桦木酸及其衍生物的合成与抗肿瘤活性的研究
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摘要
桦木酸及其衍生物具有选择性抑制肿瘤细胞生长的作用，是潜在的抗肿瘤药物。但由于桦木酸价格昂贵、水溶性差，制约了其进一步的研究开发，因此获得大量廉价的桦木酸并提高水溶性是解决问题的关键。
本文首先研究了从酸枣仁中提取分离桦木酸的工艺，得到纯度大于95％的桦木酸，收率0.15％。
其次，从白桦树皮中提取分离了大量纯度超过95％的桦木醇，然后利用琼斯氧化和硼氢化钠还原，由桦木醇合成了桦木酸。
第三，以桦木酸为母体，合成了4个桦木酰-氨基酸衍生物(桦木酰-L-丙氨酸、桦木酰-D-丙氨酸、桦木酰-β-氨基丙酸、桦木酰-γ-氨基丁酸)；以桦木酮酸为母体，合成了3个C-3位改造的桦木酮酸衍生物(3-肟-桦木酸、3-氨基-桦木酸、3-腙-桦木酸)和10个桦木酮酰-氨基酸衍生物(桦木酮酰-甘氨酸、桦木酮酰-L-丙氨酸、桦木酮酰-D-丙氨酸、桦木酮酰-β-氨基丙酸、桦木酮酰-γ-氨基丁酸、桦木酮酰-L-缬氨酸、桦木酮酰-L-谷氨酸、桦木酮酰-L-异亮氨酸、桦木酮酰-L-苯丙氨酸、桦木酮酰-L-色氨酸)。除桦木酰-L-丙氨酸、3-肟-桦木酸和3-氨基-桦木酸外，其它14个均为新化合物。
第四，利用MTT[3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide]检测法，系统地研究了桦木酸对10种肿瘤细胞的生物活性。结果表明，桦木酸能够有效地抑制KB(人口腔上皮癌细胞)等多种肿瘤细胞的生长。并测定了数十种桦木酸衍生物对KB细胞的增殖抑制率。
Betulinic acid and its derivatives were potential antitumor agents, which displayed a selective cytotoxicity on tumor cell lines but not on normal cells. Research and development of betulinic acid as a therapeutic agent has been hindered because 1) betulinic acid presently is available in very limited quantities and at a very high cost; 2) betulinic acid suffers a low water solubility, resulting in inefficient biological efficacy. It was envisioned that providing commercial quantity of betulinic acid and improving the water solubility by synthesized a number of potentially important derivatives, which may be developed as antitumor agents, could resolve the problems.First, we studied the method of extracting betulinic acid from Semen Ziziphi Spinosae and obtained fine needles betulinic acid, purity>95%, yield 0.15%.Next, betulin was extracted and isolated from the bark of Betula alba in significant quantity. After recrystallized from ethanol pure betulin(>95%)was obtained, yield 20%. Then, it was synthetically converted to betulinic acid by Jones'oxidation and sodium borohydride reduction.Third, four amino acid (L-alanine D-alanineβ-amino-propanoic acid andγ-amino-butanoic acid) conjugates of betulinic acid and ten amino acid (glycine L-alanine D-alanine,β-amino-propanoic acidγ-amino-butanoic acid L-valine L-glutamic acid L-isoleucine L-phenylalanine L-tyrptophan) conjugates of betulonic acid at C-28 carboxylic acid position, as well as three C-3 position modified derivatives of betulonic acid (3-oxime-betulinic acid 3-amino-betulinicacid 3-hydrazone-betulinic acid) were synthesized in efforts to develop potential antitumor agents. All the derivatives are new compounds except L-alanine conjugate of betulinic acid 3-oxime-betulinic acid and 3-amino- betulinic acid.Fourth, MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay showed that betulinic acid could inhibit the proliferation of ten tomor cell lines, in which the IC50 of KB (human oral epidermoid cancinoma) cells was the least. Besides, the inhibitory rates (IR%) of all derivatives were calculated against KB cell lines.
