Design,Synthesis And In Vitro Anti-inflammatory Activity Evaluation Of Novel CDK8 Inhibitors

Inflammation is the immune defense response of the body.The persistence of inflammatory factors will lead to chronic inflammation and affect the development of diseases.Inflammatory microenvironment is an important factor in the occurrence and development of all tumors.An increasing number of tumor therapy-related kinases have been identified as potential therapeutic targets for specific inflammatory diseases.Cyclin-dependent kinase(CDKs)is a multifunctional enzyme.In addition to cell cycle regulation,CDKs also have a variety of physiological functions,such as transcription,epigenetic regulation,and metabolism.Among them,the classical kinase target CDK8 is involved in the physiological functions of different tumors,such as occurrence,metabolism,drug resistance and metastasis,and is considered to be an ideal anti-tumor target.Recent reports have shown that CDKs initiate inflammatory responses by activating the activities of important pro-inflammatory transcription factors,such as agonistic nuclear factor-κB(NF-k B),signal transduction and transcriptional activator 3(STAT3)and activator protein-1(AP-1).Cyclin-dependent kinase 8(CDK8)has been found to be involved in the negative regulation of the anti-inflammatory cytokine IL-10 in bone marrow cells and the NF-κB and C/EBPβ-mediated innate immune response in multiple myeloma during the innate immune response.Therefore,CDK8 is considered to be an inflammatory mediator that plays an important role in the inflammatory signaling pathway,indicating its potential as an anti-inflammatory target.Two highly active CDK8 type II inhibitors,compounds C22 and C29,were previously reported by our group,and in this study,compounds 1-14 were synthesized by splicing two active fragments with different active sites based on the structural features of these two compounds.Compounds 1-10 were found to be more toxic and less active;Although the activities of compounds 11-14 were clearly improved,the toxicities remained substantial;Through structural analysis,the dominant structural fragment of the disubstituted phenyl group was retained,and the 2-aminopyridine structure was replaced with a pyrrolopyridine structure,leading to the synthesis of compounds 15-17,which were found to exhibit not only reduced toxicity but also increased activity.The inhibition rate of CDK8 kinase was used as the criterion to evaluate the activity of the compounds,we chose compound 16 as the final target compound to continue the study of anti-inflammatory activity in vitro.In the following anti-inflammatory study in vitro,we used LPS-induced RAW 264.7 cells as an inflammatory model to elucidate the potential anti-inflammatory mechanism of the compound.Firstly,we screened the cytotoxicity of the compounds by MTT method,and found that the target compound 16 showed low cytotoxicity in Human Colorectal Carcinoma Cells(HCT-116).Through the Cellular Thermal Shift Assay(CETSA),it was found that in HCT-116 cells,the target compound 16 can improve the thermal stability of CDK8,indicating that the compound can bind to the CDK8 target,and can also inhibit the phosphorylation of STAT1 at Ser727 mediated by CDK8 in colorectal cancer cell line HCT-116,and has no inhibitory effect on the phosphorylation at Tyr701.It indicates that it can affect the biological function of CDK8 in cells.Next,we screened the toxicity of the compound to leukemia cells in mouse macrophage(RAW264.7)by MTT,and found that the target compound 16 had no significant cytotoxicity to RAW264.7 cells.Next,we screened the anti-inflammatory activity of the compounds by Griess.The target compound 16 can better inhibit the production of nitric oxide in RAW264.7 cells after LPS stimulation,and found the compound with the best anti-inflammatory effect.Then,in order to better evaluate its anti-inflammatory activity,its effect on the expression of cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(i NOS)proteins was studied by Western Blotting experiments.It was found that the target compound 16 could inhibit the expression of COX-2 and i NOS in a dose-dependent manner.ELISA analysis showed that target compound 16 could inhibit the production of inflammatory cytokines interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in LPS-stimulated RAW264.7 cells.The results of Western Blotting showed that the target compound 16 significantly attenuated the activation of NF-κB and MAPK signaling pathways induced by LPS,and blocked the phosphorylation of IκBα,p65,p38,ERK and JNK.