To overcome resistance and ecological problems involved with conventional insecticides, there was an urgent need to discover novel potent insecticides with a new mode of action . Ryanodine receptors (RyR) are members of a family of intra-cellular Ca2+ release channel proteins present on endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) membranes, deriving the name from its high affinity with ryania [2, 3]. Ryanodine receptors insecticides had already become a focus of pesticide discovery and research because of its good insecticidal activity and high safety for nontarget creatures such as mammal [4-6]. Natural ryanodine receptors insecticides include caffeine, spiganthine, 9, 21-dehydroryanodine, ryanodol, etc. . Commercial ryanodine receptors inscticides include phthaldiamide such as flubendiamide (A) discovered by Nihon Nohyaku in 1993, anthranilic diamide such as cyantraniliprole (B) and chlorantraniliprole (C) discovered by DuPont [8-11] (Fig. 1).
In this article, we divided the chemical structure of chlorantraniliprole (Fig. 1, D) into three parts: N-pyrazinylpyrazole moiety (a), anthraniloyl moiety (b), and aliphatic moiety (c). In our previous work, o-carboxamidobenzamide compounds containing 2-(substituted phenyl)oxazole group (Fig. 1, C) in part a were synthesized, but unfortunately most of the compounds did not showed higher bioactivity than commercialized chlorantraniliprole . Based on this and encouraged by the extensive and excellent biological activityof pyrazinederivants in thefields ofmedicine , pesticide [14, 15], flavouring , etc., N-pyrazinylpyrazole was introduced into part a according to the principle of bioelectronics to give the series of E compounds (Fig. 1, E). SARs analysis revealed that E series containing N-pyrazinylpyrazole in part a exhibited inferior bioactivity compared with chlorantraniliprole containing N-pyridylpyrazole, in addition, title compounds with methoxylcarbamoyl in patrt c had the highest probability possessing high activity, so the series of F compounds (12v and 12w) containing methoxylcarbamoyl on part c and N-pyridylpyrazole in part a were further designed (Fig. 1, F and Scheme 1). Three dimensional quantitative structureactivity relationship (3D-QSAR) and action mode of title compounds were also investigated in this paper.
|Scheme 1. Synthetic route of 12a-w. Reagent and conditions: (a) NH2NH2, EtOH, 80 ℃; (b) Na, EtOH, HAc, 50 ℃; (c) P(O)X3, CH3CN; (d) NaOH, H2O, EtOH 65 ℃; (e) SOCl2, 65 ℃; (f) triphosgene, 1, 4-dioxane, 40 ℃; (g) R2NH2, CH3COOEt, 40 ℃; (h) Et3N, CH3CN, r.t.; (i) CH3CN.|
The synthesis of compounds 12a–w is shown in Scheme 1. The pure product of 2-chloro-6-hydrazinylpyrazine (2a) can easily be obtained after filtration instead of extraction and distillation by the reaction of 2, 6-dichloropyraine with 80% hydrazine hydrate at present of ethanol. Compound 4a was synthesized according the reported method for chlorantraniliprole synthesis with minor improvements . During which process, it was found that 2- hydrazinylpyrazine and 2-chloro-6-hydrazinylprazine can successfully be reacted with diethyl maleate by controlling the reaction temperature, but 3-chloro-6-hydrazinylpyrazine cannot be reacted. Compound 4a was bromized with phosphorus oxybromide to give compound 5a. Then 5a was hydrolysed with NaOH to give the corresponding carboxylic acid 6a. Compound 6a was converted into the acyl chloride 8a by treatment with thionyl chloride and pyrazolone ring was constructed at the same time. Methoxy substituted intermediates 7a can directly be obtained by refluxing compound 6a in methanol, which was not reported before.
Compound 9a was cyclized by triphosgene to give the corresponding six-membered lactam 10a at a high yield of 98%. Amine hydrochloride derivatives were firstly deprotonated with triethylamine to release their nucleophilicity, and then were used for ring opening reaction of lactam 10a to give 2-amino-5- chlorobenzamide derivatives 11. Title compounds 12a-w were synthesized with acyl chloride 8 and amine 11 throungh nucleophilic substitution reaction in the presence of organic base triethylamine.
