本研究主要是從柔軟冠形軟珊瑚Klyxum molle的有機溶劑萃取物中找尋具有生物活性的化學成分。本研究中共分離到15個eunicellin形式的化合物1–15，其中1–11為新化合物，12–15為已知的化合物，而化合物16、17則為反應所得產物。所有化合物的化學構造均由光譜數據分析(IR, MS, 1D及2D NMR)和比對文獻上已知化合物的光譜資料而確定。化合物5是首次發現具有Phenylacetyl官能基的eunicellin形式化合物。
本研究中，將所獲得的化合物1–17進行癌細胞株的細胞毒殺與抗發炎活性測試。在細胞毒殺方面，針對人類慢性白血病细胞(K562)、人類急性白血病細胞(Molt-4)和人類乳腺癌細胞(T47D)進行測試。化合物1、2及3對Molt-4有細胞毒殺活性(ED50分別為11.52 ± 2.75, 20.41 ± 2.92, 13.11 ± 3.87 μg/mL)，而化合物5對K562、Molt-4及T47D均有細胞毒殺活性(ED50分別為4.32 ± 1.38, 2.36 ± 0.34, 4.65 ± 0.93 μg/mL)。將化合物1–17進行抗發炎活性測試，化合物5在濃度為10 μg/mL下可有效抑制超氧陰離子產生與彈性蛋白酶釋放的活性(Inh %分別為81.56 ± 3.23, 89.16 ± 5.77)。

In order to search for bioactive compounds, we have studied the chemical constituents from the organic extracts of soft coral Klyxum molle. In this study, fifteen eunicellin-type diterpenoids, including eleven new compounds, klymollins K–U (1–11), along with four known compounds 12–15 were isolated. Compounds 16 and 17 were prepared by chemical synthesis. The structures of all compounds were established by spectroscopic methods and comparing the spectral data with known compounds. Compound 5 represents the first eunicellin-type compound with phenylacetyl group.
The cytotoxicity of compounds 1–17 against K562 (human erythro myeloblastoid leukemia), Molt-4 (human acute lymphoblastic leukemia), and T47D (human breast earcinoma) were determined. Compounds 1, 2, and 3 exhibited weak cytotoxicity against Molt-4 (with ED50 11.52 ± 2.75, 20.41 ± 2.92, and 13.11 ± 3.87 μg/mL). Compound 5 was found to exhibt significant cytotoxicity toward K562, Molt-4, and T47D (with ED50 4.32 ± 1.38, 2.36 ± 0.34, and 4.65 ± 0.93 μg/mL). Compound 5 also displayed significant inhibitory effects on superoxide anion generation and elastase release by human neutrophils at 10 μg/mL (with Inh % 81.56 ± 3.23, and 89.16 ± 5.77).

中文摘要 xi
英文摘要 xii
化合物1~17化學結構 xiii
第一章、緒論 1
第一節、前言 1
第二節、文獻回顧 3
第二章、生物材料與研究方法 42
第一節、研究流程 42
第二節、Klyxum molle 樣品採集時間、地點、分類地位 44
第三節、Klyxum molle 分離流程 45
第四節、化學反應步驟 47
第五節、實驗設備儀器及材料 48
第三章、化合物之結構證明 51
第一節：軟珊瑚Klyxum molle 所分離出之化合物的結構證明 51
(一)、Klymollin K (1) 構造之解析 51
(二)、Klymollin L (2) 構造之解析 61
(三)、Klymollin M (3) 構造之解析 71
(四)、Klymollin N (4) 構造之解析 81
(五)、Klymollin O (5) 構造之解析 91
(六)、Klymollin P (6) 構造之解析 101
(七)、Klymollin Q (7) 構造之解析 111
(八)、Klymollin R (8) 構造之解析 121
(九)、Klymollin S (9) 構造之解析 131
(十)、Klymollin T (10) 構造之解析 141
(十一)、Klymollin U (11) 構造之解析 151
(十二)、Klymollin E (12) 構造之解析 161
(十三)、Litophynin I (13) 構造之解析 165
(十四)、Australin C (14) 構造之解析 169
(十五)、(-)-Sclerophytin A (15) 構造之解析 173
(十六)、6-Acryloyl-(-)-sclerophytin A (16) 構造之解析 177
(十七)、6-Phenylacetyl-(-)-Sclerophytin A (17) 構造之解析 184
第二節: 化合物物理性質及圖譜數據整理 191
第四章、生物活性試驗 195
第一節、生物活性試驗方法 195
(一)、細胞毒殺活性試驗方法 195
(二)、抗發炎活性試驗方法 197
第二節、生物活性試驗結果 199
(一)、細胞毒殺活性試驗結果 199
(二)、抗發炎活性試驗結果 200
第五章、結論 201
第六章、參考文獻 204

