Design,Synthesis And Biological Evaluation Of Novel BTK PROTAC Degraders For The Treatment Of Inflammatory Diseases

The regulatory role of Bruton’s tyrosine kinase(BTK)in multiple signaling pathways in B cells and other myeloid cells has made it a promising target for the treatment of hematologic malignancies,inflammatory and autoimmune diseases.In this thesis,we firstly provided an overview of the structure and function of BTK,as well as the development of small molecule inhibitors targeting BTK.Additionally,we discussed the research progress of BTK-targeted Proteolysis-Targeting Chimeras(PROTACs).Given the current limitations of BTK PROTACs,including inadequate degradation efficiency and limited scope of indications,we hereby presented our endeavors in designing,synthesizing,and evaluating the bioactivity of novel BTK PROTACs,specifically their potential as anti-inflammatory agents.In the second section of this thesis,we presented the design,synthesis,and evaluation of anti-inflammatory properties of BTK PROTACs based on ibrutinib(I1～I23).Among them,compound 115 was identified as a highly potent BTK PROTAC with a DC50 of 3.18 nM in Ramos cells.In addition,115 could degrade BTK protein almost completely within 8 hours,which was superior to the positive control MT802.Importantly,115 was also effective in degrading BTK protein and inhibiting NF-κB activation in LPS-stimulated RAW264.7 cells,leading to a reduction in the mRNA expression and secretion of proinflammatory cytokines such as IL-1β and IL-6.Furthermore,compound 115 effectively reduced the acute inflammatory responses in a mouse zymosan-induced peritonitis(ZIP)model.The third chapter of this thesis aimed to enhance the efficiency of BTK PROTACs.We presented the design,synthesis,and analysis of the structure-activity relationships(SAR)of novel BTK PROTACs using Spebrutinib,a BTK inhibitor in clinical trials.We developed a series of covalent(Ⅱ-A1～A5)and non-covalent(Ⅱ-B1～B13,Ⅱ-C1～C13 and Ⅱ-D1～D12)BTK PROTACs through structure-based-drug-design(SBDD)strategies.Ⅱ-C5,a non-covalent BTK PROTAC,exhibited outstanding degradation potency and efficiency(DC50,4h=1.29 nM,t1/2,20 nM=0.59 h),better than that of 115(DC50,4h=18.88 nM,t1/2,20 nM=1.51 h).Molecular simulations showed that Ⅱ-C5 induced a ternary complex[BTK-PROTAC-CRBN]with a distinct spatial orientation and a larger protein-protein interaction(PPI)surface area than 115,resulting in stable incorporation of the ternary complex and enhanced degradation potency and efficiency of Ⅱ-C5.Furthermore,Ⅱ-C5 was shown to down-regulate the BTK-PLCy2-Ca2+signaling pathway activated by receptor activator of nuclear factor-KB ligand(RANKL)to prevent osteoclastogenesis and attenuate alveolar bone resorption in a mouse periodontitis model.Collectively,in this study,66 novel BTK PROTACs were synthesized and a systematic analysis of the linkers,BTK-targeting ligands,and CRBN-targeting ligands was conducted to expand the chemical space of BTK PROTACs.Among them,Ⅱ-C5 was identified as a highly potent and fast BTK PROTAC,surpassing previously reported BTK PROTACs in terms of its degradation efficiency.Furthermore,this study showed,for the first time,that BTK-targeting PROTAC degraders can be used to treat inflammatory diseases,such as peritonitis and periodontitis.Our findings further broaden the possible applications of BTK PROTACs,and compounds I15 andⅡ-C5 can be considered as promising leads for future development.