Design, Synthesis, and Herbicidal Activity of Novel Diphenyl Ether Derivatives Containing Fast Degrading Tetrahydrophthalimide

Article abstract of DOI:10.1021/acs.jafc.0c00947

To seek new protoporphyrinogen oxidase (PPO) inhibitors with better biological activity, a series of novel diphenyl ether derivatives containing tetrahydrophthalimide were designed based on the principle of substructure splicing and bioisomerization. PPO inhibition experiments exhibited that 6c is the most potential compound, with the half-maximal inhibitory concentration (IC₅₀) value of 0.00667 mg/L, showing 7 times higher activity than Oxyfluorfen (IC₅₀ = 0.0426 mg/L) against maize PPO and similar herbicidal activities to Oxyfluorfen in weeding experiments in greenhouses and field weeding experiments. In view of the inspected bioactivities, the structure-activity relationship (SAR) of this series of compounds was also discussed. Crop selection experiments demonstrate that compound 6c is safe for soybeans, maize, rice, peanuts, and cotton at a dose of 300 g ai/ha. Accumulation analysis experiments showed that the accumulation of 6c in some crops (soybeans, peanuts, and cotton) was significantly lower than Oxyfluorfen. Current work suggests that compound 6c may be developed as a new herbicide candidate in fields.

Full text of DOI:10.1021/acs.jafc.0c00947

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Agricultural and Environmental Chemistry
Design, synthesis, and herbicidal activity of novel diphenyl ether
derivatives containing fast degrading tetrahydrophthalimide
Li-Xia Zhao, Mao-Jun Jiang, Jia-Jun Hu, Yue-Li Zou,
Yuan Cheng, Tao Ren, Shuang Gao, Ying Fu, and Fei Ye
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.0c00947 • Publication Date (Web): 03 Mar 2020
Downloaded from pubs.acs.org on March 5, 2020
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Design, synthesis, and herbicidal activity of novel diphenyl ether derivatives
containing fast degrading tetrahydrophthalimide
†
†
†
†
†
†
Li-Xia Zhao , Mao-Jun Jiang , Jia-Jun Hu , Yue-Li Zou , Yuan Cheng , Tao Ren ,
†
,†
,†
Shuang Gao , Ying Fu* , Fei Ye*
†Department of Applied Chemistry, College of Science, Northeast Agricultural
University, Harbin 150030, China
* Corresponding author
Ying Fu, Email address: fuying@neau.edu.cn, Tel: 86-451-55190070
Fei Ye, Email address: yefei@neau.edu.cn, Tel: 86-451-55191507
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ABSTRACT: To seek new Protoporphyrinogen oxidase (PPO) inhibitors with
better biological active, a series of novel diphenyl ether derivatives containing
tetrahydrophthalimide were designed based on the principle of substructure splicing
and bioisomerization. PPO inhibition experiments exhibited that 6c is the most potential
compound, with the half maximal inhibitory concentration (IC ) value of 0.00667
5
0
mg/L, showed 7 times higher activity than Oxyfluorfen (IC =0.0426 mg/L) against
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0
maize PPO and similar herbicidal activities to Oxyfluorfen in weeding experiments in
greenhouses and field weeding experiments. In view of the inspected bioactivities, the
structure-activity relationship (SAR) of this series of compounds was also discussed.
Crop selection experiments demonstrates that compound 6c is safe for soybeans, maize,
rice, peanuts and cotton at a dose of 300 g ai/ha. Accumulation analysis experiments
showed that the accumulation of 6c in some crops (soybeans,peanuts and cotton) was
significantly lower than Oxyfluorfen. Current work suggests that compound 6c may be
developed as a new herbicide candidate in fields.
Keywords: PPO; Diphenyl ether; Substructure splicing; Bioisomerization;
Herbicidal activity; SAR; Cumulative analysis
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INTRODUCTION
Protoporphyrinogen oxidase (PPO) catalyzes the flavin adenine dinucleotide-
driven oxidation of protoporphyrinogen IX to form protoporphyrin IX. It is the main
target for herbicides that interfere with porphyrin synthesis. Inhibition of PPO leads to
accumulation of protoporphyrin IX in plant cytoplasm.1-4 Protoporphyrin IX produces
a large amount of single oxygen under light conditions, which causes the fatty acid
unsaturated bond peroxidation found in the membrane. This result can result in
membrane integrity and leakage damage, pigmentation and leaf necrosis, and ultimately
5
plant death. PPO inhibitor herbicides have a lot of advantages, such as low dose, broad-
spectrum weed control (including resistant biotypes), environmentally friendly, and
low toxicity to mammals.6-7 In recent years, people have become more and more keen
to study PPO inhibitor herbicides because they are very effective tools for controlling
glyphosate resistant weeds. To date, more than 100 000 PPO inhibitors have been
synthesized and are protected by more than 5000 patents.8
Among them, diphenyl ethers (DPEs) are are the most typical PPO inhibitors. As
early as 1963, Nitrofen was introduced to the market as the first PPO inhibitor.9
Subsequently, agrochemical companies began intensive research and found many
diphenyl ether herbicides with excellent activity. For example, in 1975, Dow
AgroSciences has developed an ultra-low pre-emergence and post-emergence herbicide
Oxyfluorfen,10-11 which is often used to control broadleaf weeds and certain grass weeds;
in 1982, Syngenta developed Fomesafen with good control effect on broadleaf weeds
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in soybean and peanut fields. However, DPEs have some drawbacks such as high
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usage rates and low selectivity to crops. This highlights the urgent need to modify
these PPO inhibitors.
On the one hand, substructure splicing is a widely used method for inventing novel
drugs. The use of substructure splicing or parallel synthesis to optimize specific
structures has led to a number of potential lead compounds and achieved great success
in discovering new drug candidate.14-19 On the other hand, tetrahydrophthalimide
herbicides have many advantages, such as strong selectivity, no phytotoxicity to
crops.20-21 In addition, N-Phenyl-phthalimides herbicides as another type of PPO
inhibitor also has the advantages of short residual time, fast degradation rate and no
pollution to the environment. For example, Flumioxazin has a degradation half-life of
only 10.6-32.1 days in the field site at 15 °C and 25 °C. And its degradation pathway is
mainly the opening of the imide ring, followed by the cleavage of the amide bond to
2
2
form the corresponding dicarboxylic acid and aniline. Therefore, the fast degradation
of the tetrahydrophthalimide herbicides can be attributed to the rapid degradation of the
tetrahydrophthalimide structure. These studies inspired our current hypothesis that the
introduction of a tetrahydrophthalimide structure into a diphenyl ether structure may
improve the selectivity and degradability of diphenyl ether herbicides.
In this work, a series of novel diphenyl ether derivatives containing
tetrahydrophthalimide were designed based on the principle of substructure splicing
and bioisomerization (Scheme 1). All the compounds 6a-w were synthesized and
1
13
structurally characterized by H NMR spectra, C NMR spectra and high-resolution
mass spectra. In addition, herbicidal activity, PPO inhibitory activity, structure-activity
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relationship (SAR), crop selectivity, cumulative analysis. and molecular docking of
these compounds have also been reported.
