Design、Synthesis Of Anthranilic Diamdies And Study Of Their Affinity Binding On Macrobiomolecules

Chlorantraniliprole and Cyantraniliprole are two anthranilic diamide insecticides that are acting on ryanodine receptor in insect pests. They possess unique mechanism of action, wide insecticidal spectrum, low toxicity and environmental friendly character. The resistance of field pests appears gradually under a widely and large-scale use, and the discovery of new compounds acting on ryanodine receptor is a method of solving the pests resistance issue.Insect ryanodine receptor is a macrobiomolecule across membrane, and is made up of four monomers, each 565 Kd in size, so it is very difficult to obtain its 3-D configurations for studying the affinity binding of active molecule on it. Thus, setting up suitable method of technique to study the affinity binding of active compounds on ryanodine receptors is also a research hotpot for currently discovery of insecticidal active small molecules.(1) In the dissertation, based on coordination of active fragments, acylhydrazine group possessing insecticidal activity is introduced into the main structure of anthranilic diamide, and meanwhile the skeleton benzene ring is modified by iodine atom and cyano group, a series of 2-acylamino-substituted N’-benzylbenzohydrazide derivatives with different substituted groups were designed and synthesized. The structures of all compounds were confirmed by proton nuclear magnetic resonance spectrum, and preminary bioassay showed that some new compounds possess lethal activity against Mythimna separata show, and compounds cau-lkcl, cau-lkc2 and cau-lkc5 (Figure 1) have a 100% lethal activity to Mythimna separata at the concentration 100 mg/L.(2) Through homogenization and centrifugation, the membrane proteins containing ryanodine receptors was extracted from American cockroach leg, and ryanodine receptors in extract were confirmed and determined by the Bradford method. The results showed that the ryanodine receptor content of membrane protein extract was 69 μg/mL.(3) With chlorantraniliprole-based fluorescence ligand, a novel fluorescent polarization (FP) binding assay was established to study the interaction between active compounds and insect ryanodine receptor. Chlorantraniliprole and Cyantraniliprole inhibited the binding of the chlorantraniliprole-based fluorescence ligand to extacted membranes with IC50’s 135.65 nM and 146.36 nM, but flubendiamide having different skeleton structure from chlrorantraniliprole and cyantraniliprole could not influence the binding of fluorescent ligands on ryanodine receptor(Figure 2). These results verified again that chloranitraniliprole and its analogue binds the same site on ryanodine receptor. However, flubendiamide and ryanodine bind possibly the other sites on ryanodine receptor. Furthermore, the Z’factor of estabolished assay reached 0.76, and demenstrates that this assay could be used in high throughout screening for discovery of new molecules acting on ryanodine receptor.(4) Considering of hapten characteristics, haptens were designed and synthesized through replacing methylamino group in chlorantraniliprole and cyantraniliprole with 4-aminobutyric acid(Figure 3). The monoclonal antibodies 1D2 and 3B2 for chlorantraniliprole and cyantraniliprole were obtained through immunogen preparation from hanptens, immunizing mice and antibody assessment, etc.(5) ELISA methods were developed to study the affinity binding of diamides compounds to the above obtained monoclonal antibodies with IC501.60 ng/mL for chlorantraniliprole and IC501.57 ng/mL for cyantraniliprole, and were used further to study the antibodies mimicing ryanodine receptor. The test results from ELISA showed that antibodies like ryanodine receptor could bind with some compounds having parent structures in antibody, specifically, and could also recognize other compounds at some extent. This developed method is significant to discover active compounds to ryanodine receptors.(6) Upon the specificity of monoclonal antibody from cyantraniliprole, an icELISA detemination method for cyantraniliprole was estabolished with IC50 1.57 ng/mL, detection range 0.43-6.15 ng/mL (Figure 4)and fortified recoveries 93.7-101.0% and 4.4%-100.6% in Chinese cabbage and tap water, respectively. The developed icELISA method could be employed to detect the cyantraniliprole quickly and accurately in Chinese cabbage and tap water.