白桦脂酸衍生物作为破骨细胞分化抑制剂的设计、合成与活性研究
摘要
本论文围绕白桦脂酸(BA)衍生物的设计、合成及其抑制破骨前体细胞RAW264.7分化的活性开展研究工作,以期筛选出一些高安全性、高活性的破骨细胞分化抑制剂,为新颖抗骨质疏松药物的研发提供先导化合物。
我们尝试对白桦脂酸进行了抑制RANKL诱导破骨前体细胞RAW264.7分化的活性测试,结果显示该化合物表现出中等强度的抑制活性(IC5o=20μM)。在此基础上,我们分别对白桦酯酸的A环和E环进行了结构改造和抑制破骨前体细胞分化的活性筛选：
设计、合成了系列白桦脂酸A环并五元、六元杂环衍生物。破骨前体细胞RAW264.7分化抑制活性测试结果表明,并杂环修饰提高了白桦脂酸的抑制活性,其中并吡唑衍生物13的IC50达0.1μM,相对于BA提高了近200倍。并五元杂环衍生物中,杂环上修饰亲水性基团的衍生物的活性较低,并六元杂环嘧啶环的衍生物中,修饰亲水性基团的衍生物的活性较高。对化合物13进行的细胞毒作用测试显示,化合物13抑制破骨细胞分化的活性不是由细胞毒性引起的。动物试验证明,化合物13能有效抑制骨质疏松模型鼠骨质疏松症的发生,是具有良好前景的抗骨质疏松药物先导物。
设计、合成了一些白桦脂酸E环上C-28和C-19位修饰的衍生物,考察了E环C-28和C-19位修饰对抑制破骨前体细胞RAW264.7分化活性的影响。活性测试表明,C-28位羧基改变为酯基或羟甲基后,抑制破骨前体细胞RAW264.7分化的活性下降,C-19异丙烯基上引入亲水性羟基和羧基也导致抑制活性下降,而C-19位异丙烯基的不饱和键被氢化后对抑制活性影响较小
为了方便地制备白桦脂酸衍生物,我们还研究了关键中间体桦木酮酸的高效合成方法。以商品化的天然产物白桦脂醇为原料,经IBX选择性氧化为桦木酮醛,再以脂溶性良好的四丁基高锰酸铵氧化,以高达86%的总收率获得目标产物。该方法避免使用有毒重金属氧化剂,并且IBX生成的IBA可以回收,并通过氧化再生后循环使用。
本研究工作发现了新的破骨前体细胞分化抑制剂,并且动物试验有效,为新的抗骨质疏松药物的发现提供了研究基础。但新化合物的生物利用度不高,还有待于进一步的研究探索。
With the purpose of finding more potent and safe osteoclastogenesis inhibitors which could be used as potential leads for the development of a novel type of anti-osteoporosis agents, a series of betulinic acid (BA) derivatives were designed, synthesized, and evaluated in this dissertation as inhibitors against RANKL-induced osteoclast differentiation on RAW264.7cells.
The inhibitory activity of BA on RAW264.7cells was tested and the results showed that BA is a moderate inhibitor of osteoclast differentiation (IC50=20μM). Therefore, we designed and synthesized a series of compounds by modification of A and E ring of BA, and evaluated their inhibitory activities on AW264.7cells.
A series of BA derivatives with five-member and six-member heterocyclic rings fused at C-2and C-3positions were synthesized. The inhibitory effects of all the heterocyclic ring-fused betulinic acid derivatives were evaluated against RANKL-induced osteoclast differentiation on RAW.264.7cells. The results showed that the inhibitory effect of these heterocyclic ring-fused derivatives was much stronger than that of their lead compound BA, especially the pyrazole derivative13, its IC50value was0.1μM, which was about200-fold more potent than the lead compound BA. Further, we can see that the inhibition decreased as hydrophilic substituent groups were introduced into the five-membered heterocyclic rings and the inhibition increased as hydrophilic substituent groups were introduced into the six-membered pyrimidine derivatives. To examine whether the impaired osteoclastogenesis in the presence of13is due to the decrease in viability of the precursor cells, we investigated the cytotoxicity of this compound upon osteoclast precursors RAW264.7, and the results showed that13has little cytotoxicity on osteoclasts within the effective concentrations. To examine whether13inhibits osteoclast in vivo, we used the ovariectomy mouse model to mimic menopause-induced bone loss in women. The results showed that13could prevent OVX-induced bone loss and could be used as potential leads for the development of a new type of antiosteoporosis agent.
A series of BA derivatives with modification of the C-28and C-19positions were also synthesized. Their structure-activity relationship was studied by evaluation of the inhibitory effects on RAW264.7cells. The results showed that the inhibition decreased as the C-28carboxyl group was modification to C-28methyl ester and C-28hydroxymethyl groups or C-19isopropenyl were modification to hydroxyl and carboxyl groups. The hydrogenation of C-19isopropenyl had only minimal effect on inhibition.