MATERIALS AND METHODS
Instruments and materials. All chemical reagents, such as 2-chloronicotinic acid,
4-chlorophenol, 3,4,5,6-tetrahydrophthalic anhydride, and thionyl chloride were
purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Organic solvents, such
as ethyl acetate, petroleum ether (bp 60−90 °C), dichloromethane (DCM), ethanol and
dimethylformamide (DMF) were purchased from Tianjin Fuyu Fine Chemical Co., Ltd.
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(Tianjin, China). H NMR and C NMR spectra were recorded using an AVANCE 400
MHz instrument (BRUKER Inc., Beijing, China), using CDCl or (Methyl sulfoxide-
3
d ) DMSO-d as the solvent and trimethylchlorosilane as the internal standard. High
6
6
resolution mass spectra (HRMS) were obtained on a micrOTOF-Q II 10410
spectrometer (BRUKER Inc., Beijing, China) or a Waters Xevo G2-XS QTof
spectrometer (Waters Technology Co., Ltd., Shanghai, China). The melting points
(m.p.) of the compounds 6a-w was recorded on a WRS-3 melting point apparatus
(Shanghai Yidian Scientific Instrument Co., Ltd. Shanghai, China), uncorrected. The
X-ray diffraction data of compound 6i and 7 were recorded on a BRUKER D8
VENTURE X-diffractometer (BRUKER Inc., Beijing, China).
General Synthetic Procedure for Compounds 6a-w.
Synthesis of compound 2a-w. A mixture of 2-cloronicotinic acid (1 eq),
anhydrous K CO (1.33 eq), phenol or thiophenol (1.3 eq) 1a-w and CuCl (0.25 g) was
2
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stirred in DMF at 140 °C for 10 h. The mixture was cooled to room temperature, then
poured into 100 mL of water. The pH of the aqueous solution was adjusted to 2-4 with
hydrochloric acid. The mixture was filtered off to give the desired solid products 2a-w.
Synthesis of compounds 3a-w. Thionyl chloride (3eq) and one drop of DMF in
were added to a solution of 2a-w (1eq) in dichloromethane (DCM, 40 mL). The mixture
was stirred at 60 °C for 1 h, then the solvent was removed under reduced pressure and
afforded 3a-w. The acyl chloride was used directly without further purification.
Synthesis of compound 5. 3,4,5,6-tetrahydrophthalic anhydride (1 eq) 4 and urea
(0.5 eq) were heated together at 155-160 °C until effervescence ceased and the mixture
had almost completely solidified. The product was cooled and crystallised from ethanol
to give white solid 3,4,5,6-tetrahydrophthalimide 5.
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Synthesis of compounds 6a-w. 4-dimethylaminopyridine (DMAP) (0.25 eq) was
added to a stirred solution of 3,4,5,6-tetrahydrophthalimide (1 eq) 5, 3a-w (1.3 eq) and
triethylamine (2 eq) in DCM (40 mL). The mixture was allowed to react at room
temperature until the 3,4,5,6-tetrahydrophthalimide was completely consumed and
afforded 6a-w (detected by thin layer chromatography, TLC), then the solvent was
removed in vacuo, and the solid products were purified through chromatograph on silica
gel using petroleum ether/ ethyl acetate as eluent to give pure product. The spectra data
of compounds 6a-w and the crystallographic data of compounds 6i and 7 were
displayed in the Supporting Information.
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H NMR, C NMR and HRMS of Compounds 6a-w
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2-(2-(4-chloro-3,5-dimethylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-
1
isoindole-1,3(2H)-dione (6a). H NMR (400 MHz, Chloroform-d) δ 8.28 (dd, J=5.0,
2.0 Hz, 1H, pyridine-H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.12 (dd, J=7.5, 5.0
Hz, 1H, pyridine-H), 6.78 (s, 2H, Ar-H), 2.36 (d, J=3.3 Hz, 10H, 2CH and 2CH ), 1.75
2
3
1
3
(s, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.23 (2), 162.65, 160.15,
2
150.96, 150.50, 143.56 (2), 140.96, 137.75 (2), 131.39, 121.40, 118.96 (2), 118.62,
+
21.00 (2), 20.94 (2), 20.13 (2). HRMS (ESI): calcd for C H ClN O [M+H] 411.1111,
2
2
20
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found: 411.1123.
123
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125
126
127
128
129
130
2-(2-(2,4-dichlorophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6b). H NMR (400 MHz, Chloroform-d) δ 8.25 (dd, J=5.0, 2.0 Hz, 1H,
pyridine-H), 8.04 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.44 (d, J=2.5 Hz, 1H, Ar-H),
7.29-7.21 (m, 1H, Ar-H), 7.17 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 7.07 (d, J=8.7 Hz,
1
3
1H, Ar-H), 2.37 (h, J=2.6, 2.1 Hz, 4H, 2CH ), 1.78 (p, J=3.2 Hz, 4H, 2CH ). C NMR
2
2
(101 MHz, Chloroform-d) δ 167.05 (2), 162.43, 158.74, 150.60, 147.70, 143.67, 140.97,
131.23 (2), 130.24, 128.12, 128.02, 124.29, 119.32, 119.22, 21.04 (2), 20.21 (2). HRMS
+
(ESI): calcd for C H Cl N O [M+H] 417.0408, found: 417.0408.
2
0
15
2
2
4
131
132
133
134
135
2-(2-(3,5-dimethylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6c). H NMR (400 MHz, Chloroform-d) δ 8.28 (dd, J=4.9, 2.0 Hz, 1H, pyridine-
H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.10 (dd, J=7.5, 5.0 Hz, 1H, pyridine-H),
6.86 (s, 1H, Ar-H), 6.65 (d, J=1.5 Hz, 2H, Ar-H), 2.39-2.32 (m, 4H, 2CH ), 2.31 (s, 6H,
2
1
3
2CH ), 1.74 (p, J=3.1 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.28
3
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(2), 162.9, 160.37, 152.85, 151.09, 143.52 (2), 140.86 (2), 139.53, 127.27, 119.12 (2),
118.95, 118.36, 21.35 (2), 21.01 (2), 20.12 (2). HRMS (ESI): calcd for C H N O
4
2
2
21
2
+
[M+H] 377.1501, found: 377.1492.
139
140
141
142
143
144
145
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2-(2-phenoxynicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione (6d).
1H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=4.9, 2.0 Hz, 1H, pyridine-H), 8.04
(dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.44-7.36 (m, 2H, Ar-H), 7.25-7.19 (m, 1H, Ar-
H), 7.12 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 7.08-7.00 (m, 2H, Ar-H), 2.34 (h, J=2.6,
1
3
2.1 Hz, 4H, 2CH ), 1.73 (p, J=3.2 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d)
2
2
δ 167.28 (2), 162.80, 160.18, 152.95, 150.95, 143.56 (2), 140.95, 129.67 (2), 125.38,
121.51 (2), 119.01, 118.61, 21.00 (2), 20.12 (2). HRMS (ESI): calcd for C H N O
4
2
0
17
2
+
[M+H] 349.1188, found: 349.1182.
