Synthesis and insecticidal activities of novel anthranilic diamides containing acylthiourea and acylurea

Article abstract of DOI:10.1021/jf302446c

Two series of anthranilic diamides containing acylthiourea and acylurea linkers were designed and synthesized, with changed length and flexibility of the linkers to compare to known anthranilic diamide insecticides. In total, 26 novel compounds were synthesized, and all compounds were characterized by ¹H nuclear magnetic resonance and high-resolution mass spectrometry. Their insecticidal activities against oriental armyworm (Mythimna separata), mosquito larvae (Culex pipiens pallens), and diamondback moth (Plutella xylostella) were evaluated. The larvicidal activities against oriental armyworm indicated that the introduction of acylthiourea into some structures could retain their insecticidal activity; 8 of the 15 compounds (13a-13e, 14a-14e, and 15a-15e) exhibited 100% larvicidal activity at 10 mg/L. However, the introduction of acylurea decreased the insecticidal activity; only 3 of the 11 compounds (17a-17k) exhibited 100% larvicidal activity at 200 mg/L. The whole-cell patch-clamp technique indicated that compound 13b and chlorantraniliprole exhibited similar effects on the voltage-gated calcium channel. The calcium-imaging technique was also applied to investigate the effects of compounds 13b and 15a on the intracellular calcium ion concentration ([Ca²⁺]_i), which indicated that they released stored calcium ions from endoplasmic reticulum. Experimental results denoted that several new compounds are potential activators of the insect ryanodine receptor (RyR).

Full text of DOI:10.1021/jf302446c

Article
pubs.acs.org/JAFC
Synthesis and Insecticidal Activities of Novel Anthranilic Diamides
Containing Acylthiourea and Acylurea
Ji-Feng Zhang,† Jun-Ying Xu,‡ Bao-Lei Wang,† Yu-Xin Li,*,† Li-Xia Xiong,† Yong-Qiang Li,† Yi Ma,†
and Zheng-Ming Li*,†
†State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, People’s Republic
of China
‡CNOOC Tianjin Chemical Research and Design Institute, Tianjin 300131, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two series of anthranilic diamides containing acylthiourea and acylurea linkers were designed and synthesized,
with changed length and flexibility of the linkers to compare to known anthranilic diamide insecticides. In total, 26 novel
compounds were synthesized, and all compounds were characterized by 1H nuclear magnetic resonance and high-resolution mass
spectrometry. Their insecticidal activities against oriental armyworm (Mythimna separata), mosquito larvae (Culex pipiens
pallens), and diamondback moth (Plutella xylostella) were evaluated. The larvicidal activities against oriental armyworm indicated
that the introduction of acylthiourea into some structures could retain their insecticidal activity; 8 of the 15 compounds (13a−
13e, 14a−14e, and 15a−15e) exhibited 100% larvicidal activity at 10 mg/L. However, the introduction of acylurea decreased the
insecticidal activity; only 3 of the 11 compounds (17a−17k) exhibited 100% larvicidal activity at 200 mg/L. The whole-cell
patch-clamp technique indicated that compound 13b and chlorantraniliprole exhibited similar effects on the voltage-gated
calcium channel. The calcium-imaging technique was also applied to investigate the effects of compounds 13b and 15a on the
intracellular calcium ion concentration ([Ca2+]i), which indicated that they released stored calcium ions from endoplasmic
reticulum. Experimental results denoted that several new compounds are potential activators of the insect ryanodine receptor
(RyR).
KEYWORDS: Anthranilic diamide, insecticidal activity, acylthiourea, acylurea, calcium channel
of target compounds, the whole-cell patch-clamp and calcium-
imaging techniques were used to investigate the effects of
compounds 13b and 15a on calcium channels in the central
neurons of Spodoptera exigua.
