Design, synthesis and herbicidal activity of 5-cyclopropyl-N-phenylisoxazole-4-carboxamides

Article abstract of DOI:10.1016/j.molstruc.2020.128628

4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are a type of important herbicides, and they cause bleaching symptoms by indirectly inhibiting the biosynthesis of carotenoids. In this study, thirty isoxazolamide compounds were designed based on the structure of Isoxaflutole, a commercial HPPD herbicide. Starting from 1,1-dimethoxy-N,N-dimethyl-methanamine and methyl 3-cyclopropyl-3-oxo-propanoate, the title compounds were readily prepared and their structures were determined by MS and NMR analysis. In Petri dish tests, most of the title compounds showed strong inhibitory effect on the root and stem growth of both monocotyledon and dicotyledon weeds, and it was clearly different from the symptoms caused by HPPD inhibitors. However, several of them, especially I-17, showed characteristic bleaching symptoms of HPPD herbicides and good post-emergence herbicidal activity on tested weeds in glasshouse assay. These compounds are prodrugs, and compounds undergo conversion to the active entity diketonitrile (DKN) in plant and soil. The result of molecular docking analysis revealed that the DKN moiety of I-17 excellently binds to the active sites of HPPD. The 1,3-diketone can form bidentate interaction with Fe^II, and the benzene ring can form π-π interaction with Phe 360 and Phe 403. These results indicated that the title compounds bears other herbicidal mechanism except for HPPD inhibitor. Therefore, a lead compound for the discovery of novel multi-target herbicides is provided.

Full text of DOI:10.1016/j.molstruc.2020.128628

Journal Pre-proof
Design, synthesis and herbicidal activity of 5-cyclopropyl-N-phenylisoxazole-4-
carboxamides
Xinlin Sun, Zhenmeng Ji, Shaopeng Wei, Zhiqin Ji
PII:
S0022-2860(20)30953-4
DOI:
https://doi.org/10.1016/j.molstruc.2020.128628
MOLSTR 128628
Reference:
To appear in: Journal of Molecular Structure
Received Date: 9 January 2020
Revised Date: 3 June 2020
Accepted Date: 4 June 2020
Please cite this article as: X. Sun, Z. Ji, S. Wei, Z. Ji, Design, synthesis and herbicidal activity of 5-
cyclopropyl-N-phenylisoxazole-4-carboxamides, Journal of Molecular Structure (2020), doi: https://
doi.org/10.1016/j.molstruc.2020.128628.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

CRediT author statement
Xinli Sun: Methodology, Software
Zhenmeng Ji: Visualization, Investigation.
Shaopeng Wei: Data curation, Writing- Original draft preparation.
Zhiqin Ji: Conceptualization, Validation, Writing- Reviewing and Editing.

Graphic Abstract
amide was inserted
N
group for binding to target
retained moiety for activity
O
O
O
Me
O
Me
H
O
O
S
O
S
O
in vivo
N
R
O
O
CN
DKN (active entity)
N
CF3
CF3
I-01~I-26
designed structures in this work
IFT (proherbicide)

1， Design, Synthesis and Herbicidal Activity of 5-cyclopropyl-N-phenylisoxazole-4-carboxamides
2，
3，
4，
Xinlin Suna,#, Zhenmeng Ji a,#, Shaopeng Weia,b, Zhiqin Jia,b,
*
aCollege of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100,China
bShaanxi Province Key Laboratory R&D on Botanical Pesticides, Yangling Shaanxi, 712100, China
5， #These authors are contributed equally to this work.
6， *Corresponding author: Dr. Zhiqin Ji
7， E-mail: jizhiqin@nwsuaf.edu.cn
8， Tel: +86-15309203829
9， Fax: +86-29-87092191
10，
11，
，
，
，
1，
，

12， Abstract: 4-Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors are a type of important
13， herbicides, and they cause bleaching symptoms by indirectly inhibiting the biosynthesis of carotenoids.
14， In this study, thirty isoxazolamide compounds were designed based on the structure of Isoxaflutole, a
15， commercial HPPD herbicide. Starting from 1,1-dimethoxy-N,N-dimethyl-methanamine and methyl
16， 3-cyclopropyl-3-oxo-propanoate, the title compounds were readily prepared and their structures were
17， determined by MS and NMR analysis. In Petri dish tests, most of the title compounds showed strong
18， inhibitory effect on the root and stem growth of both monocotyledon and dicotyledon weeds, and it was
19， clearly different from the symptoms caused by HPPD inhibitors. However, several of them, especially
20， I-17, showed characteristic bleaching symptoms of HPPD herbicides and good post-emergence
21， herbicidal activity on tested weeds in glasshouse assay. These compounds are prodrugs, and
22， compounds undergo conversion to the active entity diketonitrile (DKN) in plant and soil. The result of
23， molecular docking analysis revealed that the DKN moiety of I-17 excellently binds to the active sites
24， of HPPD. The 1,3-diketone can form bidentate interaction with FeII, and the benzene ring can form π-π
25， interaction with Phe 360 and Phe 403. These results indicated that the title compounds bears other
26， herbicidal mechanism except for HPPD inhibitor. Therefore, a lead compound for the discovery of
27， novel multi-target herbicides is provided.
28， KEYWORDS: 4-hydroxyphenylpyruvate dioxygenase; herbicidal activity; isoxazole; phenylamine
29， derivatives
30，
2，
，

31， 1. Introduction
32，
With the widely use of agro-chemicals, weeds resistance to commercial herbicides has become a
33， major concern to crop production worldwide[1]. Although numerous studies have demonstrated that
34， rational application of herbicide groups is helpful for delaying the evolution of herbicide-resistant
35， weeds, the development of herbicides with new mode of action is the eventual solution to address the
36， problem[2, 3]. 4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a relative new target for herbicides
37， discovered in 1990s[4]. In plants, HPPD catalyzes the biotransformation of 4-hydroxyphenylpyruvic
38， acid (HPPA) to homogentisic acid (HGA), which is an intermediate in the biosynthesis of
39， plastidquinone[5]. Plastoquinone is a co-factor of phytoene desaturase, and the inhibition of HPPD
40， finally results in a depletion of carotenoids and an absence of chloroplast development in emerging
41， foliar tissues, which is followed by necrosis and death[6]. Up to now, more than a dozen of HPPD
42， inhibitors such as Sulcotrione, Mesotrione, Topramezone, Pyrazolynate and others have been used in
43， the management of weeds[7-12]. HPPD herbicides exhibit high herbicidal activity against a variety of
44， broadleaf and grass weeds both in pre- and post-emergence treatments, and have low mammalian
45， toxicity. More important, only few weed species are resistant to HPPD herbicides[13, 14]. These good
46， features attract more attention from pesticide industry. Isoxaflutole (IFT) is a HPPD herbicide
47， developed by Rhône-Poulenc Agriculture Limited, and its herbicidal mechanism, root uptake and
48， translocation, as well as metabolism in soil and plants, have been well clarified in previous study[15].
49， IFT itself is a prodrug, and IFT undergoes conversion to the active entity diketonitrile (DKN) in plant
50， and soil. Although IFT is a highly effective herbicide, its complex structure results in a longer synthesis
51， route and high cost for production[16, 17]. Furthermore, the weed spectrum and crop selectivity of IFT
52， are also not perfect enough[18]. In view of its promising activity, screening novel herbicidal
3，
，

