Synthesis, insecticidal activities, and structure-activity relationship of phenylpyrazole derivatives containing a fluoro-substituted benzene moiety

Article abstract of DOI:10.1021/acs.jafc.9b08090

Fluorinated organic compounds represent a growing and important family of commercial chemicals. Introduction of fluorine into active ingredients has become an effective way to develop modern crop protection products. Given the particular properties of fluorine and high efficiency and selectivity of diamide insecticides, we designed and synthesized 27 anthranilic diamides analogues containing fluoro-sustituted phenylpyrazole. A preliminary bioassay indicated that most target compounds exhibited good biological activity against Mythimna separata and Plutella xylostella. Compound IIIf containing a 2,4,6-trifluoro-substituted benzene ring showed 43% insecticidal activity against M. separata at 0.1 mg L-1, while the control chlorantraniliprole was 36%. The activity of IIIe against P. xylostella at 10-5 mg L-1 was 94%, compared with that of the control being 70%. Thus, introduction of fluorine into diamide insecticides was useful for increasing activity. Insect electrophysiology studies showed that the calcium concentration in the nerve cells of third M. separata larvae was elevated by IIIf, which further confirmed that ryanodine receptor (RyR) was its potential target.

Full text of DOI:10.1021/acs.jafc.9b08090

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Functional Structure/Activity Relationships
Synthesis, Insecticidal Activities and Structure-Activity Relationship of
Phenylpyrazole Derivatives Containing Fluoro-substituted Benzene Moiety
Yangyang Zhao, Li Gao, Huangong Li, Pengwei Sun, Fanfei Meng, Yan Zhang, Yongtao
Xie, Binqiao Sun, Sha Zhou, Yi Ma, Lixia Xiong, Na Yang, Yuxin Li, and Zheng-Ming Li
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b08090 • Publication Date (Web): 14 Sep 2020
Downloaded from pubs.acs.org on September 15, 2020
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Page 1 of 33
Journal of Agricultural and Food Chemistry
1 Synthesis, Insecticidal Activities and Structure-Activity
2 Relationship of Phenylpyrazole Derivatives Containing
3 Fluoro-substituted Benzene Moiety
4
5
Yangyang Zhao,† Li Gao,† Huangong Li,† Pengwei Sun,† Fanfei Meng,† Yan Zhang,† Yongtao
Xie,§ Binqiao Sun,† Sha Zhou,† Yi Ma,† Lixia Xiong,† Na Yang,† Yuxin Li *,† and Zhengming Li*,†
6
7
†State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry,
Nankai University, Tianjin 300071, China
8
9
§ Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences,
Nanyang Technological University (NTU), Singapore 637371, Singapore
10
11
ABSTRACT
Fluorinated organic compounds represent a growing and important
12 family of commercial chemicals. The introduction of fluorine into active
13 ingredients has become an effective way to develop modern crop
14 protection products. Given the particular properties of fluorine, and high
15 efficiency and selectivity of diamide insecticides, we have designed and
16 synthesized 27 anthranilic diamides analogues containing fluro-sustituted
17 phenylpyrazole. Preliminary bioassay indicated that most target
18 compounds exhibited good biological activity against Mythimna separata
19 and Plutella xylostella. Compound IIIf containing 2, 4, 6-trifluoro
20 substituted benzene ring showed 43% insecticidal activity against M.
21 separata at 0.1 mg L-1, while the control chlorantraniliprole was 36%.
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22 The activity of IIIe against P. xylostella at 10-5 mg L−1 was 94%,
23 compared with that of the control being 70%. So the introduction of
24 fluorine into diamide insecticides was useful for increasing activity.
25 Insect electrophysiology studies showed that calcium concentration in the
26 nerve cells of third M. separata larvae was elevated by IIIf, which further
27 confirmed that ryanodine receptor (RyR) was its potential target.
