Design, Synthesis And Bioactivity Investigation Of Oleanolic Acid Derivatives

Oleanolic acid(OA)is a triterpenoid widely distributed in the nature which possesses various important bioactivities,such as antitumor,anti-HIV, hepatoprotection,and anti-inflammatory.However,the relatively weak activities and poor bioavailability hinder its clinic use.On the other hand,OA also serves as aglycon of many natural saponins,which own significantly higher activities than OA.For instance,β-hederin,a glycoside bearing a unique disaccharide at 3-OH,shows excellent inhibitory activity against many tumor cells;and this unique disaccharide,α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside,has been considered to be important to the antitumor activity of this class of saponins.In this dissertation,bioactivity and structure-activity relationship of OA and its derivatives are reviewed.Amongst the numerous structural modification of OA, synthesis of OA glycosides is relatively less reported yet.Therefore,the major work of the thesis is to synthesize the glycoside derivatives of OA and to evaluate their antitumor activities.High efficient synthetic approaches towards three classes of OA saponins (3-monodesmoside,28-monodesmoside,and 3,28-bisdesmoside)were established firstly;by these approaches,4～7 compounds were prepared for each class.Their structures were characterized by ¹H NMR,¹³C NMR,and ESI-MS.Unfortunately,in vitro assaying showed that antitumor activity of these saponins is not prominent.Next,synthesis of some antitumor natural OA saponins were carried out, includingβ-hederin,hederacolchiside A₁,lotoidoside D,lotoidoside E,giganteaside D, and oleanolic acid 3-O-(4-O-acetyl-α-L-arabinopyranoside).The synthetic products were characterized with ¹H NMR,¹³C NMR,and ESI-MS,and analytical data agreed with those of natural products.In vitro MTT assaying showed that all these synthetic saponins possess significant inhibitory activity against tumor cells.Attempts in synthesis of patrinia-glycoside B-â…¡were also made,however,only a conformational isomer of patrinia-glycoside B-â…¡was obtained,whose arabinose ring adopted the ¹C₄ form instead the normal ⁴C₁ form.This isomer did not show inhibitory effect against tumor cells,indicating that the ⁴C₁ form of arabinose is crucial to the antitumor activity of this kind of saponins.Based on the synthesis ofβ-hederin,structural modification of 28-COOH and 4'-OH ofβ-hederin was then performed to yield its derivatives for pharmacology investigation.Eleven 28-amides and 28-glycosides ofβ-hederin,together with two 4'-glycosides,were synthesized.Most of these derivatives have moderate to strong antitumor activity,especially for 28-amides YMC-27 and YMC-28,indicating this kind of saponins are valuable in further investigation.Triterpenoids including OA have significant inhibitory activity against HIV-1 protease.Unlike most clinical HIV protease inhibitors,OA does not bind the catalytic site of protease.Instead,it binds to the peptide terminus of protease monomer and prevents the monomer forming active dimer,which is called dimerization inhibition. Molecular modeling studies of triterpenoid and HIV protease monomer were very rarely reported.As the second part work of the dissertation,molecular docking of triterpenoid derivatives(OA,ursolic acid,α-hederin,etc)into HIV-1 protease monomer was performed to investigate the binding mode,and molecular dynamics simulations were then performed on the docking-derived triterpenoid-protease monomer complexes.Docking studies showed that hydrophobic interactions between polycyclic triterpene scaffold and lipophilic amino acids contributed essentially to the binding.Triterpene moiety occupied only part of dimerization site;so,C-3 substituent (carbohydrate or dicarboxylic acid)may increase the binding affinity significantly. Ligand-receptor interactions became strengthened during the molecular dynamics simulations,especially forα-hederin,possibly by induced-fit and solvation effects,etc. Based on above studies,binding mode of triterpenoid derivatives to HIV-1 protease monomer and primary structure-activity relationships were summarized.Free monomer of HIV-1 protease shows great flexibility and structural variability in N-terminus and C-terminus.Therefore,extensive molecular dynamics simulations were performed in explicit solvent to study its favorable solvated conformation and dynamics behavior in water solution.We are surprised to find that the two peptide termini,which are very flexible in short-time molecular dynamics simulation and NMR detection,usually assemble into aβ-sheet and become much less flexible,and the dimerization site disappears in this conformation.This terminalβ-sheet is stabilized by intramolecular non-bond interactions and cannot be easily disaggregated under present simulation conditions.Since the terminalβ-sheet is energy-stable,it is possibly one of the major conformations of termini after the system reaches equilibrium in aqueous solution.Therefore,it may be helpful in dimerization-dissociation mechanism study and inhibitor discovery.