Studies On The Anti-tumor Effects And Its Molecular Mechanism Of 1H-Pyrazolo[3,4-d] Pyrimidine Derivatives As Pan-RAF Kinase Inhibitors

The mitogen-activated protein kinase(MAPK)cascades consist of the RAS/RAF/MEK/ERK signal transduction play an important role in the regulation of cellular activities,including proliferation,differentiation,and survival.RAF kinase is a key kinase in this pathway,which can be activated easily in a variety of ways.Therefore,RAF kinase inhibitors have become a hot spot in the research of antitumor drugs.In this paper,a series of new compounds were designed and synthesized using 1H-pyrazolo[3,4-d]pyrimidine as a template of tyrosine kinase hinge-binding,the kinase inhibitory activity and in-vitro anti-proliferation activity of the compounds were evaluated,and the molecular mechanism of the in-vitro anti-tumor activity was investigated,which provided experimental evidence for the development of pan-RAF kinase inhibitor with higher activity and more novel structure.The main contents are summarized as follows:1.1H-pyrazolo[3,4-d]pyrimidine,a template of tyrosine kinase hinge-binding,with similar structure of the purine,was extensively chemically modified to improve the biological activity and kinase selectivity.To find potent RAF inhibitors,and encouraged by the success of Sorafenib,we designed Sorafenib analogue la by replacing the pyridine ring of Sorafenib with 1H-pyrazolo[3,4-d]pyrimidine moiety.The model suggested that 1a overlapped well with the co-crystal ligand Sorafenib in the active site of BRAFV600E.With 4,6-dichloropyrimidine-5-carbaldehyde as raw material,we synthesize a series of 1 H-pyrazolo[3,4-d]pyrimidine derivatives(1a-1t)via cycloaddition,tetrahydropyrane protection,4-phenoxy substitution,condensation reaction with aryl isocyanate,deprotection reaction etc.With 4,6-dichloropyrimidine-5-carbaldehyde as raw material,we synthesize a series of 1-methyl-1H-pyrazolo[3,4-d]pyrimidine derivatives(1u-1v)via cycloaddition,1-NH methylation,4-phenoxy substitution,condensation reaction with aryl isocyanate etc.A total of 22 compounds were synthesized.2.The enzyme activity assays of the synthesized compounds were performed,and 12 compounds exhibited inhibition rate of more than 60%at 1 μM.Compound 1v exhibited stronger inhibitory activities against BRAFV600E than the positive control Sorafenib(IC50=23.6 nM).The anti-proliferative experimental results showed that most compounds exhibited better anti-proliferative activities against four tumor cell lines(A375,HT-29,PC-3 and A549)and better selectivity towards cancer cells than normal cells(Madin-Darby canine kidney,MDCK).3.The kinase selectivity of compound 1v was performed.The results revealed that 1v had potent inhibitory activity against not only BRAFV600E but also wild-type BRAF(IC50 = 51.5 nM)and CRAF(IC50 = 8.5 nM),but almost no significant inhibitory activity against 13 other tested protein kinases even at concentration of 1000 nM(Inhibition%<30%).The results revealed that compound 1v had a very good selectivity profile and was a pan-RAF kinase inhibitor.4.The scratch-wound assay suggested that compound 1v markedly inhibits migration ability of A375 cell line with a concentration-dependent effect.Flow-activated cell sorting analysis revealed that compound 1v arrested the cell cycle of A375 cell line in the G0/G1 phase and PC-3 cell line in the G2/M phase.5.Western blot analysis showed 1v caused significant suppression of MEK phosphorylation in A375 and HT-29 cell lines and reduced the level of cell cycle protein cyclinD1.6,We performed a molecular dynamics(MD)study and free energy analysis on 1v in complex with BRAF.The binding free energy value of compound 1v and BRAFV600E was negative,indicating that there was a strong interaction between compound 1v and BRAFV600E.Free energy decomposition showed that the favorable contributors to ligand binding were van der Waals(vdW)terms,electrostatic and nonpolar salvation energies,whereas polar solvation opposed binding.