Efficient synthesis of antiviral agent uprifosbuvir enabled by new synthetic methods†

An efficient route to the HCV antiviral agent uprifosbuvir was developed in 5 steps from readily available uridine in 50% overall yield. This concise synthesis was achieved by development of several synthetic methods: (1) complexation-driven selective acyl migration/oxidation; (2) BSA-mediated cyclization to anhydrouridine; (3) hydrochlorination using FeCl3/TMDSO; (4) dynamic stereoselective phosphoramidation using a chiral nucleophilic catalyst. The new route improves the yield of uprifosbuvir 50-fold over the previous manufacturing process and expands the tool set available for synthesis of antiviral nucleotides.

0-2% LCAP of mono-piv uridine and 3-5% LCAP tris-piv uridine. Although chemically stable, the toluene solutions may begin to show signs of solid precipitation if held over several days. The precipiated solid was pure 10. Analytical samples of 10/11 were obtained via flash chromatography purification (EtOAc/hexanes) and characterized as a mixture. Compound 10: 1  . Part II -Isomerization of 10/11 mixture via BF3 complex of 11. To a vessel under N 2 was charged 10/11 toluene solution from above (275 L×305 mg/mL = 83.8 kg, 203.3 mol), followed by 6X by volume toluene (364 L) to adjust to a 12X by volume toluene solution relative to uridine (or 7X by volume relative to bis-Piv uridines at 130-150 mg/g). The batch temperature was adjusted to 20-25 °C, then 1.22 equiv BF 3 •OEt 2 (35.2 kg, 248 mol) was charged over > 30 min. The resultant slurry was aged at 40 °C for 10 h, then cooled to 20-25 °C (11 selectively formed a complex with BF 3 and precipitated from the reaction mixture. The formation of this complex was inhibited by residual water and pyridine. Both must be individually controlled below 300 ppm; Target ratio of 11/10 > 98 : 2). After adjusting the batch temperature to 0-10 °C, H 2 O (385 kg) was charged, then stirred at 0-10 °C for 1 h. The mixture was allowed to settle, then separated and the aqueous bottom layer discarded. Again, to the organic layer was charged H 2 O (385 kg), stirred at 0-10 °C for 1 h, then settled and cut and discarded the aqueous bottom layer. Typical yield of 11 in the organic phase was >99% for the step or >90% from uridine. The bis-Piv-uridine 11/10 solution will maintain > 98 : 2 ratio upon storage for at least 4 weeks when held at 0-5 °C. Typical water content of this solution is ~0.4 wt%. Typical concentration of 11 solution at this point is 130-145 mg/g, which would serve as the basis for all charges in the oxidation. Part III -TEMPO/AcOOH Oxidation of 11 to ketone 12. To a vessel was charged bis-Piv-uridine 11 solution from above (assayed 38.4 kg, 93.1 mol in a 13.5 wt% solution), then adjusted the temperature of the content to 0-5 °C. To a second vessel was charged 3.3X by volume toluene (127 L), adjusted temperature to 20-25 °C and initated a sufficient N 2 cross-sweep so as to keep O 2 composition within a safe operation level. To the second vessel was charged 0.166 equiv tetrabutylammonium bromide (TBAB) (4.98 kg, 15.45 mol), followed by 0.25 equiv TEMPO (3.6 kg, 23.3 mol), then aged for 15-30 min to make sure that TEMPO dissolved, then cooled the mixture to -10 °C. TBAB is only partially soluble in the reaction initially but completely dissolved as the reaction progressed. 1.5 equiv 40 wt% peracetic acid (27.2 kg, 143 mol) at 5 °C was added to the second vessel over 12-16 h at -10 °C. After 3.6% of the total AcOOH was charged (over 30 min), stopped the addition and aged the contents for 1 h. The remainder AcOOH and the 11 solution from the first vessel was charged simultaneously over 13.5 h at -10 °C, then rinsed with 12 L toluene. The batch was aged at -10 °C for 3 h, then sampled for reaction completion. If the reaction conversion is < 97%, aged the batch for additional 5 h. If conversion is > 97% proceeded to charge 0.332 equiv di-n-octylsulfide (8 kg, 30.9 mol) at -10 °C to quench any remaining peracetic acid. The reaction slurry was stirred for 0.5 h, warmed the batch to 0 °C and aged for 2-3 h at 0 °C until hydrogen peroxide < 100 ppm. The batch temperature was adjusted to 20 °C, and the reaction slurry was stirred for 2-3 h until ≤ 8 mg/g ketone 12 in filtered supernatant. The slurry mixture was filtered, and the cake was displacement washed with toluene (77 L) and water (77 L). The cake was slurry washed with a combined mixture of toluene (58 L) and water (58 L) for 1 h with slow agitation (4-8 rpm) before filtering. The cake was displacement washed with water (77 L), and toluene (77 L), then the wet cake was dried under vacuum at 30-35 °C for 12-48 h. Isolated yield of 12 was 34.2 kg (90% yield from 11, 83% yield from 5) with > 98% LCAP purity, containing < 0.2 wt% water, < 890 ppm toluene and < 5000 ppm acetic acid. Both the keto and keto-hydrate forms were observed by reversed phase LC/MS, whereas only the keto form was observed in CDCl 3 by NMR. Preparation of β-hydroxysilane 13 from ketone 12. A solution of ketone 12 (6.25 g, 1 equiv) in cyclopentyl methyl ether (CPME) (30 mL) was cooled to 0 °C, and to the cooled solution was added a 1 M solution of TMSCH 2 MgCl (42.6 mL, 2.5 equiv) in diethyl ether over a 15-30 min period. The resulting reaction was allowed to stir overnight at room temperature, then 2N HCl was then added. The resulting reaction was allowed to stir for 10 min at room temperature, the organic layer was separated and washed with water, brine, dried over MgSO 4 , filtered, concentrated, and purified by silica gel chromatography (hexane-ethyl acetate) to provide 6.90 g (91% yield) of alcohol 13 as foamy solid. 1   Preparation of olefin 15 from β-hydroxysilane 13. To a solution of alcohol 13 (1 g, 1 equiv) in dry acetonitrile was added trifluoroacetic anhydride (0.75 mL, 3 equiv), pyridine (0.48 mL, 3 equiv) and DMAP (49 mg, 0.2 equiv) and the resulting reaction was allowed to stir at room temperature for 15 h. Potassium fluoride (0.45 g, 3.5 equiv) was then added and the resulting reaction was heated to 70 °C and allowed to stir at this temperature for 24 h. MTBE was then added, followed by H 2 O. The organic layer was separated and washed with another portion of water, followed by an aqueous solution containing 2 equiv of K 2 CO 3 and brine. The final organic layer was concentrated in vacuo to provide crude olefin 14 (720 mg, 88% yield) as a solid. Compound 14 (0.30 g, 1 equiv) was dissolved in a mixture of MeOH (1 mL) and THF (1 mL), treated with K 2 CO 3 (0.305 g, 3 equiv) and allowed to age for 20 h at 40 °C. The reaction was then concentrated in vacuo and solvent switched to 2-MeTHF (6 mL) and treated with 4M HCl (2 equiv) in dioxane.
The resulting slurry was concentrated in vacuo to half of its volume, filtered, and the filtrate was concentrated to provide 165 mg (94% yield) of diol 15 as a white solid. The NMR spectra are in accordance with the previous report of the compound. 1 Hydrochlorination of olefin 15 to compound 4. A solution of olefin 15 (1 g, 1 equiv) and iron (III) chloride (1.35 g, 2 equiv) in water (85 mL) at 4-5 °C was deoxygenated with N 2 . Phenylsilane (3.2 mL, 6 equiv) was added followed by MeCN (1.5 mL). The reaction was allowed to stir to room temperature for 15 h. More of iron (III) chloride (0.676 g, 1 equiv) was added, stirred at rt for 24 h. More of phenylsilane (0.1 mL, 0.2 equiv) was added and the mixture was stirred for 5 h at rt. The reaction mixture was then diluted with 1:1 MTBE:hexanes (40 mL), stirred rigorously and allowed to settle. The aqueous layer was separated, treated with EDTA-disodium salt dihydrate (4.65 g, 3 equiv), saturated with NaCl, and the product was extracted with 2-MeTHF (3×50 mL). The combined organic extracts were washed with brine and concentrated in vacuo, replacing 2-MeTHF with iPrOAc.
The product was crystallized from iPrOAc-MTBE, filtered, and dried to provide 950 mg (82% yield) of compound 4 as a solid. The NMR spectra are in accordance with the previous report of the compound.