INTRODUCTION
■
For years, scientists have been dedicated in searching for
insecticides with new mechanisms to cope with the global food
shortage. Recently, the anthranilic diamides chlorantraniliprole
(Rynaxypyr; DPX-E2Y45) (compound A in Figure 1) and
cyantraniliprole (Cyazypyr) (compound B) were marketed by
Dupont, targeting at the insect ryanodine receptor,1,2 which
exhibit their action by activating the uncontrolled release of the
calcium store.1 Their broad insecticidal spectra, high efficiency,
and low toxicity aroused interests worldwide. Some modified
structures (compound C) have been reported, which mainly
focused on the 1-(3-chloropyridyl) pyrazole moiety (I), the
aliphatic amide moiety (II), and the anthraniloyl moiety
(III).2−11 Nevertheless, the modifications about the amide
bridge part as a linker of two aryl rings were seldom reported.12
In crop protection and bioactive chemicals, acylthioureas and
acylureas have been reported to display a variety of biological
activities, such as insecticidal, fungicidal, herbicidal, antimicro-
bial, antitumor, etc.13−18 With this in mind, two series of novel
anthranilic diamide derivatives (compound D) were designed
and synthesized in this paper by introducing acylthiourea and
acylurea moieties to increase the linker length and flexibility.
Their synthetic routes were shown in Schemes 1−5. Their
insecticidal activities against oriental armyworms, mosquito
larvae, and diamondback moths were tested accordingly. The
preliminary structure−activity relationship (SAR) was dis-
cussed. To provide insight to further study the biological effect
MATERIALS AND METHODS
■
Instruments. 1H nuclear magnetic resonance (NMR) spectra were
recorded at 300 MHz using a Bruker AC-P300 spectrometer or 400
MHz using a Bruker AV 400 spectrometer (Bruker Co., Switzerland)
in CDCl3 or DMSO-d6 solution with tetramethylsilane as the internal
standard, and chemical-shift values (δ) were given in parts per million
(ppm). High-resolution mass spectrometry (HRMS) data were
obtained on a Varian QFT-ESI instrument. Flash chromatography
was performed on CombiFlash Companion (Teledyne Isco, Inc.,
Lincoln, NE) with silica gel (300−400 mesh). The melting points were
determined on a X-4 binocular microscope melting point apparatus
(Beijing Tech Instruments Co., Beijing, China) and were uncorrected.
The whole-cell patch clamp was performed using a patch-clamp
amplifier (EPC-10, HEKA Electronik, Lambrecht, Germany).
Reagents were all analytically or chemically pure. All solvents and
liquid reagents were dried by standard methods in advance and
distilled before use. Chlorantraniliprole used in this work was
synthesized according to the literature only for bioassay reference.19
General Synthetic Procedure for Compounds 6a and 6b.
The title compounds 6a and 6b were prepared in our laboratory
Received: February 24, 2012
Accepted: July 19, 2012
Published: July 19, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of compounds A−D.
according to the methods reported by Dong et al. (Scheme 1), with
some modifications. The melting points and 1H NMR data of all of the
compounds were consistent with the literature.19,20
pyridyl-H), 8.31 (dd, J = 8.0, 1.5 Hz, 1H, pyridyl-H), 7.74 (dd, J = 8.0,
4.8 Hz, 1H, pyridyl-H), 7.61 (s, 1H, pyrazolyl-H) (Scheme 2).
a
Scheme 2. Synthesis of Compound 6c
a
Scheme 1. Synthesis of Title Compounds 6a and 6b
a
Reagents and conditions: (a) NaOCH3, ethyl trifluoroacetate, (b)
AcOH, reflux, and (c) KMnO4, acetone/H2O, reflux.
General Synthetic Procedure for Compounds 11a−11m. The
compounds 11a−11m were prepared in our laboratory according to
the methods reported by Dong et al. (Scheme 3 and Table 1). The
melting points and 1H NMR data of all of the compounds were
consistent with the literature.19,20
a
Reagents and conditions: (a) NH2NH2·H2O (80%), reflux, (b)
NaOEt, EtOH, reflux, (c) POBr3 or POCl3, MeCN, 80 °C, (d)
K2S2O8, H2SO4, MeCN, reflux, and (e) (i) aqueous NaOH, MeOH
and (ii) aqueous HCl.
Scheme 3. Synthesis of Compounds 11a−11m
1,1,1-Trifluoro-4-(furan-2-yl)-4-hydroxybut-3-en-2-one (8).
