against Arabidopsis thaliana HPPD (AtHPPD),

67

herbicidal activity, and structure–activity relationships (SAR) of triketone-quinoline hybrids. As

68

anticipated, many of the synthesized compounds displayed nanomolar potency toward AtHPPD.

69

In addition, some compounds exhibited promising and broad-spectrum herbicidal activity at the

70

rate of 150 g ai/ha as well as selectivity to several crops, such as maize, wheat or rice.

71 72

MATERIALS AND METHODS

73

The detailed information for the synthesis of compounds I-VI are shown in Figures 1 to 4.

74

X-ray Diffraction. Compound III-d was recrystallized from a mixture of acetone and

4


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n-hexane to afford a suitable single crystal. Light brown crystals of III-d (0.20 mm × 0.20 mm ×

76

0.20 mm) were mounted on a quartz fiber with protection oil. Cell dimensions and intensities

77

were measured at 298 K on a Bruker SMART APEX DUO area detector diffractometer (Bruker

78

AXS, Madison, WI) with graphite monochromated Mo Kα radiation (λ = 0.71073 Å); θmax =

79

28.00; 14889 measured reflections; 4658 independent reflections (Rint = 0.0484). The data sets

80

were integrated and reduced using SAINT Plus Programme.24 Data were corrected for Lorentz

81

and polarization effects and for absorption (Tmax= 0.9821; Tmin = 0.9821). The structure was

82

solved by direct method using SHELXS97 and refined with SHELXL970.25 Full-matrix

83

least-squares refinement based on F2 using the weight of 1/[σ2(Fo2) + (0.0976P)2 + 0.2989P]

84

gave final values of R1 = 0.0690, ωR2 = 0.1652, and GOF(F) = 1.128 for 265 variables, 265

85

parameters and 4658 contributing reflections.

86

maximum/minimum residual electron density = 0.614 per -0.329 e Å−3. Hydrogen atoms were

87

observed and placed at their ideal positions with a fixed value of their isotropic displacement

88

parameter.

Maximum

shift/error = 0.000, and

89

Compound III-f was recrystallized from a mixture of acetone and n-hexane to afford a

90

suitable single crystal. Light yellow crystals of III-f (0.12 mm × 0.10 mm × 0.10 mm) were

91

mounted on a quartz fiber with protection oil. Cell dimensions and intensities were measured at

92

298 K on a Bruker SMART APEX DUO area detector diffractometer with graphite

93

monochromated Mo Kα radiation (λ = 0.71073 Å); θmax = 30.00; 15593 measured reflections;

94

4396 independent reflections (Rint = 0.1064). The data sets were integrated and reduced using

95

SAINT Plus Programme.24 Data were corrected for Lorentz and polarization effects and for

96

absorption (Tmax= 0.9904; Tmin = 0.9885). The structure was solved by direct method using

5



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97

SHELXS97 and refined with SHELXL970.25 Full-matrix least-squares refinement based on F2

98

using the weight of 1/[σ2(Fo2) + (0.0886P)2 + 0.2528P] gave final values of R1 = 0.0514, ωR2 =

99

0.1438, and GOF(F) = 1.067 for 211 variables, 211 parameters and 4396 contributing reflections.

100

Maximum shift/error = 0.000, and maximum/minimum residual electron density = 0.869 per

101

-0.369 e Å−3. Hydrogen atoms were observed and placed at their ideal positions with a fixed

102

value of their isotropic displacement parameter.

103

Crystallographic data for compounds III-d and III-f have been deposited with the Cambridge

104

Crystallographic Data Centre as a supplementary publication with the deposition No. 1040193

105

and

106

http://www.ccdc.cam.ac.uk/.

977357,

respectively.

These

data

can

be

obtained

free

of

charge

from

107

Plasmid Construction, Protein Expression and Purification, activity assays, and kinetic

108

inhibition studies. AtHPPD was constructed by PCR using cDNA of HPPD in

109

pMD19-T Simple (Hangzhou BIOSCI Biotechnology Company, Hangzhou, China) as the

110

template. The primers used here were 5'-CATGCCATGGGCCACCAAAACGCCGC-3' (NcoI)

111

and 5'- CGCGGATCCTC-AGTGGTGGTGGTGGTGGTGTCCCACTAACTGTTT-3' (BamHI).

112

PCR conditions were 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 68 °C for 1.5 min. The

113

amplion was introduced into the expression vector pET-15b and subsequently transformed into

114

E. coli BL-21(DE3). The DNA sequences of the positive clones were confirmed by DNA

115

sequencing with Shanghai SANGON Company.

