Journal of Medicinal Chemistry
ARTICLE
2-Benzylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-diphosphate
(12d). 1H NMR (500 MHz, D2O): δ 3.25 (m, 2H), 4.43 (m, 1H), 4.49 (m,
1H), 4.58 (m, 1H), 4.74 (m, 2H), 6.19 (m, 1H), 6.53 (m, 1H), 7.53-7.72
(m, 5H), 8.43 (s, 1H). 31P NMR (202 MHz, D2O): δ -8.63 (m), -11.11
(m). 13C NMR (125 MHz, D2O): δ 38.64, 67.01, 73.54, 77.83, 86.78,
94.56, 101.32, 130.76, 131.66, 132.15, 139.13, 144.35, 150.93, 164.05, 174.63.
LC/ESI-MS: negative mode 509 ([M - H]-), positive mode 511
([M þ H]þ).
7.24-7.34 (m, 5H), 8.35 (d, 1H, J = 7.88 Hz). 31P NMR (202 MHz, D2O)
δ 2.84. 13C NMR (125 MHz, D2O): δ 13.39, 37.17, 65.54, 72.53, 77.99,
94.62, 100.58, 111.83, 129.62, 131.42, 132.0, 142.45, 144.70, 163.24, 173.56.
LC/ESI-MS: negative mode 443 ([M - H]-), positive mode 445
([M þ H]þ).
2-Methoxy-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate (13f).
1H NMR (500 MHz, D2O): δ 2.30 (s, 3H), 3.90-3.98 (m, 2H), 4.23
(m, 1H), 4.33 (t, 1H, J = 4.57 Hz), 4.40 (t, 1H, J = 5.04 Hz), 5.97-5.98
(m, 2H), 8.09 (d, 1H, J = 8.19 Hz). 31P NMR (202 MHz, D2O): δ 2.79.
LC/ESI-MS: negative mode 337 ([M - H]-), positive mode 339
([M þ H]þ).
2-Phenylethylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-diphosphate
1
(12e). H NMR (500 MHz, D2O): δ 3.06-3.10 (m, 2H), 3.53-3.58
(m, 1H), 3.61-3.67 (m, 1H), 4.26-4.32 (m, 2H), 4.34 (t, 2H, J = 4.57
Hz), 4.41 (t, 1H, J = 4.88 Hz), 6.0 (d, 1H, J = 4.41 Hz), 6.28 (d, 1H,
J = 7.56 Hz), 7.25-7.35 (m, 5H), 8.20 (d, 1H, J = 7.56 Hz). 31P NMR
(202 MHz, D2O) δ -8.78 (m), -11.07 (m). 13C NMR (125 MHz,
D2O): δ 13.38, 37.19, 66.96, 71.88, 77.83, 86.63, 94.53, 111.76, 129.63,
131.42, 132.01, 142.43, 144.23, 167.22, 174.52. LC/ESI-MS: negative
mode 523 ([M - H]-), positive mode 525 ([M þ H]þ).
2-Methylamino-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
(13h). 1H NMR (500 MHz, D2O): δ 2.95 (s, 3H), 3.94-3.99 (m, 2H),
4.31 (t, 1H, J = 2.20 Hz), 4.36-4.38 (m, 1H), 4.58-4.61 (m, 1H), 5.53
(d, 1H, J = 7.25 Hz), 5.98 (d, 1H, J = 7.88 Hz), 7.80 (d, 1H, J = 7.56 Hz).
31P NMR (202 MHz, D2O): δ 1.33. LC/ESI-MS: negative mode 336
([M - H]-), positive mode 338 ([M þ H]þ).
2-Methoxy-1-β-D-ribofuranosylpyrimidine-4-one-50-diphosphate (12f).
1H NMR (500 MHz, D2O þ MeOD): δ 2.20 (s, 3H), 4.19-4.23 (m, 3H),
4.36 (t, 1H, J=4.72Hz), 4.41(t, 1H,J= 5.04 Hz), 5.93 (m, 1H), 5.94 (d, 1H,
J = 2.83 Hz), 7.98 (d, 1H, J = 8.19 Hz). 31P NMR (202 MHz, D2O þ
MeOD): δ -6.63 (m), -7.43 (m). 13C NMR (125 MHz, D2O þ MeOD):
δ33.05, 60.25, 71.94, 76.72, 83.04, 91.51, 105.33, 136.82, 144.65, 169.17. LC/
ESI-MS: negative mode 417 ([M- H]-), positive mode 419 ([M þ H]þ).
2-Amino-1-β-D-ribofuranosylpyrimidine-4-one-50-diphosphate (12g).
