Pyrimidine ribonucleotides with enhanced selectivity as P2Y6 receptor agonists: Novel 4-alkyloxyimino, (S)-methanocarba, and 5′-triphosphate γ-ester modifications

Article abstract of DOI:10.1021/jm100287t

The P2Y₆ receptor is a cytoprotective G-protein-coupled receptor (GPCR) activated by UDP (EC₅₀ = 0.30 μM). We compared and combined modifications to enhance P2Y₆ receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as α,β-methylene and extension of the terminal phosphate group into γ-esters of UTP analogues. The conformationally constrained (S)-methanocarba-UDP is a full agonist (EC ₅₀ = 0.042 μM). 4-Methoxyimino modification of pyrimidine enhanced P2Y₆, preserved P2Y₂ and P2Y₄, and abolished P2Y₁₄ receptor potency, in the appropriate nucleotide. N ⁴-Benzyloxy-CDP (15, MRS2964) and N⁴-methoxy-Cp ₃U (23, MRS2957) were potent, selective P2Y₆ receptor agonists (EC₅₀ of 0.026 and 0.012 μM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-γ-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y₆ receptor potency but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N⁴-methoxycytidine 5′-triphospho-γ-[1]glucose were active (EC₅₀ of 2.47 and 0.18 μM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y₆ receptor agonists may be enhanced by modest structural changes.

Full text of DOI:10.1021/jm100287t

4488 J. Med. Chem. 2010, 53, 4488–4501
DOI: 10.1021/jm100287t
Pyrimidine Ribonucleotides with Enhanced Selectivity as P2Y6 Receptor Agonists:
Novel 4-Alkyloxyimino, (S)-Methanocarba, and 50-Triphosphate γ-Ester Modifications†
Hiroshi Maruoka,‡ Matthew O. Barrett,§ Hyojin Ko,‡,^ Dilip K. Tosh,‡ Artem Melman,‡,# Lauren E. Burianek,§
Ramachandran Balasubramanian,‡ Barkin Berk, ,¥ Stefano Costanzi, T. Kendall Harden,§ and Kenneth A. Jacobson*,‡
‡Molecular Recognition Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland, §Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina, and Laboratory of
Biological Modeling, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.
^ Present address: Gwangju Institute of Science and Technology, 216 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Republic of Korea.
# Present address: Clarkson University, Potsdam, New York. ¥ Permanent address: Yeditepe University, Istanbul, Turkey.
Received March 4, 2010
The P2Y6 receptor is a cytoprotective G-protein-coupled receptor (GPCR) activated by UDP (EC50
=
0.30 μM). We compared and combined modifications to enhance P2Y6 receptor agonist selectivity,
including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications
such as R,β-methylene and extension of the terminal phosphate group into γ-esters of UTP analogues. The
conformationally constrained (S)-methanocarba-UDP is a full agonist (EC50 = 0.042 μM). 4-Methoxy-
imino modification of pyrimidine enhanced P2Y6, preserved P2Y2 and P2Y4, and abolished P2Y14
receptor potency, in the appropriate nucleotide. N4-Benzyloxy-CDP (15, MRS2964) and N4-methoxy-
Cp3U (23, MRS2957) were potent, selective P2Y6 receptor agonists (EC50 of 0.026 and 0.012 μM,
respectively). A hydrophobicbindingregion near the nucleobasewas explored with receptor modeling and
docking. UTP-γ-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y6 receptor potency
but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methano-
carba analogue and N4-methoxycytidine 50-triphospho-γ-[1]glucose were active (EC50 of 2.47 and
0.18 μM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y6
receptor agonists may be enhanced by modest structural changes.