Sodium (1.38 g, 60 mmol) was dissolved in 30 mL of anhydrous
methanol slowly. Methanol was evaporated to dryness under reduced
pressure to give the white solid sodium methylate. Then, diethyl ether
(50 mL), ethyl trifluoroacetate (8.52 g, 60 mmol), and 2-acetylfuran
(6.60 g, 60 mmol) were successively added dropwise, stirred at room
temperature for 5 h, and filtered, which was acidized with 1 M sulfuric
Table 1. Structure of Compounds 11a−11m
compound
R1
R2
R3
1
acid to give a red liquid product. Yield = 64.9%. H NMR (400 MHz,
11a
11b
11c
11d
11e
11f
Me
Et
Me
Me
Me
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
H
CDCl3) δ: 13.67 (br s, 1H, OH), 7.67 (s, 1H, Ar−H), 7.33 (d, 1H, J =
3.6 Hz, Ar−H), 6.63 (d, 1H, J = 3.6 Hz, Ar−H), 6.48 (s, 1H, CH).
3-Chloro-2-(5-(furan-2-yl)-3-(trifluoromethyl)-1H-pyrazol-1-
yl)pyridine (9). In a three-neck 1000 mL round-bottomed flask, 1,1,1-
trifluoro-4-(furan-2-yl)-4-hydroxybut-3-en-2-one (8, 27.1 g, 130
mmol) and 3-chloro-2-hydrazinylpyridine (18.9 g, 130 mmol) were
added to 300 mL of acetic acid. The mixture was refluxed for 8 h, and
then the excess acetic acid was removed under reduced pressure. The
residue was further purified by flash column chromatography on silica
gel with petroleum ether/ethyl acetate (5:1) to give intermediate 9 as
i-Pr
i-Pr
n-Pr
H
n-Bu
Me
Me
Me
Me
Me
Me
Me
Me
11g
11h
11i
t-Bu
cyclopropyl
cyclohexyl
i-Pr
11j
1
a red oil. Yield = 42.1%. H NMR (400 MHz, CDCl3) δ: 8.58 (d, J =
11k
11l
benzyl
i-Pr
4.8 Hz, 1H, pyridyl-H), 7.96 (d, J = 8.0 Hz, 1H, pyridyl-H), 7.51 (dd, J
= 8.0, 4.8 Hz, 1H, pyridyl-H), 7.36 (s, 1H, pyrazolyl-H), 6.93 (s, 1H,
furanyl-H), 6.41−6.30 (s, 1H, furanyl-H), 6.01 (d, J = 3.4 Hz, 1H,
furanyl-H).
11m
i-amyl
H
1-(3-Chloropyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-5-
carboxylic acid (6c). To a solution of 3-chloro-2-(5-(furan-2-yl)-3-
(trifluoromethyl)-1H-pyrazol-1-yl)pyridine (9, 3.88 g, 12.4 mmol) in
acetone (35 mL) and water (35 mL), potassium permanganate (9.80
g, 62 mmol) was added in portion. When the addition was complete,
the reaction was refluxed for 30 min. The resulting mixture was
filtered. The filtrate was acidified with 2 M HCl and extracted with
ethyl acetate (50 mL × 3). The extracts were combined, washed with
water (30 mL × 2) and brine (30 mL), then dried with anhydrous
sodium sulfate, and evaporated to give compound 6c as a white solid
(1.85 g). Yield = 51.3%. Melting point (mp) = 176−179 °C. 1H NMR
(400 MHz, DMSO-d6) δ: 14.14 (s, 1H), 8.61 (dd, J = 4.8, 1.5 Hz, 1H,
General Synthetic Procedure for Compounds 13a−13e,
14a−14e, and 15a−15e. To a suspension of 1-(3-chloropyridin-2-
yl)-1H-pyrazole-5-carboxylic acid derivative (6a−6c) (1 mmol) in 25
mL of dichloromethane, oxalyl chloride (2.0 mmol) and a drop of
N,N-dimethylformamide (DMF) were added. After stirring at room
temperature for 3 h, the solution was evaporated. The resulting acyl
chloride was dissolved in 20 mL of acetonitrile and added to a solution
of potassium thiocyanate in 15 mL of acetonitrile with two drops of
polyethylene glycol-400 (PEG-400). After stirring at room temper-
ature for 30 min, the mixture was filtered to give the acyl
isothiocyanate derivatives 12a−12c, which were used without further
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a
Scheme 4. Synthesis of Compounds 13a−13e, 14a−14e, and 15a−15e
a
Reagents and conditions: (a) (i) oxalyl chloride, CH2Cl2 and (ii) KSCN, PEG-400, CH3CN and (b) 11a−11m, CH3CN.
purification, to which the 2-amino-3-methylbenzamide derivatives
(11a−11m) (1.0 mmol) were added and stirred overnight, then
filtered, and further purified by recrystallization using methanol or by a
silica-gel column eluted with petroleum ether/ethyl acetate (4:1) to
obtain the title compounds 13a−13e, 14a−14e, and 15a−15e.