28 KEYWORDS: insecticidal activity, fluorine substitution, phenylpyrazole,
29 electrophysiology study, SAR
30 INTRODUCTION
31
To counteract the emerging insect resistance and stricter environmental
32 regulations, it has become an urgent task to develop insecticides with low
33 toxicity, high activity and novel action modes.1-3 Insect ryanodine
34 receptor (RyR) controlling the release of calcium ion, is a type of ligand-
35 regulated calcium channel. It has attracted more attention in the search for
36 new crop protection compounds.4 Presently, five diamides insecticides
37 targeted at RyRs possessing high mammal safety and excellent
38 insecticidal activity have been commercialized: flubendiamide,
39 chlorantraniliprole, cyantraniliprole, cyclaniliprole and tetrachlorantrani
40 -liprole. 5-6
41
Research interests are growing in fluorinated organic compounds in
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Journal of Agricultural and Food Chemistry
42 crop protection and medicinal chemistry. The fluorine atom, with unique
43 properties such as electronegativity, size and electrostatic interactions,
44 has been applied with great success in modern agricultural chemistry.7-11
45 In year 2010-2016, since approximately 52% of the commercial
46 pesticides contained fluorine atoms, people had followed with strong
47 interest their biological activity in the crop protection.12 For example,
48 flupyradifurone (Figure 1), a new butenolide insecticide, was discovered
49 by introducing a second fluorine to the lead compound.13-14 The
50 development of tefluthrin and flubendiamide (Figure 1) also provided
51 significant illustrations of the introduction of fluorine into active
52 compounds.15, 16 When fluorine is introduced into active ingredients, new
53 chemical structures may produce unpredictable biological activity. It
54 means that introduction of fluorine could cause either an increase or a
55 decrease in the activity of a compound depending on target interaction,
56 their changed mode of action, physical and chemical properties, and
57 metabolism.11 Therefore, the introduction of fluorine into active
58 ingredients has become an effective strategy in the quest for
59 agrochemicals.
60
In the development of chlorantraniliprole, the pyridinepyrazole moiety
61 exhibits special features in terms of insecticidal activity.17-19 However,
62 compared with pyridine, the structure of benzene ring is prone to more
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63 diversified electrophilic substitution.20-21 On the basis of this
64 characteristics, we have reported N-substituted anthranilic diamides.
65 Several compounds showed promising insecticidal activities against
66 Lepidoptera, which manifested that their insecticidal activities were
67 retained after the pyridine structure was substituted by the benzene.22
68 Motivated by the aforementioned reports, we developed a new design
69 strategy (Figure 1) of introducing fluorine into benzene ring of
70 phenylpyrazole. Herein, a series of fluoro-substituted anthranilic
71 diamides analogues were designed and synthesized. Their bioactivities
72 were evaluated and the mode of action was clarified. Additionally, how
73 the different status of fluorine affect their biological results was also
74 studied and discussed.
75 MATERIALS AND METHODS
76
Instruments and Materials. All solvents were received from
77 commercial sources without further purification, and commercially
78 available reagents were used without further processing. The melting
79 points were measured by an X-4 binocular microscope melting point
80 apparatus and uncorrected. 200-300 mesh silica gel was used for
81 separation of compounds. NMR spectra data were recorded utilizing a
82 Bruker AV 400 spectrometer and processed with MestReNova 9.0.
83 Coupling constants (J) were given in hertz and multiplicities were
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Journal of Agricultural and Food Chemistry
84 abbreviated as follows: s = singlet; d = doublet; t = triplet; q = quartet; m
85 = multiple, etc. High resolution mass spectra (HRMS) data were given by
86 a Varian QFT-ESI instrument. The single-crytal structure of compound
87 was determined by X-ray crystallography and described with Olex2
88 version 1.2.9. The bioassay data in this paper were carried out with Data
89 Processing System 9.50 (DPS). In the calcium ion imaging experiment,
90 the data were analyzed using Spss Inc. version 17.0 and Graphpad Prism
91 version 7.0. The CoMFA analyses were carried out using a SYBYL 6.9
92 software.
O
S
F
I
HN
O
O
F
O
O
F
O
O
H
N
F
N
O
F
Cl
F
F
F
Cl
N
Me
F
F
F
F
Me
F
Me
F
F
F
flubendiamide
flupyradifurone
tefluthrin
R2
R2
NH
O
NH
O
Br
Br
Br
O
NH
H
N
H
H
N
F
N
N
N
R1
N
N
N
N
R1
O
Cl
O
O
Cl
N
R
R= F or Cl
B
chloranthraniliprole
A
93
94
Figure 1. Structural formulas of flupyradifurone, tefluthrin, flubendiamide and
95
design strategy of target compounds.
