Design, Synthesis, and Biological Activity of Novel Diazabicyclo Derivatives as Safeners

Article abstract of DOI:10.1021/acs.jafc.9b07449

Herbicide safeners selectively protect crops from herbicide damage without reducing the herbicidal efficiency on target weed species. The title compounds were designed by the intermediate derivatization approach and fragment splicing to exploit novel potential safeners. A total of 31 novel diazabicyclo derivatives were synthesized by the microwave-assistant method using isoxazole-4-carbonyl chloride and diazabicyclo derivatives. All synthetic compounds were confirmed by infrared, ¹H and ¹³C nuclear magnetic resonance, and high-resolution mass spectrometry. The bioassay results demonstrated that most of the title compounds could reduce the nicosulfuron phytotoxicity on maize. The glutathione S-transferase (GST) activity in vivo was assayed, and compound 4(S15) revealed an inspiring safener activity comparable to commercialized safeners isoxadifen-ethyl and BAS-145138. The molecular docking model exhibited that the competition at the active sites of target enzymes between compound 4(S15) and nicosulfuron was investigated with respect to herbicide detoxification. The current work not only provided a powerful supplement to the intermediate derivatization approach and fragment splicing in design pesticide bioactive molecules but also assisted safener development and optimization.

Full text of DOI:10.1021/acs.jafc.9b07449

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Agricultural and Environmental Chemistry
Design, Synthesis and Biological Activity of
Novel Diazabicyclo Derivatives as Safeners
Yuan-Yuan Zhang, Shuang Gao, Yong-Xuan Liu, Chen Wang, Wei Jiang, Li-Xia Zhao, Ying Fu, and Fei Ye
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07449 • Publication Date (Web): 26 Feb 2020
Downloaded from pubs.acs.org on February 27, 2020
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Design, Synthesis and Biological Activity of Novel Diazabicyclo
Derivatives as Safeners
†
#
†#
†
†
†
Yuan-Yuan Zhang , Shuang Gao , Yong-Xuan Liu , Chen Wang , Wei Jiang ,
†
†
†
Li-Xia Zhao , Ying Fu *, Fei Ye *
†Department of Applied Chemistry, College of Arts and Sciences, Northeast
Agricultural University, Harbin 150030, China
AUTHOR INFORMATION
Corresponding Authors
*
Y Fu: Tel: +86-451-55190070; E-mail: fuying@neau.edu.cn
F Ye: Tel: +86-451-55191507; E-mail: yefei@neau.edu.cn
*
ORCID
Ying Fu: 0000-0003-4265-6879
Fei Ye: 0000-0002-7731-752X
Author Contributions
#Yuan-Yuan Zhang and Shuang Gao contributed equally to this study
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ABSTRACT
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Herbicide safeners selectively protect crops from herbicide damage without
reducing the herbicidal efficiency on target weed species. The title compounds were
designed by intermediate derivatization approach and fragment splicing in order to
exploit novel potential safeners. A total of thirty-one novel diazabicyclo derivatives
were synthesized by microwave-assistant method using isoxazole-4-carbonyl chloride
1
and diazabicyclo derivatives. All synthetic compounds were confirmed by IR, H
13
NMR, C NMR, HRMS. The bioassay results demonstrated that most of the title
compounds could reduce the nicosulfuron phytotoxicity on maize. The
glutathione-S-transferase (GST) activity in vivo was assayed and compound 4 (S15)
revealed an inspiring safener activity comparable to commercialized safeners
isoxadifen-ethyl and BAS-145138. The molecular docking model exhibited that the
competition at the active sites of target enzymes between compound 4 (S15) and
nicosulfuron was investigated with respect to herbicide detoxification. The current
work not only provided a powerful supplement to the intermediate derivatization
approach and fragment splicing in design pesticide bioactive molecules, but also
assisted safener development and optimization.
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Keywords: Fragment splicing, Intermediate derivatization approach, N-substituted
diazabicyclo, Microwave-assistant Synthesis, Herbicide safener
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INTRODUCTION
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The history of agriculture has shown that humans must effectively control weeds
all times. One of the most effective and modern methods of controlling weeds in
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growing crops is the use of herbicides. The development of chemical herbicides
began with the discovery of selective herbicide 2, 4-D. So far, herbicides have entered
an efficient development stage, and the variety has been continuously updated.2
Herbicides mainly used in wheat, rice, potato, cotton, and maize fields around the
world, which greatly improve the crop production and agricultural productivity.3,4
Sulfonylurea herbicides were the most widely used herbicides in the world with
the widest range of applications and the deepest research. The sulfonylurea herbicide
nicosulfuron is a broad-spectrum, flexible and inner-absorption conducting herbicide,
which is used to control annual grasses and certain broadleaf weeds in maize or other
field crops.5-7 Nicosulfuron has a wide maize safety margin, but due to large amounts
of its application and toxigenic potential, certain maize hybrids and inbreds can be
severely injured by nicosulfuron.8,9 Some researches showed that maize seedlings can
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be severely inhibited after nicosulfuron treatment from 7 to 14 days. Nicosulfuron is
easy to cause phytotoxicity to sensitive crops and may have adverse effects on later
crops.11 For sensitive maize, it caused severe injury to lipids and changed the
ascorbate-glutathione cycle, with symptoms ranging from severe stunting of plant
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growth to death. In order to solve these serious problems, many methods have been
reported, including the development of new herbicides and so on.13-15 Other than these
methods, using herbicide safeners, is also an effective solution.
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Herbicide safeners selectively protect crops from certain herbicides without
affecting weed control effectiveness.16,17 Since the first phenomenon of herbicide
safening was discovered, the mechanisms of action of safeners have received
considerable attention. Nevertheless, due to the complexity of the interactions
between the safeners and the molecular mechanisms and the expression of the
enzyme, there is no conclusive mechanism explaining action of the available
safeners.18,19 It is widely accepted that safeners can improve crop metabolism and
detoxification by upregulating cytochrome-P450-monooxygenases (CYPs),
glutathione-S-transferases (GSTs), glutathione (GSH) and ATP-binding casette
(ABC) transporter proteins that detoxify herbicides.20-23 The use of a suitable safener
can reduce or eliminate herbicide damage to crop without affecting the herbicidal
activity. As a result, it is highly urgent to develop new safeners to provide new
solutions to weed control problems.
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Isoxadifen-ethyl was the first safener launched by Bayer CropScience AG. It
can protect maize from herbicide damage with various modes of action, which could
increase the absorption, translocation and detoxification of sulfonylurea herbicides in
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maize. Isoxadifen-ethyl can effectively alleviate the phytotoxicity of nicosulfuron to
maize and increase the plant's metabolism of nicosulfuron through
non-P450-catalyzed routes.25 BAS-145138, diazabicyclo herbicide safener, can
protect sorghum and reduce herbicide toxicity by enhancing the metabolism of lauric
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acid. Also, BAS-145138 can accelerate the metabolic rate of chlorimuron ethyl in
leaves and roots of maize without affecting the absorption of chlorimuron ethyl in
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maize. It was shown that the concentration of safety agent was linear with GSH
content and GST activity.
