Synthesis And Biological Evaluation Of 1,7-disubstituted Phthalazinones As Selective Aurora B Inhibitors

Aurora kinase are a class of serine/threonine kinases that play an important role in regulating mitotic processes.Overexpression of this kinase has been clarified in a number of solid tumor cells such as lung,breast,and colon cancer,and thus,Aurora kinase have received attention as a target for the cancer therapy.Generally speaking,selective Aurora kinase inhibitors have fewer side effects and more advantages than pan-Aurora kinase inhibitors.Therefore,based on the previous findings of our group,in order to improve the therapeutic effect of selective inhibitors,we continue to explore novel Aurora B selective inhibitors.In the previous study,the compound ZXJ-25c could inhibit Aurora A and B protein with 16.6%and 97.2%inhibition rates at the concentration of 1μM and the IC₅₀ of Aurora B kinase was 21 n M.We chose the compound ZXJ-25c as the template compound,and discussed the core part,the Linker part,the core modified part and the tail group in turn.After synthesizing four types of compounds,the optimazation of the core modified part and the tail group successfully improved the kinase inhibitory activity and anti-tumor cell proliferation activity.Compound J24 could inhibited Aurora B selectively with the IC₅₀ values of<0.2 n M,which has the highest kinase inhibitory activity among all target compounds.However,its anti-tumor cell proliferation activity was so poor that we finally selected compound J16 as the optimal compound.Compound J16 was tested to selectively inhibit Aurora B kinase over Aurora A with the IC₅₀ values of 0.27 n M by enzyme activity assay,76 times higher than template compound,and the inhibition rate of Aurora A and Aurora B protein was40%and 97.9%at the concentration of 1μM.Moreover,compound J16 was found to inhibit the growth of colon cancer cells Lovo and HT-29 with the IC₅₀ values of1.07±0.44μM and 0.79±0.33μM,respectively determined by MTT method,indicating this compound was highly targeted to colon cancer.Western blotting experiments confirmed that compound J16 could not only inhibited the phosphorylation of Aurora B protein,the expression of cycle-related proteins（Cyclin B1 and cdc2）and the anti-apoptotic protein Bcl-xl in HT-29 cells,but also promoted the expression of pro-apoptotic protein（BAD and Bax）,and all these were in a dose-dependent manner.Furthermore,flow cytometry experiments confirmed that the compound J16 could promote G2/M phase arrest and the apoptosis of HT-29 cells.Through docking simulations,we found that compound J16 could not only form a crucial hydrogen bond with the residue Ala173 in the hinge region,but also form hydrogen bonds andπ-πconjugate interactions at the outer edge of the ATP binding pocket.The results of this thesis show that 1,7-disubstituted phthalazinone compounds exhibit strong selective Aurora B kinase inhibitory activity and potent anti-tumor cell proliferation activity,indicating the introduction of substituents at the 7-position is a remarkable step,which lays the foundation for subsequent deeper activity studies,and the exploration of the other sites in the phthalazinone core has great potential for improvement of Aurora B kinase inhibitory activity.