53， compounds by the modification on the structure of IFT is an interesting program. We firstly analyzed
54， the interaction between the active DKN and its target. HPPD is a non-haem FeII-containing
55， dioxyganase, the chelating 1,3-diketone moiety of the DKN is responsible for the binding to active
56， site[19]. The ortho-Me-SO2 substituted at phenyl ring provides additionally support for the interaction.
57， In our strategy, the carbonyl between phenyl ring and isoxazole is replaced with amide while retaining
58， the crucial group for the binding to target (Figure 1). The reason for this is because various structural
59， types of amides possess good herbicidal activity in previous studies [20]. We hope that this change can
60， provide herbicidal candidates with good activity and low cost.
amide was inserted
group for binding to target
retained moiety for activity
N
O
O
O
Me
O
Me
H
O
O
S
O
S
O
in vivo
N
R
O
O
CN
DKN (active entity)
N
CF3
CF3
I-01~I-26
designed structures in this work
IFT (proherbicide)
61，
62，
，
Figure. 1. Design strategy for the title compounds
63， 2. Result and discussion
64， 2.1. Synthesis
65，
The synthetic routes for I-01~I-26 are illustrated in Scheme 1. The key intermediate,
66， 5-cyclopropylisoxazole-4-carboxylic acid (3), is prepared from 1,1-dimethoxy-N,N-dimethyl-
67， methanamine (1) and methyl 3-cyclopropyl-3-oxo-propanoate (2) according to the procedure disclosed
68， in the patent[21]. The yield of final product was strongly affected by the reaction temperature in the last
69， step. We found that the optimum temperature for the reaction was 100 oC, and the yield of final product
70， was above 80%. For the preparation of 5-cyclopropylisoxazole-4-carbonyl chloride, we examined the
71， effect of temperature on the yield, and found that the yield of acyl chloride was close to 100% at room
4，
，

72， temperature. The preparation of I-01~I-26 was carried out under ice bath condition. Because the ring of
73， isoxazole is readily opened in strong alkaline conditions, the yield of final products was strongly
74， affected by the types of alkalis and adding order of reactants used in the acylation reaction. After
75， examining the effect of different conditions on the yield, we found that adding acid chloride and
76， pyridine simultaneously to the solution of phenylamines was helpful for the stability of isoxazole.
N
N
N
O
O
H
O
O
e
a,b,c
N
HO
d
Cl
O
+
R
N
O
O
O
O
O
O
3
4
I-01~I-26
2
1
77，
78，
，
o
Reagents and conditions: (a) 60 C, 20h; (b) H2NOH-HCl, H2O, MeOH, 90 min, 60 °C;(c) concentrated HCl,
AcOH, 4 h, 100 oC; (d) oxalyl chloride, CH2Cl2, 30min, room temperature; (e) substituted benzylamines, pyridine,
CH2Cl2, 30min, 0 °C.，
79，
80，
81，
82，
Scheme 1. Synthetic routes for I-01~I-26
Starting from naphthalen-1-amine, naphthalen-2-amine, pyridin-4-amine and thiazol-2-amine,
83， other analogues, II-01~II-04, were prepared by the procedure described above (Scheme 2).
N
O
N
H
Cl
N
O
R NH2
R
O
O
II-01~II-04
4
N
II-04,
R: II-01,
II-02,
II-03,
N
S
84，
85，
，
Scheme 2. Synthetic routes for II-01~II-04
86， 2.2. Herbicidal activity
87， 2.2.1. Petri dish tests and structure-activity relationship analyses
88， The herbicidal activities of I-01~I-26 and II-01~II-04 against monocotyledon weeds such as
89， Echinochloa crusgalli (EC), Digitaria sanguinalis (DS), and dicotyledon weeds such as Amaranthus
5，
，

90， retroflexus (AR), Portulaca oleracea (PO), Abutilon theophrasti (AT) and Chenopodium album (CA)
91， were evaluated by Petri dish tests as described in literature[22]. The herbicidal activity of the title
92， compounds at 100 mg/L and 10 mg/L against tested weeds are listed in Tables 1 and 2, respectively. As
93， shown in Table 1, most of the title compounds exhibited strong inhibitory effect against the root and
94， stem growth of the tested weeds. As for 5-cyclopropyl-N-phenylisoxazole-4-carboxamides, I-16~I-24
95， showed good herbicidal activity against both monocotyledon and dicotyledon weeds, and several of
96， them exhibited nearly 100% inhibition against tested weeds. I-08~I-15 had stronger activity against
97， dicotyledon weeds than monocotyledon weeds. I-01~I-07 showed weak to moderate inhibitory effect
98， against the tested weeds, and monocotyledon weeds were less sensitive to these compounds compared
99， to dicotyledon weeds. Table 2 reports the effects of the title compounds, at the dose of 10 mg/L, on the
100， root and stem growth of 6 species of weeds. The data clearly showed that I-22~I-24 had stronger
101， inhibitory effect than other phenylamine derivatives. As for other four analogues, II-04 had better
102， herbicidal activity than II-01~II-03, and its inhibitory rate on the tested weeds are equivalent to those
103， of I-22~I-24.
104，
Table 1. Inhibitory effect of the title compounds on the growth of weeds in Petri dish tests (100 mg/L)
inhibition rate (%)
No.
R
ECa
DSa
ARa
POa
ATa
CAa
root stem root stem root stem root stem root stem root stem
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
I-11
H
2-CH3
3-CH3
4-CH3
3-OCH3
4-OCH3
4-C(CH3) 3
2-F
40
40
30
40
50
40
20
40
50
80
30
30
40
40
30
50
30
30
40
40
70
40
30
50
30
50
40
30
20
50
70
80
20
30
40
20
40
40
40
20
30
60
60
40
60
40
50
60
50
50
40
60
70
100
70
50
50
60
40
50
50
60
50
60
80
60
80
70
70
60
70
60
60
70
50
80
90
100
80
70
60
80
70
60
70
60
70
90
100
70
60
70
70
80
50
70
50
80
80
90
60
60
50
60
60
50
50
40
60
80
100
80
70
40
50
70
60
60
30
50
70
90
80
60
60
70
70
70
80
3-F
100
100
90
4-F
2-Cl
6，
，

I-12
I-13
I-14
I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
II-01
II-02
II-03
II-04
3-Cl
4-Cl
50
60
50
60
50
60
100
80
60
70
60
80
90
80
90
50
70
60
30
50
90
20b
90
60
80
70
90
80
90
90
100
70
90
100
80
100
100
70
80
100
70
80
90
90
100
60
70
70
90
100
80
70
60
50
60
70
90
30
80
100 100
100 90
90
80
70
80
70
90
80
80
70
80
90
90
80
80
60
30
40
60
100
20b
80
2-Br
40
60
50
60
80
80
80
90
70
3-Br
70
80
70
80
70
100
90
80
90
3-CF3
4-CF3
4-NO2
2,4-diF
2,4-diCl
3,4-diCl
3-Cl-4-F
3-CF3-4-F
3-CF3-4-Cl
3-CF3-4-Br
2-Br-4-CF3
-
90
100
100
80
100
90
90
100
100
100
90
100
90
80
80
100
80
100
80
100
70
100
80
90
100 90
90
70
90
100
100
90
100
80
70
60
70
80
70
80
70
70
100
90
90
80
70
70
60
80
100
90
80
90
100
100
80
90
100
100
100
60
80
100
90
100
100
100
60
90
80
90
100 100
100
60
100
50
80
100
80
80
60
70
40
50
80
70
90
80
30
50
60
70
70
70
60
70
80
80
80
90
50
40
30
40
20
50
60
-
40
60
50
50
40
60
50
-
60
70
60
80
80
80
70
-
100
30
100
30
100
20b
100
80
90
30b
100
30
100
10b
70
100 90
Isoxaflutole
20
20b
30
Butachlor
100
90
80
80
80
80
70
105，
106，
107，
aAbbreviations: EC for Echinochloa crusgalli; DS for Digitaria sanguinalis; AR for Amaranthus retroflexus; PO
b
for Portulaca oleracea , AT for Abutilon theophrasti and CA for Chenopodium album. Exhibit bleaching
symptoms.
108，
Table 2. Inhibitory effect of the title compounds on the growth of weeds in Petri dish tests (10 mg/L)
inhibition rate (%)
No.
R
ECa
DSa
ARa
POa
ATa
CAa
root stem root stem root stem root stem root stem root stem
I-01
I-02
I-03
I-04
I-05
I-06
I-07
I-08
I-09
I-10
I-11
I-12
I-13
I-14
H
2-CH3
3-CH3
4-CH3
3-OCH3
4-OCH3
4-C(CH3) 3
2-F
0
0
0
10
0
0
10
20
20
30
30
20
20
30
30
40
30
20
40
30
20
30
30
20
30
20
20
30
30
40
20
20
40
30
10
30
20
20
10
20
10
40
30
40
20
30
40
20
30
10
20
30
30
40
20
50
50
40
30
40
60
20
20
10
20
30
20
20
30
40
40
50
30
20
50
30
30
20
30
30
20
30
20
20
40
50
30
20
40
30
30
10
20
30
20
40
10
30
30
50
30
20
50
30
10
10
0
0
10
10
0
20
30
30
20
20
10
20
30
40
30
30
40
20
10
10
0
0
10
10
10
0
10
0
0
10
0
10
10
20
20
30
30
20
30
20
0
10
30
30
20
30
20
30
20
40
30
40
30
30
20
30
10
20
40
30
30
20
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
7，
，

