Preparation And Anti-tumor Activities Of The Glycosylated Artemisinin Derivatives

Artemisinin, serving as antimalarial, is generally accepted in the world. However, there are still some defects, e.g. low water-solubility and liposolubility that leads to low bioavailability and high recrudescence rate. The anticancer activites of artemisinin and its derivates may become the hot of research, and it is potent to develop a new broad-spectrum anticancer drug. Therefore, artemisinin modification and exploration of antineoplastic activity are extremely meaningful to its multiple utilizations.This project proposed pro-drug's glycosylation modification, traditional Chinese herb monomer artemisinin was glycosylated, then performed structural characterization and investigation of anticancer activites, finally exploring structure-activity relationship for presently available artemisinin analogues. Main woks are presented as following:①Synthesizing glycosylated artemisinin by chemical methods, and studying on anti-tumor activites of the compound in vitro.1)Preparing a few new sugar-residue products of artemisinin by acetylated protection one-pot.Measuring and analyzing products in 1H NMR, 13C-NMR ,LC-MS and IR methods, and ascertaining the target products. Compared this synthetic methods and methods previously reported, the methods had relatively mild reaction conditions, which was not only simpler, but also needn't extra deprotection steps.2)It showed that the water-solubility was enhanced and profited its absorption and raising the utilization rate of biology.3)MTT test showed that glycosylation artemisinin derivates had larger ability of inhibiting growth in cancer cells and lowered toxicity in normal cells. In particular, artemisinin-glucoside had the best activity on Hela cells.4)Directly observing the effection of artemisinin-glucoside on Hela cells by inverted microscope and transmission electron microscope, which the cells shape changed and the number of cells reduced. It showed that artemisinin-glucoside can induce apoptosis of Hela cells.5)Detected apoptosis by flow cytometry and western blotting for approaching mechanism of artemisinin-glucoside, which could be up-regulated bax gene and down -regulated bcl-2 gene, so as to release the cyc.Therefore, it can increase the expression caspase-3 gene and activate the caspases cascading effect, so resulted the apoptosis, the mechanisms on Hela cells was non-p53 gene way. ②Synthesizing glycosylated artemisinin by enzyme catalysis methods, and then studying on activites of anti-cancer in vitro.1)Modifying artemisinin with sugar-residue by enzyme catalysis methods at the first time in the world, and found that galactose had the maximum of efficiency, which can up to 68%. Compared enzyme catalysis and chemical methods, it was found that enzyme catalysis reaction had the characteristics of higher efficiency and stronger specific.2)Groping and optimizing the reaction condition of glycosylation by enzyme catalysis, and then obtaining the best operating conditions.3)Firstly, respecting the feature of enzyme catalysis, analyzing structure of galactosylated artemisinin with bioanalysis methods after treating it.Determining the number of sugar substitute in galactosylated artemisinin by Tricine-SDS-PAGE electrophoresis from the perspective of molecular weight. Detecting the artemisinin hydroxy position by Circular Dichroism, which showed that the position was replaced at hydroxide radical. The glucosidic bond isβ-type. Fluorescence method confirmed further that the artemisinin-residue had sugar molecule. Then confirming the structure by NMR and IR methods. At last, simulating the stereochemical structure of glycosylation artemisinin by Arguslab soft.4)MTT test showed that the galactosylated artemisinin had larger ability of inhibiting growth in cancer cells and lowered toxicity in normal cells. Moreover, it was demonstrated that galactosylated artemisinin can induce apoptosis in cancer cells by Hoecst33342\PI stain. Approaching the apoptosis mechanism by flow cytometry and western blotting, it was similar to that of the product by chemical methods.③Studying on anti-tumor activites of glycosylated artemisinin in vivo. Establishing mouse cancer of the cervix model,then studying on anti-tumor activites of glycosylated artemisinin in vivo. Compared with normal sodium, the results showed artemisinin and its glycosylated derivates can suppress growth of tumor and increase in life span of mouse.The products belonged to low-toxicity materials.Otherwise, compared with artemisinin, each index of glycosylated products raised, galactose- artemisinin by enzyme catalysis methods was best.④Base on molecular docking and three-dimensional structure-activity relationship, bioactivities of artemisinin and analogues were primarily explored, and via theoretical modeling, nonbonding effects (referring to electrostatic, steric and hydrophobic interactions) had been investigated for their influences on Fe2+-mediated artemisinin peroxy-bridge breakage and antimalarial activity.1)With Autodock, artemisinin-haemoglobin interaction modes were investigated, expecting to find potential active conformations.2)Common atoms in organic molecules were classified in terms of their fundamental chemical properties, and three-dimensional field descriptor (referring to three-dimensional holographic vector, 3D-HoVAIF) was yielded via calculating nonbonding interactions among different types of atoms.3)Based upon the active conformation of artemisinin, spatial conformations for a series of derivatives of pharmacological activities were constructed, with their structures characterized by 3D-HoVAIF.4)Genetic algorithm-partial least square (GA-PLS) was employed to build up relationship between 3D-HoVAIF descriptors and the bioactivities, and meanwhile internal-external dual-testes were used to confirm statistical significance of SAR models. This model possessed stability and ability of forecasting.5) That analyzing and interpreting bioactivities of artemisinin and analogues by the statistical model benefits structural modification of artemisinin.In sum, artemisinin modified with glycon can enhance water solubility, lower the toxicity. Moreover, some glycon modified artemisinin can raise the activity of anticancer. These works not only has actual value to artemisinin multiple utilizations, but also provide new path for artemisinin modification and reference for other drugs modified with glycon.