具有抗癌活性天然产物methyl protodioscin的全合成
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摘要
呋甾皂苷，长期以来被认为是植物体内螺甾皂苷的合成前体，其生物活性一直不为人们所认识。近年来由于对其生物活性和药理活性认识的逐步加深，呋甾皂苷越来越受到普遍关注。但是由于呋甾皂苷分子的复杂性、不稳定性以及作用机制还不很清楚，国内外迄今对其进行化学合成的报道很少。
Methyl protodioscin(1)是一种呋甾皂苷，化学名为：3-O-[α-L-鼠李糖-(1→2){α-L-鼠李糖-(1→4)}-β-D-葡萄糖]-26-O-[β-D-葡萄糖]-22-甲氧基-25(R)-呋甾-5-烯-3β，26-二醇。1974年Kawasaki等首次从新采集的薯蓣属植物Dioscorea gracillima MiQ的根中分离得到并鉴定了它的结构。长期以来，姚新生教授等一直致力于从传统中草药中寻找活性成分的研究，他们采取活性追踪分离方法，从粉荜解中同样分离得到了methyl
protodioscin并经NCI用60种人癌症细胞株筛选其体外抗癌活性。在所筛选的14个甾体皂苷化合物中，6个化合物具有较好的体外活性，其中methyl protodioscin活性最好。为了获得足够数量的样品进行进一步的生物活性研究，立项对其进行化学合成。
根据逆向合成分析，methyl protodioscin理论上可以分成三个部分：苷元部分，26-位葡萄糖部分以及3-位三糖部分。在分析苷元结构特点的基础上，我们设计了合成苷元的两条路线并进行了系统地研究。最后，利用路线2的方法，我们以薯蓣皂苷元为起始原料，经三步反应得到目标物，三步反应总收率可达到49％。即：用TBDPSCl保护薯蓣皂苷元3-位羟基、然后用DMDO选择性氧化5,6-位双键和16-位碳，所得产物不需分离纯化即可直接用于下步还原开环和乙酰化反应。整个路线步骤短，操作简单，是合成呋甾皂苷元的较理想的方法。
结构上，三糖组成非常独特，由两分子鼠李糖分别连在葡萄糖的2-和4-位构成。为此我们设计了三条合成路线并进行了摸索，最终以路线3的方法合成了三糖活性前体。与路线1和2相比，路线3巧妙地利用了葡萄糖各个羟基活性的差别以及特戊酰基具有较大空间位阻的特点，具有设计更加合理，路线短，操作简便的优点。
然后，我们用TMSOTf将葡萄糖三氯乙酰亚胺酯与苷元的26-位羟基成功相连，用N-碘代丁二酰亚胺(NIS)和催化量三氟甲磺酸(TfOH)将三糖活性前体与苷元的3-位羟基相连，所得产物再用硼氢化钠选择性还原其C_(16)-羰基成羟基并自动与C_(22)-羰基环合成半缩酮结构化合物，最后再用甲醇钠的甲醇溶液脱去糖上的全部苯甲酰基和
沈阳药科大学博士学位论文 天然产物Methyl Protodioscin的全合成及抗癌活性研究
一
特戊酚基保护基，同时 C。。－羟基甲基化成功得到陕凿皂苦 methy Protodioscin。
这样，通过两年多的努力，最终确定了以市场上易得的薯颓皂昔元为起始原料，
经过主要9步反应合成目标物的方法，总收率达到7．8％。反应中所得到的中间产物均
经过’H－N’MR、”C－N’MR和 ESIMS鉴定，并且固体产物还测定了其熔点和旋光值。所
合成的最终产物的理化性质及光谱数据等与天然得到的样品基本一致，如：L巳、
’H－N’MR、uC－N’MR、DEPT、ES恤S、熔点和旋光值。
总之，首次成功地实现了mCthy一fot。dbSCh的全合成，为 mCthyl protod1OSClll类
似物的合成研究铺平了道路。
Furostan saponins, usually regarded as the synthetic precursors of the corresponding spirostan saponins in plants, have recently been paid emphasis attention due to the increased understanding of their broad range of biological and pharmacological activities. However, there is few report about their synthesis until very recently because of their complexity, instability and non-clear functional mechanism.