I-15
I-16
I-17
I-18
I-19
I-20
I-21
I-22
I-23
I-24
I-25
I-26
II-01
II-02
II-03
II-04
3-Br
3-CF3
30
20
40
20
20
30
40
70
70
70
20
30
20
20
30
60
30
30
30
50
30
30
20
50
70
80
90
30
20
10
0
10
30
20
30
30
20
30
60
80
70
30
40
10
20
30
60
30
90
0
40
40
60
30
30
40
40
60
70
70
40
30
10
20
20
50
20
80
50
40
60
30
40
50
40
50
70
80
50
40
20
20
30
60
30b
30
40
50
40
30
50
40
70
80
70
40
30
20
30
40
60
30
80
30
50
60
50
40
60
50
80
70
80
50
40
20
20
30
60
10b
30
70
60
40
40
50
60
80
80
80
20
20
30
40
40
70
30
80
40
60
70
50
40
60
60
70
60
80
30
30
20
30
40
60
20b
80
40
50
50
40
40
60
50
80
80
80
50
40
10
30
40
60
30
50
60
60
60
50
60
60
70
60
70
50
30
20
20
40
70
20b
80
20
10
30
40
20
40
70
80
60
30
30
20
20
30
70
20b
4-CF3
4-NO2
2,4-diF
2,4-diCl
3,4-diCl
3-Cl-4-F
3-CF3-4-F
3-CF3-4-Cl
3-CF3-4-Br
2-Br-4-CF3
-
-
-
20
70
20b
-
Isoxaflutole
70
Butachlor
100
90
100
80
70
109，
110，
111，
aAbbreviations: EC for Echinochloa crusgalli; DS for Digitaria sanguinalis; AR for Amaranthus retroflexus; PO
b
for Portulaca oleracea , AT for Abutilon theophrasti and CA for Chenopodium album. Exhibit bleaching
symptoms.
，
112，
Based on the analysis of chemical structures of I-01~I-26, it was found that their herbicidal
113， activity was significantly affected by the types of substituents introduced at the benzene ring. Firstly,
114， we examined the influence of the electronic effect and position of the substituents on the activity.
115， Generally, the herbicidal activities of I-08~I-26 were stronger than those of I-02~I-07. It indicates that
116， introducing electron withdrawing groups at benzene ring is more beneficial for the herbicidal activity
117， than electron donating groups. I-08, I-11, I-14 showed weaker activity than other halogenated
118， compounds, which implies that the halogen atoms substituted at meta- and para-positions of benzene
119， ring are better for the activity than at ortho-position. The herbicidal activity of II-03 on dicotyledon
120， weeds was comparable to those of I-01, but it showed stronger inhibition than I-01 on monocotyledon
121， weeds. It reveals that the replacement of benzene with pyridine broaden the weed spectrum. Finally,
8，
，

122， II-04 showed better activity than most of other title compounds, which implies that five-membered
123， heterocyclic moiety might be a more promising structure in the follow-up study.
124，
Surprisingly, the weeds treated by the title compounds showed clearly different symptoms from
125， those treated by IFT in Petri dish tests. IFT caused characteristic bleaching symptoms, but only slightly
126， inhibitory effect on the growth of weeds was observed. As shown in Tables 1 and 2, the inhibitory rate
127， of IFT on the growth of weeds ranged from 10% to 30%. Generally, all the synthesized compounds
128， only showed inhibition against tested weeds, and some of them have better herbicidal activity than
129， Butachlor against broadleaf weeds. Unfortunately, we have not observed bleaching symptoms in the
130， weeds treated by the title compounds.
131， 2.2.2. Pre- and post-emergence herbicidal activity and structure-activity relationship analyses
132，
Pre- and post-emergence herbicidal activity of the title compounds against E. crusgalli and A.
133， theophrasti were evaluated in green house tests according to a procedure reported previously[22]. All
134， title compounds showed no pre-emergence herbicidal activity on the weeds at the application rate of
135， 150 g ai/ha. However, I-02~I-04, I-08~I-10, I-17, II-03 and II-04 exhibited post-emergence herbicidal
136， activity against both E. crusgalli and A. theophrasti, and the results are illustrated as Figure 2. The
137， inhibition rate of I-08, I-10 and I-17 on E. crusgalli were above 70%. I-17 and II-04 had excellent
138， herbicidal activity on A. theophrasti, and the inhibition rate were around 80%. The herbicidal activity
139， of I-17 on E. crusgalli was comparable to Mesotrione. The SAR revealed that the size and steric
140， hindrance of the substituted groups at benzene ring play important roles, and introducing small groups
141， such as CH3 and F are beneficial to the activity. Furthermore, the difference in activity between II-04
142， and II-03 remind us that five-member ring may be more promising than six-member ring. Because the
143， activities of II-08~II-10 were stronger than those of II-02~II-04, it can be concluded that the
9，
，

144， introduction of electron withdrawing groups at benzene ring is beneficial for the herbicidal activity.
145， The effect of the substituted positions at benzene ring on the activity is insignificant.
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
EC AT
146，
147，
148，
EC for E. crusgalli; AT for A. theophrasti
Figure 2. Post-emergence herbicidal activity of several compounds on E. crusgalli and A. theophrasti
149，
More importantly, the active title compounds caused characteristic bleaching symptoms similar to
150， that of Mesotrione. This result indicated that the title compounds take effect as HPPD inhibitors in
151， post-emergence treatments.
152， 2.3. Molecular modeling
153，
The interaction between I-17 with HPPD was studied via molecular docking method by using
154， Discovery Studio 4.0. Because I-17 is a prodrug, and the DKN formed by the ring opening of isoxazole
155， is the active entity. The 3D structure of the DKN derivative of I-17 was constructed by using
156， ChemBiodraw ultra 10.0 (Figure 3A). The compound was then opened in Discovery studio 4.0 and
157， energy minimization was carried out by CHARMm force field using ligand partial charge method CFF
158， (Consistent Force Field)[23]. The original ligand, 1TFZ, was firstly docked into AtHPPD to check the
159， docking reliability. Consequently, the DKN derivative of I-17 was docked into the same active site, and
160， twenty conformations were obtained through CDOCKER[24]. Figure 3B shows the spatial binding of
161， the molecule in the active cavity. It was found that the molecule does not face repulsions with the
10，
，

162， amino acid backbone and thus are able to occupy the binding pocket. As illustrated in Figure 3C, the
163， DKN moiety of I-17 form bidentate interaction with FeII, and the benzene ring can form π-π interaction
164， with Phe 360 and Phe 403.
165，
The results of molecular docking are partly consist with that of IFT. The two compounds have in
166， common is the chelating of the 1,3-diketone moiety with FeII in the active site of HPPD. The difference
167， is that the ortho-MeSO2 in the structure of IFT additionally supports the interaction by forming an
168， H-bridge to a molecule of water, which interacts with FeII at the same time (Figure D), whereas the π-π
169， interaction between benzene ring with Phe 360 and Phe 403 provides the additional supports for the
170， interaction in I-17.
171，
We can get more information from the 2D diagram(Figure E). Except for the two binding forces
172， mentioned above, the hydrogen atom on the amino group and the nitrogen atom on the cyano group of
173， I-17 form hydrogen bond with Phe398 and Asn261, respectively.
174，
A
CF3
O
N
O
H
N
I-17
prodrug
ring opening
in vivo
CF3
O
O
N
H
CN
Diketonitrile metabolite
active entity
175，
， ， ， ， ， ， ， ， ， ，
， ， ， ， ， ，
11，
，

