S. K. Mahto, C. S. Chow / Bioorg. Med. Chem. 16 (2008) 8795–8800
8799
by column chromatography (hexane/ethyl acetate, 8:2) to give a
yellow oil (2.27 g, 95%). 1H NMR (500 MHz, CD3Cl) d (ppm) 2.13
(s, 3H), 2.15 (s, 3H), 3.47 (s, 3H), 4.04 (m, 1H), 4.36 (m, 3H), 4.98
(m, 1H), 5.75 (d, 1H), 5.91 (dd, 1H),7.52 (d, 1H), 9.10 (s, 1H), 13C
NMR (500 MHz, CD3Cl) d (ppm) 20.86, 59.30, 62.65, 70.08, 79.40,
81.77, 88.81, 102.94, 127.23, 139.43, 145.69, 150.18, 163.06,
tary evaporator. The crude product was purified by column chro-
matography (CH2Cl2/CH3OH, 9:1) to give a yellow oil (1.62 g,
95%). 1H NMR (500 MHz, CD3Cl) d (ppm) 2.09 (s, 3H), 2.11 (s,
3H), 3.47 (s, 3H), 4.01 (d, 1H), 4.32 (m, 1H), 4.34 (m, 1H), 4.35
(m, 1H), 4.95 (m, 1H), 5.88 (m, 1H), 6.01 (d, 1H), 7.55 (d, 1H) 13C
NMR (500 MHz, CD3Cl) d (ppm) 20.89, 21.08, 58.18, 62.71, 78.84,
81.84, 90.31, 95.59, 125.96,140.82, 155.48, 165.84, 170.41,170.58
ESI-MS (ES+) calculated for C14H19N3O7 341.3, found 342.3 (MH+).
170.37, HRMS calculated for
342.1067.
C14H18N2O8 342.1063, found
4.1.3. 4-(Tetrazol-1-yl)-1-(30,50-di-O-acetyl-20-O-methyl-b-
D-
4.1.7. 20-O-Methycytidine (8)
ribofuranosyl) pyrimidine-2-(1H)-one (4)
Compound 7 (1.54 g, 4.5 mmol) was placed in a dry round-bot-
tom flask fitted with a septum. Next, 25 mL of 2 M NH3 in metha-
nol was added, and the mixture was stirred overnight at room
temperature. The mixture was dried on a rotary evaporator. The
residue was coevaporated twice with CH3OH and once with
CH3OH–CH2Cl2 under vacuum. The residue was heated to 100 °C
under a vacuum for 2 h to give 8 as a white powder (1.15 g,
99%). The NMR data matched that in the literature.25 ESI-MS
(ES+) calculated for C10H15N3O5 257.1, found 258.1 (MH+).
In a round-bottom flask containing 15 mL of pyridine, com-
pound 3 (2.05 g, 6 mmol), tetrazole (0.84 g, 12 mmol), tosyl chlo-
ride (2.29 g, 12 mmol), and diphenyl phosphate (2.25 g, 9 mmol)
were added. The mixture was stirred for 36 h at room temperature,
and then 3.75 mL of water was added. The solution was poured
over saturated NaHCO3 solution. The desired product was ex-
tracted with methylene chloride. The solvent was dried over
Na2SO4 and concentrated on a rotary evaporator. The product
was purified by column chromatography (CH2Cl2/CH3OH, 95:5)
to give a yellow foam (2.25 g, 95%). 1H NMR (500 MHz, CD3Cl) d
(ppm) 2.11 (s, 3H), 2.14 (s, 3H), 3.6 (s,3H), 4.17 (d, 1H), 4.42 (m,
2H), 4.54 (m, 1H), 4.77 (m, 1H), 5.97 (s, 1H), 7.21 (d, 1H), 8.56 (d,
1H), 9.59 (s, 1H) 13C NMR (500 MHz, CD3Cl) d (ppm) 20.75, 21.07,
59.43, 61.8, 69.24, 79.50, 81.75, 90.89, 95.16, 140.95, 147.5,
153.79, 157.69, 170.21, 170.317 ESI-MS (ES+) calculated for
4.1.8. 4-(Tetrazol-1-yl)-1-(20,30,50-Tri-O-acetyl-b-
D-
ribofuranosyl)pyrimidine-2-(1H)-one (10)
Compound 9 (20,30,50-triacetyluridine) was prepared according
to literature methods.26 Compound 9 (2.22 g, 6 mmol), tetrazole
(0.84 g, 12 mmol), tosyl chloride (2.29 g, 12 mmol), and diphenyl
phosphate (2.25 g, 9 mmol) were added to a round-bottom flask
containing 15 mL of pyridine. The mixture was stirred for 36 h at
room temperature, and then 3.75 mL of water was added. The solu-
tion was poured over a saturated sodium carbonate solution. The
desired product was extracted with methylene chloride. The sol-
vent was dried over Na2SO4 and concentrated on a rotary evapora-
tor. The product was purified by column chromatography (CH2Cl2/
CH3OH, 95:5) to give a yellow foam (2.40 g, 95%). 1H NMR
(500 MHz, CD3Cl) d (ppm) 2.07 (s, 3H), 2.12 (s, 3H), 2.14 (s, 3H),
4.40 (m, 3H), 5.27 (m, 1H), 5.48 (m, 1H), 6.09 (d, 1H), 7.24 (d,
1H), 8.38 (d, 1H), 9.60 (s, 1H), 13C NMR (500 MHz, CD3Cl) d
(ppm) 20.66, 20.68, 21.05, 62.66, 69.65, 73.99, 80.51, 90.31,
95.72,141.04, 147.69, 153.98, 157.84, 169.76, 169.76, 170.33, ESI-
MS (ES+) calculated for C16H18N6O8 422.1, found 423.1 (MH+).
