白樹仔、阿里山根節蘭化學成分、Calanquinone A全合成與其衍生物之生物活性研究

Chemical Constituents and Biological Activities on Gelonium aequoreum, Calanthe arisanensis and Total Synthesis of Calanquinone A and Its Analogues

李家琳(Chia-Lin Lee)

指導教授 ： 吳永昌 ； 張芳榮

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2009年)

摘要

白樹仔(Gelonium aequoreum)為大戟科(Euphorbiaceae)，白樹屬(Gelonium)植物；阿里山根節蘭(Calanthe arisanensis)為蘭科(Orchidaceae)，根節蘭屬(Calanthe)植物，兩者皆為台灣原生特有種。本實驗以甲醇萃取後分別進行二氯甲烷/水(白樹仔)及乙酸乙酯/水(阿里山根節蘭)的初步分離，發現白樹仔葉部二氯甲烷層與阿里山根節蘭葉部與地下部乙酸乙酯層有細胞毒殺活性(IC50< 20μg/mL)。配合生物活性導引法，從具有活性的fractions中，進行活性成分的分離純化。
由白樹仔葉部共分離得到十七個ent-abietane diterpenes化合物；gelomulide K-X (GA-1~14)、6β-acetoxy-1-one-8β,14α-dihydroxy-ent-abieta- 2(3),13(15)-diene-16,12-olide (GA-15)、gelomulide A (GA-16)與gelomulide G (GA-17)，其結構經由各式光譜、X-ray、CD與Mosher´s method解析，其中GA-1~15為新化合物，分別為8,14-epoxy或8,14-dihydroxy類型。在活性方面，只有gelomulide K (GA-1)與gelomulide M (GA-3)對人類肺癌細胞(A549)、乳癌細胞(MDA-MB-231、MCF-7)與肝癌細胞(HepG2)具有中等的細胞毒殺活性(IC50 6–18 µg/mL)。其中GA-1為此植物活性成分中最大量的化合物，進一步研究其與癌細胞週期的相關機轉，發現GA-1可以使MDA-MB-231癌細胞週期停止在G2-M 期，然後癌細胞可能進而進行凋亡或自噬作用。
由阿里山根節蘭地下部與葉部分別分離得到十一個 [calanquinones A-C (CA-1~3)、calanhydroquinones A-C (CA-4~6)、calanphenanthrene A (CA-7)、3,5-dihydroxy-2,4-dimethoxyphenanthrene (CA-8)、2,7-dihydroxy- 3,5-dimethoxy-9,10-dihydrophenanthrene (CA-9)、2,7-dihydroxy-4-methoxy -9,10-dihydrophenanthrene (CA-10)與blestriarene A (CA-11)]與八個[2,7-dihydroxy-3,5-dimethoxy-9,10-dihydrophenanthrene (CA-9)、2,7- dihydroxy-4-methoxy-9,10-dihydrophenanthrene (CA-10)、densiflorol B (CA-12)、tryptanthrin (CA-13)、(24R)-4α,24-dimethyl-5α-cholesta-7,22- dien-3β-ol (CA-14)、indirubin (CA-15)、isatin (CA-16)與indican (CA-17)]化合物其結構經由各式光譜解析，十四個為dihydro/phenanthrenes、四個為indole alkaloids與一個steroid，其中有七個dihydro/phenanthrenes (CA-1~7)為新化合物。在活性方面，新化合物calanquinone A (CA-1)對人類肺癌細胞(A549)、前列腺癌細胞(PC-3與DU145)、結腸癌細胞(HCT-8)、乳癌細胞(MCF-7)、鼻咽癌細胞(KB)與vincristine抗藥性鼻咽癌細胞(KBVIN)具有明顯的細胞毒殺活性，其IC50 0.02–0.45 µg/mL。進一步對calanquinone A (CA-1)以化學方法全合成製備，並同時製備其相關衍生物，以利我們來探討其結構與活性關係研究(structure-activity relationship, SAR)。
目前包含以全合成方式製備的calanquinone A (CA-1)，共有十九個合成相關phenanthrenes類衍生物製備完成，並進行細胞毒殺、抑制血小板凝集與抗發炎活性實驗。實驗發現此類化合物具有非常好的細胞毒殺、抑制血小板凝集與抗發炎活性，針對這些化合物我們也探討了它們的SAR。