In order to make the preparation of BA derivatives easy, we developed a highly efficient reaction system for the preparation of betulonic acid. With commercially natural product betulin as starting material, betulonic acid was prepared efficiently with a total yield of86%by a combination of selective oxidation with IBX, followed with n-Bu4NMnO4. The method is more operable and less toxic due to no heavy-metal-catalyst was used. The IBA, byproduct of IBX, could be recycled and re-oxidized to IBX.
In conclusion, we report BA heterocyclic derivatives as a series of new chemical entities for the first time. Especially13, which exhibited potent inhibition of osteoclastogenesis both in vitro and in vivo, could be used as a promising lead for the development of a new class of antiosteoporosis agents. While13had poor oral bioavailability, and how to ameliorate the bioavailability is in progressing.
我们尝试对白桦脂酸进行了抑制RANKL诱导破骨前体细胞RAW264.7分化的活性测试,结果显示该化合物表现出中等强度的抑制活性(IC5o=20μM)。在此基础上,我们分别对白桦酯酸的A环和E环进行了结构改造和抑制破骨前体细胞分化的活性筛选：
设计、合成了系列白桦脂酸A环并五元、六元杂环衍生物。破骨前体细胞RAW264.7分化抑制活性测试结果表明,并杂环修饰提高了白桦脂酸的抑制活性,其中并吡唑衍生物13的IC50达0.1μM,相对于BA提高了近200倍。并五元杂环衍生物中,杂环上修饰亲水性基团的衍生物的活性较低,并六元杂环嘧啶环的衍生物中,修饰亲水性基团的衍生物的活性较高。对化合物13进行的细胞毒作用测试显示,化合物13抑制破骨细胞分化的活性不是由细胞毒性引起的。动物试验证明,化合物13能有效抑制骨质疏松模型鼠骨质疏松症的发生,是具有良好前景的抗骨质疏松药物先导物。
设计、合成了一些白桦脂酸E环上C-28和C-19位修饰的衍生物,考察了E环C-28和C-19位修饰对抑制破骨前体细胞RAW264.7分化活性的影响。活性测试表明,C-28位羧基改变为酯基或羟甲基后,抑制破骨前体细胞RAW264.7分化的活性下降,C-19异丙烯基上引入亲水性羟基和羧基也导致抑制活性下降,而C-19位异丙烯基的不饱和键被氢化后对抑制活性影响较小
为了方便地制备白桦脂酸衍生物,我们还研究了关键中间体桦木酮酸的高效合成方法。以商品化的天然产物白桦脂醇为原料,经IBX选择性氧化为桦木酮醛,再以脂溶性良好的四丁基高锰酸铵氧化,以高达86%的总收率获得目标产物。该方法避免使用有毒重金属氧化剂,并且IBX生成的IBA可以回收,并通过氧化再生后循环使用。
本研究工作发现了新的破骨前体细胞分化抑制剂,并且动物试验有效,为新的抗骨质疏松药物的发现提供了研究基础。但新化合物的生物利用度不高,还有待于进一步的研究探索。
With the purpose of finding more potent and safe osteoclastogenesis inhibitors which could be used as potential leads for the development of a novel type of anti-osteoporosis agents, a series of betulinic acid (BA) derivatives were designed, synthesized, and evaluated in this dissertation as inhibitors against RANKL-induced osteoclast differentiation on RAW264.7cells.
The inhibitory activity of BA on RAW264.7cells was tested and the results showed that BA is a moderate inhibitor of osteoclast differentiation (IC50=20μM). Therefore, we designed and synthesized a series of compounds by modification of A and E ring of BA, and evaluated their inhibitory activities on AW264.7cells.
A series of BA derivatives with five-member and six-member heterocyclic rings fused at C-2and C-3positions were synthesized. The inhibitory effects of all the heterocyclic ring-fused betulinic acid derivatives were evaluated against RANKL-induced osteoclast differentiation on RAW.264.7cells. The results showed that the inhibitory effect of these heterocyclic ring-fused derivatives was much stronger than that of their lead compound BA, especially the pyrazole derivative13, its IC50value was0.1μM, which was about200-fold more potent than the lead compound BA. Further, we can see that the inhibition decreased as hydrophilic substituent groups were introduced into the five-membered heterocyclic rings and the inhibition increased as hydrophilic substituent groups were introduced into the six-membered pyrimidine derivatives. To examine whether the impaired osteoclastogenesis in the presence of13is due to the decrease in viability of the precursor cells, we investigated the cytotoxicity of this compound upon osteoclast precursors RAW264.7, and the results showed that13has little cytotoxicity on osteoclasts within the effective concentrations. To examine whether13inhibits osteoclast in vivo, we used the ovariectomy mouse model to mimic menopause-induced bone loss in women. The results showed that13could prevent OVX-induced bone loss and could be used as potential leads for the development of a new type of antiosteoporosis agent.