176，
，
177，
178，
，
(A) Active DKN formed via ring opening. (B) I-17 was found to fully embed into the active pocket. (C) Simulated
179，
180，
181，
182，
183，
binding mode of I-17 with AtHPPD. The key residues in the active site are shown in blue sticks, and FeII is shown
as a cyan sphere, I-17 is shown in green sticks. (D) DKN derivative of IFT and its molecular interaction with
HPPD[19]. (E) 2D diagram simulated binding mode of I-17 with AtHPPD.
Figure 3. The receptor-ligand interaction of I-17 with the HPPD active site
In general, the objective of this study is to screen high activity and low cost herbicidal compounds,
184， and the results are partly accomplished the expected goal. Firstly, the title compound is easier to
185， synthesize than IFT. Secondly, the title compounds exhibited two distinct symptoms in Petri dish test
186， and post-emergence herbicidal activity experiment, which implies that they are multi-target herbicides.
187， This characteristic might be beneficial to delay the emergence of weeds resistance to the title
12，
，

188， compounds. The disadvantage is that the two herbicidal mechanism can not take effect at the same time.
189， We suspect that this phenomena may be caused by differences in target site sensitivity, uptake and
190， translocation effects or metabolism of the chemicals in plants.
191， 3. Conclusions
192，
In summary, thirty compounds were designed based on the chemical structure of Isoxaflutole. The
193， title compounds were prepared via a simple procedure, and their structures were determined by NMR
194， and MS analysis. All the compounds were evaluated for their herbicidal activities against a panel of
195， weeds, and the SAR of them was analyzed. The post-emergence herbicidal activity of I-17 against
196， E.crusgalli and A. theophrasti is comparable to that of Mesotrione. Furthermore, the title compounds
197， bear two distinct herbicidal mechanism. In spite of several weaknesses, the research is conducive to the
198， development of novel herbicides.
199， 4. Experimental section
200， 4.1. General chemistry methods
201，
All chemical reagents were commercially available and used without further purification.
202， Precoated silica gel plates (Si60 GF254, Merck Chemical Co. Ltd) were used to monitor the progress of
203， reaction. Purification of target compounds was performed on silica gel column chromatography
1
204， (200~300 mesh, Qingdao Marine Chemical Co. Ltd, China). H and 13C NMR spectra were recorded
205， on a Bruker Avance III-500 NMR spectrometer, and the residual solvent signals were used as reference.
206， Mass spectral analysis was carried out on a Finnegan LCQ Advantage MAX LC/MS spectrometer
207， equipped with an ESI source. IR spectra were recorded on a Nicolet FT-IR 750 spectrometer (Thermo
208， Fisher Scientific, Waltham, MA, USA). The melting points were conducted on a WRS-3 apparatus, and
209， are uncorrected.
13，
，

210， 4.2. Synthesis of 5-Cyclopropylisoxazole-4-carboxylic acid
211， 1,1-Dimethoxy-N,N-dimethylmethanamine (1, 24g, 0.2 mol) and methyl 3-cyclopropyl-3-oxo-
212， propanoate (2, 28g, 0.2 mol) were mixed and heated for 20 h at 60 °C. The obtained yellow oil was
213， firstly dissolved in methanol (200 mL) and water (100 mL), and then hydroxylamine hydrochloride
214， (14g, 0.2 mol) was added. The solvents were evaporated under vacuum after the mixture was heated for
215， 90 min at 60 °C. The residue was dissolved in the mixture of acetic acid (100 mL) and concentrated
216， HCl (100mL) and refluxed for 4h. The reaction mixture was diluted with water (500 mL) and extracted
217， with ethyl acetate (200 mL×3). The organic layer was combined and washed with brine, and then dried
218， with anhydrous sodium sulfate. Ethyl acetate was evaporated under vacuum. The residue was subjected
219， to a silica gel column and eluted with the mixture of ethyl acetate and petroleum ether at the ratio of
°
1
220， 1:3 (v/v) to afford 3. Compound 3 was obtained as white solid (yield 80.13%), mp, 163-165 C; H
221， NMR (500 MHz, Chloroform-d): δ 8.53 (s, 1H), 2.91 (m, 1H), 1.38 (m, 2H), 1.3 (m, 2H). 13C NMR
222， (126 MHz, Chloroform-d): δ 179.91, 167.63, 150.62, 108.36, 10.86, 8.87. ESI-MS: m/z 152, [M-H]-.
223， 4.3. General procedure for the synthesis of 5-cyclopropyl-N-phenylisoxazole-4-carboxamides
224， (I-01~I-26
225， To a solution of substituted phenylamine (1mmol) in 20 mL of anhydrous dichloromethane, 4 (1
226， mmol) and pyridine (1 mmol) previously dissolved in 5 mL of anhydrous dichloromethane were added
ꢀII-01~II-04)
0
0
227， dropwise at 0 C. The mixture was continuously stirred at 0 C for 30 min. After completion of the
228， reaction based on TLC detection, the solution was washed with water (30 mL), saturated sodium
229， chloride solution (30 mL) and brine (30 mL), successively. The organic layer was dried over anhydrous
230， sodium sulfate. After the solvent was removed under vacuum, the residue was subjected to silica gel
231， column and eluted by ethyl acetate/petroleum ether (1:5) afford I-01.
14，
，

232， 4.3.1. 5-Cyclopropyl-N-phenylisoxazole-4-carboxamide(I-01)
°
233，
Yield 92.10%; yellow solid; mp, 89.3-91.4 C; IR (KBr, cm−1) ν 3444 (-NH-), 1649 (C=O),
1
234， 1533-1443 (C=C), 1092 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.52 (s, 1H), 8.30 (s, 1H),
235， 7.60-7.51 (m, 2H), 7.34 (t, J = 7.9 Hz, 2H), 7.20-7.14 (m, 1H), 2.86 (tt, J = 8.4, 5.1 Hz, 1H), 1.28 (tt, J
236， = 6.0, 3.3 Hz, 2H), 1.25-1.18 (m, 2H). 13C NMR (126 MHz, Chloroform-d): δ 177.05, 160.03, 148.47,
237， 137.28, 129.06, 125.04, 121.00, 111.96, 10.07, 8.61. ESI-MS: m/z 229, [M+H]+.
238， 4.3.2. 5-Cyclopropyl-N-o-tolylisoxazole-4-carboxamide (I-02)
°
239，
Yield 78.51%; Yellow solid; m.p, 125.9-127.1 C; IR (KBr, cm−1) ν 3443 (-NH-), 1639 (C=O),
240， 1543-1453 (C=C), 1122 (C-O);1H NMR (500 MHz, Chloroform-d): δ 8.44 (s, 1H), 7.81 (s, 1H), 7.40 (s,
241， 1H), 7.29 (d, J = 7.5 Hz, 2H), 7.22-7.16 (m, 1H), 2.83 (tt, J = 8.5, 5.1 Hz, 1H), 2.36 (s, 3H), 1.38-1.33
242， (m, 2H), 1.28 (dt, J = 8.9, 3.5 Hz, 2H). ESI-MS: m/z 243, [M+H] +.
243， 4.3.3. 5-Cyclopropyl-N-m-tolylisoxazole-4-carboxamide (I-03)
°
244，
Yield: 82.64%; white solid; m.p, 119.6-122.0 C; IR (KBr, cm−1) ν 3444 (-NH-), 1637 (C=O),
245， 1546-1449 (C=C), 1103-1078 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.47 (s, 1H), 7.62 (s, 1H),
246， 7.47 (s, 1H), 7.37 (dt, J = 8.1, 2.6 Hz, 1H), 7.30-7.25 (m, 1H), 7.03 (dd, J = 7.6, 3.9 Hz, 1H), 2.86 (m,
247， 1H), 2.40 (q, J = 4.1 Hz, 3H), 1.37-1.24 (m, 4H). ESI-MS: m/z 243, [M+H] +.
248， 4.3.4. 5-Cyclopropyl-N-p-tolylisoxazole-4-carboxamide(I-04)
°
249，
Yield: 80.35%; Yellow solid; m.p, 117.1-120.0 C; IR (KBr, cm−1) ν 3445 (-NH-), 1634 (C=O),
250， 1556-1451 (C=C), 1108-1088 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.47 (s, 2H), 7.55-7.42 (m,
251， 3H), 7.19 (q, J = 8.3 Hz, 3H), 2.86 (tt, J = 8.3, 5.1 Hz, 1H), 2.41-2.34 (m, 3H), 1.33 (m, 2H), 1.29-1.23
252， (m, 2H). ESI-MS: m/z 243, [M+H] +.
253， 4.3.5. 5-Cyclopropyl-N-(3-methoxyphenyl)isoxazole-4-carboxamide (I-05)
254，
Yield: 87.21%; Yellow wax; IR (KBr, cm−1) ν 3445 (-NH-), 1647 (C=O), 1556-1451 (C=C),
255， 1104 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.55 (d, J = 9.4 Hz, 2H), 7.32-7.23 (m, 1H), 7.18 (t,
256， J = 8.1 Hz, 1H), 7.06 (dd, J = 8.0, 2.0 Hz, 1H), 6.68 (dd, J = 8.3, 2.5 Hz, 1H), 3.74 (s, 3H), 2.86 (tt, J =
257， 8.4, 5.1 Hz, 1H), 1.28-1.22 (m, 2H), 1.22-1.16 (m, 2H). ESI-MS: m/z 259, [M+H]+.
258， 4.3.6. 5-Cyclopropyl-N-(4-methoxyphenyl)isoxazole-4-carboxamide (I-06)
°
259，
Yield: 77.52%; Reddish brown solid; m.p, 122.6-125.1 C; IR (KBr, cm−1) ν 3444 (-NH-), 1640
1
260， (C=O), 1545-1453 (C=C), 1107-1071 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.48 (s, 1H), 7.58
15，
，

