Design,Synthesis,Bioactivities And Docking Studies Of Indoxacarb Derivatives

Indoxacarb is the first commercialized pyrazoline-type sodium-channel blocker containing a chiral center in the angular methoxycarbonyl of oxadiazine?E.I.DuPont and Co.?.It is a mixed by the proportion of 75%S-isomers:25%R-isomers.The S-isomers with insecticidal activity is hydrolyzed by amidase into a bioactive N-demethoxyl carbonyl compound—DCJW?Decarbonmethoxyllated JW062?in vivo,which mainly block inactivation in the inactivated state by selectively targeting the recovery from slow inactivated state,resulting in preventing the transition between the inactivated and the closed state,after a prolonged depolarization.Then,block the action potential of the central nervous system,and cause insect paralysis and eventually death.Pyrethroid insecticides,which also acting on the sodium ion channel of insects,are sodium channel modulators,which promote activation and inhibit inactivation,resulting in prolonged opening of sodium channels.They showed powerful knockdown activity.Although pyrethroids and indoxacarb both affect sodium channel inactivation,pyrethroids inhibit fast inactivation,while SCBIs inhibit the recovery from fast and slow inactivation of sodium channels.Many studies showed that derivatives of cinnamic acid from plants exhibited dieversity bioactivity to insects.We postulated that an integrated molecule combining their pharmacophores,which probably will bring us some interesting biological properties unknown before.Furthermore,the new molecular entities may exert possible multiple or pleiotropic actions on fast and slow inactivated states of the sodium channel and probably showed excellent insecticidal activity.Based on the theory of pharmacophore molecular design and bioisosterism,a series of novel compounds were designed and synthesized by introducing the pyrethric acid and cinnamic acid group into indoxacarb moiety in the angular methoxycarbonyl of oxadiazine.Their insecticidal activities against S.litura F.and S.exigua were evaluated.In addition,the structure and stereochemistry-activity relationships were also discussed.According to our previous research results,adding an electron-withdrawing substituent group at the 5-position of the oxadiazine indoxacarb derivatives exhibited excellent insecticidal activity and inhibition effect to sodium ion channels of insects.So,5-Chloro-1-indanone,5-Bromo-1-indanone,and 5-Fluoro-1-indanone were selected as the starting material.The DCJW analogue G1-G4 with excellent insecticidal activity were synthesized.Then,the ester G₁-G₄ were reduced with LiAlH₄ to give key intermdiates alcohol H1-H4.H1-H4 were treated with acyl chloride in DMAP and dry DIPEA in anhydrous THF under an argon atmosphere to give the title compounds J1-J32.For the purpose of studying the relationship between chirality and biological activity,it was absolutely necessary to separate the high optical purity of target compounds with high biological activity.We selected compounds J1-J12 containing multiple chiral centers?J1-J4 containing three chiral centers,J5-J8 containing three chiral centers and cis and trans isomers,J9-J12 containing two chiral centers?,C₁ represent chiral carbon atoms in alcohol moiety and and C₂,C₃ represent chiral carbon atoms in acid moiety,respectively.Therefore,these compounds are composed of multiple different isomers.Diastereomers were separated by silica gel column chromatography?petroleum ether/ethyl acetate=8:1,v/v?,less polar isomer?J1-H-Rf to J12-H-Rf?and more polar isomer?J1-L-Rf to J12-L-Rf?,which showed different Rf values.The bioactivity of target compounds against third instar larvae of S.litura F,and S.exigua was determined by feed mixing method.The results are as follows:1)Six compounds exhibited execllent insecticidal activities against S.litura F.Among them,compounds J18 and J20 with substituent bromine and fluorine in the oxadiazine,trifluoromenthoxy in the benzene ring and cinnamic acid in the angular methoxycarbonyl of oxadiazine of indoxacarb moiety exhibited the best activity against S.litura F,and the 48h LC₅₀ were 3.43?g/mL and 2.68?g/mL espectively,are superior to the 6.08?g/mL of the indoxacarb.The compounds J5-L-Rf and J6-L-Rf with substituent chlorine and bromine in the oxadiazine,respectively,both