臺灣博碩士論文加值系統

中文摘要
先前本實驗室進行中草藥對腫瘤細胞毒性之篩選過程中，發現分離自大香葉樹的純化合物d-dicentrine有毒殺腫瘤細胞的活性，又先前的報導d-dicentrine具有抑制topoisomerase II的活性。本研究將d-dicentrine (DI)經由ethylchloroformate、pyridine的開環作用 (形成DI-7)，強鹼的水解 (形成DI-8)以及由DI-8經N-alkylayion而合成一系列的phenanthrene類的化合物 (DI-1, DI-2, DI-3, DI-4, DI-5和DI-6)，以便探討及比較其對不同腫瘤細胞之毒性作用及可能之作用機轉。細胞毒性 (MTT測試法) 之實驗結果顯示其對人類大腸癌細胞株 (COLO 201)之毒殺效力為DI-1 (IC50 = 3.9 mM) > DI-5 > DI-6 > DI-8 > DI-2 > DI (IC50 = 17.4 mM) > DI-3 > DI-4；對人類肝癌細胞株 (MS-G2) 為DI-1 (IC50 = 3.9 mM) > DI-6 > DI-5 > DI-2 > DI-8 > DI (IC50 = 16 mM) > DI-3 > DI-4；對人類口腔癌細胞株 (KB) 為DI-5 (IC50 = 3.3 mM) > DI-1 (IC50 = 3.5 mM) ~ DI-6 > DI-7 > DI-2 > DI-8 > DI (IC50 = 21.8 mM) > DI-3 > DI-4；對人類子宮頸癌細胞株 (HeLa) 為DI-1 (IC50 = 2.6 mM) > DI-7> DI-5 > DI-8 > DI-6 > DI-2 > DI-4 > DI (IC50 = 35.0 mM) ~ DI-3；而對張氏肝細胞株 (Chang liver cell) 為DI-5 (IC50 = 5.1 mM) > DI-1 (IC50 = 5.3 mM) > DI-6 > DI-2 > DI-8 > DI (IC50 = 15.3 mM) > DI-3 > DI-4。進一步針對DI，DI-1與DI-8，發現其對人類血癌細胞株 (HL-60)之毒殺效力為 DI-1 (IC50 = 11.0 mM) > DI-8 (IC50 = 18.0 mM) > DI (IC50 = 55.0 mM)；而以Annexin V染色配合流式細胞儀分析及細胞凋亡ELISA之分析發現DI，DI-1與DI-8會引起細胞凋亡；另在細胞週期分析上，發現這些化合物會引發細胞週期停滯於G2/M期。最後以HeLa 細胞核蛋白萃取物作kinetoplast DNA (KDNA)活性之分析實驗，發現DI，DI-1，DI-8有抑制topoisomerase II的活性，而DI-1 (IC50 = 2.8 mM)之抑制效力為DI之28倍。綜合上述結果，本研究認為DI-1似有進一步研發之潛力。

英文摘要
In our laboratory we have screened crude extracts and pure compounds of Chinese medicinal herbs for anti-tumor activities, and found that d-dicentrine (DI), which is a natural isoqunoline alkaloid isolated from the root of Lindera megaphylla Hemsl, has anti-tumor activities. It also has been reported that d-dicentrine can inhibit topoisomerase II activity. In the present study, a series of phenanthrene alkaloids were synthesized from d-dicentrine (DI) to study and compare their cytotoxicities to various tumor cell lines and the underlying mechanisms. The benzylic C-N bond of DI was cleaved by ethyl chloroformate in pyridine to form compound DI-7, followed by base hydrolysis with KOH in refluxing aqueous ethanol led to the form DI-8, and N-alkylation of DI-8 with various alkyl iodides in DMF to form DI-1, DI-2, DI-3, DI-4, DI-5, DI-6. The cytotoxic results obtained by MTT assay indicated that the rank order of cytotoxic potency was DI-1 (IC50 = 3.9 mM) > DI-5 > DI-6 > DI-8 > DI-2 > d-dicentrine (DI) (IC50 = 17.4 mM) > DI-3 > DI-4 in human colon adenocarcinoma cells (COLO 201)；DI-1 (IC50 = 3.9 mM) > DI-6 > DI-5 > DI-2 > DI-8 > DI (IC50 = 16 mM) > DI-3 > DI-4 in human hepatoma cells (MS-G2)；DI-5 (IC50 = 3.3 mM) > DI-1 (IC50 = 3.5 mM) ~ DI-6 > DI-7 > DI-2 > DI-8 > DI (IC50 = 21.8 mM) > DI-3 > DI-4 in human epidermoid carcinoma cells (KB)；DI-1 (IC50 = 2.6 mM) > DI-7> DI-5 > DI-8 > DI-6 > DI-2 > DI-4 > DI (IC50 = 35.0 mM) ~ DI-3 in human cervical carcinoma cells (HeLa)；and DI-5 (IC50 = 5.1 mM) > DI-1 (IC50 = 5.3 mM) > DI-6 > DI-2 > DI-8 > DI (IC50 = 15.3 mM) > DI-3 > DI-4 in Chang liver cells。Further study on DI, DI-1 and DI-8 found the cytotoxic potency of DI-1 (IC50 = 11.0 mM) > DI-8 (IC50 = 18.0 mM) > DI (IC50 = 55.0 mM) in human leukemia cells (HL-60). Furthermore, DI, DI-1, DI-8 caused apoptosis in HL-60 cells detected by Annexin V staining coupled with flow cytometry and cell death-detection ELISA assay and caused a stagnation of cells in G2/M phase measure by cell cycle analysis. Finally, the kinetoplast DNA (KDNA) decatenation assay in HeLa cell nuclear extract indicated that DI, DI-1 and DI-8 inhibited topoisomerase II activity and DI-1 (IC50 = 2.8 mM) was 28-fold more potent than DI. In conclusion, the present study suggested that DI-1 seems to have the potential for further study and development.