The insecticidal activities of 12a-w against diamondback moth and oriental armyworm were shown in Table 1. All of title compounds showed 100% insecticidal activity against diamondback moth and oriental armyworm at 100 mg/L. Title compounds 12c, 12v and 12w exhibited 100% mortality at 50 mg/L, but when the concentration was reduced to 5 mg/L, only 12w keep 100% mortality against both diamondback moth and oriental armyworm. For compound 12w with excellent activity, its insecticidal activity against Mythimnaseparata walker, Pyrausta nubilalis, Cnaphalocrocis Medinalis at concentrations below 5 mg/L were further investaged. The results were listed in Table 2, it was worth mentioning that 12w showed better activity than contrast compound chlorantraniliprole against Pyrausta nubilalis (0.1 mg/L) and Cnaphalocrocis Medinalis (2 mg/L).
In order to evaluate the SARs of title compounds systematically, according to the insecticidal activity of E series bearing N-pyrazinylpyrazole against diamondback moth at 50 mg/L, 12au were categoried and sorted into three types: H (high activity with percentage 70–100), M (moderate activity with percentage 40–70), L (low activity with percentage 0–40). As shown in Table S1 and Fig. S1 in Supporting information, seven of E series fell into H category when R2 fixed as OCH3, so 87.5% were H, similarly, 75% are H as CH3, 40% as OEt and 0% as cyclopropyl, consequengtly the order from high to low was OCH3, CH3, OEt, cyclopropyl. As a result, title compounds with methoxylcarbamoyl in patrt c had the highest probability possessing high activity, in addition, keeping R, R1, R3 same with each other, compounds with the highest activity in each series were 12c, 12d, 12e, 12f, 12k, 12l and 12n, respectively. It was found that 5/7 of these had a methoxylcarbamoyl in part a. Bulky substituent in part a could lead to a decrease activity, for example, 12m with R2 fixed as CH3 exhibited 80% mortality while 12t only showed 10% with cyclopropyl. The results analysed above did explain that the later designed 12v and 12w bearing methoxylcarbamoyl in part c and N-pyrazinylpyrazole in part a could show relatively higher activity than 12a-u and why keeping the other chemical structure consistent with each other 12w could exhibit higher actvity than chlorantraniliprole. For R3, the corresponding sequence was OCH3 > Br > Cl, for R1, it was Cl > CH3. It was also found that title compound with R and R1 fixed as Cl and Cl showed higher activity than those fixed as Cl and CH3 respectively, which was demonstrated by the sequence 12d > 12l, 12e > 12s, 12f > 12p, 12g > 12n, 12i > 12u.
Comparative molecular field analysis (CoMFA) is an effective computer implemented methodology of 3D-QSAR employing both the interactive graphics and statistical techniques for correlating structure of molecules with observed biological properties, and thus is widely used in drug design [17, 18]. The results of CoMFA computation was summarized in Table S2 (Supporting information). The cross-validated coefficient q2 obtained by leave-one-out method is 0.658, where a value over 0.5 indicates the model is a great predictor of the insecticidal activity. The results of structural alignment and isocontour diagrams of the electrostatic field contributions obtained from the CoMFA analysis were illustrated in Fig. 2, in which a postive charged group in the blue region will increase the activity but a negative charged substituent in the red region will be favorable.
|Fig. 2. (A) Superimposition of the training and test sets. (B) Electrostatic map form CoMFA model.|
Title compounds bearing an electron-withdrawing group at 3- or 4- position and an electron-donating group at 6- position of the pyrazine or pyridine ring, such as 12v, 12w, and 12c, displayed higher activity. At the 3-position of the imidazole ring (R3), compounds bearing an electron-donating groups would display higher activity, as shown in the sequence OCH3 > Br > Cl discussed above. Alkoxycarbamoyl in part c displayed higher activity because they occupy the red region, and a relatively positive-charged alkyl part in alkoxycarbamoyl was favorable for the increase of activity.
