长春碱多步修饰产物的分离与制备技术研究
|
摘要
复杂天然产物单体分离纯化以及工业化制备技术,是目前制约我国植物源天然产物单体制备产业化的瓶颈性问题,而将多种分离纯化方法有机组合是实现高纯度单体工业化制备的关键技术之一。本文以结构复杂而具高生物活性的长春碱的化学半合成产物——长春瑞滨终产物及重要中间体的分离纯化工艺研究为例,探讨其规模化制备中的分离纯化技术,并为其它天然产物单体工业化制备的创新技术研究提供技术和方法上的参考。整个研究主要包括以下内容:
1.中间体脱水长春碱的分离纯化。在系统的研究结晶法和硅胶柱层析法分离纯化脱水长春碱的工艺后,通过对比,最终确定了结晶法为适合推向生产的纯化脱水长春碱的适合的工艺方法,并确定了具体的工艺参数。实验表明:纯度为30%左右的脱水长春碱经过一次结晶后纯度可以达到65%,结晶得率为70%左右。
2.从缩环水解液中初步分离长春瑞滨。采用Sephadex LH-20柱层析和结晶法,从缩环水解液中初步分离长春瑞滨。结果显示虽然Sephadex LH-20柱层析有较高的固体回收率,但长春瑞滨与杂质未有效分离,最终确定用结晶法从缩环水解液中初步纯化长春瑞滨,该方法简便易行,可使瑞滨含量从30%提高到55%左右,适于工业化放大。
3.通过中度分离与精细分离两个阶段进一步纯化与精制长春瑞滨。使用自填的硅胶干柱,直接套用薄层层析的溶剂展开系统,可使长春瑞滨纯度从55%提高到80%以上,硅胶的负载量为12 mg样品∕g硅胶,干柱的最佳径∕高比为1∕10,产品回收率在82%;利用MCI-GEL CHP-20填料进一步对难分离的长春瑞滨结构类似物进行反相分离,确定了反相分离的最佳条件为甲醇-水(V :V =80:20)流动相常压洗脱,反相柱的载样量为10 mg样品∕g填料,经过一次MCI-GEL CHP-20柱色谱纯化再结晶后,长春瑞滨纯度可达98%以上,该反相填料能反复利用,反相柱目标物收率达97%以上。表明采取这种正相硅胶干柱与反相MCI-GEL CHP-20柱色谱相组合的新方法,大大缩短了操作时间,提高了产品回收率,可快速高效的分离纯化长春瑞滨粗产品。
本课题研究了生产长春瑞滨的高效分离与制备技术,确立了一套适合于产业化的产品分离与纯化方法。并为其它复杂天然产物单体的工业化制备提供了方法和技术参考。
Technology in mass purification and industrialized production of complex natural resources monomer is bottleneck problem, which restricts the industrialization of activated natural resources from plants. Combination of different effective technologies is one of key technologies to produce complex natural resources monomer with high purity .This paper took Vinorelbine and its intermediate Anhydrovinblastine as example, which were semi-synthesized from vinblastine with complex and activated structure, studying technology about its mass purification and prudction, to provide academic and practical reference for mass purification and industrialized production of other natural resources. Here are the main studying content:
1. Purification and separation of intermediate product Anhydrovinblastine. Comparison of different purification techniques for the Anhydrovinblastine was presented. Crystallography was selected out as the best method used to purify anhydrovinblastine in industry. Results showed that the purification of crud Anhydrovinblastine can get from 30% to 65% after the process of crystallization with the yield of 70%.
2. Preliminary separating Vinorelbine from liquid hydrolysate. Sephadex LH-20 chromatography and crystallography were taken to preliminary separate Vinorelbine in liquid hydrolysate. Results indicated that the reclaim rate of solid matter is high ,but Sephadex LH-20 chromatography could not separate Vinorelbine from impurities effectively. At last, crystallography was selected out as the best method used to purify anhydrovinblastine in liquid hydrolysate. It can heighten the content of Vinorelbine from 30% to 50%. This process is simple and convenient, and suitable to be magnified in industrialization.
