Discovery of a novel series of phenyl pyrazole inner salts based on fipronil as potential dual-target insecticides

Article abstract of DOI:10.1021/jf405512e

A series of novel phenyl pyrazole inner salt derivatives based on fipronil were designed and synthesized in the search for dual-target insecticides. These compounds were designed to target two families of nicotinic acetylcholine receptors and γ-aminobutyric acid receptors. The insecticidal activities of the new compounds against diamondback moth (Plutella xylostella) were evaluated. The results of bioassays indicated that most of the inner salts showed moderate to high activities, of which the phenyl pyrazole inner salts containing quinoline had excellent biological activity. Previous structure-activity relationship studies revealed that a suitable structure of the quaternary ammonium salts was critical for the bioactivity of phenyl pyrazole inner salts, which contribute to exposing the cationic nitrogen to bind to the receptor (for instance, nicotinic acetylcholine receptors) and possibly interact with the receptor via hydrogen bonding and cooperative π-π interaction. The present work demonstrates that the insecticidal potency of phenyl pyrazole inner salts holds promise for the development new dual-target phenyl pyrazole insecticides.

Full text of DOI:10.1021/jf405512e

Article
pubs.acs.org/JAFC
Discovery of a Novel Series of Phenyl Pyrazole Inner Salts Based on
Fipronil as Potential Dual-Target Insecticides
Dingxin Jiang,† Xiaohua Zheng,† Guang Shao,‡ Zhang Ling,† and Hanhong Xu*,†
†State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Key Laboratory of Natural Pesticide and
Chemical Biology, Ministry of Education, Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642,
People’s Republic of China
‡School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, 510275, People’s Republic of China
ABSTRACT: A series of novel phenyl pyrazole inner salt derivatives based on fipronil were designed and synthesized in the
search for dual-target insecticides. These compounds were designed to target two families of nicotinic acetylcholine receptors and
γ-aminobutyric acid receptors. The insecticidal activities of the new compounds against diamondback moth (Plutella xylostella)
were evaluated. The results of bioassays indicated that most of the inner salts showed moderate to high activities, of which the
phenyl pyrazole inner salts containing quinoline had excellent biological activity. Previous structure−activity relationship studies
revealed that a suitable structure of the quaternary ammonium salts was critical for the bioactivity of phenyl pyrazole inner salts,
which contribute to exposing the cationic nitrogen to bind to the receptor (for instance, nicotinic acetylcholine receptors) and
possibly interact with the receptor via hydrogen bonding and cooperative π−π interaction. The present work demonstrates that
the insecticidal potency of phenyl pyrazole inner salts holds promise for the development new dual-target phenyl pyrazole
insecticides.
KEYWORDS: fipronil derivatives, phenyl pyrazole inner salts, nicotinic acetylcholine receptors, γ-aminobutyric acid receptors,
dual-target insecticide
Compounds A, B, and C (Figure 1) have been synthesized
with significant bioactivities.11−14
INTRODUCTION
■
Fipronil is the first phenyl pyrazole insecticide introduced for
pest control. It is an outstanding insecticide with good
selectivity between insects and mammals that disrupts the
insect central nervous system (CNS) by blocking the passage of
chloride ions through the GABA receptor and glutamate-gated
chloride (GluCl) channels, components of the CNS.1 Fipronil
is highly toxic to insects but has only moderate acute toxicity by
oral and inhalation routes in rats.2,3 It has been widely used for
the protection of crops, such as rice and cotton, in China over
the past decade. However, it is worth noting that many insects
have developed high resistance to fipronil.4−6
To reduce insect resistance risk, many fipronil derivatives
have been synthesized7 and then commercialized, such as
ethiprole and vaniliprole (Figure 1). In 2004, Mitsubishi
Chemical reported two new fipronil derivatives, pyrafluprole
(V3039) and pyriprole (V3086) (Figure 1), with improved
synthesis method and better biological activity than fipronil. In
the meantime, RAJ Dalian Co. Ltd. developed the pesticide
flufiprole (Figure 1), which can be used to control pests on rice
and vegetables.8 However, Peng et al. found that the
Laodelphax striatellus populations resistant to fipronil had
cross-resistance to ethiprole, which has never been used in
China.9 Niu et al. reported that the resistance of fourth-instar
larvae to flufiprole increased 90.27-fold after its use to control
the diamondback moth, Plutella xylostella (L.), for 13
generations.10
Resistance of insect pests to insecticides is one of the most
serious problems in pest control, and the need for developing
new insecticides is not controversial. The dual-target
insecticides that affect two targets simultaneously always have
better bioactivity and are less prone to resistance than single-
target insecticide.15 Dual-target insecticides can modulate two
receptors, inhibit two enzymes, act on an enzyme and a
receptor, or affect an ion channel and a transporter. From the
viewpoint of molecular design, there are three approaches to
construct a dual-target insecticide molecule. A connective
molecule can simply be realized by combining two active
molecules or their pharmacophores with a linker, whereas an
integrated molecule comes into an entity either by fusing or by
merging the common structural or pharmacophoric features of
two active molecules, depending on the extent of the common
features.16
Nicotinic acetylcholine receptors (nAChRs) are thought to
be an excitatory neurotransmitter at synapses in the CNS of
insects and targets of a major class of insecticides, the
neonicotinoids.17 Generally, the quaternary ammonium salts
exhibit significant affinity to nAChRs.18−22 The quaternary
ammonium nAChR ligands, which act as agonists of the
nAChRs, might be a novel class of insecticide with decreased
In consideration of the 3-cyanogen group and 4-trifluor-
omethylsulfinyl group of phenyl pyrazole as key pharmaco-
phores, we have developed since 2000 several fipronil
derivatives by modifying the amino group on pyrazole.
