gamma-modified triphosphates of nucleoside analogues as potential antivirals and lipophilic prodrugs therof

核苷类似物在化疗中具有重要的抗病毒作用。然而，使用这些化合物也有两个主要的缺点。首先，它们需要细胞内代谢形成三磷酸核苷，这是最终的活性代谢物。其次，它们不仅可以与病毒聚合酶相互作用，还可以作为人类聚合酶的底物，这可能导致严重的毒副作用。在这里提出的项目中，我们的目标不仅是绕过三磷酸形成过程中经常限速的代谢，而且还开发了一种全新的方法，通过使用γ修饰的核苷类似物三磷酸作为假定的抗病毒药物，可能会改善病毒和人类聚合酶之间的底物选择性。在这里，我们想从三个不同的方面进行工作：1。我们将合成和研究γ修饰核苷类似物三磷酸作为病毒聚合酶的潜在底物，并与人的聚合酶进行比较。伽马修饰应该是这样构造的，即这种修饰是一个不可切割的片段，它共价地附着在三磷酸单元上。因此，这个片段也阻止了三磷酸核苷的三磷酸单元的酶降解。在最近的研究中，我们发现已知抗病毒药物3-脱氧-2,3-二脱氢胸腺嘧啶（d4T）的γ -酰基苯基修饰的三磷酸作为hiv逆转录酶的底物，导致d4TMP合并到dna链中。相反，这种γ修饰的三磷酸不是人聚合酶β的底物。有趣的是，母体化合物d4TTP也是dna聚合酶β的底物。由于这种选择性的差异，这为使用γ修饰的三磷酸类似物提供了一个全新的选择。这在可能的毒副作用的背景下是非常重要的，这些毒副作用是由三磷酸与细胞聚合酶相互作用引起的。作为γ修饰，我们一方面提出了不可切割的酰基苄基（酮）基团，这与以前使用的生物可逆性酰基苯基基团非常相似，另一方面提出了亲脂性烷基残基。由于三磷酸部分的负电荷，预计γ修饰的三磷酸不会被细胞吸收。因此，这些γ修饰的三磷酸类似物的亲脂性前药将被开发。我们的目标是引入酶可切割的掩蔽基团，例如酰基氧基苯基。水解机制的研究、细胞提取物等生物介质的稳定性研究和抗病毒试验将指导我们优化前药分子。将通过结合荧光双环核苷类似物（BCNA）和细胞孵育试验来研究新的γ修饰核苷三磷酸前体药物的细胞摄取。

Nucleoside analogues have significant impact as antiviral agents in chemotherapy. However, there are also two major drawbacks associated with the use of these compounds. First, they require an intracellular metabolism to form the nucleoside triphosphate which is the ultimately active metabolite. Second, they can not only interact with viral polymerases but can also act as substrates of human polymerases which might result in severe toxic side effects. With the project presented here we aim not only to bypass the often rate-limiting metabolism in the triphosphate formation but also to develop a entirely new approach resulting potentially in an improvement in the substrate selectivity between viral and human polymerases by using gamma-modified nucleoside analogue triphosphates as putative antiviral agents. Here we would like to work on three different aspects:1. We will synthesize and study gamma-modified nucleoside analogue triphosphates as potential substrates of viral polymerases in comparison with the human polymerases. The gamma modification should be constructed as such, that this modifcation is a non-cleavable moiety, which is covalently attached to the triphosphate unit. By that, this moiety prevents also the enzymatic degradation of the triphosphate unit of the nucleoside triphosphate. In recent studies we showed that the gamma-acyloxybenzyl-modified triphosphate of the known antiviral 3-deoxy-2,3-didehydrothymidine (d4T) was acting as a substrate of HIVs reverse transcriptase which led to the incorporation of d4TMP into DNA-strands. In contrast, this gamma-modified triphosphate was not a substrate of human polymerase beta. Interestingly, the parent compound d4TTP was also a substrate of DNA-polymerase beta. Due to this difference in selectivity this offers now a completely new option for the use of gamma-modified triphosphate analogues. This is of tremendous importance in the context of possible toxic side effects which are caused by the interaction of the triphosphate with the cellular polymerases. As gamma-modifications we propose on the one hand non-cleavable acylbenzyl-(ketone) moieties which resemble closely the previously used bioreversible acyloxybenzyl-groups and on the other hand lipophilic alkyl-residues.2. It is expected that the gamma-modified triphosphates will not be up-taken by cells due to the negative charges at the triphosphate-moiety. Therefore, lipophilic prodrugs of these gamma-modified triphosphate analogues will be developed. We aim to introduce enzyme-cleavable masking groups, e.g acyloxybenzyl-groups. Studies to elucidate the hydrolysis mechanism, stability studies in biological media such as cell extracts and antiviral tests will guide us to optimized prodrug molecules.3. The cellular up-take of the new prodrugs of the gamma-modified nucleoside triphosphates will be studied by incorporating the fluorescent bicyclic nucleoside analogue (BCNA) and cell incubation assays.