N-[(4-tert-butyl)phenyl]-N’-[3-(quinazolin-4-yloxy)phenyl] Urea Induces Non-small Cell Lung Cancer Necrosis And An Exploration Of Its Mechanisms Of Action

Diarylurea derivatives which are represented by sorafenib,as small molecule multi-kinase inhibitors,can inhibit the c-Raf and B-Raf kinase activity of downstream RAS gene in tumor cells,blocking RAF/MEK/ERK phosphorylation so that inhibit the effect of tumor cell proliferation and growth.Some small molecules also inhibit vascular endothelial growth factor receptor(VEGFR)and platelet derived growth factor receptor(PDGFR)in endothelial cells and pericytes,thus blocking the formation of tumor neovascularization.It can reach the purpose of anti-tumor.Most of non-small cell lung cancer appear K-RAS genes mutations,which serve as a potential therapeutic target.Therefore,The lead compound which taking diarylurea as pharmacophore,this paper conducts new drug research aiming at the indication of non-small cell lung cancer.This subject conducts a deep research about a series of diarylurea compounds.This paper is divided into five chapters.The first chapter is mainly to provide a comprehensive view of diarylurea compounds and the mode of death of lung cancer cells.The second chapter is to select out a lead compound 6 which its activity is more than the positive standard,compound 6 against the NCI-H460 IC50=4.97±0.39 μM.Through the MTT experiment detecting anti-tumor and anti-proliferative activity of 32 diarylurea compounds to 9 tumor cell lines.To detect the anti-tumor activity of the lead compounds at the cellular level by flow cytometry and confocal microscopy,the main experiments include:cell apoprosis,cell cycle,the detection of cell active oxygen,detection of membrane potential in cell and so on.Through those experiments,we make a conclusion that after drug stimulation,NCI-H460 do not adherence and then appear necrosis.The compound 6 can also induce apoptosis,Its apoptosis ratio compared negative reference(2.45 + 0.18)%to the dosing large concentration group(26.53+0.96)%;And the cell cycle G0/G1 phase ratio increased from the Control(70.38+2.78)%to Compd.6,20 μM(94.74+ 4.39)%,So compound 6 could block the cell cycle in G0/G1 phase;Intracellular reactive oxygen levels,and as its flow intensity of fluorescence detection results,Control(100.0+1.89)%to compd.6,μM(175.3+2.92)%;Mitochondrial membrane potential,as flow test results P1 region(mitochondrial membrane potential in the higher state),and with the increase of drug concentration of cells in the areas of P1 reduce gradually,and P2 region(mitochondrial membrane potential in low status)cell proportion rising gradually decline,so the compound 6 could reduce the mitochondrial membrane potential.The third chapter is to make a deep exploration of anti-tumor molecular mechanism of compound 6.When we receive the drug stimulation within 12 hours,the main mode of cell death is apoptosis.When it reachs above 12 hours,most of the cells will appear oncosis.From the conclusion of protein blotting analysis detecting the autophagy,we can improve that oncosis will be followed by autophagy.For another aspects,we found that compound 6 also can block signal expression of Ras/Raf/MAPK/ERK,affect the expression of related protein of cell cycle and the expression of related protein of apoptotic pathway.We use Fluorescent quantitative PCR to detect compound 6 of affecting the expression of autophagy gene and c-Raf gene,and we make a evalution of the effect of small molecule drugs and DNA.The fourth chapter,we drench drug to SD rats.At the same time,we collect blood and metabolites from the inferior orbital vein in rats,using HPLC and HRMS to make quantitative analysis of blood concentration and explore the possibility of drug metabolites.We also make experiment of water partition coefficient.The fifth chapter,based on the previous published compound 8g,adding a N-acetyl piperazine ring into the quinazoline ring forms a new compound in order to improve its water solubility.In the whole research,we found that a new compound which takes sorafenib as a template,its pharmacological activity is more than maternal.And in the experiment,we improve some disadvantages of sorafenib,therefore,our lead compound has the potential of becoming the anti-cancer drug.