Methyl protodioscin 1, is a member of furostan saponin family with the chemical name: 3-(9-[a-Z-rhamnopyranosyl-(1→2)-{a-Z,-rhamnopyranosyl -(1→4)}-β-Z)-glucopyranosyl] -26-O-[β-Z)-glucopyranosyl]-22-methoxy-25(R)-furost-5-ene-3→,26-diol. This molecule was first isolated and identified by Kawasaki and co-worker in 1974 from fresh rhizomes of Dioscorea gracillima MiQ. In continuing efforts to identify active components from traditional Chinese herbal medicine, professor Yao and co-worker also obtained this compound from the rhizome of Dioscoreaceae by repeated bioactivity-guided isolation and tested its anticancer activities in vitro with a panel of 60 human cancer cell lines performed in NCI. Among the fourteen steroidal saponins tested, 6 compounds gave good activities in vitro and methyl protodioscin showed the most potent activity. In order to get a sufficient amount of the compound for further biological investigations, we therefore initiated a program to synthesize this target molecule.
Retro synthetically, methyl protodioscin can be logically disconnected into three distinct fragments: aglycone, C26-glucopyranose and Cs-trisaccharide moiety. Based on the analysis of its structural character, two routes were designed and systemically studied to synthesize the aglycone moiety. Finally, we chose route 2 for our purpose and the title compound was readily obtained in an overall yield of 49% via three steps by using diosgenin as the starting material: that is, protecting the Ca- hydroxyl group in diosgenin with TBDPSC1, then selectively oxidizing with DMDO at both the double bond and spiroketal, and then without further purification, directly undergoing reductive cleavage and acetolysis in one step in very mild condition. Therefore, this short and simple route was an ideal method to synthesize aglycone.
Structurally, the constitution of the trisaccharide is quite unique, the sequential linkage of a-L-rhamnopyranosyl-(1→)-a-i-rhamnopyranosyl-(1→4)-bata-D-glucopyranoside led us to design three routes to synthesize it. All these three routes were studied and the title compound was obtained by methods in route 3. Compared to route 1 and 2, route 3 possessed advantages of reasonable design, short steps and simply operation because it skillfully utilized the different activity of the hydroxyl groups in glucose and the characteristic of the relatively large stereo barrier of pivaloyl chloride.
Afterwards, we successfully connected glucopyranosyl trichloroacetimidate to the C26-hydroxyl of the aglycone in the present of TMSOTf and coupled trisaccharide moiety to the C3-hydroxyl with TV-iodosuccinimide (NIS) and a catalytic amount of trifluoromethanesulfonic acid (TfOH). Selective reduction the 16-ketone of the resulting cholestan-16, 22-dioxo with NaBHU in i-PrOH was achieved, and the newly generated secondary alcohol concurrently cyclized to give the hemiketal compound. The final target furostan saponin 1 was eventually obtained by deprotection of benzoyl and pivaloyl groups, and in situ methoxylation at C22 position using NaOMe in MeOH solution.
Thus, a simple method to methyl protodioscin was established after two years effort, which starting from commercially available diosgenin through mainly 9 steps in an overall yield of 7.8%. All the intermediates were identified correctly by 'H-NMR, 13C-NMR and ESI-MS and the solid-state materials were also measured melting point and optical rotation. The analytical data for the synthesized methyl protodioscin were identical in all respects to those reported for the natural material such as IR, 'H-NMR, 13C-NMR, DEPT, ESI-MS, mp and optical rotation.
