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。这一努力导致了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N-MCT还被证明对小鼠的正痘病毒（抗病毒文献，2007,73,69-77）和牛痘病毒感染（抗病毒文献，2007,in press=doi:10.1016/ j.t Antiviral .2007.06.005）有活性。N-MCT已获得许可，体内研究仍在继续。化学合成的N-MCT-5-triphosphate帮助我们证明了其作为DNA聚合酶，特别是HIV逆转录酶的延迟链终止器的活性（J. Mol. Biol. 2005,345, 442-450）。这一特性赋予了该化合物克服HIV耐药性切除机制的独特能力。然而，N-MCT对hiv感染的细胞无活性，除非细胞转染疱疹病毒胸苷激酶（HSV-tk）。N-MCT不能被细胞胸苷激酶（c-tk）磷酸化，这促使研究人员寻找新的类似物作为激酶的底物。今年，我们完成了D-和l -异构体（光学对映体）的合成，作为N-MCT的异构体类似物，其中环丙烷环和关键的3-OH基团的融合位点都被重新定位，以方便激酶识别。这种方法导致由HSV-tk/胸苷酸激酶和细胞二磷酸激酶催化的所有三种代谢物（单磷酸、二磷酸和三磷酸）的形成增加了2倍。这本书最近出版了。化学。社会科学学报，2007,29,6216-6222)。不幸的是，该化合物仍然没有被c-tk磷酸化。令人惊讶的是，这种新类似物的l -异构体是活性对映体，更令人惊讶的是最近的两个发现：(1)尽管对5-三磷酸的合成代谢水平很高，但该化合物对HSV-1和HSV-2感染的细胞无活性；(2)该化合物对转染HSV-tk的细胞具有良好的抗HIV活性。这意味着新的5-三磷酸代谢物成功地区分了细胞聚合酶和HIV-RT，仅对后者具有选择性。未来对这种新化合物的化学合成5-三磷酸的研究（正在进行中）将确定其作用机制。由于大多数HIV RT抑制剂由于干扰细胞聚合酶而具有毒副作用，因此仅对HIV-RT而不对细胞聚合酶具有高选择性，这预示着该化合物作为一种无毒药物的前景良好。这种化合物相对容易制造，目前正在努力制造一种原核苷酸来避免HSV-tk的使用。另外，D-carba-T（碳环胸苷）作为ctk成功激活的药物的发现仍在继续。l -对映体和各种形式的核苷酸前药均显示对未转染HSV-tk的hiv感染细胞有活性。此外，这些化合物能够阻断培养细胞中多重耐药HIV-1载体的复制。最近已经合成了相应的D-和l -碳脱氧腺苷。我们关于使用双环[3.1.0]己烷核苷作为DNA成分的研究结果最近出现在报刊上（核酸杂志，2007,35,1978-1991）。通过对映的含南、含北的聚合物脱氧核苷酸的行为和退火性能的对比，表明DNA的小片段可以被塑造成特定的弯曲或曲率。这一特性正在被利用，目的是设计能够结合转录因子的短DNA片段。我们还完成了第一个凝血酶适体（g -四体）的合成，其中包含锁定的北和南鸟苷核苷。生物物理研究（CD和NMR）正在进行中

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. This effort has 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 N-MCT was also shown to be active against orthopoxvirus (Antiviral Res. 2007, 73, 69-77) and vaccinia virus infections (Antiviral Res. 2007, in press=doi:10.1016/j.antiviral.2007.06.005) in mice. N-MCT has been licensed and in vivo studies are continuing. Chemically synthesized N-MCT-5-triphosphate helped us demonstrate its activity as a delay chain terminator of DNA polymerases, and in particular, HIV reverse transcriptase (J. Mol. Biol. 2005, 345, 442-450). This property endowed the compound with the unique ability to overcome the excision mechanism of HIV resistance. However, N-MCT was inactive against HIV-infected cells unless the cells were transfected with herpes virus thymidine kinase (HSV-tk). The inability of N-MCT to be phosphorylated by cellular thymidine kinase (c-tk), prompted the search of new analogues that were investigated as substrates for kinases. This year we completed the synthesis of the D- and L-isomers (optical antipodes) as isomeric N-MCT analogues where both the fusion site of the cyclopropane ring and the critical 3-OH group were relocated to facilitate recognition by kinases. This approach resulted in a 2-fold increase in the formation of all three metabolites (mono-, di- and triphosphates) catalyzed by HSV-tk/thymidylate kinase and cellular diphosphate kinase). This work was recently published (J. Am. Chem. Soc. 2007, 129, 6216-6222). Unfortunately, the compound was still not phosphorylated by c-tk. Surprisingly, the L-isomer of this new analogue was the active enantiomer, and more surprisingly were two recent findings: (1) despite the high level anabolism to the 5-triphosphate, the compound was inactive against HSV-1 and HSV-2 infected cells; and (2) the compound was exquisitely active against HIV in HSV-tk- transfected cells. This means that new 5-triphosphate metabolite successfully discriminated between cellular polymerases and HIV-RT, being selective only against the latter. Future studies with the chemically synthesized 5-triphosphate of this new compound (in progress) will determine the mechanism of action. The high level of selectivity for just HIV-RT and not the cellular polymerases bodes well for this compound as a non-toxic agent since most HIV RT inhibitors have toxic side effects due to interference with cellular polymerases. This compound is relatively simple to make and efforts to make a pronucleotide to circumvent the use of HSV-tk are underway. Separately, the discovery of D-carba-T (carbocyclic thymidine) as a successfully kinased drug by ctk continues. Both the L-enantiomer and various forms of nucleotide prodrugs were shown to be active against HIV-infected cells without HSV-tk transfection. In addition, the compounds were able to block the replication of multi-drug resistant HIV-1 vectors in cultured cells. The corresponding D- and L-carbadeoxyadenosines have been recently synthesized. Our results on the use of bicyclo[3.1.0]hexane nucleosides as DNA components appeared recently in press (Nucleic Acids Res. 2007, 35, 1978-1991). The contrasting behavior and annealing properties of antipodal North- and South-containing olidodeoxynucleotides shows that small segments of DNA can be molded with specific bending or curvature. This property is being exploited with the aim of designing short DNA segments capable of binding to transcription factors. We have also completed the first synthesis of a thrombin aptamer (G-tetrad) incorporating locked North and South guanosine nucleosides. Biophysical studies (CD & NMR) are in progress