Novel Spiro[chromone-2,4'-piperidinyl]-4-one Derivatives As Potential Inhibitors Of Fatty Acid Synthesis In Plant Pathogenic Bacteria: Design, Synthesis And Biological Evaluatio

Spirochromanone is a kind of flavonoid compound.The compounds containing spirochromanone skeleton have various biological activities such as anti-tuberculosis,anti-arrhythmia,anti-HIV,ACC inhibitor,histamine receptor antagonist and so on.In this paper,a series of spirochromanone derivatives containing isopropanolamine moiety were obtained by using spirochromanone as the skeleton,introducing epoxide bromopropane and using different amines for epoxide ring-opening.The compounds were characterized by ¹H NMR,¹³C NMR and high-resolution mass spectrometry（HRMS）.It is expected to obtain antibacterial compounds with novel structure,clear action mode,high efficiency and low toxicity.This paper mainly includes three parts:synthesis of spirochromanone derivatives containing isopropanolamine structure and in vitro biological activity study,structural modification analysis of structure-activity relationship and the preliminary mechanism of high activity compound B₁₄ against Xanthomonas oryzae pv.oryzae.1.In this paper,a total of 58 target compounds from four series A,B,C and D were synthesized,and in vitro bioactivity of the compounds against Gram-negative bacteria Xanthomonas oryzae pv.oryzae（Xoo）,Xanthomonas axonopodis pv.citri（Xac）and Pseudomonas syringae pv.actinidiae（Psa）,and Gram-positive bacteria Clavibacter michiganense subsp.michiganse（Cmm）were tested by turbidimetric method.The bioactivity test results showed that most of the compounds showed good bioactivity,and compound B₁₄ showed the best bioactivity against Xoo,with EC₅₀ value of 1.28μg/m L,which was better than that of the control agents,bismerthiazol and thiodiazole copper(EC₅₀ value of 38.06 and 81.54μg/m L,respectively).Compound C₁demonstrated the best performance against Xac with EC₅₀ value of 0.78μg/m L,which was superior to bismerthiazol and thiodiazole copper(EC₅₀ value 53.21 and 98.46μg/m L,respectively).The EC₅₀ value of compound B₁₅ was 1.20μg/m L toward Psa,which was better than that of thiodiazole copper(EC₅₀ was 90.58μg/m L).The compound B₁₃ with EC₅₀ of 3.48μg/m L for Cmm,which was better than that of bismerthiazol and thiodiazole copper(EC₅₀ were 73.16 and 146.15μg/m L,respectively).The results of rice pot experiment showed that compound B₁₄ were 44.64%and 43.33%curative and protective activities against rice bacterial blight at the concentration with 200μg/m L,respectively.The control efficiency was higher than that of commercial pesticides bismerthiazol（BT）（33.77%and 39.20%）and thiodiazole copper（TC）（39.52%and 41.27%）which were slightly better than the control drug thiodiazole copper（39.52%and 41.27%）and bismerthiazol（33.77%and 39.20%）,respectively.In addition,the control efficiency of compound B₁₄ were improved via adding 0.1%silicone（curative and protective activities of 49.57%and 47.81%,respectively）or 0.1%orange peel essential oil（curative and protective activities of47.64%and 46.71%,respectively）,both of which were significantly better than TC and BT.2.Compound B₁₄ was selected as a lead compound to structural optimize due to its best antibacterial activities in vitro against Xoo.The following conclusions were obtained through structure-activity relationships（SAR）analysis.（1）The target compounds obtained from alkyl piperazine showed no activity at 100μg/m L,but the activity of piperazine was significantly improved after the introduction of benzyl.（2）In the absence of piperazine group,benzylamine substitution activity is higher.（3）The activity of benzylamine decreased when N-H was replaced by alkyl,and gradually decreased with the growth of the chain.（4）The structural integrity of isopropanolamine is essential for its activity.（5）The activity of spirochromanone at 6-site substitution is better than that at other sites.3.Preliminary mechanism exploration:Transmission electron microscopy（TEM）was used to detect the surface rupture of Xoo treated with compound B₁₄.Using N-phenyl-1-naphthylamine and ONPG drugs,the results indicated that the permeability of the outer membrane and inner membrane of bacteria treated with compound B₁₄ increased.By using Nile red and DAPI staining,the results exhibited that the cell membrane of bacteria treated with compound B₁₄ was damaged and the intracellular components leaked out.The lipid components of the cell membrane were decreased with increasing concentration of drug.The changes of fatty acid composition were analyzed by GC-MS.Fatty acids are synthesized through the fatty acid synthesis pathway,and encoded by genes ACC,ACP,and Fab family genes.Real-time quantitative polymerase chain reaction（RT-q PCR）assay showed that the expression levels of fatty acids related-genes were down-regulated treated with B₁₄.Subsequently,DNA binding experiments and competitive experiments implied that the mode of action between compound B₁₄ and DNA was non-covalent binding.