Anti-inflammatory Mechanism And Structure-activity Relationship Of Paeonol Etherified Urea Derivatives Based On TLR4/MyD88 Pathway

Background:Toll-like receptor 4（TLR4）is a pattern recognition receptor that recognizes lipopolysaccharide（LPS）and induces intracellular signaling to recruit adaptor protein My D88 and Toll receptor-related interferon activator（TRIF）.By activation of the My D88-dependent and non-dependent signaling pathways,NF-κB is transferred from the cytosol into the nucleus,resulting in the transcription of inflammatory cytokines,which ultimately amplify inflammatory reactions in a cascade manner.Therefore,the key proteins and inflammatory factors that regulating and interfering with the TLR4/My D88pathway are of great significance for the treatment of inflammation.Inflammation is the autoimmune response for the infection or tissue damage of the organism,which is characterized by the degeneration and necrosis of cells in local tissues.With the development of inflammatory diseases,diabetes,osteoarthritis and rheumatoid arthritis are induced and seriously threatened human health.Currently,Non-steroidal anti-inflammatory drugs（NSAIDs）are commonly used for the therapy of pain and inflammation.However,long-term use these drugs will cause the toxicity and side-effects in clinical treatment although the effectiveness is available.The toxicity and side-effects including such as myocardial infarction,hypersensitivity,gastrointestinal damage,bleeding,ulcers.Thus,the safe,effective and low-toxic anti-inflammatory candidates are still as a purpose for seeking.Paeonol exerts anti-inflammatory activity by means of the obstruction of the generation of inflammatory factors.The previous results of our group research found that:when the alkyl groups substituted the hydrogen of hydroxy at the 2-position of paeonol,the longer of the lengther of carbon chain,the stronger of the anti-inflmmatory activities of its analogue.In additional,Urea-containing compounds,such as semicarbazides,acted as 5-lipoxygenase（5-LOX）inhibitors,β-blockers or MDM2 antagonists,exhibiting antiparasite,anticonvulsant,antiviral,anticancer and antibacterial activity.Furthermore,Halogen as an electron-withdrawing group,had the capacity for improving the biological activity after introduction into the compound.The introduction of halogen in the ortho position of phenylurea would repel with oxygen atom and led to conformational constraint,it was benefit for the bonding of the urea group with hydrogen,and finally enhanced the biological activity of compound.Therefore,in terms of the theory of molecular heterozygosity,paeonol is selected as a lead compound,and a series of paeonol derivatives are obtained after the occurence of the reaction with introduction of carbon chain,phenylurea and halogen.Ultimately,drug candidants are screened on the anti-inflammatory effects through RAW264.7 macrophage model stimulated by LPS.Objective:A series of paeonol etherified urea derivatives were designed and synthesized with paeonol as a lead compound,and later intrudction of the carbon chain,phenylurea and halogen.The inflammatory factors of NO,TNFαand IL-1βin RAW264.7 macrophages stimulated by LPS were regarded as preliminary targets for candidates sceening on anti-inflammatory activity,after that a structure-activity relationship established.Candidates,conduct anti-inflammatory activity studies through q RT-PCR,phagocytosis,ROS immunofluorescence,mitochondrial membrane potential,and NF-κB protein immunofluorescence experiments.Moreover,the potential anti-inflammatory mechanism of these candidates were evaluated through the TLR4/My D88 signaling pathway.The establishment of structure-activity relationship provided a clear guidance and theoretical basis for the further design and synthesis of new paeonol derivaties as anti-inflammatory agents.Methods and results:（1）Design and synthesis of paeonol ether urea derivatives.Taking paeonol as a lead compound,a series of paeonol etherified urea derivatives were synthesized through the reaction of etherification,amino deprotection and urea formation.Their structures were confirmed by MS,¹H-NMR and ¹³C-NMR.（2）The inhibitory effect and structure-activity relationship of paeonol etherified urea derivatives on inflammatory factors were investigated by taking advantage of RAW264.7macrophages stimulated by LPS.The 50%inhibiting concentration(IC₅₀)values of these synthesized compounds and the non-lethal concentration on the cells were examined by MTT method.After those compounds pretreatment,stimulated by LPS,the cell supernatant was collected and the expression levels of NO,TNFαand IL-1βin the supernatant were measured by chemical reagent method and enzyme labeling method respectively,to clarify the anti-inflammatory ability of paeonol derivatives in vitro.To explore the structure-effect relationship.The results showed that the extension of carbon chain increased the inhibitory capacities against inflammatory factors NO,TNFαand IL-1β,as well as that of the introduction of urea phenyl and halogen.The compound b16had the strongest ability to inhibit inflammatory factors NO,TNFαand IL-1β.（3）The potential mechanism of compound b16 on anti-inflammatory activities were explored using LPS-induced RAW264.7 macrophages.The methods of q RT-PCR,phagocytosis,ROS immunofluorescence,mitochondrial membrane potential,NF-κB protein immunofluorescence and western blot assays were used to evaluated the anti-inflammatory activity of candidate b16.The results indicated that compound b16inhibited the generation of inflammatory factors of macrophage induced by LPS,reduced the production of ROS,enhanced the macrophage phagocytic capacity,reversed the decline of cell mitochondrial membrane potential,blocked the translocation of NF-κB from the cytosol into the nuclear,and downregulated the relevant proteins in the TLR4/My D88 signaling pathway.Conclusion:In summary,the introduction of long carbon chains,phenylureas and halogens brought into a lead compound for preparation of paeonol etherified urea derivatives,These derivatives exhibited apparently inhibition on the levels of LPS-induced macrophage inflammatory factors NO,TNFαand IL-1β.Compound b16 significantly inhibited inflammatory response of RAW264.7 macrophage induced by LPS.