4114 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 20
Khamnei and Torrence
were precipitated with CH3CN (50 µL) and centrifuged down
at 1000g, and 50 µL of supernatant was injected for HPLC
analysis.
evaporation in vacuo, and the residue was applied to a silica
gel column which was eluted with CHCl3/MeOH (20:1). Com-
pound 3 was obtained as a colorless amorphous glass in a yield
of 0.55 g (82%): UV λmax (MeOH) 266 nm (ꢀ ) 18 000); 1H NMR
(CDCl3) δ 1.82 (s, 3, thymine CH3), 1.85 (s, 3, thymine CH3),
2.34-2.40 (m, 4, 2′-H′s), 3.9 (m, 2, 4′-H′s), 4.2-4.4 (m, 6, 3′,5′-
H′s), 5.99-6.08 (m, 2, 1′-H′s), 7.15-7.23 (m, 5, 3 phenyl, 2
thymine 6-H′s), 7.3 (t, 2, J ) 8 Hz, phenyl), 9.39 (s, H, thymine
3-NH), 9.43 (s, H, thymine 3-NH); 31P NMR (CDCl3) δ -6.35;
HRMS (FAB) calcd for 673.1884 (M+), found 673.1857.
(Ch lor oa cetyl)sa licylic Acid . Monochloroacetyl chloride
(12 mL, 0.3 mol) in benzene (40 mL) was added to a suspension
of salicylic acid (dried over P2O5, 28 g, 0.2 mol) in benzene (120
mL). The mixture was heated on an oil bath and refluxed for
5 h or until evolution of HCl ceased. The solvent and excess
acid halide were distilled under reduced pressure. The result-
ing crystals were crystallized twice from benzene:6 EI-MS m/z
214 (M); 1H NMR (CD3OD) δ 4.5 (s, 2H, -OCOCH2Cl), 7.1 (d,
1H, J ) 8.3 Hz, phenyl), 7.3 (t, 1H, J ) 7.8 Hz, phenyl), 7.6 (t,
1H, J ) 8.3 Hz, phenyl), 8 (d, 1H, J ) 7.8 Hz, phenyl); 13C
NMR (CD3OD) δ 171.3, 170.5, 155, 138.3, 136.2, 130.7, 127.7,
112, 44.9.
(d ) Hyd r olysis of Tr iester s w ith Ra t Br a in Extr a ct.
Two rat brains (total weight 3.3 g) were washed in phosphate
buffer (0.1 M, pH 7.4) and homogenized in ice-cold buffer (13.2
mL), and the resulting suspension was centrifuged at 10000g
for 20 min at 4 °C. The supernatant was removed and kept
on ice. A solution of NADP (36 mg), glucose-6-phosphate
(163.3 mg), MgCl2‚6H2O (101.6 mg), and glucose-6-phosphate
dehydrogenase (80 U) was freshly prepared in phosphate
buffer (10 mL, 0.1 M, pH 7.4) and stored on ice. This solution
(200 µL) and 500 µL of brain supernate were added to a
solution (5 µL) of triester in phosphate buffer containing 0.5%
DMSO at 37 °C. Samples (50 µL) were removed at different
time points and added to centrifuge tubes containing aceto-
nitrile (50 µL), the protein precipitate was spun down, and 50
µL of the supernate was injected directly to the HPLC.
