| Objective:Betulin(BT)is a triterpenes compound,widely distributed in natural plants,with significant physiological activities such as anti-cancer,anti-inflammatory,anti-septicemia,anti-malarial,anti-virus,antibacterial,insect-resistant and so on.Due to the low water solubility of betulin,the results showed that the effect of physiological activities was not obvious.Therefore,it was very significant to study the structure modification of betulin.To improve anticancer activity of betulin by synergistic effect,new betulin derivatives were prepared by coupling NO donors of nitrate.In this study,we invesitigate the purity of betulin derivatives,the anti-tumor effect against 3 human tumor cell lines,and two feature parameter including the NO release and oil-water partition coefficients.The research can offer a reference for the in vivo absorption study of betulin derivatives.Methods:The positions of C-3 or/and C-28 hydroxyl groups of BT were modified by hydroxyl protection,nucleophilic substitution,deprotection and reduction.All of the compounds were purified by column chromatography.The structure of betulin derivatives was identified by IR and NMR.A RP-HPLC method was applied to the analysis on the purity of betulin derivatives.The releasing of NO of each derivative was determined by Griess method.The antitumor activity of betulin derivates was tested in vitro against three human cancer cell lines(Caucasian breast adenocarcinoma MCF 7,hepatocellular carcinoma HepG 2 and colon carcinoma HT-29)by using MTT assay.Shake-flask method and high performance liquid chromatography(HPLC)were used to determine the oil-water partition coefficients of betulin,3-2'-nitroxy propionyl betulin and 3-carbonyl-28-2'-nitrooxyacetoxy betulin.Results:Twelve of betulin derivatives were designed and synthesized.They are 3-(2-nitroxyl acetoxyl)-28-dimethyl-tert butyl siloxy betulin,3-(2-nitroxyl propionyloxy)-28-dimethyl-tert butyl siloxy betulin,3-(2-methyl-2-nitroxyl propionyloxy)-28-dimethyl-tert butyl siloxy betulin,3-(2-nitroxyl acetoxyl)betulin,3-(2-nitroxyl propionyloxy)betulin,3-carbonyl-28-2'-nitroxyl acetoxyl betulin,3-carbonyl-28-2'-nitroxyl propionyloxy betulin,3-carbonyl-28-(2-methyl-2-nitroxyl propionyloxy)betulin,3-acetoxyl-28-(2-nitroxyl acetoxyl)betulin,3-acetoxyl-28-(2-nitroxyl propionyloxy)betulin,3-acetoxyl-28-(2-methyl-2-nitroxyl propionyloxy)betulin and 3-hydroxyimino-28-(2-methyl-2-nitroxyl propionyloxy)betulin,respectively.The purity of betulin derivatives is greater than 98%.The nitric oxide release in vitro showed that betulin derivatives can release NO quickly and reach stable within 2 h.Meanwhile,the modification of betulin by NO donors on position 28 is better for NO release in vitro.The results of MTT test revealed that betulin derivatives exhibit selectivity against different cancer cells.Most of title compounds have strong inhibition effect on human colon cancer cell line HT-29.Among them,3-(2-nitroxyl propionyloxy)betulin and 3-carbonyl-28-2'-nitroxyl acetoxyl betulin are the most outstanding compounds.The 1g P values of betulin,3-2'-nitroxy propionyloxy betulin and 3-carbonyl-28-2'-nitrooxy acetyloxy betulin were-0.259,0.570 and 0.337,respectively,which showed that two derivatives of betulin have good transmembrane capacities and absorption in vivo.Conclusion:NO donors were introduced on specific location of betulin via hydroxyl protection.The NO release in vitro indicates that betulin derivatives have sustained release effect.Most of derivatives show selective cytotoxicity towards human-colon cancer cell line HT-29.3-(2-nitroxyl propionyloxy)betulin and 3-carbonyl-28-2'-nitroxyl acetoxyl betulin significantly reduce the growth of HT-29 cells.And the anti-tumor activity exhibited a time-dependent.The biology activity of betulin and its derivatives are closely related with the oil-water partition coefficients.The bioavailability of betulin can be improved by the reasonable structure modification. |
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