Wu et al.
t, J ) 6.0 Hz), 4.14 (1H, m), 3.98 (2H, m), 2.68 (1H, m), 2.52
(1H, m); 13C NMR (D2O, 75 MHz) δ 164.5, 141.9, 125.9, 89.5,
86.7, 70.6, 64.9, 39.9; 31P NMR (D2O, 121 MHz) δ -9.85 (d,
Jp,p ) 21.8 Hz), -13.38 (d, Jp,p ) 21.8 Hz). HRMS (ESI): calcd
389.0263 [M + H]+, found 389.0247 [M + H]+.
2.41 (1H, m); 13C NMR (D2O, 75 MHz) δ 163.2,156.4, 145.8,
88.9, 86.5, 70.9, 65.7, 39.1; 31P NMR (D2O, 121 MHz) δ -8.51
(d, Jp,p ) 19.8 Hz), -11.07 (d, Jp,p ) 19.8 Hz), -22.40 (t, Jp,p
)
19.8 Hz). HRMS (ESI): calcd 466.9770 [M - H]-, found
466.9774 [M - H]-.
2-(2′-Deoxy-â-D-erythro-pentofuranosyl)-(2H)-1,2,3-tri-
azole-4-carboxamide-5′-triphosphate (12). Following the
procedure described above for the synthesis of triphosphate
10, compound 9 (180 mg 0.4 mmol) was phosphorylated to yield
190 mg (NH4 salt, 89%) of 12 as a white fluffy solid. HPLC
1-(2′-Deoxy-â-D-erythro-pentofuranosyl)-(1H)-1,2,4-tri-
azole-3-carboxamide-5′-diphosphate (8). Following the
procedure described above for the synthesis of diphosphate 7,
tosylate 5 (300 mg, 0.78 mmol) and tris(tetra-n-butylammo-
nium) hydrogen pyrophosphate (1.4 g 1.57 mmol) were reacted
in dry acetonitrile (1.5 mL) for 24 h to yield 310 mg (91%) of
8 as a white fluffy solid. TLC Rf 0.25; 1H NMR (D2O, 300 MHz)
δ 8.59 (1H, s), 6.23 (1H, t, J ) 5.7 Hz), 4.12 (1H, m), 3.94 (2H,
m), 2.68 (1H, m), 2.42 (1H, m); 13C NMR (D2O, 75 MHz) δ
163.2, 156.4, 145.8, 88.8, 86.5, 71.0, 65.5, 39.1; 31P NMR (D2O,
121 MHz) δ -9.17 (d, Jp,p ) 21.3 Hz), -10.90 (d, Jp,p ) 21.3
Hz). HRMS (ESI): calcd 387.0107 [M - H]-, found 387.0104
[M - H]-.
1
11.48 min; H NMR (D2O, 300 MHz) δ 8.04 (1H, s), 6.37 (1H,
t, J ) 6.0 Hz), 4.16 (1H, m), 3.93 (2H, m), 2.91 (1H, m), 2.46
(1H, m); 13C NMR (D2O, 75 MHz) δ 164.5, 142.9, 136.3, 92.5,
86.4, 71.3, 65.9, 38.5; 31P NMR (D2O, 121 MHz) δ -8.81 (d,
Jp,p ) 19.4 Hz), -11.09 (d, Jp,p) 19.4 Hz), -22.53 (t, Jp,p
)
19.4 Hz). HRMS (ESI): calcd 466.9770 [M - H]-, found
466.9772 [M - H]-.