261， (s, 0H), 7.47 (q, J = 8.8 Hz, 2H), 6.97-6.85 (m, 2H), 3.86-3.82 (m, 3H), 2.87 (tt, J = 8.5, 5.1 Hz, 1H),
262， 1.35-1.19 (m, 4H). ESI-MS: m/z 259, [M+H] +.
263， 4.3.7. N-(4-tert-butylphenyl)-5-cyclopropylisoxazole-4-carboxamide (I-07)
°
264，
Yield: 86.27%; yellow solid; m.p, 121.8-123.4 C; IR (KBr, cm−1) ν 3443 (-NH-), 1629 (C=O),
1
265， 1548-1451 (C=C), 1100-1088 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.48 (s, 1H), 7.64 (d, J =
266， 51.0 Hz, 1H), 7.55-7.49 (m, 2H), 7.44-7.39 (m, 2H), 2.86 (m, 1H), 1.36 (d, J = 2.0 Hz, 9H), 1.35-1.25
267， (m, 4H). ESI-MS: m/z 285, [M+H] +.
268， 4.3.8. 5-Cyclopropyl-N-(2-fluorophenyl)isoxazole-4-carboxamide (I-08)
°
269，
Yield: 89.43%; yellow solid; m.p, 113.2-115.1 C; IR (KBr, cm−1) ν 3445 (-NH-), 1636 (C=O),
270， 1552-1467 (C=C), 1086 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.53 (s, 1H), 8.38 (t, J = 7.8 Hz,
271， 1H), 7.89 (s, 1H), 7.24-7.12 (m, 3H), 2.77 (tt, J = 8.2, 5.2 Hz, 1H), 1.38-1.28 (m, 4H). ESI-MS: m/z
272， 247, [M+H] +.
273， 4.3.9. 5-Cyclopropyl-N-(3-fluorophenyl)isoxazole-4-carboxamide (I-09)
°
274，
Yield: 89.43%; white solid; m.p, 113.2-115.1 C; IR (KBr, cm−1) ν 3445 (-NH-), 1641 (C=O),
1
275， 1552-1459 (C=C), 1113-1082 (C-O); H NMR (500 MHz, Chloroform-d) δ 8.50 (d, J = 8.1 Hz, 1H),
276， 7.94 (d, J = 115.6 Hz, 1H), 7.54 (m, 1H), 7.37-7.26 (m, 2H), 6.89 (qd, J = 8.0, 2.6 Hz, 1H), 2.86 (td, J =
277， 8.4, 4.1 Hz, 1H), 1.33 (m, 2H), 1.28 (m, 2H). ESI-MS: m/z 247, [M+H] +.
278， 4.3.10. 5-Cyclopropyl-N-(4-fluorophenyl)isoxazole-4-carboxamide (I-10)
°
279，
Yield: 99.59%; yellow solid; m.p, 77.5-79.6 C; IR (KBr, cm−1) ν 3445 (-NH-), 1649 (C=O),
1
280， 1527-1453 (C=C), 1126-1073 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.49 (d, J = 6.4 Hz, 1H),
281， 7.90 (d, J = 130.3 Hz, 1H), 7.53 (tq, J = 8.2, 4.3, 3.7 Hz, 2H), 7.07 (dt, J = 16.7, 8.7 Hz, 2H), 2.91-2.80
282， (m, 1H), 1.32 (m, 2H), 1.26 (m, 2H). ESI-MS: m/z 247, [M+H] +.
283， 4.3.11. N-(2-chlorophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-11)
°
284，
Yield: 93.65%; yellow solid; m.p, 99.4-101.4 C; IR (KBr, cm−1) ν 3445 (-NH-), 1644 (C=O),
1
285， 1541-1458 (C=C), 1113 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.52 (s, 1H), 8.47 (td, J = 7.6,
286， 7.0, 1.5 Hz, 1H), 8.12 (s, 1H), 7.45 (m, 1H), 7.36 (tq, J = 8.1, 1.5 Hz, 1H), 7.17-7.09 (m, 1H), 2.80 (m,
287， 1H), 1.36 (dq, J = 6.3, 3.3 Hz, 2H), 1.31 (m, 2H). ESI-MS: m/z 263, [M+H] +.
288， 4.3.12. N-(3-chlorophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-12)
16，
，

°
289，
Yield: 95.42%; Reddish brown solid; m.p, 114.6-117.0 C; IR (KBr, cm−1) ν 3445 (-NH-), 1646
290， (C=O), 1537-1432 (C=C), 1114-1079 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.54-8.48 (m, 1H),
291， 7.99 (d, J = 155.8 Hz, 1H), 7.74-7.65 (m, 1H), 7.46 (dt, J = 8.5, 2.7 Hz, 1H), 7.33-7.25 (m, 1H), 7.16
292， (m, 1H), 2.87 (m, 1H), 1.36-1.31 (m, 2H), 1.28 (m, 2H). ESI-MS: m/z 263, [M+H] +.
293， 4.3.13. N-(4-chlorophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-13)
°
294，
Yield: 93.28%; white solid; m.p, 79.7-81.8 C; IR (KBr, cm−1) ν 3445 (-NH-), 1647 (C=O),
295， 1539-1435 (C=C), 1098-1067 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.49 (s, 1H), 7.84-7.74 (m,
296， 1H), 7.59-7.53 (m, 2H), 7.38-7.33 (m, 2H), 2.86 (tt, J = 8.3, 5.1 Hz, 1H), 1.34 (m, 2H), 1.28 (m, 2H).
297， ESI-MS: m/z 263, [M+H] +.
298， 4.3.14. N-(2-bromophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-14)
°
299，
Yield: 96.40%; Reddish brown solid; m.p, 107.8-109.2 C; IR (KBr, cm−1) ν 3445 (-NH-), 1649
300， (C=O), 1543-1453 (C=C), 1082 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.53 (s, 1H), 8.49 (dd, J
301， = 8.3, 1.5 Hz, 1H), 8.09 (s, 1H), 7.63 (dd, J = 8.0, 1.4 Hz, 1H), 7.44-7.38 (m, 1H), 7.08 (td, J = 7.8, 1.6
302， Hz, 1H), 2.85 (tt, J = 8.5, 5.1 Hz, 1H), 1.39 (dt, J = 5.5, 3.2 Hz, 2H), 1.33 (dt, J = 8.5, 3.1 Hz, 2H).
303， ESI-MS: m/z 307, [M+H] +.
304， 4.3.15. N-(3-bromophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-15)
°
305，
Yield: 53.92%; Reddish brown solid; m.p, 112.2-124.3 C; IR (KBr, cm−1) ν 3443 (-NH-), 1644
306， (C=O), 1541-1438 (C=C), 1097 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.50 (d, J = 6.9 Hz, 1H),
307， 7.95-7.81 (m, 1H), 7.82-7.70 (m, 1H), 7.52 (dd, J = 8.2, 2.2 Hz, 1H), 7.35-7.31 (m, 1H), 7.28-7.23 (m,
308， 1H), 2.86 (tt, J = 8.1, 5.1 Hz, 1H), 1.34 (dq, J = 5.6, 2.5 Hz, 2H), 1.29 (m, 2H). ESI-MS: m/z 307,
309， [M+H] +.
310， 4.3.16. 5-Cyclopropyl-N-(3-(trifluoromethyl)phenyl)isoxazole-4-carboxamide (I-16)
°
311，
Yield: 67.57%; Reddish brown solid; m.p, 66.9-69.0 C; IR (KBr, cm−1) ν 3445 (-NH-), 1639
312， (C=O), 1553-1423 (C=C), 1103-1087 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.57-8.50 (m, 1H),
313， 7.94-7.87 (m, 1H), 7.81 (dt, J = 8.6, 2.5 Hz, 1H), 7.54-7.40 (m, 2H), 2.88 (tt, J = 8.3, 5.1 Hz, 1H), 1.35
314， (m, 2H), 1.32-1.25 (m, 1H). ESI-MS: m/z 297, [M+H] +.
315， 4.3.17. 5-Cyclopropyl-N-(4-(trifluoromethyl)phenyl)isoxazole-4-carboxamide (I-17)
°
316，
Yield: 95.63%; White solid; m.p, 121.5-124.1 C; IR (KBr, cm−1) ν 3445 (-NH-), 1641 (C=O),
1
317， 1540-1453 (C=C), 1107-1077 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.51 (d, J = 3.6 Hz, 1H),
17，
，

