臺灣博碩士論文加值系統

斑蝥為翹翅目莞青科斑莞青屬的昆蟲，抵禦外敵時由腳關節處分泌之有毒物質，即為斑蝥素 (Cantharidin; exo,exo-2,3-Dimethyl- 7-oxabicyclo [2,2,1] heptane-2,3-dicarboxylic anhydride)，在國外的研究中，斑蝥素具有抗癌，或當除草劑、殺蟲劑、毛髮生長刺激劑及治療傳染性疣等效果。近來有學者發現，某些具有醯亞胺-胜結構的化合物，可抑制革蘭氏陽性菌及陰性菌，而具平面結構的化合物、金剛烷及二唑皆有抗菌效果，所以我們此次實驗會探討其抗菌的活性。
本實驗主要是以斑蝥素為原料與含一級胺取代物之化合物包括：胺基酸系列、具平面結構之化合物、金剛烷，在鹼性條件下高溫 (約200 ℃) 封管反應兩小時，冷卻後進行脫水反應，利用薄層層析法 (TLC) 純化，可得一系列斑蝥胺素 (Cantharidinimide) 之衍生物；另外亦有使用酸酐系列與胺基二唑，藉由相同反應條件合成，得到一系列醯亞胺衍生物。
合成的斑蝥胺素衍生物及醯亞胺衍生物，經過核磁共振儀、紅外光光譜儀、高解析度質譜儀確定結構，並進行抗菌活性測試。結果顯示化合物13 (4-Anthracen-2-yl-2,6-dimethyl-10-oxa-4-aza-tricyclo [5.2.1.02,6]decane-3,5- dione) 對Staphylococcus及Bacillus有較好的抑制作用，但是效果仍未比Penicillin G好。

Mylabris phalerata, Meloidae family of Coleoptera, has been known since antiquity to produce a potent toxic defensive agent, now known as cantharidin (exo,exo-2,3-dimethyl-7-oxabicyclo [2.2.1] heptane-2,3- dicarboxylic anhydride), but more infamously known as Spanish Fly. In the past, Cantharidin has used as anticancer, herbicidal, pesticide and treatment of molluscum contagiosum activities.
The new analogues contain various amino acids attached to the carboxylic anhydride exhibit antibacterial activity. The compounds with triazole structure have been evaluated their antitubercular activity and the new adamantanyl derivatives have found to have antibacterial activity. In addition, some new 1,3,4-thiadiazoles containing isomeric pyridyl have been found to be active against both Gram-positive and Gram-negative bacteria.
We used cantharidin as a starting material to react with primary amine, series of amino acid, diaminotriazole and aminoadamantane, by heating 200℃ in sealed tube to form various cantharidinimide derivatives. Furthermore, we used anhydrides and aminothiadiazole in the same condition to obtain thiazolylimide derivatives.
We suppose that it might have some steric hindrance between bulky amino acids and 2,3-dimethyl group of cantharidin and gave low yields. And we found that higher polarity of amino acids didn’t react with cantharidin.
All of the cantharidinimide and thiazolylimide derivatives were measured by 1H-NMR, IR and mass spectrometry. Therefore, we would try to evaluate antibacterial activity of cantharidinimide and thiazolylimide derivatives. The results indicated that compound 13 (4-Anthracen-2-yl-2,6-dimethyl-10-oxa-4-aza-tricyclo[5.2.1.02,6]decane-3,5-dione) exhibits higher inhibitory activity against Staphylococcus and Bacillus than others, but is still less effective than penicillin G.

