Construction Of Artemisinin Derivatives Library And Its Anti-inflammatory Activity And Mechanism Of Action

Artemisinin（ART）,a natural product isolated from the traditional Chinese herb Artemisia annua,has become one of the clinical frontline drugs against malarial infections.The innate immune receptor Toll-like receptor 4（TLR4）,as one of the most important proteins in the TLR family,has been studied widely.In the previous researches,it was found that dihydroartemisinin（DHA）,an artemisinin derivative,targets the myeloid differentiation 2（MD2）directly,which is the adaptor protein of TLR4,and acts as a TLR4 antagonist.However,given its modest inhibitory effect and cellular toxicity,the structure and activity of DHA remain to be further studied and improved.In this work,we first explored the structure-activity relationships（SAR）of DHA as a TLR4antagonist.The results showed that the TLR4 antagonistic activity of DHA will disaper if its sesquiterpene endoperoxide scaffold was destroyed.Therefore,the structure optimization of DHA was focused on the C-10 of the hemiacetal.It was found that with the increasing length of the linear alkane chain at C-10,the inhibitory activity of ART analogs was increased first and then decreased,and the optimal length of the carbon chain is between 3-4 atoms.In contrast,the cellular toxicity of ART analogs was elevated with the increasing length of the linear alkane chain.Besides,the halogen substitutions at the end of the alkane chain of three carbon atoms were conducted.The corresponding TLR4 antagonistic activity of ART derivatives was decreased with the reduction of electronegativity of the substituted halogens,which indicates that the electron-rich functional group at the end of the alkane chain can improve the inhibitory activity of the derivatives.Therefore,by the comparisons between different electron-rich functional groups such as hydroxyl,heterocyclic,olefins,alkynes and so on,it was finally found that the ART derivative26q with an acetylene group as the terminal functional group exhibits the best inhibitory activity(IC₅₀=0.5±0.2μM),which is about 14-fold higher than DHA(IC₅₀=7.0±0.4μM).In addition,the 26q did not show cellular toxicity at high concentrations.Based on the above work,the second part of this paper studied the signaling pathway and the mechanism of downstream inflammatory factors of the best compound,26q.The results showed that the compound 26q can inhibit TLR4 dimerization and endocytosis induced by Lipopolysaccharide（LPS）at the initial signaling stage of cells.Further studies showed that the26q inhibits the activation of nuclear factor kappa-B（NF-κB）signaling pathway induced by LPS,not the activation of mitogen-activated protein kinase（MAPKs）,and inhibits the release of pro-inflammatory factor NO and Interleukin 1 beta（IL-1β）.Finally,mice were used to investigate the anti-inflammatory and analgesic effects of the compound 26q,and provide relevant scientific basis for the preclinical research of this type of lead compound.