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C50-Ha), 3.76 (1H, ddd, J 12.3, 5.2, 2.8, C50-Hb), 3.92 (1H, td, J 5.9, C50-Hb) 4.31–4.36 (1H, m, C40-H), 4.90 (1H, dd, J 6.4, 4.0, C20-H),
2.9, C40-H), 3.95–4.05 (2H, m, C20-H, C30-H), 5.05 (1H, d, J 5.7, 5.11–5.15 (1H, m, C30-H), 5.70 (1H, d, J 1.5, C10-H), 7.53 (2H, t, J
C30-OH), 5.18 (1H, app t, J 5.1, C50-OH), 5.51 (1H, d, J 4.8, C20- 7.6, m-Ph), 7.63 (1H, t, J 7.4, p-Ph), 7.77 (1H, d, J 7.4, C6-H), 7.89
OH), 5.81 (1H, d, J 2.9, C10-H), 7.34 (1H, d, J 7.5, C5-H), 7.52 (2H, (2H, d, J 7.2, o-Ph), 8.67 (1H, s, NH); dC (100 MHz, D2O) 25.2,
dd, J 7.1, 8.4, m-Ph), 7.63 (1H, t, J 7.4, p-Ph), 8.00 (2H, d, J 7.5, o- 27.3, 62.8, 77.2, 80.5, 83.8, 88.1, 97.0, 98.7, 114.1, 127.7, 129.1,
Ph), 8.51 (1H, d, J 7.5, C6-H), 11.25 (1H, s, NH); dC (126 MHz, 132.7, 133.4, 147.8, 163.1; m/z 413.1585 (M + H+, 100%), requires
(CD3)2SO) 60.6, 69.3, 75.3, 84.9, 90.9, 96.6, 110.0, 129.2, 133.5, 413.1573, 435.1406 (M + Na+, 40%).
133.8, 146.1, 155.4, 163.8, 168.0; m/z 346.2 (M ꢀ H, 100%) 347.2
(26) 693 (22).
50-Azido-50-deoxycytidine (19)
N4-Benzoyl-20,30-O-isopropylidenecytidine (17)
Based on a literature procedure,31 50-Azido-50-deoxy-N4-benzoyl-
20,30-O-isopropylidene cytidine (732 mg, 1.78 mmol) was placed
in a round-bottomed ask with a 1 : 1 mixture of methanol and
concentrated ammonium hydroxide (90 ml) and was stirred at
room temperature for 18 h. The solvents were removed under
vacuum, and the residue was stirred in a 9 : 1 mixture of tri-
uoroacetic acid and H2O for 3 h at room temperature before
the solvents were removed under vacuum. The residue was
dissolved in H2O and introduced to a protonated SP Sepharose®
Fast Flow cation exchange column (1.6 cm i.d. ꢃ 22 cm, 5 ml
minꢀ1 ow rate). The nucleoside was eluted with a 3% ammo-
nium hydroxide solution (diluted from 35% (w/w) ammonium
hydroxide solution) and the eluted fractions were freeze-dried.
The residue was then dissolved in methanol and ltered, and
the solvent was removed from the ltrate under vacuum to give
the solid product. (334 mg, 70%); nmax/cmꢀ1 2104 (N3), 1645
(CO), 1438; dH (400 MHz, D2O) 3.64 (1H, dd, J 13.7, 5.0, C50-Ha),
3.78 (1H, dd, J 13.7, 3.1, C50-Hb), 4.14–4.19 (1H, m, C40-H), 4.21
(1H, dd, J 6.2, 5.3, C30-H), 4.34 (1H, dd, J 5.3, 4.0, C20-H), 5.87
(1H, d, J 4.0, C10-H), 6.03 (1H, d, J 7.5, C5-H), 7.74 (1H, d, J 7.6,
C6-H); dC (100 MHz, D2O) 51.2, 69.9, 73.3, 81.5, 90.5, 96.3, 141.6,
157.4, 163.8; m/z 267.2 (M ꢀ H, 100%) 268.3 (15).