目錄 I
流程目錄 III
附表目錄 IV
附圖目錄 V
中文摘要 1
英文摘要 3
壹、緒論 5
一、研究背景： 5
二、研究目的： 9
貳、結果與討論 13
一、化學合成 13
(一) Cantharidin原料之製備 13
(二) 斑蝥胺素 (Cantharidinimide) 衍生物之合成 17
(三) 酸酐 (anhydride) 類與胺基二唑 (aminothiadiazole) 之合成 23
二、生物活性 25
參、實驗部分 28
一、儀器與試藥 28
(一) 實驗儀器 28
(二) 試藥及試劑 29
(三) 材料 30
二、化學合成部分 31
(一) Cantharidin原料之製備 31
(二) 斑蝥胺素及其類似物之合成步驟 32
三、生物活性部分 50
(一) 菌種及培養基 50
(二) 最小抑制濃度 (MIC, Minimum Inhibitory Concentration) 51
肆、結論 52
伍、參考文獻 54
陸、附圖目錄 60

1.張蘭昌，斑蝥，中藥大辭典，1981，3733-3735。
2.李時珍，斑蝥，重訂本草綱目，1994，40卷，1287-1288。
3.黃三元，斑蝥，本草備要新編，1990，507-508。
4.Yosef, R.; Carrel, J. E.; Eisner, T. Contrasting Reactions of Loggerhead Shirkes to Two Type of Chemistry Defended Insect Prey. J. Chem. Ecol., 1996, 22, 173-181.
5.Yell., J. A.; Burge, S. M.; Dean D. Cantharidin-induced acantholysis in Darier’s disease: does acantholysis initiate dyskeratosis? Brit. J. Dermatol., 1994, 313, 40-47.
6.Masafumi, M.; Graziano, M.J.; Casida, E. Endothal and Cantharidin Analogues: Relation of Structure to Herbicidal Activity and Mammalian Toxicity. J. Agr. Food Chem., 1987, 35, 823-829.
7.Silverberg, N. B.; Sidury, R.; Mancini, J. Childhood molluscum contagiosum: Experience with cantharidin therapy in 300 patients. J. Am. Acad. Dermatol., 2000, 43, 503-507.
8.Ross, G. L.; Orchard, D.C. Combination topical treatment of molluscum contagiosum with cantharidin and imiquimod 5% in children: A case series of 16 patients. Australas. J. Dermatol., 2004, 45, 100-102.
9.Woodward, R. B.; Loftfield, R. B. The Structure of Cantharidine and the Synthesis of Desoxycantharidine. J. Am. Chem. Soc., 1941, 63, 3167-3171.
10.Stork, G.; Tamelen, E. E.; Friedman, L. J.; Burgstahler, A. W. Cantharidin. A Stereospecific Total Synthesis. J. Am. Chem. Soc., 1951, 73, 4501.
11.Stork, G.; Tamelen, E. E.; Friedman, L. J.; Burgstahler, A. W. A Stereospecific Synthesis of Cantharidin. J. Am. Chem. Soc., 1953, 75, 384-392.
12.Dauben, W. G.; Krabbenhoft, H. O. Organic Reactions at High Pressure. J. Am. Chem. Soc., 1976, 98, 1992-1993.
13.Dauben, W. G.; Kessel, C. R.; Takemura, K. H. Simple, Efficient Total Synthesis of Cantharidin via a High-Pressure Diels-Alder Reaction. J. Am. Chem. Soc., 1980, 102, 6893-6894.
14.Honkanen, R. E. Cantharidin, another natural toxin that inhibits the activity of serine/threonine protein phosphatases type 1 and 2A. FEBS, 1993, 330, 283-286.
15.Eldridge, R.; Casida, J. E. Cantharidin Effects on Protein Phosphatases and the Phosphorylation State of Phosphoproteins in Mice. Toxicol. Appl. Pharm., 1995, 130, 95-100.
16.Laidley, C. W.; Cohen, E.; Casida, J. E. Protein Phosphatase in Neuroblastoma Cells: [3H]Cantharidin Binding Site in Relation to Cytotoxicity. J. Pharmacol. Exp. Ther., 1997, 280, 1152-1158.