3. Further purification of Vinorelbine, which include middling purification and high purification two steps. Normal dry-column manufactured by our-self was used to purify Vinorelbine with developer the same to TLC. After purified by dry-column flash chromatography, the purity of vinorelbine can get to 80% from 55% at the very beginning. The load of dry-column was 12 mg specimen per 1 g silica gel, the best diameter/tallness was 1/10, and the yield of vinorelbine can get to 82%. MCI-GEL CHP-20 reversed-phase chromatography was used in tandem to separate vinorelbine from impurities which were difficult to be separated. The best purification conditions of crud vinorelbine were methanol/water(V:V=80:20), The load of reversed-phase column was 10 mg specimen per 1 g MCI-GEL CHP-20 stuffing. The purity of vinorelbine can get to 98% when MCI-GEL CHP-20 reversed-phase chromatography and crystallization in sequence were used.
Technique for high efficient purification of Vinorelbine was investigated in this thesis. A series of feasible process conditions for Purification and separation of Vinorelbine and Anhydrovinblastine were obtained, which are suitable to be used in synthesis of vinorelbine in industry. It gives both academic and practical reference for mass production and purification of other natural resources monomer.
1.中间体脱水长春碱的分离纯化。在系统的研究结晶法和硅胶柱层析法分离纯化脱水长春碱的工艺后,通过对比,最终确定了结晶法为适合推向生产的纯化脱水长春碱的适合的工艺方法,并确定了具体的工艺参数。实验表明:纯度为30%左右的脱水长春碱经过一次结晶后纯度可以达到65%,结晶得率为70%左右。
2.从缩环水解液中初步分离长春瑞滨。采用Sephadex LH-20柱层析和结晶法,从缩环水解液中初步分离长春瑞滨。结果显示虽然Sephadex LH-20柱层析有较高的固体回收率,但长春瑞滨与杂质未有效分离,最终确定用结晶法从缩环水解液中初步纯化长春瑞滨,该方法简便易行,可使瑞滨含量从30%提高到55%左右,适于工业化放大。
3.通过中度分离与精细分离两个阶段进一步纯化与精制长春瑞滨。使用自填的硅胶干柱,直接套用薄层层析的溶剂展开系统,可使长春瑞滨纯度从55%提高到80%以上,硅胶的负载量为12 mg样品∕g硅胶,干柱的最佳径∕高比为1∕10,产品回收率在82%;利用MCI-GEL CHP-20填料进一步对难分离的长春瑞滨结构类似物进行反相分离,确定了反相分离的最佳条件为甲醇-水(V :V =80:20)流动相常压洗脱,反相柱的载样量为10 mg样品∕g填料,经过一次MCI-GEL CHP-20柱色谱纯化再结晶后,长春瑞滨纯度可达98%以上,该反相填料能反复利用,反相柱目标物收率达97%以上。表明采取这种正相硅胶干柱与反相MCI-GEL CHP-20柱色谱相组合的新方法,大大缩短了操作时间,提高了产品回收率,可快速高效的分离纯化长春瑞滨粗产品。
本课题研究了生产长春瑞滨的高效分离与制备技术,确立了一套适合于产业化的产品分离与纯化方法。并为其它复杂天然产物单体的工业化制备提供了方法和技术参考。
Technology in mass purification and industrialized production of complex natural resources monomer is bottleneck problem, which restricts the industrialization of activated natural resources from plants. Combination of different effective technologies is one of key technologies to produce complex natural resources monomer with high purity .This paper took Vinorelbine and its intermediate Anhydrovinblastine as example, which were semi-synthesized from vinblastine with complex and activated structure, studying technology about its mass purification and prudction, to provide academic and practical reference for mass purification and industrialized production of other natural resources. Here are the main studying content:
1. Purification and separation of intermediate product Anhydrovinblastine. Comparison of different purification techniques for the Anhydrovinblastine was presented. Crystallography was selected out as the best method used to purify anhydrovinblastine in industry. Results showed that the purification of crud Anhydrovinblastine can get from 30% to 65% after the process of crystallization with the yield of 70%.