C
15H18N6O7 394.1, found 395.1 (MH+).
4.1.4. 30,50-Diacetyl-N4,20-O-dimethylcytidine (5)
Potassium hydroxide (89%, 0.32 g, 5 mmol) and CH3NH3Cl
(0.34 g, 5 mmol) were added to a 50 mL round bottom flask. The
flask was sealed with a septum. Water (10 mL), acetonitrile
(10 mL), triethylamine (770 lL), and a solution of compound 4
(1.97 g, 5 mmol) in acetonitrile (20 mL) were added sequentially
by syringe. The mixture was stirred vigorously for 24 h. The solvent
was removed with a rotary evaporator. The crude product was puri-
fied by column chromatography (CH2Cl2/CH3OH, 9:1) to give a yel-
low oil (1.69 g, 95%). 1H NMR (500 MHz, CD3Cl) d (ppm) 2.11(s,
3H), 2.13 (s, 3H), 3.06 (s, 3H), 3.55 (s, 3H), 4.10(m, 1H), 4.40 (m,
3H), 4.88 (m, 1H), 5.75 (d, 1H), 6.00 (d, 1H), 7.50 (d, 1H), 13C NMR
(500 MHz, CD3Cl) d (ppm) 20.85, 20.99, 28.07, 59.15, 62.85, 70.43,
78.43, 78.66, 81.93, 89.93, 96.20,138.63, 164.52, 170.35, 170.52
HRMS calculated for C15H21N3O7 355.1380, found 355.1383.
4.1.9. 20,30,50-Tri-O-acetyl-N4-methylcytidine (11)
Potassium hydroxide (89%, 0.32 g, 5 mmol) and CH3NH3Cl
(0.34 g, 5 mmol) were added in a 50 mL round-bottom flask. The
flask was sealed with a septum. Water (10 mL), acetonitrile
4.1.5. N4,20-O-Dimethylcytidine (6)
(10 mL), triethylamine (770 lL), and a solution of the compound
Compound 5 (1.60 g, 4.5 mmol) was placed in a dry 50 mL
round-bottom flask. The flask was fitted with a septum. Next,
25 mL of 2 M NH3 in methanol was added, and the mixture was
stirred overnight at room temperature. The mixture was dried on
a rotary evaporator. The residue was coevaporated twice with
CH3OH and once with CH3OH/CH2Cl2 (1:1) under a vacuum. The
residue was heated to 100 °C under vacuum for 2 h to give 6 as a
white powder (1.20 g, 99%). 1H NMR (500 MHz, D20) d (ppm)
2.73 (s, 3H), 3.34 (s, 3H), 3.63 (dd, 1H), 3.75 (dd, 1H), 3.82 (m,
10 (2.11 g, 5 mmol) in acetonitrile (20 mL) were added sequen-
tially by syringe. The mixture was stirred vigorously for 24 h. The
solvent was removed with a rotary evaporator. The crude product
was purified by column chromatography (CH2Cl2/CH3OH, 9:1) to
give a yellow oil (1.82 g, 95%). 1H NMR (500 MHz, CD3Cl) d
(ppm), 2.09 (s, 3H), 2.10 (s, 3H), 2.12 (s, 3H), 4.34 (m, 2H), 5.40
(dd, 1H), 5.90 (d, 1H), 5.99 (d, 1H), 6.71 (d, 1H), 7.39 (d, 1H), (s,
3H) 8.27 (d, 1H),13C NMR (500 MHz, CD3Cl) d (ppm), 20.48,
20.53, 20.68, 63.0, 70.04, 73.41, 79.12, 89.96, 96.11, 141.03,
155.59, 166.10, 169.58, 169.71, 170.38, ESI-MS (ES+) calculated
for C16H21N3O8 383.1, found 384.1 (MH+).
1H), 3.91 (m, 1H), 4.13 (m, 1H), 5.78 (m, 2H), 7.52 (d, 1H),13
C
NMR (500 MHz, D2O) d (ppm) 27.27, 58.29, 60.54, 68.42, 82.90,
83.13, 88.31, 97.48, 139.45, 157.78, 164.59, HRMS calculated for
C11H17N3O5 271.1168, found 271.1176.
4.1.10. N4-Methylcytidine (12)
Compound 11 (1.53 g, 4 mmol) was added to a round-bottom
flask fitted with a septum. Next, 25 mL of 2 M NH3 in methanol
was added, and the mixture was stirred overnight at room temper-
ature. The mixture was dried on a rotary evaporator. The residue
was coevaporated twice with CH3OH and once with CH3OH/CH2Cl2
(1:1) under vacuum. The residue was heated at 100 °C under vac-
uum for 2 h to give 12 as a white powder (1.01 g, 99%). 1H NMR
(500 MHz, D20) d (ppm) 2.75 (s, 3H), 3.63 (dd, 1H), 3.75 (dd, 1H),
4.1.6. 30,50-Diacetyl-20-O-methycytidine (7)
Potassium hydroxide (89%, 0.32 g, 5 mmol) and NH4Cl (0.27 g,
5 mmol) were added to a round-bottom flask. The flask was sealed
with a septum. Water (10 mL), acetonitrile (10 mL), triethylamine
(770 lL), and solution of compound 4 (1.97 g, 5 mmol) in acetoni-
trile (20 mL) were added sequentially with a syringe. The mixture
was stirred vigorously for 24 h. The solvent was removed with a ro-