Journal of Medicinal Chemistry
Article
deprotection of the 3′-O-Lev group was completed in less than 10 min
(a single band on TLC), and the crude product was purified on a silica
gel column DCM/MeOH gradient, 10:(0.7−1.0)) to give 6 (735 mg,
52%) as a colorless foam: Rf = 0.42 (DCM/MeOH, 10:0.7). HPLC
analysis has shown a diastereomeric mixture (4:5 ratio): tR = 20.2 and
tR =22.2 min (∼98% pure, UV at 280 nm); conducted on Jupiter C18
of t-BuOOH (0.9 mL, ≥4.5 mmol). Before the deprotection of the 3′-
O-Lev group, a small sample of the crude product (∼11 mg) was
́
purified by HPLC on a Columbus C18, 100 Å (5 μm, 10 mm × 250
mm) column, eluting at 2.5 mL/min with 40% MeCN in water. The
HPLC analysis showed two diastereomers (45:55 ratio): tR = 26.3 min,
́
tR = 26.6 min (≥98% pure, UV at 280 nm); ACE C18, 100 Å (5 μm,
́
300Å (5 μm, 4.6 mm × 250 mm) column; eluent, solvent A 20%
4.6 mm × 250 mm); eluent, solvent A 10% MeCN in water, solvent B
MeCN; elution at 1.0 mL/min with a linear gradient of B from 0% to
95% over 45 min and 95% B for 15 min. Purified 3′-O-Lev derivative
MeCN in water, solvent B MeCN; elution at 0.8 mL/min with A for
25 min, then a linear gradient of B from 0% to 95% over 10 min, and
finally 95% B for 10 min. 1H NMR (DMSO-d6) δ = 11.68, 11.63 (2 s,
1H, NH), 7.89, 7.78 (2 s, 1H, uridine-H6), 7.33 (tt, 1H, aryl-H4, J =
7.5 Hz), 7.25−7.19 (m, 2H, aryl-H6, aryl-H5), 7.17−7.12 (m, 1H, aryl-
1
of 7 was further analyzed: H NMR (DMSO-d6) δ = 11.77 (bs, 1H,
NH), 8.08, 8.05 (2 s, 1H, uridine-H6), 7.26−7.14 (m, 1H, aryl-H5),
7.10−7.08 (m, 2H, aryl-H6, aryl-H4), 6.09 (dd, 1H, H1′, J1′‑2′ = 7.3 Hz,
J1′‑2″ = 6.4 Hz), 5.53−5.42 (m, 2H, benzyl), 5.15−5.09 (m, 1H, H3′),
4.41−4.36 (m, 2H, H4′, H5″), 4.15−4.11 (m, 1H, H5′), 3.35−3.33 (m,
2H, Lev-C3-CH2), 2.74−2.72 (m, 2H, Lev-C2-CH2), 2.41−2.24 (m,
4H, H2″, Lev-C5-CH3), 2.23−2.18 (m, 1H, H2′), 2.11 (s, 3H, aryl-C3-
CH3) ppm. 31P NMR (DMSO-d6) δ = −8.23, −8.27 (2 s,
diastereomeric mixture) ppm. HR-FABMS (m/z): [M + H]+calcd
for C22H25N2O10PI, 635.0292; found, 635.0277. Subsequent cleavage
of the 3′-O-Lev group was completed in <5 min and the crude product
was purified on a silica gel column (DCM/MeOH, 10:0.7) to give 7
(933 mg, 58%) as a colorless foam: Rf = 0.46 (DCM/MeOH, 10:0.7).
