Synthesis of 3'-amino-3'-deoxyguanosine and 3'-amino-3'-deoxyxyloguanosine monophosphate HepDirect prodrugs from guanosine.

The synthesis of 3'-amino-3'-deoxyguanosine and 3'-amino-3'-deoxyxyloguanosine monophosphate HepDirect prodrugs from guanosine is reported. Initial incorporation of N,N-dibenzylformamidino protection of the C2-amino of guanosine masked the reactivity of that group and simplified purification of subsequent analogues. The first key intermediate, 9-(2,5-bis-O-tert-butyldimethylsilyl-beta-D-ribofuranosyl)-2-N-(N,N-dibenzylformamidino)guanine (3a), was prepared in 60% yield after recycling of the undesired 3',5'-bis-O-protected byproduct (4a) by simple equilibration in methanol to a mixture of the two bis-O-protected compounds. Thus, protected, the 3'-position was manipulated to form the 3'-deoxyribo- or 3'-deoxyxylo-3'-azido derivatives (9 or 16, respectively). Further selective manipulations provided the cis-5'-monophosphate (3-chlorophenyl)-1,3-propanyl diester prodrugs (HepDirect prodrugs), 15 and 21. These HepDirect prodrugs were demonstrated to activate to their respective NTPs in rat hepatocytes.

High-throughput synthesis of HepDirect prodrugs of nucleoside monophosphates.

A high-throughput phosphoramidite method for HepDirect prodrug synthesis was optimized on seven representative nucleosides, adenosine, inosine, guanosine, uridine, cytidine, AICA-riboside, and thymidine, each on a 5 mg scale. The variables optimized included (1) reaction time, (2) reaction temperature, (3) activating agent, (4) solvent, (5) purification method, and (6) stoichiometry. Preparative HPLC with mass-based fraction collection and yield determination from an ELSD standard curve enabled high-throughput. The optimized conditions for the representative nucleosides required 6 mol equiv of phosphoramidite to nucleoside and resulted in an average HPLC determined yield of 31 +/- 14% and HPLC purity of 93 +/- 3%.

High-throughput five minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries.

The Vorbrüggen glycosylation reaction was adapted into a one-step 5 min/130 degrees C microwave assisted reaction. Triethanolamine in acetontrile containing 2% water was determined to be optimal for the neutralization of trimethylsilyl triflate allowing for direct MPLC purification of the reaction mixture. When coupled with a NH3/methanol deprotection reaction, a high-throughput method of nucleoside library synthesis was enabled. The method was demonstrated by examining the ribosylation of 48 nitrogen containing heteroaromatic bases that included 25 purines, four pyrazolopyrimidines, two 8-azapurines, one 2-azapurine, two imidazopyridines, two benzimidazoles, three imidazoles, three 1,2,4-triazoles, two pyrimidines, two 3-deazapyrimidines, one quinazolinedione, and one alloxazine. Of these, 32 yielded single regioisomer products, and six resulted in separable mixtures. Seven examples provided inseparable regioisomer mixtures of -two to three compounds (16 nucleosides), and three examples failed to yield isolable products. For the 45 single isomers isolated, the average two-step overall yield +/- SD was 26 +/- 16%, and the average purity +/- SD was 95 +/- 6%. A total of 58 different nucleosides were prepared of which 15 had not previously been accessed directly from glycosylation/deprotection of a readily available base.