Received: December 26, 2013
Revised: April 1, 2014
Accepted: April 1, 2014
Published: April 1, 2014
© 2014 American Chemical Society
3577
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of fipronil derivatives.
petroleum ether/ethyl acetate (3:1, v/v) to give clear colorless crystals
(mp 105−107 °C, yield = 72%). 1H NMR (CDCl3, 600 MHz) δ 9.22
(s, 1H, NH), 7.77 (s, 2H, ArH), 4.12 (s, 2H, CH2); 13C NMR (150
MHz, CDCl3) δ 171.37, 164.07, 141.38, 136.03, 135.58, 134.80,
134.57, 134.36, 126.59, 126.57, 126.18, 109.64, 107.90, 26.63; ESI-MS,
m/z (%) 559 [M + H]+ (40), 581 [M + Na]+ (100), 597 [M + K]+
(28).
side effects due to the reduced ability to penetrate the blood−
brain barrier.
To develop dual-target insecticides that act on GABAAR and
nAChRs, we herein report the synthesis and structure−activity
relationships (SARs) of phenyl pyrazole inner salts of fipronil
derivatives (1a−1g) and phenyl pyrazole amido (hydantoin)
derivatives (2a−2d). 1a−1g compounds, containing the phenyl
pyrazole structure of fipronil and quaternary ammonium
structure, would probably act on the two targets GABAAR
and nAChRs.23−29
General Procedure for the Synthesis of Compounds 1a−1g.
Compound 1 (3.00 g, 0.0054 mol) was dissolved in 15 mL of THF in
a 150 mL round-bottom flask equipped with a magnetic stirrer and a
calcium tube. Then 0.01 mol of tertiary amine or nitrogen heterocycles
in 10 mL of THF was dropped into the solution and stirred for 2−4 h
at room temperature. The reaction mixture was evaporated in vacuo.
The residue was purified by chromatography eluting using ethyl
acetate/methanol and afforded the desired products 1a−1g.
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(Trimethylammonio)
acetyl)amide (1a). Column chromatography (ethyl acetate/methanol
= 7:1 → 6:1, v/v) afforded compound 1a as a clear colorless crystal:
MATERIALS AND METHODS
General. All melting points were obtained with a RY-1G melting
■
point apparatus (Tianjin Tianguang Optical Instruments Co. Ltd.,
1
China) and are uncorrected. H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE 600 (600 MHz) or Bruker AC-P400Q
instrument. ESI-MS was recorded on AccuTOF CS JMS-T100CS
(JEOL), and EI-MS was recorded on DSQ (Thermo). EI-HRMS was
recorded on MAT95XP (Thermo). ESI-HRMS was recorded on LTQ
Orbitrap Elite (Thermo Fisher) or ESI-Q-TOF (Bruker Daltonics).
General Procedure for the Synthesis of Intermediate 2-
Bromo-N-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-
4-(trifluoromethylsulfinyl)-1H-pyrazol-5-yl) Acetamide (Com-
pound 1). Almost 98% fipronil (4.4 g, 0.01 mol) was dissolved in
anhydrous tetrahydrofuran (40 mL), to which sodium hydride (0.8 g
of 36% oil dispersion) was added three times below 0 °C under
nitrogen and stirred below 10 °C. After 3 h, bromoacetyl bromide (7
mL) was added and stirred at room temperature for another 3 h. The
reaction mixture was filtered and the filtrate evaporated in vacuo. The
solid residue was purified by chromatography and eluted with
1
mp 199−201 °C; yield = 89%. H NMR (600 MHz, (CD3)2CO) δ
8.05 (s, 2H, ArH), 3.95 (d, 1H, J = 15.6 Hz, CH2-1), 3.90 (d, 1H, J =
15.6 Hz, CH2-2), 3.35 (s, 9H, 3 × CH3); 13C NMR (150 MHz,
(CD3)2CO) δ 167.1, 152.6, 139.3, 136.8, 134.0, 133.8, 133.5, 128.8,
126.8, 126.6, 126.1, 124.3, 122.5, 113.6, 104.2, 68.8, 53.9; EI-MS, m/z
(%) 535 [M]+ (80); EI-HRMS for C17H13Cl2F6N5O2S [M]+, calcd
535.0066, found 535.0061.
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(Triethylammonio)-
acetyl)amide (1b). Column chromatography (ethyl acetate/methanol
= 8:1 → 6:1, v/v) afforded compound 1b as a clear colorless crystal:
mp 180−182 °C; yield = 75%. 1H NMR (600 MHz, MeOD) δ 8.03 (s,
3578
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
2H, ArH), 4.82 (s, 2H, CH2), 3.63−3.72 (m, 2H, CH2), 3.37−3.41
(m, 4H, 2CH2), 1.14−1.17 (m, 9H, 3 × CH3); 13C NMR (150 MHz,
CDCl3) δ 167.5, 152.9, 139.3, 137.5, 137.4, 135.3, 135.1, 134.8, 134.6,
127.1, 127.1, 126.4, 124.6, 124.3, 122.8, 113.4, 104.9, 61.0, 54.7, 7.8;
EI-MS, m/z (%) 577 [M]+ (30); EI-HRMS for C20H19O2N5Cl2F6S
[M]+, calcd 577.0535, found 577.0534. (The single crystal data are
available from Cambridge Crystallographic Data Centre (CCDC
975075).)