1-(3-Chloropyridin-2-yl)-3-(trifluoromethyl)-1H-pyrazole-5-
carboxamide (16). To a suspension of 3-trifluoromethyl-1-(3-
chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid (6c) (2.40 g, 8.23
mmol) in 50 mL of dichloromethane, oxalyl chloride (17.0 mmol) and
a drop of DMF were added. After stirring at room temperature for 3 h,
the solution was evaporated. The resulting acyl chloride was dissolved
in 50 mL of tetrahydrofuran (THF) and added to aqueous ammonia
(25%) (5.76 g, 41.15 mmol) at 0 °C. After stirring overnight, a large
amount of solid formed and was filtered, which was further purified by
a silica-gel column eluted with petroleum ether/ethyl acetate (2:1) to
give the title compound as a white solid (1.85 g). Yield = 77.3%. mp =
183−185 °C. 1H NMR (400 MHz, CDCl3) δ: 8.56 (d, J = 4.8 Hz, 1H,
pyridyl-H), 8.00 (d, J = 8.0 Hz, 1H, pyridyl-H), 7.53 (dd, J = 8.0, 4.8
Hz, 1H, pyridyl-H), 7.38 (s, 1H, pyrazolyl-H), 6.78 (s, 1H, NH), 5.77
(s, 1H, NH).
General Synthetic Procedure for Compounds 17a−17k. A
suspension of compound 16 (0.29 g, 1 mmol) in 20 mL of 1,2-
dichloroethane and oxalyl chloride (3.0 mmol) was heated to reflux
and kept for 5 h. Then, the mixture was evaporated, and the residue
was dissolved in acetonitrile, to which the 2-amino-3-methylbenzamide
derivatives (11a−11m) (1 mmol) were added and stirred overnight.
Then, the produced precipitate was filtered and washed with
acetonitrile (5 mL × 3) to give the title compounds 17a−17k.
Larvicidal Activity against Oriental Armyworm (M. separa-
ta). The larvicidal activities of compounds 13a−13e, 14a−14e, 15a−
15e, 17a−17k, and chlorantraniliprole were evaluated using the
reported procedure.19 The insecticidal activity against oriental
armyworms was tested by foliar application; individual corn (Tangyu
10, Zea mays L.) leaves were placed on moistened pieces of filter paper
in Petri dishes. The leaves were then sprayed with the test solution and
allowed to dry. The dishes were infested with 10 fourth-instar oriental
armyworm larvae. Percentage mortalities were evaluated 2 days after
treatment. Each treatment was replicated 3 times.
Larvicidal Activity against Mosquito Larvae (C. pipiens
pallens). The larvicidal activities of compounds 13a−13e, 14a−14e,
and chlorantraniliprole against mosquito larvae were evaluated by the
reported procedure.22 The compounds 13a−13e, 14a−14e, and
chlorantraniliprole were prepared to different concentrations by
dissolving compounds in acetone and adding distilled water. Then,
20 fourth-instar mosquito larvae were placed in 10 mL of test solution
and raised for 3 days. Each treatment was performed 3 times, and the
average value of the three tested values was calculated. The results
were expressed by death percentage.
Larvicidal Activity against Diamondback Moth (P. xylostel-
la). The larvicidal activity of compounds 15a−15e and chloran-
traniliprole was tested by the leaf-dip method. At first, a solution of
each test sample in DMF (AR, purchased from Alfa Aesar) at a
concentration of 200 mg/L was prepared and then diluted to the
required concentration with water (distilled). Leaf disks (6 × 2 cm)
were cut from fresh cabbage leaves and then sprayed with the test
solution for 3 s and allowed to dry. The resulting leaf disks were placed
individually into glass tubes. Each disk was infested with 30 s-instar
diamondback moth larvae. Percentage mortalities were evaluated 2
days after treatment. Each treatment was performed 3 times.
