This is the first report describing the synthesis and conformation of methanocarba nucleosides incorporating an endo (β-face) cyclopropyl at the 2′,3′ position of 2′,3′-didehydro-2′,3′-dideoxy carbocyclic nucleosides. These nucleoside isosteres have been shown to exist in a unique extreme eastern conformation. This prediction was confirmed by x-ray crystallography and high resolution NMR spectroscopy. As expected, the methanocarba adenosine compound was neither a substrate nor an inhibitor of adenosine deaminase. However, some of the compounds synthesized demonstrated moderate antiviral activity against HSV-1. The methanocarba adenosine and its triphosphate form were evaluated as inhibitors of HIV-1 reverse transcriptase.
Bibliographical noteFunding Information:
Purified recombinant HIV-1 reverse transcriptase was obtained from the University of Alabama at Birmingham, Center for AIDS Research, Gene Expression Core Facility (supported in part by the NIH Centers for AIDS Research program grant P30 AI27767). For the study of RNA transcription, HIV-1 reverse transcriptase activity was measured in 50-mL reactions containing 50 mM Tris (pH 8.0), 50 mM KCl, 10 mM MgCl2, 4mM b-mercaptoethanol, 3% glycerol, 1 mg/mL bovine serum albumin, 3.33 mg/mL of primed 16S rRNA from E. coli, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP, and 0.25 mM [33P] dATP (the Km concentration). The primer was annealed to the template at a ratio of 3 to 1 as described previously. After incubation, the DNA in each sample was precipitated onto glass fiber filters using a 5% trichloroacetic acid solution containing 10 mM pyrophosphate. These filters were batch washed and counted for radioactivity. Assays were done in duplicate. An initial range finding experiment was first conducted. The data from this experiment was then used to narrow the concentration range for each compound, which was then tested in smaller increments. The experiments were repeated in duplicate and these two experiments were used to calculate the average IC50 values.
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