Introduction
5-iodo-UDP (MRS2693) 3, which is a selective P2Y6 receptor
agonist, induced protection in a model of ischemia in hindleg
skeletal muscle.6 The dinucleoside triphosphate INS48823 4
activates the P2Y6 receptor in bone and increased survival of
osteoclasts.7 A role for the P2Y6 receptor in inducing phago-
cytosis in microglial cells was recently discovered, suggesting
the possible application of P2Y6 receptor ligands for the
treatment of neurodegenerative conditions.8 The P2Y6 recep-
tor is up-regulated when neurons are damaged and in intest-
inal inflammation.8,9 UDP applied topically to the cornea
reduced intraocular pressure, suggesting the use of P2Y6
receptor agonists for the treatment of ocular hypertension
and glaucoma.10 Studies of mice lacking the P2Y6 receptor
recently demonstrated a defective response to UDP in macro-
phages, endothelial cells, and vascular smooth muscle cells.11
Thus, pharmacological modulation of the P2Y6 receptor is of
increasing interest for future therapeutics.12
The P2Y receptors are G-protein-coupled receptors (GPCRs)
that respond to naturally occurring extracellular nucleotides
including ATP, ADP, UTP,a UDP, and UDP-glucose.1 Five
of the eight subtypes, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11
receptors, are preferentially coupled through Gq to activate
phospholipase C-β (PLC-β) and represent a distinct structural
subfamily.2 The remaining P2Y12, P2Y13, and P2Y14 receptors
are coupled to inhibition of adenylate cyclase. Activation of the
P2Y6 receptor by its native ligand UDP 1 (Chart 1) is associated
with vasoconstriction and cytoprotection.3-6 We reported the
effects of P2Y6 receptor agonists to counteract apoptosis induced
by tumor necrosis factor R in astrocytoma cells stably expressing
the human P2Y6 receptor and in mouse C2C12 skeletal muscle
cells and MIN6 pancreatic cells that express an endogenous
P2Y6 receptor.4-6 Furthermore, in vivo administration of
Potent P2Y6 receptor agonists, such as 3-phenacyl-UDP 5
with an EC50 value of 70 nM, have been reported.13-16
Recently, we reported that UDP also activates the P2Y14
receptor, and therefore, agonists that delineate between these
two subtypes are needed.17,18 We have adopted a structure-
based approach to developing P2Y6 receptor agonist ligands
with improved potency, selectivity, and stability. In the ab-
sence of an X-ray structure, rhodopsin-based homology
modeling has predicted a putative binding site for nucleotides
in the human P2Y6 receptor.13 The receptor recognition of the
†Presented in part at the 238th National Meeting of the American
Chemical Society, August 2009, Washington, DC; MEDI 187.
*To whom correspondence should be addressed. Phone: 301-496-
9024. Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
a Abbreviations: CDI, N,N-carbonyldiimidazole; DCC, N,N0-dicyclo-
hexylcarbodiimide; DIC, N,N0-diisopropylcarbodiimide; DMF, di-
methylformamide; HEK, human embryonic kidney; HBSS, Hank’s
balanced salt solution; HEPES, N-2-hydroxyethylpiperazine-N0-2-etha-
nesulfonic acid; HPLC, high performance liquid chromatography; PLC,
phospholipase C; SAR, structure-activity relationship; TBAP, tetra-
butylammonium dihydrogenphosphate; TEAA, triethylammoniumacet-
ate; UDP, uridine 50-diphosphate; UTP, uridine 50-triphosphate.
r
pubs.acs.org/jmc
Published on Web 05/06/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4489
Chart 1. Structures of Prototypical Agonist Ligands for Studying P2Y6 Receptors
50-diphosphate group is associated withthree cationicresidues
in the transmembrane region, which serve as counterions
and which are conserved in charge across the five Gq-coupled
P2Y receptors.2 A Monte Carlo analysis of conformations
of receptor-docked UDP molecules, followed by molecular
dynamics of the receptor-ligand complex in a lipid bilayer
model, has predicted that the South (S) conformation of the
ribose ring is preferred at the P2Y6 receptor.13 This is the only
Gq-coupled P2Y subtype at which the (S)-conformation of
theribose or a ribose-like moietyofnucleotideligandsappears
to be favored, and our comparison of the weak agonist 20-deoxy-
UDP 2 and the corresponding 20-deoxy (S)-methanocarba
analogue 613,14 supported this idea.