In conclu
Methyl protodioscin(1)是一种呋甾皂苷，化学名为：3-O-[α-L-鼠李糖-(1→2){α-L-鼠李糖-(1→4)}-β-D-葡萄糖]-26-O-[β-D-葡萄糖]-22-甲氧基-25(R)-呋甾-5-烯-3β，26-二醇。1974年Kawasaki等首次从新采集的薯蓣属植物Dioscorea gracillima MiQ的根中分离得到并鉴定了它的结构。长期以来，姚新生教授等一直致力于从传统中草药中寻找活性成分的研究，他们采取活性追踪分离方法，从粉荜解中同样分离得到了methyl
protodioscin并经NCI用60种人癌症细胞株筛选其体外抗癌活性。在所筛选的14个甾体皂苷化合物中，6个化合物具有较好的体外活性，其中methyl protodioscin活性最好。为了获得足够数量的样品进行进一步的生物活性研究，立项对其进行化学合成。
根据逆向合成分析，methyl protodioscin理论上可以分成三个部分：苷元部分，26-位葡萄糖部分以及3-位三糖部分。在分析苷元结构特点的基础上，我们设计了合成苷元的两条路线并进行了系统地研究。最后，利用路线2的方法，我们以薯蓣皂苷元为起始原料，经三步反应得到目标物，三步反应总收率可达到49％。即：用TBDPSCl保护薯蓣皂苷元3-位羟基、然后用DMDO选择性氧化5,6-位双键和16-位碳，所得产物不需分离纯化即可直接用于下步还原开环和乙酰化反应。整个路线步骤短，操作简单，是合成呋甾皂苷元的较理想的方法。
结构上，三糖组成非常独特，由两分子鼠李糖分别连在葡萄糖的2-和4-位构成。为此我们设计了三条合成路线并进行了摸索，最终以路线3的方法合成了三糖活性前体。与路线1和2相比，路线3巧妙地利用了葡萄糖各个羟基活性的差别以及特戊酰基具有较大空间位阻的特点，具有设计更加合理，路线短，操作简便的优点。
然后，我们用TMSOTf将葡萄糖三氯乙酰亚胺酯与苷元的26-位羟基成功相连，用N-碘代丁二酰亚胺(NIS)和催化量三氟甲磺酸(TfOH)将三糖活性前体与苷元的3-位羟基相连，所得产物再用硼氢化钠选择性还原其C_(16)-羰基成羟基并自动与C_(22)-羰基环合成半缩酮结构化合物，最后再用甲醇钠的甲醇溶液脱去糖上的全部苯甲酰基和
沈阳药科大学博士学位论文 天然产物Methyl Protodioscin的全合成及抗癌活性研究
一
特戊酚基保护基，同时 C。。－羟基甲基化成功得到陕凿皂苦 methy Protodioscin。
这样，通过两年多的努力，最终确定了以市场上易得的薯颓皂昔元为起始原料，
经过主要9步反应合成目标物的方法，总收率达到7．8％。反应中所得到的中间产物均
经过’H－N’MR、”C－N’MR和 ESIMS鉴定，并且固体产物还测定了其熔点和旋光值。所
合成的最终产物的理化性质及光谱数据等与天然得到的样品基本一致，如：L巳、
’H－N’MR、uC－N’MR、DEPT、ES恤S、熔点和旋光值。
总之，首次成功地实现了mCthy一fot。dbSCh的全合成，为 mCthyl protod1OSClll类
似物的合成研究铺平了道路。
Furostan saponins, usually regarded as the synthetic precursors of the corresponding spirostan saponins in plants, have recently been paid emphasis attention due to the increased understanding of their broad range of biological and pharmacological activities. However, there is few report about their synthesis until very recently because of their complexity, instability and non-clear functional mechanism.
