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
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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 In consideration of the 3-cyanogen group and 4-trifluoromethylsulfinyl group of phenyl pyrazole as key pharmacophores, we have developed since 2000 several fipronil derivatives by modifying the amino group on pyrazole. © 2014 American Chemical Society
Compounds A, B, and C (Figure 1) have been synthesized with significant bioactivities.11−14 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 singletarget 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 Received: Revised: Accepted: Published: 3577
December 26, 2013 April 1, 2014 April 1, 2014 April 1, 2014 dx.doi.org/10.1021/jf405512e | J. Agric. Food Chem. 2014, 62, 3577−3583
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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). 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: mp 199−201 °C; yield = 89%. 1H 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,
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
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MATERIALS AND METHODS
General. All melting points were obtained with a RY-1G melting point apparatus (Tianjin Tianguang Optical Instruments Co. Ltd., China) and are uncorrected. 1H 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 2Bromo-N-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)4-(trifluoromethylsulfinyl)-1H-pyrazol-5-yl) Acetamide (Compound 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 3578
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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-dimethylaminopyridine (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 colorless crystals (yield = 46%): mp 161−163 °C. 1H 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]+. N-(3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)-2-(2,4,5-trichlorophenoxy) Acetamide (2b). Compound 2b was synthesized according to the same method as used for 2a. Compound 2b was colorless crystals (yield = 34%): mp 147−149 °C. 1H 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-dichlorophenoxy) 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). N-((3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl)carbamoyl)-2-(2,4,5- trichlorophenoxy) Acetamide (2d). Compound 2d was synthesized according to the same method as used for 2c. Compound 2d was colorless crystals (yield = 37%): mp 101−103 °C. 1H 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
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).) (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: mp 221−223 °C; yield = 54%. 1H 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. (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: mp 243−245 °C; yield = 83%. 1H 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, CH21), 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: mp 225−227 °C; yield = 81%. 1H 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. (3-Cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4((trifluoromethyl)sulfinyl)-1H-pyrazol-5-yl) (2-(4-Methylpyridin-1ium-1-yl)acetyl)amide (1g). Column chromatography (ethyl acetate/methanol = 7:1 → 6:1, v/v) afforded compound 1g as a clear colorless crystal: mp 208−210 °C; yield = 43%. 1H 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); 13 C 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
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Scheme 1. Synthesis of 1a−1ga
a
Reagents and conditions: (a) bromoacetyl bromide, NaH, THF; (b) tertiary amine or nitrogen heterocyclic compounds, THF.
Scheme 2. Synthesis of 2a−2da
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.
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 acetylcholine 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
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. 3580
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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.
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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.)
compd 1a 1b 1c 1d 1e 1f 1g 2a 2b 2c 2d fipronil
NR1R2R3 (R) trimethylamine triethylamine tri-n-butylamine quinoline pyridine N,Ndimethylpyridin-4amine 4-methylpyridine 2,4-dichlorobenzene 2,4,5trichlorobenzene 2,4-dichlorobenzene 2,4,5trichlorobenzene
LC50 (μg mL−1)
95% confidence interval of LC50 (μg mL−1)
15.10 20.84 24.69 8.31 12.85 24.71
9.92−23.01 13.61−31.92 14.16−43.06 5.84−11.82 9.05−18.24 14.73−41.46
18.55 58.58 56.00
12.57−27.38 26.78−128.15 28.71−109.26
153.52 210.00
17.68−1333.19 10.93−4034.25
24.93
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.
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.
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
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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 insecticidal 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.
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 conformation, 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 6member NR1R2R3 ring is unfavorable for the insecticidal activity, and steric hindrance may weaken the activity. The bioactivity of compound 1f (NR1R2R3 = N,N-dimethylpyridin4-amine) was similar to that of fipronil, but 1g (NR1R2R3 = 4methylpyridine) 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 4pyridine 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. 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 acetylcholine binding protein (AChBP) (Figure 2).34 The fipronil-related phenyl heterocyclic compounds were effective as noncompetitive 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.
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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.
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