本文首先研究了从酸枣仁中提取分离桦木酸的工艺，得到纯度大于95％的桦木酸，收率0.15％。
其次，从白桦树皮中提取分离了大量纯度超过95％的桦木醇，然后利用琼斯氧化和硼氢化钠还原，由桦木醇合成了桦木酸。
第三，以桦木酸为母体，合成了4个桦木酰-氨基酸衍生物(桦木酰-L-丙氨酸、桦木酰-D-丙氨酸、桦木酰-β-氨基丙酸、桦木酰-γ-氨基丁酸)；以桦木酮酸为母体，合成了3个C-3位改造的桦木酮酸衍生物(3-肟-桦木酸、3-氨基-桦木酸、3-腙-桦木酸)和10个桦木酮酰-氨基酸衍生物(桦木酮酰-甘氨酸、桦木酮酰-L-丙氨酸、桦木酮酰-D-丙氨酸、桦木酮酰-β-氨基丙酸、桦木酮酰-γ-氨基丁酸、桦木酮酰-L-缬氨酸、桦木酮酰-L-谷氨酸、桦木酮酰-L-异亮氨酸、桦木酮酰-L-苯丙氨酸、桦木酮酰-L-色氨酸)。除桦木酰-L-丙氨酸、3-肟-桦木酸和3-氨基-桦木酸外，其它14个均为新化合物。
第四，利用MTT[3-(4，5-dimethylthiazol-2-yl)-2，5-diphenyltetrazolium bromide]检测法，系统地研究了桦木酸对10种肿瘤细胞的生物活性。结果表明，桦木酸能够有效地抑制KB(人口腔上皮癌细胞)等多种肿瘤细胞的生长。并测定了数十种桦木酸衍生物对KB细胞的增殖抑制率。
Betulinic acid and its derivatives were potential antitumor agents, which displayed a selective cytotoxicity on tumor cell lines but not on normal cells. Research and development of betulinic acid as a therapeutic agent has been hindered because 1) betulinic acid presently is available in very limited quantities and at a very high cost; 2) betulinic acid suffers a low water solubility, resulting in inefficient biological efficacy. It was envisioned that providing commercial quantity of betulinic acid and improving the water solubility by synthesized a number of potentially important derivatives, which may be developed as antitumor agents, could resolve the problems.First, we studied the method of extracting betulinic acid from Semen Ziziphi Spinosae and obtained fine needles betulinic acid, purity>95%, yield 0.15%.Next, betulin was extracted and isolated from the bark of Betula alba in significant quantity. After recrystallized from ethanol pure betulin(>95%)was obtained, yield 20%. Then, it was synthetically converted to betulinic acid by Jones'oxidation and sodium borohydride reduction.Third, four amino acid (L-alanine D-alanineβ-amino-propanoic acid andγ-amino-butanoic acid) conjugates of betulinic acid and ten amino acid (glycine L-alanine D-alanine,β-amino-propanoic acidγ-amino-butanoic acid L-valine L-glutamic acid L-isoleucine L-phenylalanine L-tyrptophan) conjugates of betulonic acid at C-28 carboxylic acid position, as well as three C-3 position modified derivatives of betulonic acid (3-oxime-betulinic acid 3-amino-betulinicacid 3-hydrazone-betulinic acid) were synthesized in efforts to develop potential antitumor agents. All the derivatives are new compounds except L-alanine conjugate of betulinic acid 3-oxime-betulinic acid and 3-amino- betulinic acid.Fourth, MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay showed that betulinic acid could inhibit the proliferation of ten tomor cell lines, in which the IC50 of KB (human oral epidermoid cancinoma) cells was the least. Besides, the inhibitory rates (IR%) of all derivatives were calculated against KB cell lines.
引文
[1] 欧阳高亮．细胞凋亡与肿瘤的发生发展和治疗．国外医学肿瘤学分册，2000，27(5)：266-269．
[2] 刘景生．细胞信息与调控．现化生物医学丛书．北京医科大学北京协和医科大学联合出版社，1998．
[3] 汪红，王强，余国奠．近十年抗肿瘤中药的研究进展．中国野生植物资源，2000，19(3)：7-10．
[4] 韩锐．抗癌药物研究与实验基础．北京医科大学北京协和医科大学联合出版社，1997．
[5] 王树滨，杨纯正．肿瘤化疗药物诱导细胞凋亡的研究进展．癌症，2000，19(12)：1173-1176．
[6] Sunder Ramadoss et al. Use of betulinic acid and its derivatives for inhibiting cancer growth and a method of monitoring this. US 6048847, 2000. 11.
[7] John M. Pezzuto et al. Method and composition for selectively inhibiting melanoma. US 6225353 B1, 2001.5.
[8] Tapas K. DasGupta, John M. Pezzuto. Method and eompositeon for selectively inhibiting melanoma using betulinic acid. US 5658947, 1997.8.