The Intermediate Derivatization Methods (IDM) is a new approach for
discovering and developing novel patentable leads or target compounds by chemical
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reaction utilizing various intermediates. The key to the success of the IDM is to find
the right intermediate. Successful examples such as the development of herbicides
pyribenzoxim (derivatives of bispyribac-sodium), butafenacil (derivatives of
flupropacil), and herbicide safener fenchlorazole-ethyl.29-31 Fragment splicing is also
the main strategy to design and optimize of new skeleton structures with target
biological activity. For instance, the new fungicide fluopyram was designed by
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fragment splicing of fluopicolide and flutolanil. Combining the structural features of
diphenylmethoxyacetic acid and the experimental Monsanto safener benzhydryloxy
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3
acetic acid led to the strong maize safener isoxadifen-ethyl. In connection with the
facts mentioned above, the attempt to design novel compounds with safener activity
will be valuable for alleviating the toxity of herbicides. Based on our work on
synthesis of nitrogen-containing heterocyclic compound,34,35 herein we use
N-substituted diazabicyclo as the key intermediate, then designed a series of novel
substituted diazabicyclo derivatives utilizing the IDM combined with fragment
splicing (Scheme 1). The bioassay was also conducted for evaluating the safener
activity of the title compounds. Furthermore, molecular structure comparisons and
molecular docking were performed to determine the possible detoxifcation
mechanism for the safener and to design more effective new safeners.
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MATERIALS AND METHODS
Equipment and Materials
The melting points were obtained on a Beijing Taike X-4 stage apparatus and
uncorrected. Infrared (IR) spectra were measured as KBr pellets on ALPHA-T. The
NMR spectra were recorded on a Bruker AV-400 spectrometer, using TMS as an
internal standard. The high-resolution mass spectrum (HRMS) were recorded on a
Xevo TQ spectrometer. X-ray diffraction data were collected on a Rigaku R-AXIS
RAPID
diffractometer.
The
four
isoxazole-4-carbonyl
chlorides
chloride,
chloride,
and
(5-methyl-3-phenylisoxazole-4-carbonyl
3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carbonyl
3-(2,6-dichlorophenyl)-5-methylisoxazole-4-carbonyl
chloride
3-(2-chlorophenyl)-5-methylisoxazole-4-carbonyl chloride) were purchased from
Hubei Xinmingtai Chemical Co., Ltd. Purification by column chromatography on
silica gel. Microwave activation was performed using an XH-100A focused
microwave.
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General Procedure for the Synthesis of Compounds 4
Diamine (0.05 mol), ethyl levulinate (0.05 mol) (or ethyl 4-acetobutyrate) and
montmorillonite k10 (3 g) were added to 100mL three-necked flask sequentially, the
reaction was stirred for 12 min under the microwave conditions (300 W, 25℃). The
products obtained above without reprocessing and anhydrous K CO (1.5 eq) were
2
3
dissolved in CHCl at room temperature. The isoxazole-4-carbonyl chloride (1.5 eq)
3
was added to the mixture slowly at 0℃ and the reaction was monitored by TLC.
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After the reaction was over, the solution was washed with water. The organic phase
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was dried over anhydrous MgSO . After the solvent was removed off under reduced
4
pressure, the crude products were purified by column chromatography on silica gel
eluting with ethyl acetate and petroleum ether (6:1) or recrystallized with ethyl acetate
and light petroleum ether.
3-phenyl-4-(5-methyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-methyl-isoxazolec
1
arboxamide 4 (S1). Yield: 65%; m.p. 169.9-171.0℃; H NMR (CDCl ): δ 7.69-7.28
3
(m, 5H, Ar-H), 3.92-3.87 (m, 2H, N-CH -CH -N), 3.21-2.94 (m, 2H, N-CH -CH -N),
2
2
2
2
2.76-2.67 (m, 2H, N-CH -CH -N), 2.52 (S, 3H, CH ), 2.49-2.43 (m, 2H,
2
2
3
1
3
N-CH -CH -N), 1.73 (s, 3H, CH ); C NMR (CDCl ): δ 175.21, 169.12, 160.90,
2
2
3
3
159.23, 130.69, 129.16, 129.16, 128.36, 127.11, 127.11, 112.64, 83.53, 47.52, 38.95,
+
34.06, 32.15, 22.81, 11.35; HRMS (ESI) calcd for C H N O ([M+H] ) 326.1499,
1
8
19
3
3
found 326.1495.
3-(2'-chloro-phenyl)-4-(5-methyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-methyl
1
-oxazole carboxamide 4 (S2). Yield: 51%; m.p. 219.5-220.6℃; H NMR (CDCl ): δ
3
7.55-7.39 (m, 4H, Ar-H), 3.97-3.40(m, 2H, N-CH -CH -N), 3.26-3.05 (m, 2H,
2
2
N-CH -CH -N), 2.70-2.67 (m, 2H, C-CH -CH -C=O), 2.58 (s, 3H, CH ), 2.54-2.30
2
2
2
2
3
1
3
(m, 2H, C-CH -CH -C=O), 1.66 (s, 3H, CH ); C NMR (CDCl ): δ 175.60, 169.00,
2
2
3
3
160.40, 158.86, 132.67, 131.63, 131.43, 130.27, 127.81, 127.40, 114.25, 83.74, 47.33,
+
39.26, 33.59, 32.11, 22.97, 12.37; HRMS (ESI) calcd for C H ClN O ([M+H] )
1
8
18
3
3
360.1109, found 360.1107.
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3-(2',6'-dichloro-phenyl)-4-(5-methyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-me
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thyl-isoxazole carboxamide 4 (S3). Yield: 57%; m.p. 197.4-198.3℃; H NMR
(CDCl ): δ 7.48-7.38 (m, 3H, Ar-H), 4.05-3.56 (m, 2H, N-CH -CH -N), 3.45-3.10 (m,
3
2
2
2H, N-CH -CH -N), 2.81-2.67 (m, 2H, C-CH -CH -C=O), 2.60 (s, 3H, CH ),
2
2
2
2
3
1
3
2.54-2.32 (m, 2H, C-CH -CH -C=O), 1.62 (s, 3H, CH ); C NMR (CDCl ): δ 175.66,
2
2
3
3
168.48, 159.72, 157.00, 135.71, 135.04, 131.75, 128.65, 128.20, 127.19, 114.52,
83.81, 47.20, 39.33, 33.60, 32.09, 22.80, 12.75; HRMS (ESI) calcd for
+
C H Cl N O ([M+H] ) 394.0720, found 394.0719.
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17
2
3
3
3-(2'-fluoro-6'-chloro-phenyl)-4-(5-methyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)
1
-5-methyl-isoxazolecarboxamide 4 (S4). Yield: 50%; m.p. 166.7-167.8℃; H NMR
(CDCl ): δ 7.47-7.34 (m, 3H, Ar-H), 4.02-3.50 (d, 2H, N-CH -CH -N), 3.40-2.59 (d,
3
2
2
2H, N-CH -CH -N), 2.80-2.64 (m, 2H, C-CH -CH -C=O), 2.55-2.34 (m, 2H,
2
2
2
2
1
3
C-CH -CH -C=O), 2.59 (S, 3H, CH ), 1.64 (s, 3H, CH ); C NMR (CDCl ): δ
2
2
3
3
3
175.62, 168.58, 161.87, 159.86, 159.35, 153.68, 134.68, 132.16, 125.99, 116.75,
114.67, 83.73, 47.22, 39.26, 33.61, 32.08, 22.76, 12.47; HRMS (ESI) calcd for
+
C H ClFN O ([M+H] ) 378.1015, found 378.1019.
1
8
17
3
3
3-phenyl-4-(5-methyl-9-oxa-1,4-diazabicyclo[3.4.0]nonane)-5-methyl-isoxazolec
1
arboxamide 4 (S5). Yield: 74%; m.p. 152.9-154.5℃; H NMR (CDCl ): δ 7.61-7.29
3
(m, 5H, Ar-H), 3.97-3.94 (d, 2H, N-CH -C-CH -N), 3.15-2.81 (m, 4H,
2
2
N-CH -C-CH -N), 2.53 (S, 3H, CH ), 2.52-1.76 (m, 4H, N-CH -CH -N), 1.69 (s, 3H,
2
2
3
2
2
1
3
CH ), 1.36-1.32 (d, 2H, N-CH -C-CH -N); C NMR (CDCl ): δ 172.81, 172.74,
3
2
2
3
172.72, 172.67, 163.56, 163.54, 160.13, 160.11, 130.52, 128.98, 128.15, 127.83,
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112.21, 43.99, 34.64, 33.99, 29.25, 22.53, 20.53, 11.89; HRMS (ESI) calcd for
+
1
1
1
1
1
1
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1
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C H N O ([M+Na] ) 362.1477, found 362.1475.