147
148
149
150
151
152
153
154
2-(2-(4-chlorophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6e). H NMR (400 MHz, Chloroform-d) δ 8.26 (dd, J=5.0, 2.0 Hz, 1H, pyridine-
H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.41-7.29 (m, 2H, Ar-H), 7.14 (dd, J=7.5,
4.9 Hz, 1H, pyridine-H), 7.04-6.89 (m, 2H, Ar-H), 2.35 (h, J=2.5, 2.1 Hz, 4H, 2CH2),
1
3
1.75 (p, J=3.2 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.22 (2), 162.51,
2
159.89, 151.44, 150.79, 143.62 (2), 141.00, 130.71, 129.73 (2), 122.99 (2), 119.05,
+
118.92, 20.99 (2), 20.14 (2). HRMS (ESI): calcd for C H ClN O [M+H] 383.0799,
2
0
16
2
4
found: 383.0809.
1
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2-(2-(p-tolyloxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dion (6f).
1
1H NMR (400 MHz, Chloroform-d) δ 8.26 (dd, J=5.0, 2.0 Hz, 1H, pyridine-H), 8.03
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(dd, J=7.4, 2.0 Hz, 1H, pyridine-H), 7.19 (d, J=8.3 Hz, 2H, Ar-H), 7.10 (dd, J=7.5, 5.0
Hz, 1H, pyridine-H), 6.96-6.86 (m, 2H, Ar-H), 2.39-2.28 (m, 7H, 2CH and CH ), 1.74
2
3
1
3
(p, J=3.2 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.30 (2), 162.87,
2
160.47, 151.02, 150.61, 143.53 (2), 140.92, 135.05, 130.24 (2), 121.34 (2), 118.86,
+
118.38, 21.00, 20.94 (2), 20.12 (2). HRMS (ESI): calcd for C H N O [M+H]
2
1
19
2
4
363.1344, found: 363.1338.
163
164
165
166
167
168
169
170
2-(2-(p-tolylthio)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione
1
(6g). H NMR (400 MHz, Chloroform-d) δ 8.47 (dd, J=4.8, 1.8 Hz, 1H, pyridine-H),
7.71 (dd, J=7.7, 1.8 Hz, 1H, 1H, pyridine-H), 7.40-7.31 (m, 2H, Ar-H), 7.17 (d, J=7.9
Hz, 2H, Ar-H), 7.13 (dd, J=7.7, 4.8 Hz, 1H, pyridine-H), 2.41 (dh, J=5.5, 3.4 Hz, 4H,
1
3
2CH ), 2.36 (s, 3H, CH ), 1.81 (h, J=3.8, 3.3 Hz, 4H, 2CH ). C NMR (101 MHz,
2
3
2
Chloroform-d) δ 167.06, 163.90 (2), 158.39, 151.95, 143.74 (2), 138.93, 137.60, 134.41
(2), 129.99 (2), 129.31, 126.96, 119.88, 21.34, 21.04 (2), 20.20 (2). HRMS (ESI): calcd
+
for C H N O S [M+H] 379.1116, found: 379.1112.
2
1
19
2
3
171
172
173
174
175
176
2-(2-(4-ethylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6h). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=4.9, 2.0 Hz, 1H, pyridine-
H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.24-7.16 (m, 2H, Ar-H), 7.10 (dd, J=7.5,
4.9 Hz, 1H, pyridine-H), 6.99-6.91 (m, 2H, Ar-H), 2.66 (q, J=7.6 Hz, 2H, CH ), 2.40-
2
1
3
2.28 (m, 4H, 2CH ), 1.74 (p, J=3.3 Hz, 4H, 2CH ), 1.25 (t, J=7.6 Hz, 3H, CH ). C
2
2
3
NMR (101 MHz, Chloroform-d) δ 167.29 (2), 162.89, 160.41, 151.00, 150.76, 143.54
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(2), 141.25, 140.92 (2), 129.03 (2), 121.30, 118.92, 118.40, 28.28, 21.00 (2), 20.12 (2),
+
1
15.47. HRMS (ESI): calcd for C H N O [M+H] 377.1501, found: 377.1503.
2
1
21
2
4
179
180
181
182
183
184
185
186
3-((3-(1,3-dioxo-2,3,4,5,6,7-hexahydro-1H-isoindole-2-carbonyl)pyridin-2-
1
yl)oxy)benzonitrile (6i). H NMR (400 MHz, Chloroform-d) δ 8.26 (dd, J=5.0, 2.0 Hz,
1H, pyridine-H), 8.04 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.56-7.47 (m, 2H, Ar-H),
7.40-7.35 (m, 1H, Ar-H), 7.33 (dt, J=6.9, 2.4 Hz, 1H, Ar-H), 7.19 (dd, J=7.5, 4.9 Hz,
1
3
1H, pyridine-H), 2.36 (h, J=2.5, 2.1 Hz, 4H, 2CH ), 1.77 (p, J=3.1 Hz, 4H, 2CH ). C
2
2
NMR (101 MHz, Chloroform-d) δ 167.12 (2), 162.20, 159.26, 153.15, 150.55, 143.74
(2), 141.06, 130.58, 129.03, 126.64, 125.37, 119.46, 119.28, 117.99, 113.61, 20.98 (2),
+
20.17 (2). HRMS (ESI): calcd for C H N O [M+H] 374.1141, found: 374.1147.
2
1
16
3
4
187
188
189
190
191
192
193
194
2-(2-(3-(trifluoromethyl)phenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6j). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=4.9, 2.0 Hz, 1H,
pyridine-H), 8.05 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.57-7.45 (m, 2H, Ar-H), 7.34-
7.23 (m, 2H, Ar-H), 7.17 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 2.34 (h, J=2.5 Hz, 4H,
1
3
2CH ), 1.75 (p, J=3.1 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.20
2
2
(2), 162.39, 159.52, 153.09, 150.72, 143.68 (2), 141.06, 130.16, 125.21, 122.12, 122.08,
119.22, 119.20, 118.78, 118.74, 20.96 (2), 20.11 (2). HRMS (ESI): calcd for
+
C H F N O [M+H] 417.1062, found: 417.1075.
2
1
16
3
2
4
195
196
197
2-(2-(4-methoxyphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6k). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=4.9, 2.0 Hz, 1H, pyridine-
H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.10 (dd, J=7.4, 5.0 Hz, 1H, pyridine-H),
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2
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2
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6.99-6.93 (m, 2H, Ar-H), 6.94-6.88 (m, 2H, Ar-H), 3.80 (s, 3H, OCH ), 2.36 (p, J=3.1
3
1
3
99
00
01
02
Hz, 4H, 2CH2), 1.75 (p, J=3.2 Hz, 4H, 2CH2). C NMR (101 MHz, Chloroform-d) δ
167.31 (2), 162.5, 160.60, 156.93, 150.99, 146.25 (2), 143.54, 140.92, 122.45 (2),
118.78, 118.36, 114.72 (2), 55.57, 21.01 (2), 20.13 (2). HRMS (ESI): calcd for
+
C H N O [M+H] 379.1294, found: 379.1309.
2
1
19
2
5
203
204
205
206
207
208
209
210
2-(2-(2-bromophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6l). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=5.0, 2.0 Hz, 1H, pyridine-
H), 8.07 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.61 (dd, J=8.4, 1.6 Hz, 1H, Ar-H), 7.38-
7.29 (m, 1H, Ar-H), 7.17 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 7.14-7.06 (m, 2H, Ar-H),
1
3
2.35 (h, J=2.6, 2.2 Hz, 4H, 2CH2), 1.75 (dq, J=6.2, 3.3, 2.7 Hz, 4H, 2CH2). C NMR
(101 MHz, Chloroform-d) δ 167.12 (2), 162.61, 158.93, 150.77, 150.24, 143.62 (2),
140.98, 133.55, 128.48, 126.63, 123.08, 119.34, 119.17, 116.21, 21.03 (2), 20.20 (2).