A series of BA derivatives with modification of the C-28and C-19positions were also synthesized. Their structure-activity relationship was studied by evaluation of the inhibitory effects on RAW264.7cells. The results showed that the inhibition decreased as the C-28carboxyl group was modification to C-28methyl ester and C-28hydroxymethyl groups or C-19isopropenyl were modification to hydroxyl and carboxyl groups. The hydrogenation of C-19isopropenyl had only minimal effect on inhibition.
In order to make the preparation of BA derivatives easy, we developed a highly efficient reaction system for the preparation of betulonic acid. With commercially natural product betulin as starting material, betulonic acid was prepared efficiently with a total yield of86%by a combination of selective oxidation with IBX, followed with n-Bu4NMnO4. The method is more operable and less toxic due to no heavy-metal-catalyst was used. The IBA, byproduct of IBX, could be recycled and re-oxidized to IBX.
In conclusion, we report BA heterocyclic derivatives as a series of new chemical entities for the first time. Especially13, which exhibited potent inhibition of osteoclastogenesis both in vitro and in vivo, could be used as a promising lead for the development of a new class of antiosteoporosis agents. While13had poor oral bioavailability, and how to ameliorate the bioavailability is in progressing.
引文
1. Poole K E, Compston J E. Osteoporosis and its management. Brit. Med. J.2006, 333,1251-1256.
2. Templeton K. Secondary Osteoporosis. J. Am. Acad. Orthop. Ser.2005,13, 475-486.
3.李宁华,区品中,朱汉民.中国中老年人群骨折患病率调查.中国临床康复2003,7,1284-1285.
4. Raisz L G..Pathogenesis of osteoporosis:concepts, conflicts, and prospects J. Clin. Invest.2005,15,3318-3325.
5. Boyle W J, Simonet W S, Lacey D L. Osteoclast differentiation and activation. Nature 2003,423,337-342.
6. Khosla S. Minireview:The OPG/RANKL/RANK System. Endocrinology 2001, 142,5050-5055.
7. Gogakos A I. Bone signaling pathways and treatment of osteoporosis. Expert. Rev. Endocrinol.Metab.2009,4,639-650.
8. Ahlstrom M, Pekkinen M, Riehle U, et al. Extracellular calcium regulates parathyroid hormone-related peptide expression inostenhlasts and esteoblast progenitor cells. Bone 2008,42,483-490.
9. Westendorf J J, Kahler R A, Schroeder T M. Wnt signaling in osteoblasts and bone diseases. Gene 2004,341,19-39.
10. Baron R, Rawadi G. Targeting the Wnt/beta-catenin pathway to regulate bone formation in the adult skeleton. Endocrinology 2007,148,2635-2643.
11. Yasothan U, Kar S. Osteoporosis:overview and pipeline. Nat. Rev. Drug Discov. 2008,7,725-726.
12. Komm B S, Lyttle C R. Developing a stringent preclinical selection criteria leading to an acceptable candidate for clinical evaluation. Ann. N. Y. Acad. Sci. 2001,949,317-326.
13.Heringa M. Review on raloxifene:profile of a selective estrogen receptor modulator. Int. J. Clin. Pharmacol Ther. 2003,41,331-45.
14. Barrett-Connor E, Mosca L, Collins P, et al. Effects of Raloxifene on cardiovascular Events and Breast Cancer in Postmenopausal Women. N. Engl. J. Med.2006,355,125-137.
15.张玉彬,吴梧桐,吴文俊.降钙素的研究进展.中国药学杂志1996,31,451-454.
16. Weinstein R S, Roberson P K. Manolagas S C. Giant osteoclast formation and long-term oral bisphosphonate therapy. N. Engl. J. Med.2009,360,53-62.
17. Green J, Czanner G, Reeves G, Watson J, Wise L, Beral V. Oral bisphosphonates and risk of cancer of oesophagus, stomach, and colorectum:case-control analysis within a UK primary care cohort. Brit. Med. J.2010,341, c4444.
18. Khanjar I. Bisphosphonates risks for jaw osteonecrosis. Int. J. Rhum.2010,13, 164-171.
19. Zarychanski R, Elphee E, Walton P, Johnston J. Osteonecrosis of the jaw associated with pamidronate therapy. Am. J. Hematol.2006,81,73-75.
20. Rosen C J, Bilezikian J P. Clinical review 123:anabolic therapy for osteoporosis. J. Clin. Endocrinol. Metab.2001,86,957-964.
21. Whitifield J F. Modey P, Ross V, et al. Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone. Calcified Tissue Int.1995,56,227-231.
22.陈慧.异黄酮对骨质疏松的预防作用.国外医学老年医学分册2000,21,12-14.
23. Wei M, Yang Z, Li P, Zhang Y, Sse WC. Anti-osteoporosis activity of naringin in the retinoic acid-induced osteoporosis model. Am. J. Chin. Med.2007,35, 663-667.
24. Wang Xinluan, Zhen Lizhen, Zhang Ge, Wong Man-Sau, Qin Ling, Yao Xinsheng. Osteogenic effects of flavonoid aglycones from an osteoprotective fraction of Drynaria fortunei-An in vitro efficacy study. Phytomedicine 2011,18,868-872.