O,O′-Bis(3′-a zid o-3′d eoxyth ym id in -5′-yl)-O′′-[2-[(m eth y-
loxy)ca r bon yl]p h en yl]p h osp h a te (1). Methyl salicylate
(0.165 g, 1.1 mmol) was dissolved in dry methylene chloride
(4 mL), and distilled triethylamine (500 µL, 0.363 g, 3.5 mmol)
was added to this solution. The reaction mixture was stirred
at 0 °C for 5 min, and then freshly distilled POCl3 was added
(100 µL, 0.167 g, 1 mmol) dropwise. The reaction proceeded
rapidly. After 30 min, silica gel TLC in CHCl3 showed the
methyl salicylate had disappeared. Due to the high reactivity
of methylsalicyl phosphorodichloridate, it was not isolated or
purified. Instead, 3′-azido-3′-deoxythymidine (0.75 g, 2.8
mmol) in dry pyridine (2 mL) was added dropwise to the
mixture. After 30 min at 0 °C, the solution was allowed to
warm to room temperature, and stirring was continued for
another 5 h. The solvents were evaporated in vacuo. The
residue was applied to a silica gel column which was eluted
with CHCl3/ETOH (7:3). Appropriate fractions (as determined
by TLC on silica gel plates using CHCl3/EtOAc/MeOH, 7:2.7:
0.3) were combined to give, after evaporation, compound 1 (0.5
g, 68%) as a colorless amorphous glass: UV λmax (MeOH) 267
nm (ꢀ ) 20 000); 1H NMR (CDCl3) δ 1.836 (s, 3, thymine CH3),
1.880 (s, 3, thymine CH3), 2.3-2.4 (m, 4, 2′-H′s), 3.8 (s, 3,
salicylate-CH3), 4.02-4.07 (m, 2, 4′-H′s), 4.32-4.4 (m, 4, 5′-
H′s), 4.48-4.5 (m, 2, 3′-H′s), 6.06-6.15 (m, 2, 1′-H′s), 7.2-7.5
(m, 6, 3 salicylate aromatic protons, 2 thymines 6-H′s), 7.93
(d, 1, J ) 7.8 Hz, salicylate), 9.06 (s, 1, thymine 3-NH), 9.09
(s, 1, thymine 3-NH); 31P NMR (CDCl3) δ -6.35; HRMS (FAB)
calcd for 731.1939 (M+), found 731.1945.
O,O′-Bis(3′-a zid o-3′-d eoxyt h ym id in -5′-yl)-O′′-[2-[(p h e-
n yloxy)ca r bon yl]p h en yl]p h osp h a te (2). Phenyl salicylate
(0.214 g, 1 mmol) in dry methylene chloride (2.5 mL) and dry
triethylamine (500 µL, 0.363 g, 3.5 mmol) were combined and
stirred at 0 °C. Freshly distilled POCl3 (100 µL, 0.167 g, 1
mmol) was added dropwise to this mixture. TLC using CHCl3
showed no phenyl salicylate was present in the reaction
mixture after 30 min reaction time. Dry 3′-azido-3′-deoxythy-
midine (0.75 g, 2.8 mmol) in dry pyridine (2 mL) was added
dropwise to this mixture. The reaction mixture was kept for
30 min at 0 °C and then for an additional 3-4 h at room
temperature. The solvents were evaporated in vacuo, and the
residue was applied to a silica gel column which then was
eluted with CHCl3/EtOH (100:4). Appropriate fractions (as
determined by silica gel TLC as above) were combined to give,
after evaporation, O,O′-bis(3′-azido-3′-deoxythymidin-5′-yl)-O′′-
[2-[(phenyloxy)carbonyl]phenyl]phosphate (2; 0.175 g, 22%):
UV λmax (MeOH) 266 nm (ꢀ ) 20 000); 1H NMR (CDCl3) δ 1.78
(s, 3, thymine CH3), 1.86 (s, 3, thymine CH3), 2.3-2.4 (m, 4,
2′-H′s), 3.9-4 (m, 2, 4′-H′s), 4.2-4.5 (m, 6, 5′,3′-H′s), 6.04-
6.15 (m, 2, 1′-H′s), 7.16-7.62 (m, 10, 5 phenyl, 3 salicylate, 2
thymine 6-H′s), 8.16 (d, 1, J ) 7.7 Hz, salicylate), 8.78 (s, 1,
thymine 3-NH), 8.82 (s, 1, thymine 3-NH); 31P NMR (CDCl3)
δ -6.5; HRMS (FAB) calcd for 793.2095 (M+), found 793.2115.
