合成含N-乙醯葡萄糖胺之齊墩果酸醣類分子庫及其抗癌之研究

從天然物中分離出三萜類以及固醇類的皂素具有多元的活性，包括抗感染、溶血活性、免疫調節以及細胞毒性等等。已有文獻報導，攜帶N-乙醯葡萄醣胺之三萜類皂素(N-Acetylglucosamine bearing triterpenoid saponins , ATS)，例如：lotoidoside D，這樣獨特的類型在抗腫瘤活性上有顯著的活性。為了研究ATS之結構與活性關係分析，我們利用9種不同的醣基，分別與N-乙醯葡萄醣胺的C-3′，C-4′以及C-6′位置進行醣化反應，合成27種3-O-N-乙醯葡萄醣胺之齊墩果酸的衍生物。為了備製這樣的衍生物，我們必須找出一個簡潔的合成過程，為了避免N-乙醯葡萄醣胺不同位置之羥基在醣化時所產生的選擇性問題，我們利用位向選擇保護(orthogonal protection)策略，合成6-O-tert-butyldiphenylsilyl-3-O-levulinoyl- 4-O-(2-nitrophenylacetyl)-2-(2,2,2-trichloroethoxylcarbonylamino)-2-deoxy-D-glucopyranosyl trichloroacetimidate (112)，之後醣化在benzyl oleanolate (62) 的C-3位置，獲得化合物90，然後可以使用聯胺選擇性的去除C-3′-levulinoyl基團，得到C-3′-OH之化合物，或是以3% AcCl/MeOH移除C-6′的矽基團(tert-butyldiphenylsilyl, TBDPS)，獲得C-6′-OH之化合物。而後續我們將C-3′或C-6′-OH的化合物與形成trichloroacetimidate之9種苯甲醯化的醣基，在0 oC到室溫的溫度調控下加入催化量的TMSOTf，進行醣化反應，得到C-3′或C-6′醣化之中間體。在嘗試以Zn/NH4Cl除去2-nitrophenylacetyl (NPAc)基團來得到C-4′-OH之化合物時，伴隨少量Troc基團在此環境下被去除，因而使得我們得到預期產物之產率很低。然而，化合物90TBAF/HOAc的條件下，意外獲得C-4′-NPAc基團轉移到C-6′位置的化合物149，產率為95%，所以我們以化合物149與9種不同得醣基提供者進行醣化作用，來獲得醣化在C-4′位置之3-O-N-乙醯葡萄醣胺齊墩果酸衍生物。之後將這些醣化的中間產物去除所有的保護基，得到27個3-O-N-乙醯葡萄醣胺齊墩果酸之衍生物。另外，在去除保護基的過程中，我們得到了三種N-2-methoxycarbonyl-葡萄醣胺之齊墩果酸衍生物。在生物活性方面，我們利用SRB assay來探討這類衍生物在HL-60細胞株的細胞毒性。結果發現化合物38、173以及196 在30 μM劑量下，顯現超過80%的細胞毒性。在我們初步的SAR顯示出，3-O-N-乙醯葡萄醣胺齊墩果酸之衍生物，在C-3′位置有醣基的修飾最具有活性，C-4′位置有醣基修飾則是帶有微弱的毒性，而在醣基修飾在C-6′位置，將完全失去活性。以及，在C-3′和C-4′位置修飾之醣基為D-xylose、 L-xylose 和L-arabinose 的時候，活性好於以其他醣基修飾的衍生物。而在胺基為N-2-methoxycarbonyl修飾之化合物194和196也會增加活性。

關鍵字

N-乙醯葡萄糖胺；齊墩果酸；抗癌

並列摘要

Triterpenoid and steroidal saponins isolated from natural plants have several biological activities, including anti-infection, hemolytic activity, immune-modulation, cytotoxicity, and etc. N-Acetylglucosamine bearing triterpenoid saponins (ATS), such as lotoidoside D, were reported to be a unique type of saponins with potent anti-tumor activity. In order to study the SAR of ATS, we synthesized 27 derivatives of 3-O-N-acetylglucosaminoyl oleanolic acid, with additional 9 sugars glycosylated either at 3', 4', and 6' positions of N-acetylglucosamine. It is highly essential to develop a concise procedure to prepare these derivatives. To circumvent the problem associated with selective glycosylate sugars at specific of hydroxyl groups of D-glucosamine, it was orthogonal protected to 6-O-tert- butyldiphenylsilyl-3-O-levulinoyl-4-O-(2-nitrophenylacetyl)-2-(2,2,2-trichloroethoxylcarbonylamino)-2-deoxy-D-glucopyranosyl trichloroacetimidate (112). Following by glycosylation with benzyl oleanolate at C-3-OH, compound 90 was obtained, which was selected deprotected by using hydrazine to free 3-hydroxyl group from levulinoyl group, or 3% AcCl/MeOH to free 6-hydroxyl group from 6-tert-butyldiphenylsilyl group. The free hydroxyl groups at 3' or 6' of glucosamine were further glycosylated with 9 per-benzoylated glycosyl trichloroacetimidates in the presence of TMSOTf at 0 oC to rt to afford 3' or 6' glycosylated intermedidates. In an attempt to free 4-hydroxyl group from 2-nitrophenylacetyl group by Zn in ammonia chloride, we suffered from low yield of desired product due to concomitant removal of Troc group in this condition. However, when compound 90 was subjected to TBAF/HOAc condition, we accidently obtained the 4', 6'-migration of 2-nitrophenylacetyl group in 95% yield to afford compound 149 which was glycosylated with 9 sugar donors to get the desired 4'-glycosylated products. The global deprotection of glycosylated intermediates afforded 27 3-O-N-acetylglucosaminoyl oleanolic acid derivatives. Three N-2-methoxycarbonyl D-glucosaminoyl oleanolic acid derivatives were also obtained as side products in the global deprotecting process. The effect of these derivatives on HL-60 cell line was studied via SRB assay. Compounds 38, 173, and 196 showed more than 80% of cytotoxicity at 30 miuM. The preliminary SAR results indicated that the modification at 3’ position retained most of cytotoxicity of 3-O-N-acetylglucosaminoyl oleanolic acid, modification at 4’ position have moderate cytotoxicity, and modification at 6' position completely loss the activity. D-Xylose, L-xylose and L-arabinose attached at 3' or 4' positions were found to be active among these modifications. Compounds 194 and 196 with N-2-methoxycarbonyl modification at 2'-amino position was found to increase the cytotoxicity.

並列關鍵字

N-Acetylglucosamine ； Oleanolic acid ； Anticancer Activity

參考文獻

1. Riguera, R. Isolating bioactive compounds from marine organisms. J. Mar. Biotechnol. 1997, 5, 187-193.

2. Vincken, J.-P.; Heng, L.; de Groot, A.; Gruppen, H. Saponins, classification and occurence in the plant kingdom. Phytochemistry 2007, 68, 275-297.

3. Yu, B.; Sun, J. Current synthesis of triterpene saponins. Chem.-Asian J. 2009, 4, 642-654.

4. Park, J. D.; Rhee, D. K.; Lee, Y. H. Biological activities and chemistry of saponins from Panax ginseng C. A. Meyer. Phytochem. Rev. 2005, 4, 159-175.

5. Lanzotti, V. Bioactive saponins from Allium and Aster plants. Phytochem. Rev. 2005, 4, 95-110.

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合成含N-乙醯葡萄糖胺之齊墩果酸醣類分子庫及其抗癌之研究

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