25. Wattel A, Kamel S, Prouillet C, Petit JP, Lorget F, Offord E, Brazier M. Flavonoid quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NF kappa B and AP-1.J. Cell Biochem.2004,92,285-95.
26.李灵芝.刘启兵.姜孟臣.陈虹.王舒.葛根素对体外破骨细胞性骨吸收的影响.第三军医大学学报2004,26,1830-1833.
27. Adami S, et al. Ipriglavone prevents radial bone loss in postmenopausal women with low bone mass over 2 years. Osteopetrosis Int.1997,7,119-125.
28. Meng F, Xiong Z, Sun Y, Li F. Coumarins from Cnidium monnieri and their proliferation stimulating activity on osteoblast-like UMR106 cells. Pharmazie 2004,59,643-645.
29. Tsutsumi N, Kawashima K H, Nagata A, Ujiieand H E. Effects of KCA-012 on bone metabolism in organ culture. Jpn. J. Pharmacol.1995,67,169-171.
30.江芳,王新峦,王乃利,姚新生.朝鲜淫羊藿中的木脂素类化合物及其对大鼠骨肉瘤细胞UMR106增殖及分化的影响.中草药2008,9,1281-1285.
31. Pan L, Zhou P, Zhang X, Peng S, Ding L, Qiu S X. Skelet on-rearranged pentacyclic diterpenoids possessing acyclobutane ring from Euphorbia wallichii. Org. Lett.2006,8,2775-2778.
32. Qiu S X, Dan C, Ding L, et al. A triterpene glycoside from black cohosh that inhibits osteoclastogenesis by modulating RANKL and TNFR signaling pathways. Chem. Biol.2007.14,860-869.
33. Li C, Yang Z, Li Z, Ma Y Zhang L, Zheng C, Qiu W, Wu X, Wang X, Li H, Tang J, Qian M, Li D, Wang P, Luo J, Liu M. Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating RANKL-mediated NF-kappaB and MAPK signaling pathways. J. Bone Miner. Res.2011,26,644-656.
34. Aiken C, Chen C H. Betulinic acid derivatives as HIV-1 antivirals. Trends Mol. 2005.11.31-36.
35. Yu D, Morris-Natschke S L, Lee K H. New developments in natural products-based anti-AIDS research. Med. Res. Rev.2007,27,108-132.
36. Cichewicz R H, Kouzi S A. Chemistry, biological activity, and chemo-therapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med. Res. Rev.2004,24,90-114.
37. Lo Y C, Chang Y H, Wei B L, Huang Y L, Chiou W F. Betulinic acid stimu-lates the differentiation and mineralization of osteoblastic MC3T3-E1 cells: involvement of BMP/Runx2 and beta-catenin signals. J. Agric. Food Chem. 2010,58,6643-6649.
38. Gonzalez A G, Amaro J, Fraga B M, et al.3-Oxo-6-hydroxyolean-18-en-28-oic acid from Orthopterygium huancuy. Phytochemistry,1983,22,1828-1830.
39. Pezzuto J M, Kim D S. Methods of manufacturing betulinic acid US 5804575, 1998.
40.Tomioka H, Takai K, Oshima K, et al. Selective oxidation of a primary hydroxyl in the presence of secondary one. Tetrahedron Lett.1981,22,1605-1608.
41. Kanemoto S, Oshima K, Matsubara S, et al. Transition-metal catalyzed oxidation of alcohols to aldehydes and ketones by means of Me3SiOOSiMe3. Tetrahedron Lett.1983,24,2185-2188.
42. Csuk R, Schmuck K, Schafer R. A practical synthesis of betulinic acid. Tetrahedron Lett,2006,47,8769-70.
43. Dzhemilev M M, Tolstikov G A, Pokrovskii A G, Salakhutindov N F, Tolstikova T G. RU 2317996; Chem. Abstr.2008,269455.
44. Saxena B B, Zhu L, Hao M, Kisilis E, Katdare M, Oktem O, Bomshteyn A, Rathnam P. Boc-lysinated-betulonic acid:A potent, anti-prostate cancer agent Bioorg.Med. Chem.2006,14,6349-6358.
45. a) Bal B S, Childers Jr W E, Pinnick H W. Oxidation of a,β-unsaturated aldehydes Tetrahedron 1981,37,2091. b) Barthel A, Stark S, Csuk R. Oxidative transformations of betulinol Tetrahedron,2008,64,9225-9229.
46.苏熠东,郑云红,李援朝新型氧化剂邻碘酰萘甲酸对甾体羟基选择性氧化的研究 有机化学2006,26,,199-202.
47.覃开云,苏桂发,饶万平,谭光明2-碘酰基苯甲酸(2-Iodoxybenzoic acid, IBX)在有机合成中的应用 有机化学2006,26,1623-1630.
48. Frigerio M, Santagostino M, Sputore S, A User-Friendly Entry to 2-Iodoxybenzoic Acid (IBX) J. Org. Chem.1999,64,4537-4538.