318， 7.95-7.62 (m, 5H), 2.87 (tt, J = 8.4, 5.1 Hz, 1H), 1.37 (qt, J = 5.1, 1.9 Hz, 2H), 1.34- 1.28 (m, 2H).
319， ESI-MS: m/z 297, [M+H] +.
320， 4.3.18. 5-Cyclopropyl-N-(4-(trifluoromethyl)phenyl)isoxazole-4-carboxamide (I-18)
°
321，
Yield: 50.36%; Yellow solid; m.p, 117.0-120.2 C; IR (KBr, cm−1) ν 3443 (-NH-), 1649 (C=O),
322， 1543-1459 (C=C), 1108-1076 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.57 (s, 1H), 8.33-8.27 (m,
323， 2H), 7.89-7.83 (m, 2H), 6.70-6.63 (m, 1H), 2.90 (tt, J = 8.4, 5.1 Hz, 1H), 1.38 (dt, J = 5.4, 3.0 Hz, 2H),
324， 1.34 (dt, J = 8.3, 2.9 Hz, 2H). ESI-MS: m/z 275, [M+H] +.
325， 4.3.19. 5-Cyclopropyl-N-(2,4-difluorophenyl)isoxazole-4-carboxamide (I-19)
°
326，
Yield: 81.44%; white solid; m.p,87.5-89.3 C; IR (KBr, cm−1) ν 3445 (-NH-), 1647 (C=O),
327， 1543-1440 (C=C), 1124-1067 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.52 (s, 1H), 8.36-8.28 (m,
328， 1H), 7.75 (d, J = 6.3 Hz, 1H), 6.96 (m, 2H), 2.76 (tt, J = 8.3, 5.2 Hz, 1H), 1.38-1.29 (m, 4H). ESI-MS:
329， m/z 265, [M+H] +.
330， 4.3.20. 5-Cyclopropyl-N-(2,4-dichlorophenyl)isoxazole-4-carboxamide (I-20)
°
331，
Yield: 80.36%; white solid; m.p, 85.2-87.4 C; IR (KBr, cm−1) ν 3445 (-NH-), 1641 (C=O),
1
332， 1547-1431 (C=C), 1104 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.51 (s, 1H), 8.45 (dd, J = 8.9,
333， 5.6 Hz, 1H), 8.05 (s, 1H), 7.47 (t, J = 2.6 Hz, 1H), 7.34 (dt, J = 8.9, 2.7 Hz, 1H), 2.79 (tt, J = 8.3, 5.1
334， Hz, 1H), 1.40-1.35 (m, 2H), 1.32 (m, 2H). ESI-MS: m/z 297, [M+H] +.
335， 4.3.21. 5-Cyclopropyl-N-(3,4-dichlorophenyl)isoxazole-4-carboxamide (I-21)
°
336，
Yield: 96.28%; Brown solid; m.p, 88.4-90.3 C; IR (KBr, cm−1) ν 3443 (-NH-), 1643 (C=O),
337， 1530-1434 (C=C), 1122-1089 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.51 (s, 1H), 8.02 (s, 1H),
338， 7.82 (d, J = 2.3 Hz, 1H), 7.47-7.40 (m, 2H), 2.87 (tt, J = 8.3, 5.2 Hz, 1H), 1.34 (dt, J = 5.5, 2.9 Hz, 2H),
339， 1.30 (dt, J = 8.3, 3.0 Hz, 2H). ESI-MS: m/z 297, [M+H] +.
340， 4.3.22. N-(3-chloro-4-fluorophenyl)-5-cyclopropylisoxazole-4-carboxamide (I-22)
°
341，
Yield: 97.65%; yellow solid; m.p, 83.7-85.2 C; IR (KBr, cm−1) ν 3442 (-NH-), 1642 (C=O),
342， 1540-1440 (C=C), 1110 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.51 (d, J = 8.0 Hz, 1H), 8.08 (d,
343， J = 106.3 Hz, 1H), 7.73 (m, 1H), 7.42 (m, 1H), 7.13 (dt, J = 11.8, 8.7 Hz, 1H), 2.87 (td, J = 8.4, 4.2 Hz,
344， 1H), 1.32 (m, 2H), 1.27 (m, 1H). ESI-MS: m/z 281, [M+H] +.
345， 4.3.23. 5-Cyclopropyl-N-(4-fluoro-3-(trifluoromethyl)phenyl)isoxazole-4-carboxamide (I-23)
18，
，

