Design,Synthesis And Biological Activity Evaluation Of Kinase RET And CDK4/6 Inhibitors From Two Bioactive Scaffolds

Protein kinases regulate protein phosphorylation which is one of the most vital post-translational modification,and dysfunction of protein kinases has been involved in various pathological processes of diseases,especially in cancers.Therefore protein kinases have been considered as important targets for cancer treatments.Pyrazolpyrimidine and pyrrolepyrimidine are isosteres of adenine,having great medical potential.In this thesis,we designed and synthesized a series of small molecule inhibitors targeting rearranged during transfection(RET)kianase and cyclin-dependent kinase 4/6(CDK4/6)derived from these two bioactive scaffolds,and systematically explored the structure and activity relationship(SAR)of these compounds.In the first part of this thesis,we have designed and synthesized a focus library of 92 small molecules based on pyrazolo[3,4-d]pyrimidine and pyrrolo[3,2-d]pyrimidine scaffolds targeting RET kinase.Ba/F3 cells stably transfected with KIF5B-RET and CCDC6-RET were used for bioactivity screening.Systematic SAR studies revealed two types of combinations of chemical structures among the pyrazolo[3,4-d]pyrimidines for having good bioactivities,namely combination 1,forward amide in ’Hydrogen bond’moiety and ortho substitution of ether bond in’linker’ moiety,and combination 2,reverse amide in ’Hydrogen bond’ moiety,para substitution of ether bond in’linker’moiety and 4-pyridine substituent as ’head’ motif.The representative compound of combination 2,Ⅰ-38,exhibited the best antiproliferative activities against KIF5B-RET and CCDC6-RET transformed Ba/F3 cells with IC50 values of 9 nM and 17 nM,respectively.Ⅰ-38 also showed significant potency against gatekeeper mutation V804M(IC50<50 nM).In contrast the IC50 value against parental Ba/F3 cells was up to 8030 nM,which indicated good selectivity.Furthermore,biochemical assays demonstrated that 1-38 significantly suppressed the enzyme activities of wild type RET(IC50<4 nM)and various mutations of RET(IC50<16 nM),which indicated its on-target effect.In addition,Ⅰ-38 significantly inhibited autophosphorylation of RET and its downstream signals in RET-positive cells,and suppressed tumor growth in mousexenograft models of RET-transformed Ba/F3 cells in dose-dependent manner.Taken together,our results suggest that Ⅰ-38 is a potent and selective RET kinase inhibitor with clinical potential.In the second part,through the analysis of reported CDK4/6 inhibitors,we have designed and synthesized a total of 78 novel compounds based on pyrazolo[3,4d]pyrimidine,pyrrolo[3,2-d]pyrimidine and pyrrolo[2,3-d]pyrimidine scaffolds.Among these compounds,the most active compound Ⅱ-34 was identified by phenotypic screening of three cancer cell lines,having the IC50 value aganist A3 75 cells lower than 50 nM.Ⅱ-34 displayed potent inhibition with IC50 of 111 nM in biochemical enzyme activity assay of CDK4/cyclin D3.Additionally,Ⅱ-34 significantly suppressed phosphorylation of CDK4/6 downstream protein,retinoblastoma protein(Rb),in A375 and MCF-7 cells,and induced cell cycle accumulation in G1 phase.These data establish that Ⅱ-34 is a good starting point for further development of CDK4/6 inhitors.In summary,through systematic SAR exploration of compounds derived from both bioactive scaffolds of pyrazolopyrimidine and pyrrolopyrimidine,we have discovered a potent and selective RET kinase inhibitor and a CDK4/6 kinase inhibitor,which would serve as lead compounds for the developments of targeted drugs.