[9] John M. Pezzuto et al. Method of preparing and use of prodrugs of betulinic acid derivatives. US 2002/0052352A1, 2002.5.
[10] Valentina Zuco, Rosanna Supino, Sabina C. Righetti et al. Selective eytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Letters, 175:17-25, 2002.
[11] Dasgupta et al. Use of betulinie acid and its derivatives for inhibiting malignant melanoma growth and a method of monitoring this. W096/29068, 1996.9.
[12] Toshihiro Fujioka, Yoshiki Kashiwada. Anti-aids, 11. betulinie acid and platonic acid as anti-HIV principles from syzigium claviflorum, and the anti-HIV activeity of structurally related triterpenoids. Journal of Natural Product, 1994, 57(2):243-247.
[13] I-ehen Sun, Jing-Kang, Hui-kang Wang. Anti-aids agents. 32. Synthesis and anti-HIV activity of betulin derivateives. Bioorganic & Medicinal Chemistry Letters, 1998, 8:1267-72.
[14] I-chen Sun, Hui-kang Wang, Yoshiki Kashiwada et al. Anti-aids agents. 34. Synthesis and structure-activity relationships of betulin derivatives as anti-HIV agents. J. Med. Chem., 1998, 41:4648-57.
[15] Taisei Kanamoto, Yoshiki Kashiwada, Akenji Kanbara. Anti-human immuneodefieiency virus activeity of YW-FH312(a betulinic acid derivative) a novel compound blocking viral maturation. Antimicro- bial Agents and Chemotherapy, 2001, 45(4): 1225-30.
[16] Yoshiki Kashiwada, Hui-kang Wang, Tsuneatsu Nagao. Anti-AIDS Agent. 30. Anti-HIV activity of oleanolie acid, pomolic acid, and structurally related triterpenoids. J. Nat. Prod., 1998, 61:1090 -95.
[17] Yoshiki Kashiwada, Junko Chiyo, Yasumasa Ikeshiro. Synthesis and Anti-HIV activity of 3-alkylamino-3-deoxy-betulinic acid derivatives. Chem. Pharm. Bull., 2000, 48(9):1387-90.
[18] Fhmio Hashimoto, Yoshiki Kashiwada, L Mark Cosentino. Anti-aids agents-XXVII. Synthesis and Anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorganic & Medicinal Chemistry, 1997, 5(12):2133-43.
[19] Yoshiki Kashiwada, Fumio Hashimoto, L Mark Cosentino. Betulinic acid and dihydrobetulinie acid deriva-tives as potent anti-HIV agents. J. Med Chem., 1996, 39:1016-7.
[20] Maria Del, CarmanRecio et al. Structural requirements for the anti-inflammatory activity of natural triterpenoids. Planta meal, 1995, 61:182-185.
[21] 陈惠芳．植物活性成分词典．中国医药科技出版社，2000. 10.
[22] S C Pakrashi, J Battacharyya, Sipra Mookerjee. Studies on Indian medieanai plants-ⅩⅧ. The nonaikaioidal constituents from the seeds of alangium lamarckii thw. Phytochemistry, 1968, 7:461-6.
[23] Ake Assi, et al. Betulinic acid:isolation from triphyophyllum peltatum and ancistroeladus. Medica, 1997, 63:255-7.
[24] Wolfgang Schuhly, Jorg Heilmann, Ihsan Calis, et al. New triterpenoids with antibacterial activity from Zizyphus Joazeiro. Planta Medica, 1999, 65:740-3.
[25] Andre Nick, Anthony D Wright, Topul Rali et al. Antibacterial triterpenoids from dillenia papuana and their structure-activity relationship. Phytochemistry, 1995, 40(6): 1691-5.
[26] Toshihiro Fujioka, Yoshiki Kashiwada. Anti-aids, 11. betulinic acid and platonic acid anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids, Journal of Natural Product, 1994, 57(2):243-7.
[27] Paval A, Robert M, Vitaliy V. Methods for manufacturing betulinic acid. WO01/51451A2, 19.07. 2001.
[28] Ruzichka et al. Helv. Chim. Acta., 1938, 21:1706-1715.
[29] Krasutsky, Pavel A. Methods for manufacturing betulinie acid.WO01/51451A2, 2001.7.
[30] pezzuto, John M. Improved methods of manufacturing betulinic acid. WO 98/43963, 1998. 10.
[31] Darrick S H L Kim, Zhidong Chen, Tuyen Nguyen. A concise semisynthetic approach to betulinic acid from betulin. Synthetic Communications, 1997, 27(9): 1607-12.