116

Recombinant AtHPPD was over-expressed in E. coli BL-21 (DE3) cells with pET-15b-HPPD

117

plasmid. Recombinant homogentisate 1,2-dioxygenase (HGD) from human was over-expressed

118

in E. coli BL-21 (DE3) cells with pET-28a-HGD plasmid. The cells were grown at 37 °C in

6


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119

Luria Bertani broth supplemented with 50 µg/mL of kanamycin (pET-28a plasmid) or 100

120

µg/mL antimycin (pET-15b plasmid) according to previous publication.26 Expression of the

121

AtHPPD plasmid was incubated at 37 °C for 12 h. HGD was induced by 0.5 mM Isopropyl

122

β-D-1-thiogalactopyranoside (IPTG) when bacterial grown reached an A600 of 0.6, and then the

123

cells were incubated for another 40 h at 15 °C. After

124

30 min), the pellet was resuspended in buffer (20 mM HEPES，20 mM NaCl，pH 7.0) and

125

washed twice, followed by sonication using a cell disruptor. A crude cell-free supernatant was

126

obtained by centrifugation at 20,000 x g for 30 min.

harvesting by centrifugation (5000 x g,

127

AtHPPD was purified in two chromatographic steps. The crude cell-free supernatant was

128

loaded onto a Ni-NTA column (Qiagen, Canada), equilibrated with 20 mM HEPES, pH 7.0.

129

Then, HPPD was eluted with 20 mM HEPES, pH 7.0, 150 mM NaCl, and 250 mM imidazole.

130

The fractions containing HPPD were concentrated and the buffer was exchanged for 20 mM

131

HEPES, pH 7.0 by ultrafiltration in Ultrafree filter devices (Millipore, MA). To further purify

132

the recombinant HPPD, anion exchange chromatography was carried out on Q resin

133

(Amersham-Pharmacia Biotech, Germany) in 20 mM HEPES, pH 7.0. Elution of the

134

recombinant HPPD was carried out in a linear gradient from 0 to 250 mM NaCl. Again, the

135

HPPD-containing fractions were collected and the buffer was exchanged to 20 mM HEPES, pH

136

7.0.

137

The in vitro activity and inhibition of AtHPPD were measured by a modification of coupled

138

enzyme assay methods previously reported in the literature.27 Assays were performed in

139

96-well plates at 30 °C using a UV/visible plate reader (Bio-tech, Vermont) to monitor the

140

formation of maleylacetoacetate at 318 nm (ε330 = 13 500/M/cm). The reaction mixture in a

7



141

total assay volume of 200 µL contained appropriate amounts of HPPA, 100 µM FeSO4, 2 mM

142

sodium ascorbate, 20 mM HEPES buffer (pH 7.0), HPPD and HGD. Before assays were

143

conducted, all reaction components were preequilibrated at 30 °C for at least 10 min. The

144

amount of HGD activity was predetermined to be in large excess of the HPPD activity to

145

ensure that the reaction was tightly coupled (the Km of HGD for HGA was 25 µM). Each

146

experiment was repeated at least three duplicates and the values were averaged. HPPD

147

inhibitors were dissolved in dimethyl sulfoxide (DMSO) for stock solution and diluted to

148

various concentrations with reaction buffer just before using. The inhibition constant (Ki), the

149

indication of an inhibitor’s potency, was obtained from the Dixon plot of plotting 1/v against

150

concentration of inhibitor at certain concentrations of substrate. Bovine serum albumin is

151

usually added up to 0.5% of the total reaction volume for coating of the target enzyme during

152

the incubation. In our assay, no obvious effect from bovine serum albumin on the activity of

153

the compounds has been found, which indicated that the new compounds selectively inhibited

154

the target enzyme but did not interact with bovine serum albumin.

155

Computational Modeling and CoMSIA Analysis: The crystal structure of AtHPPD was

156

taken from PDB data bank (PDB ID: 1TFZ). Compounds were constructed and optimized by

157

using SYBYL 7.0 (Tripos Inc.) and Gasteiger-Huckel charges were calculated for them.

158

Docking calculations were performed on the two molecules by using AutoDock 4.0. The protein

159

and ligand structures were prepared with AutoDock Tools. A total of 256 runs were launched for

160

each molecule. Each docked structure was scored by the built-in scoring function and was

161

clustered by 0.8 Å of RMSD criterions. The best binding modes were determined by docking

162

scores and also the comparison with available complex crystal structure of DAS645 with

8


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AtHPPD (PDB entry: 1TG5) as reference.28 Standard Amber ff99 force field parameters were

164

assigned to protein, and general AMBER force field (gaff) was assigned to ligands. The partial

165

atomic charges of ligands were calculated using the AM1-BCC method and the system was

166

solvated in an octahedral box of TIP3P water with the crystallographic water molecules kept.

167

The edge of the box was at least 8 Å from the solute, appropriate counterions were added to the

168

system to preserve neutrality. In each step, energy minimization was first performed by using the

169

steepest descent algorithm for 1,000 steps and then the conjugated gradient algorithm for

170

another 2,000 steps.

171

We selected the majority of the new molecules which show different activities to perform

172

3D-QSAR. All molecular modeling and 3D-QSAR studies were performed using SYBYL7.3

173

with TRIPOS Force Field. The 3D structures of all compounds were built by using default

174

setting of SYBYL, and the molecules were subjected for energy minimization at a gradient of

175

1.0 kcal/mol with delta energy change of 0.05 cal/mol. The CoMFA descriptors, steric and

176

electrostatic field energies were calculated using the SYBYL default parameters: 2.0 Å rid

177

points spacing, an sp3 carbon probe atom with +1 charge and a minimum σ(column filting) of

178

2.0 kcal/mol, and the energy cutoff of 30.0 kcal/mol.

