5’-胺基,去氧-阿拉伯糖尿苷之合成與其5’醯胺化衍生物分子庫於癌症細胞的直接毒性分析

Synthesis of 5’-amino,deoxy -ara-Uridine and development of in situ cytotoxic screening with amide bond forming reaction

裴 鍇(Kai Pei)

指導教授 ： 俞鐘山

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2007年)

摘要

以HBTU輔助的醯胺化反應與組合化學的方法，於液相建立起類似尿苷與阿拉伯糖尿苷5’衍生物等分子庫。並在沒有分離純化的情況下，一鍋化於多孔盤進行細胞毒性分析（MTT試驗）。藉分子庫與細胞實驗的直接結合，快速篩選有潛力的抗癌藥物結構。
有機合成的路徑上，依文獻合成出5’去氧胺基化尿苷，並略修改用於阿拉伯糖尿苷的案例：
1.將阿拉伯糖尿苷5'OH以TBDMS保護、2',3'OH以Ac保護
2.去掉5'的TBDMS後於5’OH引入離去基tosyl (TsO-)
3.將離去基置換azido(N3)後去2',3' Ac保護
4.通入氫氣以Pd/C催化，使azido還原
5.得5’去氧胺基化阿拉伯糖尿苷
分子庫建立過程中，發現結構類似2-苯甲酰基苯甲酸的羧酸與耦合胺基後之醯胺衍生物會有環化的現象。細胞毒性分析方面，選用人類肺癌A549（Cisplatin, IC50為12μM）與乳癌MCF7細胞株（Cisplatin, IC50為19μM）等貼附型細胞進行實驗。
依照醯胺化衍生物分子庫一鍋化於細胞毒性篩選的結果，純化出有活性的化合物再進行分析：Fenbufen與正丁胺耦合之產物IC50值皆約100μM。月桂酸與5’去氧胺基化尿苷耦合之產物IC50值為100μM於MCF7，無明顯毒性於A549。這表示分子庫與細胞實驗的直接結合獲得了初步的成功。

關鍵字

分子庫 ； 液相 ； 阿拉伯糖尿苷 ； 醯胺 ； 一鍋化 ； 貼附型細胞 ； MTT實驗

並列摘要

Using HBTU based amide bond forming reaction and Combinatorial Chemistry could build libraries of Uridine/ara-Uridine 5’ derivatives analogs in solution phase. Then the process of cell assay（MTT assay）in situ in the microtiter plate was carried out without isolation or purification. The potent structures of Anti-cancer drug were screened rapidly by the combination of library and in situ cytotoxic assay.
Our scheme of organic synthesis was following literature to get the 5’-amino,deoxy Uridine. And some steps were changed for ara-Uridine case：
1. ara-Uridine 5'OH and 2',3'OH were protected by TBDMS and Ac.
2. After 5’ TBDMS was removed, a leaving group tosyl (TsO-) was made on 5'OH.
3. 5’ tosyl was replaced by azido(N3) and then 2',3' Ac was removed.
4. 5’azido was reduced by hydrogen gas and Pd/C.
5. 5’-amino,deoxy ara-Uridine was gotten.
When setting up the library, we found that the amide derivatives from 2-Benzoylbenzoic acid analog coupling with amine would cyclize. In cytotoxic assay, human lung cancer cell line A549（Cisplatin, IC50＝12μM）and breast cancer cell line MCF7（Cisplatin, IC50＝19μM）were adherent and selected for testing.
According to the result of in situ cytotoxic screening with HBTU based amide bond forming reaction, the active compounds were purified for the next assay：the amide derivative from Fenbufen coupling with n-butyl amine was 100μM（IC50 value）in both Cell lines and from Lauric acid coupling with 5’-amino; deoxy Uridine was 100μM（IC50 value）in MCF7 but seemed no toxicity in A549. It showed that the combination of library and in situ cell assay received fundamental success.

並列關鍵字

library ； solution phase ； ara-Uridine ； amide ； in situ ； adherent cell ； MTT assay ； HBTU

參考文獻

2.Gareth Thomas Medicinal chemistry : an introduction,2000,Wiley.

4.L. A. Thompson and J. A. Ellman, Chem. Rev., 1996, 96, 555–600.

5.Dolle, R.E. J. Comb. Chem, 2003, 5, 693–753.

6.Boger, D.L., Desharnais, J. & Capps, K. Angew. Chem., 2003, 42, 4138–4176.

8.C.-Y.Wu, C.-F. Chang, J. S.-Y. Chen, S.-T. Lee, C.-H.Wong and C.-H.Lin, Angew. Chem., Int. Ed., 2003, 42, 4661–4664.