63.4; ESI-MS, m/z (%) 569 [M]+ (100); ESI-HRMS for
C20H11Cl2F6N5O2S [M + H]+, calcd 569.9987, found 570.0012.
General Procedure for the Synthesis of Compounds 2a−2d.
N-(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)-2-(2,4-dichlorophenoxy)
Acetamide (2a). 2,4-Dichlorophenoxyacetyl chloride was synthesized
according to a method from the literature.30 Almost 98% fipronil (4.4
g, 0.01 mol), triethylamine (0.013 mol), and N,N-dimethylaminopyr-
idine (DMAP) (0.001 mol) dissolved in chloroform (15 mL) were
added to 2,4-dichlorophenoxyacetic chloride (2.14 g, 0.009 mol). The
reaction mixture was stirred at reflux temperature for about 3 h and
monitored by TLC. After cooling to room temperature, the reaction
mixture was filtered, and dichloromethane (20 mL) was added and
washed with 1 M HCl (10 mL) and water (2 × 20 mL). The organic
extracts were dried with anhydrous sodium sulfate and then
evaporated in vacuo. The solid residue was purified by column
chromatography by petroleum ether and ethyl acetate to give clear
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(Tributylammonio)-
acetyl)amide (1c). Column chromatography (ethyl acetate/methanol
= 10:1 → 7:1, v/v) afforded compound 1c as a clear colorless crystal:
1
mp 221−223 °C; yield = 54%. H NMR (600 MHz, (CD3)2SO) δ
8.27 (s-like, 1H, ArH), 8.26 (s-like, 1H, ArH), 3.75 (d, 1H, J = 15.6
Hz, CH2-1), 3.70 (d, 1H, J = 15.6 Hz, CH2-2), 3.23−3.25 (m, 6H, 3 ×
CH2), 1.45−1.49 (m, 6H, 3 × CH2), 1.11−1.45 (m, 6H, 3 × CH2)
1.11 (t, 9H, J = 7.2 Hz, 3 × CH3); 13C NMR (150 MHz, (CD3)2SO) δ
206.4, 166.6, 151.2, 137.7, 135.2, 135.1, 126.2, 125.3, 112.7, 60.6, 58.1,
30.6, 23.1, 19.1, 13.4; ESI-MS, m/z (%) 662 [M + H]+ (80), 684 [M +
Na]+ (100); ESI-HRMS for C27H32O4N5Cl2F6S [M + HCOO]−, calcd
706.14617, found 706.14694.
1
colorless crystals (yield = 46%): mp 161−163 °C. H NMR (400
MHz, DMSO-d6) δ 8.35, 8.31 (2H, s, CF3C6H2Cl2), 7.57 (1H, d, J =
2.0 Hz, 3-C6H3Cl2), 7.34 (1H, dd, J = 2.0, 8.0 Hz, 5-C6H3Cl2), 6.91
(1H, d, J = 8.0 Hz, 6-C6H3Cl2), 4.90, 4.85 (2H, d, J = 16.0 Hz, CH2);
ESI-MS, m/z (%) 638 (100) [M + H]+, 641 [M + H + 2]+, 643 [M +
H + 4]+.
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(Quinolin-1-ium-1-yl)-
acetyl)amide (1d). Column chromatography (ethyl acetate/methanol
= 12:1 → 10:1, v/v) afforded compound 1d as a clear colorless crystal:
N-(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)-2-(2,4,5-trichlorophe-
noxy) Acetamide (2b). Compound 2b was synthesized according to
the same method as used for 2a. Compound 2b was colorless crystals
1
mp 243−245 °C; yield = 83%. H NMR (600 MHz, MeOD) δ 9.15
(dd, 1H, J = 6.0, 1.2 Hz, quinoline), 8.93 (d, 1H, J = 7.8 Hz,
quinoline), 8.24 (d, 1H, J = 8.4 Hz, quinoline), 8.11−8.12 (m, 2H,
quinoline), 7.97−7.94 (m, 2H, quinoline), 7.86 (d, 1H, J = 8.4 Hz,
quinoline), 7.84 (d, 1H, J = 8.4 Hz, quinoline), 7.60 (d, 1H, J = 1.2 Hz,
Ar-H), 7.57 (d, 1H, J = 1.8 Hz, Ar-H), 5.57 (d, 1H, J = 16.8 Hz, CH2-
1), 5.51 (d, 1H, J = 16.8 Hz, CH2-2); 13C NMR (150 MHz, MeOD) δ
169.2, 152.2, 151.0, 148.3, 140.4, 138.5, 136.8, 136.6, 131.4, 131.1,
130.8, 126.8, 126.5, 122.3, 120.2, 113.3, 62.7; ESI-MS, m/z (%) 606
[M + H]+ (100), 628 [M + Na]+ (70); ESI-HRMS for
C24H14O3N5Cl2F6S [M + CH3O]−, calcd 636.01041, found 636.01105.
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(Pyridine-1-ium-1-yl)-
acetyl)amide (1e). Column chromatography (ethyl acetate/methanol
= 9:1 → 7:1, v/v) afforded compound 1e as a clear colorless crystal:
1
(yield = 34%): mp 147−149 °C. H NMR (400 MHz, DMSO-d6) δ
11.63 (1H, s, NH), 8.40, 8.36 (2H, s, CF3C6H2Cl2), 7.84, 7.25 (2H, s,
6,3-C6H2Cl3), 4.98 (2H, s, CH2); ESI-MS, m/z (%) 672 (100) [M +
H]+, 675 [M + H + 2]+.