2-(2′,3′-O-Methoxymethylidene-â-D-ribofuranosyl)-(2H)-
1,2,3-triazole-4-carboxamide-5′-tosylate (15). Compound
1339 (250 mg, 1.0 mmol) and p-toluenesulfonic acid (210 mg,
1.1 mmol) were stirred in THF (5 mL) at room temperature,
trimethyl orthoformate (0.55 mL, 5.0 mmol) was added via
syringe, and the solution was stirred for 2 h. Pyridine (0.5 mL)
was added and the solvent was removed in vacuo. The residue
was then dissolved in dry CH2Cl2 (5 mL), and TsCl (230 mg,
1.2 mmol) and 4-DMAP (160 mg, 1.3 mmol) were added. The
reaction mixture was stirred for 24 h and purification on silica
gel eluting with hexane/ethyl acetate/methanol (10:9:1) yielded
310 mg (70%) of 15 as a white solid. TLC Rf 0.7 (ethyl acetate/
2-(2′-Deoxy-â-D-erythro-pentofuranosyl)-(2H)-1,2,3-tri-
azole-4-carboxamide-5′-diphosphate (9). Following the
procedure described above for the synthesis of diphosphate 7,
tosylate 6 (320 mg, 0.84 mmol) and tris(tetra-n-butylammo-
nium) hydrogen pyrophosphate (1.5 g, 1.7 mmol) were reacted
in anhydrous acetonitrile (1.5 mL) for 96 h to yield 260 mg
(70%) of 9 as a white fluffy solid. TLC Rf 0.26; 1H NMR (D2O,
300 MHz) δ 8.01 (1H, s), 6.35 (1H, t, J ) 6.0 Hz), 4.13 (1H,
m), 3.88 (2H, m), 2.89 (1H, m), 2.44 (1H, m); 13C NMR (D2O,
75 MHz) δ 164.4, 142.9, 136.2, 92.5, 86.4, 71.4, 65.7, 38.4; 31
P
NMR (D2O, 121 MHz) δ -9.83 (d, Jp,p ) 20.6 Hz), -11.00 (d,
Jp,p ) 20.6 Hz). HRMS (ESI): calcd 389.0263 [M + H]+, found
389.0263 [M + H]+.
1
methanol, 10:1); mp 122-125 °C; H NMR for diastereomer
(DMSO-d6, 300 MHz) δ 8.09, 8.10 (1H, s), 7.89 (1H, s), 7.61
(1H, s), 7.58 (2H, d), 7.35 (2H, d), 6.33 (1H, d, J ) 6.0 Hz),
6.03, 6.14 (1H, s), 5.20 (2H, m), 4.49 (1H, m), 4.06 (2H, m),
3.21, 3.29 (3H, s), 2.39 (3H, s); MS (ESI): 463 [M + Na]+. Anal.
Calcd for C17H20O8N4S: C, 46.36; H, 4.58; N, 12.72. Found:
C, 46.74; H, 4.55; N, 12.61.
1-(2′-Deoxy-â-D-erythro-pentofuranosyl)-(1H)-1,2,3-tri-
azole-4-carboxamide-5′-triphosphate (10). A 10-mL solu-
tion of 83 mM triethanolamine/17 mM MgCl2/67 mM KCl
containing compound 7 (180 mg, 0.4 mmol), ATP (220 mg, 0.4
mmol), and PEP (152 mg, 0.8 mmol) was adjusted to pH 7.6
with 6 M NaOH. To this solution were added NDPK (100 unit)
and pyruvate kinase (200 unit), and the mixture was incubated
at 37 °C for 4 h. The reaction was monitored by analytical
HPLC to ensure complete conversion of compound 7 to the
corresponding triphosphate. To this mixture ADP (180 mg, 0.4
mmol) was added and incubation was continued for another 1
h to consume the excess PEP before filtration through a 0.2-
µm syringe disk and drying by lyophilization.
The residual solid obtained was dissolved in 10 mL of 1 M
NH4HCO3 (pH 8.5, with concentrated NH4OH) and applied to
a Bio-Rad boronate affinity gel column (2.5 cm × 5.5 cm, in 1
M NH4HCO3 , pH 8.5). The column was eluted with the same
buffer at 1 mL/min and the fractions containing the first two-
column volumes were pooled and dried by lyophilization.
Excess NH4HCO3 was removed by repeated lyophilization from
deionized water after adjustment to pH 7.2 with CO2.
