Article pubs.acs.org/JAFC
Synthesis and Insecticidal Evaluation of Novel N‑Pyridylpyrazolecarboxamides Containing an Amino Acid Methyl Ester and Their Analogues Ming-Zhen Mao,‡ Yu-Xin Li,† Yun-Yun Zhou,† Xiu-Lan Zhang,† Qiao-Xia Liu,† Feng-Juan Di,† Hai-Bin Song,† Li-Xia Xiong,† Yong-Qiang Li,† and Zheng-Ming Li*,† †
State Key Laboratory of Elemento-Organic Chemistry, Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People’s Republic of China ‡ Xi’an Modern Chemistry Research Institute, Xi’an 710065, People’s Republic of China S Supporting Information *
ABSTRACT: On the basis of the commercial insecticide chlorantraniliprole, a series of novel N-pyridylpyrazolecarboxamides containing an amino acid methyl ester and their analogues were designed and synthesized. Their chemical structures were established on the basis of corresponding 1 H NMR spectroscopy, 13C NMR spectroscopy, elemental analysis, and single-crystal X-ray diffraction analysis. The insecticidal activities of the new compounds against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) were evaluated. The results of bioassays indicated that most of the compounds showed moderate to high activities at the tested concentration, of which the compounds 6 containing a methyl acrylate substructure had excellent larvicidal activity; for example, 6a displayed 100% larvicidal activity against P. xylostella at the concentration of 0.005 mg/L, whereas the activities of both compounds 6g and 6h against M. separata were 100% at 2.5 mg/L. The calcium imaging technique experiment results showed that novel compound 6 could elevate the calcium concentration in the cytoplasm. Furthermore, this study also provided evidence that compound 6h activates inositol 1,4,5-trisphosphate (IP3) sensitive intracellular calcium release channels in the endoplasmic reticulum of Spodoptera exigua third-instar larva neurons. KEYWORDS: insecticidal activity, N-pyridylpyrazolecarboxamides, amino acid methyl ester, methyl acrylate substructure, calcium channel
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INTRODUCTION The discovery of novel insecticides with a new mode of action is crucial to overcoming resistance and ecobiological problems associated with some conventional insecticides. In recent years, DuPont has discovered chlorantraniliprole1 (Figure1A), which has an anthranilic diamide structure, exhibits exceptional broadspectrum activity, high potency, and low mammalian toxicity, and has proved itself to be a selective activator of the insect ryanodine receptor.2 Owing to their prominent insecticidal activity, unique mode of action, and good environmental profiles, anthranilic diamides and their chemical synthesis have recently attracted considerable attention in the field of novel agricultural insecticides. Most of the modifications in chlorantraniliprole structure preserve the anthranilic amide moiety,3−8 indicating that the anthranilic amide is a key pharmacophore in this kind of compound. However, structural modifications of the amides in the ortho-position, such as cyano,9 hydrazone,10 and heterocyclic groups, were also reported.11,12 Recently, a novel pyrazolecarboxamide with a thiadiazol group in the orthoposition (Figure1B, JS9117) was reported with high insecticidal activity.13 Compound C (Figure1) containing an oxazoline group reported by Kang et al. showed good larvicidal activity against beet armyworm (Spodoptera exigua),14 indicating the ortho-position is still attractive in further modification. Inspired by these studies, we speculated that the introduction of oxazoline-4-carboxylate moiety into N-pyridylpyrazolecarbox© 2014 American Chemical Society
amides at the ortho-position (Figure1D) could improve biological properties. To our disappointment, these compounds showed low larvicidal activity against oriental armyworm (Mythimna separata). Interestingly, an elimination product of the amino acid methyl ester containing methyl acrylate substructure (Figure1E) showed excellent insecticidal activities against oriental armyworm and diamondback moth. Building on these developments, we designed and synthesized a series of novel N-pyridylpyrazolecarboxamides containing an amino acid methyl ester and analogue substructures in the ortho-position (Figure1E). The larvicidal activities against oriental armyworm and diamondback moth were evaluated, and the structure− activity relationships were also discussed. To further explore the mode of action for the target compounds, the effect of some target compounds on intracellular calcium ion concentration ([Ca2+]i) in the central neurons isolated from the third instar of S. exigua was studied by calcium imaging techniques. The present study also explored the possible effect of novel compounds on the calcium signaling pathway in the central neurons. Received: September 26, 2013 Accepted: January 16, 2014 Published: January 16, 2014 1536
dx.doi.org/10.1021/jf500003d | J. Agric. Food Chem. 2014, 62, 1536−1542
Journal of Agricultural and Food Chemistry
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Figure 1. Chemical structures of compounds A−E.