179

Herbicidal Activities. The post-emergent herbicidal activities of compounds I-VI, against

180

Echinochloa crus-galli (EC), Setaria faberi (SF), Digitaria sanguinalis (DS), Amaranthus

181

retroflexus (AR), Eclipta prostrata (EP) and Abutilon juncea (AJ) were evaluated according to

182

the previously reported procedure;14-16, 29 the commercial triketone herbicide mesotrione was

183

selected as a positive control. All test compounds were formulated as 100 g/L emulsified

184

concentrates by using DMF as solvent and Tween-80 as emulsification reagent. The

9



185

concentrates were diluted with water to the required concentration and applied to pot-grown

186

plants in a greenhouse. The soil used was a clay soil, pH 6.5, 1.6% organic matter, 37.3% clay

187

particles, and CEC 12.1 mol/kg. The rate of application (g ai/ha) was calculated by the total

188

amount of active ingredient in the formulation divided by the surface area of the pot. Plastic pots

189

with a diameter of 9 cm were filled with soil to a depth of 8 cm. Approximately 20 seeds of the

190

tested weeds were sown in the soil at the depth of 1-3 cm and grown at the temperature of 15-30

191

°C in a greenhouse. The air relative humidity was 50%. The diluted formulation solutions were

192

applied for post-emergence treatment. Broadleaf weeds were treated at the 2-leaf stage and

193

monocotyledon weeds were treated at the 1-leaf stage. The post-emergence application rate was

194

150 g ai/ha. Untreated seedlings were used as the control group and the solvent (DMF +

195

Tween-80)-treated seedlings were used as the solvent control group. Herbicidal activity was

196

evaluated visually at 15 days post-treatment (Tables 1 and 2), with three replicates per treatment.

197

Crop Selectivity. The conventional rice, soybean, cotton, wheat, canola and maize were

198

planted separately in pots (12 cm diam.) containing test soil and grown in a greenhouse at 20-25

199

°C.23 After the plants had reached the 4-leaf stage, the spraying treatment was conducted at the

200

dosage of 150 g ai/ha. The visual injury and growth state of the individual plants were observed

201

at regular intervals. After 15 days, the final results of crop safety were determined (Table 3),

202

using three replicates per treatment.

203 204

RESULTS AND DISCUSSION

205

Chemistry. According to the substituents at R2, the target compounds I-VI were prepared

206

by four different synthetic routes. When R2 is a hydrogen atom, the synthetic details for

10


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207

compound I are shown in Figure 1; when R2 are alkoxy or aryloxy groups, the synthetic routes

208

for compounds II and III are outlined in Figure 2; when R2 are methyl or trifluoromethyl groups,

209

the detail synthetic routes for compounds IV and V are shown in Figure 3; when R2 is a cyano

210

group, the detail synthetic routes for compounds VI are outlined in Figure 4. The lead

211

compound I can be obtained by a three-step synthetic route using the commercially available

212

quinoline-3-carboxylic acid 1 as the starting material (Figure 1). The carboxylic acid 1 was very

213

sensitive to SOCl2, POCl3, PCl5, and PCl3; because of the high reactivity of the hydrogen at the

214

2 position on the quinoline ring. To our delight that, by using 1.2 equivalents of oxalyl chloride

215

as the chloroformylation reagent, CH2Cl2 as the reaction solvent, the corresponding acid

216

chloride can be successfully synthesized. The acid chloride thus obtained was very unstable, so

217

it was used in the next step without further purification. Subsequently, the acid chloride reacted

218

with 1,3-cyclohexanedione, the enol ester 2 can be obtained in a yield of 78%. Finally, by using

219

acetone cyanohydrin as the Fries catalyst, the lead compound I was synthesized in a yield of

220

66%.

221

Since no carboxylic acids 7a-q are commercially available, the reported methods were

222

applied to synthesize them.30-32 As seen in Figure 2, the target compounds II and III were

223

synthesized by an eight-step synthetic route using (substituted) anilines 3a-q as the starting

224

materials.

225

N-phenylacetamides 4a-q can be synthesized in high yields. Subsequently, the corresponding

226

intermediates 5a-q were obtained in yields of 30-80% via Vilsmeier-Haack reaction. It was

227

found that, the substitutions of R3 have a big impact on reactivity at this reaction step, if R3 were

228

electron-withdrawing groups, the reaction yields were very low or did not react at all. 5a-q were

After

reaction

with

acetic

anhydride,

the

11


corresponding

(substituted)


229

then oxidized by iodine in methanol, compounds 6a-q were synthesized in yields of 70-98%.

230

Various carboxylic acids 7a-q were prepared by a three step one-pot method. More specifically,

231

6a-q first reacted with sodium alkoxides or aryloxides, and then water was added to the reaction

232

solution to hydrolyze the methoxycarbonyl group. After treating the residue with citric acid, the

233

corresponding 7a-q were obtained in yields of 64-98%. Followed by chloroformylation and

234

esterification reaction, the enol esters 8a-l and 9a-t were obtained in yields of 42-90%. Finally,

235

by using the same method as the synthesis of I, the title compounds II and III can be afforded in

236

yields of 52-84%.

