Design,Synthesis And Biological Evaluation Of Novel P21-Activated Kinase 4 Inhibitors

Malignant tumors has become the first cause of death of urban residents in China,the development of anticancer drugs has been the preferential themes of Chinese Science and Technology Development Plan.It has been shown that the dysregulation of protein kinase signaling pathway in tumor cells is the main cause of tumorigenesis and tumor metastasis.Therefore,in recent years,protein kinase inhibitors have been the hot topic and attractive interest for antitumor drug development.p21-activated kinases(PAKs)are a class of serine/threonine protein kinase,which is a central node of many intracellular signaling pathways.PAK4 plays an important role in the regulation of cytoskeleton,apoptosis and cell migration.In recent years,PAK4 gene amplification and over-expression were found in a variety of tumor cells such as gastric cancer,breast cancer,colon cancer etc,in particular,it plays important role in tumor invasion and metastasis process,therefore,PAK4 has become a new antitumor drug target.In this thesis,we chose PAK4 as the research subject to design and synthesis novel small molecular inhibitors in order to develop novel PAK4 inhibitors,additionally to provide chemical probe for exploring the PAKs signal transduction mechanism.The main work of this thesis are as follow:Compound 7915461 is a specific PAK4 inhibitor previously discovered by our team which could inhibit the migration and invasion of gastric cancer cell line SGC7901,we synthesized the biotin-labeled compound 7915461 in order to explore the mechanism of PAK4 inhibition of this compound by biotin-avidin binding experiments,and the results showed that compound 7915461 is able to interact with full-length PAK4 and the C-terminal of PAK4 protein,while the N-terminal of PAK4 could not interact with compound 7915461.From these results we conclude that compound 7915461 is an ATP-competitive inhibitor of PAK4,and this work lay the foundation for further study on the mechanism of our synthesized PAK4 inhibitors.Based on the structural features and the molecular docking mode of compound 7915461,We designed and synthesized four series of novel pyrimidone derivatives.And all target compounds was confirmed by MS and 1H-NMR spectroscopy,with 83 unreported.Using experimental(X-ray,H-NMR)and theoretical(ADF calculation)methods,we identified the most stable tautomer and the different tautomeric situation in solvents of important intermediate N-phenyl-2-thiobarbituric acid and the target compound GA03.We found that N-phenyl-2-thiobarbituric acid exists in triketo form in the solid state and in CDC13 solution while tautomerization was observed in DMSO-d6,DMF-d7 and CD3OD solution.And target compound GA03 exists in enol form in solid state and in DMSO-d6 solution whereas in CD3OD solution tautomerization occurs.We also identified the methylation site of N-phenyl-2-thiobarbituric acid by X-ray diffraction and ADF calculation finding that the methylated product to be the most energetically stable one.Additionally,we also identified the methylation site of some methylated target compounds and the molecular structure of important intermediate 6-amino-1-phenyl-2-thioxo-2,3-dihydro-pyrimidin-4-one by NOESY spectrascopy.To explore the PAK4 kinase inhibitory activity of our target compounds,we determined the inhibitory rate of 67 target compounds by Kinase-Glo(?) Luminescent Kinase Assay.The results show that the PAK4 inhibitory activity of some synthesized derivatives have improved greatly compared to lead compound.27 compounds showed better activity than lead compound and 18 compounds showed better activity than positive control staurosporine.And the structure-activity relationships are summarized to give direction to further optimization.We also tested the anti-migration activity of 93 target compounds on gastric cancer cell lines SGC7901 and MKN45 at 20 μM concentration.And some of the target compounds showed comparative activity to lead compound.One compound GB20 showed better activity than lead compound.The anti-migration activity of our synthesized compounds showed certain correlation to PAK4 kinase inhibition activity.In order to provide positive control to follow-up study with specific PAK4 kinase inhibitory activity,we synthesized a carbonylamimo pyrrolopyrazole PAK4 inhibitor 456054 developed by Pfizer through 12-step reactions.And the target compound was confirmed by MS and 1H-NMR spectra.The total yield was 0.56%.Epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor Gefitinib is one of the most popular anti-cancer drugs in china.We analyzed the existing synthesis route of Gefitinib in literature and chose an environment-friendly route.Now the Laboratory Scale test has been completed.The total yield was 28.9%.Further optimization of each step is under way.To date,there are very few specific PAK4 inhibitors reported.In this thesis,we design and synthesized a series of novel PAK4 inhibitors based on the previous work of our team.Preliminary biological evaluation indicated that some compounds had better activity than lead compound.The structure-activity relationship of the target compounds were briefly summarized to give directions to further optimization.In a word,this thesis lays substantial foundation for development of specific PAK4 inhibitor.