N-((3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)carbamoyl)-2-(2,4-di-
chlorophenoxy) Acetamide (2c). 2-(2,4-Dichlorophenoxy) acetamide
and 2-(2,4-dichlorophenoxy) acetyl isocyanate was synthesized
according to a method from the literature.30 2-(2,4-Dichlorophenoxy)
acetyl isocyanate (2.5 g, 0.01 mol) was dissolved in anhydrous diethyl
ether (50 mL), to which fipronil (4.4 g, 0.01 mol) dissolved in
anhydrous diethyl ether (20 mL) was added below 0 °C, stirred at
room temperature, and monitored by TLC. The reaction mixture was
filtered and the filtrate evaporated in vacuo. The solid residue was
purified by chromatography and eluted with petroleum ether/ethyl
acetate to give colorless crystals (yield = 57%): mp 142−144 °C. 1H
NMR (400 MHz, CDCl3) δ 10.96, 8.94 (2H, s, 2NH), 7.86−7.83 (2H,
s, CF3C6H2Cl2), 7.46 (1H, d, J = 2.2 Hz, 3-C6H3Cl2), 7.27 (1H, dd, J =
2.2, 8.0 Hz, 5-C6H3Cl2), 6.85 (1H, d, J = 8.0 Hz, 6-C6H3Cl2), 4.64
(2H, s, CH2); ESI-MS, m/z (%) 685 [M + H + 4]+ (50), 684 [M + H
+ 2]+ (100), 681 [M + H]+ (80).
1
mp 225−227 °C; yield = 81%. H NMR (600 MHz, (CD3)2SO) δ
8.96 (s-like, 2H, pyridine), 8.63−8.64 (m, 1H, pyridine), 8.41−8.35
(m, 2H, pyridine), 8.14 (brs, 2H, benzene), 4.25 (brs, 2H, CH2); 13C
NMR (150 MHz, (CD3)2SO) δ 146.3, 135.5, 134.9, 127.7, 127.5,
127.2, 126.9, 126.8, 125.5, 124.9, 124.6, 123.1, 121.2, 119.4, 111.0,
61.9; ESI-MS, m/z (%) 556 [M + H]+ (100); ESI-HRMS for
C19H8O2N5Cl2F6S [M − H]−, calcd 553.96854, found 553.96906.
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(4-(Dimethylamino)-
pyridine-1-ium-1-yl)acetyl)amide (1f). Column chromatography
(ethyl acetate/methanol = 8:1 → 7:1, v/v) afforded compound 1f as
a clear colorless crystal: mp 214−216 °C; yield = 62%. 1H NMR (600
MHz, (CD3)2SO) δ 8.11 (s, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 7.98 (d,
2H, J = 7.8 Hz, pyridine-H), 6.78 (d, 2H, J = 7.8 Hz, pyridine-H), 4.71
(d, 1H, J = 16.2 Hz, CH2-1), 4.64 (d, 1H, J = 16.8 Hz, CH2-2), 3.12 (s,
6H, 2 × NCH3); 13C NMR (150 MHz, (CD3)2SO) δ 169.6, 156.9,
155.4, 151.7, 143.0, 139.1, 137.5, 135.2, 135.0, 127.4, 125.6, 125.2,
125.0, 123.1, 121.3, 112.8, 106.9, 106.1, 60.5; ESI-MS, m/z (%) 599
[M + H]+ (10); ESI-HRMS for C22H15O4N6Cl2F6S [M + HCOO]−,
calcd 643.01622, found 643.01703.
N-((3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)carbamoyl)-2-(2,4,5- tri-
chlorophenoxy) Acetamide (2d). Compound 2d was synthesized
according to the same method as used for 2c. Compound 2d was
1
colorless crystals (yield = 37%): mp 101−103 °C. H NMR (400
MHz, CDCl3) δ 10.87, 8.85 (2H, s, 2NH), 7.81, 7.78 (2H, s,
CF3C6H2Cl2), 7.51 (1H, s, 6-C6H2Cl3), 6.97 (1H, s, 3-C6H2Cl3), 4.60
(2H, s, CH2); ESI-MS, m/z (%) 717 [M + H + 2]+ (100), 715 [M +
H]+ (60).
Bioactivity Assay against Plutella xylostella. Rearing Methods
for P. xylostella. Healthy larvae of P. xylostella were collected from the
experimental farm of Dongguan Agricultural Science Research Center,
Guangdong, China, and were reared on Chinese cabbage (Brassica
rapa) under cage conditions of 24−29 °C, 70−80% relative humidity,
and photoperiod 16:8 h light/dark for the third-instar larvae.