1. Ruggieri, G. D. “Drugs from the sea” Science 1976, 194, 491–497.
2. Anake Kijjoa ,and Pichan Sawangwong “Drugs and Cosmetics from the Sea” Mar. Drugs 2004, 2, 73–82.
3. Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.; Gerwick, W. H. “Marine natural products as anticancer drugs” Mol. Cancer. Ther. 2005, 4, 333–342.
4. Bowden, B. F.; Coll, J. C.; Patalinghug, W.; Skelton, B. W.; Vasilescu, I.; White, A. H. Aust. J. Chem. 1987, 40, 2085–2096.
5. Coval, S. J.; Cross, S.; Bernardinelli, G.; Jefford, C. W. “Brianthein V, a New Cytotoxic and Antiviral Diterpene Isolated from Briareum asbestinum” J. Nat. Prod. 1988, 51, 981–984.
6. Neve, J. E.; McCool, B. J.; Bowden, B. F. “Excavatolides N－T, New Briarane Diterpenes from the Western Australian Gorgonian Briareum excavatum” Aust. J. Chem. 1999, 52, 359–366.
7. Shin, J.; Park, M.; Fenical, W. “The junceellolides new anti- inflammatory diterpenoids of the briarane class from the Chinese gorgonian Junceella fragilis” Tetrahedron 1989, 45, 1633–1638.
8. Wratten, S. J.; Fenical, W.; Faulkner, D. J. “Ptilosarcone, the toxin from the sea pen Ptilosarcus gurneyi” Tetrahedron Lett. 1977, 18, 1559–1562.
9. Kobayashi, J.; Cheng, J.-F.; Nakamura, H.; Ohizumi, Y.; Tomotake, Y.; Matsuzaki, T.; Grace, K. J. S.; Jacobs, R. S.; Kato, Y.; Brinen, L. S.; Clardy, J. “Structure and stereochemistry of brianolide, a new antiinflammatory diterpenoid from the Okinawan gorgonian Briareum sp.” Cell. Mol. Life. Sci. 1991, 47, 501–502.
10. Grode, S. H.; James, T. R., Jr; Cardellina, J. H.,II.; Onan, K.D. “Molecular Structures of the Briantheins, New Insecticidal Diterpenes from Briareum polyanthes” J. Org. Chem. 1983, 48, 5203–5207.
11. Hassan, H. M.; Khanfar, M. A.; Elnagar, A. Y.; Mohammed, R.; Shaala, L. A.; Youssef, D. T. A.; Hifnawy, M. S.; Sayed, K. A. El “Pachycladins A－E, Prostate Cancer Invasion and Migration Inhibitory Eunicellin-Based Diterpenoids from the Red Sea Soft Coral Cladiella pachyclados” J. Nat. Prod. 2010, 73, 848–853.
12. Rodriguez, A. D.; Cobar, O. M. “The Briarellins, New Eunicellin-Based Diterpenoids from A Caribbean Gorgonian, Briareum asbestinum” Tetrahedron, 1995, 51, 6869–6880.
