印度茄化學成分暨Aurantiamide acetate、Brazilein及其衍生物全合成與生物活性之研究

Chemical Constitutents and Biological Activities of Solanum violaceum and Total Synthesis of Aurantiamide acetate, Brazilein and their Analogues

顏巧婷(Chiao-Ting Yen)

指導教授 ： 吳永昌

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2013年)

摘要

本論文主要分為兩大部分：一為天然物印度茄（Solanum violaceum）化學成分分離與生物活性評估；二為 aurantiamide acete 與 brazilein 之全合成及探討相關衍生物細胞毒殺與抗發炎活性。
印度茄（S. violaceum）為茄科（Solanaceae）茄屬（Solanum）植物。本實驗以甲醇萃取全株植物後再以乙酸乙酯與水進行分配萃取，發現印度茄乙酸乙酯層有細胞毒殺活性（IC50 < 20 μg/mL）。配合生物活性導引法，從具有細胞毒殺活性的 fractions 中，進行活性成分分離與純化，共分離得到 17 個化合物：indiosides G-L（SI-1~6）、yamogenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside （SI-7）、borassoside D （SI-8）、borassoside E （SI-9）、3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol （SI-10）、 sitosterol 3-O-β-D-glucopyranoside （SI-11）、7α-hydroxysitosterol-3-O-β-D-glucopyranoside （SI-12）、dumoside （SI-13）、pregna-5,16-dien-3α-ol-20-one-3-O-β-chacotrioside （SI-14）、tricalysioside U （SI-15）、N-p-coumaroyltyramine （SI-16） 與 trans-N-feruloyloctopamine（SI-17），其中化合物 SI-1~6 為新化合物。將分離得到的化合物進行細胞毒殺與抗發炎活性篩選，活性篩選結果顯示 22β-O-spirostanoids 毒殺能力優於 22α-O-spirostanoids；3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol (SI-10) 抑制人類嗜中性白血球之elastase release 較 PMSF (positive control) 高五倍之多。
根據本實驗室過去研究發現，aurantiamide acetate與brazilein具優越細胞毒殺與抗發炎活性。前者為自石竹科（Caryophyllaceae）瞿麥屬（Dianthus）植物瞿麥（Dianthus superbus）分離而得；後者則自豆科（Leguminosae）蘇木屬（Caesalpinia）植物蘇木（Caesalpinia sappan L.）分離而得。
合成一系列aurantiamide衍生物及其類似物，實驗結果顯示： benzoate類化合物中具Fmoc取代者有好的抗癌活性，化合物Au-14c對人類口腔癌細胞（Ca9-22）有明顯毒殺活性（IC50為0.4 μM），且細胞毒殺效果較doxorubicin高出3倍之多；acetate類化合物中具Fmoc取代者對elastase release有抑制活性，且在Fmoc-acetate取代的dipeptides中，擁有芳香環胺基酸 (Au-5b, Au-6b, Au-9b ~ Au-12b, Au-25b, Au-26b) 取代的抑制效果更優於直鏈胺基酸 (Au-13b, Au-14b)。
Brazilein全合成與活性衍生物研究，實驗過程先利用酸與鹼進行aldol condensation得到3,11-dehydrohomoisoflavanone，以氧化劑（H2O2）進行氧化反應，先得到中間物epoxide後，再以還原劑進行還原反應產生diol，進一步環化後可得具特殊四環骨架4,6’-cyclohomoisoflavanoid的化合物。最後再脫去保護基並利用碘再次進行氧化反應，成功得到目標產物brazilein。以半合成方式製備衍生物，針對brazilein上的3個OH官能基進行修飾，以研究是否能夠提高brazilein之細胞毒殺與抗發炎活性。在所有的中間物與衍生物中，化合物CS-9的毒殺效果優於目標產物brazilein；化合物Bz-1b 對抑制superoxide anion generation 和 elastase release 具有顯著活性，其IC50 分別為1.2 和1.9 μM。
以上來自天然或合成之化合物皆進行細胞毒殺與抗發炎篩選，並找到數個有效之化合物可繼續進行相關藥理機轉之研究，期望這些化合物經深入研究後可做為未來發展癌症與抗發炎用藥。

關鍵字

印度茄 ； Auanrtiamide acetate ； Brazilein ； 抗癌 ； 抗發炎

並列摘要

The thesis contains two topics; one is chemical constitutents and biological activities of Solanum violaceum, and the other is total synthesis of aurantiamide acete and brazilein together with their derivatives related to cytotoxicity and anti-inflammatory effect.
S. violaceum is Solanaceae family and solanum genus. The S. violaceum MeOH extract was partitioned with EtOAc and H2O (1:1), and the EtOAC layer was active against various human cancer cell lines with IC50 < 20 μg/mL. Bioassay-guided fractionation of the EtOAC layer of S. violaceum methanolic extract led to the isolation of 17 compounds, indiosides G-L (SI-1~6), yamogenin 3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside (SI-7), borassoside D (SI-8), borassoside E (SI-9), 3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol (SI-10), sitosterol 3-O-β-D-glucopyranoside (SI-11), 7α-hydroxysitosterol-3-O-β-D-glucopyranoside (SI-12), dumoside (SI-13), pregna-5,16-dien-3α-ol-20-one-3-O-β-chacotrioside (SI-14), tricalysioside U (SI-15), N-p-coumaroyltyramine (SI-16), and trans-N-feruloyloctopamine (SI-17). Among them, compounds SI-1~6 are new compounds. The isolates were evaluated against various cancer cell lines and toward human neutrophils elastase release and superoxide anion generation. The cytotoxic data showed that 22β-O-spirostanoids are better than 22α-O-spirostanoids; 3-O-chacotriosyl-25(S)-spirost-5-en-3β-ol (SI-10) was 5-fold more potent than PMSF in the anti-human neutrophils elastase release assay.
Aurantiamide acetate and brazilein, which were found in our previous studies, showed promising cytotoxic and anti-inflammatory activities. The former substance was isolated from Dianthus superbus (family: Caryophllaceae, genus: Dianthus) and the latter was acquired from Caesalpinia sappan L. (family: Leguminosae, genus: Caesalpinia).
The bio-data of the synthesized aurantiamide derivatives showed that the Fmoc-benzoate dipeptide (Au-14c) inhibited Ca9-22 cancer cell line with an IC50 value of 0.4 μM which were 3-fold more potent than doxorubicin, and the Fmoc-acetate dipeptides with aromatic residues (Au-5b, Au-6b, Au-9b ~ Au-12b, Au-25b, and Au-26b) were more potent than those with aliphatic ones (Au-13b and Au-14b) in the anti-human neutrophils elastase release assay.
Further investigation focused on the total synthesis of brazilein and the preparation of its derivatives. The target brazilein was prepared by construction of the benzylidene moiety via acid-catalyzed aldol condensation with aryl aldehydes, and followed by oxidation, reduction, and cyclolization. All newly synthesized compounds, including structurally related intermediates, were assayed for in vitro cytotoxicity against four human cancer cell lines and for anti-inflammatory action in terms of superoxide anion generation and elastase release by human neutrophils in response to fMLP/CB. Among all the analogs, compound CS-9 enhanced cytotoxic activity in comparison with brazilein, and Bz-1b showed the highest potency against both superoxide anion generation and elastase release with significant IC50 values of 1.2 and 1.9 μM, respectively.
We found several compounds originated from nature or synthesize are active in the cytotoxic and anti-inflammatory assays, and we wish to further deal with the mechanism to discover new drugs.

並列關鍵字

Solanum violaceum ； Auanrtiamide acetate ； Brazilein ； anticancer ； anti-inflammatory

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