2. Preliminary separating Vinorelbine from liquid hydrolysate. Sephadex LH-20 chromatography and crystallography were taken to preliminary separate Vinorelbine in liquid hydrolysate. Results indicated that the reclaim rate of solid matter is high ,but Sephadex LH-20 chromatography could not separate Vinorelbine from impurities effectively. At last, crystallography was selected out as the best method used to purify anhydrovinblastine in liquid hydrolysate. It can heighten the content of Vinorelbine from 30% to 50%. This process is simple and convenient, and suitable to be magnified in industrialization.
3. Further purification of Vinorelbine, which include middling purification and high purification two steps. Normal dry-column manufactured by our-self was used to purify Vinorelbine with developer the same to TLC. After purified by dry-column flash chromatography, the purity of vinorelbine can get to 80% from 55% at the very beginning. The load of dry-column was 12 mg specimen per 1 g silica gel, the best diameter/tallness was 1/10, and the yield of vinorelbine can get to 82%. MCI-GEL CHP-20 reversed-phase chromatography was used in tandem to separate vinorelbine from impurities which were difficult to be separated. The best purification conditions of crud vinorelbine were methanol/water(V:V=80:20), The load of reversed-phase column was 10 mg specimen per 1 g MCI-GEL CHP-20 stuffing. The purity of vinorelbine can get to 98% when MCI-GEL CHP-20 reversed-phase chromatography and crystallization in sequence were used.
Technique for high efficient purification of Vinorelbine was investigated in this thesis. A series of feasible process conditions for Purification and separation of Vinorelbine and Anhydrovinblastine were obtained, which are suitable to be used in synthesis of vinorelbine in industry. It gives both academic and practical reference for mass production and purification of other natural resources monomer.
引文
[1] Sunsannw G.. Ralf T. Bioactive agents from source: trends in discovery and application[J]. Advance in Biochemical Engineering, 1999, 6(4): 105-154
[2]潘启超.抗癌药研究进展[J].中国新药杂志,1994,3(3):15
[3]郝小江,沈月毛.我国未来的天然药物研究--药学科学前沿与发展方向[M].北京:中国医药科技出版社,2000.85-90
[4] Pacli. Taxol for ovarian cancer[J]. The Medical, 1993, 35(896):39
[5]徐任生,陈仲良.中草药有效成分提取与分离[M].上海:上海科学技术出版社,1983.280-283
[6]谭天伟.天然产物分离新技术[J].化工进展,2003,22(7):665-667
[7]冯艳菊,王林,郭亚军,等.天然产物特殊分离技术的研究进展[J].应用化工,2006,35(7):545-548
[8]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用[J].中药材,2005,8(23):239-241
[9]魏云,曹学丽.值得关注的分离科学技术--逆流色谱技术[J].现代科学技术--中药现代化,2001,3(5):17-18
[10]果德安,陈道峰,邱峰.我国中药基础研究现状与未来应重点研究的领域--药学科学前沿与发展方向[M].北京:中国医药科技出版社,2000.117-123
[11] Uffelie O F. Alkaloids with tumor inhibiting action from catharanthus roseus [J]. Pharm Weekbl, 1969, 104(16): 321-330
[12]丁亚芳,包永明,安利佳.长春碱类抗肿瘤药物的研究进展[J].中国医药工业杂志,2005,36(7):425-428
[13] Fahy J.Modifications in the upper or velbenamine part of the vinca alkaloids have major implications for tubulin interacting ctivities [J]. Curr Pharm Des. 2001. 7(13): 1181-l197
[14] Chabner B A, Calabresi P. Goodman and Gilman's the Pharmacological Basis ofTherapeutics [J]. New York: McGraw-Hill Publishing Co.1996: 1225-1289
[15] GoodboA E, Watson C D, Misawa M. Extraction of alkaloids of Catharanthus roseus tissue [P]. US, 4831133, 1989.