In an adapted literature procedure,29 N4-benzoylcytidine (2.79 g,
8.03 mmol), 2,2-dimethoxypropane (11 ml), 4 A molecular
sieves, and tosic acid monohydrate (0.45 g, 2.37 mmol) were
˚
placed in a round-bottomed ask with DMF (44 ml). The
ꢂ
mixture was heated at 40 C for 2.5 h, before Amberlyst® A-21
anion exchanger (1.19 g) was added and heating was continued
for a further 30 min. The mixture was ltered through Celite®,
the solvent was then removed under vacuum from the ltrate,
and the residue was recrystallised from water to yield the
product (2.375 g, 76%); mp 163–165 C; nmax/cmꢀ1 1652 (CO),
ꢂ
1482, 1303; dH (400 MHz, CDCl3) 1.37 (3H, s, CH3a), 1.63 (3H, s,
CH3b), 3.54 (1H, br s, C50-OH), 3.80–3.89 (1H, m, C50-Ha), 3.97
(1H, dd, J 12.2, 2.3, C50-Hb), 4.40 (1H, app q, J 3.0, C40-H), 5.09
(1H, dd, J 6.4, 3.3, C30-H), 5.25 (1H, dd, J 6.6, 2.6, C20-H), 5.55
(1H, d, J 2.7, C10-H), 7.52 (2H, d, J 7.8, m-Ph), 7.63 (1H, t, J 7.4,
p-Ph), 7.81 (1H, d, J 7.4, C6-H), 7.89 (2H, d, J 7.5, o-Ph), 8.77 (1H,
s, NH); dC (126 MHz, CDCl3) 25.2, 27.3, 62.9, 80.5, 83.6, 88.1,
99.0, 100.0, 112.8, 114.1, 127.3, 127.6, 128.6, 129.1, 133.4, 143.3,
166.1; m/z 388.1500 (M + H+, 20%), requires 388.1509, 410.1325
(M + Na+, 100).
N4-Benzoyl-20,30-O-isopropylidene-50-O-
toluenesulfonylcytidine
50-Amino-50-deoxycytidine, hydrochloride salt (20$2HCl)
N4-Benzoyl-20,30-O-isopropylidenecytidine (2.10 g, 5.42 mmol),
4-toluenesulfonyl chloride (2.44 g, 12.9 mmol), pyridine (20 ml)
and DCM (40 ml) were placed in a round-bottomed ask and
heated at reux for 2.5 h. The solution was then diluted with
chloroform (120 ml) and washed with hydrochloric acid (0.5 M,
5 ꢃ 50 ml) and saturated sodium hydrogencarbonate solution
(2 ꢃ 50 ml). The organic layer was dried over MgSO4 and the
solvent was removed under vacuum to yield the crude product,
which was used in the next reaction without further purica-
tion. (1.53 g).
50-Azido-50-deoxycytidine (400 mg, 1.49 mmol) was placed in a
ask with triphenylphosphine (790 mg, 3.01 mmol) and pyri-
dine (6 ml). The solution was stirred at room temperature for 3
h before further triphenylphosphine (791 mg, 3.02) and pyri-
dine (6 ml) were added. The solution was stirred for an addi-
tional 3 h at room temperature before ammonia solution (50 ml,
35% w/w) was added, whereupon immediate precipitation was
observed. The mixture was stirred overnight before being
diluted with water (100 ml) and extracted with chloroform (3 ꢃ
100 ml). Residual chloroform was removed from the aqueous
layer under vacuum, and the aqueous extracts were lyophilised.
The resulting solid was dissolved in ethanol (60 ml) and
hydrogen chloride gas was bubbled through the solution. The
50-Azido-50-deoxy-N4-benzoyl-20,30-O-isopropylidenecytidine
(18)
Adapting
a
literature procedure,30 N4-benzoyl-20,30-O-iso- addition of diethyl ether (300 ml) precipitated the product,
propylidene-50-O-toluenesulfonylcytidine (1.08 g, 1.99 mmol) which was isolated by ltration, washed with a small quantity of
and sodium azide (579 mg, 8.91 mmol) were placed in a ask diethyl ether, and dried under vacuum overnight (382 mg, 81%);
with DMSO (10 ml) and heated at 70 ꢂC for 2 h 20 min. The mp 190–191 ꢂC (decomp.) (from ethanol); nmax/cmꢀ1 3118, 3046,
solution was then added to H2O (550 ml) and le to stir for 18 h. 1715, 1673, 1404; dH (400 MHz, D2O) 3.34 (1H, dd, J 13.6, 9.4,
The precipitated product was then isolated by vacuum ltration. C50-Ha), 3.47 (1H, dd, J 13.6, 3.0, C50-Hb), 4.18–4.31 (2H, m, C30-
(430 mg, 52%); mp 163–165 ꢂC; nmax/cmꢀ1 3354, 1640 (CO), H, C40-H), 4.51 (1H, dd, J 3.4, 5.5, C20-H), 5.77 (1H, d, J 3.4, C10-
1485; dH (400 MHz, CDCl3) 1.37 (3H, s, CH3a), 1.60 (3H, s, CH3b), H), 6.24 (1H, d, J 7.9, C5-H), 7.87 (1H, d, J 7.9, C6-H); dC
3.65 (1H, dd, J 12.8, 4.4, C50-Ha), 3.76, (1H, dd, J 12.8, 6.8, (100 MHz, D2O) 41.9, 71.4, 73.5, 80.2, 94.1, 95.8, 137.8, 145.9,
38668 | RSC Adv., 2014, 4, 38663–38671
This journal is © The Royal Society of Chemistry 2014