Synthesis And Anticancer Activity Study Of Bioactive Natural Saponins And Their Analogues

Natural saponins, distributed widely in higher plants and certain marine organisms, possess versatile biological activities including anticancer, anti-inflammatory, antifungal, antiparasitic, antiviral, hepatoprotective, neuroprotective and antimutagenic effects. Some of them represented glycoconjugate templates in drug design and development. This dissertation is focusing on the synthesis, cytotoxic activities as well as the preliminary mechanism study of the naturally derived anticancer saponins and their analogues.In Chapter one, the bioactivities of the representative saponins and their structure-activity relationship (SAR) was reviewed briefly. Furthermore, several classic methodologies and strategies developed for the successful preparation of saponins bearing different structure were also described.In Chapter two, Indioside E, a potent anticancer natural saponin, was facilely synthesized via a transglycosylation strategy using the trisaccharide imidate, which was readily prepared from D-xylose, L-rhamnose and IPTG. Based on this convenient approach, its five congeners bearing the same sugar residue and varied aglycones (e.g., tigogenin, cholesterol, sitosterol, taraxerol and lupeol) were efficiently synthesized. The cell killing effects of the synthesized Indioside E and its analogues on four cancer cells (K562, KB, MCF-7, and U87) and three normal cell lines (HL7702, H9C2, and EVC304) was assayed using MTT method. Both Indioside E and its derivative2, substituted with tigogenin as aglycone, displayed strong anticancer activity with IC50ranging from1.32to3.77μM, but saponin2had a lower cytotoxicity to normal HL7702cell. These results indicated that the spirostane-type aglycone part of1was of value for its antiproliferative activity and cholesterol, sitosterol, taraxerol and lupeol replacement will decrease its cytotoxicity. Time-lapse microscopy, LDH release, PI staining, and immunocytochemical investigations demonstrated that the cell death caused by neosaponin2was through oncotic necrosis involving plasma membrance perturbation and destruction of cytoskeleton. Furthermore, the double bond in the aglycone of Indioside E may be a good active spot to modify in the next work.In Chapter three, two anticancer oleanolic acid saponins, Spinasaponin A methyl ester and Calenduloside G methyl ester, was efficiently synthesized for the first time and the approach was achieved in a total of17steps with about12%overall yield from cheap starting materials. To investigate the relationship of their cytotoxic effects with the varied aglycones and find novel glycosides possessing excellent antiproliferative activity, four structurally modified congeners with ursolic acid and glycyrrhetinic acid as aglycones were synthesized based on this developed strategy. In vitro cytotoxic activity of those synthesized saponins against three cancer cells (MCF-7, HepG-2, and K562) was investigated using MTT method. Calenduloside G methyl ester displayed the most strong anticancer activity against the three tested cancer cells with IC50ranging from1.87to2.36μM. Compared to Calenduloside G methyl ester, Spinasaponin A methyl ester was less cytotoxic to MCF-7and HepG-2with IC50of4.52and8.36μM, respectively. Meanwhile, their four derivatives bearing ursolic acid and glycyrrhetinic acid as the aglycone did not show the antiproliferative effect against the three cancer cells. These results indicated that the oleanolic acid aglycone of Spinasaponin A methyl ester and Calenduloside G methyl ester was of value for their cytotoxicity, and slight modification in the aglycone would decrease their anticancer effects dramatically. Calenduloside G methyl ester-induced apoptosis on MCF-7cells in a dose-dependent manner was detected by confocal micrographs using DAPI staining and flow cytometry using Annixin V and PI double staining. In addition, Calenduloside G methyl ester-induced apoptosis on MCF-7cells was associated with ROS generation and loss of the mitochondria membrane potential (ΔΨm).In Chapter four, a facile synthesis of biotinylated-chacotriose,-rhamnose and-glucose were described. Through combination of the biotinylated carbohydrates and QD-streptavidin, a method for in situ observation of RBLR on cancer cell surface was established. In addition, different cancer cells expressed different levels of RBLR. Increased RBLR expression or higher numbers of rhamnose moieties in the structure of solasodine rhamnosides could mediate more effective cancer cell elimination. This study provided a method to help design the most anticancer sugar moiety, which could be used to modify the other natural aglycones. Furthermore, the method could help design chemotherapy agents by introducing rhamnose moiety into their structures to increase the cell uptake, cancer targeting effects and prognosis of the chemotherapy sensitivity to cancer cells.