+
HRMS (ESI): calcd for C H BrN O [M+H] 427.0293, found: 427.0295.
2
0
16
2
4
211
212
213
214
215
216
217
218
2-(2-((4-methoxyphenyl)thio)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6m). H NMR (400 MHz, Chloroform-d) δ 8.46 (dd, J=4.8, 1.8 Hz, 1H,
pyridine-H), 7.70 (dd, J=7.7, 1.7 Hz, 1H, pyridine-H), 7.47-7.36 (m, 2H, Ar-H), 7.11
(dd, J=7.7, 4.8 Hz, 1H, pyridine-H), 6.97-6.84 (m, 2H, Ar-H), 3.82 (s, 3H, OCH ), 2.42
3
1
3
(h, J=3.3 Hz, 4H, 2CH2), 1.82 (h, J=3.6, 3.2 Hz, 4H, 2CH2). C NMR (101 MHz,
Chloroform-d) δ 167.08 (2), 163.91, 160.32, 159.04, 151.87, 143.75 (2), 137.61, 136.61
(2), 128.87, 120.69, 119.65, 114.79 (2), 55.32, 21.05 (2), 20.22 (2). HRMS (ESI): calcd
+
for C H N O S [M+H] 395.1066, found: 395.1069.
2
1
19
2
4
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225
226
2-(2-(2,4-dimethylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6n). H NMR (400 MHz, Chloroform-d) δ 8.25 (dd, J=5.0, 1.9 Hz, 1H, pyridine-
H), 8.02 (dd, J=7.4, 1.8 Hz, 1H, pyridine-H), 7.13-7.04 (m, 2H, Ar-H), 7.01 (dd, J=8.2,
2.1 Hz, 1H, pyridine-H), 6.81 (d, J=8.1 Hz, 1H, Ar-H), 2.37 (p, J=3.1 Hz, 4H, 2CH2),
1
3
2.31 (s, 3H, CH ), 2.04 (s, 3H, CH ), 1.76 (p, J=3.1 Hz, 4H, 2CH2). C NMR (101
3
3
MHz, Chloroform-d) δ 167.20 (2), 163.03, 160.06, 151.13, 149.10, 143.54(2), 140.76,
135.26, 131.95, 130.58, 127.68, 121.34, 118.81, 118.16, 21.00 (2), 20.88, 20.14 (2),
+
16.35. HRMS (ESI): calcd for C H N O [M+H] 377.1501, found: 377.1509.
2
2
21
2
4
227
228
229
230
231
232
233
234
2-(2-(4-fluoro-2-methylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6o). H NMR (400 MHz, Chloroform-d) δ 8.25 (dd, J=5.0, 2.0 Hz, 1H,
pyridine-H), 8.02 (dd, J=7.4, 1.9 Hz, 1H, pyridine-H), 7.11 (dd, J=7.5, 5.0 Hz, 1H,
pyridine-H), 6.95 (dt, J=9.1, 1.6 Hz, 1H, Ar-H), 6.92-6.85 (m, 2H, Ar-H), 2.47-2.28 (m,
1
3
4H, 2CH ), 2.07 (s, 3H, CH ), 1.77 (p, J=3.3 Hz, 4H, 2CH ). C NMR (101 MHz,
2
3
2
Chloroform-d) δ 167.17 (2), 162.79, 161.11, 159.77, 158.69, 150.92 (2), 147.19, 143.62,
140.79, 133.04, 122.88, 118.48, 117.76, 113.77, 21.01 (2), 20.16 (2), 16.58. HRMS
+
(ESI): calcd for C H FN O [M+H] 381.1251, found: 381.1253.
2
1
18
2
4
235
236
237
238
239
2-(2-(2-methoxy-4-methylphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6p). H NMR (400 MHz, Chloroform-d) δ 8.24 (dd, J=5.0, 2.0 Hz, 1H,
pyridine-H), 8.02 (dd, J=7.4, 2.0 Hz, 1H, pyridine-H), 7.07 (dd, J=7.5, 4.9 Hz, 1H,
pyridine-H), 6.88 (d, J=7.9 Hz, 1H, Ar-H), 6.80-6.72 (m, 2H, Ar-H), 3.68 (s, 3H, OCH3),
1
3
2.38 (p, J=3.1 Hz, 4H, 2CH ), 2.35 (s, 3H, CH ), 1.76 (p, J=3.3 Hz, 4H, 2CH ). C
2
3
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NMR (101 MHz, Chloroform-d) δ 167.17 (2), 163.15, 160.18, 151.40, 151.00, 143.38
41
42
(2), 140.67, 139.41, 136.45, 122.40, 121.31, 118.14, 113.73, 55.72, 21.49, 21.06 (2),
+
20.12 (2). HRMS (ESI): calcd for C H N O [M+H] 393.1450, found: 393.1441.
2
2
21
2
5
243
244
245
246
247
248
249
250
2-(2-(4-fluorophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6q). H NMR (400 MHz, Chloroform-d) δ 8.26 (dd, J=4.9, 2.0 Hz, 1H, pyridine-
H), 8.03 (dd, J=7.5, 1.9 Hz, 1H, pyridine-H), 7.13 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H),
7.10-7.04 (m, 2H, Ar-H), 7.04-6.97 (m, 2H, Ar-H), 2.35 (h, J=3.3 Hz, 4H, 2CH ), 1.76
2
1
3
(h, J=3.6 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.23 (2), 162.60,
2
161.14, 160.14, 158.72, 150.80 (2), 148.69, 143.59, 140.96 (2), 123.04, 118.94, 116.44
+
(2), 20.99 (2), 20.13 (2). HRMS (ESI): calcd for C H FN O [M+H] 367.1094, found:
2
0
16
2
4
367.1092.
251
252
253
254
255
256
257
258
2-(2-(4-iodophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6r). H NMR (400 MHz, Chloroform-d) δ 8.26 (dd, J=4.9, 2.0 Hz, 1H, pyridine-
H), 8.04 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.74-7.63 (m, 2H, Ar-H), 7.14 (dd, J=7.5,
5.0 Hz, 1H, pyridine-H), 6.87-6.75 (m, 2H, Ar-H), 2.34 (h, J=2.5, 2.1 Hz, 4H, 2CH2),
1
3
1.76 (h, J=3.8, 3.3 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.19 (2),
2
162.48, 159.76, 152.85, 150.78, 143.60 (2), 141.01, 138.69 (2), 123.82 (2), 119.08,
+
118.93, 89.33, 20.98 (2), 20.12 (2). HRMS (ESI): calcd for C H IN O [M+H]
2
0
16
2
4
475.0154, found: 475.0147.