13. Chill, L.; Berrer, N.; Benayahu, Y.; Kashman, Y. “Eunicellin Diterpenes from two Kenyan Soft Corals” J. Nat. Prod. 2005, 68, 19–25.
14. Hsu, F.-J.; Chen, B.-W.; Wen., Z.-H.; Huang, C.-Y.; Dai, C.-F.; Su, J.-H.; Wu, Y.-C.; Sheu, J.-H. “Klymollins A－H, Bioactive Eunicellin-Based Diterpenoids from the Formosan Soft Coral Klyxum molle” J. Nat. Prod. 2011, 74, 2467−2471.
15. 陳威錚 “台灣產軟珊瑚Klyxum simplex和印尼產海綿Halichondria sp. 所含化學成份之研究” 國立中山大學海洋資源學系碩士論文，2005年。
16. Wu, S.-L.; Su, J.-H.; Wen, Z.-H.; Hsu, C.-H.; Chen, B.-W.; Dai, C.-F.; Kuo, Y.-H.; Sheu, J.-H. “Simplexins A－I, Eunicellin-Based Diterpenoids from the Soft Coral Klyxum simplex” J. Nat. Prod. 2009, 72, 994–1000.
17. Wu, S.-L.; Su, J.-H.; Yi, L.; Chen, B.-W.; Huang, C.-Y.; Wen, Z.-H.; Kuo, Y.-H.; Sheu, J.-H.; “Simplexins J－O, Eunicellin-Based Diterpenoids from a Dongsha Atoll Soft Coral Klyxum simplex” Bull. Chem. Soc. Jpn. 2011, 84, 626–632.
18. Chen, B.-W.; Wu, Y.-C.; Chiang, Michael Y.; Su, J.-H.; Wang, W.-H.; Fan, T.-Y.; Sheu, J.-H. “Eunicellin-based Diterpenoids from the Cultured Soft Coral Klyxum simplex” Tetrahedron 2009, 65, 7016–7022.
19. Chen, B.-W.; Chao, C.-H.; Su, J.-H.; Tsai, C.-W.; Wang, W.-H.; Wen, Z.-H.; Huang, C.-Y.; Sung, P.-J.; Wu, Y.-C.; Sheu, J.-H.; “Klysimplexins I－T, Eunicellin-based Diterpenoids from the Cultured Soft Coral Klyxum simplex” Org. Biomol. Chem., 2011, 9, 834–844.
20. Chen, B.-W.; Huang, C.-Y.; Wen, Z.-H.; Su, J.-H.; Wang, W.-H.; Sung, P.-J.; Wu, Y.-C.; Sheu, J.-H. “Klysimplexins U－X, Eunicellin-Based Diterpenoids from the Cultured Soft Coral Klyxum simplex” Bull. Chem. Soc. Jpn., 2011, 84, 1237-1242.
21. Chen, B.-W.; Chao, C.-H.; Su, J.-H.; Wen, Z.-H.; Sung, P.-J.; Sheu, J.-H. “Anti-inflammatory Eunicellin-based Diterpenoids from the Cultured Soft Coral Klyxum simplex” Org & Biomol Chem. 2010, 8, 2363–2366.
22. Bowden, B. F.; Coll, J. C.; Dai, M. C.; “Studies of Australian Soft Corals. XLIII. The Structure Elucidation of a New Diterpene from Alcyonium molle” Australian J. Chem. 1989, 42, 665–673.
23. Su, J.; Zheng, Y.; Zeng, L.; Pordesimo, Eva O.; Schmitz, F. J.; Hossain, M. B.; Helm, D. V. D. “Patagonicol: A Diterpenoid from the Chinese Soft Coral Alcyonium patagonicum” J. Nat. Prod. 1993, 56, 1601–1604.
24. Kyeremeh, K.; Baddeley, T. C.; Stein, B. K.; Jaspars, M. “A Homologous Series of Eunicellin-Based Diterpenes from Acalycigorgia sp. Characterised by Tandem Mass Spectrometry” Tetrahedron 2006, 62, 8770–8778.
