Studies On The Synthesis? Release Mechanism And In Vitro Antitumor Activity Of The Prodrug With Combretastatin Base On GSH Or Nitroreductase

Cancer is one of the most common life-threatening diseases in the world.Although various approaches for cancer therapy have been developed,several barries to improving effectiveness and avoiding toxicity still remain.Theranostic prodrugs is an emerging technology in biomedicine,which can serve the dual purposes of therapy and diagnosis.Combretastatin is a broad-spectrum and effective natural product,but the cancer therpy application has been limited by toxicity and poor solubility.Thus,two new Combretastatin prodrugs containing a 2,4-Dinitrobenzene-sulfonyl,a4-Nitrobenyl and a coumarin were designed and synthesized.The release mechanisms and antitumor mechanisms of the two compounds were studied by means of HPLC;fluorescence microscopy;flow cytometry and Western Blot.The main points and results of this thesis are as follows:Firstly,the two target compounds were designed and synthesized by four steps including Pechmann reaction,hydrolysis,nucleophilic reaction.The synthesis of two compounds has realized the combination of Combretastatin,coumarin and responsive group.Six compounds were obtained.And all the compounds were characterized by MS,NMR;Secondly,the release mechanisms of two target compounds were studied by means of fluorescence spectrometry and HPLC.In the two compounds,coumarin serves as the reporter of release process via the change of fluorescence signal.The two compounds showed good response to GSH or Nitroreductase,in conclusion,Combretastatin were released from the prodrugs.The release mechanism of two compounds in T-24 cells was studied by the fluorescence microscopy.The fluorescentsignal of the coumarin could be detected in T-24 cells.The results showed that the compounds can release fluorescent molecule(coumarin)and Combretastatin in human bladder cancer T-24 cells.In addition,the two compounds exhibited different inhibitory activities on different cell lines.And the two compounds exhibited potent cytotoxicity against human bladder cancer T-24 cells,human cervical carcinomas Hela cells and human gastric cancer MGC-803 cells in colony formation assays.The results showed the antitumor activity was similar to that of Combretastatin.The toxicity of the two compounds to human liver HL-7702 cells was lower than that of Combretastatin.Besides,the compound 4b has better antitumor activity under anaerobic condition than under normal oxygen condition,which confirmed it was sensitive to Nitroreductase.Finally,the antitumor mechanism of the two compounds was studied by means of flow cytometry.The results showed that the cell cycle were arrested in G2/M phase.The rate of apoptosis has increased with increasing concentration of the two compounds.And the two compounds activitied Caspase 3/8.The compound 4b promoted the cell apoptosis from early apoptosis to late apoptosis and the cell cycle changed from G2/M phase to S phase under the different oxygen concentrations.Moreover,the two compounds inhibited the expression of Tubulin and VEGFR-1protein.These results suggested that the mechanisms of the two compounds are caused by multiple pathway.In summary,two prodrugs of Combretastatin realized drug and fluorescent molecule release processes.The two compounds showed much higher cytotoxicity towards cancer cells than that of normal cells.This work may provide new approachs for designing theranostic anticancer prodrug.