Table 1. Potencies of (S)-Methanocarba Analogues 6, 7, and 9 in
Relation to Known Uridine 50-Diphosphate Derivatives for Activation
of the Human P2Y6 Receptora
We recently reported the first synthetic route to the enantio-
merically pure (S)-methanocarba-uridine,19 and in this study
we examined the hypothesis that the (S)-conformation is
preferred at the P2Y6 receptor in the more potent 20-hydrox-
ynucleotide series. Modifications of the pyrimidine ring
of UDP, including 5-iodo and 4-alkyloxyimino, and of the
phosphate moiety, including R,β-methylene, and extension of
the terminal phosphate group were compared and combined
in an effort to further enhance selectivity for the P2Y6
receptor.14,16 Chemical and metabolic instability of mononu-
cleotides is well-documented.20 Mononucleotides are de-
graded enzymatically by ectonucleotidases into the lower
nucleotides and eventually into the nucleoside. Chemical
degradation of nucleotides is also evident at pH ranges less
than 4 or greater than 10. The degradation products of
nucleotides may interact with multiple P2Y receptors or with
adenosine receptors and lead to side effects. Since various
dinucleotides, such as diuridine triphosphates, show greater
stability than analogues of mononucleotides,21,22 we hypothe-
sized that blockade of the terminal phosphate with an alkyl or
aryl group may also increase its stability.
EC50, μMb
hP2Y6
compd
modification
structure
1f
UDP
dUDP
0.30 ( 0.06
1.72 ( 0.76
0.015 ( 0.002
2
R = H
X = 5-I
3c
5c
6c,d
7d
8c,e
9
5-I-UDP
phenacyl-UDP
X = 3-Ph-COCH2 0.070 ( 0.006
(S)-methanocarba-dUDP
(S)-methanocarba-UDP
(N)-methanocarba-UDP
(S)-methanocarba
U-cyclic-30,50-diphosphate
R,β-CH2-UDP
R = H
0.23 ( 0.05
0.042 ( 0.008
NE
NE
10c,f
11f
Y = CH2
0.70 ( 0.11
X = 5-I, Y = CH2 0.13 ( 0.02
R,β-CH2-5-I-UDP
a Unless otherwise noted, R = uridine and X = O. NE: no effect at
10 μM. b Agonist potencies reflect stimulation of phospholipase C in
1321N1 human astrocytoma cells stably expressing the human P2Y6
receptor. Potencies are presented in the form of EC50 values, which
represent the concentration of agonist at which 50% of the maximal
effect is achieved. These values were determined using a four-parameter
logistic equation and the GraphPad software package (GraphPad, San
Diego, CA). The results are presented as the mean ( standard error and
are the average of three to six different experiments with each molecule.
c Values reported in literature: 3, Besada et al.;14 5, El-Tayeb et al.;8 6
and 8, Costanzi et al.;13 10, Ko et al.16 d 6, MRS2633; 7, MRS2795.
e Structure of 8 given in Chart 1. f EC50 values33 (μM) reported in
activation of the hP2Y14 receptor expressed in HEK-293 cells: 1, 0.16 (
0.04; 10, 0.011 ( 0.005; 11, 0.14 ( 0.07.
Results
Chemical Synthesis. Known (1-6, 8, 10, and 11) and newly
synthesized derivatives of UDP (7, 9) and CDP (12-16) were
assembled and compared (Tables 1 and 2). Novel analogues
of UTP and Up3U (18, 20, and 22-33) also were included in
this structure-activity relationship (SAR) analysis (Table 3).
The chemical synthesis of ribonucleoside 50-diphosphate
and triphosphate derivatives (Scheme 1) was performed by

4490 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
Table 2. Potencies of N4-Alkyloxycytidine 50-Diphosphate Derivatives for Activation of the Human P2Y2, P2Y4, and P2Y6 Receptorsa
EC50, μMb
compd
modification
structure
Z = N-OCH3
hP2Y2
hP2Y4
hP2Y6
12c
13
14
15
16
N4-OMe-CDP
3.60 ( 1.08
0.28 ( 0.015
3.51 ( 1.52
2.13 ( 0.64
>10d
6.45 ( 1.55
0.18 ( 0.02
6.23 ( 1.88
1.15 ( 0.15
NE
0.070 ( 0.007
0.104 ( 0.023
2.55 ( 0.70
0.026 ( 0.002
0.678 ( 0.018
N4-OEt-CDP
Z = N-OCH2CH3
Z = N-OC(CH3)3
Z = N-OCH2Ph
N4-OtBu-CDP
N4-OBn-CDP
R,β-CH2-N4-OMe-CDP
Y = CH2, Z = N-OCH3
a ND: not determined. NE: no effect at 10 μM. b Agonist potencies reflect stimulation of phospholipase C in 1321N1 human astrocytoma cells stably
expressing the human P2Y2, P2Y4, or P2Y6 receptor. Potencies are presented in the form of EC50 values, which represent the concentration of agonist at
which 50% of the maximal effect is achieved. These values were determined using a four-parameter logistic equation and the GraphPadsoftware package
(GraphPad, San Diego, CA). The results are presented as the mean ( standard error and are the average of three to six different experiments with each
molecule. c EC50 value33 reported in activation of the hP2Y14 receptor expressed in HEK-293 cells: 12, 3.32 ( 1.62 μM. d e50% effect at 10 μM.