Electrophysiological Recording and Calcium Imaging. The
currents of calcium (ICa) were recorded using the reported
procedure.23 Calibration of the fluorescence signal was achieved
using the method by Iakahashi et al., with some modifications.24 The
attached neurons were rinsed in standard physiological saline [150
mM NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM N-2-
hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), buffered
to pH 6.9] and then incubated in the dark at 28 °C in standard
external saline containing the dye fluo-3-AM (Sigma, 10 μM) for 45
min or incubated in the dark at 28 °C in external saline [150 mM
NaCl, 4 mM KCl, 2 mM MgCl2, 2 mM ethylene glycol bis(2-
aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 10 mM
HEPES, buffered to pH 6.9] containing the dye fluo-5N (Invitrogen, 5
μM) for 5 h. After dye loading, cells were again rinsed in physiological
saline twice.
For full depletion of thapsigargin-sensitive stores, cells were
incubated with 1 μM thapsigargin for 10 min. Calcium ratio imaging
studies were conducted using the imaging system coupled to an
inverted fluorescence microscope with a Fluor 40× oil immersion
objective (Olympus IX71). Cells which excited at 488 and 530 nm
under fluorescence emission were acquired using a charge-coupled
device (CCD) camera (Image Pri-6.0).
Each experiment was repeated at least 6 times. The data were
analyzed using SPSS, Inc., version 17.0, and Microcal Origin, version
8.0 (Origin Lab Corp., Northampton, MA). Results were expressed as
the mean standard deviation (SD) (n = number of cells). Statistical
significance was determined by Student’s paired or unpaired t tests.
Fluorescence values were expressed as F/F0, with F0 being the resting
(or baseline) fluorescence and F being the change in fluorescence from
baseline after the drug application.
RESULTS AND DISCUSSION
■
Synthesis. The carboxylic acids 6a and 6b were prepared
using the procedure reported by Dong et al. (Scheme 1),19,20
with some modifications, for the synthesis of intermediates.
With the reported procedure, intermediate 2 was prepared in
69% yield by refluxing 2,3-dichloropyridine (1) and hydrazine
hydrate (50%) in ethanol for 36 h. In our experiments,
hydrazine hydrate (80%) was used to directly reflux with
compound 1 for 5−6 h without solvent ethanol. By the
improvement, compound 2 could be obtained in a 93% high
yield.
The title compounds 13a−13e, 14a−14e, and 15a−15e
were synthesized, as shown in Scheme 4. Carboxylic acid (6a−
6c) was treated with oxalyl chloride and then added to
potassium thiocyanate in acetonitrile with two drops of PEG-
400. The corresponding acyl isothiocyanate derivative was
filtered after stirring at room temperature for 30 min and
reacted with 1 equiv of amines 11a−11m to afford the title
compounds 13a−13e, 14a−14e, and 15a−15e, without further
purification. Unlike the reported procedure that a reflux
condition was usually needed,14 this reaction was worked out
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a
Scheme 5. Synthesis of Compounds 17a−17k
a
Reagents and conditions: (a) (i) oxalyl chloride, CH2Cl2 and (ii) NH3·H2O and (b) (i) oxalyl chloride, ClCH2CH2Cl, reflux and (ii) 11a−11m,
CH3CN.
Table 2. Insecticidal Activities of Compounds 13a−13e, 14a−14e, 15a−15e, 17a−17k, and Chlorantraniliprole against Oriental
Armyworms
larvicidal activity (%) at a concentration of mg/L
compound
R1
R2
R3
R4
Cl
200
100
50
20
10
5
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
15a
15b
15c
15d
15e
17a
17b
17c
17d
17e
17f
Me
i-Pr
i-Pr
i-Pr
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Cl
Cl
Br
H
100
100
100
100
60
100
100
80
100
100
40
100
100
40
Cl
100
40
0
Cl
Cl
100
100
100
100
i-amyl
H
Cl
Me
Cl
Br
Cl
Cl
H
Br
100
100
100
0
100
100
100
100
100
100
100
100
100
100
100
100
50
0
i-Pr
Br
t-Bu
Br
40
cyclohexyl
i-amyl
Br
Br
100
100
100
40
30
100
100
Me
Cl
Cl
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Br
Cl
H
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
100
100
100
100
100
100
50
0
Et
cyclohexyl
cyclopropyl
benzyl
Me
100
100
100
20
100
100
20
100
100
100
100
100
0
30
cyclopropyl
n-Bu
40
t-Bu
60
cyclohexyl
i-Pr
0
70
17g
17h
17i
cyclopropyl
n-Pr
100
0
0
cyclopropyl
cyclopropyl
n-Pr
Me
H
100
30
80
17j
Cl
H
17k
Me
60
chlorantraniliprole
100
100
100
100
100
100
at room temperature under the catalysis of a small amount of
PEG-400 with high yield.
