Carbocyclic Nucleoside Isosteres as Potential Antitumor and Antiviral Agents

双环[3.1.0]己烷模板作为构建新型构象锁定碳环核苷的平台的研究继续产生许多有趣的化合物。我们使用这些修饰的核苷的目的是：(1)确定参与核苷、核苷酸和寡核苷酸生物化学的酶的构象偏好；(2)根据我们对其生化作用机制的了解，开发出可能的抗肿瘤/抗病毒药物。这些努力导致了n -甲烷氨基（N-MCT）的发现，这是一种对疱疹病毒1和2以及与卡波西肉瘤相关的人类疱疹病毒8有活性的化合物。今年，我们承包了N-MCT （65 g）的半准备合成，用于未来的体内毒理学和药理学研究。去年发现的d -碳- t（碳环胸腺嘧啶）作为一种成功激酶核苷，作为一种动力学延迟链终止剂，对hiv感染的细胞有效，这导致了对其作用机制的全面研究。D-carba-TTP被HIV-RT有效地结合；然而，下一个脱氧核苷酸三磷酸被缓慢地添加到D-carba-TMP的引物末端。在DNA模板中，可以检测到一些停顿，这表明d -碳- dtpp在一定程度上被纳入。然而，与DNA模板相比，D-carba-dTTP似乎没有与正常的dTTP有效竞争，因为大多数产物是全长的。RNA模板比DNA模板有更多的停顿，这表明D-carba-dTPP能够更好地与RNA模板的TTP竞争。由于d -碳- t能够在细胞培养中抑制病毒复制，这些数据表明，大多数抑制活性发生在第一链合成过程中。此外，D-carba-T能够阻断培养细胞中多重耐药HIV-1载体的复制（J. Med. Chem.）。待提交)。在双环[3.1.0]己烷假糖模板上合成了北、南两个构象锁定的嘌呤霉素类似物。产物的最终组装是通过相应的南北3-叠氮嘌呤碳核苷与fmoc保护的4-甲氧基- l -酪氨酸的1-羟基苯并三唑酯的Staudinger-Vilarrasa偶联完成的。化学)。携带新核苷的寡核苷酸的合成被锁定在一个特定的环皱化是一个越来越感兴趣的领域。制备了携带腺嘌呤和鸟嘌呤双环[3.1.0]己烷假糖的寡核苷酸。用北双环或南双环[3.1.0]己烷假糖衍生物取代一个腺嘌呤或鸟嘌呤对双链DNA结构的整体稳定性影响不大。研究了三种携带一个修饰双环[3.1.0]己烷鸟嘌呤残基的凝血酶结合适体衍生物。用双环鸟嘌呤假糖取代凝血酶结合适体的一个鸟嘌呤残基不会改变适体的反平行四联体结构，但对四联体的稳定性有较大影响。同型鸟嘌呤被锁在南构象上的同型鸟嘌呤假糖取代，保持了稳定性。将反取向的鸟嘌呤转变为锁定在同步构象中的鸟嘌呤假糖，会引起四联体的强烈不稳定（手稿正在准备中）。十多年来，我们的实验室一直在研究建立在双环[3.1.0]己烷模板上的锁定碳环核苷的性质（Marquez， V. E. the properties of locked Methanocarba nucleosides in Biochemistry, Biotechnology and Medicine，第12章）。见：《核苷在生物化学、生物技术和医学中的应用》，P. Herdewijn Ed. Wiley-VCH, 2008, pp 307-341)。使用这种模板的缺点之一是O4氧的损失及其在头型效应中的作用。为了在糖基C-N键附近的等效位置重新引入一个氧，我们合成了两个氧环[3.1.0]己烷核苷，试图恢复头异构效应。更重要的是，这两种结构冻结了氧孤对轨道相对于C-N反键轨道的取向，从而增强（反周面）或减弱（间扭式）异头效应的强度。这使我们能够研究氧孤对离域进入反键C-N键轨道的后果，这种现象也被称为异头效应。用Gaussian 98和NBO5.0研究了两种分子中p轨道分量最高的氧孤对与相邻的C-N键之间的二阶微扰相互作用。分子的几何形状在B3LYP/6-31G*的理论水平上进行了优化。在反周平面取向的化合物中，孤对反键轨道相互作用最强，键长预期更大。这种轨道相互作用在质子化物质中增加得更多，使C-N键延长0.034。为了帮助将这些结果与实验数据联系起来，我们测量了这些分子在pH 2（37℃）下的酸稳定性。歧异效应明显降低的化合物（间扭式）非常稳定，半衰期为52.8 min，而具有强歧异效应的反周面处理的化合物，C-N键断裂，半衰期为6.46 min（核酸研讨会系列第52期，2008,543-544）。在此期间首次合成了对映体纯形式的核糖样南双环[3.1.0]己烷核苷。这是通过在一个敏感的双环[3.1.0]己烷体系上使用几个官能团变换来完成的。d -核糖被转化为甲醇醇，随后羟基被转化为腈，并发生构型反转。腈基随后分两个阶段还原为5′-羟甲基。然后在叔碳（C1）上附加一个酯基，作为核碱基前体转化为氨基（J. Org）。化学。在出版社)。

The investigation of the bicyclo[3.1.0]hexane template as a platform for the construction of novel conformationally locked carbocyclic nucleosides continues to yield many interesting compounds. We use these modified nucleosides with the intent of: (1) determining the conformational preferences of enzymes involved in the biochemistry of nucleosides, nucleotides, and oligonucleotides; and (2) developing selected compounds as possible antitumor/antiviral drugs based on our understanding of their biochemical mechanism of action. These efforts have resulted in the discovery of N-methanocarbathymidine (N-MCT), a compound active against herpes viruses 1 and 2, as well as human herpesvirus 8 associated with Kaposi sarcoma. This year we contracted a semi-prep synthesis of N-MCT (65 g) to use for future toxicological and pharmacological studies in vivo. Last years discovery of D-carba-T (carbocyclic thymidine) as a successfully kinased nucleoside that functions as a kinetic delayed chain terminator and is effective against HIV-infected cells led to a full investigation of its mechanism of action. D-carba-TTP is efficiently incorporated by HIV-RT; however, the next deoxynucleotide triphosphate is added slowly to D-carba-TMP at the primer terminus. With a DNA template, some pausing was detectable, suggesting that D-carba-dTPP is incorporated to some extent. However, with the DNA template, D-carba-dTTP did not appear to compete effectively with the normal dTTP, since most of the products are full length. With the RNA template, there was more pausing than with the DNA template, suggesting that D-carba-dTPP is better able to compete with TTP with an RNA template. Since D-carba-T is able to inhibit viral replication in cell culture, these data suggest that most of the inhibitory activity occurs during first strand synthesis. In addition, D-carba-T was able to block the replication of multi-drug resistant HIV-1 vectors in cultured cells (J. Med. Chem. to be submitted). Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North and South 3-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine (manuscript submitted to J. Org. Chem.). The synthesis of oligonucleotides carrying novel nucleosides that are locked toward one specific ring puckering is a field of increasing interest. Oligonucleotides carrying adenine and guanine bicyclo[3.1.0]hexane pseudosugars have been prepared. The substitution of one adenine or guanine with either North- or South-bicyclo[3.1.0]hexane pseudosugar derivatives have little effect on the global stability of a duplex DNA structure. Three derivatives of the thrombin binding aptamer carrying one modified bicyclo[3.1.0]hexane guanine residue were studied. The replacement of one guanine residue of the thrombin binding aptamer with a bicyclic guanine pseudosugar does not change the antiparallel quadruplex structure of the aptamer but it has a strong influence in the stability of the quadruplex. The substitution of a syn-guanine with equivalent syn-guanine pseudosugar locked in the South-conformation maintains the stability. Changing a guanine that is in the anti orientation for a guanine pseudosugar locked in a syn conformation induces a strong destabilization of the quadruplex (manuscript in preparation). For more than a decade our laboratory has been studying the properties of locked carbocyclic nucleosides built on a bicyclo[3.1.0]hexane template (Marquez, V. E. The properties of Locked Methanocarba Nucleosides in Biochemistry, Biotechnology and Medicine, Chapter 12. In: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, P. Herdewijn Ed. Wiley-VCH, 2008, pp 307-341). One of the drawbacks of using such a template is the loss of the O4 oxygen and its role in the anomeric effect. To reintroduce an oxygen in an equivalent position adjacent to the glycosyl C-N bond, we have synthesized two oxobicyclo[3.1.0]hexane nucleosides in an attempt to reinstate the anomeric effect. Even more importantly, these two structures freeze the orientation of the oxygens lone pair orbitals in relation to the C-N antibonding orbital to either augment (antiperiplanar) or diminish (gauche) the strength of the anomeric effect. This allowed us to study the consequences of delocalizing the oxygens lone pair into the antibonding C-N bond orbital, a phenomenon also known as the anomeric effect. Using Gaussian 98 and NBO5.0 the second order perturbation interaction between the oxygen lone pair with the highest p orbital component and the adjacent C-N bond was studied for both molecules. The geometries of the molecules were optimized at the B3LYP/6-31G* level of theory. The strongest lone pair-antibonding orbital interaction was observed for the compound with the antiperiplanar orientation and expectedly the bond length was greater. This orbital interaction increases even more in the protonated species and lengthens the C-N bond by 0.034 . To help correlate these results with experimental data we measured the acid stability of these molecules at pH 2 (37 C). The compound for which the anomeric effect was significantly reduced (gauche) was quite stable and had a half-life of 52.8 min, while for the antiperiplanar disposed compound with a strong anomeric effect, the C-N bond was cleaved with a half-life of 6.46 min (Nucleic Acids Symposium Series No. 52, 2008, 543-544). The first synthesis of the ribo-like South bicyclo[3.1.0]hexane nucleoside in enantiomerically pure form was achieved during this period. This was accomplished through the use of several functional group transformations on a sensitive bicyclo[3.1.0]hexane system. D-Ribose was transformed into a methanocarba alcohol followed by conversion of the OH group to a nitrile with inversion of configuration. The nitrile group was subsequently reduced in two stages to the 5'-hydroxymethyl group. An ester group was then appended to a tertiary carbon (C1), which was transformed to an amino group as a nucleobase precursor (J. Org. Chem. in press).