Design,Synthesis,Biological Evaluation And Molecular Modeling Of Novel 1H-pyrazolo[3,4-d] Pyrimidine Derivatives As BRAF^V600E And VEGFR-2 Dual Inhibitors

The mitogen-activated protein kinase(MAPK)is one of the most important intracellular signal pathways.BRAF,one of three RAF isoforms,has the highest mutation frequency in three subtypes.And BRAFV600E leads to continued abnormal activation of downstream signaling pathway,which is crucial for tumor growth,proliferation,invasion and metastasis.However,VEGFR-2 plays an important role in tumor angiogenesis.Its activation by VEGF initiates downstream signaling,ultimately leading to angiogenesis,tumor proliferation and tumor migration.Related studies have shown that BRAFV600E and VEGFR-2 have a synergistic effect in the development and progression of tumors.Based on the crystal structure of Sorafenib in complex with VEGFR-2 and our previous studies,a series of novel 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed,synthesized and evaluated,which provide experimental evidence for the discovery of novel BRAFV600E/VEGFR-2 dual inhibitors.The main research contents are summarized as follows:1.Based on our previous study,compound 1 was further optimized.We reserve the 1H-pyrazolo[3,4-d]pyrimidine ring as scaffold,and cyclized the urea group of 1 into the benzimidazole ring or benzoxazole ring to yield new compounds in order to improve inhibitory activities of BRAFV600E and VEGFR-2 kinases.2.The newly designed 40 1H-pyrazolo[3,4-d]pyrimidine derivatives(7-11)were synthesized with 4,6-dichloro-5-pyrimidinecarbaldehyde as starting material,by the cyclization,1-NH alkylation,4-phenoxy substitution,hydrazine reduction,and aryl isothiocyanate condensation reaction.3.At a concentration of 1 ?M,6 compounds inhibit both BRAFV600E and VEGFR-2 strongly(Inhibition>80%).And 8u(IC50:BRAFV600E = 171.5 nM,VEGFR-2 = 77.9 nM)was slightly weaker than Sorafenib.Cell anti-proliferative experiments showed that most of compounds exerted strong activity on A3 75,HT-29 and HUVEC cell lines.4.The preliminary structure-activity relationship counld be summarized:(1)for the NH alkyl substitutions of 1H-pyrazolo[3,4-d]pyrimidine,the methyl group was the best.(2)The benzoxazole ring showed weaker inhibitory activities than the benzimidazole ring,and the methylated benzimidazole ring showed stronger inhibitory activities than the unmethylated analogue.(3)Between the terminal benzene ring and the amino group,the carbon chain elongation leads to weak activity.(4)The opposite substitution of the terminal benzene ring is optimal for activity.5.The kinase selectivity profile showed that compound 8u had moderate inhibitory activity against CRAF(inhibition = 87.6%),almost poor or no significant inhibitory activity against wild-type(WT)BRAF and 15 other tested protein kinases(Inhibition<60%),which revealed that compound 8u had a very good selectivity profile and was a BRAFV600E/VEGFR-2 kinases dual inhibitor.6.Cell migration assay showed that compound 8u markedly inhibits the migration ability of A375 and HUVEC cell lines with a concentration-dependent effect.Flow-activated cell sorting analysis revealed that compound 8u arrested both A375 and HUVEC cell lines in G0/G1 phase.7.We performed molecular dynamics(MD)study and binding free energy analysis of compound 8u in complexs with BRAFV600E and VEGFR-2 respectively.The binding free energy of compound 8u with BRAFV600E and VEGFR-2 are all negative,indicating that compound 8u can strongly interact with them.Free energy decomposition illustrated that the van der waals(vdW)terms,electrostatic and nonpolar salvation energies contributed to ligand binding,whereas polar salvation opposed binding.