237

The key intermediates 11a and 11b were prepared by reaction of methyl acetoacetate 10a or

238

ethyl trifluoroacetoacetate 10b with o-toluidine 3j in the presence of 4-methylbenzenesulfonic

239

acid as the catalyst (Figure 3). Then, 11a and 11b reacted with N,N-Dimethylformamide (DMF)

240

and POCl3 in dichloroethane (DCE), the corresponding 12a and 12b were obtained in yields of

241

90% and 50%, respectively. Alkaline hydrolysis of 12a and 12b with LiOH.H2O in the presence

242

of methanol and water furnished the hydroxyacids 13a and 13b. The synthesis of the target

243

compounds IV and V from 13a and 13b were the same as the preparation of compound I.

244

Similarly, compounds VI can be obtained by a six-step reaction route using methyl

245

2-chloro-8-methylquinoline-3-carboxylate 6j as the starting material, after cyanation reaction

246

with CuCN, the key intermediate 16 was synthesized in a yield of 88%. Then followed by

247

hydrolysis, chloroformylation, enol esterification, and rearrangement reactions, the target

248

compounds VI can be successfully obtained.

249 250

The chemical structures of all of the synthetic triketone-quinoline hybrids (I-VI) were confirmed by 1H NMR,

13

C NMR spectroscopic and HRMS spectrometric data. Furthermore,

12


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251


the structures of compounds III-d and III-f were verified by X-ray analysis (Figure 5).

252

HPPD Inhibition and SAR. The Ki value of the designed lead compound I against AtHPPD

253

was about 0.075 µM, suitable worth for further optimization (Table 1). Molecular modeling was

254

performed to guide us in rational structural optimization. Because no crystal structure of

255

compound I with AtHPPD available, we used the reported binding mode of DAS645 with

256

AtHPPD (PDB entry: 1TG5) as reference.33 There were two major interactions of compound I

257

with AtHPPD active site (Figure 6). The triketone part of compound I can form a bidentate

258

interaction with the active site ironII, while the quinoline motif can form π-π interaction with

259

Phe360 and Phe403. Furthermore, there was no significant interaction of compound I with

260

active site of AtHPPD (around 4Å residues); which indicated that a variety of substituent groups

261

might be introduced to compound I to increase its interaction with the enzyme. To obtain new

262

compounds with improved AtHPPD inhibitory activity we then designed compounds II-VI

263

based on the core structure on lead compound I.

264

The substituents at R1 have a significant impact on the activity (Table 1). In most cases,

265

compounds with a 5,5-di-CH3 at R1 had decreased HPPD inhibitory activity compared to lead

266

compound I. Although the Ki value of compound II-i was 0.041 µM, it showed a slightly potent

267

activity than compound I, however, its activity is still far from enough when compared with

268

mesotrione. For further optimization, compound II-g was chosen as a representative to

269

investigate the binding modes. The trikeone part of compound II-g can form a bidentate

270

association with the ferrous iron active site and the quinoline ring can form π-π interaction with

271

Phe360 and Phe403, respectively (panel A of Figure 7). When the two methyl groups were

272

introduced to the 5-positions of the 1,3-cyclohexane ring, the distances between the methyl

13



273

groups and the surrounding residues would be reduced to lead to steric hindrance. The distances

274

from two methyl groups on the cyclohexanedione ring of compound II-g to residues Asn261,

275

Ser246, Lys400, is 3.0 Å, 2.9 Å and 3.1 Å, respectively (left picture of Figure 7). The loss of

276

activity might due to the spatial repellence caused by the methyl groups at 5-positions of the

277

1,3-cyclohexane ring. Therefore, we proposed that the removal of steric hindrance at this

278

position may be favorable to activity. After one methyl group was removed from this position,

279

the activity of compound II-k was greatly improved compared with its parent compound II-j,

280

however, its activity was still inadequate. This suggests that removing the remaining methyl

281

group would further improve the activity of these molecules.

282

Based on the above analysis, another methyl group was also removed from the 5-position of

283

the 1,3-cyclohexane ring of compound II-j. The Ki value of compound III-a was as low as

284

0.009 µM, about 10 times more potent than its parent compound II-k, slightly more potent than

285

commercial mesotrione (Ki = 0.013 µM). Encouraged by these results, we further optimized the

286

structure and studied the activity of this series of compounds. As the steric effects at R1 had a

287

big impact on activity, compounds III-b—III-d were synthesized to test the contribution of R2

288

on activity. The sterically bulk groups at R2 were also detrimental to activity, for example,

289

compound III-a with a methoxy group at R2 displayed enhance activity relative to compounds

290

III-b—III-d (III-a > III-b > III-c > III-d). To understand the structural basis, compound III-d

291

was used as a representative for further binding modes investigation. The distance of benzyl

292

group between Phe371 is 3.0 Å, showing strong steric repulsion effect (Panel B of Figure 7).