Methyl protodioscin 1, is a member of furostan saponin family with the chemical name: 3-(9-[a-Z-rhamnopyranosyl-(1→2)-{a-Z,-rhamnopyranosyl -(1→4)}-β-Z)-glucopyranosyl] -26-O-[β-Z)-glucopyranosyl]-22-methoxy-25(R)-furost-5-ene-3→,26-diol. This molecule was first isolated and identified by Kawasaki and co-worker in 1974 from fresh rhizomes of Dioscorea gracillima MiQ. In continuing efforts to identify active components from traditional Chinese herbal medicine, professor Yao and co-worker also obtained this compound from the rhizome of Dioscoreaceae by repeated bioactivity-guided isolation and tested its anticancer activities in vitro with a panel of 60 human cancer cell lines performed in NCI. Among the fourteen steroidal saponins tested, 6 compounds gave good activities in vitro and methyl protodioscin showed the most potent activity. In order to get a sufficient amount of the compound for further biological investigations, we therefore initiated a program to synthesize this target molecule.
Retro synthetically, methyl protodioscin can be logically disconnected into three distinct fragments: aglycone, C26-glucopyranose and Cs-trisaccharide moiety. Based on the analysis of its structural character, two routes were designed and systemically studied to synthesize the aglycone moiety. Finally, we chose route 2 for our purpose and the title compound was readily obtained in an overall yield of 49% via three steps by using diosgenin as the starting material: that is, protecting the Ca- hydroxyl group in diosgenin with TBDPSC1, then selectively oxidizing with DMDO at both the double bond and spiroketal, and then without further purification, directly undergoing reductive cleavage and acetolysis in one step in very mild condition. Therefore, this short and simple route was an ideal method to synthesize aglycone.
Structurally, the constitution of the trisaccharide is quite unique, the sequential linkage of a-L-rhamnopyranosyl-(1→)-a-i-rhamnopyranosyl-(1→4)-bata-D-glucopyranoside led us to design three routes to synthesize it. All these three routes were studied and the title compound was obtained by methods in route 3. Compared to route 1 and 2, route 3 possessed advantages of reasonable design, short steps and simply operation because it skillfully utilized the different activity of the hydroxyl groups in glucose and the characteristic of the relatively large stereo barrier of pivaloyl chloride.
Afterwards, we successfully connected glucopyranosyl trichloroacetimidate to the C26-hydroxyl of the aglycone in the present of TMSOTf and coupled trisaccharide moiety to the C3-hydroxyl with TV-iodosuccinimide (NIS) and a catalytic amount of trifluoromethanesulfonic acid (TfOH). Selective reduction the 16-ketone of the resulting cholestan-16, 22-dioxo with NaBHU in i-PrOH was achieved, and the newly generated secondary alcohol concurrently cyclized to give the hemiketal compound. The final target furostan saponin 1 was eventually obtained by deprotection of benzoyl and pivaloyl groups, and in situ methoxylation at C22 position using NaOMe in MeOH solution.
Thus, a simple method to methyl protodioscin was established after two years effort, which starting from commercially available diosgenin through mainly 9 steps in an overall yield of 7.8%. All the intermediates were identified correctly by 'H-NMR, 13C-NMR and ESI-MS and the solid-state materials were also measured melting point and optical rotation. The analytical data for the synthesized methyl protodioscin were identical in all respects to those reported for the natural material such as IR, 'H-NMR, 13C-NMR, DEPT, ESI-MS, mp and optical rotation.
In conclu
引文
1. Marker R E, Lopez J. Steroidal sapogenins. No. 163. The biogenesis of steroidal sapogenins in plants. J Am Chem Soc, 1947, 69: 2383-2385.
2. Schreiber K, Ripperger H. Jurubine, a novel type of steroidal saponin with (25S)-3β-amino-5 α-furostane-22 α, 26-diol O(26)-β-D-glucopyranoside structure from Solanum paniculatum L. Tetrahedron Lett, 1966, 48: 5997-6002.
3. Inoue K, Shimomura K, Kobayashi S, Sankawa U, Ebizuka Y. Conversion of furostanol glycoside to spirostanol glycoside by β-glucosidase in Costus speciosus. Phytochemistry, 1996, 41(3): 725-727.
