Journal of Medicinal Chemistry
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7.5% aqueous copper sulfate pentahydrate solution (4.0 μL, 1.2 μmol,
1.5 equiv). The reaction mixture was stirred overnight at rt, solvent
was evaporated, and the residue was purified by semipreparative
HPLC as described above to obtain 27 (1.64 mg, 34% yield) as a
yellow/orange solid. HRMS-EI found: 1399.2106 (M − H+)−.
C51H58N10O27S2P3 requires 1399.2104.
amine hydrochloride (87, 617 mg, 3.0 mmol, 2 equiv) in pyridine (4
mL) was stirred at 100 °C overnight. The reaction mixture was
evaporated, and the residue was evaporated twice with toluene,
triturated with chloroform, and filtered. The filtrate was evaporated,
and the residue was purified by flash chromatography (chloroform−
methanol, gradient of 3−10%) to afford N4-(4-phenylbutyloxy)-
cytidine (97) (0.430 g, 1.10 mmol, 73%). 1H NMR (400 MHz,
MeOD): 7.52 (d, J1 = 8.20 Hz, 1H), 7.20−7.17 (m, 2H), 7.15−7.05
(m, 3H), 5.83 (d, J1 = 4.84 Hz, 1H), 5.63 (d, J1 = 8.12 Hz, 1H), 4.13
(t, J1 = 5.20 Hz, 1H), 4.09 (m, 1H), 4.00−3.92 (m, 3H), 3.78 (dd, J1 =
12.16 Hz, J1 = 2.72 Hz, 1H), 3.68 (dd, J1 =12.20 Hz, J2 = 3.16 Hz, 1H),
2.60 (t, J1 = 6.88 Hz, 2H), 1.70−1.60 (m, 4H). 13C NMR (400 MHz,
MeOD): 149.2, 147.6, 142.0, 137.1, 135.0, 128.0, 127.9, 125.4, 95.0,
88.9, 84.8, 74.4, 73.7, 69.9, 60.9, 35.1, 27.7, 27.3. m/z (M + ESI MS)
found, 392.1813; calcd for C19H26N3O6, 392.1816.
N4-(3-((4-O-CH2CONH-(CH2)2(BODIPY-1H-1,2,3-Triazol-4-yl))-
phenyl)propyloxy)cytidine-5′-triphosphate Triethylammo-
nium Salt (28). To a mixture of BODIPY 630/650 azide (124,
Supporting Information, 1.0 mg, 1.0 μmol, 1 equiv) and alkyne 26 (2.0
t
mg, 1.8 μmol, 1.2 equiv) in a 200 μL of (1:1) mixture of BuOH and
water, and 20 μL of DMF, were added tris[(1-benzyl-1H-1,2,3-triazol-
4-yl)methyl] amine (TBTA, 0.05 mg) and freshly prepared 1 M
aqueous sodium ascorbate solution (1.1 μL, 1.6 μmol, 1 equiv)
followed by 7.5% aqueous copper sulfate pentahydrate solution (2.5
μL, 0.8 μmol, 0.8 equiv). The bright-blue color reaction mixture was
stirred overnight at rt, solvent was evaporated, and the residue was
purified by semipreparative HPLC as described above to obtain
desired triphosphate 28 as blue-colored solids. HRMS-EI m/z (M +
ES MS) for 28 found, 1388.3648 (M + H+)+; calcd for
C64H68N11O20BF2P3S, 1388.3631; analytical HPLC (system A: 7.91
min). Fluorescence absorption and emission spectra indicated max
values at 634 and 650 nm, respectively (Supporting Information), as
measured in solution in a cuvette using a SpectraMax M5 reader
(Molecular Devices, Sunnyvale, CA).
N4-(3-((4-tert-Butyloxy-carbonylmethoxy)phenyl)-
propyloxy)cytidine (115). Compound 115 (45 mg, 88.5 μmol,
65%) was obtained as a white solid from cytidine (29 mg, 0.12 mmol)
and N4-(3-(4-tert-butyloxy-carbonylmethoxy)phenyl)propyloxyamine
hydrochloride (91) (79 mg, 0.25 mmol) using the above procedure.