Assessment of Bioactivity on P. xylostella. The bioactivities of
phenyl pyrazole derivatives and fipronil against the third-instar larvae
of P. xylostella were determined by the leaf disk-dipping assay. Leaves
of Chinese cabbage grown in the greenhouse were collected, and disks
(5.5 cm diameter) were punched from each leaf. The compounds were
dissolved in acetone and suspended in distilled water containing
Triton X-100. Leaf disks were dipped in each test solution for 30 s and
allowed to dry for 2 h. The treated leaf disks were placed into Petri
(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-
((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(4-Methylpyridin-1-
ium-1-yl)acetyl)amide (1g). Column chromatography (ethyl ac-
etate/methanol = 7:1 → 6:1, v/v) afforded compound 1g as a clear
1
colorless crystal: mp 208−210 °C; yield = 43%. H NMR (600 MHz,
(CD3)2CO) δ 8.97 (d, 2H, J = 6.6 Hz, pyridine-H), 8.05 (d, 2H, J =
6.0 Hz, pyridine-H), 8.00 (d, 2H, J = 0.6 Hz, Ar-H), 5.88 (d, 1H, J =
16.2 Hz, CH2-1), 5.79 (d, 1H, J = 16.8 Hz, CH2-2), 2.09 (s, 3H, CH3);
13C NMR (150 MHz, (CD3)2CO) δ 166.8, 161.0, 145.9, 137.3, 136.8,
136.5, 134.5, 128.7, 128.2, 127.2, 127.0, 126.0, 124.2, 122.4, 112.3,
3579
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
a
Scheme 1. Synthesis of 1a−1g
a
Reagents and conditions: (a) bromoacetyl bromide, NaH, THF; (b) tertiary amine or nitrogen heterocyclic compounds, THF.
a
Scheme 2. Synthesis of 2a−2d
a
Reagents and conditions: (a) SOCl2; (b) fipronil, triethylamine, DMAP, CHCl3; (c) NH3·H2O; (d) (COCl)2, 1,2-dichloroethane; (e) fipronil,
anhydrous diethyl ether.
dishes (9 cm diameter). Then, 10 P. xylostella larvae were introduced
into each dish. Distilled water containing an acetone−Triton X-100
solution but not the test compound was used as the control. Petri
dishes were kept in an incubator at 26 °C and 85% relative humidity
under a photoperiod of 16:8 h light/dark. All treatments were
replicated five times. Mortalities were determined 24 h after treatment.
The death rate of each treatment group was confirmed. The LC50
value was calculated by the SPSS software.
Docking Study. The Musca domestica GABA receptor subunit
sequence was obtained from the NCBI database (GenBank
AAC23602.1). The sequence and structure of the nicotinic acetylcho-
line receptor (nAChR) were obtained from the RSCB protein data
bank at 4 Å resolutions (PDB ID 2BG9). The amino acid sequence of
the subunit was edited to remove the extracellular region and residues
in the loop between transmembrane (TM) domains 3 and 4 (TM3−
TM4 loop). Using the sequence analysis program above, sequence
alignment was carried out. The sequence and structure of the nicotinic
3580
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
acetylcholine receptor (nAChR) were obtained from the RSCB
protein data bank at 4 Å resolutions (PDB ID 3C79). The models of
the transmembrane region of the GABA receptor subunit were built by
homology modeling using the MODELER program with the default
parameters. In the model building, we employed an optimization
method involving conjugate gradients and molecular dynamics to
minimize violations of the spatial restraints. All molecule modeling was
done using Discovery Studio 2.5 on SGI workstation. The initial
structures of these compounds were built and energetically minimized.
To investigate how the insecticide anchored at the putative binding
cavity, fipronil and phenyl pyrazole inner salts were docked into the
ion channel pore formed by the second transmembrane segments of
the homooligomeric receptor. Binding site interactions of compounds
and AChBP are simulated with Aplysia californica AChBP on the basis
of its structure cocrystallized with bound IMI (Protein Data Bank code
3C79). The A−E subunit interface of the AChBP−IMI complex is
arbitrarily used for modeling because the homomeric AChBP
pentamer has five identical binding pockets localized at subunit
interfacial regions. The compound is docked into the ligand-binding
pocket of IMI. All molecule dockings were done using the CDOCK
program with the default parameters. The docking complexes were
solvated in the water layer in a truncated octahedral periodic box.
Then, the complexes were minimized using the molecular dynamics
simulation.
Figure 2. Model of compound 1d in the GABAA receptor β3
homopentamer binding site. It shows a model of the ion channel with
five helices and compounds docked into the putative binding site,
which it clearly fills to block the pore. Compounds interact with
resides of TM2 and contact A2, T6, and L9 coinciding with mutation
experiments.
RESULTS AND DISCUSSION
■
Synthesis. The synthesis of compounds 1a−1g and 2a−2d
was outlined in Schemes 1 and 2. Fipronil was reacted with
Table 1. 24 h Effect of Phenyl Pyrazole Inner Salts against
Third-Instar Larvae of Diamondback Moth (Plutella
xyllostella L.)
LC50
95% confidence interval of
NR1R2R3 (R)
(μg mL−1
)
LC50 (μg mL−1
)
Figure 3. Simulated binding site interactions of compound 1d with the
Aplysia californica AChBP structural surrogate of the extracellular
domain of the nAChR. The structure of Aplysia californica AChBP
cocrystallized with bound IMI (Protein Data Bank code 3C79) shows
the binding site of IMI was located in the interface of the AChBP
subunit. Simulated docking shows docking compounds in the same
site formed π−π stacking interactions and hydrogen bonding.
compd
1a
1b
1c
1d
1e
1f
trimethylamine
triethylamine
tri-n-butylamine
quinoline
15.10
20.84
24.69
8.31
9.92−23.01
13.61−31.92
14.16−43.06
5.84−11.82
9.05−18.24
14.73−41.46
pyridine
12.85
24.71
N,N-
dimethylpyridin-4-
amine
1g
2a
2b
4-methylpyridine
18.55
58.58
56.00
12.57−27.38
26.78−128.15
28.71−109.26
SAR. The preliminary insecticidal activities of compounds
1a−1g and 2a−2d were assessed by the leaf disk-dipping assay,
and the results are listed in Table 1. Due to the low insecticidal
activities of 2a−2d, the SAR study does not include the
compounds 2a−2d.