1-(2′,3′-O-Methoxymethylidene-â-D-ribofuranosyl)-
(1H)-1,2,3-triazole-4-carboxamide-5′-tosylate (16). Com-
pound 1439 (250 mg, 1.0 mmol) and pyridine hydrochloride (140
mg, 1.2 mmol) were stirred in dioxane (10 mL) at room
temperature, trimethyl orthoformate (0.55 mL, 5.0 mmol) was
added via syringe, and the mixture was stirred at room
temperature. After 24 h, the reaction mixture was passed
through a silica gel column and eluted with dichloromethane/
methanol (20:1). The partially purified compound was then
dissolved in anhydrous pyridine (5 mL), and TsCl (210 mg,
1.1 mmol) and 4-DMAP (150 mg, 1.2 mmol) were added. The
reaction mixture was stirred at room temperature. After 2
days, silica gel (1.0 g, 60-200 mesh) was added to the reaction
mixture and the solvent was evaporated to dryness. Methanol
(5 mL) was added to the residue and evaporated to dryness
again and the procedure was repeated three times to remove
pyridine. Then the residue was applied to a flash silica gel
column and eluted with hexane/acetone (1:1) to yield 230 mg
(52%) of 16 as a white waxy solid. TLC Rf 0.4 (hexane/acetone
Final purification of the compound was performed on a Q
-
Sepharose FF anion exchange column (2.5 cm × 20 cm, HCO3
form) by a linear gradient elution with NH4HCO3 (0.05-0.5
M, pH 7.8) at a flow rate of 5 mL/min over 2 h with UV
detection at 230 nm. The desired fractions were dried by
lyophilization to yield a white fluffy solid (120 mg, NH4+ salt,
58%). HPLC 10.74 min; 1H NMR (D2O, 300 MHz) δ 8.64 (1H,
s), 6.42 (1H, t, J ) 6.0 Hz), 4.14 (1H, m), 4.01 (2H, m), 2.68
(1H), 2.50 (1H, m); 13C NMR (D2O, 75 MHz) δ 164.5, 141.9,
125.9, 89.5, 86.6, 70.4, 65.2, 39.9; 31P NMR (D2O, 121 MHz) δ
-6.26 (d, Jp,p ) 19.4 Hz), -10.97 (d, Jp,p ) 19.4 Hz), -21.88
(t, Jp,p ) 19.4 Hz). HRMS (ESI): calcd 466.9770 [M - H]-,
found 466.9766 [M - H]-.
1
1:1); H NMR for diastereomer (DMSO-d6, 300 MHz) δ 8.63,
8.66 (1H, s), 7.93 (1H, s), 7.62 (2H, d), 7.58 (1H, s), 7.37 (2H,
d), 6.42 (1H, t, J ) 1.8 Hz), 6.05, 6.16 (1H, s), 5.33 (1H, m),
5.07 (1H, m), 4.47 (1H, m), 4.25 (1H, m), 3.98 (1H, m), 3.21,
3.30 (3, s), 2.38 (3H, s). HRMS (EI): calcd 440.1002 [M]+, found
440.0998 [M]+.
2-(â-D-Ribofuranosyl)-(2H)-1,2,3-triazole-4-carbox-
amide-5′-tosylate (17). Tosylate 15 obtained from compound
13 (400 mg 1.64 mmol) through protection in THF (10 mL)
with p-toluenesulfonic acid (340 mg, 1.8 mmol) and trimethyl
orthoformate (0.9 mL, 8.2 mmol) and subsequent reaction in
dichloromethane (10 mL) with TsCl (350 mg, 1.8 mmol) and
4-DMAP (240 mg, 2.0 mmol) was dissolved in a 1:1 (v/v)
1-(2′-Deoxy-â-D-erythro-pentofuranosyl)-(1H)-1,2,4-tri-
azole-3-carboxamide-5′-triphosphate (11). Following the
procedure described above for the synthesis of triphosphate
10, compound 8 (180 mg, 0.4 mmol) was phosphorylated to
(38) Witkowski, J. T.; Fuertes, M.; Cook, P. D.; Robins, R. K. J.
Carbohydr. Nucleosides Nucleotides 1975, 2, 1-36.
(39) Lehmkuhl, F. A.; Witkowski, J. T.; Robins, R. K. J. Heterocyclic
Chem. 1972, 9, 1195-1201.
+
yield 120 mg (NH4 salt, 58%) of 11 as a white fluffy solid.
HPLC 9.23 min; 1H NMR (D2O, 300 MHz) δ 8.59 (1H, s), 6.23
(1H, t, J ) 5.7 Hz), 4.14 (1H, m), 4.00 (2H, m), 2.68 (1H, m),
3864 J. Org. Chem., Vol. 68, No. 10, 2003