compounds 17a−17k, when R2 = Me, the two active proton
signals of CONHCO and NHCO on the carbonyl acylurea
moiety appeared at 11.55−11.46 and 9.93−9.72 ppm,
respectively. Whereas their values shifted to 11.54−11.52 and
11.16−11.11 ppm, respectively, when R2 = H (17h and 17j) in
DMSO-d6.
SAR. Larvicidal Activities against Oriental Armyworms (M.
separata). The larvicidal activities of compounds 13a−13e,
14a−14e, 15a−15e, 17a−17k, and commercial chlorantranili-
prole against oriental armyworms were summarized in Table 2.
In general, most of the compounds exist in moderate to good
pesticidal activities. Particularly, eight compounds (13a−13e,
14a−14e, and 15a−15e) exhibited 100% larvicidal activities at
10 mg/L against oriental armyworm. From the activities against
oriental armyworms, we could obviously find that the
compounds containing the acylthiourea moiety (13a−13e,
14a−14e, and 15a−15e) gave higher insecticidal activities than
the corresponding acylurea derivatives (17a−17k). When
compounds 15a and 15d are compared to compounds 17a
The title compounds 17a−17k were synthesized, as shown in
Scheme 5. Carboxylic acid 6c was converted to amide 16 by
reacting with oxalyl chloride and then aqueous ammonia.
Compound 16 was then refluxed with oxalyl chloride in 1,2-
dichloroethane to give its corresponding acyl isocyanate
derivative, which coupled with amines 11a−11m to afford
the title compounds 17a−17k.
1
The H NMR data of acylthiourea and acylurea derivatives
were characteristic in all of the target compounds. In
compounds 13a−13e and 14a−14e, the two active proton
signals of NHCS and NHCO on carbonyl thiourea moiety were
observed in DMSO-d6 at 12.15−12.04 and 11.48−11.37 ppm,
respectively. In compounds 15a−15e, the introduction of a
trifluoromethyl group increased the hydrophobicity of the
1
whole molecule; therefore, the H NMR determination was
carried out in CDCl3. The chemical shifts of these two active
protons were at 11.34−11.21 and 10.10−9.77 ppm. In
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and 17b, we could find that one atom difference between
acylthiourea and acylurea can cause a great change in its
insecticidal activity. When different substitutions in the aliphatic
amide moiety of title compounds 13a−13e, 14a−14e and
15a−15e are compared, it was found that large substituents,
such as i-amyl and cyclohexyl (13e, 14d, 14e, and 15c)
decreased the insecticidal activity against oriental armyworms.
Furthermore, compounds with the i-propyl and t-butyl
substituents showed the best larvicidal activity against ariental
armyworms in our research, which was consistent with the
results reported in previous SARs.2,25
Table 4. Insecticidal Activities of Compounds 15a−15e and
Chlorantraniliprole against Diamondback Moths
larvicidal activity (%) at a concentration of mg/L
compound
20
1
0.1
0.01
0.001
15a
15b
15c
15d
15e
100
100
30
100
100
0
100
100
71
86
43
29
100
100
100
100
100
100
100
100
100
100
100
100
71
57
chlorantraniliprole
100
Biological Assay. Insecticidal activities against oriental
armyworms (Mythimna separata), mosquito larvae (Culex
pipiens pallens), and diamondback moths (Plutella xylostella)
were performed on test organisms reared in a greenhouse. The
respectively, but still higher than that of chlorantraniliprole
(0.000 012 3 mg/mL; Table 5).