293

On the basis of above observation, the methoxy group at R2 was retained and another set of

294

compounds III-e-III-t were synthesized to further investigate their HPPD inhibitory activity

14


Page 14 of 40

Page 15 of 40


295

(Figure 2). All newly synthesized compounds displayed inhibitory constants at the nanomolar

296

range, some of them even showing slightly more potential than mesotrione (Table 1). The

297

position of a single group at R3, has a big influence on activity. For example, when a methyl

298

group was introduced at 6-position (III-f), 7-positon (III-g) or 8-positon (III-a) on quinoline

299

ring, compound III-a would have better activity than compounds III-g and III-f (III-a > III-g >

300

III-f). The similar SAR can also be applied to those with methoxy-substituted analogues (III-i >

301

III-h) and chloro-substituted analogues (III-k > III-j). It appeared that the steric bulky groups

302

at R3 were also detrimental to activity (III-a > III-m > III-n). As to the multi-substituted

303

analogues, compounds with substituents at 7,8 positions showed improved activity relative to

304

those equivalent substitutions at 5,8 and 6,8 positions (III-q > III-p > III-o). It seemed that the

305

electron-withdrawing groups at R3 would produce enhanced effects compared to those with

306

electron-donating groups, although there may be few exceptions.

307

Herbicidal Activity and SAR. The post-emergence herbicidal activity of the newly prepared

308

compounds were tested against monocotyledon weeds (E. crus-galli, S. faberii, and D.

309

sanguinalis) and broadleaf weeds (A. retroflexus, E. prostrata, and A. juncea) in the greenhouse

310

experiments. The commercial herbicide mesotrione was used as a control; the results are listed

311

in Table 1 and Table 2. The treated weeds had developed unique whitening symptoms in light,

312

which indicated that these compounds inhibited HPPD in planta. Some of the synthesized

313

compounds displayed promising and broad-spectrum herbicidal activities at the rate of 150 g

314

ai/ha. Compounds III-l even exhibited broader spectrum of weeds control (inhibition > 80%)

315

than mesotrione at the rate of 75 g ai/ha.

316

In most cases, introducing substitutions at R1 were detrimental to herbicidal activity (Table 1).

15



317

Notably, compounds with sterically bulky groups at R2 also displayed decreased herbicidal

318

activity effects (III-a > III-6b > III-c > III-d). These results were consistent with in vitro

319

HPPD inhibitory activity. Furthermore, an interesting structure-activity relationships was

320

observed with respect to the R3 substitution. For example, when a single substitution was

321

introduce at different positions on the quinoline ring, compounds with substituents at 7 position

322

would have broader-spectrum of weeds control than those at 8- and 6- positions (III-g > III-a >

323

III-f). It seemed that bulky groups on the quinoline ring were favorable to herbicidal activity

324

(III-n > III-m > III-a). In most case, compounds with electron-withdrawing groups at R3 were

325

found to exhibit higher herbicidal activity than those with electron-donating groups (III-k >

326

III-g). Surprisingly, compounds II-d and III-l with methylthio group at R3 also displayed

327

excellent herbicidal activity at the rate of 150 ai/ha, even at a dosage as low as 37.5 g ai/ha

328

compound III-1 still showed over 80% control against tested broadleaf weeds and over 65%

329

inhibition against monocotyledon weeds (Table 2). A possible explanation for the enhanced

330

herbicidal activity of compounds II-d and III-l is that, the unique properties of methylthio group

331

make compounds more easily absorbed by plants.33

332

In this work, some of the synthesized compounds had good HPPD-inhibiting activity,

333

however, their herbicidal activities were not satisfactory. For example, compound III-a (Ki =

334

0.009 µM) showed a slightly higher HPPD inhibitory activity than mesotrione (Ki = 0.013 µM),

335

however, its herbicidal activity still could not compete with mesotrione. As far as we know,

336

there are many reasons for a compound having good in vitro inhibitory activity without

337

displaying promising herbicidal activity, such as, the absorption, distribution, metabolism, and

338

excretion (ADME) properties. As there is a methoxy group at the 2- position on quinoline ring

16


Page 16 of 40

Page 17 of 40


339

of compound III-a. We inferred that methoxy group of compound III-a might be easily

340

metabolized by plants.34 This because the unique chemical properties of quinoline make its 2-

341

position very active compared with other positions. Therefore a series of compounds with

342

different metabolism blocking groups were synthesized to verify this hypothesis (Figure 3 and

343

4). As indicated in Table 1, among the newly prepared compounds IV and VI, compounds with

344

electron-withdrawing group at R2 were found to detrimental to herbicidal activity. Promisingly,

345

compounds IV-a and IV-b displayed significantly enhanced herbicidal activity than their parent

346

compound III-a and II-k, respectively. Therefore, the SAR of R2 can be summarized as follows:

347

CH3 > CF3> OCH3 > CN > OCH2H3, OCH2CH2H3 >OCH2Ph.

348

Crop Selectivity. As mentioned in the introduction, crop selectivity is one of the main

349

concerns in herbicides discovery. Compounds III-l, III-m, and III-n with promising herbicidal

350

activity were chose as representatives for further crop selectivity studies. Maize displayed high

351

tolerance to compound III-l after post-emergence application at the rate of 150 g ai/ha (Table 3).

