蒽菎類及查耳酮類衍生物之合成與細胞毒活性評估之研究

Design, Synthesis and Cytotoxic Activities of Anthraquinones and Chalcones

鄧志煌(Chi-Huang Teng)

指導教授 ： 林忠男

高雄醫學大學/藥學院/藥學研究所/博士(2006年)

摘要

天然界中蒽菎類及查耳酮類衍生物具有多樣化的藥理活性。其中蒽菎類衍生物能藉由apoptosis，造成細胞死亡，在先前研究中已做了一系列蒽菎類衍生物在細胞毒活性上的評估，為了更加了解其中結構及活性之間的關係，我們繼續合成了21個蒽菎類衍生物。而查耳酮衍生物在之前的研究中發現查耳酮化合物2',5'-dimethoxy-4-hydroxychalcone (29)具有顯著的抗癌活性，針對此化合物作修飾，希望能獲得更加具有活性的化合物，我們合成了一系列的查耳酮衍生物，將這些蒽菎及查耳酮衍生物，進行細胞毒活性的研究並且利用蒽菎類衍生物做DNA開環作用實驗，另外也對這些化合物進行人類富含血小板血漿對epinephrine所引起之凝集的抑制活性研究，以及合成之查耳酮衍生物結合分子模擬去預測其與cyclooxygenase (COX)是否有作用關係。
根據實驗結果蒽菎類衍生物在細胞毒活性中3-[3-(4-Methylpiperazinopropoxy)]-9,10-anthraquinone (14)對大腸癌、乳癌細胞有顯著的細胞毒活性，化合物14加入培養72個小時以後，發現sub-G1的peak增加及DNA斷片，由此結果可知加入化合物14後細胞引起apoptosis而死亡。而查耳酮衍生物細胞毒活性實驗結果，含硫衍生物2',5'-Dimethoxy-2-thienylchalcone (32)，2',5'-Dimethoxy-3-thienylchalcone (33)，2',5'-Dimethoxy-2-(5-methylthienyl)chalcone (35)對乳癌也有顯著的細胞毒活性，化合物35對大腸癌更是具有顯著的抑制活性，化合物35加入培養72個小時後sub-G1的peak增加，且有nuclear斷片產生，亦推測其可使細胞引起apoptosis而死亡。
DNA scission實驗中，發現已報告之合成化合物1-Hydroxy-3-[3-(diethylamino)-2-hydroxypropoxy]-9,10-anthraquinone (44)在銅離子濃度300、200、100 ?嵱的存在下具有使DNA開環的作用，實驗中加入的一價銅箝合劑(neocuproine)後就無開環作用，可知其斷裂和一價銅有關係，一價銅為其DNA斷裂重要的中間產物，在反應中若加入KI及catalase，DNA開環完全被抑制，加入superoxide dismutase則無抑制DNA開環的效果，由此結果推測其有將Cu (II)還原成Cu (I)，產生H2O2及OH．來使DNA斷裂。
在抑制血小板凝集活性實驗中可知化合物9、17、42、43可以抑制第二相凝集反應和在高濃度時可以抑制第一相凝集反應，表示與血小板細胞膜上的接受體作用以及部份經由thromboxane生合成之抑制，由分子模擬結果推測，查耳酮類化合物Ligscore2越高及JAIN值大於等於2.46時，會有抑制COX活性的效果，其中化合物30-38有較高的Ligscore2值及大於2.46的JAIN值，推測其可能具有抑制COX的活性，而抑制血小板凝集實驗中化合物32、33、35可以抑制第二相凝集反應，由此結果可推測其有抑制COX的效果，而其真正的活性及作用機轉仍在進行中。由分子模擬結果預測合成之查耳酮衍生物30-38應具有抑制COX活性。

關鍵字

蒽菎 ； 查耳酮 ； 細胞毒

並列摘要

Natural anthraquinones and chalcones exhibited various biological activities. Some anthraquinones mediated their cytotoxicity through apoptosis. In previous reports, a series of anthraquinones were evaluated for their cytotoxicity. In order to understand the structure and cytotoxicity relationship, we continued to design and synthesize twenty one anthraquinone derivatives. In a previous study, we found that chalcone, 2',5'-dimethoxy-4-hydroxychalcone (29) had significantly cytotoxic activity, therefore we had also to design and synthesize a series of chalcones, all of the compounds of anthraquinone and chalcone were evaluated for their cytotoxic activity and DNA strand scission activity. Anthraquinones were also evaluated for inhibitory effect on the platelet aggregation induced by epinephrine in human PRP. We also predicted their anti-cycloxygenase activity by molecular docking.
Compound 3-[3-(4-Methylpiperazinopropoxy)]-9,10-anthraquinone (14) showed significantly cytotoxic activity against HT-29 and MCF-7. MCF-7 were treated with various concentrations of 14 for different time periods. The appearance of cells with low DNA stainability formed a “sub-G1 peak” and DNA fragmentation in MCF-7 cells was significantly observed after 72 hrs. These compounds might induce cell death by apoptosis. Among chalcone derivatives, the chalcones which have thio moiety, 2',5'-Dimethoxy-2-thienylchalcone (32), 2',5'-Dimethoxy-3-thienylchalcone (33) and 2',5'-Dimethoxy-2-(5-methylthienyl)chalcone (35) had potent cytotoxic activity against MCF-7. Compound 35 showed significantly cytotoxic activity against colorectal cancer cell line. A “sub-G1 peak” increased and nuclear fragmentation in MCF-7 cells was significantly observed after 72 hrs. We suggested that these compounds might also induce cell death by apoptosis.
1-Hydroxy-3-[3-(diethylamino)-2-hydroxypropoxy]-9,10-anthraqunione (44) mediated DNA breakage in the presence of Cu (II) (300, 200, 100 ?嵱). The convertion of supercoiled DNA to the relaxed form was inhibited with neocuproine, a Cu (I)-specific sequestering agent. The result suggested that Cu (I) was the essential intermediate in the 44 mediated DNA cleavage reaction. Compound 44 in the presence of Cu (II) induced DNA degradation was completely inhibited by the addition of KI and catalase while superoxide dismutase (SOD) did not protect the DNA from strand breakage. We suggested that compound 44 induce H2O2 and OH• generation in the presence of Cu (II) and subsequent damage to cellular DNA.
In epinephrine induced platelet aggregation of human PRP, compounds 9, 17, 42 and 43 prevented secondary aggregations and suppressed the primary aggregation at high concentrations induced by epinephrine. We supposed that these compounds might partially inhibit the thromboxane synthesis. In the molecular docking, compounds with the high Ligscore2 and the Jain score more than 2.46, revealed anti-cyclooxygenase activity. The synthetic chalcones 30-38 had high Ligscore2 and their Jain score also more than 2.46. It suggested that they might reveal anti-cyclooxyganase activity. In epinephrine induced platelet aggregation of human PRP, compounds 32, 33 and 35 prevented secondary aggregations. We suggested that these compounds might partially inhibit the thromboxane synthesis.

並列關鍵字

anthraquinone ； chalcone ； cytotoxic activities

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