trifluoromenthyl,in the benzene ring,and pyrethric acid of cyhalothrin,tefluthrin or bifenfluthrin in the angular methoxycarbonyl of oxadiazine of indoxacarb moiety LC₅₀ are 11.10?g/mL and 24.38?g/mL.Besides,the LC₅₀ of J31 and J17were 28.54?g/mL and 27.45?g/mL respectively.2)Four compounds exhibited execllent insecticidal activities against S.exigua.The 48h LC₅₀ of J18 and J20 were 1.48?g/mL and 2?g/mL,which equal to indoxacarb?1.96?g/mL?.while J5-L-Rf and J6-L-Rf LC₅₀ were 9.48?g/mL and 11.81?g/mL respectively.The other compounds had no obvious or even no activity against S.exigua.3)The structure and stereochemistry-activity relationship showed that compounds containing pyrethric acid and cinnamic acid displayed the best bioacitivties.In general,the activity order of the substituted derivatives is cinnamic acid>pyrethric acid>chloroacetic acid>2-thiophene acetic acid>other acids.The introduce groups can improve the molecular stability,modify the electric distribution,and then improve the pharmacodynamic activity.The insecticidal activities of the more polar diastereoisomers of J5 and J6?J5-L-Rf and J6-L-Rf?far better than their low polar diastereoisomers?J5-H-Rf and J6-H-Rf?.It may contribute to receptor binding by interaction with a chirally defined accessory site.The structure-activity studies further reveal the absolute stereochemistry of the chiral carbon atoms in the angular methoxycarbonyl of oxadiazine of indoxacarb moiety and acid moiety that are critical the insecticidal activities.4)The representative compounds J5 and J18 were selected as molecular docking ligands.The results showed that the carbonyl oxygen in the amide group and trifluoromethyl substituted benzene ring are attracted by the sodium ion NaIII.The central oxadiazine ring is close to the pore axis,while large terminal groups extend towards interfaces between repeat domains.The pyrethric acid moiety accepted an H-bond from N²ⁱ¹⁵ and interacted with many hydrophobic residues in helices IS6,IP1 and IIS6,including F^1p44,M^1p47,T^1p48,L¹ⁱ¹⁸,L¹ⁱ²¹,I¹ⁱ²²,V²ⁱ¹¹,V²ⁱ¹²,and L²ⁱ¹⁹.Three of these(L¹ⁱ¹⁸,I¹ⁱ²² and N²ⁱ¹⁵)interact with deltamethrin in the model of the pyrethroid receptor site PyR2,which is based on the open potassium channel X-ray structure,whereas V²ⁱ¹² and I¹ⁱ²² interact with PyR2 bound DDT.Importantly,deltamethrin and DDT reach the PyR2 site from the lipid-exposed side of the I/II domain interface,while compound J5 reaches the four residues in the PyR2 site from the inner-pore side of the same interface.The hydrophobic5-membered ring of J5 fused with the chlorine-substituted aromatic ring bind in the II/III domain interface and enjoy hydrophobic interactions with L^2o13,L^2p47,C^2p48,V²ⁱ¹¹,V²ⁱ¹⁸,L²ⁱ¹⁹,F^3p49,I3i11 and I³ⁱ¹².Two these(V²ⁱ¹⁸ and I³ⁱ¹²)interact with deltamethrin in the PyR1 model and four residues(L^2p47,L^2o13,I³ⁱ¹² and V²ⁱ¹⁸)interact with tau-fluvalinate,which is bound in the PyR1 site of the bumble bee sodium channel.Importantly,while tau-fluvalinate reaches the PyR1 site from the lipid-exposed side of the II/III domain interface,compound J5 reaches the four residues in the PyR1 site from the inner-pore side of the same interface.The trifluoromethyl group of J5 binds between helices IIIP1 and IVS6 and infects with residues F⁴ⁱ¹⁵,S^4p49 and T^3p48.The binding mode of compound J₁₈general resembles that of J5.A common feature is location of the oxadiazine ring close to the pore axis,interaction of the amide carbonyl oxygen with NaIII,binding of two large terminal moieties in domain interfaces I/II and II/III where they interact with residues that contribute to the PyR1 and PyR2 receptor sites.However,the aromatic ring does not interact with NaIII(L¹ⁱ¹⁸?L¹ⁱ²¹?I¹ⁱ²²?L²ⁱ¹⁹?F^1p44?L²ⁱ¹⁹?V²ⁱ¹¹).Specific list of ligand-sensing residues overlaps with,but does not coincide with that for J5.Compounds J5-L-Rf,J6-L-Rf,J18 and J20 exhibit excellent insecticidal activity and knockdown activity.To this end,we identified several indoxacarb derivatives that exhibit excellent insecticidal activity against third-instar larvae of S.litura F.and S.exigua,which also revealed the relationship of stereochemistry and biological activity.These findings open up new avenues towards innovating new eco-friendly insecticides as sodium channel blockers and/or modulators.