96 Scheme 1. Synthesis of Compounds 3a-e
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R2
O
NH
O
O
O
O
OH
NH2
1.Triphosgene, THF, RT
2.R2-NH2, THF
NH2
NH
R1
R1
R1
1a-b
3a-e
2a-b
3.SOCl2, DCE
R
1 = Cl, R2 = CH3
3d R1 = Br, R2 = i-propyl
3a
1 = Cl, R2 = i-propyl
1 = Br, R2 = CH3
R
1 = Cl, R2 = CH2CF2H
3b
3c
R
3e
R
97
98 Scheme 2. Synthesis of Compounds 9a-l
O
NH
Br
NH2
NH2
HN
O
O
POBr3, TEA
CH3CN
(1)NaNO2, con HCl
N
Na, diethyl maleate
EtOH
N
N
(2)SnCl2.2H2O
O
O
Rx
Rx
Rx
Rx
4
5
6
7
Br
Br
O
O
LiOH.H2O
MeOH, H2O
N
N
con H2SO4, K2S2O8
N
N
1
O
HO
or KMnO4
6
2
5
3
Rx
Rx
4
9
8
(4-9)i
(4-9)j
: Rx= 2-F,4-F,6-F
(4-9)a
(4-9)b
(4-9)e
: Rx= 2-F
: Rx= 2-F,4-F
: Rx= 3-F
(4-9)f
: Rx= 2-F,5-F
: Rx= 2-Cl,4-F,5-F
(4-9)c
: Rx= 4-F
: Rx= 2-F,3-F
(4-9)g
:Rx= 2-F,6-F
:Rx= 2-F,4-F,5-F
(4-9)k
: Rx= 2-Cl,4-F,6-F
(4-9)h
(4-9)l
(4-9)d
: Rx= 2-Cl,4-F,6-Cl
99
100 Scheme 3. Synthesis of Title Compounds I-IV
R2
O
Br
NH
Br
O
H
N
N
(1). (COCl)2, DMF, DCM
3a-e
N
N
1
N
HO
R1
(2).
, DIPEA, THF
O
2
6
3
Rx
9a-l
5
Rx
4
I-IV
101
102
Scheme 1 showed synthesis and chemical structures of compounds 3a-
103 e. 2-Amino-3-methylbenzamide derivatives 3a, 3c were synthesized
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104 using the reported methods.23 2-Amino-3-methylbenzamide derivatives
105 3b, 3d and 3e were prepared by referring to the literatures with some
106 modifications.24-25
107 General Synthetic Procedure for Compound 3e.
108
To a stirred solution of absolute THF (30 mL), 2-amino-5-chloro-3-
109 methylbenzoic acid (16.2 mmol) and triphosgene (2.4 g, 8.1 mmol) was
110 added in turn. After the reaction was completed, the solvent was
111 evaporated in vacuo. The residue was filled with methanol (30 mL), and
112 further stirred for 0.5 h. Intermediate 2e was filtrated without further
113 purification.
114
A solution of 2e (14 mmol) in THF (25 mL) was stirred at sealed tube,
115 2, 2-difluoroethan-1-amine was added. Then the mixture was heated to
116 80 °C. After the reaction was completed, the solvent was removed in
117 vacuo. The residue was treated with saturated NaHCO3 solution and
118 extracted with ethyl acetate. The combined organic extracts were dried
119 over anhydrous Na2SO4. After evaporation of the solvent, the crude
120 product was purified by column chromatography to obtain product 3e.
121 General Synthetic Procedure for Compounds 5a-l.
122
To a stirred solution of concentrated HCl (50 mL) at -5 °C, substituted
123 aniline (72.0 mmol) was added. Then the mixture was treated with
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124 aqueous solution of sodium nitrate (5.47 g, 79.2 mmol) and stirred at -
125 5 °C for 1 h. The concentrated HCl solution of tin(II) chloride (32.50 g,
126 144 mmol) was slowly added and the mixture was stirred at -5 °C for 4 h.
127 After the reaction was completed, the produced precipitate was filtered.
128 The filter cake was treated with 200 mL water, then the pH of aqueous
129 solution was adjusted to 13 with 6 N NaOH solution. The precipitated
130 solid was filtered, and allowed to air-dry overnight to afford compounds
131 5a-l.
132 General Synthetic Procedure for Compounds 6a-l.
133
To 50 mL of absolute ethanol in a round-bottomed flask, sodium (1.29
134 g, 56.0 mmol) was slowly added. Then the compounds 5a-l (28.0 mmol)
135 was added, and the mixture was allowed to reflux. After 30 minutes of
136 reflux, mixture was treated with the diethyl maleate (5 mL, 0.3 mol). The
137 resulting solution was allowed to reflux for 3 h. The glacial acetic acid
138 (30 g, 30.8 mol) was added. After removal of solvent under reduced
139 presure, the residue was diluted with 50 mL of ethyl acetate and washed
140 with water and saturated NaHCO3 solution successively. The solvent was
141 removed in vacuo, the crude product was purified by column
142 chromatography to obtain products 6a-l.