°
346，
Yield: 79.62%; white solid; m.p, 88.7-90.6 C; IR (KBr, cm−1) ν 3443 (-NH-), 1632 (C=O),
347， 1527-1430 (C=C), 1100-1079 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.49 (s, 1H), 7.87 (dd, J =
348， 6.1, 2.7 Hz, 1H), 7.82 (dt, J = 8.8, 3.5 Hz, 1H), 7.76 (s, 1H), 7.25 (t, J = 9.3 Hz, 1H), 2.88 (tt, J = 8.3,
349， 5.1 Hz, 1H), 1.36 (dt, J = 5.7, 3.1 Hz, 2H), 1.32 (dt, J = 8.6, 3.0 Hz, 2H). ESI-MS: m/z 315, [M+H] +.
350， 4.3.24. N-(4-chloro-3-(trifluoromethyl)phenyl)-5-cyclopropylisoxazole-4-carboxamide (I-24)
°
351，
Yield: 60.60%; white solid; m.p, 127.2-129.5 C; IR (KBr, cm−1) ν 3445 (-NH-), 1645 (C=O),
352， 1542-1443 (C=C), 1117-1087 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.51 (s, 1H), 7.96-7.87 (m,
353， 2H), 7.82 (dd, J = 8.7, 2.6 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 2.88 (tt, J = 8.3, 5.2 Hz, 1H), 1.35 (dt, J =
354， 5.5, 2.9 Hz, 2H), 1.31 (m, 2H). ESI-MS: m/z 331, [M+H] +.
355， 4.3.25. N-(4-bromo-3-methylphenyl)-5-cyclopropylisoxazole-4-carboxamide (I-25)
°
356，
Yield: 62.50%; Yellow solid; m.p, 97.3-99.8 C; IR (KBr, cm−1) ν 3443 (-NH-), 1635 (C=O),
1
357， 1537-1434 (C=C), 1083 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.48 (s, 1H), 7.75 (s, 1H),
358， 7.55-7.50 (m, 2H), 7.28 (dd, J = 8.6, 2.7 Hz, 1H), 2.86 (tt, J = 8.4, 5.1 Hz, 1H), 2.42 (s, 3H), 1.34 (tt, J
359， = 5.8, 2.6 Hz, 2H), 1.29 (m, 2H). ESI-MS: m/z 321, [M+H] +.
360， 4.3.26. N-(2-bromo-4-(trifluoromethyl)phenyl)-5-cyclopropylisoxazole-4-carboxamide (I-26)
°
361，
Yield: 53.48%; white solid; m.p, 110.2-111.5 C; IR (KBr, cm−1) ν 3442 (-NH-), 1642 (C=O),
362， 1540-1443 (C=C), 1112 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.69 (d, J = 8.7 Hz, 1H), 8.53 (s,
363， 1H), 8.22 (s, 1H), 7.90 (d, J = 2.0 Hz, 1H), 7.67 (dd, J = 8.7, 2.0 Hz, 1H), 2.85 (tt, J = 8.4, 5.1 Hz, 1H),
364， 1.41 (tt, J = 5.9, 2.8 Hz, 2H), 1.36 (m, 2H). ESI-MS: m/z 375, [M+H] +.
365， 4.3.27. 5-Cyclopropyl-N-(naphthalen-1-yl)isoxazole-4-carboxamide (II-01)
°
366，
Yield: 85.64%; Reddish brown solid; m.p, 115.8-117.2 C; IR (KBr, cm−1) ν 3413 (-NH-), 1600
367， (C=O), 1537-1437 (C=C), 1130-1100 (C-O); 1H NMR (500 MHz, DMSO-d6): δ 10.25 (s, 1H), 9.20 (s,
368， 1H), 8.07-8.02 (m, 1H), 8.00-7.95 (m, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.63 (d, J = 7.3 Hz, 1H), 7.57 (qd,
369， J = 7.8, 7.3, 4.6 Hz, 3H), 3.00-2.92 (m, 1H), 1.22 (m, 2H), 1.17 (dt, J = 5.3, 3.0 Hz, 2H). 13C NMR
370， (126 MHz, DMSO-d6): δ 176.99, 160.68, 149.92, 133.45, 129.46, 128.58, 126.88, 126.63, 126.56,
371， 126.02, 124.28, 123.78, 111.96, 10.28, 8.80. ESI-MS: m/z 279, [M+H] +.
372， 4.3.28. 5-Cyclopropyl-N-(naphthalen-2-yl)isoxazole-4-carboxamide (II-02)
°
373，
Yield: 79.13%; white solid; m.p, 78.2-81.0 C; IR (KBr, cm−1) ν 3444 (-NH-), 1600 (C=O),
1
374， 1537-1430 (C=C), 1126 (C-O); H NMR (500 MHz, Chloroform-d): δ 8.86 (s, 1H), 8.61 (s, 1H), 8.13
19，
，

375， (d, J = 2.2 Hz, 1H), 7.78-7.73 (m, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.69-7.64 (m, 1H), 7.52 (dd, J = 8.8,
376， 2.2 Hz, 1H), 7.47-7.40 (m, 2H), 2.89 (tt, J = 8.4, 5.1 Hz, 1H), 1.24 (tt, J = 6.0, 3.5 Hz, 2H), 1.20-1.13
377， (m, 2H). 13C NMR (126 MHz, Chloroform-d) δ 177.11, 160.08, 148.20, 134.48, 133.32, 130.60, 128.44,
378， 127.29, 126.31, 125.11, 120.44, 118.01, 111.57, 9.94 , 8.41. ESI-MS: m/z 279, [M+H] +.
379， 4.3.29. 5-Cyclopropyl-N-(pyridin-4-yl)isoxazole-4-carboxamide(II-03)
380，
Yield: 70.35%; yellow solid; m.p, 89.8-91.8°C; IR (KBr, cm−1) ν 3447 (-NH-), 1633 (C=O),
381， 1504-1414 (C=C), 1123 (C-O); 1H NMR (500 MHz, Chloroform-d): δ 8.59 (d, J = 5.4 Hz, 2H), 8.54 (s,
382， 1H), 7.98 (s, 1H), 7.65-7.58 (m, 2H), 2.92-2.85 (m, 1H), 1.37 (dt, J = 5.4, 3.0 Hz, 2H), 1.33 (dt, J = 8.6,
383， 3.1 Hz, 2H). ESI-MS: m/z 230, [M+H] +.
384， 4.3.30. 5-Cyclopropyl-N-(thiazol-2-yl)isoxazole-4-carboxamide (II-04)
°
385，
Yield: 55.32%; white solid; m.p, 157.9-159.9 C; IR (KBr, cm−1) ν 3445 (-NH-), 1596 (C=O),
386， 1443 (C=C), 1128-1076 (C-O); 1H NMR (500 MHz, Chloroform-d):δ 8.61 (s, 1H), 7.37 (d, J = 3.6 Hz,
387， 1H), 7.31 (s, 1H), 7.09 (d, J = 3.6 Hz, 1H), 3.04 (tt, J = 8.4, 5.1 Hz, 1H), 1.40 (tt, J = 6.0, 3.6 Hz, 2H),
388， 1.32 (tt, J = 6.3, 2.5 Hz, 2H). 13C NMR (126 MHz, Chloroform-d): δ 179.22, 159.86, 159.39, 148.31,
389， 136.55, 114.24, 110.15, 10.77, 8.99. ESI-MS: m/z 236, [M+H] +.
390， 4.4. Herbicidal activity assay
391， 4.4.1. Petri dish tests
392，
Seeds of monocotyledon weeds such as Echinochloa crusgalli and Digitaria sanguinalis, and
393， dicotyledon weeds such as Amaranthus retroflexus, Portulaca oleracea and Abutilon theophrasti
394， were collected from campus of Northwest A&F University in 2017. The germinated seeds were placed
395， in Petri dishes (90 mm diameter) containing two layers of filter paper, and impregnated with 5 mL of
396， the solutions of tested compounds at 100 mg/L and 10 mg/L, respectively. Water was used as blank
397， control, isoxaflutole and butachlor were used as positive control. Then the Petri dishes were placed in a
20，
，

°
398， light incubator at 25 C, light intensity of 300 Lux. The growth inhibition rate of root and stem were
399， observed after 5 days.
400， 4.4.2 Pre- and post-emergence herbicidal activity
401，
Pre- and post-emergence herbicidal activities of the title compounds against E. crusgalli and A.
402， theophrasti were evaluated in glasshouse according to a procedure reported previously[22]. All tested
403， compounds were firstly dissolved in DMSO to the concentration of 100 g/L. The solutions were then
404， diluted with 0.1% Tween-80 to desired concentrations before using. The soil used was a mixed soil
405， (33.3% garden soil and 66.7% seedling substrate). Plastic pots with an inner diameter of 7.5 cm were
406， filled with the above soil to three-fourths of their height. About 20 seeds of the tested weeds were sown
407， in the pot and covered with soil to a thickness of 0.2 cm and grown at temperatures from 15 to 30 °C in
408， a glasshouse. For pre-emergence treatments, the diluted test solutions (150 g ai/ha) were sprayed on the
409， surface of soil 24 h after the seeds were sown. For post-emergence treatment, the weeds were treated
410， with the solutions of tested compounds (150 g ai/ha) at three-leaf stage. The seedlings treated with the
411， diluted solution of DMSO and Tween-80 were used as the control groups. Each treatment was
412， performed in 4 replicates. IFT were used as positive control. After 15 days of treatment, the herbicidal
413， activity was evaluated visually.
414， 4.5. Molecular docking protocol
415，
The 3D structure of the DKN derivative of I-17, the representative compound, was constructed by
416， using ChemBiodraw ultra 10.0. It was then opened in Discovery studio 4.0 and energy minimization
417， was carried out by CHARMm force field using ligand partial charge method CFF (Consistent Force
418， Field)[23].Minimization was carried out until energy gradient of 0.01 was reached. The CDOCKER
419， was used for docking of all compounds. A representative AtHPPD co-crystallized with NTBC (PDB ID:
21，
，

