Thymidine Analogues as Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 22 5289
r-D-Thymidine 5′-Monophosphate (9). A solution of R-D-
thymidine (150 mg, 0.62 mmol) in trimethyl phosphate (6.2 mL)
was cooled to 0 °C. POCl3 (369 µL, 4.03 mmol) was added
dropwise, and the mixture was stirred for 4 h at 0 °C and for 30
min at room temperature. The mixture was poured into ice-water
(12 mL), neutralized with concentrated NH4OH, and evaporated
to dryness. The resulting residue was purified by column chroma-
tography (iPrOH/NH4OH/H2O, 77.5:15:2.5). Further purification
was performed by HPLC (C-18, CH3CN/MeOH/0.05% HCOOH
in H2O, 45:45:10, 3 mL/min). After lyophilization of the collected
pure fractions, compound 9 was obtained (123 mg, 62%) as a white
4.34 (1H, m, H-4′), 5.47 (1H, d, J ) 3.3 Hz, 3′-OH), 6.18 (1H, dd,
J ) 3.6 and 8.1, H-1′), 7.10 (1H, t, J ) 7.2 Hz, arom H), 7.32
(2H, t, J ) 7.5 Hz, 2 arom H), 7.77 (2H, d, J ) 1.2 Hz, H-6 +
N(5′)H), 9.63 (1H, br s, N(ar)H), 11.26 (1H, br s, N(3)H). 13C NMR
(75 MHz, DMSO-d6): δ 13.03 (5-CH3), under DMSO (C-2′), 46.18
(C-5′), 71.58 (C-3′), 85.40 (C-1′), 86.57 (C-4′), 109.51 (C-5), 123.83
(arom C), 124.97 (arom C), 129.29 (arom C), 137.68 (C-6), 139.84
(arom C), 151.21 (C-4), 164.55 (C-2), 181.41 (CdS). HRMS (ESI-
MS) for C17H21N4O4S [M + H]+ found, 377.1279; calcd, 377.1283.
Anal. (C17H20N4O4S) C, H, N.
N-(5′-Deoxy-r-D-thymidin-5′-yl)-N′-(3-trifluoromethyl-4-chlo-
robenzyl)thiourea (23). 4-Chloro-3-trifluoromethylbenzylamine (65
mg, 0.31 mmol) was added at 0 °C to a stirred solution of 1,1′-
thiocarbonyldiimidazole (61 mg, 0.34 mmol) and imidazole (6.3
mg, 0.09 mmol) in 4 mL of acetonitrile. After 10 min at 0 °C, the
mixture was allowed to stir for 3 h at room temperature. A solution
of 49 (75 mg, 0.31 mmol) in 2 mL of DMF was added, and the
reaction mixture was stirred at room temperature overnight. The
reaction mixture was evaporated to dryness and purified by column
chromatography (CH2Cl2/MeOH, 99:1) to obtain compound 23 (114
1
powder. H NMR (300 MHz, D2O): δ 1.81 (3H, d, J ) 0.9 Hz,
5-CH3), 2.02-2.09 (2H, ddd, J ) 3.0 and 14.7 Hz, H-2′), 2.66-
2.76 (1H, m, H-2′′), 3.78 (2H, app t, J ) 5.1 Hz, H-5′ and H-5′′),
4.43 (2H, m, H-3′ and H-4′), 6.12 (1H, dd, J ) 3.0 and 7.2 Hz,
H-1′), 7.68 (1H, d, J ) 0.9 Hz, H-6). 31P NMR (500 MHz, D2O):
δ 2.97. 13C NMR (75 MHz, D2O): δ 11.73 (5-CH3), 39.49 (C-2′),
64.67 (C-5′), 71.20 (C-3′), 87.38 and 87.81 (C-4′ and C-1′), 110.75
(C-5), 138.39 (C-6), 151.76 (C-4), 166.97. HRMS (ESI-MS) for
C10H14N2O8PNa [M + Na]+ found, 345.0477; calcd, 345.0464.
Anal. (C10H14N2O8P) C, H, N.
1
mg, 75%). H NMR (300 MHz, DMSO-d6): δ 1.77 (3H, d, J )
5′-Azido-5′-deoxy-r-D-thymidine (48). To a solution of R-D-
thymidine 47 (886 mg, 3.66 mmol) in pyridine (13.5 mL) at
-78 °C, methanesulfonyl chloride (256 µL, 0.41 mmol) was added.