壹、中文摘要--------------------------------------------------1
貳、英文摘要--------------------------------------------------3
參、緒論------------------------------------------------------5
肆、合成部分--------------------------------------------------13
(一) 合成實驗儀器和材料-----------------------------------13
(二) 合成流程---------------------------------------------15
(三) 合成步驟及結果---------------------------------------24
伍、生物活性部分----------------------------------------------31
(一) 試藥-------------------------------------------------31
(二) 儀器-------------------------------------------------34
(三) 實驗方法---------------------------------------------35
陸、實驗結果--------------------------------------------------41
柒、討論------------------------------------------------------46
捌、參考文獻--------------------------------------------------53
玖、附表------------------------------------------------------60
拾、附圖------------------------------------------------------62

參考文獻
[1] Mushinski, J. F., Davidson, W. F., Morse, H. C. (1987) Activation of cellular oncogene in human and mouse leukemia-lymphomas : in murine B lymphocytic neoplasm. Cancer Investigation 5 : 345-368.
[2] Kroeger, P. E. and Rowe, T. C. (1992) Analysis of topoisomerase I and topoisomerase II cleavage sites on the drosphila actin and Hsp 70 shock genes. Biochemistry 31 : 2492-2501.
[3] Wang, J. C. (1971) Interaction between DNA and Escherichia coli protein w. Journal of Molecular Biology 55 : 523-525.
[4] Wang, J. C. (1985) DNA topoisomerase. Annual Review of Biochemistry 54 : 665-697.
[5] Gellert, M. (1981) DNA topoisomerase. Annual Review of Biochemistry 50 : 879-910.
[6] Wang, J. C. (1987) Recent studies of DNA topoisomerases. Biochimica et Biophysica Acta 909 : 1-9.
[7] Osheroff, N., Zechierich, E. L. and Gale, K. C. (1991) Catalytic function of DNA topoisomerase II. BioEssays 13 : 269-275.
[8] Wang, J. C. (1991) DNA topoisomerase : Why so many ? Journal of Biological Chemistry 266 : 6659-6662.
[9] Chempoux, J. J. (1978) Mechanism of the reaction catalyzed by the DNA unntwisting enzyme to 3’-terminus of the nicked DNA. Journal of Molecular Biology 118 : 441-446.
[10] Sander, M. and Hsieh, T. (1983) Double strand DNA cleavage by type II DNA topoisomerase from Drosophlia melanogaster. Journal of Biological Chemistry 258 : 8421-8428.
[11] Ross, W. E., Glaubiger, D. L. and Kohn, K. W. (1979) Qualitative and qantitative aspects of intercalator-induced DNA strand breaks. Biochimica et Biophysica Acta 562 : 41-50.
[12] Ross, W. E., Glaubiger, D. L. and Kohn, K. W. (1978) Protein-associated DNA breaks in cells treated with adriamycin or ellipiticine. Biochimica et Biophysica Acta 519 : 23-30.
[13] Yves, D., Pharm,D., Mark, R. F., Akira, F., Richard, B., Eric, S., Glenda, K. and Kurt, W. K. (1994) Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors. Cancer Investigation 12 : 530-542.