For the alkyl quaternary ammonium 1a−1c, the activities
decreased from 15.10 μg mL−1 (LC50 value, 24 h) to 24.69 μg
mL−1 (LC50 value, 24 h) against P. xylostella, respectively,
whereas the bulk of R1, R2, and R3 increased. The activity of
compound 1a, 15.10 μg mL−1 (24 h LC50 value), was better
than that of fipronil, 24.93 μg mL−1 (24 h LC50 value), against
P. xylostella, indicating that the steric hindrance of NR1R2R3 is
unfavorable for the insecticidal activity of the phenyl pyrazole
inner salts.
2,4-dichlorobenzene
2,4,5-
trichlorobenzene
2c
2,4-dichlorobenzene
153.52
210.00
17.68−1333.19
10.93−4034.25
2d
2,4,5-
trichlorobenzene
fipronil
24.93
16.55−37.54
bromoacetyl bromide in the presence of sodium hydride to
afford the key intermediate 1. Compounds 1a−1g were
obtained via the reaction of intermediate 1 and tertiary amine
or nitrogen heterocyclic compounds in the presence of
tetrahydrofuran. Compounds 2a−2d were obtained by using
the classical methodology.
3581
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
Compounds 1d−1g are heterocyclic quaternary ammoniums.
It is clear that the insecticidal activity obviously increased when
the ring of NR1R2R3 was enlarged. For instance, compounds 1d
(10-member bicyclic aromatic ring, 8.31 μg mL−1, 24 h LC50
value) and 1e (6-member ring, 12.85 μg mL−1, 24 h LC50
value) have better activity than fipronil (24.93 μg mL−1, 24 h
LC50 value) against P. xylostella. This result is opposite to
traditional agonists, as the increase of the bulk of R1, R2, and R3
weakens the activity significantly.31 This may confirm the effect
of the unique heterocyclic quaternary ammonium conforma-
tion, possibly due to the influence of the size of cycle on the
conformation. Therefore, an appropriate conformation is
critical for the interaction between cationic nitrogen and
receptor.
With the pyridine group considered to be the key
pharmacophore in 1e, compounds 1f and 1g were synthesized.
The bioassay data on compounds 1e, 1f, and 1g revealed that a
large substituent at the 4-position of the 6-member NR1R2R3
ring is not preferred. A substitution at the 4-position of the 6-
member NR1R2R3 ring is unfavorable for the insecticidal
activity, and steric hindrance may weaken the activity. The
bioactivity of compound 1f (NR1R2R3 = N,N-dimethylpyridin-
4-amine) was similar to that of fipronil, but 1g (NR1R2R3 = 4-
methylpyridine) was more active than fipronil with an
insecticidal activity of 18.55 μg mL−1 (24 h LC50 value).
These results suggested that the substituent at the 4-position of
the 6-member NR1R2R3 ring might bind to the receptor as an
H-bond acceptor.
Furthermore, the influence of the substituted groups of the 4-
pyridine ring on insecticidal activity was explored. As compared
to compound 1e (12.85 μg mL−1, 24 h LC50 value) without any
substituted group, the electron-donating group (1f, 4-N(CH3)2,
24.71 μg mL−1, 24 h LC50 value, and 1g, 4-CH3, 18.55 μg mL−1,
24 h LC50 value) at the 4-position was unfavorable for
insecticidal activity.
Because of the special conformation of the heterocyclic
quaternary ammonium structure, substitution at the 4-position
of the pyridine ring was unfavorable for cooperative π−π
interaction.32 This may confirm the effect of the special
heterocyclic quaternary ammonium conformation.
The structure of A. californica AChBP cocrystallized with
bound IMI (Protein Data Bank code 3C79) shows the binding
site of IMI was located in the interface of the AChBP subunit.
Simulated docking shows docking compounds into the same
site formed π−π stacking interactions and hydrogen bonding
(Figure 3).35 The interaction was not the same with IMI.
In summary, by selecting phenyl pyrazole inner salts of
fipronil derivatives as the lead compounds, a series of phenyl
pyrazole inner salts were designed and synthesized to search for
a new dual-target insecticide with low side effects. Extensive
SAR studies revealed that a suitable NR1R2R3 was critical for
the insecticidal activity, which might be beneficial to expose the
cationic nitrogen to bind to the receptor and possibly interact
with the receptor via hydrogen bonding and cooperative π−π
interaction. More importantly, compound 1d, a new type of
quinoline quaternary ammonium salt, showed potent insecti-
cidal activity, which would be very valuable for the further
development of dual-target insecticides. The action mechanism
of dual-target phenyl pyrazole inner salts deserves further study.
AUTHOR INFORMATION
Corresponding Author
*(Hanhong Xu) Tel.: +86-20-85285127; E-mail: hhxu@scau.
edu.cn.
■
Funding
This research was supported by the National Natural Science
Foundation of China (No. 31171871), the Guangdong Natural
Science Foundation (No. S2011010001141), and the Research
Foundation for Talented Scholars (No. (2010)79).
Notes
The authors declare no competing financial interest.