關鍵字

白樹仔 ； 阿里山根節蘭 ； Calanquinone A

並列摘要

Gelonium aequoreum (family: Euphorbiaceae, genus: Gelonium) and Calanthe arisanensis (family: Orchidaceae, genus: Calanthe) are endemic in Taiwan. The MeOH extracts of Formosan plants were partitioned with CH2Cl2/H2O (G. aequoreum) and EtOAc/H2O (C. arisanensis), respectively. Based on cytotoxicity screening, we found that a CH2Cl2 extract of G. aequoreum leaves and EtOAc extracts from C. arisanensis leaves and underground part were active against various human cancer cell lines with IC50< 20 μg/mL. Bioassay-guided chromatographic fractionation of these extracts led to isolation of pure active compounds.
Seventeen ent-abietane diterpenes [gelomulide K-X (GA-1~14), 6 β-acetoxy-1-one-8β,14α-dihydroxy-ent-abieta-2(3),13(15)-diene-16,12-olide (GA-15), gelomulide A (GA-16), and gelomulide G (GA-17)] were isolated from the leaves of G. aequoreum. Their structures were identified by spectroscopic methods, and stereochemistries were confirmed by X-ray crystallographic analysis, CD spectral data, and Mosher’s method. Among seventeen compounds, GA-1~15 were new compounds, including 8,14-epoxy and 8,14-dihydroxy types. In cytotoxicity, gelomulide K (GA-1) and gelomulide M (GA-3) showed moderate cytotoxicity (IC50 6–18 µg/mL) against lung (A549), breast (MDA-MB-231 and MCF-7), and liver (HepG2) cancer cell lines. GA-1 is the major and active component of this plant. GA-1 induced MDA-MB-231 cancer cell cycle arrest in G2-M phase and cancer cells possibly undergo apoptosis or autophagy.
Eleven compounds [calanquinones A-C (CA-1~3), calanhydroquinones A-C (CA-4~6), calanphenanthrene A (CA-7), 3,5-dihydroxy-2,4- dimethoxyphenanthrene (CA-8), 2,7-dihydroxy-3,5-dimethoxy-9,10-dihydro -phenanthrene (CA-9), 2,7-dihydroxy-4-methoxy-9,10-dihydro -phenanthrene (CA-10), blestriarene A (CA-11)] and eight compounds [2,7-dihydroxy-3,5-dimethoxy-9,10-dihydrophenanthrene (CA-9), 2,7- dihydroxy-4-methoxy-9,10-dihydrophenanthrene (CA-10), densiflorol B (CA-12), tryptanthrin (CA-13), (24R)-4α,24-dimethyl-5α-cholesta-7,22-dien- 3β-ol (CA-14), indirubin (CA-15), isatin (CA-16), and indican (CA-17)] were isolated from underground part and leaves of C. arisanensis, respectively. The structures, including fourteen dihydro/phenanthrenes, four indole alkaloids, and one steroid were identified on the basis of spectroscopic data. Seven dihydro/phenanthrenes (CA-1~7) were new. Calanquinone A (CA-1) showed the highest potency (IC50 0.02–0.45 µg/mL) against human lung (A549), prostate (PC-3 and DU145), colon (HCT-8), breast (MCF-7), nasopharyngeal (KB), and vincristine-resistant nasopharyngeal (KBVIN) cancer cell lines. Furthermore, we total synthesized the natural product, calanquinone A (CA-1), and its analogues to evaluate their structure-activity relationship (SAR).
Presently, nineteen synthetic analogues, including synthetic calanquinone A (CA-1) have been finished and subjected to cytotoxicity, anti-platelet aggregation, and anti-inflammatory bioassays. Some of them showed significant effects on these bioassays. Their SAR will be reported herein.

並列關鍵字

Gelonium aequoreum ； Calanthe arisanensis ； Calanquinone A

參考文獻

1.Chen, L. A.; Hsia, H. M.; Kuo, N. F.; Wang, C. W.; Wang, S. M.; Hsieh, K. C. Flora of Taiwan 1993, 3, 474-475.

2.Boufford, D. E.; Hsieh, C.-F.; Huang, T.-C.; Kuoh, C.-S.; Ohashi, H.; Su, H.-J.; Yeh, H.-Y.; Hsiao, J.-L.; Hsiao, S.-H. Flora of Taiwan 2000, 5, 776-791.

3.Talapatra, S. K.; Das, G.; Talapatra, B. Stereostructures and molecular conformations of six diterpene lactones from Gelonium multiflorum. Phytochemistry 1989, 28, 1181-1185, and 3581-3582.

4.Chakravarty, A. K.; Pal, B. C.; Guittet, E.; Ahond, A. Structure of a new diterpene lactone from Gelonium multiflorum by 2D NMR. Indian J. Chem. Sect. B 1991, 30, 3-6.

5.Talapatra, B.; Das, G.; Das, A. K.; Biswas, K.; Talapatra, S. K. Stereostructures and conformations of four diterpene lactones from Gelonium multiflorum. Phytochemistry 1998, 49, 1353 -1359.