[14] Nelso, E. M., Tewey, K. M., Liu, L. F. (1984) Mechanism of antitumor drug action : Poisoning of mammalian DNA topoisomerase II on DNA by 4’-(9-acridinylamino)-methanesulfon-m-amiside. Proceedings of the National Academy of Sciences, USA 81 : 1361-1365.
[15] Kerr J. K.,Wyllie, A. H and Currie A. R. (1972) Apoptosis : a basic biological phenomenon with wide ranging implicdatons in tissue kinetics. British Journal of Cancer 26 : 1790-1794.
[16] Thompson, C. B. (1995) Apotosis in the pathogenesis and treatment of diseases. Science 267 : 1457-1462.
[17] Raff, M.C (1992) Social controls on cell survial and cell death. Nature 356 : 397-399.
[18] Bishop, C.J and Whiting, V.A. (1983) The role of natural killer cells in the intravascular death of intravenously injected murine tumor cells. British journal of Cancer 48 : 441-444.
[19] Wyllie, A. H., Kerr, J. F and Currie, A. R. (1980) Cell death : The significance of apoptosis. International Review of Cytology 68 : 251-306
[20] Haskett, C. (1992) Resolution of acute inflammation and the role of apoptosis in tissue fate of granulocytes. Clinical Science 83 : 639-649.
[21] Lennon S. V., Martin S. J and Cotter T. G. (1991) Dose-dependent induction of apoptosis in human tumor cell lines by widely divering stimuli. Cell Proliferation 24 : 203-214.
[22] Nicholson D. W and Thornberry N. (1997) Caspase : killer protease. Thrend in Biochemical Sciences 22 : 299-306.
[23] Ashkennazi, A and Dikit, V. M. (1998) Death receptor signaling and modulation. Science 281 : 1305-1308.
[24] Adams, J. M and Corry, S. (1998) The Bcl-2 protein family: Arbiters of cell survial. Science 281 : 1322-1331.
[25] Dixon, S. C., Soriano, B. J., Lush, R. M., Borner, M. M. and Figg, W. D. (1997) Apotosis : its role in the development of malignancies and its potential as a novel therapeutic target. Annals of Pharmacotherapy 31 : 76-82.
[26] Kerr, J.F., Winterford, C.M. and Harmon, B. V. (1994) Apotosis. Its significance in cancer and cancer therapy. Cancer 73 : 2013-2026.
[27] Mc Donnell, T. J, Meyn, R. E. and Robertson, L. E. (1995) Implications of apototic cell death regulation in cancer therapy. Seminars in Cancer Biology 6 : 53-60.
[28] Martin, S. J. and Green, D. R. (1994) Apotosis as a goal of cancer therapy. Current Opinion in Oncology 6 : 616-621.
[29] Dixon S. C., Soriano B. J., Lush R. M., Borner M. M and Figg W. D (1997) Apoptosis : its role in the develoment of malignancies and its potential as a novel therapeutic target. Annals of Pharmacotheraphy 31 : 76-82.
[30] Debatin K. M. (2000) Activation of apoptosis pathways by anticancer treatment. Toxicology Letters 41-48
[31] Fisher, R. P. (1997) Cdks and cyclins in transition. Current Opinion in Gene Development 7 : 32-38.
[32] Morgan, D. O. (1995) Principles of Cdk regulation. Nature 374 : 131-134.
[33] Hunter, T and Pines, J. (1994) Cyclins and cancer II : cyclin D and cdk inhibitors come of age. Cell 79 : 573-582.
[34] Sherr C. J and Roberts J. M. (1995) Inhibitors of mammalian G1 cyclin-dependent kinase. Genes & Develoment 9 : 1149-1163.
[35] Chen, C. C., Huang, Y. L., Ou, J.C., Su, M. J., Teng, C. M. (1991) Bioactive principles from the roots of lindera megaphylla. Planta Medica 57: 406-408.
[36] Yu, S.M., Chen, C. C., Ko, F. N., Huang., T. F., Teng, C. M.(1992) Dicentrine, a novel antiplatelet agent inhibiting thromboxane formation and increasing the cyclic amp level of rabbit platelets. Biochemical Pharmacology 43 : 323-329.
[37] Teng, C. M., Yu, S. M., Ko, F. N., Chen, C.C., Huang, Y. L., Huang, T. F. (1996) Dicentrine, a novel a1-adrenoceptor antagonist, isolated from Lindera megaphylla. British Journal of Pharmacology 43 : 323-329.
[38] Tsai, T.J., Lin, R.H., Chang, C.C., Chen, Y. M., Chen, C. F., Ko, F.N., Teng, C. M. (1995) Vasodilator agents modulate rat glomerular mesangial cell growth and collagen synthesis. Nephron 70 : 91-99.