standard phosphorylation methods using the unprotected
nucleoside precursors.23,24 Various 50-diphosphate derivatives
were then extended to form dinucleoside triphosphates or other
50-triphosphate γ-esters (Scheme 2). (S)-Methanocarba nucleo-
tides were synthesized as shown in Scheme 3. The synthesis of
several of the pyrimidine nucleotides was reported in previous
studies of the P2Y receptor (3, 5, 6, 8, 10, 19, 21, and 26).14-16
5-Iodouridine 34 was available from commercial sources,
while the 4-methoxyimino derivative 36 and (S)-methano-
carbauridine 41 were prepared as described,19,25 and com-
pounds 37-39 were prepared by the same procedure as 36.
Compounds 34 and 36 were converted to the R,β-methylene
diphosphonate analogues 11 and 16, respectively, by a car-
bodiimide condensation. N4-Alkoxycytidines 36-39 were
converted to the corresponding 50-diphosphates 12-15 and
N4-methoxycytidine 36 to the 50-triphosphate 20 using phos-
phorus oxychloride followed by either phosphate or pyro-
phosphate.24 Various 50-diphosphates (1, 3, 5, 10, and 12)
were converted to the corresponding 50-triphosphate γ-esters
by condensation with the appropriate monophosphate deri-
vative in DMF.
Nucleotides 18 and 28 derived from (S)-methanocarba-
uridine 40 were prepared by similar phosphorylation of the
unprotected nucleoside (Scheme 3).24 The 30,50-cyclic-dipho-
sphate 9 was isolated as a side product in the synthesis of 28.
A similar cyclic diphosphate was isolated following phos-
phorylation of other uridine analogues.14
Pharmacological Evaluation. The nucleotide analogues
(Tables 1-3) were assayed for capacity to promote activation
of phospholipase C (PLC) in 1321N1 human astrocytoma
cells stably expressing the human P2Y6 receptor.26 Receptor
selectivity of the novel analogues of CDP and UTP in Tables 2
and 3 was assessed by quantification of PLC activation in
1321N1 cells stably expressing either the P2Y2 receptor or the
P2Y4 receptor. Previously reported data for several analogues
(2-6, 8, and 10) were included for comparison.
orthe previously reported(S)-methanocarba-20-deoxy-UDP6
(5-fold). In contrast, the North (N)-methanocarba conforma-
tionally locked UDP analogue 8 was previously found to be
inactive at the P2Y6 receptor, confirming a strong conforma-
tional preferrence in P2Y6 receptor recognition.13,14 The (S)-
methanocarbauridine 30,50-cyclic-diphosphate 9 was also in-
active at the P2Y6 receptor.
The carbon-bridged analogue R,β-methylene-UDP 10 was
recently reported to be nearly as potent as UDP at the P2Y6
receptor.16 Compound 11 contained the 50-R,β-methylene-
diphosphonate modification, which would increase the sta-
bility of a nucleotide in biological systems, and the 5-iodo
modification (as in 3), both of which were previously noted
to preserve potency at the P2Y6 receptor.14 This combination
of modifications resulted in a molecule 11 that potently
activated the P2Y6 receptor with an EC50 value of 127 nM.
Introduction of the 4-methoxyimino modification of
UDP, i.e., N4-methoxy-CDP 12, resulted in high potency
at the P2Y6 receptor (EC50 = 70 nM), which represents a
4-fold gain of potency over UDP (Table 2, Figure 1A).
