the clinical phase than that provided by the alternative
chemical approach.
concentrated in vacuo to afford the benzyloxycarbonyl
alanine trifluoroester as a solid in quantitative yield. 1H NMR
(400 MHz, CDCl3) δ 1.44 (d, 3H, J ) 6.40 Hz), 4.18-4.35
(m, 3H), 5.11 (d, 2H part of ABC system), 5.21 (br s, 1H),
7.33-7.36 (m, 5H). 13C NMR (100 MHz, CDCl3) δ 19.3,
50.7, 62.0 (2JCF ) 36.7 Hz), 68.3, 124.3 (1JCF ) 124.3),
129.13, 129.3, 129.6, 137.1, 156.6, 172.6.
Experimental Section
General. Commercial reagents and solvents of the highest
purity available were used without further purification. For
large-scale reactions, the water content in THF was <0.02%
(determined by Karl Fisher analysis). The di-tert-butyldi-
carbonate (mp 23 °C) was stored between 30 and 35 °C for
melting before using. Chirazyme L-2 enzyme (lipase B from
Candida antarctica immobilized on a microporous acrylic
resin) was purchased from Roche Molecular Biochemicals,
Germany.
TLC was performed on Analtech precoated HLF-254
plates and visualized using light or applying a solution of
AMC (6.25 g of ammonium molybdate, 2.5 g of cerium (IV)
sulfate in 250 mL of 10% aqueous H2SO4) followed by
heating.
Chiral HPLC was performed using a Chiralpak AS
column (4.6 mm × 250 mm) (Chiral Technologies, Exton,
PA) eluted with 35% EtOH/heptane mobile phase (40 °C,
0.7 mL/min; UV detection at 210 nm).
Reversed-phase HPLC was performed on a YMC ODS-
AQ S5 120A column (YMC Inc, Wilmington, NC) 4.6 mm
× 150 mm at 1 mL/min flow rate using the following
conditions: 100% solvent A for 9.0 min, followed by a linear
gradient to 100% solvent B in 16 min (A ) 7.0 mM Na2-
SO4 in 0.02% aqueous H3PO4; B ) 85% MeCN and 15%
solvent A), at a flow rate of 1.0 mL/min and 25 °C; UV
detection at 220 nm.
Carbamic Acid [2-{4-[3S-(Aminocarbonyl)-1H-1,2,4-
triazol-1yl]-1-r-D-ribofuranosyl}-1-methyl-2-oxyethyl]-
phenyl Methyl Ester, 4. One-Pot Acetylation Procedure.
Cbz-Ala-OH (27.5 g, 0.12 mol) in THF (287 mL), pyridine
(7.7 g, 0.1 mol), acetone oxime (9.5 g, 0.13 mol), and di-
tert-butyl carbonate (29.6 g, 0.14 mol) were placed in a
jacketed flask and stirred under nitrogen at 22 °C for 20 h.
THF (550 mL), ribavirin (20 g, 0.082 mol), and lipase
Chirazyme L-2 (16 g, 20 g/L) were then added, and the
mixture was heated at 60 °C with stirring. After 45 h the
enzyme was removed by filtration, and the solution was
concentrated and dried under vacuum overnight to give a
sticky oily solid. This solid was dissolved in THF (350 mL),
and MTBE (600 mL) was added in portions under reflux.
The product precipitated gradually as the refluxing pro-
ceeded. Following filtration 31.5 g (85.6%) of 4 was
1
obtained. H NMR (400 MHz, DMSO-d6) δ 1.23 (d, 3H, J
) 7.6 Hz), 4.13-3.38 (m, 3H), 4.36-4.29 (m, 3H), 5.01 (d,
2H, JABC ) 2.0 Hz), 5.42 (d, 1H, J ) 6.0 Hz), 5.70 (d, 1H,
J ) 5.20 Hz), 5.91 (d, 1H, J ) 3.20), 7.33-7.28 (m, 5H),
7.66 (s, 1H), 7.77 (d, 1H, J ) 7.60), 7.88 (s, 1H), 8.83 (s,
1H). 13C (100 MHz, DMSO-d6) δ 18.56, 51.01, 66.31, 67.09,
71.96, 75.82, 83.19, 93.04, 129.19, 129.28, 129.81, 138.33,
146.80, 146.94, 157.24, 158.95, 161.78, 174.11. FAB HRMS
calcd for C29H24N5O8 (MH+) m/z 450.1625, found 450.1623.