17.Sheppeck, J. E.; Gauss, C. M.; Chamberlin, A. R. Inhibition of the Ser-Thr Phosphatases PP1 and PP2A by Naturally Occurring Toxins. Bioorg. Med. Chem., 1997, 5, 1739-1750.
18.Kanpp, J.; Boknik, P.; Lonck, B. The effect of the protein phosphatases inhibitor cantharidin on β-adrenoceptor-mediated vasorelaxation. Brit. J. Pharmacol., 1997, 120, 421-428.
19.Kanpp, J.; Boknik, Huke, S. The mechanism of action of cantharidin in smooth muscle. Brit. J. Pharmacol., 1998, 123, 911-919.
20.Kanpp, J.; Boknik, Luss, I. The Protein Phosphatase Inhibitor Cantharidin Alters Vascular Endothelial Cell Permeability. J. Pharmacol. Exp. Ther., 1999, 189, 1480-1486.
21.Hong, S. J. Inhibition of mouse neuromuscular transmission and contractile function by okadaic acid and cantharidin. Brit. J. Pharmacol., 2000, 130, 1211-1218.
22.Neumann, J.; Herzig, S.; Boknik, P. On the Cardiac Contractile, Biochemical and Electrophysiological Effects of Cantharidin, a Phosphatase Inhibitor. J. Pharmacol. Exp. Ther., 1995, 274, 530-539.
23.Linck, B.; Boknik, P.; Knapp, J. Effects of cantharidin on force of contraction and phosphatase activity in nonfailing and failing human hearts. Brit. J. Pharmacol., 1996, 119, 545-550.
24.Rabkin, S. W.; Friesen, J. M.; Ferris, J. A. J.; Fung, H. Y. M. A Model of Cardiac Arrhythmias and Sudden Death: Cantharidin-Induced Toxic Cardiomyopathy. J. Pharmacol. Exp. Ther., 1979, 210, 43-50.
25.Krautheim, A.; Brechlin, P.; Becker, K. Hamster pancreatic beta cell lines with altered sensitivity towards apoptoic signaling by phosphatase inhibitors. Brit. J. Pharmacol., 2000, 129, 687-694.
26.Morana, S. J.; Wolf, C. M.; Jason, J. L. The Involvement of Protein Phosphatases in the Activation of ICE/CED-3 Protease, Intracellular Acidification, DNA Digestion, and Apoptosis. J. Biol. Chem., 1996, 271, 18263-18271.
27.Wang, C. C.; Wu, C. H.; Hsieh, K. J. Cytotoxic effects of cantharidin on the growth of normal and carcinoma cells. Toxicology, 2000, 147, 77-87.
28.劉建；高建輝；劉曉秋，班蝥素及其衍生物的研究進展，中藥材，2003，26卷，453-455。
29.Lin, P. Y.; Shi, S. J.; Hsu, F. L.; Chen , C. F. New Cantharidinimides from Cantharidin and 2-Arylethylamines: Efficient Synthesis under High Pressure. J. Chin. Chem. Soc., 1998, 45, 323-326.
30.Lin, P. Y.; Shi, S. J.; Lin, L. H.; Chen, H. C.; Hsu, F. L. Synthesis of Novel N-Pyridylcantharidinimides by Using High Pressure. J. Chin. Chem. Soc., 2001, 48, 49-53.
31.Lin, P. Y.; Shi, S. J.; Hsu, F. L. Synthesis of Cantharidinimide Derivatives via the Mitsunobu Reaction. New Taipei J. Med., 1999, 1, 20-22.
32.Tsauer, W.; Lin, J. G.; Lin, P. Y. The Effects of Cantharidin Analogues on Xanthine Oxidase. Anticancer Res., 1997, 17, 2095-2098.
33.Lin, P. Y.; Shi, S. J.; Shu, H. L.; Chen, H. F.; Lin, C. C.; Liu, P. C.; Wang, L. F. Bioorg. Chem., 2000, 28, 266-272.