49. Frigerio M, Santagostino M, Sputore S, et al. Oxidation of Alcohols with o-Iodoxybenzoic Acid in DMSO:A New Insight into an Old Hypervalent Iodine Reagent J. Org. Chem.1995,60,7272-7276.
50.Collman J P, Slaughter L M, Eberspacher T A, et al. Mechanism of dihydrogen cleavage by high-valent metal oxo compounds:experimental and computational studies Inorg.Chem.2001,40,6272-6280.
51. Khosravi I, Yazdanbakhsh M, Preparation and characterization of novel oxo-centered basic p-chlorobenzoic bridging trinuclear complexes. J. Serb. Chem. Soc.,2010,75,929-934.
52. Su J T. Goddard W A. Enhancing 2-Iodoxybenzoic Acid Reactivity by Exploiting a Hypervalent Twist. J. Am. Chem. Soc.2005,127,14146-14147.
53. Ladziata M, Zhdankin V V. Hypervalent iodine(Ⅴ) reagents in organic synthesis ARKIVOC 2005,2006(ix),26-58.
54. Jesse D. A Simple and Advantageous Protocol for the Oxidation of Alcohols with o-Iodoxybenzoic Acid (IBX). Org.lett.2002,12,3001-3003
55. SALA T, SARGENT M V, Tetrabutylammonium permanganate:an efficient oxidant for organic substrates. J. Chem. Soc. Chem. Commun..1978,6,253-254.
56. Evers M, Poujade C, Soler F, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. J. Med. Chem.1996,39,1056-68.
57. Huang L, Ho P, Leeb K H and Chen C H. Synthesis and anti-HIV activity of bi-functional betulinic acid derivatives. Bioorg. Med. Chem.2006,14, 2279-2289.
58. Kommera H. Small structural changes of pentacycliclupane type triterpenoid derivatives lead to significant differences in their anticancer properties. Eur. J. Med. Chem.2010,45,3346-3353.
59. Urban M, Sarek J, Kvasnica M, Tislerova I, Hajduch M. Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic Activity. J. Nat. Prod.2007,70,526-532.
60. Kumar V, Rani N, Aggarwal P, et al. Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives. Bioorg. Med. Chem. Lett. 2008,18,5058-5062.
61.易必新,叶海,张鹏,张灿培美曲塞二钠的合成 中国医药工业杂志2006,37,798-800.
62. Pozo O J, Van Eenoo P, Deventer K et al. Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry, Steroids,2009,74,837-852.
63. Makino K, Kim H S, Kurasawa Y. Synthesis of Pyrazoles and condensed Pyrazoles. J. Heterocycl. Chem.1999,36,321-332.
64. Makino K, Kim H S, Kurasawa Y. Synthesis of Pyrazoles. J. Heterocycl. Chem. 1998,35,489-496.
65. Pevarello P, Varasi M. An improved synthesis of muscimol. Syn. Commun.1992, 22,1939-1948.
66. Fujita M, Kobori T, Hiyaama T, Holm A. On the regioselecivity in the Hurdmori reaction, Heterocycles 1993,36,33-36
67.李润涛葛泽梅王欣(译)杂环化学-结构、反应、合成与应用,化学工业出版社361页
68. Kamal A, Dastagiri D, Ramaiah M J, et al. Synthesis and apoptosis inducing ability of new anilino substituted pyrimidine sulfonamides as potential anticancer agents. Eur. J. Med. Chem.2011,46,5817-24.
69. Maggi A, Ciana P. Reporter mice and drug discovery and development. Nat. Rev. Drug Discov.2005,4,249
70.尚庆刘宗超鲁晓波骨质疏松动物模型特点的综述 中国实用医学2008,3,193-195
71. Evers M, Poujade C L, Soler F, et al. Betulinic Acid Derivatives:A New Class of Human Immunodeficiency Virus Type 1 Specific Inhibitors with a New Mode of Action. J. Med. Chem.1996,39,1056-1068
72. Sun I C, Chen C H, Kashiwada Y, et al. Anti-AIDS Agents 49.Synthesis, Anti-HIV, and Anti-Fusion Activities of IC9564Analogues Based on Betulinic Acid. J. Med. Chem.2002,45,4271-4275.
73. Kim J Y, Koo H M, Kim D. Development of C-20 Modified Betulinic Acid Derivatives as Antitumor Agents. Bioorg.Med.Chem.Lett.2001,11,2405-2408.
74. Hashimoto F, Kashiwada Y, Cosentino L M, et al. Anti-AIDS Agents--ⅩⅩⅦ. Synthesis and Anti-HIV Activity of Betulinic Acid and Dihydrobetulinic Acid Derivatives. Bioorg.Med.Chem.1997,5,2133-2143
75. Qian K, Yu D, Chen C H, et al. Anti-AIDS Agents.78.Design, Synthesis, Metabolic Stability Assessment, and Antiviral Evaluation of Novel Betulinic Acid Derivatives as Potent Anti-Human Immunodeficiency Virus (HIV) Agents. J. Med. Chem.2009,52,3248-3258.