[16] Cullinan G J, Gerzon K. Derivatives of 4-desacetyl vinblastine C-3 carboxyhydrazide [P]. US, 4166810, 1979. (CA, 92: 76761, 1982.)
[17] Ades E W,Cullina G J. Cytotoxic compositions of transferring coupled to vinca alkaloids [P]. US, 4522750, 1985. (CA, 104: 15079, 1986.)
[18] Verdier-Pinard P, Gares M, Wright M.Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals [J]. Biochem Pharmacol, 1999, 58(6): 959-971
[19] Zhou X J, Ranmani R. Preclinical pharmacology of vinca alkaloids [J]. Drugs, 1992, 44(4): 1-16
[20] Hill B. Vinflunine a second generation novel vinca alkaloid with a distinctive pharmacological profile now in clinical development and prospects for future mitotic blockers[J]. Curr Pharm Des, 2001, 7(13): l199-1212
[21] Lobert S, Vulevic B, Correia J J. Interaction ofvinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine [J]. Biochemistry, 1996, 35(21): 6806-6814
[22] Goodbody A E, Tsuyoshi E,Vukovic J, et a1. Enzymatic preparation of 3’,4’-anhydrovinblastine [P]. US, 4918011, 1990; JP, 63119690, 1988.
[23] Goodbody A E,Vukovic J.Production of alkaloid dimers using ferric ion [P]. US, 4778885. 1988.
[24]陈永江,陈洪明,李莉等.酒石酸长春瑞宾的合成[J].中国医药工业杂志,1999,30(1):6-8
[25] Kutney J P, Choi L S, Nakano J, et a1. Process of synthesis of 3’,4'-anhydrovinblastine. Vinblastine and vincristine [P]. US, 5047528, 1991. (CA, 116: 129354, 1992.)
[26] Endo T. Vinblastine synthesis [P]. US, 5034320, 199l; JP, 63258590, 1988. (CA,111: 113750, 1989.)
[27] Kuboyama T, Yokoshima S, Tokuyama H, et a1. Stereocontrolled total synthesis of (+) -vincristine [J]. Proceedings of the National Academy of Sciences of the USA, 2004, 101(33): l1966-11970
[28] Paschal G C. Thompson G L. Vindesine synthesis [P]. US, 4210584, 1980. (CA, 94:15949, 1981.)
[29] Van Heugen J C, De Graeve J, Zorza G, et al. New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urin and faeces [J]. Journal of Chromatography, 2001, 926(A): 11-20
[30]金美春.酒石酸长春瑞滨相关杂质研究:[硕士学位论文].保存地点:浙江大学图书馆,2006.
[31]李晓蕾,任其龙,杨亦文,等.抗肿瘤药长春碱的提取纯化与分析方法研究概况[J].中国医药工业杂志,2004,25(4):247-250
[32]丁贤儒,倪坤仪,曹海,等.长春花中长春碱含量测定方法的研究[J].中国药科大学报,1995,26(3): 157-159
[33]黄宗玉,龚青,唐湘江.长春碱类药物的反相高效液相色谱法测定[J].中草药,28(4):185-186
[34] Potier P, Mangeney P, Langlois N, et al. Process for the synthesis of vinblastine and leurosidine [P]. US, 4305875, 1981.
[35]祖元刚.一种长春碱和长春新碱的纯化方法[P].CN,1724539A,2006.
[36]祖元刚.一种连续中压柱层析制备高纯度硫酸长春碱的方法[P].CN,1724538A,2006.
[37]李晓蕾.抗肿瘤药长春碱的提取分离工艺研究:[硕士学位论文].保存地点:浙江大学图书馆,2004.
[38]罗猛.长春碱高效提取纯化的工艺研究:[硕士学位论文].保存地点:东北林业大学图书馆,2006.