34.Lin, L. H.; Huang, H. S.; Lin, C. C.; Lee, L. W.; Lin, P. Y. Effects of Cantharidinimides on Human Carcinoma Cells. Chem. Pharm. Bull., 2004 (in press)
35.Cros, E.; Planas, M.; Bardaji, E. Synthesis of N-Tetrachlorophthaloyl (TCP) —Protected Amino Acids under Microwave Irradiation (MWI). Synthesis, 2001, 9, 1313-1320.
36.McCluskey, A.; Walkom, C.; Bowyer, M. C.; Ackland, S. P.; Gardiner, E.; Sakoff, A. Cantharimides: A New Class of Modified Cantharidin Analogues Inhibiting Protein Phosphatases 1 and 2A. Bioorg. Med. Chem. Lett., 2001, 11, 2941-2946.
37.Miller, C. T.; Weragoda, R.; Izbicka, E.; Iverson, B. L. The Synthesis and Screening of 1,4,5,8-Naphthalenetetracarboxylic Diimide-Peptide Conjugates with Antibacterial Activity. Bioorg. Med. Chem., 2001, 9, 2015-2024.
38.Zamani, K.; Faghihia, K.; Mehranjanib, M. S. Synthesis of Some New 2,5-Disubstituted 1,3,4-Thiadiazoles Containing Isomeric Pyridyl as Potent Antimicrobial Agents. Pol. J. Pharmacol., 2003, 55, 1111—1117.
39.Pandeya, S. N.; Sriram, D.; Nath, G.; Clercq, E. Synthesis, Antibacterial, Antifungal and Anti-HIV Evaluation of Schiff and Mannich Bases of Isatin and its Derivatives with Triazole. Arzneim.-Forsch.-Drug Res., 2000, 50, 55-59.
40.Bhat, A. R.; Bhat, G. V.; Shenoy, G. G. Synthesis and in-vitro antimicrobial activity of new 1,2,4-triazoles. J. Pharm. Pharmacol., 2001, 53, 267-272.
41.Calıs, .; Yarım, M.; Kksala, M.; zalp, M. Synthesis and Antimicrobial Activity Evaluation of some New Adamantane Derivatives. Arznelm.-Forsch.-Drug Res., 2002, 52, 778-781.
42.Orzeszko, A.; Gralewska, R.; Starosciak, B. J.; Kazimierczuk, Z. Synthesis and antimicrobial activity of new adamantine derivatives. Acta Biochim. Pol., 2000, 47, 87-94.
43.Grogan, C. H.; Rice, L. M. Bicycle Imide and Isoindolines. J. Med. Chem., 1963, 6, 802-805.
44.Salama, R. B.; Hammouda, Y.; Gassim, I. Isolation of cantharidin from Cyaneolytta sapphirina. J. Pharm. Pharmacol., 1974, 26, 268-269.
45.Zehnder, M.; Thewalt, U. Struktur von Cantharidin, C10H12O4. Helv. Chim. Acta, 1977, 60, 740-742.
46.Paleo, M. R.; Calaza, M. I.; Sardina, F. J. Enantiospecific Synthesis of N-(9-Phenylfluoren-9-yl)-α-amino Ketones. J. Org. Chem., 1997, 62, 6862-6869.
47.Chang, H.; Kim, K.; Ree, M,; Lee, K. W. An NMR study on polycondensation reactions of 3,5-diamino-1,2,4-triazole with dianhydrides nd diacyl chlorides. Macromol. Chem. Phys., 1999, 200, 422-430.
48.Lin, L. H.; Lee, L. W.; Sheu, S. Y.; Lin, P. Y.; Study on the Stevioside Analogues of Steviolbioside, Steviol, and Isosteviol 19-Alkyl Amide Dimers: Synthesis and Cytotoxic and Antibacterial Activity, Chem. Pharm. Bull., 2004, submitted (accepted).