143 General Synthetic Procedure for Compounds 7a-l.
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144
Compounds 6a-l (12.0 mmol) was added to acetonitrile (30 mL) at
145 0 °C. Then triethylamine (13.2 mmol) and the phosphorus oxybromide
146 (13.2 mmol) was slowly added in turn. The mixture was allowed to heat
147 at 50 °C. After completion, the solvent was removed. The residue was
148 diluted with 50 mL of ethyl acetate and washed with saturated NaHCO3
149 solution. The solvent was removed under reduced pressure, and the crude
150 product was purified by column chromatography to afford intermediates
151 7a-l.
152 General Synthetic Procedure for Compounds 8(a, c, d, e, g, h, i, j, k,
153 l).
154
A mixture of compounds 7(a, c, d, e, g, h, i, j, k, l) (10 mmol), sulfuric
155 acid (98%, 1.1 mL, 20 mmol) and K2S2O8 (1.3 g, 5 mmol) in 25 mL of
156 acetonitrile was stirred at 75 °C for 2 h. Then K2S2O8 (1.3 g, 5 mmol) was
157 added and the mixture was allowed to reflux for another 2 h. The reaction
158 mixture was filtered. The combined organic layers were diluted with
159 ethyl acetate (60 mL) and washed with H2O, saturated NaHCO3, and
160 brine sequentially. After removed of the solvent under reduced pressure,
161 the crude product was purified by flash chromatography to give the pure
162 products 8(a, c, d, e, g, h, i, j, k, l).
163 General Synthetic Procedure for Compounds 8b, 8f.
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164
To a solution of compounds 7b, 7f (5.0 mmol) in acetone (20 mL),
165 KMnO4 (3.2 g, 20.0 mmol) was added at 0 °C. The reaction was allowed
166 to warm to room temperature and stirred for 12 h. After the reaction was
167 completed, the reaction mixture was filtered. The combined organic
168 layers were concentrated under reduced pressure. The crude product was
169 purified by column chromatography on silica-gel to give target
170 compounds 8b, 8f.
171 General Synthetic Procedure for Compounds 9a-l.
172
To a stirred suspension of compound 8a-l (1.0 mmol) in methanol (10
173 mL), an aqueous solution (5 mL) of Lithium hydroxide (6.0 mmol) was
174 added. The reaction mixture was stirred at room temperature for 12 h.
175 After the reaction was completed, the methanol was removed in vacuo,
176 and the residue was diluted with 30 mL of H2O, the aqueous solution was
177 acidified to pH 2 by 1N HCl. The precipitation was collected by filtration,
178 and then dried to give the pure products 9a-l.
179 General Synthetic Procedure for Title Compounds I-IV
180
To a stirred solution of anhydrous CH2Cl2 (4 mL), compounds 9a-l (0.3
181 mmol) and oxalyl chloride (114.2 mg, 0.9 mmol) was added successively,
182 together with one drop of DMF at room temperature. After the reaction
183 was completed, the solvent was evaporated. The residue was diluted with
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184 anhydrous THF (4 mL) and added to a solution of diisopropylethylamine
185 (46.5 mg, 0.36 mmol) along with 3a-e (0.3 mmol) in anhydrous THF (4
186 mL) at 0 °C. The reaction was allowed to warm to room temperature and
187 stirred for 12 h. After removed of the solvent in vacuo, the crude product
188 was purified on silica gel to obtain the target compounds I-IV, among
189 which, compounds Ia and IIi were reported by patent
1
190 WO 2008010897 A2, but there were no detailed H NMR and HRMS
191 data.
192
X-ray Diffraction. Compound IIIf was recrystallized by slow
193 evaporation from a mixture of methanol and water. A colorless crystal
194 with dimensions of 0.200 × 0.180 × 0.100 mm was suitable for X-ray
195 diffraction study. Cell dimensions and intensity were measuredat at 113(2)
196 K on a Bruker SMART 1000 CCD diffractometer. The crystal structure
197 was solved by direct methods with SHELXL-97 program.26
198
Biological Assay. Larvicidal activity against M. separata and P.
199 xylostella. All biological assays were performed on test organisms
200 prepared in the laboratory, and Abbott’s formula was used to corrected
201 mortality rates.27-28 The insecticidal activities of the compounds I-IV and
202 chlorantraniliprole against M. separata were tested with the reported
203 procedure.29 Preliminary bioassay results were assembled in Table 1 and
204 LC95 and LC50 values of chlorantraniliprole and IIIf against M. separata
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205 were summarized in Table 2. Using the method previously reported, 30 the
206 insecticidal activity of chlorantraniliprole and I-IV against P. xylostella
207 were also tested, the results were summarized in Table 3.
