| Bis-arylurea compounds are small molecule multi-kinase inhibitors,the drug on liver cancer,non-small cell lung cancer and melanoma have a certain effect,mainly for non-surgical removal of advanced kidney cancer,liver cancer and lung cancer treatment.However,Sorafenib as the representative of bis-arylurea compounds listed drugs poor water-soluble,more toxic with more side effects.Therefore,the development of sorafenib structure analogues of innovative tumor drugs,to obtain better efficacy of new drugs,is of great significance.Quinazoline bis-arylurea compounds also have a wide range of physiological activities,are a lot of tumor target enzyme inhibitors,have become a research focus of anti-tumor drugs.In this study,a series of quinazoline bis-arylurea derivatives have been synthesized and studied antitumor activity from cell level,molecular level,gene level and pharmacokinetics.This paper consists five chapters.Chapter 1 summarizes the bladder cancer and its treatment,the biological activity of quinazoline compounds and the way of cell death.Chapter 2 studies the anti-tumor activity on a series of novel quinazoline bis-arylurea compounds at the cellular level.First,we used MTT assay to test in vitro proliferation inhibitory activity of eight tumor cell lines(Bel-7402,MGC-803,HCT-116,HCC-827,T24,4T1,B16 and RT4),and two human normal tissue cell lines(WI-38 and HCV-29)were tested for cytotoxicity.We found that compared with other types of cell lines,the vast majority of quinazoline bis-arylurea derivatives are very sensitive to bladder cancer cell lines,and five of these compounds(1j,1f,1g,2d,2f)against T24 cells IC50 value were 3.52μ±0.91,2.85±0.47,1.2 6±0.12 μM and 2.36±0.17 μM,their proliferation in vitro inhibitory activities exceed the positive reference(Gemcitabine:IC50=4.73μM).Therefore,we conclude that compound Ij is much more active in the proliferation of bladder cancer cell lines than in the positive reference,so we set the compound as an indication of bladder cancer,continued its in-depth study.In addition,we used the compound 1j as the representative,using the fluorescent staining(calcein and PI fluorescence staining),flow cytometry(FITC Annexin V/Pl double staining)proved that compound 1j can induce apoptosis,the apoptosis ratio of dosing group(59.51 ± 3.62)%;And the cell cycle G0/G1 phase ratio increased from the Control(51.13 ± 2.37)%to Compd.1j,8 μM(66.50 ± 3.13)%,So compound 1j 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±1.95)%to compd.1j,8 μM(149.7± 2.56)%;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 1j could reduce the mitochondrial membrane potential.In conclusion compound 1j can induce bladder cancer cells apoptosis,block the cell cycle,lift the intracellular ros level,decrease of the mitochondrial membrane potential and calcium outflow.Tumor cells function is broken down,thus suppressing the proliferation of bladder cancer cells.Chapter 3,in this chapter we used protein immune-blotting(western blot)to detect the expression of related proteins in multiple pathways,Including cell cycle-related protein expression detection;Raf/MEK/ERK pathway-related protein expression detection;programmed necrotic pathway-related protein expression detection;PI3K/Akt/mTOR pathway-related protein expression detection;autophagy-related protein test results;.We conclude that compound 1j can up-regulate the expression of autophagy-associated protein LC3B,thereby inducing autophagy and death of bladder cancer cells to achieve the purpose of inhibiting tumor cells.Chapter 4,At the gene level,we used agarose gel electrophoresis and fluorescence quantitative PCR to detect the antitumor activity of compound 1j.We conclude that compound 1j disrupts its activity by covalent interaction with DNA molecules.In addition,the expression of Raf gene and RIP1 gene was detected.By detecting the expression of Raf gene and RIP1 gene,it was found that the expression of C-Raf gene was down-regulated with the increase of drug concentration and the expression of RIP1 gene expression was up-regulated,consistent with the results of protein imprinting experiments.Chapter 5,in the fifth chapter,we tested the plasma concentration of rats after oral administration,and obtained the relevant pharmacokinetic parameters of compound 1j from the experimental resultsIn summary,we screen out the lead compound 1j in some of the column compounds and has a strong inhibitory activity in vitro(more positive for sorafenib and gemcitabine),we study and characterize its molecular mechanism through cell level,molecular level,gene level,pharmacokinetics.Thus compound 1j has the potential to become a clinical chemotherapeutic agent. |
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