Title compounds 12c, 12d, 12e and chlorantraniliprole at a concentration of 100 mg/L were applied to neurons of beet armyworm, after which the effect of that on free cytosolic calcium concentration ([Ca2+]c) in beet armyworm neurons was shown in Fig. 3A. It was observed that the application of 12c, 12d and 12e can induce a rapid increase of [Ca2+]c in single neuron. Previous studies suggested that calcium signal was generated by calcium ion store present on ER in neurons of insects. Ryanodine receptors, which locates at ER membranes of insect nerve cells, is the ligand-gated calcium channel regraluting release of calcium ion . It was reported that the transfer of calcium from ER to cytosol in insect neurons can indirectly evoke the muscle contraction . In view of the description above, the action mode could be correctly conjectured (Fig. 3B). The combination of tested compounds with RyR resulted in the sustained release of calcium ion from ER to cytoplasm, which induced the stress contraction of insect muscle fiber. Ultimately, pests died from symptoms of body contraction, paralysis, feeding cessation, etc. . It was concluded that the target of E series of compounds is the ligand-gated calcium channel present on ER, and thus they may be RyR activators.
|Fig. 3. (A) Effect of 12c, 12d, 12e and chlorantraniliprole (100 mg/L) on [Ca2+]c in beet armyworm neurons. (B) Schematic illuatration of the molecular target and action mode of E series of compounds. (C) Effect of Ca2+ in extracellular fluid on [Ca2+]c in beet armyworm neurons after the application of 12e (100 mg/L). (D) Effect of the application of 12e (100 mg/L) and caffeine (30 mmol/L) during different time intervals on [Ca2+]c in beet armyworm neurons.|
To explore the effect of E series of compounds on voltage-gated calcium channel present on cytomembrane of neurons. Title compound 12e (100 mg/L) was applied to beet armyworm neurons at the presence and absence of Ca2+ in extracellular fluid and its effect on [Ca2+]c was recorded (Fig. 3C). After beet armyworm neurons were loaded by incubation with 100 mL Fluo-3-AM for 3 h, F/F0 (%) reached a maximum of about 109% at 5 min after application under both calcium-rich and calcium-free conditon in extracellular fluid. As a result, title compound 12e cannot activate the voltage-gated calcium channel present on cytomembrane of neurons and have no molecular target on it.
After beet armyworm neurons were loaded by incubation with 100 mL Fluo-3-AM for 3 h, under calcium-free condition in extracellular fluid, the effect of the application of 12e (100 mg/L) and caffeine (30 mmol/L) during different intervals on [Ca2+]c in beet armyworm neurons was recorded (Fig. 3D). It was observed that the prior application of 12e had no effect on [Ca2+]c change of caffeine application and the subsequent [Ca2+]c change of 12e application was not affected by prior application of caffeine neither. If the molecular target of caffeine was occupied by the priorly applied 12e, a rise on [Ca2+]c would not be observed, in other words, title compound 12e has a molecular target on RyR different from natural ryanodine receptors insecticide caffeine.
In summary, we synthesized 21 novel anthranilic diamide derivatives containing pyrazinyl, 2 derivatives containing methoxylcarbamoyl in the aliphatic part of chlorantraniliprole. Bioassays indicated that all of the title compounds displayed 100% mortality against diamondback moth and oriental armyworm at 100 mg/L, based on analysis, we obtained title compound 12w with better insecticidal activity than chlorantraniliprole against Pyrausta nubilalis (0.1 mg/L) and Cnaphalocrocis Medinalis (2 mg/L). The 3D-QSAR results provided significant information for the structure optimization of anthranilic diamide insecticides, especially, 12w can serve as lead compound to screen highly active pesticide and have the value of further research. The calcium fluorescence measurements results revealed that E series compounds containing pyrazinyl may possess a molecular target different from caffeine on RyR rather than the voltage-gated calcium channel present on cytomembrane.Acknowledgment
This work was supported by the National Natural Science Foundation of China (Nos. 20772060 and 21172114).Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.cclet.2018.05.023.
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