Design,Synthesis And Evaluation Of Anti-inflammatory Activity Of Novel Paeonol Derivatives

T-Lak cell-derived protein kinase（TOPK）,also known as PDZ-binding kinase（PBK）,was identified as a new member of the mitogen-activated protein kinase（MAPK）family and is highly expressed in various actively dividing cells.As the upstream protein of MAPKs,TOPK is closely related to the occurrence of various inflammations,DNA damage,apoptosis and tumor formation,and has been confirmed to be involved in the transduction of multiple signaling pathways in vivo.Although in recent years,it has been reported that the inhibition of TOPK is beneficial to the prevention and treatment of various inflammations,and it has recently been regarded as an effective therapeutic target for the treatment of skin inflammation.However,the intrinsic relationship and mechanism of TOPK activity and skin inflammation have not been reported.In this study,we adopted a fragment growth method to modify the structure of paeonol.First of all,the urea moiety is widely used in drug optimization,because the urea function can form multiple stable hydrogen bonds with proteins or receptor targets.The ortho position introduces a urea linker that can modulate drug potency,selectivity,and improve drug properties.Furthermore,38 compounds（A-D）were further obtained by substitution with different types of amines,heterocyclic/cyclic amines,alkylamines and anilines based on the balance of activity and toxicity.In the synthetic scheme,we use paeonol as raw material,firstly nitrify the 5-position of paeonol to introduce a nitro group,and further catalyze hydrogenation and reduction to amino group,and the obtained intermediate 5-aminopaeonol has the effect of triphosgene.Isocyanate is generated under low pressure,and finally reacts with amine to obtain the target compound.All compounds were first tested for their anti-inflammatory activity,as assessed by LPS-induced NO production in Raw264.7 cells.Cytotoxicity was also evaluated against the two cell lines used in the experiments,the RAW264.7 and HaCaT cell lines;liver toxicity was also evaluated by LO2 cells.For antiproliferative activity,a hyperproliferation model of LPS-stimulated HaCat cells was established to evaluate.Among them,compound B12 was found to be the best compound（Raw264.7 IC50=2.14μM）,low toxicity（MTT IC50>50 μM）and dose-dependent anti-HaCaT hyperproliferation of cells.In addition,preliminary studies on the target validation and mechanism of action of the compounds were carried out.In the CETASA experiment,the binding of the target compound and the target protein was demonstrated by gradient denaturation followed by immunoblotting,and then the binding of the target compound was confirmed in immunoblotting.The compounds can interfere with the function of TOPK,thereby inhibiting the TOPK-p38/JNK signaling pathway and the phosphorylation and expression of skin inflammation-related proteins in the TOPK downstream pathway,as well as inhibiting the overexpression of inflammatory cytokines.At the same time,the drugability of the compound was also studied,its solubility and other properties were predicted by computer ADMET,and its stability was evaluated in human and mouse liver microsomes.In vivo experiments found that the target compound acts on the imiquimod-induced mouse psoriasis-like skin inflammation model,which is a model that widely mimics clinical psoriasis symptoms.All significantly reduced redness,scaling,skin thickening,and PASI scores in a dose-dependent manner.HE histopathological analysis of skin tissue revealed that imiquimod-induced mice exhibited psoriatic lesions,including loss of the granular layer,epidermal hyperplasia,thickening of the acanthoid layer,parakeratosis,and inflammatory cell infiltration.Compound B12 successfully reduced scale,thickness,and erythema in psoriasis-like mice,and histopathologically attenuated hyperkeratosis,acanthocyte proliferation,and inflammatory cell infiltration.Secondly,the study of tissue protein found that the mice induced by imiquimod all overexpressed various related proteins.After compound B12 treatment,the expression levels of each protein were significantly decreased in a dose-dependent manner.Notably,compound B12 inhibited the expression of the proliferation-related protein STAT3 and the precancerous factor PCNA in skin tissue.In addition,the inflammatory factor IL-1β in the skin tissue samples was also detected,and the target compound simultaneously inhibited the expression of the inflammatory factor.In total,we designed and synthesized 38 compounds by modifying the natural product paeonol,and evaluated their anti-inflammatory activities.At the same time,the structure-activity relationship was sorted out.The alkylamine substitution did not significantly improve the activity of paeonol,while the aniline-substituted compounds improved significantly compared with benzylamine and phenethylamine substitution.Among them,trifluoromethyl-substituted compounds Compound B12 had the best anti-inflammatory activity with IC50 of 2.14 μM for NO inhibition in RAW264.7 cells.The mechanism of action was initially discussed,and finally the in vivo experiments were carried out to verify the in vivo pharmacodynamic activity of the compounds.