Design, Synthesis And Antitumor Activities Of Oleanane Derivatives

Pentacyclic triterpenoids are a profound family with a variety of biological activities including inhibition of human immunodeficiency virus,antibacterial,anti-inflammatory and antitumor properties.Some of them have been shown to exhibit excellent antitumor activity with low toxic effect,and would be a suitable leading compound for antitumor drugs.To improve the efficacy of natural triterpenoids against tumor cell growth and investigate their antitumor mechanism in leukemia cells,boswellic acid acetate（BAA）and 18β-glycyrrhetinic acid（GA）which was the main active principle of the plant Boswellia serrata and Glycyrrhizae radix respectively,were taken as the leading compounds,structure modification was done,and eighty-one pentacyclic triterpenoid compounds were designed and synthesized.They were divided into six series according to their structures:3-hydroxy （acyloxy）-11-oxo-urs-12-ene-24-oic acids and its related ester compounds（BA-1～BA-8）,3-hydroxy （acyloxy）-11-oxo-olean（urs）-12-ene-24-oic acid（ester）compounds（BA-9～BA-12）, 3-hydroxyimino（acyloxyimino or alkoxyimino）-11-oxo-olean-12-ene-29-oic acids and its related ester compounds（GA-1～GA-15）,A ring modification of GA derivatives（GB-1～GB-8）, 3-oxo（acetyloxy or hydroxy）-（11-oxo-）olean-12-ene-29-oic acid amide compounds（GC-1～GC-35）, 3-hydroxy（acetyloxy or oxo）-（11-oxo-）olean-12-ene-29-oic acids and its related methyl ester compounds（GD-1～GD-11）,and the structures were confirmed by the application of ¹H-NMR.¹³C-NMR,IR and MS spectral data.In which 54 compounds have never been reported in literatures.During the preparation of 3-hydroxyimino（alkoxyimino）-glycyrrhetinic acid esters,two products were gotten in one reaction,the one having an alkylated oxime at position C₃ and esterified carboxyl group at position C₂₉,the other with only esterified carboxyl group at C₂₉ and without alkylation of the oxime at position C₃.So the main target compound could be obtained by controlling the amount of alkyl halide and the reaction time,and purified on one silica gel column.In this thesis,some characteristics about ¹H-NMR and ¹³C-NMR spectral data of these triterpenoid compounds were summarized.In ¹H-NMR spectra,the chemical shift of H-3 was displayed at 4.08～4.18ppm for BA derivatives with free 3-hydroxyl group,and it was seen at 5.30～5.35ppm for 3-acetyloxy compounds;while the corresponding proton was appeared at 3.03～3.26ppm and 4.48～4.55ppm for GA derivatives,so the configuration of C-3 could be affirmed,α-OH andβ-H for BAs,α-H andβ-OH for GAs.The structure assignment for the compounds with alkoxyimino group at position C₃ and esterification at position C₂₉was supported by the corresponding proton chemical shift.The class of double bond and the position of substituted group linked could be judged by ¹³C-NMR spectral data.When it was 11-oxo-12-ene-oleanane,the chemical shiftδ198.8～202.4ppm,δ127.4～128.6ppm,δ169.3～170.9ppm were the C-11,C-12,C-13 respectively.In the ¹³C-NMR spectra of the product of Clemmensen reduction reaction,the peak of C₁₁-keto was disappeared,and the chemical shift of C₁₂and C₁₃was downfielded to 122.1～122.6ppm and 144.3～145.1ppm respectively.The results obtained from ¹³C-NMR spectra indicated that the chemical shift of C-29 was depressed in turn while it was free carboxyl group,carboxylic ester,amide,theδvalue was 178.1～182.0ppm,176.3～176.9ppm, 173.5～174.8ppm respectively.The results of cell growth inhibitory effect and apoptosis induction ability in human leukemia HL-60 cells for BA derivatives were shown that the compound BA-9 with a replacement of acetyl group in BAA by a propionyl group having comparative cell growth inhibitory and apoptotic effect with BAA and stronger than compounds with removed acetyl group.It was shown that it might be needed a suitable space hindrance and lipsoluble group at the position C₃ for BA compounds.The antiproliferative effects of these GA derivatives in HL-60 cells were determined by direct cell counting,and the majority of these GA derivatives showed preferable activity against HL-60 cells growth.Among them,compounds GB-2,GB-3,GB-7,GB-8,GC-1, GC-6,GC-8,GC-10,GC-14,GC-15,GC-16,GC-17,GC-18,GC-20,GC-21,GC-23,GC-26, GC-31,GC-34 possessed evidently improved antiproliferative effects(GI₅₀＜10μmol/L) than the mother compound glycyrrhetinic acid with GI₅₀value of 63.2μmol/L.In all the compounds,GB-8 showed the strongest activity against HL-60 cells growth,GI₅₀= 0.89μmol/L.From the primary structure-activity relationships of HL-60 cells growth inhibition,we can conclude that:①compounds with a acetyloxy group at the C₃ position were more effective than the compounds with a keto or a hydroxyl group at the C₃ position;②11-deoxoglycyrrhetinic acid derivatives were more effective than 11-oxo compounds;③compounds with an amide at the C₂₉position were more effective than the compounds with a free carboxyl group or a carboxylic ester at the position C₂₉,and among the amides,the compounds introduced N-methyl piperazine were most effective;④compounds with A ring modification,especially 2-cyano-1,2-ene-3-oxo-glycyrrhetinic acid derivatives showed a significantly increased antiproliferative effects.The apoptosis-inducing activities of those compounds with significant cell growth inhibition activity were evaluated using AO/EB double staining assay and fluorescenceactivated cell sorting analysis.HL-60 cells were treated with GB-7 or GB-8 at 10μmol/L for 6h,the percentage of apoptosis cells was 90%.It was shown by preliminary antitumor mechanism research that glycyrrhetinic acid derivatives might induce apoptosis through the death receptor pathway.