2
59
60
2-(2-(3-bromophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
2
dione (6s). H NMR (400 MHz, DMSO-d ) δ 8.32 (dd, J=4.9, 1.9 Hz, 1H, pyridine-H),
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8.12 (dd, J=7.5, 1.9 Hz, 1H, pyridine-H), 7.44 (dt, J=8.1, 1.4 Hz, 1H, Ar-H), 7.39 (t,
J=8.0 Hz, 1H, Ar-H), 7.32 (dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 7.26 (t, J=2.1 Hz, 1H,
Ar-H), 7.06 (ddd, J=8.0, 2.3, 1.2 Hz, 1H, Ar-H), 2.27 (t, J=3.1 Hz, 4H, 2CH ), 1.67 (p,
2
1
3
J=3.0 Hz, 4H, 2CH ). C NMR (101 MHz, DMSO-d ) δ 167.57 (2), 162.29, 159.26,
2
6
154.24, 150.83, 143.59 (2), 141.08, 131.96, 128.50, 124.72, 122.07, 120.97, 120.09,
+
119.77, 21.00 (2), 20.11 (2). HRMS (ESI): calcd for C H BrN O [M+H] 427.0293,
2
0
16
2
4
found: 427.0304.
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269
270
271
272
273
274
275
2-(2-(4-(methylthio)phenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6t). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=5.0, 2.0 Hz, 1H,
pyridine-H), 8.04 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.31-7.27 (m, 2H, Ar-H), 7.12
(dd, J=7.5, 4.9 Hz, 1H, pyridine-H), 7.01-6.94 (m, 2H, Ar-H), 2.48 (s, 3H, SCH ), 2.35
3
1
3
(p, J=3.1 Hz, 4H, 2CH ), 1.75 (p, J=3.3 Hz, 4H, 2CH ). C NMR (101 MHz,
2
2
Chloroform-d) δ 167.26 (2), 162.69, 160.18, 150.91, 150.63, 143.58 (2), 141.00, 135.13,
128.25 (2), 122.09 (2), 118.97, 118.67, 21.00, 20.13 (2), 16.49 (2). HRMS (ESI): calcd
+
for C H N O S [M+H] 395.1066, found: 395.1076.
2
1
19
2
4
276
277
278
279
280
281
2-(2-(2-ethoxyphenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-
1
dione (6u). H NMR (400 MHz, Chloroform-d) δ 8.24 (dd, J=4.9, 2.0 Hz, 1H), pyridine-
H, 8.03 (dd, J=7.4, 1.9 Hz, 1H, pyridine-H), 7.21-7.14 (m, 1H, Ar-H), 7.09 (dd, J=7.5,
4.9 Hz, 1H, Ar-H), 7.01 (dd, J=8.3, 1.8 Hz, 1H, pyridine-H), 6.98-6.92 (m, 2H, Ar-H),
3.94 (q, J=7.0 Hz, 2H, CH ), 2.38 (h, J=2.6, 2.2 Hz, 4H, 2CH ), 1.76 (p, J=3.2 Hz, 4H,
2
2
1
3
2CH ), 1.16 (t, J=7.0 Hz, 3H, CH ). C NMR (101 MHz, Chloroform-d) δ 167.18 (2),
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163.07, 160.04, 151.06, 150.84, 143.44 (2), 142.20, 140.69, 126.38, 122.64, 120.84,
118.75, 118.26, 114.08, 64.39, 21.06 (2), 20.15 (2), 14.60. HRMS (ESI): calcd for
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84
+
C H N O [M+H] 393.1450, found: 393.1440.
2
2
21
2
5
285
286
287
288
289
290
291
292
2-(2-((4-fluorophenyl)thio)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6v). H NMR (400 MHz, Chloroform-d) δ 8.47 (dd, J=4.8, 1.8 Hz, 1H,
pyridine-H), 7.73 (dd, J=7.7, 1.8 Hz, 1H, pyridine-H), 7.50-7.38 (m, 2H, Ar-H), 7.15
(dd, J=7.7, 4.8 Hz, 1H, pyridine-H), 7.11-7.04 (m, 2H, Ar-H), 2.42 (h, J=2.5, 2.0 Hz,
1
3
4H, 2CH ), 1.82 (p, J=3.3 Hz, 4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.07
2
2
(2), 163.80, 161.96, 158.27, 151.92, 143.83 (2), 137.79, 136.86 (2), 129.04, 125.62,
120.00, 116.47 (2), 21.03 (2), 20.23 (2). HRMS (ESI): calcd for C H F N O S
2
0
16
3
2
3
+
[M+H] 383.0866, found: 383.0869.
293
294
295
296
297
298
299
2-(2-(3,4,5-trifluorophenoxy)nicotinoyl)-4,5,6,7-tetrahydro-1H-isoindole-
1
1,3(2H)-dione (6w). H NMR (400 MHz, Chloroform-d) δ 8.27 (dd, J=4.9, 2.0 Hz, 1H,
pyridine-H), 8.03 (dd, J=7.5, 2.0 Hz, 1H, pyridine-H), 7.19 (dd, J=7.5, 5.0 Hz, 1H,
pyridine-H), 6.84-6.67 (m, 2H, Ar-H), 2.37 (h, J=2.6 Hz, 4H, 2CH ), 1.78 (p, J=3.2 Hz,
2
1
3
4H, 2CH ). C NMR (101 MHz, Chloroform-d) δ 167.11 (2), 162.09, 159.21, 150.57,
2
143.74, 141.09 (2), 119.50, 119.16, 107.19 (2), 107.12, 107.02, 106.95 (2), 20.98 (2),
+
20.17 (2). HRMS (ESI): calcd for C H F N O [M+H] 403.0906, found: 403.0908.
2
0
14
3
2
4
300
301
302
PPO inhibitory experiments in vitro
The preparation of maize protoporphyrinogen IX oxidase used the reported
2
3
method. The catalytic rate of maize PPO is calculated by measuring the change in
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concentration of the enzyme product protoporphyrin IX over a certain period of time.
The concentration of enzyme product protoporphyrin IX was determined by the
absorbance monitored by a UV-visible spectrophotometer at a wavelength of 410 nm.24
The enzymatic reaction was carried out in a 200 μl system consisting of 150 μl test
buffer solution, 25 μl reaction buffer solution, 10 μl enzyme sample, 10 μl different
concentrations of inhibitor solution and 5 μl protoporphyrinogen IX solution. The
inhibition rate of the test compounds to the enzyme at different concentrations was
calculated by eq 1.
풂 ― 풃
풂
I=
(1)
Where I is the inhibition rate, a is the enzyme catalyzed rate without inhibitor and
b is the catalytic rate of the enzyme after the addition of the inhibitor. The inhibition
rate at different concentrations was measured by fixing the concentration of the
substrate and changing the concentration of the inhibitor. Each experiment was repeated
three times and Oxyfluorfen was used as a control. Then, SPSS 20.0 (Chicago, Illinois,
USA) was used to analyze the linear relationship between inhibitor concentration and
inhibition rate at a fixed substrate concentration. Finally, this linear relationship was
used to determine the IC value. In addition, SPSS 20.0 was also used to perform
5
0
significance test.