Effects of Compounds 13b and 15a on Calcium
Channels of Neurons from S. exigua. Figure 2 shows the
change rate of peak current amplitude versus recording time in
0.1 and 0.001 mmol/L compound 13b- and 0.1 mmol/L
chlorantraniliprole-treated and control neurons. The peak
bioassay was replicated at 25
1 °C according to statistical
requirements. Assessments were made on a dead/alive basis,
and mortality rates were corrected applying Abbott’s formula.21
Evaluation was based on a percentage scale of 0−100, where 0
equals no activity and 100 equals total kill. Error of the
experiments was 5%. For comparative purposes, chlorantrani-
liprole was tested as a reference. The insecticidal activity was
summarized in Tables 2−4.
currents were reduced to 54.71
4.95% (n = 6), 74.63
5.37% (n = 5), 39.43 5.02% (n = 8), and 85.44 1.14% (n =
6) of the initial value by the end of the 10 min recording, when
the neurons were treated with 0.1 and 0.001 mmol/L
compound 13b, 0.1 mmol/L chlorantraniliprole, and the
control, respectively. The peak current was reduced to 54.71
4.95% (n = 6) when the neurons were treated with 0.1
mmol/L compound 13b. In comparison to the control (85.44
1.14%), compound 13b at 0.001 mmol/L has a weak effect
Larvicidal Activities against Mosquito Larvae (C. pipiens
pallens). The larvicidal activities of compounds 13a−13e and
14a−14e against mosquito larvae were shown in Table 3. Most
Table 3. Insecticidal Activities of Compounds 13a−13e,
14a−14e, and Chlorantraniliprole against Mosquito Larvae
on Ca2+ currents. The recorded peak currents of calcium
(L)
channels treated with compound 13b were in a concentration-
dependent manner (Figure 2 and Table 6).
larvicidal activity (%) at a concentration of
mg/L
There was a significant change in maximal value of the peak
current when the neurons were held at −70 mV with the
treatment of 0.1 mmol/L compound 13b (Figure 3). The
calcium current decreased when compound 13b was injected in
the surrounding of the neuron cells. The maximal value of ICa
compound
2
1
0.5
20
13a
13b
13c
13d
13e
14a
14b
14c
14d
14e
70
40
30
90
60
20
(−2.1303
0.0237 nA) shifted to the negative direction by
10
approximately 10 mV after the neurons were treated with
compound 13b for 1 min. By the end of the 5 and 10 min
recordings, ICa decreased to −1.6356 0.0641 and −1.2738
0.882 nA, respectively, and the I−V curves were also shifted to
the negative direction by approximately 10 mV during the
recording. Unlike the results for ICa described above, the
maximal value of ICa did not shift to a negative direction when
the neurons were treated with 0.001 mM compound 13b, from
which we concluded that, when the concentration was ≤0.001
mM, there was no effect on calcium channels in the central
neurons of S. exigua third larvae.
60
20
70
90
90
30
30
100
40
20
chlorantraniliprole
100
100
100
of the compounds tested showed good larvicidal activities,
while compounds with big substituents i-amyl and cyclohexyl in
the aliphatic amide moiety revealed somewhat less activity.
Larvicidal Activity against Diamondback Moths (P.
xylostella). The larvicidal activities against diamondback
moths of compounds 15a−15e and chlorantraniliprole were
shown in Table 4. From it, we can see that most of the
compounds tested showed excellent larvicidal activities.
Compounds 15d and 15e showed the best activities (both
100% at 0.01 mg/L). Similarly, compound 15e with a large
substituent (R2 = benzyl) also showed good larvicidal activity
against diamondback moths.
The toxicity profile (LC50 values) of compounds 15d and
15e, which were found to be the most active insecticides of
these two series of compounds against diamondback moths
(100% at 0.01 mg/L), was shown in Table 5. The LC50 values
of compounds 15d and 15e were 0.000 25 and 0.000 65 mg/L,
These results indicated that Ca2+ channels of S. exigua
(L)
neurons were modulated by compound 13b and partly closed
after the action. Although there was no notable change on
activation voltage, the negatively shifted I−V relationship
curves indicate that voltage dependence was influenced by
compound 13b. All of these results suggest that the Ca2+
(L)
channels of S. exigua neurons were the possible target of
compound 13b.