Scheme 1a
a
Reagents and conditions: (i) CH3CN, pyridine, CH3SO2Cl, room temperature; (ii) CH3CN, L-cysteine methyl ester hydrochloride, Et3N, room temperature, 10 min, then reflux; (iii) CH3CN, L-serine methyl ester hydrochloride, Et3N, room temperature, 10 min, then reflux; (iv) THF, SOCl2, 0 °C; (v) CH2Cl2, CH3SO2Cl, Et3N, 0 °C, then room temperature.
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Scheme 2. Proposed Formation of 6a, 7a, and 8a
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added to quench the excess amount of thionyl chloride. The resulting mixture was extracted with ethyl acetate (3 × 20 mL), and then the combined organic extracts were washed with brine and dried with magnesium sulfate. The solvent was evaporated in a vacuum, and the residue was purified on silica gel with petroleum/ethyl acetate (v/v, 4:1) to give 6a.21 X-ray Diffraction. The crystal structure of compound 6g was determined, and X-ray intensity data were recorded on a Bruker SMART 1000 CCD diffraction meter using graphite monochromated Mo Kα radiation (λ = 0.71073 Å). All calculations were refined anisotropically. All hydrogen atoms were located from a difference Fourier map and were placed at calculated positions and were included in the refinements in the riding mode with isotropic thermal parameters. Biological Assay. Insecticidal activities against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) were performed on test organisms reared in a greenhouse. The 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.22 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, chlorantraniliprole was tested as control under the same conditions. Larvicidal Activity against Oriental Armyworm (M. separata). The insecticidal activities of compounds 4a−8a and chlorantraniliprole were evaluated using the reported procedure.23 For the foliar armyworm tests, individual corn 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 performed three times. Larvicidal Activity against Diamondback Moth (P. xylostella). The larvicidal activities of compounds 4a−8a and chlorantraniliprole were tested by the leafdip method. First, a solution of each test sample in DMSO (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 cm × 2 cm) were cut from fresh cabbage leaves and then were 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 second-
MATERIALS AND METHODS
Instruments. The measurement of melting points was conducted on a Yanaco MP-500 micromelting point apparatus without calibration. 1H NMR spectra were recorded on a Bruker AC-400 instrument in CDCl3 as solvent and Me4Si as internal standard. Chemical shifts were reported as δ values in parts per million (ppm). Elemental analysis was performed on a Yanaco CHN Corder MF-3 automatic elemental analyzer. All chemical reagents and solvents were of analytical grade. Compounds 1a−c were synthesized according to the method reported by our previous work.15−17 2-Amino-5-substuituted-3methylbenzoic acids (2a−c) were prepared by referring to the known procedure.15,16 The intermediates 3a−h were synthesized according to the literature.18 The title compounds were prepared as shown in Scheme 1. General Procedure for the Synthesis of Compounds 4 and 5.19 L-Cysteine methyl ester hydrochloride (1.0 mmol) was added to 20 mL of acetonitrile under stirring, followed by the addition of triethylamine (1.2 mmol). After stirring for 0.5 h at room temperature, 3a (1.0 mmol) was added and the resulting mixture was refluxed for a further 12 h (monitored by TLC). After completion of the reaction, the solvent was evaporated in vacuum and the residue was purified on silica gel, eluting with petroleum/ethyl acetate (v/v, 1:1), and 4a was obtained. Compounds 5a−f were prepared following the same procedures described in the preparation of compound 4a using L-serine methyl ester hydrochloride instead of L-cysteine methyl ester hydrochloride. General Procedure for the Synthesis of Compounds 6−8.20 To an ice-cold solution of 5 (1 mmol) in 20 mL of dichloromethane were added successively triethylamine (3 mmol) and methanesulfonyl chloride (1.5 mmol) with stirring. Then the reaction mixture was allowed to warm to room temperature and stirred until the reaction completed, monitored by TLC. After completion, the mixture was then washed with dilute hydrochloric acid solution and brine and dried with magnesium sulfate. The solvent was evaporated in a vacuum, the residue was purified on silica gel with petroleum/ethyl acetate (v/v, 4:1), and 6a, 7a, and 8a were obtained. Compound 5 (1 mmol) was dissolved in 20 mL of tetrahydrofuran and cooled in an ice bath. With constant agitation, thionyl chloride (2 mmol) was added dropwise, and the reaction mixture was stirred at 0 °C until the reaction completed, monitored by TLC. After the reaction, saturated sodium bicarbonate solution (30 mL) was then 1538