Herbicidal Activities
To evaluate the post-emergence activity of compounds 6a-w and 7, three
representative broadleaf weeds Abutilon theophrasti Medicus (At), Capsella bursa-
pastoris (Linn.) Medic (Cb), Portulaca oleraceaL. (Po), and three representative
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25
monocotyledon weeds, Setaria viridis (L.) Beauv (Sv), Poa annua L. (Pa) Echinochloa
9
26
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crusgalli (L.) Beauv (Ec), were evaluated according to the reported method. Use the
commonly applied PPO herbicides Oxyfluorfen as a control. Prior to the experiment,
10 mg of compounds 6a-w or 7 dissolved in 200 μl DCM was added to a 10 ml
volumetric flask. Next, 300 μl of 1% Tween-80 was added as an emulsifying agent and
added water to volume to 10ml. The spray dose (active ingredient, g ai/ha) was
calculated by dividing the mass of the spray inhibitor by the upper surface area of the
plot. Before planting, select a pot with a diameter of 8 cm and fill the pot with 8-10 cm
deep soil. Approximately 10-30 test weed seeds were sown in soil at a depth of 1-3 cm.
During the experiment, the temperature was maintained at 15-30 °C and the air
humidity was maintained at approximately 50%. Monocotyledonous weeds at the leaf
stage and broadleaf weeds at the two leaves stage are treated with a diluted inhibitor
emulsion. The inhibitor-free solvent (DCM + Tween-80) was used as a solvent control
group. The results of the herbicidal activity were visually evaluated within 30 days after
the treatment (Table 1), and each treatment was repeated three times.
Crop Safety
In order to test the crop safety of the most potential compound 6c, crop safety of
compound 6c against six common crops (soybean, peanut, cotton, maize, wheat, and
rice) was evaluated in post-emergence crop safety assessment experiments. The seeds
of the crop were sown in a plastic pot of 8 cm in diameter and grown at room
temperature at 15-30 °C. The four-leaf stage crop was treated with compound 6c at a
dose of 75-300 g ai/ha. The safety of the compound 6c against the crop was evaluated
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30 days after the spraying. The recorded data are shown in Table 2, using Oxyfluorfen
as a control, and each evaluation experiment was repeated three times.
Molecular docking
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360
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Sketch module of SYBYL 6.9 (Tripos, Inc., St. Louis, MO) was used to construct
the 3D structures of compound 6c and Oxyfluorfen. The charges of compound 6c and
Oxyfluorfen were calculated by Gasteiger-Huckel method. Subsequently, compound
6c and Oxyfluorfen were optimized by power method, and then used for docking. The
crystal structure of tobacco PPO enzyme (mtPPO) [Protein Data Bank (PDB) ID: 1SEZ]
was obtained from the PDB. Before the docking, the crystal of the enzyme was
optimized (removed all heteroatoms and water molecules, repaired amino acid residues
and added polar hydrogen). The OMN module is the best inhibitor binding site due to
the presence of an PPO inhibitor Fluazolate at the OMN module in the crystal structure
of the enzyme. AutoDock 4.2 was used to calculate the interaction between the
compound and the OMN module. The optimal binding modes was screened by
comparison with eutectic ligands in mtPPO and docking scores.
362
Cumulative concentration analysis
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365
In order to further verify whether compound 6c is safe for crops, the cumulative
concentration of compound 6c in 6 common crops was determined, and Oxyfluorfen
was used as a control.
366
Collection of samples. When the crops grow to four-leaf stage, they were treated
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with 6c or Oxyfluorfen at the dose of 300g ai/ha. The stems and leaves of these crops
were collected at 2 h and 1 days after spraying of the herbicide. Subsequently, the
collected stems and leaves were cut with scissors and stored at -20 ° C until analysis.25-26
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Analytical method. A modified QuEChERS method was used for sample
pretreatmen27-28t. A 10 g sample was placed in a 50 ml centrifuge tube and homogenized
using a homogenizer. Then, 10 ml of acetonitrile was added to a centrifuge tube and the
centrifuge tube was sonicated for 30 min at 30 °C.29-30 After adding 4 g of anhydrous
magnesium sulfate and 1 g of sodium chloride to the centrifuge tube, the centrifuge tube
was shaken for one minute and centrifuged at 4000 rpm for 5 minutes. Subsequently, 1
ml of the supernatant was added to a 2 ml centrifuge tube containing 150 mg of
anhydrous magnesium sulfate and 40 mg of N-propylethylenediamine. The centrifuge
tube was shaken for 1 minute, and then centrifuged at 15000 rpm for 20 minutes at 4 °
C31-32. Finally, the supernatant was filtered through a 0.22 μm organic filter and
3
3
transferred to an LC vial for chromatographic analysis .
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383
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385
386
387
LC Analysis. Compounds 6c and Oxyfluorfen were both analyzed by high
performance liquid chromatography (LC-600E-2487,Waters Corporation, America)
equipped with a C18 column (ZORBAX SB-C18，5μm，2.1 mm×50 mm，Agilent,
America). The mobile phase of compound 6c was acetonitrile / water (7: 3, v / v), and
the mobile phase of Oxyfluorfen was acetonitrile / water (7.5: 2.5, v / v). The injection
volume was 10 μl, the flow rate was 3 ml / min, and the temperature was maintained at
30 °C.
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Linearity and recovery. Acetonitrile was used to configure 6 different herbicide
solutions (0.01-10mg/L). Draw the standard curve of concentration and peak area
2
according to the relationship between these 6 concentrations and peak area. The R of
the final drawn standard curves are all greater than 0.99, indicating a good linear
relationship. The linear relationship was shown in Table 3. Recovery was determined
by adding 0.1 mg/kg to 6 crops. After three repeated experiments, the average recovery
was between 70.55%-116.77%. The added recovery rates of the six crops were shown
in Table 3.
Field trial
397
398
399
400
401
402
403
Field bioassays is important for screening new pesticides. To further study the
potential herbicidal activity of compound 6c, a field test was performed in Foshan,
Guangdong, China, according to the Chinese pesticide standard GB/T17980.42–2000.
Weeds grown under wild conditions were selected for testing of control effects. The
2
area for treating weeds was 20 m , and the treatment doses were 75, 150, 300, and 600
g ai/ha. The herbicidal activity was visually inspected within 3-10 days after the
treatment34-36
.
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406
407
408
RESULTS AND DISCUSSION
Synthesis. The synthetic route of the final product 6a-w and the unexpected
product 7 were shown in Scheme 2. In short, commercially available various types of
phenol or thiophenol 1a-w were firstly reacted with 2-chloronicotinic acid in DMF,
which afforded the intermediate phenoxynicotinic acid or phenylthionicotinic acid 2a-
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4
09
w. Subsequent treatment of 2a-w with thionyl chloride in DCM affordeds the
corresponding active intermediate phenoxynicotinoyl chloride or phenylthionicotinic
chloride 3a-w, which was used for further reaction to obtain the target products.
Commercially available 4 and urea were stirred directly in a round bottom flask to give
intermediate 5. Next, 3a-w and 5 were used to synthesize the final product 6a-w.