Figure 4 illustrated the change of [Ca2+]i versus the recording
time when the neurons were treated with compounds 13b, 15a,
and chlorantraniliprole. The peak of [Ca2+]i was 116.863
2.33% (n = 10), 112.408
1.26% (n = 8), and 123.292
2.17% (n = 21) of the initial value by the end of the 10 min
recording when the cells were treated with 1000 mg/L
compound 13b, 1000 mg/L compound 15a, and 1000 mg/L
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Table 5. LC50 Values of Compounds 15d, 15e, and Chlorantraniliprole against Diamondback Moths
compound
y = a + bx
LC50 (mg/L)
R
95% confidence interval
15d
15e
y =11.04 + 1.68x
y =12.90 + 2.48x
y = 11.50 + 1.32x
0.00025
0.9974
0.9723
0.9995
0.00023−0.00028
0.00053−0.00081
0.00065
chlorantraniliprole
0.0000123
1.16 × 10−5−1.31 × 10−5
Figure 3. Current−voltage relationship curves of whole-cell calcium
channels recorded in neurons of S. exigua (treated with 0.1 mmol/L
compound 13b and chlorantraniliprole) at different intervals (in
minutes).
Figure 2. Variation of the peak current for the whole-cell calcium
channels in neurons (treated with different concentrations of
compound 13b and chlorantraniliprole) at different times during
patch-clamp recording in comparison to the peak value at 0 min.
chlorantraniliprole, respectively. In comparison to the control
(99.91 2.56%), compounds 13b and 15a induced a [Ca2+]i
increase without extracellular Ca2+. It indicated that compounds
13b and 15a could activate the calcium release channel in the
endoplasmic reticulum (ER) membrane. Figure 3 also indicated
that the recorded [Ca2+]i (F/F0) had a good positive
correlation with bioactivities.
There were two kinds of calcium release channels in the ER
membrane, namely, RyR and IP3R Ca2+ channels.26 To test
which pathway was involved in the elevation of [Ca2+]i, the
primary cultured neurons were dyed, loading with fluo-5N,
then treated with heparin (10 mg/mL, a competitive antagonist
of IP3) for 20 min, and incubated with 1 μM thapsigargin for 10
min. When external Ca2+ was free, IP3 receptors were blocked
using heparin and the intracellular calcium store was depleted
with thapsigargin; the decrease of [Ca2+]i was only attributed to
compound 13b (1000 mg/L). These data indicated that RyRs
would be the possible action target of this series of novel
compounds.
Figure 4. Change of [Ca2+]i versus recording time when the neurons
were treated with 1000 mg/L compounds 13b, 15a, and
chlorantraniliprole.
In summary, two novel series of anthranilic diamides
containing acylthiourea and acylurea moieties were synthesized,
Table 6. Analysis of the Peak Current Change Rate with Time in the Neurons of S. exigua (Treated by Compound 13b and
a
Chlorantraniliprole)
time (min)
0.1 mM compound 13b
0.001 mM compound 13b
0.1 mM chlorantraniliprole
100
control
0
1
100
100
100
b
c
b
b
c
91.492 3.47
104.964 1.34
89.62 2.16
104.47 2.33
102.75 2.45
b
b
b
2
84.128 2.16
97.215 7.58
74.01 8.01
b
b
b
c
3
80.727 4.77
95.803 7.26
69.57 9.43
101.76 2.7
b
b
b
c
4
74.437 5.31
90.134 6.41
63.36 7.71
99.66 1.64
98.06 1.01
90.85 0.65
85.44 1.14
b
b
b
c
5
70.252 8.48
84.442 5.48
55.79 4.73
b
b
b
b
b
8
61.013 3.28
81.209 3.69
44.31 2.62
b
b
b
10
54.715 4.95
74.632 6.12
39.685 5.02
a
b
c
Values are the mean SD. Significant difference at p < 0.01. Significant difference at p < 0.05.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H NMR, HRMS, and melting point data for compounds 13a−
13e, 14a−14e, 15a−15e, and 17a−17k. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: liyx128@nankai.edu.cn (Y.-X.L.); nkzml@vip.163.com
(Z.-M.L.).
■
Funding
This work has been supported by the National Basic Research
Program of China (973 Project 2010CB126106) and the
National Natural Science Foundation of China (NNSFC)
(31000861). We are also grateful for the financial support from
the National Key Technologies Research and Development
Program (2011BAE06B05), the Fundamental Research Funds
for the Central Universities, and the Natural Science
Foundation Project of Tianjin Ministry of Science and
Technology of China (11JCYBJC04500).
Notes
The authors declare no competing financial interest.
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