25. Fusetani, N.; Nagata, H.; Hirota, H.; Tsuyuki, T. “Astrogorgiadiol and Astrogorgin, Inhibitors of Cell Division in Fertilized starfish Eggs, from a Gorgonian Astrogorgia sp.” Tetrahedron Lett. 1989, 50, 7079–7082.
26. Lin, Y.; Bewely, C. A.; Faulkner, D. J. “The Valdivones, Antiinammatory Diterpene Esters from the South African Soft Coral Alcyonium valdivae ” Tetrahedron 1993, 49, 7977–7984.
27. Rodriguez, A. D.; Cobar, O. M. “Studies on the Minor Constituents of the Caribbean Gorgonian Octocoral Briareum asbestinum Pallas. Isolation and Structure Determination of the Eunicellin-Based Diterpenoids Briarellins E－I” Chem. Pharm. Bull. 1995, 43, 1853–1858.
28. Ospina, C. A.; Rodriguez, A. D.; Ortega-Barria, E.; Capson, T. L. “Briarellins J－P and Polyanthellin A: New Eunicellin-Based Diterpenes from the Gorgonian Coral Briareum polyanthrs and Their Antimalarial Activity” J. Nat. Prod. 2003, 66, 357–363.
29. Ospina, C. A.; Rodriguez, A. D. “Bioactive Compounds from the Gorgonian Briareum polyanthes. Correction of the structures of Four Asbestinane-Type Diterpenes” J. Nat. Prod. 2006, 69, 1721–1727.
30. Chen, Y.-H.; Tai, C.-Y.; Hwang, T.-L.; Weng, C.-F.; Li, J.-J.; Fang, L.-S.; Wang, W.-H.; Wu, Y.-C.; Sung, P.-J.; “Cladielloides A and B: New Eunicellin-Type Diterpenoids from an Indonesian Octocoral Cladiella sp.” Mar. Drugs 2010, 8, 2936–2945.
31. Tai, C.-Y.; Chen, Y.-H.; Hwang, T.-L.; Fang, L.-S.; Wang, W.-H.; Liu, M.-C.; Su, J.-H.; Wu, Y.-C.; Sung, P.-J.; “Cladielloides C and D: Novel Eunicellin-Based Diterpenoids from an Indonesian Octocoral Cladiella sp.” Bull. Chem. Soc. Jpn., 2011, 84, 531–536.
32. Chen, Y.-H.; Tai, C.-Y.; Su, Y.-D.; Chang, Y.-C.; Lu, M.-C.; Weng, C.-F.; Su, J.-H.; Hwang, T.-L.; Wu, Y.-C.; Sung, P.-J.; “Discovery of New Eunicellins from an Indonesian Octocoral Cladiella sp.” Mar. Drugs 2011, 9, 934–943.
33. Ahmed, A. F.; Wu, M.-H.; Wang, G.-H.; Wu, Y.-C.; Sheu, J.-H. “Eunicellin-Based Diterpenoids, Australins A－D, Isolated from the Soft Coral Cladiella australis” J. Nat. Prod. 2005, 68, 1051–1055.
34. Rao, C. B.; Rao, D. S.; Satyanarayana, C.; Rao, D. V.; Kassuhlke, K. E.; Faulkner, D. J. “New Cladlellane Diterpenes from the Soft Coral Cladiella Australis of the Andaman and Nicobar Islands” J. Nat. Prod. 1994, 57, 574–580.
35. Chen, B.-W.; Chang, S.-M.; Huang, C.-Y.; Chao, C.-H.; Su, J.-H.; Wen, Z.-H.; Hsu, C.-H.; Dai, C.-F.; Wu, Y.-C.; Sheu, J.-H. “Hirsutalins A-H, Eunicellin-Based Diterpenoids from the Soft Coral Cladiella hirsute” J. Nat. Prod. 2010, 73, 1785–1791.
