潛質抗癌藥物MTH1抑制劑之設計合成 與生物活性評估

Abstract

現代癌症的治療通常以癌細胞的基因缺陷為標靶。近年的研究提出以MTH1蛋白作為靶點可以提供一種更通用的抗癌策略。因為MTH1這種特殊的蛋白在正常細胞中無關緊要，但對癌細胞的存活是至關重要的。本研究的目的為設計與合成MTH1抑制劑，並探究其作為抗癌藥物的可行性。 在研究的第一部分中，MTH1抑制劑TH287被選作先導化合物，並以構象限制為設計理念，通過環化、氯化、Suzuki反應、氫化和甲基化等步驟，合成五個四氫吡啶並[3,4-d]嘧啶-2-胺衍生物，期待可以改善代謝穩定性。然而，這一系列的化合物均失去抑制活性。 研究的第二部分的設計策略為，通過高通量篩選得到的化合物23作為先導化合物，再通過改造和優化結構，經過環化、Vilsmeier-Haack-Arnold反應和親核取代等反應，合成了十五個以嘧啶-2,4-二胺為母核的化合物，以獲得更好的抑制活性。所有的化合物均通過核磁共振光譜和質譜進行結構鑒定，它們的生物活性通過酵素抑制實驗與細胞毒殺實驗評估。根據酵素抑制實驗結果，主結構中去掉乙基的化合物均顯示出更好的抑制效果。另外，將主結構中的甲基替換成環丙基，酵素抑制活性有所下降。本研究中，化合物38c (N4環丙基-6-（哌啶-1-基）嘧啶-2,4-二胺) 表現出最好的酵素抑制活性(MTH1 IC50 = 6.5 nM)，可能適合繼續發展成有潛力的抗癌候選藥物。然而，在細胞毒殺實驗中， 只有化合物38c顯示出微弱的活性，其他具有良好的MTH1抑制效果的化合物均未表現出細胞毒殺性。一個可能的解釋是這類化合物不能將氧化的核苷酸(8-oxo G)片段引入DNA。

Modern cancer treatment often targets on genetic defects in cancers. Current researches provide a more generic anti-cancer strategy by targeting MTH1 protein, which is crucial for cancer survival but non-essential to normal cells. The objective of this study is to design and synthesize MTH1 inhibitors as potential cancer treatment for further evaluation. In the first part of this study, reported MTH1 inhibitor TH287 was chosen as lead, and was modified employing the concept of conformational restriction, expecting to improve metabolic stability. In this way, five tetrahydropyrido[3,4-d]pyrimidin-2-amine derivatives were synthesized through cyclization, chlorination, Suzuki coupling, hydrogenation and methylation. But this series of compounds completely lose inhibitory potency against MTH1 enzyme. In the second part, the designing strategy is to increase potency of a moderate hit found by high-throughput screening through structural modification and optimization. And 15 compounds with pyrimidine-2,4-diamine as main structure were synthesized through cyclization, Vilsmeier-Haack-Arnold reaction and nucleophilic substitution. The structures of these compounds were characterized by NMR spectroscopy and mass spectroscopy. And their biological activities were evaluated through MTH1 enzyme activity assay and cytotoxic assay. According to the result obtained from enzymatic assay, all the compounds without ethyl group showed significantly enhanced inhibitory activity than those with ethyl group in main structure. And the replacement of methyl group with cyclopropyl group led to decrease in potency. Among the compounds in this series, compound 38c (N4-cyclopropyl-6-(piperidin-1-yl)pyrimidine-2,4-diamine) exhibits the best inhibitory potency with an IC50 value of 6.5 nM. However, from the result of cytotoxic assay, only 38c showed moderate activity with an IC50 value of 23.0 ± 1.8 μM, the other compounds with potent MTH1 inhibitory activity failed to kill cancer cells. A possible explanation to the lack of toxicity might be inability to increase oxidized (8-oxo G) lesions in DNA.