[32] John M Pezzuto, Darrick S. H. L. Kim. Method of manufacturing betulinic acid. US 5804575, 1998.9.
[33] Darriek S H L Kim, John M. Pezzuto, Emily Pisha. Synthesis of betulinic acid derivatives with activity against human melanoma. Bioorganic &Medicinal Chemistry Letters, 1998, 8:1707-12.
[34] Tong-shuang Li, Jian-xin Wang and Xue-jing Zheng. Sipmle synthesis of allobetulin, 28-oxyallo- betulin and related biomarkers from betulin and betulinic acid catalysed by solid acids, J. Chem. Soc. Perkin Trans., 1998, 1:3957-65.
[35] M Jaggi et al. Antiangiogenic activity of betulinie acid and its derivatives. US 6228850B 1,2001.5.
[36] Hyeh-Jean, Hee-Byung thai, So-Young Park and Dardek S H L Kim. Preparation of amino acid conjugates of betulinic acid with activity against human melanoma. Bioorganic & Medicinal Chemistry Letters, 1999, 9:1201-1204.
[37] Simone Fulda, Irmela Jeremias, Hans H Steiner et al. Betulinic acid :a new cytotoxic against malignant brain-tumor cell. Int. J. Cancer, 1999, 82:435-441.
[38] Yasuhiro Noda, Toyo Kaiya, Kohfuku Kohda et al. Enhanced cytotoxicity of some cultured in low pH media: possibility of a new mode of cell killing. Chem. Pharm. Bull., 1997, 45(10): 1665-70.
[39] P R Wachsberger, R Burd, M L Wahl et al. Betulinic acid sensitization of low pH adapted human melanoma cell to hyperthermia. Int. J. Hyperthermia, 2002, 18(2): 153-64.
[40] Simone Fulda, Irmela Jeremias, Hans H Steiner et al. Betulinie acid:a new cytotoxic agent against malignant brain-tumor cells, Int. d. Cancer, 1999, 82:435-441.
[41] J C P Steele, D C Warhurst, G C Kirby et al. In vitro and in vivo evaluation of betulinie acid as an antimalarial. Phytotherapy Research, 1999, 13:115-9.
[42] Kang-Ju Chou, Hua-chang Fang, Hsiao-min Chung et al. Effect of betulinic acid on intraeellular-free Ca~(2+) levels in madin darby canine kidney cells. European Journal of Phamacology, 2000, 408: 99-106.
[43] Takao Konoshima, Midori Takasaki, Mutsuo Kozuka. Studies on inhibitors of skin-tumor promotion, 1. Inhibitor effects of troterpenes from euptelea on Epstein-Barr virus activation. Journal of Natural Products, 1987, 50(6):1167-70.
[44] Kouzi S A, Chantterjee P, Pezzuto J M et al. Microbial transformations of the antimelanoma agent betulinic acid. J. Nat. Prod., 2000, 63(12):1653-7.
[45] Kouzi S A, Chantterjee P, Pezzuto J M et al. Biotransformation of antimelanoma agent betulinie acid by bacillus megaterium ATCC 13368. Environ Microbiol, 2000, 66(9):3850-5.
[46] Chowdhury A, Mandal S, Mittra B et al. Betulinie acid, a potent inhibitor of eukatyotie topoisomerase Ⅰ: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives. Med. Sci. Monit., 2002, 8(7):254-65.
[47] Berridge M V, Tan A S. Characterization of Cellar Reduction of 3-(4, 5-Dimethyl-2-Thiazyl)-2, 5-Diphenyl-2H-Tetrazolium Bromide (MTT): Subcellar Lo-calization, Substrate Dependence, and Involvement of Mitochondrial Eleetr- on Transport in MTT Reduction. Archives of Biochemistry and Biophysics, 1993, 303(2):474-482.
[48] Joseph Meletiadis, Jacques F G M Meis. Comparison of NCCLS and 3-(4, 5-Dimethyl-2-Thiazyl)-2, 5-Diphenyl-2H-Tetrazolium Bromide (MTT)Methods of In Vitro Susceptibility Testing of Filamen- tous Fungi and Development of a New Simplified Method. Journal of Clinical Microbiology, 2000, 38(8):2949-2954.