The HPLC analysis showed a mixture of diastereomers: tR = 18.2 min,
3
H3), 6.03, 5.98 (2 t, 1H, H1′, JH,H = 7.0 Hz), 5.22−5.19 (m, 2H,
benzyl), 5.42 (d, 1H, C3′- OH, J = 4.6 Hz), 4.36−4.23 (m, 2H, H5″,
H5′), 4.22−4.18 (m, 1H, H3′), 3.94−3.89 (m, 1H, H4′), 2.23−2.12
(m, 1H, H2″), 2.10−2.05 (m, 1H, H2′) ppm. 31P NMR (DMSO-d6) δ
= −9.18, −9.33 (2 s, diastereomeric mixture) ppm. HR-FABMS (m/
z): [M + Li]+calcd for C16H16N2O8PILi, 528.9849; found, 528.9837.
Diastereomers of 6 were separated by preparative HPLC on a
́
Columbus C18, 100 Å (5 μm, 10 mm × 250 mm) column, eluting at
2.6 mL/min with 20% MeCN in water. The amount of 59 mg of the
diastereomeric mixture used for the separation gave 19.1 mg of 6-fast
and 17.2 mg of 6-slow. Analytical data of diastereomer 6-fast are as
1
́
tR = 18.8 min (≥98% pure, UV at 280 nm); ACE C18, 100 Å (5 μm,
follows: H NMR (DMSO-d6) δ = 11.69 (s, 1H, NH), 7.98 (s, 1H,
uridine-H6), 7.35 (tt, 1H, aryl-H4, 3JHH = 7.8 Hz, 4JHH = 1.0 Hz), 7.27,
4.6 mm × 250 mm) column; eluent, solvent A 10% MeCN in water,
solvent B MeCN; elution at 1.0 mL/min with a linear gradient of B
from 0% to 95% over 45 min and 95% B for 15 min. 1H NMR
(DMSO-d6) δ = 11.73, 11.68 (2 s, 1H, NH), 7.99, 7.86 (2 s, 1H,
uridine-H6), 7.25−7.21 (m, 1H, aryl-H5), 7.12−7.07 (m, 2H, aryl-H6,
aryl-H4), 6.08 (dd, 1H, H1′, 2J1′‑2′ = 7.6 Hz, 2J1′‑2″ = 6.0 Hz), 6.05 (dd,
3
7.25 (2 d, 1H, aryl-H6, J = 1.5 Hz), 7.18 (tt, 1H, aryl-H5, JHH = 7.5
Hz, 4JHH = 1.0 Hz), 7.14, 7.12 (2 d, 1H, aryl-H3, 3JHH = 7.6 Hz, 4JHH
=
1,0 Hz), 6.07 (dd, 1H, H1′, J1′−2′ = 7.4 Hz, J1′−2″ = 6.5 Hz), 5.55−5.44
(m, 2H, benzyl), 5.41 (bd, 1H, C3′-OH, J = 4.4 Hz), 4.39 (ddd, 1H,
2
3
3
H5″, JHH = 11.6 Hz, JHH = 7.1 Hz, JHP = 3.7 Hz), 4.29 (ddd, 1H,
H5′, 2JHH = 11.6 Hz, 3JHH = 6.7 Hz, 3JHP = 4.6 Hz), 4.18−4.16 (m, 1H,
H3′), 3.94−3.91 (m, 1H, H4′), 2.19−2.16 (m, 1H, H2″), 2.09−2.05
(m, 1H, H2′) ppm. 13C NMR (DMSO-d6) δ = 160.77 (C4), 150.38
(C2), 149.35 (C2-aryl), 144.33 (C6), 129.62 (C4-aryl), 126.0 (C6-
aryl), 124.31 (C5-aryl), 120.93 (C1-aryl), 118.0 (C3-aryl), 109.42
(C5), 84.75 (C1′), 84.32 (C4′), 69.92 (C3′), 68.86 (C-benzyl), 67.64
(C5′), 38.72 (C2′) ppm. 31P NMR (DMSO-d6) δ = −9.19 ppm.