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21
3
3
3-(2'-chloro-phenyl)-4-(5-methyl-9-oxa-1,4-diazabicyclo[3.4.0]nonane)-oxazole
1
carboxamide 4 (S6). Yield: 66%; m.p. 181.7-183.1℃; H NMR (CDCl ): δ 7.53-7.40
3
(m, 4H, Ar-H), 4.04-2.71 (m, 4H, N-CH -CH -CH -N), 2.58 (s, 3H, CH ), 2.39-1.71
2
2
2
3
(m, 4H, C-CH -CH -C=O), 1.63 (s, 3H, CH3), 1.51-1.47 (m, 2H,
2
2
1
3
N-CH -CH -CH -N); C NMR (CDCl ): δ 172.67, 163.64, 132.91, 131.55, 131.43,
2
2
2
3
131.43, 130.14, 130.14, 127.71, 127.38, 127.38, 113.40, 44.58, 35.00, 33.79, 29.25,
+
23.37, 19.37, 12.25; HRMS (ESI) calcd for C H ClN O ([M+H] ) 374.1266, found
1
9
20
3
3
374.1268.
3-(2',6'-dichloro-phenyl)-4-(5-methyl-9-oxa-1,4-diazabicyclo[3.4.0]nonane)-5-m
1
ethyl-isoxazole carboxamide 4 (S7). Yield: 71%; m.p. 200-201℃; H NMR (CDCl ):
3
δ 7.47-7.34 (m, 3H, Ar-H), 3.98-2.67 (m, 4H, N-CH -CH -CH -N), 2.60 (s, 3H, CH ),
2
2
2
3
2.39-2.25 (m, 2H, C-CH -CH -C=O), 1.57 (s, 3H, CH ), 1.51-1.49 (m, 2H,
2
2
3
1
3
C-CH -CH -C=O), 0.85-0.81 (m, 2H, N-CH -CH -CH -N); C NMR (CDCl ): δ
2
2
2
2
2
3
172.59, 163.37, 156.56, 135.53, 135.37, 131.80, 131.80, 128.80, 128.80, 128.27,
127.04, 113.63, 44.46, 35.05, 33.64, 29.21, 23.51, 19.17, 12.43; HRMS (ESI) calcd
+
for C H Cl N O ([M+H] ) 408.0876, found 408.0873.
1
9
19
2
3
3
3-(2'-fluoro-6'-chloro-phenyl)-4-(5-methyl-9-oxa-1,4-diazabicyclo[3.4.0]nonane)
1
-5-methyl-isoxazolecarboxamide 4 (S8). Yield: 60%; m.p. 183.0-183.8℃; H NMR
(CDCl ): δ 7.47-7.12 (m, 3H,
Ar-H), 4.02-3.98 (m, 2H, N-CH -C-CH -N),
2 2
3
3.44-3.20 (m, 2H, N-CH -C-CH -N), 2.85-2.69 (m, 2H, N-CH -C-CH -N), 2.62 (S,
2
2
2
2
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191
192
3H, CH ), 2.36-1.67 (m, 4H, N-CH -CH -N), 1.61 (s, 3H, CH ), 1.54-1.51 (m, 2H,
3 2 2 3
1
3
N-CH -C-CH -N); C NMR (CDCl ): δ 172.55, 163.48, 161.98, 159.44, 134.81,
2
2
3
134.77, 132.30, 128.01, 125.98, 114.72, 114.50, 113.71, 44.49, 36.00, 33.70, 29.19,
+
23.47, 19.13, 12.28; HRMS (ESI) calcd for C H ClFN O ([M+Na] ) 414.0989,
1
9
19
3
3
found 414.0991.
3-phenyl-4-(2
or
3,5-dimethyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-methyl-isoxazole formamide 4
1
(S9). Yield: 61%; m.p. 156.5-157.5℃; H NMR (CDCl ): δ 7.69-7.48 (m, 5H, Ar-H),
3
4.07-4.05 (m, 1H, N-CH-CH -N), 2.90-2.86 (m, 2H, N-CH-CH -N), 2.74-2.67 (m,
2
2
2H, C-CH -CH -C=O), 2.53 (s, 3H, CH ), 2.44-2.32 (m, 2H, C-CH -CH -C=O), 1.78
2
2
3
2
2
1
3
(s, 3H, CH ), 0.96-0.94 (d, J=8, 3H, CH ); C NMR (CDCl ): δ 175.47, 169.48,
3
3
3
160.98, 159.45, 130.68, 129.18, 129.18, 127.35, 127.35, 127.35, 112.58, 83.83, 54.66,
47.91, 36.53, 32.54, 24.57, 20.81, 11.78; HRMS (ESI) calcd for C H N O
1
9
21
3
3
+
([M+H] ) 340.1656, found 340.1653.
3-(2'-chloro-phenyl)-4-(2
or
3,5-dimethyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-methyl-isoxazolecarboxamide 4
1
(S10). Yield: 42%; m.p. 170.4-172.3℃; H NMR (CDCl ): δ 7.56-7.38 (m, 4H,
3
Ar-H), 4.08-4.04 (m, 1H, N-CH-CH -N), 3.63-3.09 (m, 2H, N-CH-CH -N), 2.77-2.61
2
2
(m, 2H, C-CH -CH -C=O), 2.59 (s, 3H, CH ), 2.56-2.29 (m, 2H, C-CH -CH -C=O),
2
2
3
2
2
1
3
1.70 (s, 3H, CH ), 1.06 (s, 3H, CH ); C NMR (CDCl ): δ 175.76, 169.51, 160.50,
3
3
3
158.79, 132.88, 131.63, 131.47, 130.32, 127.89, 127.39, 114.00, 83.96, 54.49, 48.21,
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+
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
93
36.13, 32.55, 24.52, 20.63, 12.25; HRMS (ESI) calcd for C H ClN O ([M+H] )
19 20 3 3
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
374.1266, found 374.1262.
3-(2',6'-dichloro-phenyl)-4-(2
or
3,5-dimethyl-8-oxa-1,4-diazabicyclo[3.3.0]octane
1
Alkyl)-5-methyl-isoxazolecarboxamide 4 (S11). Yield: 55%; m.p. 162.1-163.8℃; H
NMR (CDCl ): δ 7.52-7.40 (m, 3H, Ar-H), 4.03-4.00 (m, 1H, N-CH-CH -N),
3
2
3.81-3.14 (m, 2H, N-CH-CH -N), 2.63 (s, 3H, CH ), 2.48-2.40 (m, 2H,
2
3
C-CH -CH -C=O), 1.94-1.62 (m, 2H, C-CH -CH -C=O), 1.41 (s, 3H, CH ), 0.99 (s,
2
2
2
2
3
3H, CH3); 13C NMR (CDCl ): δ 170.46, 168.39, 159.66, 155.51, 136.30, 135.45,
3
131.84, 131.84, 128.70, 128.45, 127.26, 115.12, 78.84, 52.82, 46.22, 35.01, 30.08,
+
21.64, 17.55, 12.08; HRMS (ESI) calcd for C H Cl N O ([M+H] ) 408.0876, found
1
9
19
2
3
3
408.0881.