1H NMR (400 MHz, MeOD): 7.16 (d, J1 = 8.24 Hz, 1H), 7.06 (d, J1 =
8.36 Hz, 2H), 6.76 (d, J1 = 8.52 Hz, 1H), 5.82 (d, J1 = 5.20 Hz, 1H),
5.55 (d, J1 = 8.24 Hz, 1H), 4.46 (s, 2H), 4.13−4.10 (m, 2H), 3.94−
3.91 (m, 3H), 3.76 (d, J1 = 12.08 Hz, 1H), 3.66 (dd, J1 =12.12 Hz, J2
=3.00 Hz, 1H), 2.58 (t, J1 = 7.08 Hz, 2H), 2.01−1.86(m, 2H), 1.42(s,
9H). 13C NMR (400 MHz, MeOD): 169.0, 156.2, 150.1, 144.7, 134.7,
131.4, 129.1, 114.2, 97.6, 88.4, 84.6, 82.0, 73.4, 72.8, 70.3, 65.4, 61.3,
30.8, 30.4, 27.0. m/z (M + ESI MS) found, 508.2294; calcd for
C24H34N3O9, 508.2290.
General Synthesis of P1-(N4-Phenylpropoxycytidine-5′-)P4-
(uridine-5′-)-tetraphosphate Triethylammonium Salt (30).
Uridine 5′-monophosphate disodium salt (4.4 mg, 11.0 μmol, 2
equiv) and N4-(3-phenylpropoxy)cytidine-5′-triphosphate triethylam-
monium salt 2 (4.5 mg, 5.4 μmol, 1 equiv) were converted to the
tributylammonium salts by treatment with ion-exchange resin
(DOWEX 50WX2-200 (H)) and tributylamine. After removal of the
water, the obtained uridine monophosphate dibutylammonium salt
and the tributylammonium salt of N4-phenylpropoxycytidine-5′-
triphosphate 2 (4.5 mg, 5.5 μmol, 1 equiv) were dried under high
vacuum for 1 h in separate vials. DIC (1 μL, 5.5 μmol, 1 equiv) was
added to a solution of N4-(3-phenylpropoxy)cytidine-5′-triphosphate
tributylammonium salt 2 in DMF (200 μL). After stirring the reaction
mixture at rt for 3 h, a solution of the uridine 5′-monophosphate
tributylammonium salt (11.0 μmol) and MgCl2 (1.0 mg, 11.0 μmol, 2
equiv) in DMF (100 μL) were added. The reaction mixture was stirred
at rt overnight. After removal of the solvent, the MgCl2 was removed
by treatment with ion-exchange resin (DOWEX 50WX2-200 (H)) and
ammonia bicarbonate or tri-n-butylamine, and the residue was purified
by a semipreparative HPLC purification using system C. Compound
N4-(3-(((But-3-ynyl-1-amino)-4-methoxycarbonyl)phenyl)-
propyloxy)cytidine (116). N4 -(3-((4-tert-Butyloxy-
carbonylmethoxy)phenyl)propyloxy)cytidine 115 (44 mg, 0.12
mmol) was dissolved in 1.0 mL of THF and treated with 2.0 mL of
1 N NaOH. The reaction mixture was stirred at 50 °C for 2 h. The
mixture was cooled to rt and neutralized by adding 1N HCl. After
removal of the solvent, N4-(3-((4-methoxycarbonate)phenyl)-
propyloxy)cytidine (31 mg, 68 μmol, 56%) was isolated after column
chromatography. 1H NMR (400 MHz, MeOD): 7.17 (d, J1 = 8.24 Hz,
1H), 7.09 (d, J1 = 8.44 Hz, 2H), 6.81 (d, J1 = 8.64 Hz, 1H), 5.82 (d, J1
= 5.44 Hz, 1H), 5.55 (d, J1 = 8.24 Hz, 1H), 4.57 (s, 2H), 4.12 (t, J1 =
5.40 Hz, 1H), 4.08 (m, 1H), 3.95−3.90 (m, 3H), 3.74 (dd, J1 = 12.08
Hz, J1 = 2.88 Hz, 1H), 3.66 (dd, J1 = 12.16 Hz, J2 = 3.40 Hz, 1H), 2.60
(t, J1 = 7.40 Hz, 2H), 1.95−1.86 (m, 2H). 13C NMR (400 MHz,
MeOD): 156.2, 150.1, 144.7, 134.7, 131.3, 129.0, 114.1, 97.4, 88.3,
84.7, 73.3, 72.7, 70.3, 64.6, 61.3, 30.8, 30.4, 27.0. m/z (M + ESI MS)
found, 442.1665; calcd for C20H26N3O9, 452.1664.