The reaction mixture was stirred for 1 h at 0 °C. The reaction was
quenched by adding saturated aqueous NaHCO3 solution and
extracted with CH2Cl2 three times, dried over MgSO4, and
evaporated. The residue was purified by column chromatography
(CH2Cl2/MeOH, 98:2) to give the mesylated compound (916 mg,
78%). 1H NMR (300 MHz, DMSO-d6): 1.75 (3H, s, 5-CH3), 1.95
(1H, m, H-2′), 2.55 (1H, m, H-2′′), 3.19 (3H, s, CH3SO2), 4.12-
4.26 (3H, m, H-5′, H-5′′ and H-3′), 4.33 (1H, m, H-4′), 5.58 (1H,
br s, 3′-OH), 6.12 (1H, dd, J ) 4.4 and 7.6 Hz, H-1′), 7.73 (1H, s,
H-6), 11.27 (1H, br s, N(3)H). HRMS (ESI-MS) for C11H17N2O7S
[M + H]+ found, 321.0759; calcd, 321.0756.
A solution of 5′-mesylated R-D-thymidine (916 mg, 2.88 mmol)
and NaN3 (1.87 g, 29 mmol) in DMF (50 mL) was heated to 60 °C
overnight. The reaction mixture was evaporated in vacuo. The
residue was resolved in CH2Cl2 and washed with H2O. The organic
layer was dried over MgSO4, evaporated, and purified by column
chromatography (CH2Cl2/MeOH, 98:2) to afford compound 48 (672
mg, 87%). 1H NMR (300 MHz, DMSO-d6): 1.76 (3H, s, 5-CH3),
1.93 (1H, m, H-2′), 2.51 (1H, m, H-2′′), 3.49 (2H, m, H-5′ and
H-5′′), 4.12 (1H, dd, H-3′), 4.24 (1H, m, H-4′), 5.50 (1H, br s,
3′-OH), 6.13 (1H, dd, J ) 4.5 and 7.5 Hz, H-1′), 7.72 (1H, s, H-6),
11.26 (1H, br s, N(3)H). HRMS (ESI-MS) for C10H14N5O4 [M +
H]+ found, 268.1045; calcd, 268.1046. Anal. (C10H13N5O4) C, H,
N.
5′-Amino-5′-deoxy-r-D-thymidine (49). A solution of azide 48
(531 mg, 1.99 mmol) in methanol (30 mL) was hydrogenated under
atmospheric pressure for 6 h in the presence of 10% Pd/C (53.1
mg). The catalyst was removed by filtration through Celite and
the filtrate was evaporated to yield pure amine 49 (471 mg, 98%).
1H NMR (300 MHz, DMSO-d6): 1.74 (3H, s, 5-CH3), 1.85 (1H,
m, H-2′), 2.49 (1H, m, H-2′′), 3.43 (2H, m, H-5′ and H-5′′), 4.02
(1H, m, H-3′), 4.14 (1H, m, H-4′), 5.27 (1H, br s, 3′-OH), 6.05
(1H, dd, J ) 3.3 and 7.5 Hz, H-1′), 7.69 (1H, s, H-6), 11.24 (1H,
br s, N(3)H). HRMS (ESI-MS) for C10H16N3O4 [M + H]+ found,
242.1134; calcd, 242.1141. Anal. (C10H15N3O4‚1/2H2O) H, N. C:
calcd, 47.99; found, 48.46.
N-(5′-Deoxy-r-D-thymidin-5′-yl)-N′-phenylthiourea (10). For
the synthesis of compound 10, compound 49 (54 mg, 0.22 mmol)
was dissolved in DMF (2 mL). At 0 °C, phenyl isothiocyanate (36
mg, 0.26 mmol) was added, and the reaction mixture was allowed
to stir at room temperature during 3 h. After completion of the
reaction, the reaction mixture was evaporated to dryness and the
residue was purified by column chromatography (CH2Cl2/MeOH,
97:3) to obtain the pure final compound 10 (69 mg, 83%). 1H NMR
(300 MHz, DMSO-d6): δ 1.77 (3H, d, J ) 1.2 Hz, 5-CH3), 1.89-
1.98 (1H, ddd, J ) 3.3 and 14.1 Hz, H-2′), 2.53-2.63 (1H, m,
H-2′′), 3.49 (1H, m, H-5′), 3.64 (1H, m, H-5′′), 4.22 (1H, m, H-3′),
0.9 Hz, 5-CH3), 1.87-1.95 (1H, ddd, J ) 2.9 and 14.4 Hz, H-2′),
2.50-2.60 (1H, m, H-2′′), 3.47 (1H, m, H-5′), 3.55 (1H, m, H-5′′),
4.18 (1H, m, H-3′), 4.26 (1H, m, H-4′), 4.71 (2H, d, J ) 4.8 Hz,
CH2NH), 5.41 (1H, d, J ) 3.0 Hz, 3′-OH), 6.14 (1H, dd, J ) 3.2
and 7.6 Hz, H-1′), 7.56 (1H, dd, J ) 2.0 and 8.8 Hz, arom H),
7.65 (1H, d, J ) 8.7 Hz, arom H), 7.73 (2H, app s, arom H and
H-6), 7.78 (1H, t, J ) 4.7 Hz, N(5′)H), 8.14 (1H, d, J ) 4.9 Hz,
CH2NH), 11.20 (1H, br s, N(3)H). 13C NMR (75 MHz, DMSO-
d6): δ 13.00 (5-CH3), 31.47 (CH2NH), 46.48 (C-2′), 46.58 (C-5′),
71.58 (C-3′), 85.46 (C-1′), 86.98 (C-4′), 109.44 (C-5), 127.10 (arom
C), 127.17 (arom C), 129.49 (arom C), 132.14 (arom C), 133.57
(arom C), 137.58 (C-6), 140.54 (arom C), 151.19 (C-4), 164.52
(C-2), 182.28 (CdS). HRMS (ESI-MS) for C19H21N4O4SClF3 [M
+ H]+ found; calcd, 493.0924. Anal. (C19H20N4O4SClF3) C, H, N.