208 RESULTS AND DISCUSSION
209
Synthesis. Compounds 3a-e were prepared using the reported
210 procedure with some modifications as shown in scheme 1.23-25 Synthesis
211 of compounds 9a-l was shown in Scheme 2.21, 24 Initially, substituted
212 aniline 4a-l was converted to the diazo compound by treatment with
213 sodium nitrite and reduced by stannous chloride to obtain the
214 intermediates 5a-l, which underwent cyclization with diethyl maleate to
215 give the intermediates 6a-l. The reaction of compounds 6a-l with
216 phosphorus oxybromide afforded the bromine substituted compounds 7a-
217 l, which was then oxidized to give the intermediates 8a-l. Then the
218 saponifcation of the ester intermediates 8a-l afforded compound 9a-l. The
219 synthesis of title compounds I-IV (Scheme 3) was completed by
220 converting compounds 9a-l to the acyl chlorides with oxalyl chloride,
221 coupled with compounds 3a-e.21
222 Table 1. Insecticidal Activities of Chlorantraniliprole and
223 Compounds I-IV against M. separata
224
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insecticidal activity (%) at a concentration of (mg L-1)
Compd
Ia
R1
Cl
Cl
Cl
Br
Cl
Cl
Cl
Br
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Br
Cl
Br
Cl
Cl
Cl
Br
Cl
Br
Cl
Cl
R2
i-propyl
CH3
Rx
50
25
100
60
10
50
5
2.5
1
0.5
0.25
0.1
0.05
2-F
100
100
50
Ib
3-F
Ic
i-propyl
CH3
3-F
Id
4-F
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
60
100
100
100
100
100
100
65
Ie
i-propyl
CH3
4-F
100
100
10
IIa
2-F,3-F
2-F,3-F
2-F,4-F
2-F,4-F
2-F,4-F
2-F,5-F
2-F,5-F
2-F,5-F
2-F,6-F
2-F,4-F,5-F
2-F,4-F,5-F
2-F,4-F,5-F
2-F,4-F,5-F
2-F,4-F,5-F
2-F,4-F,6-F
2-F,4-F,6-F
IIb
IIc
i-propyl
CH3
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
40
100
100
100
100
40
100
100
100
60
100
100
100
100
60
100
100
45
10
IId
IIe
CF2HCH3
i-propyl
CH3
100
IIf
IIg
CF2HCH3
i-propyl
CH3
IIh
IIi
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
50
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
55
60
100
60
IIIa
IIIb
IIIc
IIId
IIIe
IIIf
IIIg
IVa
IVb
IVc
IVd
IVe
IVf
CH3
50
60
CF2HCH3
CH3
100
30
i-propyl
i-propyl
CH3
100
100
35
55
100
100
43
20
i-propyl
i-propyl
i-propyl
CH3
2-Cl,4-F,5-F 100
2-Cl,4-F,5-F 100
2-Cl,4-F,6-F 100
2-Cl,4-F,6-F 100
70
100
100
100
45
60
100
40
CH3
45
2-Cl,4-F,6-Cl
CH3
100
2-Cl,4-F,6-Cl
i-propyl
100
Chlorantraniliprole
100
100
100
100
100
36
20
225 Table 2. LC50 Values of Chlorantraniliprole and Compound IIIf
226 against M. separata
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compund
y = a + bx
R
LC50(mg L-1)
0.0936
LC95(mg L-1)