76. Genet C, Strehle A, Schmidt C and Boudjelal G et al. Structure-activity relationship study of betulinic acid, a novel and selective TGR5 agonist, and its synthetic derivatives:potential impact in diabetes. J. Med. Chem.2010,53, 178-90.
2. Templeton K. Secondary Osteoporosis. J. Am. Acad. Orthop. Ser.2005,13, 475-486.
3.李宁华,区品中,朱汉民.中国中老年人群骨折患病率调查.中国临床康复2003,7,1284-1285.
4. Raisz L G..Pathogenesis of osteoporosis:concepts, conflicts, and prospects J. Clin. Invest.2005,15,3318-3325.
5. Boyle W J, Simonet W S, Lacey D L. Osteoclast differentiation and activation. Nature 2003,423,337-342.
6. Khosla S. Minireview:The OPG/RANKL/RANK System. Endocrinology 2001, 142,5050-5055.
7. Gogakos A I. Bone signaling pathways and treatment of osteoporosis. Expert. Rev. Endocrinol.Metab.2009,4,639-650.
8. Ahlstrom M, Pekkinen M, Riehle U, et al. Extracellular calcium regulates parathyroid hormone-related peptide expression inostenhlasts and esteoblast progenitor cells. Bone 2008,42,483-490.
9. Westendorf J J, Kahler R A, Schroeder T M. Wnt signaling in osteoblasts and bone diseases. Gene 2004,341,19-39.
10. Baron R, Rawadi G. Targeting the Wnt/beta-catenin pathway to regulate bone formation in the adult skeleton. Endocrinology 2007,148,2635-2643.
11. Yasothan U, Kar S. Osteoporosis:overview and pipeline. Nat. Rev. Drug Discov. 2008,7,725-726.
12. Komm B S, Lyttle C R. Developing a stringent preclinical selection criteria leading to an acceptable candidate for clinical evaluation. Ann. N. Y. Acad. Sci. 2001,949,317-326.
13.Heringa M. Review on raloxifene:profile of a selective estrogen receptor modulator. Int. J. Clin. Pharmacol Ther. 2003,41,331-45.
14. Barrett-Connor E, Mosca L, Collins P, et al. Effects of Raloxifene on cardiovascular Events and Breast Cancer in Postmenopausal Women. N. Engl. J. Med.2006,355,125-137.
15.张玉彬,吴梧桐,吴文俊.降钙素的研究进展.中国药学杂志1996,31,451-454.
16. Weinstein R S, Roberson P K. Manolagas S C. Giant osteoclast formation and long-term oral bisphosphonate therapy. N. Engl. J. Med.2009,360,53-62.
17. Green J, Czanner G, Reeves G, Watson J, Wise L, Beral V. Oral bisphosphonates and risk of cancer of oesophagus, stomach, and colorectum:case-control analysis within a UK primary care cohort. Brit. Med. J.2010,341, c4444.
18. Khanjar I. Bisphosphonates risks for jaw osteonecrosis. Int. J. Rhum.2010,13, 164-171.
19. Zarychanski R, Elphee E, Walton P, Johnston J. Osteonecrosis of the jaw associated with pamidronate therapy. Am. J. Hematol.2006,81,73-75.
20. Rosen C J, Bilezikian J P. Clinical review 123:anabolic therapy for osteoporosis. J. Clin. Endocrinol. Metab.2001,86,957-964.
21. Whitifield J F. Modey P, Ross V, et al. Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone. Calcified Tissue Int.1995,56,227-231.
22.陈慧.异黄酮对骨质疏松的预防作用.国外医学老年医学分册2000,21,12-14.
23. Wei M, Yang Z, Li P, Zhang Y, Sse WC. Anti-osteoporosis activity of naringin in the retinoic acid-induced osteoporosis model. Am. J. Chin. Med.2007,35, 663-667.
24. Wang Xinluan, Zhen Lizhen, Zhang Ge, Wong Man-Sau, Qin Ling, Yao Xinsheng. Osteogenic effects of flavonoid aglycones from an osteoprotective fraction of Drynaria fortunei-An in vitro efficacy study. Phytomedicine 2011,18,868-872.
25. Wattel A, Kamel S, Prouillet C, Petit JP, Lorget F, Offord E, Brazier M. Flavonoid quercetin decreases osteoclastic differentiation induced by RANKL via a mechanism involving NF kappa B and AP-1.J. Cell Biochem.2004,92,285-95.
26.李灵芝.刘启兵.姜孟臣.陈虹.王舒.葛根素对体外破骨细胞性骨吸收的影响.第三军医大学学报2004,26,1830-1833.
27. Adami S, et al. Ipriglavone prevents radial bone loss in postmenopausal women with low bone mass over 2 years. Osteopetrosis Int.1997,7,119-125.
28. Meng F, Xiong Z, Sun Y, Li F. Coumarins from Cnidium monnieri and their proliferation stimulating activity on osteoblast-like UMR106 cells. Pharmazie 2004,59,643-645.