1H NMR (500 MHz, D2O): δ 4.05-4.13 (m, 2H), 4.22 (m, 1H), 4.25
(m, 1H), 4.37 (m, 1H), 5.63 (d, 1H, J = 6.62 Hz), 6.01 (m, 1H), 8.10 (d,
1H, J = 7.88 Hz). 31P NMR (202 MHz, D2O): δ -9.79 (m). LC/ESI-
MS: negative mode 402 ([M - H]-), positive mode 404 ([M þ H]þ).
2-Methylamino-1-β-D-ribofuranosylpyrimidine-4-one-50-diphosphate
3-Phenacyl-50-uridylic Acid (1,1-Chloro-1-phosphonomethyl-1-
1
phosphonyl)anhydride (14b). H NMR (500 MHz, MeOD): δ 4.18
(m, 1H), 4.29-4.37 (m, 3H), 4.44 (q, 1H, J = 4.30 Hz), 5.44 (s, 2H), 6.02-
6.05 (m, 2H), 7.57-8.22 (m, 5H), 8.10 (d, 1H, J = 8.19 Hz). 31P NMR
(202 MHz, MeOD): δ6.38, -1.89, -10.72. 13C NMR (125 MHz, MeOD):
δ 8.70, 43.30, 60.30, 66.37, 71.35, 76.11, 85.37, 91.15, 102.65, 129.42, 130.27,
135.29, 136.60, 141.76, 152.98, 159.32. LC/ESI-MS: negative mode 670, 670
([M - H]-), positive mode 686, 689 ([M þ NH4þ þ H]þ).
4-Thio-50-uridylic Acid (1,1-Chloro-1-phosphonomethyl-1-phos-
1
phonyl)anhydride (14d). H NMR (500 MHz, D2O): δ 3.98-4.11
(m, 2H), 4.26 (q, 1H, J = 2.31 Hz), 4.33 (t, 1H, J = 4.88 Hz), 4.38 (t, 1H,
J = 4.88 Hz), 5.93 (d, 1H, J = 4.41 Hz), 6.65 (d, 1H, J = 7.56 Hz), 7.97 (d,
1H, J = 7.56 Hz). 31P NMR (202 MHz, D2O): δ 10.09, 1.21, -7.98. LC/
ESI-MS: negative mode 565 ([M - H]-).
1
(12h). H NMR (500 MHz, D2O): δ 2.93 (s, 3H), 3.99 (m, 3H), 4.19
3-Phenacyl-50-uridylic Acid (1,1-Difluoro-1-phosphonomethyl-1-
(m, 1H), 4.34 (m, 1H), 5.57 (d, 1H, J = 6.62 Hz), 5.95 (d, 1H, J = 7.56 Hz),
7.78 (d, 1H, J=7.88Hz). 31P NMR (202 MHz, D2O): δ-9.21, -9.83. LC/
ESI-MS: negative mode 416 ([M -H]-), positive mode 418 ([M þ H]þ).
2-Methylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
(13a). 1H NMR (500 MHz, D2O): δ 2.22 (s, 3H), 4.05-4.09 (m, 2H),
4.32 (m, 1H), 4.37 (m, 1H), 4.44 (m, 1H), 6.03 (d, 1H, J = 4.09 Hz), 6.33
(d, 1H, J =7.56Hz), 8.34(d, 1H, J =7.56Hz). 31P NMR (202 MHz, D2O):
δ 1.06. LC/ESI-MS: negative mode 353 ([M - H]-), positive mode 355
([M þ H]þ).
1
phosphonyl)anhydride (14f). H NMR (500 MHz, MeOD): δ 4.18
(m, 1H), 4.31-4.34 (m, 3H), 4.41 (t, 1H, J = 4.41 Hz), 5.44 (d, 2H, J =
1.26 Hz), 6.02-6.08 (m, 2H), 7.57-8.09 (m, 5H), 8.10 (d, 1H, J = 7.25
Hz). 31P NMR (202 MHz, MeOD): δ 2.66, -5.51, -10.99. 13C NMR
(125 MHz, MeOD): δ 11.82, 43.41, 66.44, 71.72, 76.11, 85.40, 88.26,
91.15, 102.73, 129.43, 130.28, 135.29, 136.59, 141.69, 152.99, 164.94.
LC/ESI-MS: negative mode 635 ([M - H]-).
4-Thio-50-uridylic Acid (1,1-Difluoro-1-phosphonomethyl-1-phos-
2-Ethylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
(13b). 1H NMR (500 MHz, D2O): δ1.41 (t, 3H, J= 7.40 Hz), 3.35 (q, 2H,
J = 7.35 Hz), 4.10 (m, 2H), 4.27 (m, 1H), 4.34 (t, 1H, J = 4.88 Hz), 4.38
(t, 1H, J = 5.20 Hz), 5.96 (d, 1H, J = 8.19 Hz), 5.98 (d, 1H, J = 5.04 Hz),
8.02 (d, 1H, J=7.88Hz). 31P NMR (202 MHz, D2O): δ0.63. LC/ESI-MS:
negative mode 367 ([M - H]-), positive mode 369 ([M þ H]þ).