0.3201
IIIf
chlorantraniliprole
y = 8.1688 + 3.0808x
y = 7.6052 + 2.6878x
0.9672
0.9668
0.1073
0.4392
227 Table 3. Insecticidal Activities of Chlorantraniliprole and
228 Compounds Ie, IIc, IIe, IIIa, IIIc, IIIe, IIIf, IVa, IVd against P.
229 xylostella
insecticidal activity (%) at a concentration of (mg L-1)
compound
10
5
0.5
100
100
100
100
100
100
100
100
100
100
5 x 10-2
100
100
100
100
100
100
90
5 x 10-3
100
100
100
90
1 x 10-4
95
1 x 10-5
80
Ie
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
IIc
100
100
85
90
IIe
90
IIIa
70
IIIc
100
100
70
100
100
50
90
IIIe
94
IIIf
IVa
100
100
100
100
94
100
80
90
70
IVd
Chlorantraniliprole
90
84
230
231
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232
Figure 2. Crystal structure of compound IIIf. (CCDC-1962419)
233
Crystal Structure Analysis. The single-crytal structure of compound
234 IIIf was described in Figure 2. From the crystal structure data, we could
235 find that the average bond lengths and bond angles of ring system
236 (benzene ring, pyrazole ring) were normal ranges.31-32 It was observed
237 that the dihedral angel between benzene ring (C14-C19) and pyrazole
238 ring (N4/N3/C11/C12/C13) was 66.82°, indicating two rings were
239 nonplanar in the molecular structure. Under the influence of
240 delocalization of the lone pair electron on the amine of pyrazole, it may
241 have an effect on the benzene ring. The bond lengths of F(1)-C(19), F(2)-
242 C(17), F(3)-C(15) were 1.345(3), 1.358(3) and 1.356(3) Å, respectively.
243 The packing structure demonstrated that there was a weak interaction
244 between the benzene ring and the pyridine ring of the adjacent molecules.
245 It played an important role in stabilizing the crystal structure.
246
Structure-Activity Relationship. Larvicidal Activity against M.
247 separata. The larvicidal activities of I-IV against M. separata were
248 described in Table 1. The bioactivity results revealed that most of the
249 compounds showed good larvicidal activities against M. separata.
250 Compounds Ie, IIc, IIe and IIIf exhibited the same good larvicidal
251 activity against M. separata at 0.5 mg L-1, while IIIf exhibited excellent
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252 larvicidal activity (0.1 mg L-1, 43%), which was a little higher than that of
253 the chlorantraniliprole (0.1 mg L-1, 36%).
254
The synthesized compounds can be subdivided into four series: 1)
255 monofluoro substituted phenylpyrazole derivatives, 2) di-fluorine
256 substituted phenylpyrazole derivatives, 3) tri-fluorine substituted
257 phenylpyrazole derivatives, 4) chlorine and fluorine substituted
258 phenylpyrazole derivatives. As for the monofluoro substituted
259 compounds Ia-Ie, the larvicidal activities of Ia (Rx = 2-F), Ic (Rx = 3-F),
260 and Ie (Rx = 4-F) were 50%, 0%, and 100% respectively at 10 mg L-1.
261 The results show that the position of the fluorine on the benzene ring had
262 a significant impact on biological activity, and the sequence of the
263 biological activity was Ie (Rx = 4-F) > Ia (Rx = 2-F) > Ic (Rx = 3-F).
264 Within the group of di-fluorine substituted compounds, 2, 3- difluoro (IIa,
265 IIb), 2, 4- difluoro (IIc, IId, IIe), 2, 5- difluoro (IIf, IIg, IIh) and 2, 6-
266 difluoro (IIi) displayed different larvicidal activities. Generally, the trend
267 of the larvicidal activities was IIc, IIe (Rx= 2-F, 4-F) > IIi (Rx= 2-F, 6-
268 F) > IIf, IIh (Rx= 2-F, 5-F) > IIb (Rx= 2-F, 3-F). Compounds IIc, IIe
269 (Rx= 2-F, 4-F) showed 45% and 10% larvicidal activities at 0.25 mg L-1,
270 respectively, higher than those against M. separata. These results clearly
271 indicated that the fluoro-substitution in the 2-position and 4-position of
272 benzene ring had a notable impact on the biological activity. The
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273 biological activity of tri-fluorine substituted derivatives indicated that the
274 introduction of fluorine atoms into the 5-position of the benzene ring
275 reduced the larvicidal activity. For instance, the larvicidal activity of IIIa
276 (Rx= 2-F, 4-F, 5-F) was 50% at 0.5 mg L-1, while that of IIc (Rx= 2-F, 4-
277 F) was 100%. By contrast, the introduction of fluorine atoms into the 6-
278 positions of the benzene ring improved larvicidal activity. Compared to
279 the standard chlorantraniliprole, IIIf (Rx= 2-F, 4-F, 6-F) exhibited better
280 larvicidal activity (43%, 0.1 mg L−1).