Large-Scale Synthesis of 4. Cbz-Ala-OH (24.8 kg, 111.1
mol) acetone oxime (8.5 kg, 116.3 mol), di-tert-butyl
dicarbonate (28.3 kg, 129.7 mol), and pyridine (6.9 kg, 87.2
mol) were dissolved in THF (260 L) and stirred at 22 °C.
After 24 h ribavirin (18.0 kg, 73.7 mol), Chirazyme L-2 (14.4
kg), and THF (495 L) were added, and the mixture was
agitated at 55-60 °C for 24 h. Celite was added to the reactor
to assist the filtration, the reaction mixture was filtered, the
filtrate was clarified by recycling through a 15 µm in-line
cartridge filter and concentrated to about 216 L by atmo-
spheric pressure distillation. After cooling to 40-50 °C
MTBE (250 L) was slowly added. Following precipitation,
the mixture was cooled to 25 °C over 1 h, and the precipitated
product was filtered. The filter-cake was dried under vacuum
at 50 °C for 12-18 h. It was then resuspended in 15 vols of
water and stirred at 45 °C for about 7 h to remove unreacted
ribavirin. The suspension was then filtered and dried in an
air-draft oven at about 45 °C to give 27.5 kg of 4 (83% yield,
99.3% purity, >99.9% de).
NMR spectra were recorded on a Bruker AVANCE 400
spectrometer using TMS (0.00 ppm) as an internal standard.
High-resolution MS was performed on JEOL JMS-HX110A
spectrometer.
O-(N-Benzyloxycarbonyl-L-alanyl)acetoxime, 2. Cbz-
Ala-OH (50 g, 0.22 mol), acetone oxime (19.64 g, 0.27 mol),
and di-tert-butylcarbonate (53.77 g, 0.24 mol) were dissolved
in THF (450 mL) and pyridine (13 mL) and incubated at 20
°C for 20 h. EtOAc (300 mL) and water (300 mL) were
added to the reaction mixture, the organic layer was
separated, and the aqueous layer was extracted twice with
EtOAc (2 × 100 mL). The combined organic extracts were
successively washed with 5% aqueous K2CO3, water, and
brine (300 mL each), dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo to provide 62.81 g (100% yield)
of 2 as a solid. 1H NMR (400 MHz, CDCl3) δ 1.44 (d, 3H,
J ) 7.2 Hz), 1.95 (s, 3H), 2.01 (s, 3H), 4.50 (t, 1 H, J )
7.4), 5.08 (s, 2H), 5.5-5.48 (br, 1H), 7.30-7.33 (m, 5H).
13C NMR (100 MHz, CDCl3) δ 18.3, 20.0, 23.1, 49.9, 68.1,
129.07, 129.1, 129.5, 137.2, 156.5, 166.2, 171.6
Benzyloxycarbonyl Alanine Trifluoroester (Cbz-Ala-
TFA). Trifluoroethanol (3.0 g, 30 mmol), pyridine (1.4 g,
18 mmol), and di-tert-butyl carbonate (6 g, 27.5 mmol) were
added to a solution of Cbz-Ala-OH (5.58 g, 25 mmol) in
THF (50 mL). After stirring for 22 h at room temperature
the reaction mixture was poured into water and extracted
with EtOAc. The combined organic extracts were washed
with 5% aqueous K2CO3, water, 5% aqueous H2SO4, and
brine, dried over Na2SO4, and filtered. The solution was
Acknowledgment
We thank Jian Ning for the development of the chiral
HPLC methods.
Received for review September 13, 2002.
OP0255938
Vol. 7, No. 6, 2003 / Organic Process Research & Development
•
953