3-(2'-fluoro-6'-chloro-phenyl)-4-(2
or
3,5-dimethyl-8-oxa-1,4-diazabicyclo[3.3.0]octane)-5-methyl-isoxazolecarboxamide 4
1
(S12). Yield: 39%; m.p. 187.3-187.9℃; H NMR (CDCl ): δ 7.48-7.14 (m, 3H,
3
Ar-H), 4.08-4.17 (m, 1H, N-CH-CH -N), 3.73-3.20 (m, 2H, N-CH-CH -N), 2.61 (s,
2
2
3H, CH ), 1.60-1.15 (m, 4H, C-CH -CH -C=O), 1.68 (s, 3H, CH ), 1.13 (s, 3H, CH );
3
2
2
3
3
1
3
C NMR (CDCl ): δ 175.81, 169.21, 160.01, 132.30, 132.21, 126.07, 126.04, 114.72,
3
114.50, 83.93, 54.47, 48.28, 36.01, 32.56, 30.99, 28.29, 24.37, 20.73, 12.36; HRMS
+
(ESI) calcd for C H ClFN O ([M+H] ) 392.1172, found 392.1175.
1
9
19
3
3
3-phenyl-4-(3,3,6-trimethyl-9-oxa-1,5-diazabicyclo[3.4.0]nonane)-5-methyl-isox
1
azole formamide 4 (S13). Yield: 81%; m.p. 135.1-137℃; H NMR (CDCl ): δ
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236
7.64-7.45 (m, 5H, Ar-H), 3.75-3.02 (m, 2H, N-CH -CH -N), 2.79-2.76 (m, 2H,
2 2
N-CH -CH -N), 2.53 (s, 3H, CH ), 2.52-2.48 (m, 2H, C-CH -CH -C=O), 2.44-2.38
2
2
3
2
2
1
3
(m, 2H, C-CH -CH -C=O), 1.60 (s, 3H, CH ), 0.81-0.49 (m, 3H, CH ); C NMR
2
2
3
3
(CDCl ): δ 172.23, 165.19, 130.51, 129.08, 129.08, 129.08, 128.23, 127.58, 112.55,
3
55.68, 46.09, 35.31, 30.31, 30.31, 29.66, 24.50, 21.54, 21.52, 21.39, 17.67, 11.70,
+
11.70; HRMS (ESI) calcd for C H N O ([M+H] ) 368.1969, found 368.1966.
2
1
25
3
3
3-(2'-chloro-phenyl)-4-(3,3,6-trimethyl-9-oxa-1,5-diazabicyclo[3.4.0]nonane)-5-
1
methyl-isoxazolecarboxamide 4 (S14). Yield: 70%; m.p. 174.9-176.3℃; H NMR
(CDCl ): δ 7.53-7.37 (m, 4H, Ar-H), 3.75-3.11 (m, 2H, N-CH -CH -N), 2.93-2.82 (m,
3
2
2
2H, N-CH -CH -N), 2.54 (s, 3H, CH ), 2.51-2.30 (m, 4H, C-CH -CH -C=O), 1.52 (s,
2
2
3
2
2
1
3
3H, CH ), 0.86 (s, 3H, CH ), 0.48 (s, 3H, CH ); C NMR (CDCl ): δ 172.34, 164.96,
3
3
3
3
133.05, 131.58, 131.58, 130.44, 127.79, 127.33, 113.93, 77.26, 77.20, 55.92, 46.25,
34.80, 30.43, 29.63, 24.58, 24.58, 21.20, 17.39, 12.23; HRMS (ESI) calcd for
+
C H ClN O ([M+H] ) 402.1579, found 402.1577.
2
1
24
3
3
3-(2',6'-dichloro-phenyl)-4-(3,3,6-trimethyl-9-oxa-1,5-diazabicyclo[3.4.0]nonan
1
e alkyl)-5-methyl-isoxazolecarboxamide 4 (S15). Yield: 78%; m.p. 218.2-219.2℃; H
NMR (CDCl ): δ 7.48-7.35 (m, 3H, Ar-H), 3.77-3.21 (m, 2H, N-CH -CH -N),
3
2
2
2.92-2.85 (m, 2H, N-CH -CH -N), 2.57 (s, 3H, CH ), 2.53-2.26 (m, 4H,
2
2
3
1
3
C-CH -CH -C=O), 1.48 (s, 3H, CH ), 0.92 (s, 3H, CH ), 0.61 (s, 3H, CH ); C NMR
2
2
3
3
3
(CDCl ): δ 172.35, 169.49, 169.47, 164.45, 156.56, 136.14, 135.18, 131.74, 128.93,
3
128.27, 127.22, 114.21, 55.86, 46.37, 34.87, 30.52, 29.60, 24.59, 21.37, 17.29, 12.50;
+
HRMS (ESI) calcd for C H Cl N O ([M+H] ) 436.1189, found 436.1187.
2
1
23
2
3
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3-(2'-fluoro-6'-chloro-phenyl)-4-(3,3,6-trimethyl-9-oxa-1,5-diazabicyclo[3.4.0]n
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38
39
40
41
42
43
44
45
46
47
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50
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52
53
54
55
56
57
onane)-5-methyl-isoxazole carboxamide 4 (S16). Yield: 62%; m.p. 198.6-200.3℃; H
NMR (CDCl ): δ 7.45-7.11 (m, 3H, Ar-H), 3.78-3.21 (m, 2H, N-CH -CH -N),
3
2
2
2.92-2.86 (m, 2H, N-CH -CH -N), 2.58 (s, 3H, CH ), 2.56-2.27 (m, 4H,
2
2
3
1
3
C-CH -CH -C=O), 1.50 (s, 3H, CH ), 0.92 (s, 3H, CH ), 0.64 (s, 3H, CH ); C NMR
2
2
3
3
3
(CDCl ): δ 172.34, 164.56, 135.01, 132.19, 132.10, 126.20, 126.16, 114.68, 114.46,
3
114.38, 77.26, 77.20, 55.96, 46.28, 34.85, 30.48, 29.61, 24.66, 21.56, 17.19, 12.34;
+
HRMS (ESI) calcd for C H ClFN O ([M+H] ) 420.1485, found 420.1486.
2
1
23
3
3
3-phenyl-4-(6-methyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane)-5-methyl-isoxazolec
1
arboxamide 4 (S17). Yield: 63%; m.p. 127.4-128.7℃; H NMR (CDCl ): δ 7.71-7.46
3
(m, 5H, Ar-H), 4.24-3.18 (m, 2H, N-CH -CH -N), 3.13-2.93 (m, 2H, N-CH -CH -N),
2
2
2
2
1
3
2.52 (S, 3H, CH ), 2.49-1.86 (m, 4H, N-CH -CH -N), 1.71 (s, 3H, CH ); C NMR
3
2
2
3
(CDCl ): δ 168.93, 167.57, 161.33, 159.26, 130.65, 129.12, 129.12, 128.39, 127.19,
3
127.19, 113.12, 78.42, 45.35, 39.86, 32.90, 30.47, 22.18, 17.52, 11.78; HRMS (ESI)
+
calcd for C H N O ([M+H] ) 340.1656, found 340.1656.
1
9
21
3
3
3-(2'-chloro-phenyl)-4-(6-methyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane) oxazole
1
carboxamide 4 (S18). Yield: 48%; m.p. 172.6-174.1℃; H NMR (CDCl ): δ 7.55-7.38
3
(m, 4H, Ar-H), 4,26-3.11 (m, 4H, N-CH -CH -N), 3.04-3.01 (m, 2H,
2
2
C-CH -CH -CH -C=O), 2.57 (s, 3H, CH ), 2.52-1.81 (m, 2H, C-CH -CH -CH -C=O),
2
2
2
3
2
2
2
1
3
1.65 (s, 3H, CH ), 1.60-1.53 (m, 2H, C-CH -CH -CH -C=O); C NMR (CDCl ): δ
3
2
2
2
3
168.81, 167.67, 160.83, 159.05, 132.67, 131.59, 131.48, 130.19, 127.81, 127.35,
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278
279
114.64, 78.53, 45.30, 40.05, 32.81, 30.53, 22.03, 17.47, 12.39; HRMS (ESI) calcd for
+
C H ClN O ([M+H] ) 374.1266, found 374.1269.