N-(3,4-Dichlorophenyl)-N′-(5′-deoxy-r-D-thymidin-5′-yl)-
urea (26). Urea 26 was synthesized from 49 (85 mg, 0.35 mmol)
and 3,4-dichlorophenyl isocyanate (79 mg, 0.42 mmol) using the
same procedure as described for the synthesis of 10. After
purification by column chromatography (CH2Cl2/MeOH, 97:3),
1
compound 26 (113 mg, 75%) was obtained. H NMR (300 MHz,
DMSO-d6): δ 1.78 (3H, s, 5-CH3), 1.89-1.97 (1H, ddd, J ) 3.2
and 14.3 Hz, H-2′), 2.51-2.61 (1H, m, H-2′′), 3.09 (1H, m, H-5′),
3.21 (1H, m, H-5′′), 4.16 (2H, m, H-3′ and H-4′), 5.42 (1H, d, J )
3.3 Hz, 3′-OH), 6.15 (1H, dd, J ) 3.6 and 7.8, H-1′), 6.44 (1H, t,
J ) 5.1 Hz, N(5′)H), 7.22 (1H, dd, J ) 2.4 and 9.0 Hz, arom H),
7.43 (1H, d, J ) 9.0 Hz, arom H), 7.75 (1H, d, J ) 0.9 Hz, H-6),
7.82 (1H, d, J ) 2.4 Hz, arom H), 8.92 (1H, br s, N(ar)H), 11.24
(1H, br s, N(3)H). 13C NMR (75 MHz, DMSO-d6): δ 12.99 (5-
CH3), 41.77 (C-2′), 49.28 (C-5′), 71.57 (C-3′), 85.35 (C-1′), 87.32
(C-4′), 109.54 (C-5), 118.40 (arom C), 119.36 (arom C), 122.97
(arom C), 131.12 (arom C), 131.61 (arom C), 137.60 (C-6), 141.33
(arom C), 151.21 (C-4), 155.56 (CdO), 164.51 (C-2). HRMS (ESI-
MS) for C17H18N4O5Cl2Na [M + Na]+ found, 451.0548; calcd,
451.0552. Anal. (C17H18Cl2N4O5) C, H, N.
5′-Azido-5′-deoxy-r-D-thymidine 3′-Methanesulfonate (50).
Compound 48 (400 mg, 1.5 mmol) was dissolved in pyridine (5
mL), and methanesulfonyl chloride (150 µL, 1.95 mmol) was added
at 0 °C. After 2 h, the reaction was quenched by saturated NaHCO3
solution (5 mL) and extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were dried with MgSO4 and evaporated
1
to dryness to obtain pure compound 50 (420 mg, 81%). H NMR
(300 MHz, DMSO-d6): δ 1.77 (3H, d, J ) 1.0 Hz, 5-CH3), 2.32-
2.40 (1H, ddd, J ) 3.6 and 15.0 Hz, H-2′), 2.81-2.91 (1H, m,
H-2′′), 3.27 (3H, s, SO2CH3), 3.52 (2H, d, J ) 5.1 Hz, H-5′ and
H-5′′), 4.70 (1H, m, H-4′), 5.17 (1H, m, H-3′), 6.16 (1H, dd, J )
3.6 and 6.9, H-1′), 7.52 (1H, d, J ) 1.1 Hz, H-6), 11.31 (1H, br s,
N(3)H). HRMS (ESI-MS) for C11H15N5O6S [M + H]+ found,
346.0818; calcd, 346.0821.
5′-Azido-3′,5′-dideoxy-2′,3′-didehydro-r-D-thymidine (51). To
a solution of mesylate ester 50 (420 mg, 1.21 mmol) in THF (15