Initially, methanol was used as a solvent to synthesize target compounds 6a-w, but
unfortunately, the resulting compound was not desired compounds 6a-w. However,
what is exciting is that the single crystal structure of resulting compound was cultured
and characterized. The single crystal structure of resulting compound was shown in the
Figure 1A (compound 7). Based on the observation of the single crystal structure of
compound 7, methanol may react with 6e to cause cleavage of the
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tetrahydrophthalimide amide bond . Therefore, various solvents were tried, and finally
the desired compound was synthesized using DCM as a solvent. In addition, the deacid
reagent has a great influence on the yield of the final product 6a-w. The initial synthesis
of compounds 6a-w was synthesized using K CO as the deacid reagent, but the yield
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of compounds 6a-w was less than 30%. Fortunately, when Et N was used instead of
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K CO as the deacid reagent, the yield of the compounds 6a-w was significantly
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increased. Finally, compounds 6a-w was synthesized using DCM as a solvent, K CO3
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as a deacid reagent and DMAP as a catalyst. All synthesized target compounds 6a-w
(Table 4) were purified by column chromatography.
Spectral characterization. All the compounds 6a-w were synthesized and
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structurally characterized by H NMR spectra, C NMR spectra and HRMS. Take the
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result of the spectrum of the compound 6i as an example. As shown in Figure 2, H
NMR spectra indicated that the signals at δ 1.77 ppm and δ 2.35 ppm related to
hydrogen on the six-membered ring of tetrahydrophthalimide. The 7 hydrogen signals
at δ 8.26-7.19 ppm related to 7 hydrogens on the phenoxypyridine of compound 6i. The
13C NMR spectra of the compound 6i was shown in Figure 3. The C at δ 167.12 ppm
related to the two C=O of tetrahydrophthalimide. The signal at δ 162.20 ppm related to
another C=O of compound 6i. The signal at δ 143.74 ppm related to C=C of
tetrahydrophthalimide. The 12 signals at δ 159.26 ppm, δ 153.15 ppm, δ 150.55 ppm,
δ 141.06 pm, δ 130.58 ppm, δ 129.03 ppm, δ 126.64 ppm, δ 125.37 ppm, δ 119.46 ppm,
δ 119.28 ppm, δ 117.99 ppm, δ 113.61 ppm related to 11 C on the phenoxypyridine
structure and C on the CN. The signals observed at δ 20.98 ppm, δ 20.17 ppm are
characteristic of the four CH of tetrahydrohydrophthalimide. The HRMS of compound
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6i was shown in Figure 4. The [M + H] of compound 6i was calculated to be 374.1141
by ChemDraw 15.1(CambridgeSoft, USA), and the actual detected signal was 374.1150.
The error is within the specified range. In order to further determine the structure of the
synthesized compounds, the single crystal of compound 6i was cultivated and
characterized (Figure 1B). The skeleton structure obtained by single crystal diffraction
is as expected.
PPO Inhibition Activity and SAR. The IC values against maize PPO of the
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synthesized compounds 6a-w and 7 are listed in Table 1. Use the commonly applied
PPO herbicides Oxyfluorfen as herbicide control. As shown in Table 1, compounds 6c,
6l, 6n, 6r and 6t showed higher PPO inhibitory activity than Oxyfluorfen. Compound
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6c is the most promising compound with an IC value of 0.00667 mg / L for maize
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PPO. This result indicates that the PPO inhibitory activity of compound 6c is 7 times
that of Oxyfluorfen (IC =0.0426 mg/L). Based on Table 1, it can be found that the
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compounds 6c (X=O, R , R =CH , IC =0.00667 mg/L), 6n (X=O, R , R =CH ,
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IC =0.0180 mg/L) and 6t (X=O, R =SCH , IC =0.0106 mg/L) have excellent PPO
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inhibitory. And they have a common feature that the substituent of the compound on
the benzene ring are weak electron-donating groups. Correspondingly, the PPO
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inhibitory activities of compounds 6k (X=O, R OCH , IC =1.190 mg/L) and 6u (X=O,
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R =OCH CH , IC =2.779 mg/L) with strong electron-donating groups are relatively
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low. Therefore, the introduction of weak electron-donating groups on the benzene ring
of the compound is beneficial to increase the PPO inhibitory activity of the compound.
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Unexpectedly, the compounds 6l (X=O, R =Br, IC =0.0183 mg/L) and 6r (X=O, R =I,
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IC =0.0366 mg/L) whose substituents on the benzene ring are not weak electron
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donating groups but weakly electron withdrawing groups also exhibited excellent PPO
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inhibitory activity. Moreover, compounds 6j (X=O, R =CF , IC =0.788 mg/L) and 6w
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(X=O, R , R , R =F, IC =3.426 mg/L) with strong electron withdrawing groups on the
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phenyl ring exhibited very low activity. Thus, the introduction of electron withdrawing
groups on the benzene ring of the compound can also increase the PPO inhibitory
activity of the compound. Combining the above analysis, a group that introduces weak
electron effect groups in the benzene ring of the compound can increase the PPO
inhibitory activity of the compound.
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In addition, when the phenoxypyridine structure of the compound is changed into
phenylthiopyridine, the PPO inhibitory activity of the compound is significantly
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reduced. For example, 6q (X=O, R =F) and 6v (X=S, R =F) have the same substituent
group on the phenyl ring, but the PPO inhibitory activity of 6q (IC =0.253 mg/L) is 7
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times higher than 6v (IC =1.950 mg/L). Since the compound 6f (X=O, R =CH ,
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IC =0.193 mg/L) has weak electron effect groups methyl on the benzene ring, it
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exhibits a good PPO inhibitory activity. However, after replacing the phenoxypyridine
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structure of compound 6f with phenylthiopyridine (obtained 6g, X=S, R =CH ,
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IC =2.151 mg/L), its PPO inhibitory activity was reduced to less than one tenth of the
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original. Thus, it is advantageous to retain the phenoxypyridine structure of the
compound and introduce weak electron effect groups on the benzene ring to enhance
PPO inhibitory activity of the compound.
Herbicidal Activities and SAR. To study the post-emergence herbicidal activity
of compounds 6a-w and 7, three representative broadleaf weeds Abutilon theophrasti
Medicus (At), Capsella bursa-pastoris (Linn.) Medic (Cb), Portulaca oleraceaL. (Po),
and three representative monocotyledon weeds, Setaria viridis (L.) Beauv (Sv), Poa
annua L. (Pa), Echinochloa crusgalli (L.) Beauv (Ec) were selected for post-emergence
activity experiments. The commonly used PPO inhibitor herbicide Oxyfluorfen was
used as control. After spraying the compounds 6a-w for 1-2 days, most of the tested
weeds developed burn symptoms, then bleached and died. This result indicates that
these synthetic compounds are PPO inhibitors.
In most cases, the herbicidal activity of these synthetic compounds is consistent
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with the enzyme inhibitory activity against maize PPO. For instance, a compound
having weak electron effect groups on a benzene ring (6c, 6l, 6n, 6t) always exhibits
higher herbicidal activity than a compound having strong electron effect groups on a
benzene ring (6j, 6u, 6v, 6w), and a compound containing a phenoxypyridine structure
(6f) has higher herbicidal activity than a compound containing a phenylthiopyridine
structure (6g) (Figure 5).
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As shown in Table 1, more than half of the compounds have a strong inhibitory
activity against Sv (Inhibition rate ≥ 80%). Even at doses as low as 37.5 g ai/ha,
compounds 6c, 6l, 6n, 6r, 6s, 6t also displayed more than 90% inhibition against Sv.