4. Tschesche R. Uber sarsaparilloside, ein saponinderivat mit bisglykosidischer furostanol-struktur. Tetrahedron Lett, 1967, 29: 2785-2790.
5. Kawasaki T, Komori T, Miyahara K, Nohara T, Hosokawa I, Mihashi K. Furostanol bisglycosides corresponding to dioscin and gracillin. Chem Pharm Bull, 1974, 22(9): 2164-2175.
6.a) Kawano K, Sakai K, Sato H. A bitter principle of Asparagus: Isolation and structure of furostanol saponin in Asparagus storage root. Agric Biol Chem, 1975, 39(10): 1999-2002. b) Sato H, Sakamura S. A bitter principle of Tomato seeds: Isolation and structure of a new furostanol saponin. Agric Biol Chem, 1973, 37(2): 225-231.
7. Gupta R K, Jain D C, Thakur R S. Furostanol glycosides from Trigonella Foenum-Graecum seeds. Phytochemistry, 1985, 24(10): 2399-2401.
8.a) Konishi T, Shoji J. Studies on the constituents of Asparagi radix, I. On the structures of furostanol oligosides of Asparagus cochinchinensis (Loureio) Merrill. Chem Pharm Bull, 1979, 27(12): 3086-3094. b) 丁怡，杨崇仁，小百部的甾体皂苷成分．药学学报，1990, 25(7): 509.
9. Yu B, Liao J C, Zhang J B, Hui Y Z. The first synthetic route to furostan saponins. Tetrahedron Lett, 2001, 42: 77-79.
10. Sari O P, Pant G, Hostettmann K. Potent molluscicides from Asparagus. Pharmazie, 1984, 39: 581-582.
11. Yoshi J, Dev S. Chemistry of Ayurvedic crude drugs: part Ⅷ-Shatavari-2: Structure elucidation of bioactive Shatavarin-Ⅰ & other glycosides. Indian J Chem, 1988, 27B: 12-16.
12. Wang Y F, Li X C, Yang H Y. Inhibitory effects of some steroidal saponins on human spermatozoa in vitro. Planta Med, 1996, 62: 130-132.
13. Dong J X, Han K Y. A new active steroidal saponin from Anemarrhena asphodeloides. Planta Med,
1991, 57(5): 460-462.
14. Kamel M S, Ohtani K, Kurokawa T. Studies on Balanites aegyptiaca fruits, an antidiabetic Egyptian folk medicine. Chem Pharm Bull, 1991, 39(5): 1229-1233.
15. Ori K, Mimaki Y, Mito K, Sashida Y, Nikaido T, Ohmoto T, Masuko A. Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii. Phytochemistry, 1992, 31(8): 2767-2775.
16. Hu K, Dong A J, Yao X S, Kobayashi H, Iwasaki S. Antineoplastic agent: Ⅱ. Four furostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca. Planta Med, 1997, 63(2): 161-165.
17. Namba T, Huang X L, Shu Y Z, Huang S L, Hattori M, Kakiuchi N, Wang Q, Xu G J. Chronotropic effect of the metholic extracts of the plants of the Paris species and steroidal glycosides isolated from P. vietnamensis on spontaneous beating of myocardial cells. Planta Med, 1989, 55: 501-505.
18. Paczkowski C, Wojciechowski Z A. Adv Exp Med Biol, 1996, 404: 37.
19. Weymouth-Wilson A C. The role of carbohydrates in biologically active natural products. Nat Prod Rep, 1997: 99-110.
2. Schreiber K, Ripperger H. Jurubine, a novel type of steroidal saponin with (25S)-3β-amino-5 α-furostane-22 α, 26-diol O(26)-β-D-glucopyranoside structure from Solanum paniculatum L. Tetrahedron Lett, 1966, 48: 5997-6002.