281
Compounds (IVa-IVf) with a chlorine atom in the 2-position, or two
282 chlorine atoms in the 2-position and 6-position of benzene ring exhibited
283 different degree of inhibitory activities. For instance, compounds IVa,
284 IVb (Rx= 2-Cl, 4-F, 5-F) and IVc, IVd (Rx= 2-Cl, 4-F, 6-F) substituted
285 with a chlorine atom at 2-position on the benzene ring showed 70%, 0%,
286 60% and 100% larvicidal activity at 1.0 mg L-1, respectively. However,
287 compounds IVe, IVf (Rx= 2-Cl, 4-F, 6-Cl) substituted with two chlorine
288 atoms at 2-position and 6-position on the benzene ring, showed 100%,
289 45% larvicidal activity at 2.5 mg L-1. The above biological data also
290 showed that the larvicidal activities of these compounds were reduced,
291 compared to the corresponding fluoro-substituted compounds.
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292
From Table 2, the LC50 and LC95 values of IIIf were 0.0936 and 0.3201
293 mg L-1. It indicated that IIIf showed almost the same biological activity
294 as that of the commercialized chlorantraniliprole (0.1073, 0.4392 mg L-1).
295
Larvicidal Activity against P. xylostella. The larvicidal activities of Ie,
296 IIc, IIe, IIIa, IIIc, IIIe, IIIf, IVa, IVd and chlorantraniliprole against P.
297 xylostella were summarized in Table 3. The results showed that these
298 compounds exhibited good activities. The excellent larvicidal activity
299 (50% at 10-4 mg L-1) of IIIf was consist with the activity against M.
300 separata (0.1 mg L-1, 43%). It was also worth noting that IIc, IIe, IIIc
301 and IIIe showed 100%, 100%, 60%, 55% activity against M. separata at
302 0.5 mg L-1, respectively, however, they showed excellent activities
303 against P. xylostella, which exhibited 90%, 90%, 90%, 94% mortality at
304 the very low concentration of 10-5 mg L−1, respectively.
305
306
307
Figure 3. Effects of different concentrations of IIIf on calcium ion concentration in
the central neurons of M. separata. (A. B. C).
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308
309
310
311
312
Figure 4. When the neurons were preincubated by 2.5 μM 2-APB, effects of IIIf (5
mg L−1) on calcium ion concentration in the central neurons of M. separata (A).
When the neurons were preincubated by 0.5 μM Ryanodine, effects of IIIf (5 mg L−1)
on calcium ion concentration in the central neurons of M. separata (B).
313
Mode of action. To investigate the larvicidal mechanism of these
314 compounds, calcium imaging experiment was done according to previous
315 reports.33-34 In this way, we explored the effects of IIIf on calcium ion
316 concentration after M. separata was dyed by loading with fluo-3 AM.
317 From figure 3 (A. B. C), we could find that when central neurons of th M.
318 separata were processed with different concentrations of IIIf from 1, 5,
319 50 mg L−1, respectively, there is a significant change in calcium ion
320 concentration versus recording time. It revealed that the peak of calcium
321 ion concentration increased to 102.03 ± 0.75%, (n = 13), 104.25 ± 1.71%,
322 (n = 7), 106.75 ± 2.45%, (n = 3), respectively, after the neurons were
323 processed with different concentrations of IIIf for < 10s. Figure 3 also
324 showed that under the same concentration conditions, the free calcium
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325 released from the calcium store by chlorantraniliprole and IIIf was
326 consistent within the error range, indicating that the activity levels of the
327 two were similar.
328
As we known, RyRs and 1, 4, 5-trisphosphate receptor (IP3R) Ca2+
329 channels were major calcium release channels in the endoplasmic
330 reticulum membrane. To understand which channel was involved in the
331 release of calcium ions promoted by such compounds. 2-
332 Aminoethoxydiphenyl borate (2-APB) and Ryanodine were used to treat
333 the central neurons of M. separata, respectively. After central neurons of
334 M. separata were treated with 2-APB, IP3R was blocked. Figure 4 A
335 revealed that treatment of the neurons with 2-APB had little effect on the
336 calaium increase in internal calcium, the increase of calcium ion
337 concentration was possibly attributed to the RyR in the endoplasmic
338 reticulum. After the neurons had been treated with Ryanodine, the effect
339 of IIIf (5 mg L−1) on F/F0 increased to 107.96 ± 6.27%, (n = 15) (Figure
340 4 B), which showed that they have a similar mechanism. From the above
341 results, we could speculate that the target of compound IIIf most
342 probably was RyR. IIIf could be considered as a possible candidate for
343 RyR modulators, which could cause the release of intracellular calcium
344 ions and eventually lead to the death of insects.