352

The promising results indicated that, compound III-l might be developed as a potential

353

herbicidal for maize fields. Most promisingly, compound III-m was found to be 'selective' to

354

maize, rice, and wheat at the dosage of 150 g ai/ha, however, the commercial mesotrione was

355

found to be 'nonseletive' to rice and wheat at the same rate. These results showed that there is a

356

great potential for III-m to be developed as a herbicide for weeds control in maize, rice, and

357

wheat fields. Furthermore, compound III-n was safe for maize at the dosage of 150 g ai/ha,

358

indicating that III-n might be developed as a potential herbicide for maize field.

359

CoMFA analysis. To further understand the substituent effects on AtHPPD inhibition, we

360

performed a brief CoMFA analysis of 31 representative compounds with SYBYL.29 The

17



361

predicted CoMFA model was established with the conventional coefficient r2 = 0.974 and the

362

cross-validated coefficient q2 = 0.806, the predicted non-cross-validated coefficient r2(pred) =

363

0.854. The calculated activity values of the representative compounds are shown in Table 4, and

364

the plots of the predicted versus the actual activity values for the selected compounds are shown.

365

The contour map of the steric contributions was marked in green and yellow areas (Figure 8A).

366

The yellow contour means a bulky substituent in this area is unfavorable for the inhibition

367

activity of AtHPPD. For example, the compound II-b (R1 = 5,5-diCH3, Ki =0.304 µM) is less

368

active than compound III-i (R1=H, Ki = 0.029 µM), although they have the same R2 and R3

369

groups, which is similar as II-e and III-m, II-f and III-n, II-j and II-k. On the contrary, the

370

green region highlights positions where a bulky group would be favorable for HPPD inhibition

371

activity. The electrostatic contour map is shown in Figure 9B. The blue color represents that the

372

electro-positive group will be advantageous to the bioactivity of HPPD inhibitors. The

373

electro-negative groups is marked by red area, which are closed to the region of R2 group and

374

the 7,8-positions on the quinoline ring. So we can infer that the compound which bears an

375

electro-withdrawing group at R2 and R3 (7 and 8 position) will show higher activity. The

376

experimental values are consistent with our calculation results, for example, the activity of III-r

377

(R3 = 7-F-8-CH3, Ki = 0.011 µM) > III-s (R3 = 7-Cl-8-CH3, Ki = 0.014 µM) > III-t (R3 =

378

7-Cl-8-CH3, Ki = 0.014 µM), III-b (R3 = OCH2H3, Ki = 0.025 µM) > III-c (R3 = O-n-Pr, Ki =

379

0.171 µM) > III-d (R3 = OCH2Ph, Ki = 0.213 µM) and the activity of compounds VI-a and VI-b

380

are higher than most of other compounds in Table 4, because of the cyano group at R2 position.

381

In conclusion, a series of novel triketone-quinoline hybrids were rationally designed and

382

identified as potent HPPD inhibitors for herbicide discovery. Most of the synthesized

18


Page 18 of 40

Page 19 of 40


383

compounds displayed excellent HPPD inhibitory activity, and some of them were even superior

384

to the commercial herbicide mesotrione. To our delight that, compounds III-l, III-m and III-n

385

displayed promising herbicidal activity at the rate of 37.5-150 g ai/ha. Especially, compound

386

III-l displayed broader spectrum of weed control than mesotrione. In addition, compounds III-l

387

and III-n were highly ‘selective’ to maize, III-m was safe for maize, rice, and wheat by

388

post-emergence application at the rate of 150 g ai/ha. These results indicated that

389

triketone-quinoline hybrids could be novel lead compounds for novel herbicides discovery.

390

Further field trials and structure optimization of compounds III-l, III-m and III-n are

391

underway.

392 393

SUPPORTING INFORMATIONupporting Information

394

The detailed information for the preparation and the analytical data of compounds I-VI are

395

shown in the Supporting Information. These materials are available free of charge via internet at

396

http://pubs.acs.org.

397 398

ACKNOWLEDGMENTS

399

The authors are very grateful to the National Key Technologies R&D Program of China (No.

400

2011BAE06B03) and National Natural Science Foundation of China (No.21332004 and

401

21372093).

402 403

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24


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Page 25 of 40


Figure captions: Figure 1. Synthetic route for lead compound I. Reagents and conditions: (a) (COCl)2, CH2Cl2, -5 °C; (b) 1,3-cyclohexanedione, CH2Cl2, 0 °C; (c) acetone cyanohydrin, Et3N, CH2Cl2, RT. Figure 2. Synthetic route for compounds II and III. Reagents and conditions: (a) (CH3C)2O, Et3N, CH2Cl2, 0 °C-RT. (b) DMF, POCl3, 0-80 °C; (c) K2CO3, CH3OH, I2, reflux; (d) Na, alcohols, reflux; (e) H2O, citric acid; (f) (COCl)2, CH2Cl2, -5 °C; (g) (substituted) 1,3-cyclohexanedione, CH2Cl2, 0 °C; (h) acetone cyanohydrin, Et3N, CH2Cl2, RT. Figure 3. Synthetic route for target compounds IV-V. Reagents and conditions: (a) p-toluenesulfonic acid (p-TSA), cyclohexane, reflux; (b) DMF, POCl3, DCE, 70-80 °C; (c) LiOH·H2O, methanol, H2O, reflux; (d) citric acid;