dx.doi.org/10.1021/jf500003d | J. Agric. Food Chem. 2014, 62, 1536−1542
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instar diamondback moth larvae. Percentage mortalities were evaluated 2 days after treatment. Each treatment was performed in triplicate. Calcium Imaging. Calibration of the fluorescence signal was obtained using the procedure of Iakahashi et al. with minor modifications.24,25 The attached neurons were rinsed in standard physiological saline [(mM) 150 NaCl, 4 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, buffered to pH 7.0] and then incubated in the dark at 28 °C in standard external saline containing the dye Fluo-3 AM (Sigma, 10 μM). After dye loading, cells were again rinsed in physiological saline twice. To block IP3Rs, cells were incubated with heparin 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 excited at 488 and 530 nm under fluorescence emission were acquired by using a CCD (Image Pri-6.0). Each experiment was replicated at least six times. The data were analyzed by using SPSS Inc., version 17.0, and Microcal Origin, version 8.0 (Origin Lab Corp., Northampton, MA, USA). Results were expressed as the mean ± SD (n = number of cells). Statistical significance was determined by Student’s paired or unpaired t test. Fluorescence values were expressed as F/F0, with F0 being the resting (or baseline) fluorescence and F the change in fluorescence from baseline after drug application.26
Figure 2. Crystal structure of compound 6g.
some compounds have excellent larvicidal activities against oriental armyworm. Compounds 4a and 5a−h containing an amino acid methyl ester displayed moderate activities against oriental armyworm; the insecticidal activities were below 100% at 100 mg/L except for compound 4a, which showed 60% at 50 mg/L. Compound 7a containing oxazoline-4-carboxylate also showed low larvicidal activity against oriental armyworm; the mortality was only 60% at 200 mg/L. The larvicidal activity of mesylation product 8a at a concentration of 10 mg L−1 was 50%, much higher than that of 5a, indicating that the introduction of a mesyl in the hydroxyl group has a positive effect on larvicidal activities. The elimination products containing methyl acrylate substructure 6a−h showed excellent insecticidal activities against oriental armyworm, especially compounds 6g and 6h, which showed 100% larvicidal activities at the concentration of 2.5 mg/L. Activities varied significantly depending upon the types of substituents on the 3-position of pyrazole. Compared with 3-Br and 3-CF3 in pyrazole, compounds with 3-OCH2CF3 substituents showed higher insecticidal activities against oriental armyworm, which suggests that the introduction of the 2,2,2-trifluoroethoxy groups in the 3-position of pyrazole has a positive effect on the larvicidal activities. The substituents on the aromatic ring showed no significant effect on the activity. The larvicidal activity of compounds 6a−h against diamondback moth was evaluated as shown in Table 2. Most of them had excellent larvicidal activity against diamondback moth; all of the compounds showed 100% activity at 1 mg/L. Compounds with 3-Br substituents on the 3-position of pyrazole showed higher insecticidal activities against diamondback moth, which was different from the activities against oriental armyworm. For instance, the larvicidal activities of 6a− c at 0.01 mg/L were 100, 57, and 43%, respectively, whereas 6d−h had no more than 43% at the same dosage. In particular, compound 6a had 100% mortality at the concentration of 0.005 mg/L, equal to that of chlorantraniliprole, but its LC50 was 0.0008 mg/mL, still higher than that of chlorantraniliprole (0.00009 mg/mL, Table 3). Previously, the neurons of S. exigua were used for studying the mechanism of diamide insecticides, and we also found the increase of [Ca2+]i was positive for the insecticidal activity against oriental armyworm. To determine whether compounds 6a−h might be involved in calcium signaling transduction, we examined the effects of several novel compounds on the central neurons of S. exigua on the calcium homeostasis with calcium imaging technique after neuron loading with Fluo-3 AM. Figure 3 shows the change of [Ca2+]i versus recording time when the neurons were treated with compounds 6a, 6d, 6g, and 6h in the