36. Sarma, N. S.; Chavakula, R.; Rao, I. N.; Kadirvelraj, R.; Row, T. N. G.; Saito, I. “Crystal and Molecular structure of Sclerophytin F methyl ether from the Soft Coral Cladiella krempfi” J. Nat. Prod. 1993, 56, 1977–1980.
37. Tai, C.-J.; Su, J.-H.; Huang, M.-S.; Wen, Z.-H.; Dai, C.-F.; Sheu, J.-H. “Bioactive Eunicellin-Based Diterpenoids from the Soft Coral Cladiella krempfi” Mar. Drugs 2011, 9, 2036–2045.
38. Ciavatta, M. L.; Manzo, E.; Mollo, E,; Mattia, C. A.; Tedesco, C.; Irace, C.; Guo, Y.-W.; Li, X.-B.; Cimino, G.; Gavagnin, M. “Tritoniopsins A-D, Cladiellane-Based Diterpenes from the South China Sea Nudibranch Tritoniopsis elegans and Its Prey Cladiella krempfi” J. Nat. Prod. 2011, 74, 1902–1907.
39. Yamada, K.; Ogata, N.; Ryu, K.; Miyamoto, T.; Komori, T.; Higuchi, R. “Bioactive Terpenoids from Octocorallia. 3. A New Eunicellin-Based Diterpenoid from the Soft Coral Cladiella sphaeroides” J. Nat. Prod. 1997, 60, 393–396.
40. Chen, S.-P.; Sung, P.-J.; Duh, C.-Y.; Dai, C. F.; Sheu, J.-H. “Junceol A, a New Sesquiterpenoid from the Sea Pen Virgularia juncea” J. Nat. Prod. 2001, 64, 1241–1242.
41. Ketzinel, S.; Rudi, A.; Schleyer, M.; Benayahu, Y.; Kashman, Y. “Sarcodictyin A and Two Novel Diterpenoid Glycosides, Eleuthosides A and B, from the Soft Coral Eleutherobia aurea” J. Nat. Prod. 1996, 59, 873–875.
42. De Rosa, S. ; Cimino, G.; De Giulio, A.; Milone, A.; Crispino, A.; Iodice, C. “A New Bioactive Eunicellin-Type Ditepene from the Gorgonian Eunicella cavolini” Nat. Prod. Lett. 1995, 7, 259–265.
43. Mancini, I.; Guella, G.; Zibrowius, H.; Pietra, F. “Configuration, Conformation, and Reactivity of Highly Functionalized Eunicellane Diterpenes Isolated from the Gorgonians Eunicella cavolinii and Eunicella singularis from Marseille” Helv. Chim. Acta. 2000, 83, 1561–1575.
44. Ortega, M. J.; Zubia, E.; Salva, J. “A New Cladiellane Diterpenoid from Eunicella labiata” J. Nat. Prod. 1997, 60, 485–487.
45. Roussis, V.; Fenical, W.; Vagias, C.; Kornprobst, J.-M.; Miralles, J. “Labiatamides A, B, and other Eunicellan Diterpenoids from the Senegalese Gorgonian Eunicella labiata” Tetrahedron 1996, 52, 2735–2742.
46. Kakonikos, C.; Vagias, C.; Roussis, V.; Roussakis, C.; Kornprobst, J.-M. “New Eunicellin Type Diterpenoids from the Gorgonian Coral Eunicella labiata” Nat. Prod. Lett. 1999, 13, 89–95.
47. Kennard, O.; Watson, D. G.; Sanseverino, L. R. D. “The Crystal and Molecular Structure of Eunicellin Dibromide, C28H42O9Br2” Acta Cryst. 1970, B26, 1038.
48. Ortega, M. J.; Zubia, E.; He, H.-Y.; Salva, J. “New Eunicellin-Based Diterpene from the Gorgonian Eunicella verrucosa” Tetrahedron 1993, 49, 7823–7828.
49. Ortega, M. J.; Zubia, E.; Salva, J. “Structure and Absolute Configuration of Palmonine F, A New Eunicellin-Based Diterpene from the Gorgonian Eunicella verrucosa” J. Nat. Prod. 1994, 57, 1584–1586.
