Studies On Synthesis, Characterization And In-vitro Cytotoxicity Of Hypoxia-selective5,10-dioxy-indeno[1,2-b]quinoxalin-11-one Oxime Ethers

The hypoxia environment of tumor provides a target for tumor therapy. Bio-reducingdrugs can selectively inhibit cell growth and tumor hypoxia. Nitrogen oxides compound isone of the representative and promising compounds. Quinoxaline nitrogen oxides indene(1,2-b) quinoxaline-5,10-dioxane-11-one belonging to a nitrogen oxide, has hypoxia activity.However, its poor solubility results in the low diffusion ability, limiting its applications.Taken the poor fat-solubility of indene(1,2-b)quinoxaline-5,10-dioxane-11-one intoconsideration, we used1,2-diaminobenzene and1,2,3-Indantrione hydrate as raw materials.The indene(1,2-b)quinoxaline-5,10-dioxane-11-one oximes was obtained after condensation,oximation, etherification, oxidation and amination reaction. We investigated the effects ofsolvents, reagents and other reaction conditions on the yield. The structures of the synthesizedcompounds and intermediates were proved by mass-spectrometry, IR spectroscopy andNMR-spectroscopy. The MTT assay was used to investigate the in vitro cell cytotoxicity ofindene(1,2-b)quinoxaline-5,10-dioxane-11oxime ethers to Hela cell lines (Human cervicalcancer cell line), A549cell lines (Human lung adenocarcinoma epithelial cell line) andMCF-7cell lines (Human breast cancer cell line) under normal and hypoxia conditionsrespectively. We utilized PI single staining assay and Annexin V/PI double staining assay todetect the effect of II-a on the blockade and apoptosis of Hela cell lines. Comet assay wasused to investigate the the damage of IV-a to the DNA of Hela cells under normoxic andhypoxic conditions. We also explored the mechanism how the indene(1,2-b)quinoxaline-5,10-dioxane-11oxime ethers produced hypoxia selectivity.The results showed that: the introduction of oxime ether chain and terminal groupinduced the fat-solubility increasment three-fold and water-solubility incensement2-fold. TheMTT assay results showed the indene(1,2-b)quinoxaline-5,10-dioxane-11-oxime ethershowed high cytotoxicity and some hypoxia selectivity. The IC50of compounds IV-a inhypoxic conditions for A549cells reached2.34μmol/L and the hypoxia cytotoxicity ratioreached2.7. The introduction of oxime ether chain and terminal group could increase thecytotoxicity of cells and hypoxia selectivity. When terminal group is the same, the shorterlength of the side chain of the compounds leads to the high hypoxia selective activity of thecompounds. When the length of the side chain is the same, the sequence of differentend-substituents is piperidine> pyrrolidine> dibutylamine> morpholine. The cell cycle andapoptosis experimental results showed that the compounds II-a cell could arrested Hela cells in G0/G1phase, leading to the apoptosis of cells. Moreover, with the increasing concentrationof the compounds, the cell cycle and apoptosis phenomenon becomes more obvious. Whenthe concentration of II-a increased from5μmol/L rose to10μmol/L, the G0/G1phaseproportion of Hela cells increased from57.33%to68.83%, the proportion of early apoptosisincreased from31.25%to55.62%, the proportion of late apoptosis increased from0.13%to2.20%. The comet experimental results showed that the damage of compound IV-a to theDNA in Hela cells is more pronounced in hypoxia conditions than in normoxic conditions.Under normoxic conditions, tail DNA percentage of control group was0.01%, dosing groupwas3.63%. Under hypoxic conditions, tail DNA percentage of control group was0.01, dosinggroup was7.43%. The hydroxyl radicals released by Indene(1,2-b)quinoxaline-5,10-dioxane-11-oxime ethers in hypoxic environment may caused the damage of DNA in Helacells, which in turn caused Hela cells cell cycle arrest and apoptosis.