Synthesis And Optimization Of Isoliquiritigenin Analogues And Their Mechanism Of Anti-acute Hepatitis Research

Research BackgroundLiver disease is a common and extremely harmful disease that exists all over the world.More and more evidences show that the mechanism of liver disease is inseparable from the body-mediated immune regulation.In the initiation of innate immune responses against pathogen-associated molecule patterns（PAMPs）and damaged-associated molecular patterns（DAMPs）,pattern recognition receptors（PRR）were of great importance in recognizing specific components of antigen and triggering immune responses to regulate the balance of host’s internal environment.TLRs（Tolllike receptors）are a class of natural immune receptors that not only facilitates the body to eliminate pathogens and promote a series of immune responses,but is also related to the production of many diseases.Among the many subtype families of TLRs,TLR2 has attracted more attention for its potent pre-clinical research value.Once TLR2 combined with its ligand Pam3CSK4,MyD88 were recruited to initiate downstream pathways,resulting in the activation of NF-κB and MAPK signaling pathway.As a result,pro-inflammatory cytokines were released and conducted inflammatory respone,such as TNF-α,IL-6,IL-1β.The appropriate amount of inflammatory response mediated by TLR2 is beneficial,and overexpression can lead to many inflammatory diseases,such as hepatocellular carcinoma.Thus,inhibition of TLR2 may be an important target for the treatment of inflammatory diseases.In recent years,there were certain inhibitors of TLR2 had been found.However,most of them had moderate TLR2 inhibitory activity or unstable structure,which limited their subsequent researches.Research purposesScreening a natural product library of 800 compounds yield isoliquiritigenin as an effective anti-inflammatory agent,while its activity accompanied by toxicity.In our study,we uses the natural product isoliquiritigenin as the lead compound to synthesize a series of isoliquiritin derivatives,aiming to find a small molecule anti-inflammatory compound with high activity and low toxicity that can inhibit TLR2 and evaluate its anti-acute hepatitis-induced liver injury effect.The structure of the synthesized compounds were confirmed by 1H-NMR,13C-NMR and HR-ESI-MS.Research content（1）A series of new structural interior derivatives were synthesized according to the backbone of isoliquiritigenin.The synthesis optimization ideas are as follows,explore the influence of the different substituents of the left and right benzene ring in the core structure on the activity and toxicity,such as changing its hydrophilicity,electronic effects,steric hindrance and so on.Moreoover,the middle chain-like part in the core structure is also focused,and the isoliquiritigenin derivative with a new structure is obtained by forming a five-membered or six-membered nitrogen-containing heterocycle.（2）Using murine primary peritoneal macrophages cells,the pharmacodynamic activity was determined by the NO signal that inhibits Pam3CSK4 activation,and then the cytotoxicity of the compound was monitored by MTT method.Moreover,we analyzed the polarization of M1 phenotype macrophage by detecting the specific marker CD86 and CD llb with flow cytometry.At the same time,the Pam3CSK4mediated inflammatory factor inhibition was investigated by using different cell lines such as Raw264.7,BMDC and PBMC,TLR2 protein expression were quantified by Western Blot.（3）Acute hepatitis mouse model was established by intravenous injection of concanavalin A.Through comparative analysis of serum alanine aminotransferase expression levels in mice,macrophage differentiation and liver tissue sections to study the optimal anti-inflammatory compounds to reduce liver injury induced by acute hepatitis.Analysis conclusionWe found the preferred compound B14（SMU-B14）by structural modification,synthesis and novel structural analysis,which showed significant TLR2 inhibition in murine primary peritoneal macrophages cells.To be precise,B14 inhibits the nitric oxide（NO）signal produced by Pam3CSK4-activated TLR2 pathway in murine primary peritoneal macrophages cells with lower toxicity compared with isoliquiritigenin.It also found that B14 has a strong inhibitory effect to the Pam3CSK4-induced inflammatory factor TNF-α in Raw264.7 and the blood mononuclear cell line（PBMC）.In addition,we observed a dose-inhibiting effect of B14 on TLR2 at the protein level.It is worth noting that SMU-B14 significantly reduced the expression of CD86 and CD11b at 10 μM,indicating that the polarization of the Ml phenotype was hindered.From above experiments,we can conclude that SMU-B14 reduces the release of inflammatory cytokines induced by TLR2 agonist both in human and murine cell lines,and impairs inflammation by reducing the inflammatory macrophage M1 phenotype.At the same time,in vivo experiments show that SMU-B14 can reduce the expression level of alanine aminotransferase in the serum,reduce the accumulation of neutrophils and inhibit the polarization of macrophages to the M1 phenotype,and ultimately reduce the liver injury induced by acute hepatitis.