1
9
20
3
3
3-(2',6'-dichloro-phenyl)-4-(6-methyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane)-5-m
1
ethyl-isoxazole carboxamide 4 (S19). Yield: 54%; m.p. 244.1-245.6℃; H NMR
(CDCl ): δ 7.48-7.35 (m, 3H, Ar-H), 4,35-3.19 (m, 4H, N-CH -CH -N), 3.01-2.97 (m,
3
2
2
2H, C-CH -CH -CH -C=O), 2.60 (s, 3H, CH3), 2.51-1.88 (m, 2H,
2
2
2
C-CH -CH -CH -C=O), 1.59 (s, 3H, CH ), 1.56-1.48 (m, 2H, C-CH -CH -CH -C=O);
2
2
2
3
2
2
2
1
3
C NMR (CDCl ): δ 168.47, 167.66, 160.16, 157.11, 135.95, 134.80, 131.71, 128.81,
3
127.97, 127.22, 114.91, 78.58, 45.23, 40.10, 32.91, 30.56, 21.87, 17.46, 12.74; HRMS
+
(ESI) calcd for C H Cl N O ([M+H] ) 408.0876, found 408.0872.
1
9
19
2
3
3
3-(2'-fluoro-6'-chloro-phenyl)-4-(6-methyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane)
1
-5-methyl-isoxazolecarboxamide 4 (S20). Yield: 39%; m.p. 236.3-237.8℃; H NMR
(CDCl ): δ 7.45-7.12 (m, 3H, Ar-H), 4.35-3.52 (m, 2H, N-CH -CH -N), 3.26-3.22 (m,
3
2
2
2H, N-CH -CH -N), 3.03-3.00 (m, 2H, C-CH -CH -CH -C=O), 2.60 (s, 3H, CH ),
2
2
2
2
2
3
2.49-1.91 (m, 2H, C-CH -CH -CH -C=O), 1.63 (s, 3H, CH ), 1.54-1.53 (m, 2H,
2
2
2
3
1
3
C-CH -CH -CH -C=O); C NMR (CDCl ): δ 168.54, 167.69, 160.36, 153.82,
2
2
2
3
134.74, 134.70, 132.20, 132.10, 125.98, 125.94, 114.66, 78.54, 45.19, 40.08, 32.93,
+
30.58, 21.85, 17.48, 12.49; HRMS (ESI) calcd for C H ClFN O ([M+H] )
1
9
19
3
3
392.1172, found 392.1176.
3-phenyl-4-(6-methyl-10-oxa-1,5-diazabicycl
[4.4.0]decane)-5-methyl-isoxazolecarboxamide
4
(S21). Yield: 59%; m.p.
1
163.5-164.9℃; H NMR (CDCl ): δ 7.67-7.50 (m, 5H, Ar-H), 4.50-2.60 (m, 4H,
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2
2
2
2
2
3
3
80
N-CH -CH -CH -N), 2.52 (s, 3H, CH ), 2.33-1.42 (m, 6H, C-CH -CH -CH -C=O),
2 2 2 3 2 2 2
1
3
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
1.28 (s, 3H, CH ), 1.06-0.69 (m, 2H, N-CH -CH -CH -N); C NMR (CDCl ): δ
3 2 2 2 3
173.55, 161.98, 160.18, 130.50, 130.43, 129.07, 129.07, 128.98, 128.95, 127.81,
113.40, 111.19, 36.10, 33.46, 32.00, 29.73, 29.57, 16.92, 12.89, 11.79; HRMS(ESI)
+
calcd for C H N O ([M+H] ) 354.1812, found 354.1819.
2
0
23
3
3
3-(2'-chloro-phenyl)-4-(6-methyl-10-oxa-1,5-diazabicyclo[4.4.0]decane) oxazole
1
carboxamide 4 (S22). Yield: 43%; m.p. 197.7-198.7℃; H NMR (CDCl ): δ 7.55-7.40
3
(m, 4H, Ar-H), 4.61-2.98 (m, 4H, N-CH -CH -CH -N), 2.90-2.79 (m, 2H,
2
2
2
C-CH -CH -CH -C=O), 2.61 (s, 3H, CH ), 2.55-2.35 (m, 2H, C-CH -CH -CH -C=O),
2
2
2
3
2
2
2
1.79-1.64 (m, 2H, N-CH -CH -CH -N), 1.44-1.27 (m, 2H, C-CH -CH -CH -C=O),
2
2
2
2
2
2
1.88 (s, 3H, CH3); 13C NMR (CDCl ): δ 170.31, 169.01, 162.37, 159.42, 133.04,
3
131.60, 131.46, 130.02, 130.02, 127.90, 127.43, 114.74, 43.21, 33.81, 32.61, 31.73,
+
24.68, 22.67, 16.90, 12.30; HRMS (ESI) calcd for C H ClN O ([M+H] ) 388.1422,
2
0
22
3
3
found 388.1427.
3-(2',6'-dichloro-phenyl)-4-(6-methyl-10-oxa-1,5-diazabicyclo[4.4.0]decane)-5-
1
methyl-isoxazole carboxamide 4 (S23). Yield: 47%; m.p. 232.1-233.5℃; H NMR
(CDCl ): δ 7.48-7.38 (m, 3H, Ar-H), 4.61-2.92 (m, 4H, N-CH -CH -CH -N), 2.61 (s,
3
2
2
2
3H, CH ), 2.41-2.23 (m, 4H, C-CH -CH -CH -C=O), 1.88 (s, 3H, CH ), 1.66-1.65 (m,
3
2
2
2
3
2H, N-CH -CH -CH -N), 1.60-1.58 (m, 2H, C-CH -CH -CH -C=O); 13C NMR
2
2
2
2
2
2
(CDCl ): δ 169.42, 161.74, 157.01, 135.87, 135.76, 135.48, 131.62, 128.61, 128.55,
3
128.20, 127.32, 115.01, 43.62, 33.89, 32.82, 31.90, 25.25, 22.89, 16.91, 12.55; HRMS
+
(ESI) calcd for C H Cl N O ([M+H] ) 422.1033, found 422.1035.
2
0
21
2
3
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322
323
3-(2'-fluoro-6'-chloro-phenyl)-4-(6-methyl-10-oxa-1,5-diazabicyclo[4.4.0]decan
1
e)-5-methyl-isoxazolecarboxamide 4 (S24). Yield: 31%; m.p. 183.7-185.6℃; H NMR
(CDCl ): δ 7.44-7.14 (m, 3H, Ar-H), 4.64-3.54 (m, 4H, N-CH -CH -CH -N), 2.59 (s,
3
2
2
2
3H, CH ), 3.14-2.86 (m, 4H, C-CH -CH -CH -C=O), 2.42-2.16 (m, 2H,
3
2
2
2
C-CH -CH -CH -C=O), 1.88 (s, 3H, CH ), 1.82-1.21 (m, 2H, N-CH -CH -CH -N);
2
2
2
3
2
2
2
1
3
C NMR (CDCl ): δ 170.49, 169.28, 162.04, 161.89, 159.53, 153.94, 134.97, 132.10,
3
125.87, 115.15, 114.67, 114.45, 43.38, 33.88, 32.69, 31.88, 24.98, 22.78, 16.85,
+
12.34; HRMS (ESI) calcd for C H ClFN O ([M+H] ) 406.1328, found 406.1331.