[39] Huang, R. L., Chen, C. C., Ou, J. C., Hu, C. P., Chen, C. P. and Chang, C. (1998) Anti-tumor effects of d-dicentrine from the root of Lindera megaphylla. Planta Medica 64 : 212-215.
[40] Woo, S. H., Sun, N. J., Cassady, J. M. and Snapka, R. M. (1999) Topoisomerase II inhibition by aporphine alkaloids. Biochemical Pharmacology 57 : 1141-1145.
[41] Dong, U. L., Kinuko, I., Miyoko, K., Narao, T and Wolfgang, W. (1991) Degradation of some phathalideisoquinolines with ethyl chloroformate-stereochemical aspects. Chemical & Pharmaceutical Bulletin 39 : 1944-1948.
[42] Geisow M. J., Fritsche U., Hexham J. M., Dash B., Johnson T. (1986) A consensus amino acid seqence repeat in Torpedo and mammalian Ca2+-dependent membrane-binding protein. Nature 320 : 636-638.
[43] England M. V., Niceland L. W., Ramackers F. C. S. (1996) A novel assay measure loss of plasma membrane asymmetry during apoptosis of adherant cells in culture. Cytometry 24 : 131-139.
[44] Reutelingsperger C. P and Heerde W. L. Annexin V, the regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis (1997) Cellular and Molecular Life Sciences 53 : 527-532.
[45] Reutelingsperger C. P., Hornstra G., Hemkeer H. C. (1985) Isolation and partial purification of human umbilical cord. European Journal of Biochemistry 151: 625-629.
[46] Vermes I., Haanen C., Steffens-Nakken H., Reutelingsperger C. P. (1995) A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labbled Annexin V. Journal of Immunological Methods (1995) 184 : 39-51.
[47] Polzar B., Zanotti S., Stephan H., Rauch F., Peitsch M. C., Irmler M., Tschopp J., Mannherz H. G. (1994) Distribution of deoxyribonuclease in rat tissues and its correlation to cellular turnover and apoptosis. European Journal of Cell Biology 4 : 200-210.
[48] Bertrand R., Sarang M., Jenkin J., Kerrigan D., Pommier Y. (1991) Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression. Cancer Research 51 : 6280-6285.
[49] Solary E., Bertrand R., Jenkins J., Pommier Y. (1992) Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells. Experimental Cell Research 203 : 495-504.
[50] Bertrand R., Solary E., Jenkins J., Pommier Y. (1993) Apoptosis and its modulayion in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors. Expermental Cell Research 207 : 388-397.
[51] Solany E., Bertrand R., Kohn K. W., Pommier Y. (1993) Differenaial induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors. Blood 81 : 1359-1368.
[52] Castedo, L., Tojo, G. (1990) The alkaloids. Phenanthrene Alkaloids 39 : 99-138.
[53] Oberhammer F., Wilson J. W., Dive C., Morris I. D., and Hickman J. A., Wakeling A. E., Walker P. R and Sikorska M. (1993) Apoptotic death in epithelial cells : cleavage of DNA to 300 and / or 50 kb fragments prior to or in the absence of internucleosomal fragmentation.The EMBO Journal 12 : 3679-3684.
[54] Chen, G, L and Liu, L. F. (1986) DNA topoisomerase as therapeutic targets in cancer chemtherapy. Annual reports in medicinal chemistry 21 : 257-262.
[55] Baguley, B. C. (1991) DNA intercalating anti-tumor agents. Anti-cancer drug design 6 : 1-35.
[56] Zhang, H., Darpa, P and Lui, L. F. (1990) A model for tumor cell killing by topoisomerase posions. Cancer Cells 2 : 23-32.
[57] Lambert B and Le Pacq J. B. (1987) Pharmacology of DNA binding drugs in DNA ligand interactions, Ed. Guschlbauer W and Saenger W., Plenum Press, pp 141-157.
[58] Chen G. L., Yang L., Rowe T. C., Halligan B. D., Tewey K. M and Liu L. F. (1984) Nonintercalative antitumor drugs interfere with the breakage-reunion reaction of mammalian DNA topoisomerase II. The Journal of Biological Chemistry 259 : 13560-13566.
[59] Ross W., Rowe T., Glisson B and Liu L. F. (1984) Role of topoisomerase II in mediating epipodophyllotoxin-induced DNA cleavage. Cancer Research 44 : 5857-5860.
[60] Chow K. C., Macdonald T. L and Ross W. E. (1988) DNA binding by epipodophyllotoxins and N-acyl anthracyclines: implications for mechanism of topoisomerase II inhibition. Molecular Pharmacology 34 : 467-473.