Design,Synthesis And Anti-tumor Activity Of Novel Selective ACK1 Inhibitors

Activated Cdc42-associated kinase1（ACK1）,also known as TNK2,is a non-receptor tyrosine kinase（NRTK）.ACK1 shuttles between the cytoplasm and the nucleus to quickly transmit extracellular signals from receptor tyrosine kinase（RTK）to intracellular effectors.Studies have found that ACK1 gene amplification and mutations have been observed in a variety of human malignancies（such as:lung cancer,prostate cancer,gastric cancer,pancreatic cancer,breast cancer,and ovarian cancer）.Abnormally activated ACK1 can not only promote the activation of key pro-survival kinases and receptors by phosphorylation of different tyrosine residues,but also eliminate tumor suppressor factors from cancer cells to cause cancer.Recently,we and other research groups found that ACK1 can mediate non-small cell lung cancer resistance to third-generation EGFR inhibitors（such as osimertinib and ASK120067）through bypass activation.Therefore,inhibiting the activity of ACK1 can be used as potential strategies to overcome clinical resistance.However,currently no selective ACK1 inhibitor has entered clinical studies.Therefore,there is an urgent need to design and synthesize ACK1 inhibitors with novel frameworks and high selectivity.In this paper,on the basis of the reported ACK1 inhibitor R-9b,we used molecular simulation docking to analyze the binding mode of the compound and the protein.Through the strategy of backbone transition,we identified the pyrimidopyridone compound 13 as the lead molecule(IC₅₀ value is 0.4 n M).Subsequently,the structure optimization and structure-activity relationship study of compound 13 were carried out,and a series of new ACK1 inhibitors of pyrimidinopridones were designed and synthesized.Most compounds have strong inhibitory activity on ACK1(IC₅₀ value is 0.3-2.1 n M).Western blot experiments also showed that representative compounds 13j can significantly inhibit the phosphorylation level of ACK1 and its downstream protein AKT,but the kinase selectivity of compound13j is poor.In order to further improve the selectivity of inhibitors,we analyzed the ACK1 protein and SRC protein overlap analysis and found that ACK1 has a larger back pocket.From this we designed and synthesized the compound 13ze-13zk,of which the representative compound 13zj has better inhibitory activity against ACK1,IC₅₀is 2.1n M.When screening 468 kinases,compound 13zj also showed good kinase selectivity,with selectivity coefficients S（35）,S（10）and S（1）of 0.159,0.087 and 0.015,respectively.In addition,when the concentration of compound 13zj is 10μM,some off-target kinase inhibitory activities are tested:CSF1R(IC₅₀=100.8 n M),ABL(IC₅₀=1.68μM),DDR1(IC₅₀=1.68μM),DDR2(IC₅₀=1.68μM),ZAK(IC₅₀=71.2 n M),RET(IC₅₀=27.7 n M),PDGFRB(IC₅₀=59.04 n M),KIT(IC₅₀=1.23μM),SRC(IC₅₀=218.7 n M).Preliminary studies have shown that compounds 13zj can also significantly inhibit the phosphorylation level of ACK1 and its downstream protein AKT.In this thesis,through rational drug design and structural optimization strategies,our studies provide a new ACK1 inhibitor 13zj with good biological activity and pharmacokinetic properties.Further in vivo research is still in progress.The research of this subject will provide a certain research foundation for the research and development of ACK1 inhibitors and overcoming the resistance of EGFR third-generation inhibitors.