1
30 (1.0 mg, 0.8 μmol, 16%) was obtained as a white solid. H NMR
The above isolated acid (50 mg, 0.1 mmol), DIEA (0.128 mL, 0.93
mmol, 8.5 equiv), and 6.0 mL of DMF were added to round-bottom
flask. Then 4-amino-1-butyne (13.5 μL, 0.16 mmol, 1.5 equiv) and 1.0
mL of DMF were added to the above mixture, which was then stirred
for 10 min at rt. PyBOP (57 mg, 0.11 mmol, 1.1 equiv) was added, and
the mixture was stirred overnight. Solvent was removed, and 116 (45.1
mg, 89.9 μmol, 80%) was isolated following column chromatography.
1H NMR (400 MHz, MeOD): 7.17 (d, J1 = 8.28 Hz, 1H), 7.11 (d, J1 =
8.56 Hz, 2H), 6.86 (d, J1 = 8.64 Hz, 2H), 5.82 (d, J1 = 5.44 Hz, 1H)
5.53 (d, J1 = 8.24 Hz, 1H), 4.43 (s, 2H), 4.15−4.00 (m, 2H), 3.95 (t, J1
= 6.36 Hz, 2H), 3.91 (q, J1 = 3.32 Hz, 1H), 3.75 (dd, J1 = 12.16 Hz, J1
= 2.88 Hz, 1H), 3.66 (dd, J1 = 12.12 Hz, J2 = 3.36 Hz, 1H), 3.38 (t, J1
= 7.08 Hz, 2H), 2.62 (t, J1 = 7.40 Hz, 2H), 2.37 (td, J1 = 7.08 Hz, J1 =
2.64 Hz, 2H), 2.25 (t, J1 = 2.64 Hz, lH), 2.01−1.85 (m, 2H). 13C
NMR (400 MHz, MeOD): 170.11, 155.98, 150.03, 144.68, 135.27,
131.37, 12 129.5, 123.9, 114.7, 114.5, 112.1, 111.9, 97.4, 88.3, 84.7,
73.3, 72.5, 70.3, 61.3, 31.4, 30.1. m/z (M + H ESI MS) found,
503.2139; calcd for C24H31N4O8, 503.2136.
(D2O) δ 7.93 (d, J = 7.9 Hz, 1H), 7.35−7.30 (m, J = 8.3 Hz, 4H),
7.25−7.21 (m, 2H), 6.02−5.91 (m, 2H), 5.78 (d, J = 7.9 Hz, 1H),
4.42−4.33 (m, 4H), 4.30−4.16 (m, 6H), 4.05 (t, J = 4.8 Hz, 2H), 2.74
(t, J = 8.3 Hz, 2H), 2.05−1.79 (m, 2H). 31P NMR (D2O) δ −11.51
(br), −23.22 (br). HRMS-EI found: 922.0742 (M − H+)−.
C27H36N5O23P4 requires 922.0752; analytical HPLC (system A: 7.92
min).
P1 -(N4 -(3-Phenylpropoxy)cytidine-5′-)P4 -(N4 -(3-
phenylpropoxy)cytidine-5′-)-tetraphosphate Triethylammo-
nium Salt (31). Compound 31 (0.5 mg, 0.3 μmol, 10%) was
obtained as a white solid using N4-(3-phenylpropoxy)cytidine-5′-
triphosphate triethylammonium salt (3.0 mg, 3.0 μmol) and N4-(3-
phenylpropoxy)cytidine-5′-monophosphate diethylammonium salt
1
(3.0 mg, 6.1 μmol) from the previous procedure. H NMR (D2O) δ
7.45−7.10 (m, 12H), 5.95−5.87 (m, 2H), 5.79−5.63 (m, 2H), 4.39−
4.29 (m, 4H), 4.27−4.16 (m, 6H), 4.06−4.00 (m, 4H), 2.80−2.65 (m,
4H), 2.05−1.93 (m, 4H). HRMS-EI found: 1055.1593 (M − H+)−.
C36H47N6O23P4 requires 1055.1643; analytical HPLC (system A: 9.10
min).
Procedures for Phospholipase C Assay. Stable cell lines for
study of the human (h) P2Y2, P2Y4, and P2Y6Rs were produced by
retroviral expression of the individual receptors in 1321N1 human
astrocytoma cells, which do not natively express P2YRs.31 Agonist-
stimulated [3H]inositol phosphate accumulation was quantified in cells
plated at 20000 cells/well on 96-well plates two days prior to assay.
Synthesis of compounds 40−96 is described in the Supporting
Information.
General Procedure for the Synthesis of N4-Alkoxycytidine
analogues (97−110, 113) N4-(4-Phenylbutyloxy)cytidine (97).
A suspension of cytidine (360 mg, 1.5 mmol) and 4-phenylbutoxy-
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dx.doi.org/10.1021/jm500367e | J. Med. Chem. 2014, 57, 3874−3883