2-Propylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
1
phonyl)anhydride (14g). H NMR (500 MHz, D2O): δ 4.25-4.30
(m, 3H), 4.37-4.40 (m, 2H), 5.95 (d, 1H, J = 4.09 Hz), 6.66 (d, 1H, J =
7.88 Hz), 7.85 (d, 1H, J = 7.56 Hz). 31P NMR (202 MHz, D2O): δ 3.13,
-2.07, -10.90 (d, J = 29.53 Hz). 13C NMR (125 MHz, D2O): δ 67.56,
72.21, 76.95, 82.87, 86.25, 91.74, 116.79, 139.57, 152.20, 193.66. LC/
ESI-MS: negative mode 533 ([M - H]-).
5-Bromo-1-β-D-ribofuranosyl(3H)pyrimidine-2,4-dione 50-(γ-phenylami-
no)triphosphate (16). 1H NMR (500 MHz, D2O þ MeOD þ NaOD): δ
3.34-3.36 (m, 2H), 3.94 (t, 1H, J = 5.20 Hz), 4.05-4.08 (m, 2H), 5.80 (d,
1H, J = 5.04 Hz), 6.92-7.31 (m, 5H), 7.92 (s, 1H). 31P NMR (202 MHz,
D2O þ MeOD þ NaOD): δ -9.38 (d, J = 19.68 Hz), -10.58 (d, J = 17.22
Hz), -21.74 (t, J = 18.45 Hz). 13C NMR (125 MHz, D2O þ MeOD þ
NaOD): δ 68.72, 73.43, 78.05, 86.10, 93.09, 101.50, 120.26, 120.32, 123.13,
131.99, 142.72, 161.90, 174.17. LC/ESI-MS: negative mode 636 and 639
([M - H]-), positive mode 655 ([M þ NH4þ þ H]þ).
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1
(13c). H NMR (500 MHz, D2O): δ H NMR (500 MHz, D2O þ
MeOD): δ 0.99 (t, 3H, J = 7.40 Hz), 1.72 (m, 2H), 3.02-3.06 (m, 2H),
4.06 (m, 2H), 4.25 (m, 1H), 4.42 (m, 2H), 5.49 (d, 1H, J = 4.41 Hz),
6.02-6.10 (m, 1H), 8.06 (d, 1H, J = 8.19 Hz). 31P NMR (202 MHz,
D2O þ MeOD): δ 1.97. LC/ESI-MS: negative mode 381 ([M - H]-),
positive mode 383 ([M þ H]þ).
2-Benzylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
1
(13d). H NMR (500 MHz, D2O þ MeOD): δ 3.88-3.97 (m, 2H),
4.21 (m, 1H), 4.29-4.32 (m, 1H), 4.55 (m, 1H), 4.84 (m, 2H),
5.94 (d, 1H, J = 5.35 Hz), 6.28 (d, 1H, J = 7.88 Hz), 7.30-7.51 (m,
5H), 8.38 (d, 1H, J = 7.88 Hz). 31P NMR (202 MHz, D2O): δ 0.27.
LC/ESI-MS: negative mode 429 ([M - H]-), positive mode 431
([M þ H]þ).
Determination of IP Accumulation at P2Y2, P2Y4, and P2Y6 Receptors.
Assays were performed using 1321N1 astrocytoma cells recombinantly
expressing the human P2Y2, P2Y4, or P2Y6 receptor subtype, respectively,
as previously described.18,37A scintillation proximity assay in a 96-well plate
format was applied18 determining IP production induced by activation of the
Gq-coupled receptors by the test compounds. Data were analyzed with
PRISM 4.0 (GraphPad Software Inc., San Diego, CA). Three separate
experiments were performed, and each data point was determined in
triplicate.
2-Phenylethylthio-1-β-D-ribofuranosylpyrimidine-4-one-50-monophosphate
1
(13e). H NMR (500 MHz, D2O): δ 3.06-3.10 (m, 2H), 3.53-3.64
(m, 2H), 3.96-4.06 (m, 2H), 4.28 (m, 1H), 4.34 (t, 1H, J = 4.72 Hz), 4.37
(t, 1H, J = 4.57 Hz), 6.0 (d, 1H, J = 4.72 Hz), 6.29 (d, 1H, J = 7.88 Hz),
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dx.doi.org/10.1021/jm1016297 |J. Med. Chem. 2011, 54, 2878–2890