(e) (COCl)2,

CH2Cl2, 0 °C; (g) (substituted) 1,3-cyclohexanedione, Et3N, CH2Cl2, 0 °C; (h) acetone cyanohydrin, Et3N, CH2Cl2, RT. Figure 4. Synthetic route for target compounds VI. Reagents and conditions: (a) CuCN, DMF, reflux; (b) LiOH·H2O, methanol, H2O, reflux; (c) citric acid; (d) (COCl)2, CH2Cl2, 0 °C; (e) (substituted) 1,3-cyclohexanedione, Et3N, CH2Cl2, 0 °C; (f) acetone cyanohydrin, Et3N, CH2Cl2, RT. Figure 5. X-ray crystal structures for compounds III-d and III-f. Figure 6. Simulated binding mode of lead compound I with AtHPPD and the designed compounds II-III. The ferrous iron is shown in cyan sphere, the structure of compound I is shown in yellow sticks, and the key residues surrounding the active site are shown in light blue.

25



Figure 7. Simulated binding mode of compounds II-g (A) and III-t (B) with AtHPPD. The ferrous iron is shown as a cyan sphere, the structures of compounds II-g and III-t are shown in yellow sticks, and the key residues surrounding the active site are shown in light blue. Figure 8. A: CoMFA map for steric contribution. Compound IV-b is shown inside the filed. Green polyhedra represents the sterically favored areas where more bulky substituents favor activity and yellow polyhedra represents the disfavored areas where less bulky substituents favor activity. B: CoMFA map for electrostatic contribution. Compound IV-b is shown inside the filed. Blue contours mean the increase of positive charge in these areas will promote the activity; on the contrary, it will enhance the activity if we increase negative charges in the red contours areas. Compound IV-b is shown inside the filed. C: The alignment of 24 compounds of training set.

26


Page 26 of 40

Page 27 of 40


Table 1: Chemical Structures and Post-Emergent Herbicidal Activitya of Compounds I-IV, and Their Inhibitory Activities Against AtHPPD. Compd. I II-a II-b II-c II-d II-e II-f II-g II-h II-i II-j II-k II-l III-a III-b III-c III-d III-e III-f III-g III-h III-i III-j III-k III-l III-m III-n III-o III-p III-q III-r III-s III-t IV-a IV-b V VI-a VI-b

R

1

2

R

R

% Inhibition

3

H H H 5,5-diCH3 OCH3 6-OCH3 5,5-diCH3 OCH3 7-OCH3 5,5-diCH3 OCH3 6-Cl 5,5-diCH3 OCH3 7-SH3 5,5-diCH3 OCH3 8-CH2CH3 5,5-diCH3 OCH3 8-CH(CH3)2 5,5-diCH3 OCH3 7-F-8-CH3 5,5-diCH3 OCH3 7-Cl-8-CH3 5,5-diCH3 OCH3 7-Br-8-CH3 8-CH3 5,5-diCH3 OCH3 5-CH3 OCH3 8-CH3 6,6-diCH3 OCH3 8-CH3 H OCH3 8-CH3 H OCH2H3 8-CH3 H O-n-Pr 8-CH3 H OCH2Ph 8-CH3 H OCH3 H H OCH3 6-CH3 7-CH3 H OCH3 H OCH3 6-OCH3 H OCH3 7-OCH3 H OCH3 6-Cl H OCH3 7-Cl H OCH3 7-SCH3 H OCH3 8-CH2CH3 H OCH3 8-CH(CH3)2 H OCH3 5,8-diCH3 H OCH3 6,8-diCH3 H OCH3 7,8-diCH3 H OCH3 7-F-8-CH3 H OCH3 7-Cl-8-CH3 H OCH3 7-Br-8-CH3 8-CH3 H CH3 5-CH3 CH3 8-CH3 H CF3 8-CH3 H CN 8-CH3 5-CH3 CN 8-CH3

ECb SFb DSb ARb EPb 0 0 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 30 0 75 70 80 0 0 0 40 40 50 40 45 0 45 0 0 0 0 70 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 50 50 0 0 0 30 30 0 0 0 40 20 0 0 0 0 30 0 0 0 0 30 0 0 0 0 0 15 0 0 30 20 0 0 0 0 0 0 0 0 60 60 0 0 0 0 0 30 0 0 80 50 0 0 0 0 0 0 0 0 80 55 90 85 90 90 90 75 80 60 80 90 85 75 70 90 100 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 40 50 0 80 80 70 60 0 90 80 0 0 0 70 30 75 60 0 60 90 80 85 80 90 90 50 30 0 60 50 0 0 0 0 30 0 0 0 0 0 27