2
0
21
3
3
3-(2'-chloro-phenyl)-4-(2
or
3,5-dimethyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane)-5-methyl-isoxazolecarboxamide 4
1
(S25). Yield: 33%; m.p. 223.3-224.5℃; H NMR (CDCl ): δ 7.56-7.39 (m, 4H,
3
Ar-H), 4.34-4.32 (m, 1H, N-CH-CH -N), 3.45-3.11 (m, 2H, N-CH-CH -N), 3.04-3.01
2
2
(m, 2H, C-CH -CH -CH -C=O), 2.58 (s, 3H, CH ), 2.38-1.89 (m, 2H,
2
2
2
3
C-CH -CH -CH -C=O), 1.71 (s, 3H, CH ), 1.15 (s, 3H, CH ), 1.48-1.17 (m, 2H,
2
2
2
3
3
1
3
C-CH -CH -CH -C=O); C NMR (CDCl ): δ 169.07, 168.16, 161.12, 158.95,
2
2
2
3
132.82, 131.60, 131.50, 130.25, 127.82, 127.36, 114.56, 78.78, 51.49, 49.53, 33.30,
+
29.91, 24.43, 20.37, 16.85, 12.26; HRMS (ESI) calcd for C H ClN O ([M+H] )
2
0
22
3
3
388.1422, found 388.1428.
3-(2',6'-dichloro-phenyl)-4-(2
or
3,5-dimethyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane
1
alkyl)-5-methyl-isoxazolecarboxamide 4 (S26). Yield: 38%; m.p. 187.3-189.3℃; H
NMR (CDCl ): δ 7.50-7.40 (m, 3H, Ar-H), 4.03-4.00 (m, 1H, N-CH-CH -N),
3
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3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
24
3.81-3.15 (m, 3H, N-CH-CH -N), 2.69-2.64 (m, 2H, C-CH -CH -CH -C=O), 2.63 (s,
2 2 2 2
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
3H, CH ), 2.62-1.62 (m, 4H, C-CH -CH -CH -C=O), 1.41 (s, 3H, CH ), 0.99-1.01 (d,
3 2 2 2 3
1
3
J=8, 3H, CH ); C NMR (CDCl ): δ 170.47, 168.38, 159.67, 155.51, 136.29, 135.45,
3
3
131.84, 131.84, 128.70, 128.45, 127.26, 115.12, 78.84, 52.82, 46.24, 30.01, 30.09,
+
21.66, 17.56, 12.09; HRMS (ESI) calcd for C H Cl N O ([M+H] ) 422.1033, found
2
0
21
2
3
3
422.1035.
3-(2'-fluoro-6'-chloro-phenyl)-4-(2
or
3,5-dimethyl-9-oxa-1,4-diazabicyclo[4.3.0]nonane)-5-methyl-isoxazole carboxamide
1
4 (S27). Yield: 21%; m.p. 226.5-227.7℃; H NMR (CDCl ): δ 7.44-7.14 (m, 3H,
3
Ar-H), 4.35-4.34 (m, 1H, N-CH-CH -N), 3.68-3.19 (m, 2H, N-CH-CH -N), 3.01-2.98
2
2
(m, 2H, C-CH -CH -CH -C=O), 2.60 (s, 3H, CH ), 2.47-1.84 (m, 2H,
2
2
2
3
C-CH -CH -CH -C=O), 1.44-1.25 (m, 2H, C-CH -CH -CH -C=O), 1.68 (s, 3H, CH ),
2
2
2
2
2
2
3
1.23 (s, 3H, CH3); 13C NMR (CDCl ): δ 169.05, 161.94, 160.72, 159.42, 153.74,
3
134.78, 132.32, 126.04, 116.75, 114.84, 114.68, 78.91, 51.46, 50.01, 33.28, 29.77,
+
24.13, 20.32, 16.74, 12.44; HRMS (ESI) calcd for C H ClFN O ([M+H] )
2
0
21
3
3
406.1328, found 406.1331.
3-phenyl-4-(3,3,6-trimethyl-10-oxa-1,5-diazabicyclo[4.4.0]decane)-5-methyl-iso
1
xazole formamide 4 (S28). Yield: 79%; m.p. 178.8-179.5℃; H NMR (CDCl ): δ
3
7.70-7.44 (m, 5H, Ar-H), 4.42-2.63 (m, 2H, N-CH -CH -N), 2.49 (s, 3H, CH ),
2
2
3
2.43-2.31 (m, 2H, C-CH -CH -CH -C=O), 2.04-1.94 (m, 2H, N-CH -CH -N),
2
2
2
2
2
2.01-1.91 (m, 4H, C-CH -CH -CH -C=O), 2.05 (s, 3H, CH ), 0.89-0.39 (m, 6H, CH );
2
2
2
3
3
1
3
C NMR (CDCl ): δ 169.09, 130.44, 129.12, 129.10, 129.04, 128.53, 127.55, 127.49,
3
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365
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113.50, 54.47, 54.44, 54.38, 54.35, 46.00, 33.63, 33.63, 32.20, 26.64, 25.71, 17.11,
+
11.76, 11.71; HRMS (ESI) calcd for C H N O ([M+H] ) 382.2125, found
2
2
27
3
3
382.2123.
3-(2'-chloro-phenyl)-4-(3,3,6-trimethyl-10-oxa-1,5-diazabicyclo[4.4.0]decane)-5
1
-methyl-isoxazolecarboxamide 4 (S29). Yield: 65%; m.p. 111.5-113.5℃; H NMR
(CDCl ): δ 7.54-7.35 (m, 4H, Ar-H), 4.43-2.92 (m, 2H, N-CH -CH -N), 2.55 (s, 3H,
3
2
2
CH ), 2.52-1.99 (m, 6H, C-CH -CH -CH -C=O), 1.95 (s, 3H, CH ), 1.69-1.50 (m, 2H,
3
2
2
2
3
1
3
N-CH -CH -N), 0.86-0.83 (d, J=12, 6H, CH ); C NMR (CDCl ): δ 169.41, 162.66,
2
2
3
3
158.21, 133.40, 131.37, 131.37, 130.38, 130.38, 127.92, 127.03, 127.03, 115.05,
55.07, 45.84, 33.30, 32.46, 32.06, 26.67, 25.87, 25.25, 16.95, 11.87; HRMS (ESI)
+
calcd for C H ClN O ([M+H] ) 416.1735, found 416.1732.
2
2
26
3
3
3-(2',6'-dichloro-phenyl)-4-(3,3,6-trimethyl-10-oxa-1,5-diazabicyclo[4.4.0]decan
1
e alkyl)-5-methyl-isoxazolecarboxamide 4 (S30). Yield: 72%; m.p. 177.2-178.6℃; H
NMR (CDCl ): δ 7.48-7.37 (m, 3H, Ar-H), 4.46-3.08 (m, 2H, N-CH -CH -N), 2.59 (s,
3
2
2
3H, CH ), 2.56-2.08 (m, 6H, C-CH -CH -CH -C=O), 1.95 (s, 3H, CH ), 1.64-1.39 (m,
3
2
2
2
3
1
3
2H, N-CH -CH -N), 0.95-0.89 (d, J=24, 6H, CH ); C NMR (CDCl ): δ 170.15,
2
2
3
3
169.58, 162.18, 155.92, 136.07, 135.57, 131.77, 131.77, 128.73, 128.27, 127.46,
115.34, 76.43, 55.35, 45.83, 33.30, 32.33, 26.94, 26.02, 25.81, 16.91, 12.08; HRMS
+
(ESI) calcd for C H Cl N O ([M+H] ) 450.1346, found 450.1348.
