Carbocyclic Nucleoside Isosteres as Potential Antitumor

常规核苷的糖部分的固有灵活性已经通过用刚性碳环（即双环[3.1.0]己烷）替代糖部分而被阻止，所述刚性碳环模拟常规核苷在伪旋转循环中的北（N）或南（S）构象。我们的主要目标是系统地探测一系列的核苷/核苷酸结合酶的构象刚性N和S基板，以了解首选的结合模式。第二个目的是构建修饰的核酸，其掺入这些固定单元中的一些以分别增强或破坏与S和N构象相关的典型B-或A-DNA构象。胸苷的构象锁定对映体，N和S methanocarba胸苷（N-MCT和S-MCT），提供了一个明确的例子，有效的构象歧视的两个关键酶，控制生物活性的结果。我们已经证明，在用HSV-1胸苷激酶（TK）转染的鼠癌细胞（38 MC）中，S-MCT是比N-MCT更好的疱疹病毒（HSV-1）胸苷激酶（TK）底物。另一方面，令人惊讶的是，尽管细胞中存在较高水平的S-MCT三磷酸，但仅观察到N-MCT的DNA掺入。我们相信，这一发现代表了第一个明确的证明，激酶和聚合酶有不同的和相反的底物构象偏好。由于N-MCT和其他5-取代的尿嘧啶碳环类似物是非常有效的抗疱疹（例如5-溴）和抗水痘带状疱疹（例如5-溴乙烯基）药物，并且双环[3.1.0]己烷模板的合成相当困难，我们开始了一个项目，以开发一种简化的方法来合成这些化合物。通过开发两种新的分子内环丙烷化方法来满足挑战，其中整个双环结构在一个步骤中构建为N和S系列。对于这种化学，我们在形成双环[3.1.0]己烷系统后加入了脂肪酶催化的拆分步骤，这将保证进一步生物学研究所需的对映体纯化合物的充足供应。相对于我们的第二个目标，我们已经合成了一系列短的寡脱氧核苷酸（ODNs），其对应于自身互补的EcoR 1识别序列[ds（5 '-CGCGAATTCGCG-3'）]，其中中间的A已经被锁定的腺苷取代。这些序列是当前核磁共振和晶体学研究的主题，以检测弯曲或扭结的形成。关于使用含有刚性S和N脱碱基位点的短ODN作为DNA甲基转移酶抑制剂的研究已经完成并发表（Wang等人，J. Am. 2000，122，12422）。

The inherent flexibility of the sugar moiety of conventional nucleosides has been arrested by replacing the sugar moiety with a rigid carbocyclic ring (i.e. bicyclo[3.1.0]hexane) that mimics either the North (N) or South (S) conformations of conventional nucleosides in the pseudorotational cycle. Our main objective has been to systematically probe a series of nucleoside/nucleotide binding enzymes with sets of conformationally rigid N and S substrates to learn about the preferred mode of binding. A secondary objective has been to construct modified nucleic acids that incorporate some of these fixed units to either reinforce or disrupt the typical B- or A-DNA conformations associated with S and N conformations, respectively. The conformationally locked antipodes of thymidine, N and S methanocarba thymidines (N-MCT and S-MCT), provided a clear example of efficient conformational discrimination by two critical enzymes that control the outcome of biological activity. We have demonstrated that S-MCT is a much better substrate than N-MCT for the herpes (HSV-1) thymidine kinase (TK) in murine cancer cells (38MC) transfected with HSV-TK. Surprisingly, on the other hand, DNA incorporation was observed only for N-MCT, despite the higher levels of S-MCT triphosphate present in the cell. We believe that this finding represents the first clear demonstration that kinases and polymerases have different and opposing substrate conformational preferences. Since N-MCT and other 5-substituted uracil carbocyclic analogues are extremely potent anti-herpes (e.g. 5-bromo) and anti-varicela zoster (e.g. 5-bromovinyl) agents, and synthesis of the bicyclo[3.1.0]hexane template is rather difficult, we embarked on a project to develop a simplified approach for the synthesis of these compounds. The challenged was met by developing two novel intramolecular cyclopropanation approaches where the entire bicyclic structure is constructed in one step for both N and S series. To this chemistry, we added the inclusion of a lipase-catalyzed resolution step following the formation of the bicyclo[3.1.0]hexane system which will guarantee an ample supply of enatiomerically pure compounds required for further biological studies. Relative to our second goal, we have synthesized a series of short oligodeoxynucleotides (ODNs) corrresponding to the self-complementary EcoR1 recognition sequence [ds(5'-CGCGAATTCGCG-3')] where the middle A's have been replaced by locked adenosines. These sequences are the subject of current NMR and crystallographic studies to detect the formation of bends or kinks. The study on the use of short ODNs containing rigid S and N abasic sites as inhibitors of DNA methyltransferase was completed and published (Wang et al. J. Am. Chem. Soc. 2000, 122, 12422).