REFERENCES
■
(1) Moffat, A. S. New chemicals seek to outwit insect pests. Science
1993, 261, 550−551.
(2) Ikeda, T.; Zhao, X.; Nagata, K.; Kono, Y.; Shono, T.; Yeh, J. Z.
Fipronil modulation of γ-aminobutyric acid (A) receptors in rat dorsal
root ganglion neurons. J. Pharmacol. Exp. Ther. 2001, 296, 914−921.
(3) Hainzl, D.; Casida, J. E. Fipronil insecticide: novel photochemical
desulfinylation with retention of neurotoxicity. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 12764−12767.
(4) Li, A.; Yang, Y.; Wu, S.; Li, C.; Wu, Y. Investigation of resistance
mechanisms to fipronil in diamondback moth (Lepidoptera
Plutellidae). J. Econ. Entomol. 2006, 99, 914−919.
(5) Kristensen, M.; Hansen, K. K.; Jensen, K. M. Cross-resistance
between dieldrin and fipronil in German cockroach (Dictyoptera
Blattellidae). J. Econ. Entomol. 2005, 98, 1305−1310.
(6) Tang, J.; Li, J.; Shao, Y.; Yang, B. J.; Liu, Z. W. Fipronil resistance
in the white backed planthopper (Sogatella furcifera): possible
resistance mechanisms and cross-resistance. Pest Manage. Sci. 2010,
66, 121−125.
On the contrary, 1e with pyridine for NR1R2R3 exhibited
excellent insecticidal activity (12.85 μg mL−1, 24 h LC50 value)
and may have a special conformation to bind to receptors.
Inspired by this result, we introduced quinoline to the fipronil
molecule and obtained 1d with a rather good insecticidal
activity (1d, 8.31 μg mL−1, 24 h LC50 value). The insecticidal
potency of 1d and 1e indicated that a quinoline or pyridine ring
at a proper position of NR1R2R3 was preferred, which might
contribute to affinity with a suitable conformation and/or
hydrogen bonding and cooperative π−π interaction.33
Docking Study. To clarify the binding mode of our
synthesized phenyl pyrazole inner salts, compounds were
docked in the active site of the GABAA receptor recombinant
β3 homopentamer and the crystal structures of the acetylcho-
line binding protein (AChBP) (Figure 2).34 The fipronil-related
phenyl heterocyclic compounds were effective as noncompe-
titive antagonists to the housefly GABA receptor. Several
observations suggest that the noncompetitive antagonists
interact with the same position of the M2 transmembrane
segment region on the cytoplasmic side [A]. As a channel
blocker, the fipronil-related noncompetitive antagonists interact
with the ion channel associated with the β3-homopentamer.
(7) Ozoe, Y.; Yagi, K.; Nakamura, M.; Akamatsu, M.; Miyake, T.;
Matsumura, F. Fipronil-related heterocyclic compounds: structure-
activity relationships for interaction with γ-aminobutyric acid-and
voltage-gated ion channels and insecticidal action. Pestic. Biochem.
Physiol. 2000, 66, 92−104.
(8) Zhang, Y. B.; Zhang, Y. New Progress of Pesticide in World (in
Chinese); Chemical Industry: Beijing, China, 2006; p 152.
(9) Peng, Y. Q.; Gao, C. F.; Ma, C. Y.; Mao, Y. X.; Shen, J. L. Risk
assessment and genetic analysis of fipronil resistance and insecticide
susceptibility in the small brown planthopper, Laodelphax striatellus
(Homoptera: Delphacidae) (in Chinese). Chin. J. Rice Sci. 2009, 23,
645−652.
(10) Niu, H. T.; Luo, W. C.; Zong, J. P.; Wei, S. J.; Wang, H. Y.; Pan,
Z. X. Realized heritability of resistance to butene-fipronil in
3582
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583

Journal of Agricultural and Food Chemistry
Article
diamondback moth, Plutella xylostella (in Chinese). Acta Phytophyla-
cica Sinica 2008, 35, 165−168.
(11) Jiang, D. X.; Lu, X. L.; Hu, S.; Zhang, X. B.; Xu, H. H. A new
derivative of fipronil: effect of adding a glycinyl group to the 5-amine
of pyrazole on phloem mobility and insecticidal activity. Pestic.
Biochem. Physiol. 2009, 95, 126−130.
(12) Zheng, X. H.; Jiang, D. X.; Liu, Z. C.; Xu, H. H. Synthesis,
characterization and bioactivity of fipronil derivatives as a lead for new
insecticide. Indian J. Chem. Technol. 2010, 17, 215−219.
(13) Xu, H. H.; Jiang, D. X.; Hu, S.; Zhang, X. W.; Liu, Z. C.; Zheng,
X. H. Fipronil-GABA conjugate − a phloem mobile insecticide against
sucking insect. 4th Pan Pacific Conference on Pesticide Science, Hawaii,
USA; American Chemical Society: Washington DC, 2008; p 39.
(14) Xu, H. H.; Jiang, D. X.; Hu, S. Synthesis of guided pesticide and
their biological activities. 11th IUPAC International Congress of Pesticide
Chemistry, Kobe, Japan; Pesticide Science Society of Japan: Tokyo,
Japan, 2006; p 26.
2-(4-chlorophenyl) butyramide derivatives. Chem. Res. Chinese Univ.
2010, 26, 929−932.
(31) Lin, S. W.; Sun, Q.; Ge, Z. M.; Wang, X.; Ye, J.; Li, R. T.