2
2
25
2
3
3
3-(2'-fluoro-6'-chloro-phenyl)-4-(3,3,6-trimethyl-10-oxa-1,5-diazabicyclo[4.4.0]
decane)-5-methyl-isoxazole carboxamide 4 (S31). Yield: 54%; m.p. 120.9-121.9℃;
1
H NMR (CDCl ): δ 7.48-7.12 (m, 3H, Ar-H), 4.47-3.00 (m, 2H, N-CH -CH -N),
3
2
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3
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3
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2.58 (s, 3H, CH ), 2.55-2.00 (m, 6H, C-CH -CH -CH -C=O), 1.95 (s, 3H, CH ),
3 2 2 2 3
1
3
69
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1.66-1.43 (m, 2H, N-CH -CH -N), 0.94-0.87 (d, J=28, 6H, CH ); C NMR (CDCl ):
2 2 3 3
δ 169.53, 162.22, 159.49, 153.31, 135.33, 132.26, 125.93, 125.89, 117.08, 116.90,
115.43, 114.37, 55.20, 45.83, 33.21, 32.57, 32.27, 26.75, 25.97, 25.42, 16.91, 12.03;
+
HRMS (ESI) calcd for C H ClFN O ([M+H] ) 434.1641, found 434.1638.
2
2
25
3
3
X-ray Crystal Structure of Compound 4 (S14)
A crystal of compound 4 (S14), with dimensions of 0.15 mm × 0.12 mm × 0.10
mm, was measured at 293 K on a Rigaku R-AXIS-RAPID area detector
diffractometer (Japan) with graphite-monochromated Mo-Kα radiation (λ=0.71073
Å). θmax=28.318; 22323 measured reflections; 5167 independent reflections
(R =0.0227). The structure was solved by direct methods using SHELXS 97 and
int
2
refined with SHELXL 97. Full-matrix least-squares refinement based on F using the
2
2
2
weight of ω=1/[σ (F )+(0.0702P) +0.0736P] gave final values of R =0.0585,
o
1
3
3
ωR =0.1696 with I >2σ(I), Δρ =1.053 e Å and Δρ =-0.493e Å . Crystallographic
2
max
min
data has been deposited at the Cambridge Crystallographic Data Centre as
supplementary publication number 1912712.
Biological Assay
Maize seeds (Kennian 1, Shuangji Seed Industry Co., Ltd., Jilin) were moistened
with warm water for 30 min, and then soaked with 0.6% carbendazim about 30 min.
The seeds were immersed in a solution comprising a mixture of the title compounds
(10 mg/kg) at 26.5 °C for 12 h, and then germinated in an incubator for 24 h. The
injury recovery rate (IRR) of the growth indices (root length, plant height, root and
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plant fresh weights) were calculated after treated with nicosulfuron for 7 d. IRR was
3
3
91
92
calculated as follows:
Treated with safener and nicosulfuron - Treated with nicosulfuron
Injury Recovery Rate (%) 
 100%
Contrast - Treated with nicosulfuron
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
GST Enzyme Extraction and Assay in vivo
The GST was extracted and determined as described by Jablnkai.36 All test
procedures were carried out at 0-4°C. To measure the GST activity, 200 mg frozen
maize seedling tissue was ground into powder under liquid nitrogen in 1 mL of
enzyme extracting solution. The homogenate was centrifuged for 20 minutes, and the
supernatant was taken. 1 mM CDNB and 1 mM GSH were added to the enzyme to be
measured. The reaction mixture was measured through spectrophotometry at 340 nm
for 6 minutes. The absorbance of the crude enzyme solution at 595 nm was
determined by Coomassie Brilliant Blue method.37
Statistical Analysis
Each reported value represents the mean±standard deviation (SD) of data from at
least three replicates of each assay. For the determinations of growth indexes, three
different plant batches were repeated. Statistical analyses were conducted in SPSS 20
(IBM Corp., Armonk, NY, USA). Significant differences between treatment means
were identified by Duncan’s tests at p < 0.05.
Docking Study
The docking method was based on the reported method in references.32,35 The
three-dimensional structure of compound 4 (S15) was generated in the Sketch module
of SYBYL-X 2.0. Gasteiger-Huckel charges were calculated and the molecule was
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4
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optimized. The crystal structure of acetolactate synthase (ALS) was adopted from the
Protein Data Bank (PDB ID 3EA4). Docking modeling used the “CDOCKER”
method in Accelrys Discovery Studio 3.0. Before docking, the CHARMm force field
was applied to the protein structure and water and certain co-crystallized small
molecules were removed. The active site for the docking studies was determined with
a subset region 13.0 Å from the center of the known ligand. The Top Hits was set to
13
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15
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19
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22
23
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3
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100, the remaining parameters were used “CDOCKER” as the default value. The
binding energy of the small molecule-receptor protein complex was used as an
evaluation index, with the largest negative representation of the most stable
conformation. Based on the molecular docking results, the essential amino acids in the
binding pocket were then confirmed by site-directed mutagenesis.
RESULTS AND DISCUSSION
Chemistry
The synthetic route of compounds 4 was outlined in Scheme 2. The synthesis of
compounds 4 was performed by reacting isoxazole-4-carbonyl chloride with
diazabicyclo derivatives in CHCl for stirring 1-2 h with 21%~81% yields (Table 1).
3
As shown in Table 1, the structure of the substituent greatly affected the yields. The
yields of compounds 4 (S1)-4 (S16) which were obtained by the ethyl levulinate were
significantly better than those of compounds 4 (S17)-4 (S31) with ethyl acetobutylate
1
as the raw material. For compounds 4 (S1)-4 (S16), when the substituent R was
2
five-membered ring, the target compounds with substitution at R being -(CH ) -
2
3
showed better yields than the substituent being -(CH ) -. So the yields of compounds
2
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451
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453
454
455
4 (S5)-4 (S7) were better than others, which were 66%-74%, this may due to the
six-membered ring had a more stable structure than the five-membered ring. Notably,
3
4
the yields of compound 4 (S4), with F substituent at R and Cl substituent at R , were
the lowest among all compounds. This was because the five-membered ring was
unstable, and the more obvious steric hindrance effect caused by
1
2
electron-withdrawing group F. When the substituents R and R were the same, the
yields of compounds 4 (S9)-4 (S12) were generally higher than those of compounds 4
(S13)-4 (S16). Presumably, the six-membered ring was more stable. For dihalogen
3
substituted compounds, the yields of 4 (S12) and 4 (S16), with F atom at R and Cl
4
atom at R , were the lowest among all compounds with 39% and 62%, respectively.
This was most likely because introduction of halogen, the electron-withdrawing
groups such as F, Cl had a large steric hindrance, resulting in decreased yields.
3
4
Meanwhile, when the R and R positions were replaced by different halogen groups,
which made the structure not symmetrical and had poor stability, thus the yields were
lower.
1
Data presented in Table 1 revealed that when the substituent -(CH ) - was at R
2
3
3
4
position could find the same conclusion. Compounds with no substituents (R =R =H)
showed the highest yields. However, the yields of the compounds decreased when the
substituent was halogen. For example, the yield of compound 4 (S28) was 79%, but
compound 4 (S31) was only obtained in 54% yield. The main reason for this
phenomenon may probably because the H atom with a small steric effect, which was
favorable for the progress of the reaction. As a general trend, phenyl groups bearing
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electron-withdrawing groups (Cl; F), in the final compounds gave bad yields.
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Interestingly, when Cl atoms were placed both on the R position and R position of
benzene ring, compounds showed greatly enhanced yields. For example, the yield of
3
4
compound 4 (S30) increased to 72%. The result showed that when R position and R
position were both replaced by the same atom, the structure of the compound had
good symmetry and can improve the reaction yield.
Biological Activity and Structure-Activity Relationships (SARs).
The safener activity of the target compounds 4 were evaluated in the greenhouse
environment. All the novel compounds were evaluated for their protection of maize
(Kennian 1) in vivo against the injury of nicosulfuron at the concentration of 60
2
g.a.i/hm (Table 2). The results indicated that BAS-145138 showed better recovery
rates than isoxadifen-ethyl. Most of the target compounds showed excellent growth
index recovery, indicating the successful design of the title compounds.