Preparation of 16 by methylation of 12 with MeMgBr / MnCl2. To a vessel under nitrogen was charged anhydrous
anisole (450 kg) and anhydrous manganese dichloride (particle size < 100 µm) (89 kg, 706 mol, 3.2 equiv, pre-milled to small particle size of D90 = 32 µm). The slurry was cooled to 0 °C and methyl magnesium bromide (275 kg, 3.0 M in 2-MeTHF, 657 mol, 3 equiv) was slowly charged while keeping at 0 °C. The mixture was agitated for 6 h (note: aging is important for generation of MeMnCl) at 20-30 °C and then cooled to -15 °C followed by charge of 2-MeTHF (388 kg). Solid ketone 12 was charged at -15 -0 °C slowly over 12 h (note: lower temperature and slow charge are important for good d.r.), in total of 91.5 kg solid (90.0 kg after correcting for wt%, 219 mol). After aging for additional 3 h, HPLC analysis indicated complete consumption of ketone 12 (0.1% left) and the product d.r. of 95-97:5-3 (19-32 : 1). The batch was inverse quenched into a mixture of 35% HCl (123 kg), water (1100 kg) and 2-MeTHF (150 kg) 0-10 °C. The reaction vessel was rinsed with additional 2-MeTHF (360 kg) and combined into the quench. After layer cut, the organic layer was washed with 15% brine (500 kg), then 20% brine (500 kg). The organic layer was then concentrated under vacuum to about 8X volume affording 764 kg of solution at 11.4% wt of the 16, corresponding to 81.7 kg desired stereoisomer 16 or 93% yield. This solution was used directly in the next step.