[39]胥彬.抗癌症药物研究的一些新进展[J].癌症,1990,9(3):226-228
[40]罗猛,付玉杰,祖元刚,等.反相高效液相色谱法快速测定长春花中4种生物碱[J].分析化学,2005,33(1):87-89
[41]朱丽,徐为公,赵广荣.注射用重酒石酸长春瑞滨稳定性研究[J].中国药业,2006,15(10):9-10
[42] Potier P. Synthesis of the antitumor dimeric indole alkaloids from Catharanthus species [J]. Journal of Natural Products, 1980, 43(1): 72-87
[43]赵维民,张天佑.制备色谱技术——在天然产物分离中的应用[M].北京:科学出版社,2000.100-105
[44]袁珂,吕洁丽,殷明文.海南含羞草中黄酮碳苷类化学成分的研究[J].药学学报,2006,41(5):435-438
[45]王慧春,张成总.干柱色谱法分离大黄酚和大黄素甲醚[J].青岛大学学报,2006,24(1):60-61
[46]秦箐.快速低压干柱柱色谱技术的改进及其在刺苋分离提纯中的应用[J].蛇志,2000,12(2):76-77
[47]杨凌,何克江,杨义,等.一种组合式色谱干柱[P].CN,1552500,2004.
[2]潘启超.抗癌药研究进展[J].中国新药杂志,1994,3(3):15
[3]郝小江,沈月毛.我国未来的天然药物研究--药学科学前沿与发展方向[M].北京:中国医药科技出版社,2000.85-90
[4] Pacli. Taxol for ovarian cancer[J]. The Medical, 1993, 35(896):39
[5]徐任生,陈仲良.中草药有效成分提取与分离[M].上海:上海科学技术出版社,1983.280-283
[6]谭天伟.天然产物分离新技术[J].化工进展,2003,22(7):665-667
[7]冯艳菊,王林,郭亚军,等.天然产物特殊分离技术的研究进展[J].应用化工,2006,35(7):545-548
[8]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用[J].中药材,2005,8(23):239-241
[9]魏云,曹学丽.值得关注的分离科学技术--逆流色谱技术[J].现代科学技术--中药现代化,2001,3(5):17-18
[10]果德安,陈道峰,邱峰.我国中药基础研究现状与未来应重点研究的领域--药学科学前沿与发展方向[M].北京:中国医药科技出版社,2000.117-123
[11] Uffelie O F. Alkaloids with tumor inhibiting action from catharanthus roseus [J]. Pharm Weekbl, 1969, 104(16): 321-330
[12]丁亚芳,包永明,安利佳.长春碱类抗肿瘤药物的研究进展[J].中国医药工业杂志,2005,36(7):425-428
[13] Fahy J.Modifications in the upper or velbenamine part of the vinca alkaloids have major implications for tubulin interacting ctivities [J]. Curr Pharm Des. 2001. 7(13): 1181-l197
[14] Chabner B A, Calabresi P. Goodman and Gilman's the Pharmacological Basis ofTherapeutics [J]. New York: McGraw-Hill Publishing Co.1996: 1225-1289
[15] GoodboA E, Watson C D, Misawa M. Extraction of alkaloids of Catharanthus roseus tissue [P]. US, 4831133, 1989.
[16] Cullinan G J, Gerzon K. Derivatives of 4-desacetyl vinblastine C-3 carboxyhydrazide [P]. US, 4166810, 1979. (CA, 92: 76761, 1982.)
[17] Ades E W,Cullina G J. Cytotoxic compositions of transferring coupled to vinca alkaloids [P]. US, 4522750, 1985. (CA, 104: 15079, 1986.)
[18] Verdier-Pinard P, Gares M, Wright M.Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals [J]. Biochem Pharmacol, 1999, 58(6): 959-971
[19] Zhou X J, Ranmani R. Preclinical pharmacology of vinca alkaloids [J]. Drugs, 1992, 44(4): 1-16
[20] Hill B. Vinflunine a second generation novel vinca alkaloid with a distinctive pharmacological profile now in clinical development and prospects for future mitotic blockers[J]. Curr Pharm Des, 2001, 7(13): l199-1212
[21] Lobert S, Vulevic B, Correia J J. Interaction ofvinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine [J]. Biochemistry, 1996, 35(21): 6806-6814
[22] Goodbody A E, Tsuyoshi E,Vukovic J, et a1. Enzymatic preparation of 3’,4’-anhydrovinblastine [P]. US, 4918011, 1990; JP, 63119690, 1988.
