Design, Synthesis And Anti-tumor Activity Of Pentacyclic Triterpenoids

18β-Glycyrrhetinic acid and boswellic acids, which belong to pentacyclic triterpenoids, exhibit various biological activities, such as ant-inflammation, anti-ulcer and anti-virus. They are also associated with inhibiting proliferation, inducing apoptosis and preventing invasion of tumor cells.To improve the efficacy of 18β-glycyrrhetinic acid and boswellic acids against tumor cells growth and investigate their antitumor mechanism in leukemia cells,18β-glycyrrhetinic acid (GA) and 11-oxo-boswellic acid acetate (AKBA) were taken as the leading compounds and structural modification was done in the paper. Modification of ring C by converting 11-oxo-12-en to 12-en,12-oxo,12-oxo-9(11)-en,9(11)-en and 9(11),12-diene, introduction of a 3-oxo,2-hydroxymethylene-3-oxo, isoxazole,2-cyano-3-oxo,2-cyano-3-oxo-len,2-cyano-1-en-3-one in the A-ring, and replacement at carboxylic group with a nitrogen heterocycle, piperidine,4-piperidyl piperidine,4-methyl piperazine, or piperazine gave one hundred and twenty two pentacyclic triterpenoids, including 68 novel compounds which have never been reported in literatures. They were divided into seven groups according to different structures in ring C:11-oxo-18p-olean-12-en derivatives (YA-01-YA-25),18p-olean-12-en derivatives (YB-01-YB-29), 12-oxo-18β-olean derivatives (YC-01-YC-11),12-oxo-18p-olean-9(11)-en derivatives (YD-01-YD-1),18p-olean-9(11)-en derivatives (YE-01-YE-12),18β-olean-9(11),12-dien derivatives and 18β-olean-11,13(18)-dien derivatives (YF-01-YF-13), ursane derivatives (YG-01-YG-13), and their chemical structures were confirmed by the application of 1H-NMR,13C-NMR, IR and MS spectral data.Two synthetic strategies were adopted to synthesize these target compounds. The strategy of modifying C ring, forming amides and modifying A ring in turn was utilized to obtain 11-oxo-18β-olean-12-en derivatives(YA) and 18β-olean-12-en derivatives (YB). The strategy of modifying C ring, modifying A ring and forming amides in turn was utilized to synthesize other target compounds. Two different synthetic routes were taken to modify the A ring:one route including oxidizing 3-hydroxyl to 3-oxo, introducing 2-hydroxylmethylene, forming isoxazole, cleaving isoxazole ring to form 2-cyano and dehydrogenation to form 1-en was carried to obtain target compounds with different structures of A ring; the other route including formingα,β-unsturated ketone by 2-iodoxybenzoic acid (IBX), iodine substitution and cyano substitution at 2 position was carried to form the structures of 2-iodine-3-oxo-1-en and 2-cyano-3-oxo-1-en in A ring.Some characteristics about 1H-NMR spectra data of these triterpenoids were summarized in this paper. The characteristic peaks between 5.00～7.00 ppm were correlated to the structure of C ring. The single peak of H-12 deriving from 11-oxo-12-en functionality appeared at high field (5.60～5.70 ppm) compared to the single peak of H-11 (5.80-6.10 ppm) deriving from 12-oxo-9(11)-en functionality. And The triplet peak of H-12 deriving from 12-en functionality appeared at high field compared to the triplet peak of H-11 deriving from 9(11)-en functionality. The characteristic peaks of 1H-NMR spectra were also correlated to the structure of A ring.The antiproliferative effects of these target compounds in HL-60 cells and PC-3 cells were determined by direct cell counting and MTT. The majority of these derivatives showed preferable activity against HL-60 and PC-3 cells growth. The BA derivatives exhibited better activities than GA derivatives. The results reveal that 1) the structure of C ring had great influence to antitumor activity. The 12-oxo-9(11)-en derivatives exhibited better activities than other derivatives.2) introducing 2-cyano-3-oxo-1-en can significantly improve the antitumor activity.3) the amide derivatives had better activies than GA and similar activities as their methyl esters. Among them, the compounds substituted by 4-piperidyl piperidine showed better activities and the compounds substituted by morpholine showed low activities.4) the compounds combined modification of C ring, A ring and carboxylic acid had the best activities. For example, the GI50 of Methyl 2-cyano-3,12-dioxo-18β-olean-1,9(11)-dien-30-oate (YD-06) was 0.4μM and worthy of further study.The apoptosis-inducing activities of YA-06 and YD-06 with significant cell growth inhibition activities were evaluated by using AO/EB double staining assay and fluorescence-activated cell sorting analysis. HL-60 cells were treated with 6μmol/L YA-06 or 0.6μmol/L YD-06 for 12h and apoptotic rate was over 50%. The antitumour mechanism study indicated that1) both extracellular pathway and intracellular pathway were involved in their apoptosis induction.2) YA-06 and YD-06 depleted intracellular GSH levels and exogenous GSH reversed apoptosis induction by them in HL-60 cells. UV Spectrophotometric analysis revealed that both of them interacted with GSH in extracellular. These data suggested that their apoptotic activities might be related with their conjugation with GSH.