Compound 12 was 47-fold selective for activation of PLC
via the P2Y6 receptor in comparison to inhibition of cyclic
AMP accumulation in HEK-293 cells expressing the Gi-
coupled human P2Y14 receptor.18 The native agonist for
P2Y14, UDP-glucose, did not tolerate the 4-methoxyimino
substitution.27 However, the 4-methoxyimino modification
of the pyrimidine ring in the 50-triphosphate derivative 20
produced a gain of potency (EC50 = 80 nM) of >100-fold
over UTP 17 at the P2Y6 receptor (Table 3). The 4-methoxy-
imino modification of 20 was also well-tolerated at the P2Y2
and P2Y4 receptors, with EC50 values of 50 nM. Thus,
compound 20 was a nonselective agonist at these three
P2Y receptor subtypes.
In the series of 4-alkyloxyimino 50-diphosphate analogues,
the progression from MeO to larger O-alkyl substituents,
12-15, first decreased then increased the P2Y6 receptor
potency. The sterically bulky O-tert-butyl group of 14 was
not well tolerated. However, the O-benzyl analogue 15 was
The enantiomerically pure (S)-methanocarba-UDP 7 was
more potent at the P2Y6 receptor than either UDP 1 (7-fold)

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4491
Table 3. Potencies of Nucleotide (50-Triphosphate) Derivatives for Activation of the Human P2Y2, P2Y4, and P2Y6 Receptorsa
EC50, μMb
compd
modification
structure
hP2Y2
hP2Y4
hP2Y6
>10e
17
18
19c
20
21c
22
23
UTP
0.060 ( 0.00
0.08 ( 0.02
0.016 ( 0.01
0.05 ( 0.00
1.31 ( 0.21
>10e
0.090 ( 0.01
0.30 ( 0.10
0.085 ( 0.005
0.05 ( 0.02
0.87 ( 0.11
NE
(S)-methanocarba-UTP
(N)-methanocarba-UTP
N4-OMe-CTP
1.37 ( 0.05
NE
Z = N-OCH3
R0 = [50]uridine
R0 = [50]uridine, Y = CH2
Z = N-OCH3
0.08 ( 0.02
0.27 ( 0.07
1.30 ( 0.18
0.012 ( 0.0024
Up3U
R,β-CH2-Up3U
N4-OMe-Cp3U
0.17 ( 0.04
0.79 ( 0.12
R0 = [50]uridine
Z = N-OCH3
24
N4-OMe-Cp3(N4-OMe)C
2.12 ( 0.66
0.56 ( 0.04
0.063 ( 0.002
R0 = [50](N4-methoxy)cytidine
R0 = cyclohexyl
25
26
27
28d
29
30
31
32
33
Up3-O-cyclohexyl
Up3-O-phenyl
Up3-gluc
2.28 ( 0.1
>10e
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
6.99 ( 0.18
2.13 ( 0.18
>10e
R0 = phenyl
(S)-methanocarba-Up3-gluc
5-I-Up3-gluc
NE
NE
2.47 ( 0.39
4.69 ( 1.68
>10e
>10e
X = 5-I
3-phenacyl-Up3-gluc
R,β-CH2-Up3-gluc
N4-OMe-Cp3-gluc
N4-OMe-Cp3-O-Ph
X = 3-Ph-COCH2
Y = CH2
Z = N-OCH3
NE
NE
>10e
>10e
0.18 ( 0.04
3.49 ( 1.63
R0 = phenyl, Z = N-OCH3
a NE: no effect at 10 μM. ND: not determined. b Agonist potencies reflect stimulation of phospholipase C in 1321N1 human astrocytoma cells stably
expressing the human P2Y2, P2Y4, or P2Y6 receptor. Potencies are presented in the form of EC50 values, which represent the concentration of agonist at
which 50% of the maximal effect is achieved. These values were determined using a four-parameter logistic equation and the GraphPadsoftware package
(GraphPad, San Diego, CA). The results are presented as the mean ( standard error and are the average of three to six different experiments with each
molecule. c Values reported in literature: 19, Kim et al.;26 21, Ko et al.16 d Structure of 28:
e e50% effect at 10 μM.
the most potent P2Y6 receptor agonist in the series of 4-imino
50-diphosphate analogues with 82- and 44-fold selectivity vs
P2Y2 and P2Y4 receptors, respectively. Curiously, the O-ethyl
analogue 13 was potent at P2Y2 and P2Y4 receptors and was
thus a nonselective hP2Y2/4/6 agonist. The potency-enhancing
4-methoxyimino modification was combined with R,β-methy-
lene substitutions in compound 16. However, there was no
enhancement by the 4-methoxyimino modification, as 10 and
16 were equipotent at the P2Y6 receptor.
