Synthesis And Anti-tumor Activity Research Of The Glycosylated Aromatic Nitrogen Mustard Derivatives

Hepatocellular carcinoma is one of the most common digestive system tumors in China, which is malignant, invasive, and metastatic. The available therapies such as , surgery, radiotherapy and chemotherapy can not achieve ideal effectiveness. Howerver, the chemotherapy is a major therapy for the extrahepatic metastases and intrahepatic diffusion.In present,the anti-hepatocellular carcinoma drugs have some shortages,such as low efficiency, poor selectivity, high toxicity for normal tissues.The key point to solve these questions is to find and definite the effective target to drugs,and synthesis specific drugs targeting at the known molecule or gene.The key point to develop anti-hepatocellular carcinoma targeting drugs is to find specific targets for hepatocellular carcinoma cells.Now some molecules ,which have some relations with hepatocellular carcinoma ,will be become new target points,such as specificness gene , receptor or enzyme ,and so on .We can design and synthesis some drugs by the targets and maybe discover some original targeting drugs for anti-hepatocellular carcinoma treatment.The nitrogen mustard is an important anticancer drug in the clinic tumor treatment and belongs to biological alkylating agent. Although it is effective in anti- hepatocellular carcinoma therapy, it has some shortages. In order to elevate the specificity of nitrogen mustard for tumor cells and decrease the toxicity for normal cells, two glycosylated aromatic nitrogen mustard derivatives were synthesized to test their antitumor activities. Glucose and lactose were converted into glycosyl donors-trichloroacetimidate; the obtained glycosyl donors were glycosylated with p-nitrophenol (glycosyl donors) to formβ-glucosyl p- nitrobenzene andβ- lactosyl p- nitrobenzene that were protected by acetyl in a stereoselective manner; the two products were reduced by zinc dust and then treated with ethylene oxide, afforded two glycosylated nitrogen mustard derivatives that were protected by acetyl; the last step was to deacetylate and then afforded the two target compounds that could be used as prodrugs for further Anti-tumor research.Glycosylated aromatic nitrogen mustard derivatives as the prodrugs of targeted therapy in the present study. Anti-tumor activities are tested to select prodrugs that will be used in molecule targeted therapy.The main results obtained from our research are as follows:1 Glycosyl donors-trichloroacetimidate was selected as the method of glycoside synthesis through a great number of screening experiments, for the first time in the world, two glycosylated aromatic nitrogen mustard derivatives: benzene nitrogen mustard glucoside derivate and benzene nitrogen mustard lactose derivate were synthesized. The cost of the synthesis was much lower and the yield was much higher. Two glycosylated aromatic nitrogen mustard derivatives were proved to be high purity after being tested by HPLC, and their structures were identified by NMR.2 Inhibition to normal cells of 3T3 and Vero cells by both benzene nitrogen mustard glucoside derivate 7a and benzene nitrogen mustard lactose derivate 7b is much lower compared with nitrogen mustard , which provides a priority for their application in targeted therapy.3 The results from tumor growth inhibition by benzene nitrogen mustard glucoside derivate 7a and benzene nitrogen mustard lactose derivate 7b using 7721 hepatumor cells showed that the two derivates can inhibit the growth of 7721 cells significantly, and 7b can inhibit the growth of 7721 cells more significantly than 7a. The IC50 of benzene nitrogen mustard glucoside derivate 7a and benzene nitrogen mustard lactose derivate 7b using 7721 hepatumor cells are 64μg/ml and 45μg/ml ,because benzene nitrogen mustard lactose derivate 7b has galactosyl-ligand, which can bind to galactosyl-receptor on the surface of hepatumor cells, so hepatocellular carcinoma is the target cells for 7b.4 The antitumor mechanisms of two glycosylated aromatic nitrogen mustard derivatives were explored by 7721cell line in vitro at morphological, cell cycle and gene level. The results showed that both benzene nitrogen mustard glucoside derivate 7a and benzene nitrogen mustard lactose derivate 7b can induce the apoptosis of 7721 cells, and flow cytometery results demonstrated that these two nitrogen mustard derivatives can induce 7721 cells to cease at G0/G1 phase, and inhibit 7721 cells entering S phase and DNA synthesis of 7721 cells. The molecule mechanisms of the two nitrogen mustard derivatives in inducing apoptosis of 7721 cells may be that it can upregulate the expression of bax gene and downregulate the expression of bcl-2 gene and enhance the releasing of cyto-c to strengthen the activities of caspase-3 and finally resulted in 7721 cell apoptosis.