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R2
NH
O
*
H
N
* *Br
*
*
*
N
*
1
*
*
*
*
N
R
*
O
*
*
*
*
*
*
*
R
*
x
*
345
346
Figure 5. The asterisk skeleton of title compounds
347 Table 4. Summary of CoMFA Analysis.
q2
r2
F
Contribution(%)
Steric
44.9
Electrostatic
55.1
CoMFA
0.61
0.923
26.968
348
349
350
Figure 6. Contour maps of steric field (A) and electrostatic field (B) from CoMFA
model.
351
CoMFA Analysis. The CoMFA could be used for fast prediction of
352 QSAR of new molecules,35-37 so it is widely used in the design of
353 medicines and agrochemicals. Based on the asterisked atoms of template
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354 molecule (Figure 5), CoMFA analyses for the compounds I-IV were
355 performed. By the CoMFA module of SYBYL 6.9 software, structure-
356 activity relationship research was conducted on 27 compounds
357 synthesized by our group, among which Ia-Ie, IIa-IIb, IId-IIi, IIIa, IIIc-
358 IIIg, IVa-IVd, IVf were the training set, and IIc, IIIb, IVe were the test
359 set. As shown in Table 4, a predictive CoMFA model was established
360 with the conventional correlation coefficient r2 = 0.923 and the cross-
361 validated coefficient q2 = 0.61. The contributions of steric and
362 electrostatic fields were 44.9% and 55.1% respectively. It shows that the
363 main factor to impact on larvicidal activity was the charge distributions of
364 substituted groups, followed by hydrophobic factor and steric hindrance
365 of substituted groups.
366
Through the CoMFA model, the isocontour diagrams of the steric and
367 electrostatic field contributions were obtained. In Figure 6 A, the green
368 region representing the steric contour was mainly located around the 5-
369 position of the benzene ring. It meant that a small group here would be
370 better for larvicidal activity, for example, compounds Ie, IIc, IIe, IIIf had
371 higher larvicidal activity when there was a small group in 5-position. For
372 the electrostatic field contour map (Figure 6 B), red contour indicated that
373 the increase in positive charge on the 2 and 4 positions of the benzene
374 ring is accompanied by an increase in insecticidal activity. Preliminary
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375 bioassay also showed that the title compounds bearing electron-
376 withdrawing groups at the 2, 4 or 6 positions of the benzene rings, such as
377 IIc, IIe and IIIf, exhibited higher activity. As expected from the fluorine
378 position on the periodic table of elements, it possesses some extreme
379 properties, in particular, ultimate electronegativity. So fluorine plays an
380 important role in activity. These results also provide a new idea for the
381 structural optimization of target compounds.
382
In conclusion, 27 phenylpyrazole derivatives containing fluoro-
383 substituted benzene moiety were designed and synthesized. The
384 bioactivity results exhibited most compounds displayed good insecticidal
385 activities against M. separata and P. xylostella. From the preliminary
386 SAR, we found that the number and position of fluorine on the benzene
387 ring showed different influences on insecticidal activity, indicating that
388 the introduction of fluorine into diamide insecticides was an effective
389 strategy for the development of pesticides. Promisingly, compound IIIf
390 (Rx= 2-F, 4-F, 6-F) exhibited 43% insecticidal activity against M.
391 separata at 0.1 mg L-1, while the control chlorantraniliprole was 36%.
392 The activity of IIIe (Rx= 2-F, 4-F, 5-F) against P. xylostella was 94% at
393 10-5 mg L−1, while the control was 70%. Insect electrophysiology studies
394 proved that RyR in the central neurons of M. separata was possible target
395 of these compounds. More importantly, compared to some reported
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396 compounds, fluoro-substituted phenylpyrazole derivatives were more
397 simplified and showed better insecticidal activities against M. separata
398 and P. xylostella. So that they could be used as lead compounds for the
399 development of new anthranilic diamide insecticides.
400 ASSOCIATED CONTENT
401 Supporting Information
402 Data and characterization of intermediates 3e, 5a-l, 6a-l, 7a-l, 8a-l, 9a-l
403 and target compounds Ia-e, IIa-i, IIIa-g, IVa-f.
404 AUTHOR INFORMATION
405 Corresponding Authors
406 *(Zheng-Ming Li) E-mail: nkzml@vip.163.com. Phone: 0086-22-
407 23503732.
408 * (Yu-Xin Li) E-mail: liyx128@nankai.edu.cn. Phone: 0086-22-
409 23503798.
410 Funding
411 This project was supported by the National Key Research and
412 Development Program of China (2018YFD0200100, 2017YFD0200505)
413 and National Natural Science Foundation of China (No 31972287,
414 21602118)
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415 Notes
416 The authors declare no competing financial interest.
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