Discovery of a Series of 2'-α-Fluoro,2'-β-bromo-ribonucleosides and Their Phosphoramidate Prodrugs as Potent Pan-Genotypic Inhibitors of Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) nucleoside inhibitors display pan-genotypic activity, a high barrier to the selection of resistant virus, and are some of the most potent direct-acting agents with durable sustained virologic response in humans. Herein, we report, the discovery of β-d-2'-Br,2'-F-uridine phosphoramidate diastereomers 27 and 28, as nontoxic pan-genotypic anti-HCV agents. Extensive profiling of these two phosphorous diastereomers was performed to select one for in-depth preclinical profiling. The 5'-triphosphate formed from these phosphoramidates selectively inhibited HCV NS5B polymerase with no inhibition of human polymerases and cellular mitochondrial RNA polymerase up to 100 μM. Both are nontoxic by a variety of measures and display good stability in human blood and favorable metabolism in human intestinal microsomes and liver microsomes. Ultimately, a preliminary oral pharmacokinetics study in male beagles showed that 28 is superior to 27 and is an attractive candidate for further studies to establish its potential value as a new clinical anti-HCV agent.

Figure 1.

Selected potent HCV antiviral agents and targeted β-d-2′-deoxy-2′-β-bromo-2′-α-fluoro nucleoside prodrugs.

Figure 2.

α/β anomer assignment for nucleoside 12.

Figure 3.

ORTEP drawing of nucleoside 12 from X-ray crystal analysis.

Figure 4.

Cellular uptakes of 27, 28, and SOF in Huh-7 cells and primary human hepatocytes (pmol/10⁶ cells).

Figure 5.

Cellular egress profiles of prodrug 27, 28, and SOF in Huh-7 cells and primary human hepatocytes (pmol/10⁶ cells).

Scheme 1.. Synthesis of 2-Br,2-F Lactone and Mesylate 10^a

^aReagents and conditions: (a) TBDPSCl, imidazole, dimethylformamide, room temperature (rt), 24 h, 85%; (b) NFSI, LiHMDS, tetrahydrofuran (THF), −78 °C, 1 h, 29%; (c) NBS, LiHMDS, THF, −78 °C, 40 min, β-anomer 29% and α-anomer 31%; (d) Li(t-BuO)₃AlH, THF, 2 h, 0 °C to rt, 90%; (e) trimethylsulfonyl chloride (MsCl), Et₃N, dichloromethane (DCM), 0 °C to rt, 1 h, 99%.

Scheme 2.. Syntheses of 2′-α-F,2′-β-Br Pyrimidine Nucleosides and Their Corresponding Prodrugs^a

^aReagents and conditions: (a) (i) Uracil or N⁴-benzoyl cytosine, N,O-bis(trimethylsilyl)acetamide (BSA), 1,2-dichloroethane (DCE), 60 °C, 30 min; (ii) TMSOTf, DCE, 80 °C, 5 h, 55–57%; (b) 1 M tetra-N-butylammonium fluoride (TBAF) in THF, THF, 0 °C to rt, 1 h, U analog: β-anomer 21%, α-anomer 46%, C analog: β-anomer 24%, α-anomer: 46%; (c) NMI, THF, rt, 4 h, 45%; (d) NH₃, MeOH, overnight, 93%; (e) 13, t-BuMgCl, THF, 0 °C to rt, 3 h, 21%.

Scheme 3.. Syntheses of 2′-α-F,2′-β-Br Adenosine Nucleoside 21 and Its Prodrug 22^a

^aReagents and conditions: (a) bis-N-Boc adenine, diisopropyl azodicarboxylate (DIAD), triphenylphosphine (PPh₃), THF, rt, 24 h, 39%; (b) 1 M TBAF in THF, THF, 0 °C to rt, 1 h, β-anomer 44%, α-anomer: 25%; (c) 1 M BCl₃, DCM, −78 °C to rt, 1 h, 69%; (d) (i) 13, NMI, THF, 0 °C to rt, 3 h; (ii) 50% TFA-H₂O, 0 °C to rt, overnight, 40% over two steps.

Scheme 4.. Syntheses of 2′-α-F,2′-β-Br Guanine Nucleoside 25 and Its Prodrug 26^a

^aReagents and conditions: (a) 6-O-Bn-bis-N-Boc guanine, DIAD, PPh₃, THF, 0 °C to rt, 24 h, 40%; (b) 1 M TBAF in THF, THF, 0 °C to rt, 1 h, β-anomer 60%, α-anomer: 19%; (c) 1 M BCl₃, DCM, −20 °C to rt, 2 h, 65%; (d) (i) 13, NMI, THF, 0 °C to rt, 3 h; (ii) 1 M BCl₃, DCM, −78 °C to rt, 3 h, 20% over two steps.

Scheme 5.. Syntheses of Diastereomers 27 and 28^a

^aReagents and conditions: (a) t-BuMgCl, THF, −5 to 4 °C, 16 h, 68%.