三萜類皂苷衍生物之合成和生物活性探討

Synthesis and Biological Activity of Triterpenoid Saponins

林佑諭(You-Yu Lin)

指導教授 ： 梁碧惠

國立臺灣大學/醫學院/藥學研究所/博士(2020年)

https://doi.org/10.6342/NTU202001866

摘要

第一部分
天然物中部份三萜類的皂素具有抗癌活性，根據文獻以及我們先前的研究，齊墩果酸帶有乙醯葡萄糖胺之皂素具有良好的抗癌活性，在葡萄糖胺上的二號位置胺基位置修飾成醯胺基或是胺甲醯基或氨基甲醯胺基可以大幅提升抗癌活性，為了進一步討論醯胺基和胺甲醯基的碳鏈長度影響抗癌活性的關係，此研究合成了一系列碳鏈超過十個碳的醯胺基修飾的皂素衍生物和超過三個碳鏈的胺甲醯基修飾的皂素衍生物。另一方面也設計以刺囊酸、常春藤皂苷元或皂皮酸三種不同的三萜類為核心的皂素衍生物，確認齊墩果酸基團在抗癌活性的重要性以及三萜類結構與活性的關係。
用來測試抗癌活性的癌細胞包含之前研究的血癌細胞HL-60之外，還新增了前列腺癌細胞PC-3和大腸癌細胞HT-29，結果顯示此類化合物對於實質固態瘤PC-3和HT-29的抗癌效果普遍都弱於血癌細胞HL-60，而在HL-60的結果中，改變齊墩果酸三萜類的部分並未改善活性，而以七個碳到九個碳長度的修飾化合物則有較好的活性，而其中以七個碳的胺甲醯基修飾化合物有最好的活性(IC50 = 0.76 μM)，根據此實驗結果顯示過長或過短的碳鏈都會降低其活性，考慮到化合物在細胞分布的不同會影響活性，我們設計並合成出含疊氮基團或末端炔類的皂素化合物，在分別與細胞培養之後，再給予市售的DBCO-488或是fluorogenic azido-coumarin來進行click chemistry，並透過細胞影像實驗觀察化合物在細胞的分布，此實驗結果顯示化合物主要分布在細胞質，符合在粒線體活性實驗看到活性的結果相符，故推測此類化合物作用在粒線體而進一步引起癌細胞凋亡。

第二部分
Chikusetusaponin IV的結構是由葡萄醣醛酸和齊墩果酸所組成，並被報導具有抑制玻尿酸分解脢的活性，但葡萄醣醛酸和三萜類合成上卻相對困難，因為葡萄醣醛酸六號的羧酸為拉電子基，會導致中間體的形成更不穩定，進而降低反應性，為了改善其選擇性和產率，我們合成了不同保護策略的葡萄醣醛酸來測試其反應性和選擇性，採用單一酯類的保護基，可藉由鄰基效應來增加β選擇性，但此類保護基會降低反應性而且會產生orthoester，為了避免orthoester，改採用立體障礙較大的路易士酸B(PhF5)3活化苯甲醯基保護的葡萄醣醛酸，得到良好的產率60%，且只有β連接的產物。另外，為增加葡萄糖醛酸之反應活性，以ether-type的保護基-2,3,4-tri-O-TBS保護的葡萄醣醛酸，產率改善到97% (α/β = 1/3.5)。因α/β位向選擇性下降，故保留二號位保護基的鄰基效應並同時提高葡萄醣醛酸的反應性，將3、4號位保護基換成苯甲基，而二號位則是使用立體障礙大的三甲基乙醯基的酯類保護，產率改善到90%且只有β連接的產物。為了釐清donor活性和產率的關係，利用與帶有三個苯甲醯基的donor 130(定義相對反應活性值(RRV)為1)共同反應來取得不同donor的RRV，RRV大小的結果表示donor帶有越多苯甲基或TBS的保護基可以提升反應活性，透過分析RRV和產率的關聯性，發現增加RRV可以提高donor與齊墩果酸或皂皮酸的葡萄醣醛酸化產率，這些研究成果解析了RRV和葡萄醣醛酸化產率的關係，也同時提供不同葡萄醣醛酸 donors的相對反應活性的數值來優化葡萄醣醛酸化，更多有關donor和acceptor反應活性和產率關係的研究還在進行中，希望未來能利用此研究來優化葡萄醣醛酸化反應，進而發展玻尿酸水解酶抑制劑。