As shown in Table 2 most of the synthesized compounds improved recovery rate
of growth index. Among the series, compound 4 (S15) exhibited the best activity
against nicosulfuron, even better than both two commercialized safeners BAS-145138
and isoxadifen-ethyl. The IRR of compound 4 (S15) on root length, root fresh weight,
plant height and plant fresh weight were 88.4%, 81.8%, 76.6% and 71.6%,
respectively, which verified that compound 4 (S15) was the best among all target
compounds. The substituent on benzene ring played a key role in the biological
activity. Herein, we found that different substituents exhibited diverse biological
1
2
activity. For the same substitutions of R and R , the target compounds with
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4
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4
4
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4
4
4
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substitution at R and R being Cl displayed better biological activity than those with
3
4
H at R and R positions. For example, compound 4 (S23) exhibited 2.3 times higher
recovery of root length than its corresponding compound 4 (S21). In terms of overall
biological activity, the introduction of F atom was not conducive to the increase in
safety activity. For example, compounds 4 (S4) and 4 (S8) showed lower biological
activity in the same series of compounds. In addition, it was also found that the
difference in root length injury recovery rate was more obvious in biological
3
4
indicators. For example, the IRR of root length of compound 4 (S22) (R =H; R =Cl)
3
4
was about 3.5 times that of compound 4 (S24) (R =F; R =Cl). The IRR of root length
3
4
was only 0.4% for compound 4 (S31) (R =F; R =Cl), while it was 27.2% for
3
4
compound 4 (S29) (R =H; R =Cl). The results indicated that the F atom on the
benzene ring induced the biological activity descend.
3
4
For the same substituents in the benzene ring (R and R ), the target compounds
with different substitutions of the N-substituted diazabicyclo affected the biological
1
2
activity considerably. To be specific, R and R affected the safening activities
1
significantly. The target compounds 4 with ethyl at the R position revealed a better
biological activity compared with the corresponding methyl substitution. As
anticipated, the recovery of root length of compound 4 (S28) was 84.0%, which was
better than that of compound 4 (S13) with 20.6%. Compounds with
2
3,3-dimethylpentane groups at R exhibited better biological activity than that of the
compound with an isopentane group. Replacement of propyl group by ethyl group
diminished the protective effects of compounds 4.
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00
The SAR at the R2 can be summarized as following (Scheme 3):
3,3-dimethylpentane>propyl>isopentane>ethyl. The SAR results suggested that the
structure of compound 4 (S15) was more similar to the combination of
isoxadifen-ethyl and BAS-145138, indicating that the structure-activity correlations
were useful methods for discovering bioactivity because they provided favorable
information about substituents that are necessary for the required biological activity.
GST Activity
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
GST is present in all plant tissues and plays an important role in the process of
safener detoxification. Here we aimed to investigate the specific effect of compounds
4 on GST activity, compound 4 (S15) with the best biological activity was selected for
the experimental study on detoxification mechanism, and commercialized safener
BAS-145138 was used as control. It turned out that GST activity increased obviously
in vivo (Table 3).
The GST activity of maize treated by compound 4 (S15) was similar to that of
BAS-145138. There was a significant effect on GST activity after treatment with
BAS-145138 and compound 4 (S15). The GST activity reached the maximum value
when the concentration of BAS-145138 was 5 mg/kg, and then gradually decreased. It
turned out that high concentrations were not conducive to GST activity. Similarly,
compound 4 (S15) also exhibited a negative correlation at 50 mg/kg. This is due to the
high concentration, compound 4 (S15) acted as xenobiotics against maize. When the
concentration of compound 4 (S15) was 10 mg/kg, the largest GST activity value in
the root was attained. Therefore, it can be inferred that compound 4 (S15) can induce
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540
541
542
543
the GST activity to the greatest extent, thereby enhancing its detoxification effect on
nicosulfuron in maize.
Molecular Structure Comparisons
Molecular structure comparisons provided an increased understanding of lead
compounds and demonstrated promise in drug discovery. Comparing the
physicochemical properties of compound 4 (S15), isoxadifen-ethyl and BAS-145138
(Table 4), it was observed that log p and aromatic rings (ARs) of compound 4 (S15)
were similar to that of isoxadifen-ethyl, rotatable bonds (RBs) and the
electronegativity of compound 4 (S15) were similar to BAS-145138. Moreover,
hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs) of compound 4
(S15) were similar to both kinds of safeners. According to the investigated features,
the compound 4 (S15)/safener BAS-145138 were quite similar at the molecular level.
As shown in Figure 2, the visual assessment of the molecular overlay revealed
BAS-145138 and compound 4 (S15) were perfectly aligned in common skeleton. This
indicated that, on the basis of its physicochemical properties, compound 4 (S15) and
BAS-145138 may share the similar mechanism of action. Compound 4 (S15) has
great potential for the future development of novel safener.
Molecular Docking Studies
Molecular docking is a perfect way for exploring the binding modes of two
interacting molecules.39,40 To further investigate the interaction between these
safeners and target enzyme of herbicides, molecular docking experiment was
conducted (Figure 3). The representative compound 4 (S15), the most potent
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compound of this series, revealed excellent shape complementarity between ligand
and the binding pocket. As shown in Figure 3, among all three safeners interact with
ARG-377 and TRP-574, compound 4 (S15) had the binding mode similar to that of
BAS-145138 as we expected. BAS-145138 and TRP-574 have one π-alkyl
interaction, while compound 4 (S15) has three π-alkyl interactions, which suggested
that compound 4 (S15) had a stronger binding effect and was more likely to occupy
the acetolactate synthase (ALS) binding site. The molecular docking models showed
that nicosulfuron prevented the substrate from binding to the active site by effectively
blocking the active pocket inlet.41,42 Nicosulfuron had three H-bond interactions and a
π-π interaction with ARG-377, and had a π-π interaction with TRP-574, which in turn
occupied the ALS binding site. (Figure 3D). In contrast, compound 4 (S15) only
partially blocked the entrance of the channel, and small substrate molecules could still
enter the channel and catalyzed the active site. To our delight and amazement, the
minimum distance between the oxygen atom and residue of nicosulfuron was 2.0 Å,
while the corresponding distances of compound 4 (S15) was only 1.8 Å. (Figure 4A
and 4B). The shorter the distance between the oxygen atom and the binding residue in
compound, the easier it is to combine. This result indicated that when compound 4
(S15) was combined or previously applied with nicosulfuron, it can compete with
nicosulfuron on the active site of ALS by preventing herbicide action or by reaching
the target active site. Based on docking research and combined with biometric results,
compound 4 (S15) might be as a potential candidate for novel safeners discovery.
CONCLUSIONS
45
46
47
48
49
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51
52
53
54
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57
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60
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575
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577
578
579
580
581
582
583
In conclusion, this series of novel diazabicyclo derivatives with different
substituents were designed and synthesized via intermediate derivatization methods
and fragment splicing. The bioassay showed that most of target compounds exhibited
different safening activity to sensitive maize from nicosulfuron injury. In particular,
compound 4 (S15) displayed the best activity against nicosulfuron and improved the
tolerance of maize by enhancing GST activity. The molecular structure comparisons
indicated that compound 4 (S15) was quite similar to safener BAS-145138 at
molecular level. Molecular docking revealed that the promising safener efficacy may
be attributed to the competition between compound 4 (S15) and nicosulfuron in the
active pocket of ALS. The current work will provide a new insight for the design of
new safeners in the future.
ACKNOWLEDGEMENT
This work was supported by the National Nature Science Foundation of China
(Nos. 31572042 and 31801784) and the Natural Science Foundation of Heilongjiang
Province (No. ZD2017002). The authors thank Prof. Jia-Zhong Li of Lanzhou
University for assistance with the molecular docking analyses.
Supporting Information - Brief descriptions in non-sentence format listing the
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contents of the files supplied as Supporting Information.
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