Synthesis and structure-analgesic activity relationships of a novel series
of monospirocyclopiperazinium salts (MSPZ). Bioorg. Med. Chem. Lett.
2011, 21, 940−943.
(32) Ye, Z. J.; Xia, S.; Shao, X. H.; Cheng, J. G.; Xu, X. Y.; Xu, Z. P.
Design, synthesis, crystal structure analysis, and insecticidal evaluation
of phenylazoneonicotinoid. J. Agric. Food Chem. 2011, 59, 10615−
10623.
(33) Wang, Y. L.; Cheng, J. G.; Qian, X. H.; Li, Z. Actions between
neonicotinoids and key residues of insect nAChR based on an ab initio
quantum chemistry study: hydrogen bonding and cooperative π-π
interaction. Bioorg. Med. Chem. 2007, 15, 2624−2630.
(34) Chen, L.; Durkin, K. A.; Casida, J. E. Structural model for γ-
aminobutyric acid receptor noncompetitive antagonist binding: widely
diverse structures fit the same site. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 5185−5190.
(15) Guo, Y. S.; Guo, Z. R. Design of multiple targeted drugs (in
Chinese). Acta Pharm. Sinica 2009, 44, 276−281.
(35) Ohno, I.; Tomizawa, M.; Durkin, K. A.; Casida, J. E.; Kagabu, S.
Bis-neonicotinoid insecticides: observed and predicted binding
interactions with the nicotinic receptor. Bioorg. Med. Chem. Lett.
2009, 19, 3449−3452.
(16) Guo, Z. R. Strategy of molecular drug design: dual-target drug
design (in Chinese). Acta Pharm. Sinica 2009, 44, 209−218.
(17) Jones, A. K.; Sattelle, D. B. Diversity of insect nicotinic
acetylcholine receptor subunits. Adv. Exp. Med. Biol. 2010, 683, 25−43.
(18) Kizawa, Y.; Takayanagi, I. Nicotinic stimulant, DMPP (1,1-
dimethyl-4-phenylpiperazinium iodide) on the isolated bronchial
smooth muscle preparation of guinea-pig. Gen. Pharmacol. 1984, 15,
149−150.
(19) Lippiello, P. M.; Fernandes, K. G. The binding of L-[3H]
nicotine to a single class of high affinity sites in rat brain membranes.
Mol. Pharmacol. 1986, 29, 448−454.
(20) Romanelli, M. N.; Manetti, D.; Scapecchi, S.; Borea, P. A.; Dei,
S.; Bartolini, A. Structure-affinity relationships of a unique nicotinic
ligand: N(1)-dimethyl-N(4)-phenylpiperazinium iodide (DMPP). J.
Med. Chem. 2001, 44, 3946−3955.
(21) Simsek, R.; Chang, F. J.; Lee, M.; Dukat, M.; Damaj, M. I.;
Martin, B. R. Quaternary ammonium 3-(aminoethoxy)pyridines as
antinociceptive agents. Bioorg. Med. Chem. Lett. 2003, 13, 2917−2920.
(22) Gundisch, D.; Andra, M.; Munoz, L.; Tilotta, M. C. Synthesis
̈
̈
and evaluation of phenylcarbamate derivatives as ligands for nicotinic
acetylcholine receptors. Bioorg. Med. Chem. 2004, 12, 4953−4962.
(23) Toda, N.; Tago, K.; Marumoto, S.; Takami, K.; Ori, M.;
Yamada, N. A conformational restriction approach to the development
of dual inhibitors of acetylcholinesterase and serotonin transporter as
potential agents for Alzheimer’s disease. Bioorg. Med. Chem. 2003, 11,
4389−4415.
(24) Kogen, H.; Toda, N.; Tago, K.; Marumoto, S.; Takami, K.; Ori,
M. Design and synthesis of dual inhibitors of acetylcholinesterase and
serotonin transporter targeting potential agents for Alzheimer’s
disease. Org. Lett. 2002, 4, 3359−3362.
(25) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti,
V.; Recanatini, M. Multi-target-directed ligands to combat neuro-
degenerative diseases. J. Med. Chem. 2008, 51, 347−372.
(26) Meunier, B. Hybrid molecules with a dual mode of action:
dream or reality? Acc. Chem. Res. 2008, 41, 69−77.
(27) Woo, L. W. L.; Bubert, C.; Purohit, A.; Potter, B. V. L. Hybrid
dual aromatase-steroid sulfatase inhibitors with exquisite picomolar
inhibitory activity. ACS Med. Chem. Lett. 2011, 2, 243−247.
(28) Wenzel, N. I.; Chavain, N.; Wang, Y.; Friebolin, W.; Maes, L.;
Pradines, B. Antimalarial versus cytotoxic properties of dual drugs
derived from 4-aminoquinolines and Mannich bases: interaction with
DNA. J. Med. Chem. 2010, 53, 3214−3226.
(29) Detsi, A.; Bouloumbasi, D.; Prousis, K. C.; Koufaki, M.;
Athanasellis, G.; Melagraki, G. Design and synthesis of novel
quinolinone-3-aminoamides and their alpha-lipoic acid adducts as
antioxidant and anti-inflammatory agents. J. Med. Chem. 2007, 50,
2450−2458.
(30) Liu, L.; Xue, S. J.; Bu, H. F.; Chen, L. Synthesis and biological
activities of 2,4-dichlorophenoxyacetyl(thio)urea and S-(+)-3-methyl-
3583
dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583