Design,Synthesis,Biological Activities And Mechanism Study Of Harmine Derivatives Against Rhizoctonia Solani

?-carboline alkaloids,including harmine and its derivatives,is mainly isolated from the genus Peganum,which have a wide range of pesticidal activities.Therefore,harmine and its derivatives could be used as the potent lead compounds for the synthesis and development of new green pesticides.Rice sheath blight,caused by the phytopathogenic fungus Rhizoctonia solani,is one of the most important rice diseases worldwide.Although a few fungicides are available in the control of rice sheath blight,this critical disease has not been effectively controlled while the intensive and continuous use of the same fungicide might increase the risk of resistance development of the pathogen.Therefore,it is necessary to develop new green fungicides against rice sheath blight.Here,we used group splicing method and analogous synthesis method to introduce the sub-activity structure of pesticide into the harmine scaffold,or optimized the ?-carboline scaffold,and evaluated the bioactivity of the synthesized compounds against rice sheath blight and Sf9 cells.Finally,The preliminarily study of the fungicidal mechanism of high active compounds I-54,III-14 and IV-43 was carried out,and the results were as follows.(1)The ?-carboline oxazoline derivatives I-1~I-66 and ?-carboline acylguanidine derivatives II-1~II-8 were obtained using group splicing method and analogous synthesis method,respectively.The results showed that all the derivatives showed good fungicidal activity with the EC50 values ranged from 4.24 to 303.56 ?g/m L,which was significantly higher than that of the harmine(EC50 = 520.23 ?g/m L).Some compounds,such as compounds I-4,I-5,I-7,I-11,I-38,I-39,I-45,I-62 and II-2,showed excellent fungicidal activity.The EC50 values of these compounds were 18.09,13.02,15.33,10.53,13.54,14.35,4.24,18.16,and 13.71 ?g/m L,respectively,which were superior fungicidal activity compared with commercially fungicide validamycin A(EC50 = 197.59 ?g/m L).Significantly,compound I-45 was nearly 50-fold more potent than validamycin A.In vivobioassay results showed that compound I-45 displayed good protection effect and curative effect against R.solani,which was superior to validamycin A.Meanwhile,cytotoxic activity results showed that most of the compounds had strong cytotoxicity against Sf9 cells,especially compounds I-6,I-7,I-11,I-14,I-16,I-23 and I-58,which displayed superior fungicidal activity compared with harmine and validamycin A.Furthermore,compounds I-6 and I-58 could induce cells apoptosis and cell cycle arrest and stimulate the Sf-caspase-1 activation in Sf9 cells,and significantly inhibit larvae growth of Spodoptera litura with decreasing the weight of larvae and pupae.(2)The compound 2,3,4,5-tetrahydrothiopyran[4,3-b]indole(III-7)was found with the superior fungicidal activity(EC50 = 38.58 ?g/m L)through the optimization on C ring of the harmine scaffold,which was nearly 10-fold higher than that of harmine(EC50 = 318.56?g/m L).The effects of different substituent groups at different positions with III-7 as the lead compound were investigated.The fungicidal results showed that most of the derivatives exhibited good activity against R.solani with the EC50 values ranged from 2.35 to 241.52 ?g/m L.Especially,compounds III-13,III-14 and III-15 displayed excellent fungicidal activity with the EC50 values of 2.94,2.35 and 3.79 ?g/m L,respectively,which were significantly more potent than validamycin A(EC50 = 183 ?g/m L).The detached leaf assay and in vivo bioassay results showed that compounds III-13,III-14 and III-15 displayed good protection effect and curative effect against R.solani,which were as or more potent than validamycin A.(3)The lead compound scaffold was further simplified,and the C ring of ?-carboline was removed to obtained indole derivatives.The fungicidal activity results showed that the lead compound indole exhibited good fungicidal activity against R.solani.The inhibition rate reached 100% and 87.68% respectively at the high concentrations(100 and 50 ?g/m L),while the fungicidal activities were weak at the low concentrations(10,5 and 1 ?g/m L).The effects of different substituent groups at different positions of indole scaffold were investigated.The results showed that most of these compounds displayed better fungicidal activity than that of indole,especially compounds IV-14,IV-15,IV-16,IV-41,IV-43,IV-44 and IV-54 with EC50 values of 3.53,2.15,2.74,4.99,0.62,1.25,and 7.25 ?g/m L,respectively,which were more potent than validamycin A(EC50 = 183 ?g/m L).This results indicated that o-chlorobenzoyl substitution at 1 position,fluorine or chlorine substitution at4 position and chlorine substitution at 5 position on indole scaffold were favourable for enhancing fungicidal activity.Based on this structure-activity relationship(SAR)correlations,The compounds IV-73~IV-74 were synthesized and the bioassay showed that the fungicidal activities of these compounds were reduced after 1-position substitution.Additionally,the compounds V-1~V-22 were obtained using group splicing and the results showed that most of these compound displayed superior fungicidal activity compared with indole.The detached leaf assay and in vivo bioassay results showed that compounds IV-43,IV-44,IV-54,IV-73,V-7 and V-17 displayed good protection effect and curative effect against R.solani.Especially compound IV-43 displayed the best efficacy in vivo and superior fungicidal activity compared with validamycin A.(4)The compounds I-45,III-14 and IV-43 with the best efficacy were selected to further detect the fungicidal mechanism against R.solani.The results showed that these three compounds could inhibit the sclerotia formation.Among them,compound IV-43 displayed the highest inhibitory activities against sclerotia formation and germination of R.solani.The scanning electron microscopy results showed that the surface of the mycelial was rough and some parts were contracted in the presence of compounds I-45,III-14 and IV-43.The transmission electron microscopy morphology showed that compounds I-45,III-14 and IV-43 could cause mitochondrion swollen with the reduction of ridge and vacuoles appeared.The cell wall became thicker and the plasmolysis occurred obviously in the treatment with compound I-45.Hoechst staining results showed that compounds I-45,III-14 and IV-43 could significantly reduce the average number of nuclei in mycelial single cell.Rhodamine staining showed that compounds I-45,III-14 and IV-43 could destroy the mitochondrial membrane.The electrical conductivity results showed that the cell membrane permeability increased after treatment with compound I-45.The active oxygen results showed that the active oxygen free radicals in mycelium increased after treatment withcompound III-14.The above results indicated that the fungicidal mechanisms of compounds I-45,III-14 and IV-43 were different.