AJb 0 0 30 0 80 15 30 0 0 0 0 50 50 80 0 0 0 100 0 80 0 30 95 95 100 100 100 0 0 0 90 80 70 80 95 80 30 0

AtHPPD Inhibition Ki (µM)c 0.075±0.001 0.055±0.005 0.304±0.008 0.055±0.001 0.341±0.005 0.222±0.009 0.132±0.009 0.626±0.210 0.291±0.015 0.041±0.004 0.298±0.007 0.099±0.003 0.074±0.009 0.009±0.001 0.025±0.002 0.171±0.008 0.213±0.006 0.062±0.003 0.030±0.002 0.024±0.001 0.076±0.005 0.029±0.005 0.072±0.008 0.011±0.001 0.009±0.001 0.017±0.001 0.035±0.007 0.039±0.001 0.028±0.002 0.016±0.001 0.011±0.001 0.014±0.001 0.021±0.001 0.007±0.001 0.007±0.001 0.020±0.003 0.007±0.001 0.008±0.001


mesotrione

a

85

50

75

95

95

Page 28 of 40

95

0.013±0.001

b

Herbicidal Activity was tested at the rate of 150 g ai/ha. Abbreviations: EC for Echinochloa

crus-galli; SF for Setaria faberii; DS for Digitaria sanguinalis; AR for Amaranthus retroflexu; EP for Eclipta prostrata and AJ for Abutilon juncea. cInhibition constant of the enzyme reaction.

28


Page 29 of 40


Table 2. Post-Emergent Herbicidal Activity of Compounds III-l to III-n, and IV-b. % Inhibition dosage (g ai/ha) Compd. a a EC SF DS a AR a EP a AJ a 75 80 80 80 80 80 90 III-l 37.5 70 65 70 80 80 85 75 70 65 40 80 80 90 III-m 37.5 60 45 30 80 80 80 75 80 60 60 80 90 85 III-n 37.5 70 45 30 80 80 80 75 80 75 70 85 80 80 IV-b 37.5 60 50 30 75 75 70 75 80 30 70 80 90 90 mesotrione 37.5 50 30 55 80 90 85 a

Abbreviations: EC for Echinochloa crus-galli; SF for Setaria faberii; DS for Digitaria

sanguinalis; AR for Amaranthus retroflexu; EP for Eclipta prostrata and AJ for Abutilon juncea.

29



Table 3. Crop Selectivity of Selected Compounds Post Emergencea. % Injury Compd. maize rice wheat soybean cotton canola 5 40 30 30 40 80 III-l 0 0 0 25 20 55 III-m 0 30 20 30 40 80 III-n 10 50 40 55 70 100 mesotrione a

The experiments were conducted at the rate of 150 g ai/ha.

30


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Table 4. Comparison of experimental pKi and calculated pKi. 8.5

cal

8

pK i

Page 31 of 40

7.5

7 6.5

6 6

6.5

7

7.5

pK i

Compd.

pKia

Exp. 6.517 II-b 6.467 II-d 6.654 II-e 6.879 II-f 7.387 II-i 6.526 II-j 7.004 II-k 7.131 II-l 7.602 III-b 6.767 III-c 6.672 III-d 7.208 III-e 7.523 III-f 7.620 III-g 7.119 III-h 7.538 III-i a pKi = -logKi.

8

8.5

exp

Compd.

Cal. 6.528 6.446 6.719 6.593 7.048 6.642 7.363 7.137 7.670 6.762 6.649 7.317 7.377 7.596 7.089 7.483

III-i III-j III-k III-l III-m III-n III-o III-p III-q III-r III-s III-t IV-b V VI-a VI-b

pKia Expl 7.538 7.143 7.959 8.046 7.770 7.456 7.409 7.553 7.796 7.959 7.854 7.678 8.097 7.699 8.155 8.097

31


Cal. 7.483 7.143 7.862 7.989 7.552 7.481 7.456 7.543 7.927 7.871 7.948 7.875 8.061 7.738 8.174 8.061


Figure 1.

32


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O NH2 R3

a

H N R3

3a-q

O

O f

c

H3CO

Cl N

d,e 3

R3

R2

R3 7a-t

N

b O

4a-q

N

HO

O

Cl

5a-q

6a-q

R2

OH O N

O

g

R

h R3

R1

R2 N

R1 O

R3

O 8a-l: R1 = 5,5-di-CH3; 5-CH3; 6,6-di-CH3 9a-t: R1 = H;

Figure 2

33


II: R1 = 5,5-di-CH3; 5-CH3; 6,6-di-CH3 III: R1= H


Figure 3

34


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Page 35 of 40


O

Cl

O N

H3CO

a

H3CO

CN N

O b,c

HO

N

16 OH O

CN N

f R1

O

17

VI-a: R1 = H; VI-b: R1 = CH3

Figure 4

35


N

O

d,e

R1 6j

CN

O

CN

O 18a: R1 = H; 18b: R1 = CH3


Figure 5

36


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Page 37 of 40


Figure 6

37



Figure 7

38


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Page 39 of 40


Figure 8

39



TOC Graphic:

40


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Synthesis and Herbicidal Activity of Triketone ... - ACS Publications

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