[2] 刘景生．细胞信息与调控．现化生物医学丛书．北京医科大学北京协和医科大学联合出版社，1998．
[3] 汪红，王强，余国奠．近十年抗肿瘤中药的研究进展．中国野生植物资源，2000，19(3)：7-10．
[4] 韩锐．抗癌药物研究与实验基础．北京医科大学北京协和医科大学联合出版社，1997．
[5] 王树滨，杨纯正．肿瘤化疗药物诱导细胞凋亡的研究进展．癌症，2000，19(12)：1173-1176．
[6] Sunder Ramadoss et al. Use of betulinic acid and its derivatives for inhibiting cancer growth and a method of monitoring this. US 6048847, 2000. 11.
[7] John M. Pezzuto et al. Method and composition for selectively inhibiting melanoma. US 6225353 B1, 2001.5.
[8] Tapas K. DasGupta, John M. Pezzuto. Method and eompositeon for selectively inhibiting melanoma using betulinic acid. US 5658947, 1997.8.
[9] John M. Pezzuto et al. Method of preparing and use of prodrugs of betulinic acid derivatives. US 2002/0052352A1, 2002.5.
[10] Valentina Zuco, Rosanna Supino, Sabina C. Righetti et al. Selective eytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Letters, 175:17-25, 2002.
[11] Dasgupta et al. Use of betulinie acid and its derivatives for inhibiting malignant melanoma growth and a method of monitoring this. W096/29068, 1996.9.
[12] Toshihiro Fujioka, Yoshiki Kashiwada. Anti-aids, 11. betulinie acid and platonic acid as anti-HIV principles from syzigium claviflorum, and the anti-HIV activeity of structurally related triterpenoids. Journal of Natural Product, 1994, 57(2):243-247.
[13] I-ehen Sun, Jing-Kang, Hui-kang Wang. Anti-aids agents. 32. Synthesis and anti-HIV activity of betulin derivateives. Bioorganic & Medicinal Chemistry Letters, 1998, 8:1267-72.
[14] I-chen Sun, Hui-kang Wang, Yoshiki Kashiwada et al. Anti-aids agents. 34. Synthesis and structure-activity relationships of betulin derivatives as anti-HIV agents. J. Med. Chem., 1998, 41:4648-57.
[15] Taisei Kanamoto, Yoshiki Kashiwada, Akenji Kanbara. Anti-human immuneodefieiency virus activeity of YW-FH312(a betulinic acid derivative) a novel compound blocking viral maturation. Antimicro- bial Agents and Chemotherapy, 2001, 45(4): 1225-30.
[16] Yoshiki Kashiwada, Hui-kang Wang, Tsuneatsu Nagao. Anti-AIDS Agent. 30. Anti-HIV activity of oleanolie acid, pomolic acid, and structurally related triterpenoids. J. Nat. Prod., 1998, 61:1090 -95.
[17] Yoshiki Kashiwada, Junko Chiyo, Yasumasa Ikeshiro. Synthesis and Anti-HIV activity of 3-alkylamino-3-deoxy-betulinic acid derivatives. Chem. Pharm. Bull., 2000, 48(9):1387-90.
[18] Fhmio Hashimoto, Yoshiki Kashiwada, L Mark Cosentino. Anti-aids agents-XXVII. Synthesis and Anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives. Bioorganic & Medicinal Chemistry, 1997, 5(12):2133-43.
[19] Yoshiki Kashiwada, Fumio Hashimoto, L Mark Cosentino. Betulinic acid and dihydrobetulinie acid deriva-tives as potent anti-HIV agents. J. Med Chem., 1996, 39:1016-7.
[20] Maria Del, CarmanRecio et al. Structural requirements for the anti-inflammatory activity of natural triterpenoids. Planta meal, 1995, 61:182-185.
[21] 陈惠芳．植物活性成分词典．中国医药科技出版社，2000. 10.
[22] S C Pakrashi, J Battacharyya, Sipra Mookerjee. Studies on Indian medieanai plants-ⅩⅧ. The nonaikaioidal constituents from the seeds of alangium lamarckii thw. Phytochemistry, 1968, 7:461-6.
[23] Ake Assi, et al. Betulinic acid:isolation from triphyophyllum peltatum and ancistroeladus. Medica, 1997, 63:255-7.
[24] Wolfgang Schuhly, Jorg Heilmann, Ihsan Calis, et al. New triterpenoids with antibacterial activity from Zizyphus Joazeiro. Planta Medica, 1999, 65:740-3.
[25] Andre Nick, Anthony D Wright, Topul Rali et al. Antibacterial triterpenoids from dillenia papuana and their structure-activity relationship. Phytochemistry, 1995, 40(6): 1691-5.