Although compounds 6c, 6l, 6n, 6r, 6s, 6t shown strong inhibition against Sv, only 6c,
6n has broad-spectrum herbicidal activity. Compounds 6c and 6n showed greater than
60% inhibition of the six test weeds at a dose of 150 g ai/ha. As shown in Figure 6,
compound 6c showed over 80% inhibition of the tested weeds even though the
compound 6c treatment dose was as low as 37.5 g ai/ha. This herbicidal activity is
comparable to Oxyfluorfen. In addition, at the dose of 37.5 g ai/ha, compound 6c has a
better inhibitory effect on At than Oxyfluorfen (Figure 6). But compound 7 did not
show favorable activity in the herbicidal activity test and the PPO inhibition experiment,
and therefore no further studies were carried out.
Crop Selectivity of compound 6c. Whether herbicide is safe for crops is an
important indicator in the development of herbicides. In order to test the crop safety of
the most potential compound 6c, crop safety of compound 6c against six common crops
(soybean, peanut, cotton, maize, wheat, and rice) was evaluated in post-emergence crop
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safety assessment experiments. As shown in Table 2, compound 6c was nonselective
(inhibition >10%) for wheat even at doses as low as 75 g ai/ha. Therefore, so it cannot
be used to develop potential herbicides that control weed in wheat fields. But what is
exciting is that even at a dose of 300 g ai/ha, maize, cotton, soybeans, rice and peanuts
still show tolerance to compound 6c, which indicated that compound 6c might be
developed as a potential herbicide used for weed control in rice, cotton, soybeans, maize
and peanuts field. Correspondingly, Oxyfluorfen is only selective for rice, maize and
wheat in the post-emergence safety experiment. Thus, the selectivity of the compound
6c is significantly better than that of the Oxyfluorfen.
Docking study. The best way to predict the interaction of small molecules with
receptor binding pockets at the molecular level is molecular docking.39-42 In order to
understand the mechanism by which compound 6c and Oxyfluorfen acted on the target
site of PPO, molecular docking experiments were carried out. As shown in Figure 7,
the results showed that compound 6c and Oxyfluorfen had similar interactions with the
surrounding amino acid residues, but the interaction of compound 6c with amino acid
residues was stronger than that of Oxyfluorfen. As shown in Figure 7, both compound
6c and Oxyfluorfen form π-π stacking interaction with the amino acid residue PHE-392
and hydrogen bond with amino acid residue AGR-98. In organisms, the amino acid
residue PHE-392 and the amino acid residue AGR-98 are important groups involved in
the catalysis of porphyrins.43-44 The inhibitor will compete with the substrate porphyrin
for binding to these two amino acid residues. Thus, the inhibitory activity of the
inhibitor is related to the tightness of the binding of the inhibitor to these two amino
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acid residues. As show in Figure 7A, compound 6c forms two hydrogen bonds with
the surrounding amino acid residues AGR-98, but Oxyfluorfen form only one hydrogen
bond with amino acid residue AGR-98. Therefore, compound 6c acts more tightly on
the active site than Oxyfluorfen, indicating that 6c may have a stronger PPO inhibitory
activity than Oxyfluorfen. This result is consistent with post-emergence herbicidal
activity (Figure 6) and maize PPO inhibitory activity (Table 1).
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Accumulated concentration. The accumulated concentrations of 6c and
Oxyfluorfen in 6 crops were shown in Table 5. As shown in Table 5, two hours after
spraying, the accumulation amount of compound 6c in the stems and leaves of wheat
was 9.264 mg/kg. The accumulation of 6c in wheat was significantly higher than that
in other crops, which may be the reason why compound 6c is not safe for wheat.
Correspondingly, the cumulative concentration of Oxyfluorfen in wheat is 1.848 mg/kg.
Even after one day, the accumulation concentration of 6c in the stems and leaves of
wheat was still significantly higher than that of crops. Therefore, compound 6c cannot
be used as a potential herbicide in wheat fields. This result is consistent with the results
of previous safety tests. But excitingly, the accumulated concentration of compound 6c
in cotton, soybean, and peanut is significantly lower than that of Oxyfluorfen in these
crops. This result indicates that compound 6c is significantly better than Oxyfluorfen
for the safety of soybean, peanut and cotton. In addition, the accumulation
concentration of compound 6c in the stems and leaves of rice and corn was significantly
higher than the accumulation concentration of Oxyfluorfen in the stems and leaves of
rice and corn. However, in the crop safety test of 6c, both rice and corn showed certain
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tolerance to 6c. And the accumulation concentration of 6c in rice and corn was
significantly lower than cotton. In general, compound 6c showed safety to five crops of
rice, cotton, soybean, corn, and peanut (One day after spraying, the accumulated
concentration of stems and leaves was as low as a few tenths of a mg/kg), and it is worth
exciting that the accumulated concentration of 6c in some crops is significantly lower
than Oxyfluorfen.
Field test results.
The results of the field tests were shown in Table 6. Frustratingly, 75g ai/ha of 6c
inhibited only 25% of field weeds, and then weeds started to grow again. Fortunately,
with the increase of spraying concentration, the inhibition rate of 6c against weeds
gradually increased. Although the inhibitory rate of 6c against field weeds was not as
high as Oxyfluorfen at the beginning, the inhibitory rate of 6c against field weeds
gradually increased over time. When the application dose of 6c was increased to 300g
ai/ha, the inhibition rate on weeds reached 80% after 10 days. And The inhibition rate
of 6c on field weeds was better than that of Oxyfluorfen. When the field weeds were
treated with 600g ai/ha of 6c, the field weeds in 6c were as high as 98% 10 days after
spraying (Figure 8). In short, when the spraying dose of 6c is not less than 300g ai/ha,
the inhibitory rate of compound 6c on field weeds was equivalent to the commercial
herbicide Oxyfluorfen.
In conclusion, a series of novel diphenyl ether derivatives containing
tetrahydrophthalimide were designed, synthesized and their structures were
characterized. The SAR results indicate that retaining the phenoxypyridine structure of
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the compound and introducing weak electron effect groups on the benzene ring can
enhances the activity. Excitingly, compound 6c showed 7 times higher PPO inhibitory
activity than Oxyfluorfen and similar herbicidal activities to Oxyfluorfen in weeding
experiments in greenhouses and field weeding experiments. Docking studies have
shown that compound 6c acts more tightly on the active site than Oxyfluorfen.
Furthermore, the selectivity of compound 6c was significantly better than that of
Oxyfluorfen. Compound 6c is safe for cotton, maize, soybean, rice, and peanut even at
a dose of 300g ai/ha. Accumulation analysis experiments showed that the accumulation
of 6c in some crops (soybeans, peanuts and cotton) was significantly lower than
Oxyfluorfen. Current work suggests that compound 6c may be developed as a new
herbicide candidate in fields.
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ACKNOWLEDGMENTS
The authors are grateful to Prof. Jia-Zhong Li (Lanzhou University) for assistance
with molecular docking analyses, and Prof. Chun-Hong Teng (Agronomy College,
Northeast Agricultural University) for assistance with bioactivity assay.
FUNDING
This research was supported by the National Natural Science Foundation of China
(31572042), the Natural Science Foundation of Heilongjiang Province of China
(ZD2017002), the Heilongjiang Province Postdoctoral Research Startup Fund (LBH-
Q19083), the Research Science Foundation in Technology Innovation of Harbin
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