O,O′-Bis(3′-azido-3′deoxyth ym idin -5′-yl)-O′′-ph en ylph os-
p h a te (3). Phenyl phosphorodichloridate (150 µL, 0.21 g, 1
mmol) was added dropwise to a solution of 3′-azido-3′-deox-
ythymidine (75 mg, 2.8 mmol) in a mixture of dry pyridine (2
mL) and methylene chloride (1 mL) at 0 °C. After overnight
reaction at room temperature, solvents were removed by
[[(2,3,4,6-Tetr a -O-ben zyl-D-glu cop yr a n osyl)oxy]ca r bo-
n yl]-2-(1-ch lor oa cetoxy)ben zen e (4) a n d [[(2,3,4,6-Tetr a -
O-ben zyl-D-glu copyr an osyl)oxy]car bon yl]ph en ol (5). (Chlo-
roacetyl)salicylic acid (7.3 g, 34 mmol, dried over P2O5) was
mixed with thionyl chloride (40 mL, 65.6 g, 556 mmol), and
the solution was refluxed until evolution of hydrogen chloride
had practically ceased (4 h). The excess of thionyl chloride
was evaporated in vacuo, and the final traces were removed
from the residue by azeotroping7 with benzene. The product
was dissolved in dry methylene chloride (25 mL), and the
resultant solution was added dropwise to a cold solution of
2,3,4,6-tetra-O-benzyl-D-glucopyranose (10 g, 18 mmol) in
triethylamine (5.6 mL, 4 g, 40 mmol) and methylene chloride
(60 mL). The reaction mixture was kept at room temperature
for 4 h. After the reaction was completed, an excess of thiourea
(10.6 g, 140 mmol) in MeOH was added directly to this
mixture.5 The mixture was stirred at 50 °C for 48 h until the
reaction was complete as determined by TLC [silica gel TLC
in hexane/EtOAc (9:1)]. Appropriate fractions were combined
to give, after evaporation, [[(2,3,4,6-tetra-O-benzyl-D-glucopy-
ranosyl)oxy]carbonyl]phenol (5) (5 g, 7.5 mmol): CI-MS m/z
1
678 (M + NH4), 156 (base); H NMR (CDCl3) δ 3.66-3.8 (m,
6, 2-6 glucose-H’s), 4.47-4.94 (m, 8, benzyl -CH2-), 5.89 (d,
1, J ) 7 Hz, glucose H-1), 6.88 (t, 1, J ) 7.6 Hz, salicylate),
6.99 (d, 1, J ) 8.3 Hz, salicylate), 7.16-7.31 (m, 20, benzyl
H’s), 7.49 (t, 1, J ) 7.8 Hz, salicylate), 7.8 (d, 1, J ) 8 Hz,
salicylate), 10.5 (s, 1, salicylate hydroxyl).
O,O′-Bis(2′,3′-dideoxyth ym idin -5′-yl)-O′′-[[[(2,3,4,6-tetr a-
O -b e n zy l-D-g lu c o p y r a n o s y l)o x y ]c a r b o n y l]p h e n y l]-
p h osp h a te (6). POCl3 (100 µL, 0.167 g, 1.1 mmol) was added
to a cold solution of [[(2,3,4,6-tetra-O-benzyl-D-glucopyranosyl)-
oxy]carbonyl]phenol (5) (0.726 g, 1.1 mmol) in methylene
chloride (4 mL) and triethylamine (500 µL, 0.363 g, 3.5 mmol).
The mixture was stirred and kept at 0 °C until there was no
sign of starting material by TLC analysis (CHCl3). Dry
dideoxythymidine (0.61 g, 2.7 mmol) in dry pyridine (2.7 mL)
was added to this mixture at 0 °C. The reaction mixture was
stirred and kept at 4 °C for 30 min and then for another 3 h
at room temperature. The solvent was evaporated, and the
residue was applied to a silica gel column which was eluted
with CHCl3/MeOH (9:1). Appropriate [as determined by silica
gel TLC in CHCl3/MeOH (19:1)] fractions were combined to
give, after evaporation, compound 6 as a colorless glass in 47%
yield (600 mg, 0.52 mmol): 1H NMR (CDCl3) δ 1.83-2.3 (m,
14, thymine CH3’s, 2′,3′-H’s), 3.6-3.7 (m, 6, 2-6 glucose-H’s),
4.2-4.9 (m, 14, benzyl-CH2-, 4′,5′-H’s), 5.82 (d, 1, J ) 7 Hz,
glucose H-1), 5.99 (dd, 1, J ) 6, 4.6 Hz, 1′-H’s), 6.07 (dd, 1, J
) 6.1, 4.1 Hz, 1′-H’s), 7.16-7.32 (m, 22, salicylate, benzyl H’s),
7.46-7.49 (m, 3, salicylate, thymine 6-H’s), 7.89 (d, 1, J ) 7.6
Hz, salicylate), 9.01 (s, 1, thymine-NH), 9.07 (s, 1, thymine-
NH); 31P NMR (CDCl3) δ -6.58.
Bis(2′,3′-dideoxyth ym idin -5′-yl) D-Glu copyr an osyl P h os-
p h a te (7). Compound 5 (0.4 g, 0.35 mmol) was dissolved in
acetone/methanol (1:1, 100 mL). To this mixture was added