420， 1TFZ) was taken from the PDB data bank. The water molecules were deleted and hydrogen atoms were
421， added. Finally protein was refined with CHARMm in DS 4.0 at physiological pH. To validate the
422， docking reliability, co-crystalized ligand (DAS869) was first re-docked to the binding site of HPPD.
423， Consequently, the DKN derivative of I-17 was docked into the same active site, and twenty
424， conformations of it were obtained through CDOCKER[24]. The conformation with lowest energy was
425， selected as the most probable binding conformation. PYMOL was used to analyze the binding mode.
426， Declare of Competing Interest
427，
The authors declare that they have no conflict of interest.
428， Acknowledgements
429，
This work was financially supported by the National Natural Science Foundation of China (No.，
430， 31872011).
431， Appendix A. Supplementary material
432，
Supplementary data to this article can be found online.
433， References
434， [1] A. Forouzesh, E. Zand, S. Soufizadeh, S. Samadi Foroushani, E. Barriuso, Classification of
435， herbicides according to chemical family for weed resistance management strategies - an update, Weed
436， Research 55(4) (2015) 334-358.
437， [2] H. Abul, B. David, P. Terry, D. Harmohinder, Resistance of Wild Radish (Raphanus raphanistrum)
438， to Acetolactate Synthase-Inhibiting Herbicides in the Western Australia Wheat Belt, Weed Technology
439， (2001).
440， [3] L.A. Schmidt, R.E. Talbert, M. Mcclelland, Management of Acetolactate Synthase (ALS)–Resistant
441， Common Cocklebur (Xanthium strumarium) in Soybean1, Weed Technology 18 (2009) 665-674.
22，
，

442， [4] A. Schulz, O. Ort, P. Beyer, H. Kleinig, SC-0051, a 2-benzoyl-cyclohexane-1,3-dione bleaching
443， herbicide, is a potent inhibitor of the enzyme p-hydroxyphenylpyruvate dioxygenase, Febs Letters
444， 318(2) (1993) 162-166.
445， [5] C.S. Wu, J.-L. Huang, Y.-S. Sun, D.-Y. Yang, Mode of Action of 4-Hydroxyphenylpyruvate
446， Dioxygenase Inhibition by Triketone-type Inhibitors, Journal of Medicinal Chemistry 45(11) (2002)
447， 2222-2228.
448， [6] G. Meazza, B.E. Scheffler, M.R. Tellez, A.M. Rimando, J.G. Romagni, S.O. Duke, D. Nanayakkara,
449， I.A. Khan, E.A. Abourashed, F.E. Dayan, The inhibitory activity of natural products on plant
450， p-hydroxyphenylpyruvate dioxygenase, Phytochemistry 60(3) (2002) 281-288.
451， [7] R. Beaudegnies, A.J.F. Edmunds, T.E.M. Fraser, R.G. Hall, T.R. Hawkes, G. Mitchell, J. Schaetzer,
452， S. Wendeborn, J. Wibley, Herbicidal 4-hydroxyphenylpyruvate dioxygenase inhibitors—A review of
453， the triketone chemistry story from a Syngenta perspective, Bioorganic & Medicinal Chemistry 17(12)
454， (2009) 4134-4152.
455， [8] F. Ndikuryayo, B. Moosavi, W.-C. Yang, G.-F. Yang, 4-Hydroxyphenylpyruvate Dioxygenase
456， Inhibitors: From Chemical Biology to Agrochemicals, Journal of Agricultural and Food Chemistry
457， 65(39) (2017) 8523-8537.
458， [9] Y. Fu, S.-Q. Zhang, Y.-X. Liu, J.-Y. Wang, F.Y.J.I. Crops, Products, Design, synthesis, SAR and
459， molecular docking of novel green niacin-triketone HPPD inhibitor, 137 (2019) 566-575.
460， [10] B. He, J. Dong, H.-Y. Lin, M.-Y. Wang, X.-K. Li, B.-F. Zheng, Q. Chen, G.-F. Hao, W.-C. Yang,
461， G.-F.
Yang,
Pyrazole–Isoindoline-1,3-dione
Hybrid:
A
Promising
Scaffold
for
462， 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors, Journal of Agricultural and Food Chemistry 67(39)
463， (2019) 10844-10852.
23，
，

464， [11] X.-H. Liu, L. Qiao, Z.-W. Zhai, P.-P. Cai, C.L. Cantrell, C.-X. Tan, J.-Q. Weng, L. Han, H.-K. Wu,
465， Novel 4-pyrazole carboxamide derivatives containing flexible chain motif: design, synthesis and
466， antifungal activity, 75(11) (2019) 2892-2900.
467， [12] Y. Fu, Y.-X. Liu, K.-H. Yi, M.-Q. Li, J.-Z. Li, F. Ye, Quantitative Structure Activity Relationship
468， Studies and Molecular Dynamics Simulations of 2-(Aryloxyacetyl)cyclohexane-1,3-Diones Derivatives
469， as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors, 7(556) (2019).
470， [13] N.E. Hausman, S. Singh, P.J. Tranel, D.E. Riechers, S.S. Kaundun, N.D. Polge, D.A. Thomas, A.G.
471， Hager, Resistance to HPPD-inhibiting herbicides in a population of waterhemp (Amaranthus
472， tuberculatus) from Illinois, United States, pest management science 67(3) (2011) 258-261.
473， [14] N. Sridevi, G.A. S., W.P. S., T.C. R., P.D. E., R. Jeroen, J. Mithila, Physiological and Molecular
474， Characterization of Hydroxyphenylpyruvate Dioxygenase (HPPD)-inhibitor Resistance in Palmer
475， Amaranth (Amaranthus palmeri S.Wats.), Frontiers in Plant Science 8 (2017).
476， [15] K.E. Pallett, S.M. Cramp, J.P. Little, P. Veerasekaran, A.J. Crudace, A.E. Slater, Isoxaflutole: the
477， background to its discovery and the basis of its herbicidal properties, Pest Management Science 57(2)
478， (2001) 133-142.
479， [16] P.A. Cain, S.M. Cramp, G.M. Little, B.M. Luscombe.Preparation of benzoylisoxazoles as
480， herbicides.EP527036A1.1993
481， [17] D. Bernard, A. Viauvy.Process for the preparation of 2-(alkylthio)benzoic acids and esters from
482， their corresponding 2-nitro or 2-halo precursors and alkylthiols or their salts.WO9902489A1.1999
483， [18] H. Ahrens, G. Lange, T. Mueller, C. Rosinger, L. Willms, A. van Almsick,
484， 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors in Combination with Safeners: Solutions for
485， Modern and Sustainable Agriculture, Angewandte Chemie-International Edition 52(36) (2013)
24，
，

486， 9388-9398.
487， [19] P. Jeschke, Propesticides and their use as agrochemicals, Pest Management Science 72(2) (2016)
488， 210-225.
489， [20] K. Matsunari, H. Sugiyama, H. Sadohara, K. Motojima, Synthesis and herbicidal activity of
490， N-alkyl-N-(substituted benzyl)-4-halo-2-alkenamides, Journal of Pesticide Science 24(1) (1999) 1-6.
491， [21] C.H. Fotsch, M.D. Bartberger, M. Haraldsson, D. St. Jean, L. Johansson, M. Nilsson, L.
492， Sutin.Preparation of azole compounds as 11β-HSD1 inhibitors.WO2008011453A2.2008
493， [22] Y.-y. Cao, D.-j. Mao, W.-w. Wang, X.-h. Du, Kresoxim-methyl Derivatives: Synthesis and
494， Herbicidal Activities of (Pyridinylphenoxymethylene)phenyl Methoxyiminoacetates, Journal of
495， Agricultural and Food Chemistry 65(30) (2017) 6114-6121.
496， [23] J. Penna-Coutinho, W.A. Cortopassi, A.A. Oliveira, T.C. Costa Franca, A.U. Krettli, Antimalarial
497， Activity of Potential Inhibitors of Plasmodium falciparum Lactate Dehydrogenase Enzyme Selected by
498， Docking Studies, Plos One 6(7) (2011) e21237.
499， [24] A. Nafees, A. Sirajudheen, T.H.T.J.F.i. Chemistry, Docking Based 3D-QSAR Study of Tricyclic
500， Guanidine Analogues of Batzelladine K As Anti-Malarial Agents, Frontiers in chemistry 5 (2017) 36-.
501， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ， ，
25，
，

Highlights
ꢀ
ꢀ
ꢀ
ꢀ
Strong inhibitory effect on the growth of weeds were observed in Petri dish tests
Characteristic bleaching effect was observed in post-emergence treatments
Excellent binding with HPPD was observed in molecular docking analysis
A potential lead compound for multi-target herbicides

Declaration of interests
×
ꢀ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: