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；以及(2)根据我们对其生化作用机制的了解，开发可能的抗肿瘤/抗病毒药物。这一努力的结果是发现了N-甲基卡巴夫定(N-MCT)，这是一种对疱疹病毒1和2以及与卡波西肉瘤有关的人类疱疹病毒8具有活性的化合物。今年，N-MCT还被证明对小鼠的正痘病毒(抗病毒研究报告2007年，73，69-77)和牛痘病毒感染(抗病毒研究报告2007，出版：DOI：10.1016/j.antiviral.2007.06.005)也是有效的。N-MCT已经获得许可，体内研究仍在继续。化学合成的N-MCT-5-三磷酸帮助我们展示了它作为DNA聚合酶延迟链终止子的活性，特别是HIV逆转录酶(J.Mol.比奥尔。2005、345、442-450)。这一性质赋予了该化合物独特的能力，以克服艾滋病毒耐药性的切除机制。然而，N-MCT对HIV感染的细胞没有活性，除非细胞被疱疹病毒胸苷激酶(HSV-tk)感染。N-MCT不能被细胞胸苷激酶(c-tk)磷酸化，促使人们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，作为异构体的N-MCT类似物，其中环丙烷环的融合点和关键的3-OH基团都被重新定位，以便于被激动酶识别。这一方法导致由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。这项工作是最近出版的(J.Am化学。SoC。2007、129、6216-6222)。不幸的是，该化合物仍未被c-tk磷酸化。令人惊讶的是，这种新类似物的L异构体是活性的对映体，更令人惊讶的是最近的两个发现：(1)尽管对5-三磷酸有高水平的合成代谢，但该化合物对单纯疱疹病毒-1和单纯疱疹病毒-2感染的细胞没有活性；(2)该化合物在单纯疱疹病毒-tk感染细胞中对艾滋病毒具有极强的活性。这意味着新的5-三磷酸代谢产物成功地区分了细胞聚合酶和HIV-RT，只对后者具有选择性。未来对这种新化合物的化学合成的5-三磷酸的研究(正在进行中)将确定其作用机制。仅对HIV-RT而不是细胞聚合酶具有高水平的选择性，这对该化合物作为无毒药物来说是个好兆头，因为大多数HIV RT抑制剂都有毒副作用，因为干扰细胞聚合酶。这种化合物的制造相对简单，目前正在努力制造原核苷酸以绕过HSV-tk的使用。另外，CTK继续发现D-Carba-T(碳环胸苷)作为一种成功的激活剂。L对映体和不同形式的核苷酸前药对未转导单纯疱疹病毒-tk的HIV感染细胞均有抑制作用。此外，这些化合物能够阻止多药耐药HIV-1载体在培养细胞中的复制。最近合成了相应的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组分的结果最近发表在PRESS(核酸研究报告2007，35,1978-1991)上。对极北和南的脱氧核苷酸的对比行为和退火性表明，小片段的DNA可以用特定的弯曲或曲率来模塑。人们正在利用这一特性，目的是设计能够与转录因子结合的短DNA片段。我们还完成了首次合成凝血酶适配子(G-四元体)，其中包括锁定的南北鸟苷核苷。生物物理研究(CD和核磁共振)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