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RESULTS AND DISCUSSION Chemistry. Compounds 5a−h were prepared by treating compounds 3a−h with L-serine methyl ester hydrochloride using triethylamine as alkali in acetonitrile, affording the desired product in good yields. Compound 4a was prepared following the same procedures described in the preparation of compounds 5a−h using L-cysteine methyl ester hydrochloride instead of L-serine methyl ester hydrochloride. In the initial step,20 we attempted to treat 5a with methanesulfonyl chloride in the presence of triethylamine to prepare 7a. Surprisingly, the elimination product 6a was obtained as the main product, together with compounds 7a and 8a in low yields as shown in Scheme 1. On the basis of the literature previously reported, a mechanism that can possibly explain the formation of 6a, 7a, and 8a was proposed and is illustrated in Scheme 2. In the presence of methanesulfonyl chloride in dichoromethane, compound 5a first undergoes initial mesylation to give the intermediate 8a and then generates nitrogen anion 9 and carbanion 11. The nitrogen anion 9 subsequent isomerizes to 10, which undergoes intramolecular nucleophilic attack to form the oxazoline ring closure compound 7a, whereas compound 6a is obtained by elimination of 11. Due to the low insecticidal activities of 7a, the synthesis of this derivative was not further processed. Another alternative synthetic route was explored to prepare the target compound 6a21 by using thionyl chloride in tetrahydrofuran at 0 °C, which resulted in high yields and purity of 6a. Crystal Structure Analysis. Compound 6g was recrystallized from a mixture of dichloromethane and petroleum ether (60−90 °C) to give colorless crystals (0.22 mm × 0.14 mm × 0.12 mm) suitable for X-ray single crystal diffraction as shown in Figure 2 with the following crystallographic parameters: a = 15.005(2) Å, b = 15.005(2) Å, c = 21.630 (4) Å, α = 90°, β = 90°, γ = 120°, V = 4217.8 (12) Å3, Z = 6, Dc = 1.553 g cm−3, μ = 0.486 mm−1, F(000) = 2004, R = 0.0498, T = 113 (2) K, wR = 0.0988, final R factor = 4.63%. Structure−Activity Relationship (SAR). The larvicidal activity of compounds 4a−8a against oriental armyworms is summarized in Table 1. The bioassay results indicated that 1539
dx.doi.org/10.1021/jf500003d | J. Agric. Food Chem. 2014, 62, 1536−1542
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Table 1. Insecticidal Activities of Compounds 4a, 5a−h, 6a−h, 7a, and 8a and Chlorantraniliprole against Oriental Armyworm larvicidal activity (%) at a concentration of compd
200 mg/L
100 mg/L
50 mg/L
10 mg/L
5 mg/L
2.5 mg/L
4a 5a 5b 5c 5d 5e 5f 5g 5h 6a 6b 6c 6d 6e 6f 6g 6h 7a 8a chlorantraniliprole
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 60 100 100
100 80 50 40 60 30 80 70 100 100 100 100 100 100 100 100 100
60
100 100
1 mg/L
40 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100
100 80 40 73.3 60 50 100 100
60
100 100
40 40
100 100
50 100
100
100
100
Table 2. Insecticidal Activities of Compounds 6a−h and Chlorantraniliprole against Diamondback Moth larvicidal activity (%) at a concentration of compd
10 mg/L
1 mg/L
0.1 mg/L
0.01 mg/L
0.005 mg/L
0.001 mg/L
0.0005 mg/L
6a 6b 6c 6d 6e 6f 6g 6h chlorantraniliprole
100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100
100 86 71 100 100 43 43 86 100
100 57 43 43 29 0 0 29 100
100
57
29
100
90
80
Table 3. LC 50 Values of Compound 6a and Chlorantraniliprole against Diamondback Moth compd
y = a + bx
LC50 (mg/L)
R
6a chlorantraniliprole
y = 13.28 + 2.65x y = 9.66 + 1.07x
0.0008 0.00009
0.99 0.93
absence of extracellular calcium. Application of 1000 mg/L compound 6 and chlorantraniliprole to isolated S. exigua neurons caused an increase in the cytosolic calcium concentration. The peak of [Ca2+]i was increased to 104.13 ± 2.88% (n = 9), 103.6 ± 3.10% (n = 16), 106.00 ± 2.17% (n = 12), 106.59 ± 2.41% (n = 15), and 115.42 ± 2.37% (n = 12) of the initial value after the neurons were treated with compounds 6a, 6d, 6g, and 6h for