[26] Toshihiro Fujioka, Yoshiki Kashiwada. Anti-aids, 11. betulinic acid and platonic acid anti-HIV principles from syzigium claviflorum and the anti-HIV activity of structurally related triterpenoids, Journal of Natural Product, 1994, 57(2):243-7.
[27] Paval A, Robert M, Vitaliy V. Methods for manufacturing betulinic acid. WO01/51451A2, 19.07. 2001.
[28] Ruzichka et al. Helv. Chim. Acta., 1938, 21:1706-1715.
[29] Krasutsky, Pavel A. Methods for manufacturing betulinie acid.WO01/51451A2, 2001.7.
[30] pezzuto, John M. Improved methods of manufacturing betulinic acid. WO 98/43963, 1998. 10.
[31] Darrick S H L Kim, Zhidong Chen, Tuyen Nguyen. A concise semisynthetic approach to betulinic acid from betulin. Synthetic Communications, 1997, 27(9): 1607-12.
[32] John M Pezzuto, Darrick S. H. L. Kim. Method of manufacturing betulinic acid. US 5804575, 1998.9.
[33] Darriek S H L Kim, John M. Pezzuto, Emily Pisha. Synthesis of betulinic acid derivatives with activity against human melanoma. Bioorganic &Medicinal Chemistry Letters, 1998, 8:1707-12.
[34] Tong-shuang Li, Jian-xin Wang and Xue-jing Zheng. Sipmle synthesis of allobetulin, 28-oxyallo- betulin and related biomarkers from betulin and betulinic acid catalysed by solid acids, J. Chem. Soc. Perkin Trans., 1998, 1:3957-65.
[35] M Jaggi et al. Antiangiogenic activity of betulinie acid and its derivatives. US 6228850B 1,2001.5.
[36] Hyeh-Jean, Hee-Byung thai, So-Young Park and Dardek S H L Kim. Preparation of amino acid conjugates of betulinic acid with activity against human melanoma. Bioorganic & Medicinal Chemistry Letters, 1999, 9:1201-1204.
[37] Simone Fulda, Irmela Jeremias, Hans H Steiner et al. Betulinic acid :a new cytotoxic against malignant brain-tumor cell. Int. J. Cancer, 1999, 82:435-441.
[38] Yasuhiro Noda, Toyo Kaiya, Kohfuku Kohda et al. Enhanced cytotoxicity of some cultured in low pH media: possibility of a new mode of cell killing. Chem. Pharm. Bull., 1997, 45(10): 1665-70.
[39] P R Wachsberger, R Burd, M L Wahl et al. Betulinic acid sensitization of low pH adapted human melanoma cell to hyperthermia. Int. J. Hyperthermia, 2002, 18(2): 153-64.
[40] Simone Fulda, Irmela Jeremias, Hans H Steiner et al. Betulinie acid:a new cytotoxic agent against malignant brain-tumor cells, Int. d. Cancer, 1999, 82:435-441.
[41] J C P Steele, D C Warhurst, G C Kirby et al. In vitro and in vivo evaluation of betulinie acid as an antimalarial. Phytotherapy Research, 1999, 13:115-9.
[42] Kang-Ju Chou, Hua-chang Fang, Hsiao-min Chung et al. Effect of betulinic acid on intraeellular-free Ca~(2+) levels in madin darby canine kidney cells. European Journal of Phamacology, 2000, 408: 99-106.
[43] Takao Konoshima, Midori Takasaki, Mutsuo Kozuka. Studies on inhibitors of skin-tumor promotion, 1. Inhibitor effects of troterpenes from euptelea on Epstein-Barr virus activation. Journal of Natural Products, 1987, 50(6):1167-70.
[44] Kouzi S A, Chantterjee P, Pezzuto J M et al. Microbial transformations of the antimelanoma agent betulinic acid. J. Nat. Prod., 2000, 63(12):1653-7.
[45] Kouzi S A, Chantterjee P, Pezzuto J M et al. Biotransformation of antimelanoma agent betulinie acid by bacillus megaterium ATCC 13368. Environ Microbiol, 2000, 66(9):3850-5.
[46] Chowdhury A, Mandal S, Mittra B et al. Betulinie acid, a potent inhibitor of eukatyotie topoisomerase Ⅰ: identification of the inhibitory step, the major functional group responsible and development of more potent derivatives. Med. Sci. Monit., 2002, 8(7):254-65.
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