50. Ochi, M.; Yamada, K.; Futatsugi, K.; Kotsuki, H.; Shibata, K. “Litophynin D and E, Two new Diterpenoids from a soft coral Litophyton sp.” Chem. Lett. 1990, 12, 2183–2186.
51. Miyamoto, T.; Yamada, K.; Ikeda, N.; Komori, T.; Higuchi, R. “Bioactive Terpenoids from Octocorallia, I. Bioactive Diterpenoids: Litophynols A and B from the Mucus of the Soft Coral Litophyton sp.” J. Nat. Prod. 1994, 57, 1212–1219.
52. Ochi, M.; Yamada, K.; Futatsugi, K.; Kotsuji, H.; “Litophynin F, G and H, Three new Diterpenoids from a soft coral Litophyton sp.” Heterocycles, 1991, 1, 29–31.
53. Seo, Y.; Rho, J.-R.; Cho, K. W.; Shin, J. “Isolation of Diterpenoids of Cladiellane Class from Gorgonians of the Genus Muricella” J. Nat. Prod. 1997, 60, 171–174.
54. Wang, G.-H.; Sheu, J.-H.; Chiang, M. Y.; Lee, T.-J. “Pachyclavulariaenines A－C, Three Novel Diterpenoids from the Soft Coral Pachyclavularia violacea” Tetrahedron Lett. 2001, 42, 2333–2336.
55. Wang, G.-H.; Sheu, J.-H.; Duh, C.-Y.; Chiang, M. Y. “Pachyaclavulariaenones D－G, New Diterpenoids from the Soft Coral Pachyclavlaria violacea” J. Nat. Prod. 2002, 65, 1475–1478.
56. Alam, M.; Sharma, P.; Zektzer, A. S.; Martin, G. E.; Ji, X.; Helm, D. V. D. “Sclerophytin C－F: Isolation and Structures of Four New Diterpenes from the Soft Coral Sclerophytum capitalis” J. Org. Chem. 1989, 54, 1896–1900.
57. Sharma, P.; Alam, M. “Sclerophytin A and B. Isolation and Structures of Novel Cytotoxic Diterpenes from the Marine Coral Sclerophytum capitalis” J. Chem. Soc., Perkin Trans. 1 1988, 2537–2540.
58. Kusumi, T.; Uchida, H.; Ishitsuka, M. O.; Yamamoto, H.; Kakisawa, H. “Alcyonin, a new cladiellane diterpene from the soft coral Sinularia flexibilis” Chem. Lett. 1988, 6, 1077–1078.
59. Bloor, S. J.; Schmitz, F. J.; Hossain, M. B.; Helm, D. V. D. “Diterpenoids from the Gorgonian Solenopodium stechei” J. Org. Chem. 1992, 57, 1205–1216.
60. Su, J.-H.; Huang, H.-C.; Chao, C.-H.; Yan, L.-Y.; Wu, Y.-C.; Wu, C.-C.; Sheu, J.-H. “Vigulariol, a New Metabolite from the Sea Pen Vigularia juncea” Bull. Chem. Soc. Jpn. 2005, 78, 877–879.
61. Charles L. Cantrell; Tiansheng Lu; Frank R. Fronczek; Nikolaus H. Fischer. “Antimycobacterial Cycloartanes from Borrichia frutescens” J. Nat. Prod. 1996, 59, 1131–1136.
62. Hwang T.-L.; Leu Y.-L.; Kao S.-H.; Tang M.-C.; Chang H.-L. “Viscolin, a new chalcone from Viscum coloratum, inhibits human neutrophilsuperoxide anion and elastase release via a cAMP-dependent pathway” Free Radical Biology & Medicine. 2006, 41, 1433–1441.
63. Neish, A. C. “Biosynthesis of Pungenin from 14C-Labelled Compounds by Colorado Spruce” Can. J. Bot. 1959, 37, 1085–1100.