Analytical data of diastereomer 6-slow are as follows: 1H NMR
(DMSO-d6) δ = 11.71 (s, 1H, NH), 7.99 (s, 1H, uridine-H6), 7.37 (tt,
2
2
1H, H1′, J1′‑2′ = 7.8 Hz, J1′‑2″ = 5.8 Hz), 5.51−5.39 (m, 2H, benzyl),
5.41 (d, 1H, C3′-OH′, J = 3.7 Hz), 4.34−4.26 (m, 1H, H5″, H3′),
4.22−4.18 (m, 1H, H5′), 3.93−3.89 (m, 1H, H4′), 2.25−2.19 (m, 4H,
H2″,), 2.23 (s, 3H, C3-aryl-CH3), 2.10−2.05 (m, 1H, H2″) ppm. 13C
NMR (DMSO-d6) δ = 162.87 (C4), 150.31 (C2), 148.55, 148.51 (C2-
aryl), 141.23, 141.18 (C6), 132.86 (C5-aryl), 129.18 (C4-aryl), 126.33
(C6-aryl), 120.73, 120.66 (C1-aryl), 117.35 (C3-aryl), 109.67 (C5),
84.75, 84.65 (C1′), 84.32, 84.22 (C4′), 69.98 (C3′), 68.82 (C-benzyl),
67.64 (C5′), 38.72 (C2′), 21.11 (CH3-aryl) ppm.31P NMR (DMSO-
d6) δ = −8.89, −8.93 (2 s, diastereomeric mixture) ppm. HR-FABMS
(m/z): [M + H]+calcd for C16H19N2O8PI, 536.9924; found, 536.9899.
Diastereomers were separated using preparative HPLC on a Columbus
3
4
1H, aryl-H4, J
= 7.7 Hz, JHH = 1.1 Hz), 7.28 (dd, 1H, aryl-H6,
HH
3JHH = 7.1 Hz, JH,H = 1.5 Hz), 7.20 (tt, 1H, aryl-H5, JHH = 7.6 Hz,
4
3
4JHH = 1.0 Hz), 7.15 (dd, 1H, aryl-H3, JHH = 7.6 Hz, 4JHH = 1,0 Hz),
3
́
C18, 100 Å (5 μm, 10 mm × 250 mm) column; elution was with 22%
6.10 (dd, 1H, H1′, J1′−2′ = 7.5 Hz, J1′−2″ = 6.4 Hz), 5.53−5.45 (m, 2H,
benzyl), 5.43 (d, 1H, C3′-OH, J = 4.5 Hz), 4.35−4.27 (m, 2H, H5″ and
H5′), 4.22−4.18 (m, 1H, H3′), 3.94−3.90 (m, 1H, H4′), 2.22−2.17
(m, 1H, H2″), 2.10−2.05 (m, 1H, H2′) ppm. 13C NMR (DMSO-d6) δ
= 160.81 (C4), 150.42 (C2), 149.39 (C2-aryl), 144.41 (C6), 129.59
(C4-aryl), 126.12 (C6-aryl), 124.29 (C5-aryl), 120.87 (C1-aryl),
118.90 (C3-aryl), 110.02 (C5), 84.83 (C1′), 84.29 (C4′), 70.13 (C3′),
68.90 (C-benzyl), 67.76 (C5′), 38.59 (C2′) ppm. 31P NMR (DMSO-
d6) δ = −9.32 ppm.
Method II. General procedure B was conducted with IUdR 1 (1.12
g, 3.16 mmol) dissolved in 15 mL of DMF, DIPEA (1.14 mL, 0.84 g,
6.5 mmol), and newly distilled chlorophosphite 15 (604 mg, 3.83
mmol) diluted with 6 mL of dry THF. The mixture was split into three
(2 mL) portions. The oxidation was with a solution of t-BuOOH (0.86
mL, ≥ 4.3 mmol). The time of phosphitylation was 2 h, and the
oxidation was carried out for 1 h. All three phosphotriesters, purified
on a silica gel column (DCM/MeOH gradient, 10:(0.7−1.0)), were
collected in the form of a colorless foam: 5′,3′-O,O′-dicycloSal-5-
IUdRMP 9 (Rf = 0.72), 350 mg (16%); 3′-O-cycloSal-IUdRMP 12 (Rf
= 0.58), 611 mg (37%); 5′-O-cycloSal-5-IUdRMP 6 (Rf = 0.42), 759
mg (46%). The analytical data of the product 6 were identical to those
reported above for 6 obtained using method I.