關鍵字

齊墩果酸皂素 ； 抗癌活性 ； 細胞質 ； 粒線體 ； 葡萄醣醛酸化 ； 相對反應活性值

並列摘要

Part 1
3-Glucosamine bearing oleanolic acid (OA), a nature product from Albizia subdimidiata and Acacia tenuifolia, showed great cytotoxicity in the cancer cells. To improve its cytotoxic activity, oleanolic saponin derivatives with different N-modification in glycone or different aglycone were synthesized. In the glycone moiety, the N-modification contains different carbon chain length acyl, alkoxycarbonyl, and alkylcarbamoyl group. On the other hand, the change of aglycone moiety includes oleanolic acid, hederagenin, echinocystic acid, and quillaic acid. These compounds were synthesized and evaluated against HL-60, PC-3, and HT29 tumor cancer cells by the MTT assay. These compounds have better cytotoxicity for leukemia cell (HL-60) than for solid tumors (PC-3 and HT29). Different aglycones did not ameliorate the anticancer activity, but the glycone modification displayed great results and the 2ʹ-N-heptoxycarbonyl derivative was found to be the most cytotoxic (IC50 = 0.76 μM) against HL-60 cells. Images obtained in a cellar uptake assay clearly showed that compounds 117 and 120 enter the cells and located mainly in the cytosol. A mitochondrial membrane potential assay found that compound 71 induced mitochondria damage, and this result coincided with compound distribution on cytosol from cell image. These findings suggest that the apoptosis caused by the compounds synthesized proceeds via perturbation of the mitochondria.

Part 2
Chikusetusaponin IV containing glucuronic acid and oleanolic acid was reported to inhibit hyaluronidase activity. Synthesis of chikusetusaponin IV is challenge, because glycosylation between glucuronic acid and triterpene usually is low yield. The 6-carboxylic group of glucuronic acid, as a donor, destabilizes the oxocarbenium dromation which results in decrease of anomeric reactivity. Fine-tuning the protecting groups for glucuronic acid donor would be an accessible strategy to achieve high yield and b-selectivity of glycosylation product. Usually, disarmed protective group can induce neighboring group anticipation to impart β-selectivity. Unfortunately, these disarmed-protected donors gave low yield of glucuronidations along with orthoester formation. To minimized orthoester formation, we used a bulkier promotor—B(PhF5)3 to activate perbenzoylated glucuronyl donor gave the best yield of 60% with only β-linked product. We also tried 2,3,4-tri-O-TBDMS groups on the glucuronyl donor and it was found such modifications enhanced reactivity through conformation change and these silyl-protective strategies improved yield to 97% (α/β = 1/3.5). It then turned into to preserve neighboring group anticipation ability by tuning various 2-O-etser groups, and 3,4-positions were protected by di-O-benzyl groups. 2-O-pivaloyl bearing donor showed the best glucuronidation yield to 90% with only β-linked product. To investigate the relationship of donor’s reactivity and yields, we used the competitive experiments to define the donor’s relative reactivity value related to tri-benzyl protecting donor 130 (RRV = 1). The reactivity order indicated the donor with more armed protecting groups such as Bn or TBS, possessed higher reactivity. With RRVs in hand, we investigated the correlation between RRVs and yields and found that increasing RRVs could improve the yields of glucuronidation of OA or QA acceptors. The discovery gives deeply insight to relationship between RRVs and yields and quantifies the effect of protecting groups on reactivity of trichloroacetimidate glucuronide donors. More studies about the relationship between the donor and the acceptor's yields and reactivities are still in progress. Fine-tuning the glucuronidation conditions would enable us to develop the hyaluronidase inhibitor in the future.

並列關鍵字

oleanolic saponin ； cytotoxic activity ； cytosol ； mitochondria ； glucuronidation ； relative reactivity value

參考文獻

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(3) Pollier, J.; Goossens, A. Oleanolic acid; Phytochemistry 2012, 77, 10.

(4) Sporn, M. B.; Liby, K. T.; Yore, M. M.; Fu, L.; Lopchuk, J. M.; Gribble, G. W. New Synthetic Triterpenoids: Potent Agents for Prevention and Treatment of Tissue Injury Caused by Inflammatory and Oxidative Stress; J. Nat. Prod. 2011, 74, 537.

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