29. Tsutsumi N, Kawashima K H, Nagata A, Ujiieand H E. Effects of KCA-012 on bone metabolism in organ culture. Jpn. J. Pharmacol.1995,67,169-171.
30.江芳,王新峦,王乃利,姚新生.朝鲜淫羊藿中的木脂素类化合物及其对大鼠骨肉瘤细胞UMR106增殖及分化的影响.中草药2008,9,1281-1285.
31. Pan L, Zhou P, Zhang X, Peng S, Ding L, Qiu S X. Skelet on-rearranged pentacyclic diterpenoids possessing acyclobutane ring from Euphorbia wallichii. Org. Lett.2006,8,2775-2778.
32. Qiu S X, Dan C, Ding L, et al. A triterpene glycoside from black cohosh that inhibits osteoclastogenesis by modulating RANKL and TNFR signaling pathways. Chem. Biol.2007.14,860-869.
33. Li C, Yang Z, Li Z, Ma Y Zhang L, Zheng C, Qiu W, Wu X, Wang X, Li H, Tang J, Qian M, Li D, Wang P, Luo J, Liu M. Maslinic acid suppresses osteoclastogenesis and prevents ovariectomy-induced bone loss by regulating RANKL-mediated NF-kappaB and MAPK signaling pathways. J. Bone Miner. Res.2011,26,644-656.
34. Aiken C, Chen C H. Betulinic acid derivatives as HIV-1 antivirals. Trends Mol. 2005.11.31-36.
35. Yu D, Morris-Natschke S L, Lee K H. New developments in natural products-based anti-AIDS research. Med. Res. Rev.2007,27,108-132.
36. Cichewicz R H, Kouzi S A. Chemistry, biological activity, and chemo-therapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med. Res. Rev.2004,24,90-114.
37. Lo Y C, Chang Y H, Wei B L, Huang Y L, Chiou W F. Betulinic acid stimu-lates the differentiation and mineralization of osteoblastic MC3T3-E1 cells: involvement of BMP/Runx2 and beta-catenin signals. J. Agric. Food Chem. 2010,58,6643-6649.
38. Gonzalez A G, Amaro J, Fraga B M, et al.3-Oxo-6-hydroxyolean-18-en-28-oic acid from Orthopterygium huancuy. Phytochemistry,1983,22,1828-1830.
39. Pezzuto J M, Kim D S. Methods of manufacturing betulinic acid US 5804575, 1998.
40.Tomioka H, Takai K, Oshima K, et al. Selective oxidation of a primary hydroxyl in the presence of secondary one. Tetrahedron Lett.1981,22,1605-1608.
41. Kanemoto S, Oshima K, Matsubara S, et al. Transition-metal catalyzed oxidation of alcohols to aldehydes and ketones by means of Me3SiOOSiMe3. Tetrahedron Lett.1983,24,2185-2188.
42. Csuk R, Schmuck K, Schafer R. A practical synthesis of betulinic acid. Tetrahedron Lett,2006,47,8769-70.
43. Dzhemilev M M, Tolstikov G A, Pokrovskii A G, Salakhutindov N F, Tolstikova T G. RU 2317996; Chem. Abstr.2008,269455.
44. Saxena B B, Zhu L, Hao M, Kisilis E, Katdare M, Oktem O, Bomshteyn A, Rathnam P. Boc-lysinated-betulonic acid:A potent, anti-prostate cancer agent Bioorg.Med. Chem.2006,14,6349-6358.
45. a) Bal B S, Childers Jr W E, Pinnick H W. Oxidation of a,β-unsaturated aldehydes Tetrahedron 1981,37,2091. b) Barthel A, Stark S, Csuk R. Oxidative transformations of betulinol Tetrahedron,2008,64,9225-9229.
46.苏熠东,郑云红,李援朝新型氧化剂邻碘酰萘甲酸对甾体羟基选择性氧化的研究 有机化学2006,26,,199-202.
47.覃开云,苏桂发,饶万平,谭光明2-碘酰基苯甲酸(2-Iodoxybenzoic acid, IBX)在有机合成中的应用 有机化学2006,26,1623-1630.
48. Frigerio M, Santagostino M, Sputore S, A User-Friendly Entry to 2-Iodoxybenzoic Acid (IBX) J. Org. Chem.1999,64,4537-4538.
49. Frigerio M, Santagostino M, Sputore S, et al. Oxidation of Alcohols with o-Iodoxybenzoic Acid in DMSO:A New Insight into an Old Hypervalent Iodine Reagent J. Org. Chem.1995,60,7272-7276.
50.Collman J P, Slaughter L M, Eberspacher T A, et al. Mechanism of dihydrogen cleavage by high-valent metal oxo compounds:experimental and computational studies Inorg.Chem.2001,40,6272-6280.
51. Khosravi I, Yazdanbakhsh M, Preparation and characterization of novel oxo-centered basic p-chlorobenzoic bridging trinuclear complexes. J. Serb. Chem. Soc.,2010,75,929-934.