Up3U 21 slightly favored activation of the P2Y6 receptor,
in comparison to the P2Y2 and P2Y4 receptors, with an EC50
value of 270 nM.16 Introduction of a carbon bridge in the
corresponding R,β-methylene analogue 22 reduced P2Y6
receptor potency 5-fold but with increased selectivity. Given
the P2Y6 receptor potency of extended phosphate deriva-
tives, such as dinucleoside triphosphates related to Up3U 21
(including 4), we tested other γ-phosphoesters of UTP for
P2Y6 receptor activity, including alkyl and aryl esters and
Up3-sugar derivatives. The analogue of Up3U containing a
single 4-methoxyimino modification 23 displayed 22-fold
enhanced potency at the P2Y6 receptor with an EC50 value
of 12 nM and selectivity versus the P2Y2 and P2Y4 receptors
of 14- and 66-fold, respectively. The γ-cyclohexyl 25 and
γ-phenyl 26 esters of UTP displayed moderate potency at the
P2Y6 receptor, with EC50 values of 6.99 and 2.13 μM,
respectively. However, Up3-[1]glucose 27 only weakly activated
the P2Y6 receptor, and an EC50 value was not determined. The
introduction of the potency enhancing (S)-methanocarba modi-
fication in the corresponding ring-constrained analogue 28
increased potency, as predicted. Nevertheless, only a moderate
potency of 2.47 μM was attained.
Combinations of Up3-glucose with other known modifica-
tions that are tolerated or favored at the P2Y6 receptor were
evaluated. Introduction of the 5-iodo modification in 29 only
slightly enhanced potency in comparison to 27. The corres-
ponding 3-phenacyl 30 and R,β-methylene 31 analogues were
only weakly active at the P2Y6 receptor. However, the P2Y6
receptor potency of the corresponding N4-methoxycytidine

4492 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
Scheme 1. Synthesis of Various Pyrimidine Ribonucleoside 50-Diphosphate and Triphosphate Derivativesa
a Reagents and conditions: (a) DCC, methylene bisphosphonate, rt, overnight, 11% for 11, 48% for 16; (b) RONH2, pyridine, 90 ꢀC, overnight,
55-100%; (c) POCl3, Proton Sponge, PO(OMe)3, 0 ꢀC, 2 h, followed by tributylammonium phosphate, rt, 0.5 h (for 12-15), 14-47%; (d) POCl3,
Proton Sponge, PO(OMe)3, 0 ꢀC, 2 h, followed by tributylammonium pyrophosphate, rt, 0.5 h, 31% (for 20).
analogue 32 was greatly increased with an EC50 value of 180 nM,
which also displayed >100-fold selectivity versus P2Y2 and
P2Y4 receptors. This finding suggested another analogue,
N4-methoxycytidine 50-triphosphate γ-phenyl ester 33, but
anadditiveeffect onpotency was absent, asit weaklyactivated
the P2Y6 receptor (EC50 = 3.49 nM).
The stabilityin the presence of cell membranes was25, 22 >
12 >21, 26 > 17, 27 > 15, 23 > 31. The potent P2Y6 receptor
agonist 12 was more stable than the related N4-benzyloxycy-
tidine 50-diphosphate 15. The γ-cyclohexyl ester 25 was more
stable than either Up3U 21, which has a uridyl group at this
terminal position, or the γ-phenyl ester 26. The R,β-methylene
analogue of Up3U 22 was more stable than the parent Up3U
21 in the presence of cell membranes, which may indicate that
a R,β-methylene bridge blocks enzymatic breakdown, as has
been observed for other P2 receptor agonists containing
phosphonates.16,28,29 However, the introduction of a methy-
lene bridge into Up3U unfortunately also reduced potency at
the P2Y6 receptor 5-fold.