MeCN in water at 2.5 mL/min flow rate. From a total of 66 mg of
diastereomeric 7 used for separation, 14.1 mg of 7-fast and 12.2 mg of
7-slow were isolated. Diastereomer 7-fast eluted within 72−76 min
and 7-slow within 79−81 min after the injection, and each isomer was
collected in ∼9 mL of eluent. A solvent was evaporated to dryness in a
high vacuum. The product residue was reconstituted in MeCN and
analyzed one more time using analytical HPLC. Each diastereomer
was found to be ≥97% pure (UV at 220 and 280 nm). Analytical data
of 7-fast are as follows: 1H NMR (DMSO-d6) δ = 11.70 (s, 1H, NH),
7.98 (s, 1H, uridine-H6), 7.23−7.20 (m, 1H, aryl-H5), 7.17 (dd, 1H,
3
4
aryl-H4, JHH = 7.6 Hz, JH,H = 1.5 Hz), 7.12−7.06 (m, 1H, aryl-H6),
6.06 (dd, 1H, H1′, J1′2″ = 6.6 Hz, J1′‑2″ = 5.8 Hz), 5.51−5.43 (m, 3H,
benzyl, C3′-OH), 4.38−4.33 (m, 1H, H5″), 4.27−4.22 (m, 1H, H5′),
4.17−4.15 (m, 1H, H3′), 3.92−3.90 (m, 1H, H4′), 2.21−2.19 (m, 3H,
C3-aryl-CH3), 2.18−2.13 (m, 1H, H2″), 2.10−2.05 (m, 1H, H2′) ppm.
31P NMR (DMSO-d6) δ = −8.89 ppm. Analytical data of 7-slow are as
1
follows: H NMR (DMSO-d6) δ = 11.69 (s, 1H, NH), 7.88 (s, 1H,
3
4
uridine-H6), 7.24 (dd, 1H, aryl-H5, JHH = 7.8 Hz, JH,H = 1.2
Hz),7.09−7.06 (m, 2H, aryl-H6, aryl-H4), 6.07 (dd, 1H, H1′, J1′,2′ = 6.5
Hz, J1′,2″ = 4.8 Hz), 5.49−5.38 (m, 3H, benzyl, C3′-OH), 4.33−4.25
(m, 2H, H5″, H5′), 4.21−4.17 (m, 1H, H3′), 3.92−3.89 (m, 1H, H4′),
2.24−2.18 (m, 4H, H2″, C3-aryl-CH3), 2.11−2.06 (m, 1H, H2′) ppm.
31P NMR (DMSO-d6) δ = −8.94 ppm.
5-Iodo-5′-O-cyclo(3-methylsaligenyl)-2′-deoxyuridine
Monophosphate (7). Method I. General procedure C with 3′-O-
levulinyl IUdR 4 (1.36 g, 3.0 mmol), DIPEA (1.5 mL, 1.11 g, 8.6
mmol), and the crude chlorophosphite 16 (1.04 g, ∼6 mmol) was
conducted in 27 mL of dry MeCN. The reaction time was extended to
4 h (TLC monitoring). The oxidation was carried out with a solution
Method II. General procedure B was conducted with IUdR 1 (2.51
g, 7.09 mmol) dissolved in 18 mL of DMF and DIPEA (1.54 mL, 1.14
g, 8.8 mmol). The crude chlorophosphite 16 (1.39 g, ∼8 mmol) was
dissolved in 6 mL of dry THF added in 3 × 2 mL portions. A solution
2660
dx.doi.org/10.1021/jm201482p | J. Med. Chem. 2012, 55, 2649−2671