52. Su J T. Goddard W A. Enhancing 2-Iodoxybenzoic Acid Reactivity by Exploiting a Hypervalent Twist. J. Am. Chem. Soc.2005,127,14146-14147.
53. Ladziata M, Zhdankin V V. Hypervalent iodine(Ⅴ) reagents in organic synthesis ARKIVOC 2005,2006(ix),26-58.
54. Jesse D. A Simple and Advantageous Protocol for the Oxidation of Alcohols with o-Iodoxybenzoic Acid (IBX). Org.lett.2002,12,3001-3003
55. SALA T, SARGENT M V, Tetrabutylammonium permanganate:an efficient oxidant for organic substrates. J. Chem. Soc. Chem. Commun..1978,6,253-254.
56. Evers M, Poujade C, Soler F, et al. Betulinic acid derivatives:a new class of human immunodeficiency virus type 1 specific inhibitors with a new mode of action. J. Med. Chem.1996,39,1056-68.
57. Huang L, Ho P, Leeb K H and Chen C H. Synthesis and anti-HIV activity of bi-functional betulinic acid derivatives. Bioorg. Med. Chem.2006,14, 2279-2289.
58. Kommera H. Small structural changes of pentacycliclupane type triterpenoid derivatives lead to significant differences in their anticancer properties. Eur. J. Med. Chem.2010,45,3346-3353.
59. Urban M, Sarek J, Kvasnica M, Tislerova I, Hajduch M. Triterpenoid Pyrazines and Benzopyrazines with Cytotoxic Activity. J. Nat. Prod.2007,70,526-532.
60. Kumar V, Rani N, Aggarwal P, et al. Synthesis and cytotoxic activity of heterocyclic ring-substituted betulinic acid derivatives. Bioorg. Med. Chem. Lett. 2008,18,5058-5062.
61.易必新,叶海,张鹏,张灿培美曲塞二钠的合成 中国医药工业杂志2006,37,798-800.
62. Pozo O J, Van Eenoo P, Deventer K et al. Detection and structural investigation of metabolites of stanozolol in human urine by liquid chromatography tandem mass spectrometry, Steroids,2009,74,837-852.
63. Makino K, Kim H S, Kurasawa Y. Synthesis of Pyrazoles and condensed Pyrazoles. J. Heterocycl. Chem.1999,36,321-332.
64. Makino K, Kim H S, Kurasawa Y. Synthesis of Pyrazoles. J. Heterocycl. Chem. 1998,35,489-496.
65. Pevarello P, Varasi M. An improved synthesis of muscimol. Syn. Commun.1992, 22,1939-1948.
66. Fujita M, Kobori T, Hiyaama T, Holm A. On the regioselecivity in the Hurdmori reaction, Heterocycles 1993,36,33-36
67.李润涛葛泽梅王欣(译)杂环化学-结构、反应、合成与应用,化学工业出版社361页
68. Kamal A, Dastagiri D, Ramaiah M J, et al. Synthesis and apoptosis inducing ability of new anilino substituted pyrimidine sulfonamides as potential anticancer agents. Eur. J. Med. Chem.2011,46,5817-24.
69. Maggi A, Ciana P. Reporter mice and drug discovery and development. Nat. Rev. Drug Discov.2005,4,249
70.尚庆刘宗超鲁晓波骨质疏松动物模型特点的综述 中国实用医学2008,3,193-195
71. Evers M, Poujade C L, Soler F, et al. Betulinic Acid Derivatives:A New Class of Human Immunodeficiency Virus Type 1 Specific Inhibitors with a New Mode of Action. J. Med. Chem.1996,39,1056-1068
72. Sun I C, Chen C H, Kashiwada Y, et al. Anti-AIDS Agents 49.Synthesis, Anti-HIV, and Anti-Fusion Activities of IC9564Analogues Based on Betulinic Acid. J. Med. Chem.2002,45,4271-4275.
73. Kim J Y, Koo H M, Kim D. Development of C-20 Modified Betulinic Acid Derivatives as Antitumor Agents. Bioorg.Med.Chem.Lett.2001,11,2405-2408.
74. Hashimoto F, Kashiwada Y, Cosentino L M, et al. Anti-AIDS Agents--ⅩⅩⅦ. Synthesis and Anti-HIV Activity of Betulinic Acid and Dihydrobetulinic Acid Derivatives. Bioorg.Med.Chem.1997,5,2133-2143
75. Qian K, Yu D, Chen C H, et al. Anti-AIDS Agents.78.Design, Synthesis, Metabolic Stability Assessment, and Antiviral Evaluation of Novel Betulinic Acid Derivatives as Potent Anti-Human Immunodeficiency Virus (HIV) Agents. J. Med. Chem.2009,52,3248-3258.
76. Genet C, Strehle A, Schmidt C and Boudjelal G et al. Structure-activity relationship study of betulinic acid, a novel and selective TGR5 agonist, and its synthetic derivatives:potential impact in diabetes. J. Med. Chem.2010,53, 178-90.