[23] Goodbody A E,Vukovic J.Production of alkaloid dimers using ferric ion [P]. US, 4778885. 1988.
[24]陈永江,陈洪明,李莉等.酒石酸长春瑞宾的合成[J].中国医药工业杂志,1999,30(1):6-8
[25] Kutney J P, Choi L S, Nakano J, et a1. Process of synthesis of 3’,4'-anhydrovinblastine. Vinblastine and vincristine [P]. US, 5047528, 1991. (CA, 116: 129354, 1992.)
[26] Endo T. Vinblastine synthesis [P]. US, 5034320, 199l; JP, 63258590, 1988. (CA,111: 113750, 1989.)
[27] Kuboyama T, Yokoshima S, Tokuyama H, et a1. Stereocontrolled total synthesis of (+) -vincristine [J]. Proceedings of the National Academy of Sciences of the USA, 2004, 101(33): l1966-11970
[28] Paschal G C. Thompson G L. Vindesine synthesis [P]. US, 4210584, 1980. (CA, 94:15949, 1981.)
[29] Van Heugen J C, De Graeve J, Zorza G, et al. New sensitive liquid chromatography method coupled with tandem mass spectrometric detection for the clinical analysis of vinorelbine and its metabolites in blood, plasma, urin and faeces [J]. Journal of Chromatography, 2001, 926(A): 11-20
[30]金美春.酒石酸长春瑞滨相关杂质研究:[硕士学位论文].保存地点:浙江大学图书馆,2006.
[31]李晓蕾,任其龙,杨亦文,等.抗肿瘤药长春碱的提取纯化与分析方法研究概况[J].中国医药工业杂志,2004,25(4):247-250
[32]丁贤儒,倪坤仪,曹海,等.长春花中长春碱含量测定方法的研究[J].中国药科大学报,1995,26(3): 157-159
[33]黄宗玉,龚青,唐湘江.长春碱类药物的反相高效液相色谱法测定[J].中草药,28(4):185-186
[34] Potier P, Mangeney P, Langlois N, et al. Process for the synthesis of vinblastine and leurosidine [P]. US, 4305875, 1981.
[35]祖元刚.一种长春碱和长春新碱的纯化方法[P].CN,1724539A,2006.
[36]祖元刚.一种连续中压柱层析制备高纯度硫酸长春碱的方法[P].CN,1724538A,2006.
[37]李晓蕾.抗肿瘤药长春碱的提取分离工艺研究:[硕士学位论文].保存地点:浙江大学图书馆,2004.
[38]罗猛.长春碱高效提取纯化的工艺研究:[硕士学位论文].保存地点:东北林业大学图书馆,2006.
[39]胥彬.抗癌症药物研究的一些新进展[J].癌症,1990,9(3):226-228
[40]罗猛,付玉杰,祖元刚,等.反相高效液相色谱法快速测定长春花中4种生物碱[J].分析化学,2005,33(1):87-89
[41]朱丽,徐为公,赵广荣.注射用重酒石酸长春瑞滨稳定性研究[J].中国药业,2006,15(10):9-10
[42] Potier P. Synthesis of the antitumor dimeric indole alkaloids from Catharanthus species [J]. Journal of Natural Products, 1980, 43(1): 72-87
[43]赵维民,张天佑.制备色谱技术——在天然产物分离中的应用[M].北京:科学出版社,2000.100-105
[44]袁珂,吕洁丽,殷明文.海南含羞草中黄酮碳苷类化学成分的研究[J].药学学报,2006,41(5):435-438
[45]王慧春,张成总.干柱色谱法分离大黄酚和大黄素甲醚[J].青岛大学学报,2006,24(1):60-61
[46]秦箐.快速低压干柱柱色谱技术的改进及其在刺苋分离提纯中的应用[J].蛇志,2000,12(2):76-77
[47]杨凌,何克江,杨义,等.一种组合式色谱干柱[P].CN,1552500,2004.