3. Inoue K, Shimomura K, Kobayashi S, Sankawa U, Ebizuka Y. Conversion of furostanol glycoside to spirostanol glycoside by β-glucosidase in Costus speciosus. Phytochemistry, 1996, 41(3): 725-727.
4. Tschesche R. Uber sarsaparilloside, ein saponinderivat mit bisglykosidischer furostanol-struktur. Tetrahedron Lett, 1967, 29: 2785-2790.
5. Kawasaki T, Komori T, Miyahara K, Nohara T, Hosokawa I, Mihashi K. Furostanol bisglycosides corresponding to dioscin and gracillin. Chem Pharm Bull, 1974, 22(9): 2164-2175.
6.a) Kawano K, Sakai K, Sato H. A bitter principle of Asparagus: Isolation and structure of furostanol saponin in Asparagus storage root. Agric Biol Chem, 1975, 39(10): 1999-2002. b) Sato H, Sakamura S. A bitter principle of Tomato seeds: Isolation and structure of a new furostanol saponin. Agric Biol Chem, 1973, 37(2): 225-231.
7. Gupta R K, Jain D C, Thakur R S. Furostanol glycosides from Trigonella Foenum-Graecum seeds. Phytochemistry, 1985, 24(10): 2399-2401.
8.a) Konishi T, Shoji J. Studies on the constituents of Asparagi radix, I. On the structures of furostanol oligosides of Asparagus cochinchinensis (Loureio) Merrill. Chem Pharm Bull, 1979, 27(12): 3086-3094. b) 丁怡，杨崇仁，小百部的甾体皂苷成分．药学学报，1990, 25(7): 509.
9. Yu B, Liao J C, Zhang J B, Hui Y Z. The first synthetic route to furostan saponins. Tetrahedron Lett, 2001, 42: 77-79.
10. Sari O P, Pant G, Hostettmann K. Potent molluscicides from Asparagus. Pharmazie, 1984, 39: 581-582.
11. Yoshi J, Dev S. Chemistry of Ayurvedic crude drugs: part Ⅷ-Shatavari-2: Structure elucidation of bioactive Shatavarin-Ⅰ & other glycosides. Indian J Chem, 1988, 27B: 12-16.
12. Wang Y F, Li X C, Yang H Y. Inhibitory effects of some steroidal saponins on human spermatozoa in vitro. Planta Med, 1996, 62: 130-132.
13. Dong J X, Han K Y. A new active steroidal saponin from Anemarrhena asphodeloides. Planta Med,
1991, 57(5): 460-462.
14. Kamel M S, Ohtani K, Kurokawa T. Studies on Balanites aegyptiaca fruits, an antidiabetic Egyptian folk medicine. Chem Pharm Bull, 1991, 39(5): 1229-1233.
15. Ori K, Mimaki Y, Mito K, Sashida Y, Nikaido T, Ohmoto T, Masuko A. Jatropham derivatives and steroidal saponins from the bulbs of Lilium hansonii. Phytochemistry, 1992, 31(8): 2767-2775.
16. Hu K, Dong A J, Yao X S, Kobayashi H, Iwasaki S. Antineoplastic agent: Ⅱ. Four furostanol glycosides from rhizomes of Dioscorea collettii var. hypoglauca. Planta Med, 1997, 63(2): 161-165.
17. Namba T, Huang X L, Shu Y Z, Huang S L, Hattori M, Kakiuchi N, Wang Q, Xu G J. Chronotropic effect of the metholic extracts of the plants of the Paris species and steroidal glycosides isolated from P. vietnamensis on spontaneous beating of myocardial cells. Planta Med, 1989, 55: 501-505.
18. Paczkowski C, Wojciechowski Z A. Adv Exp Med Biol, 1996, 404: 37.
19. Weymouth-Wilson A C. The role of carbohydrates in biologically active natural products. Nat Prod Rep, 1997: 99-110.