Formation of anhydrouridine 19 from alcohol 16.
To a crude solution of alcohol 16 (9.37 g assay, 22.0 mmol, 1.0 equiv) in anisole-2-Me-THF (~55 mL total volume, <30 wt% Me-THF) obtained in the previous step, was added 37% aq HCl (0.018 mL, 1 mol%) and the mixture was warmed to 75 °C. N,O-Bis(trimethylsilyl)acetamide (13.4 g, 3.0 equiv) was added slowly over 1 h and stirred for additional 8 h at 75 °C. The reaction temperature was adjusted to 60 °C and MeOH (100 mL) was added followed by DBU (2.51 g, 0.75 equiv). After 90 min at 65 °C, the batch was seeded and stirred for additional 10 h. The batch was cooled to <40 °C, concentrated to 75 mL volume, solventswitched at constant volume with 75 mL iPrOH, and cooled to 10 °C. The slurry was filtered and the cake was washed with 1:1 mixture of anisole/iPrOH (30 mL) and iPrOH (90 mL). The cake was dried under nitrogen to provide anhydrouridine 19 (4.62 g, 87%). The NMR spectra of compound 19 are in accordance with the previous report of the compound. 3 Analytical sample of the TMS ether impurity 20 could be isolated by flash chromatography on silica gel using hexane-ethyl acetate as eluent. 1    Na 2 SO 4 (0.504 kg), mixed, and then cut away the aqueous layer. Added CUNO-5 carbon (45 g), filtered, and rinsed the cake with 2-MeTHF (2.9 L). The filtrate was concentrated to 6.3 L volume at 55 °C with vacuum distillation, then distilled with methyl isobutyl ketone (4.5 L) at 55 °C and 6.3 L constant volume. The batch was seeded and distillation with methyl isobutyl ketone (8.1 L) was continued at 55 °C and 6.3 L constant volume. n-Heptane (1.8 L) was added over 2 h at 55 °C, the slurry was cooled to 20 °C and filtered. The cake was washed with a 5:2 mixture of methyl isobutyl ketone and n-heptane (3×1.8 L) and dried with nitrogen sweep to provide 881 g (85% yield) of compound 4 as a solid. The NMR spectra are in accordance with the previous report of the compound. 2 Preparation of alanine ester 21b solution. To a slurry of D-alanine (22 kg, 1.0 equiv) in iPrOH (123 L) was added TMS-Cl (40.2 kg, 1.5 equiv) over 30 min keping the temperature below 50 °C. The mixture was stirred at 70 °C for 12 h. Cooled to 45 °C and Et 3 N (2.6 kg, 0.11 equiv) was added to pH 3-5. The batch was concentrated to 88 L volume while maintaining batch temperature at 50 °C (~100 torr). Added iPrOAc (44 L) and solvent switched at constant 130 L volume at 50 °C using more iPrOAc (450 L). Added iPrOAc (79 L) followed by Et 3 N (25.1 kg, 1.0 equiv) to pH>9.
The slurry was cooled to 0 °C, filtered, and the cake was washed with iPrOAc (123 L). The combined filtrates were concentrated to 50-60 wt% using wiped film evaporator at 40 mm Hg and 40 °C on the jacket. The concentrated product was then distilled using wiped film evaporator at 10 mmHg and 65 °C on the jacket to provide 29.2 kg (80%) assay yield of compound 21b as ~50 wt% solution in iPrOAc that was stored at +5 °C and used directly in the next step.

Preparation of chlorophosphoramidate 22 solution.
In the first vessel, phenyl dichlorophosphate (22.4 kg, 1.0 equiv) was dissolved in dry iPrOAc (76 L) and the solution was cooled to -20 °C. In the second vessel, a mixture of alanine ester 21b (1.02-1.05 equiv), prepared above, and Et 3 N (11.3 kg, 1.05 equiv) was cooled to -20 °C and then added over 2 h to the first vessel at < -10 °C. After 1 h at < -10 °C, the slurry was filtered under nitrogen and the cake was washed with dry iPrOAc (76 kg). The filtrates were combined and concentrated to ~35 wt% using vacuum distillation at 20 °C to provide 29.2 kg (90%) assay yield of 22 that was used directly in the next step.
The chlorophosphoramidate 22 solution in iPrOAc prepared above (1.2 equiv) was added over 1 h maintaining the temperature at -10 °C. The mixture was stirred at -10 °C for 24 h. iPrOAc (80 mL) was added and the mixture was warmed to 0 °C. A 10% aq solution of NaHSO 4 (40 mL) was added and the mixture was stirred at 30 °C for 1 h. The lower phase was cut away. An aqueous solution of 5% NaHCO 3 and 5% Na 2 SO 4 (60 mL) was added and the mixture was stirred at 50 °C for 10 h. The lower phase was cut away. The organic phase was washed with 10% aq NaCl (60 mL) at 50 °C and then concentrated under vacuum at 50 °C to 160 mL volume. While maintaining the batch volume at 160 mL, the mixture was distilled with iPrOH (400 mL). The mixture was warmed to 70 °C, filtered hot, and the