Design,Synthesis And Bioactivity Evaluation Of TANK-binding Kinase 1 Inhibitors

TANK binding kinase 1（TBK1）,a crucial serine/threonine kinase,functions as a key node protein in several cell signaling pathways and integrates a variety of upstream signals and directly modulates the function of numerous downstream targets.TBK1 is central to multiple biological processes that promote tumorigenesis and immune inflammatory reaction,including cell division,autophagy,innate immune response.Detailed understanding of its active mechanism and exploration of the application of its inhibitors in various diseases is required.First,TBK1 can induce cell mitosis,which makes it as a crucial factor in the developmental process of tumor.The unlimited proliferation of tumor cells requires the support of mitosis,therefore,TBK1 has a pro-tumor effect.TBK1 is located in the downstream of the transmembrane protein stimulator of interferon genes（STING）in the type I interferon（IFN-I）response pathway.In the immune response caused by virus,STING senses the presence of nucleic acids from intracellular pathogen infection and in turn initiates a downstream signaling cascade that includes TBK1-mediated activation of IRF3,resulting in IFN-I and cytokine production,and produces the antiviral response.Spontaneous T cell responses against tumors occur frequently and have prognostic value in patients.The major mechanism for innate immune sensing of cancer occurs via the host STING pathway.As a key protein in this pathway,TBK1 plays an important role in anti-tumor immunity.Intratumoral injection of synthetic cyclic dinucleotides to activate STING resulted in primary tumor regression,which implicates TBK1 as a potential anti-tumor protein.However,TBK1 has shown different effects in dendritic cells（DC）.DC-specific deletion of TBK1 causes T cell activation and autoimmune symptoms leading to enhanced antitumor immunity in animal model.TBK1 was essential in cells that contain mutant KRAS.Suppression of TBK1 induced apoptosis specifically in human cancer cell lines harboring oncogenic KRAS.TBK1 has been shown to promote the intracellular degradation pathway,including autophagy that is often deregulated in human cancers.Though it has gradually become a potential target for cancer immunotherapy,highly effective and selective TBK1 inhibitors are still under development.Currently,no selective TBK1 inhibitors have entered clinic trials.Therefore,this thesis is aimed to design and synthesis of novel and efficient TBK1 inhibitors and exploration of their potential application in cancer immunotherapy.From the reported structural scaffolds of TBK1 inhibitors,we chose compound2-1,a well-studied TBK1 inhibitor bearing 2-aminopyrimidine skeleton,as the reference compound.Three series of analogues were designed and synthesized,including compounds by 1)altering the side chains attached to piperazine N and changing the piperazinyl ring,2)designing double-target inhibitors and 3)synthesizing degraders（PROTAC）.A total of 24 new compounds were obtained and characterized.Through the introduction of side chains on the piperazine,we got compound 3-1～3-11.We found the inhibitory activity against TBK1 was greatly increased by introducing chains that have small steric hindrance with more hydrogen bond binding sites.An IC₅₀ value of 0.1 n M was determined for compound 3-10against TBK1,which is 200-fold more potent than compound 2-1.Compound 3-14 is a TBK1/HDAC dual target inhibitor,which an IC₅₀ value of 0.6 n M against TBK1,33-fold more potent than compounds 2-1.We also designed several PROTAC compounds（compounds 3-15 to 3-23）to degrade TBK1 protein.Among the five compounds containing PEG link chains,compounds 3-16 and 3-20 showed IC₅₀values 7.2 n M and 7.4 n M,respectively.These results will be valuable for further studies on both TBK1-mediated mechanism and treatment application.