In acidic solution, the CDP derivatives 12 and 15 and the
dinucleoside triphosphates were of enhanced chemical sta-
bility. Also, the γ-phenyl phosphoester 26 was relatively
stable. However, the Up3U analogue 22 containing a R,β-
methylene bridge was significantly less stable than the parent
Up3U 21. Likewise, the Up3-[1]glucose analogue 31 contain-
ing a R,β-methylene bridge was less stable than the parent
Up3-[1]glucose 27, which itself was very labile. Since both of
the glucose derivatives 27 and 31 were rapidly degraded, the
introduction of glucose was not a favored modification.
Molecular Modeling. A docking of the semirigid (S)-
methanocarba analogue 7 in complex with our rhodopsin-
based P2Y6 receptor model13 (Figure 2A) suggested that 7
Selected nucleotide analogues were also examined for the
ability to raise levels of intracellular calcium ions in 1321N1
human astrocytoma cells expressing the human P2Y6 receptor
(Figure 1B). The rank order of potency was 23, 3 > 21, 24,
which was similar to the order of potency in PLC activation.
Stability of Nucleotide Derivatives. The chemical and enzy-
matic instability of nucleotide analogues in biological systems
is a major limitation in their use as pharmacological probes.
Therefore, to assess their potential for broad use, selected
nucleotide derivatives were evaluated for stability during pro-
longed exposure to two different conditions. The derivatives
were incubated at 37 ꢀC in aqueous medium at low pH or in the
presence of 1321N1 astrocytoma membranes, which are
known to contain ectonucleotidases, as a representative mam-
malian cell of the central nervous system. At regular intervals
aliquots were taken for HPLC analysis (Table 4 and Support-
ing Information). The injection sample was prediluted with
5 mM tetrabutylammonium dihydrogen phosphate (TBAP) to
allow complete equilibration with the mobile phase in order to
avoid the early HPLC elution at around 1 min.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4493
Scheme 2. Synthesis of Various Pyrimidine Ribonucleoside 50-Triphosphates and Dinucleoside Triphosphatesa
a Reagents and conditions: (a) DIC, MgCl2, glucose-1-monophosphate tributylammonium salt, DMF, rt, overnight, 9-80%; (b) DIC, MgCl2,
respective monophosphate tributylammonium salt, e.g., uridine 50-monophosphate, 35, cyclohexylmonophosphate16 or phenylmonophosphate, in
DMF, rt, overnight, 9-80%. Unless otherwise noted, X = H and Y, Z = O.
Scheme 3. Synthesis of Uracil Ribonucleoside Phosphate Derivatives Constrained with a (S)-Methanocarba Ringa
a Reagents and conditions: (a) POCl3, Proton Sponge, PO(OMe)3, 0 ꢀC, 2 h; then tributylammonium phosphate, rt, 0.5 h, 45%; (b) POCl3, Proton
Sponge, PO(OMe)3, 0 ꢀC, 2 h; then tributylammonium pyrophosphate, rt, 0.5 h, 56%; (c) CDI, DMF, rt, 1 h, then glucose-1-monophosphate
tributylammonium salt, overnight, 23% for 28, 51% for 9.
establishes with the receptor the same putative interactions
characteristic of UDP. In particular, three cationic residues
located in transmembrane domain 3 (TM3), TM6, and TM7
coordinate the diphosphate of 7, namely, R103(3.29),
K259(6.55), and R287(7.39), while the base is coordinated
by Y33(1.39) and S291(7.43), located in TM1 and TM7,
respectively (see Experimental Section for residue indexing).
The higher potency of 7 compared to UDP is induced by the
rigid methanocarba scaffold, which constrains the ribose in
the bioactive conformation, likely with a consequent gain in
the entropic component of the binding affinity. A hydrogen
bond is possibly formed between the ribose 20-hydroxy group
and the uracil 2-oxo group of 7 (Figure 2A). Furthermore, as
shown in Figure 2A, the molecular dynamics simulation

4494 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
predicted the access of several water molecules to the receptor
binding pocket from the extracellular milieu. These molecules
surround the portion of the ligand exposed to the extracellular
opening of the binding site, including the 5 position of the uracil
ring. For entropic reasons, substituents capable of displacing
these water molecules from the binding cavity to the bulk of the
solvent are expected to enhance the affinity of the compounds.
The increase in affinity noted upon the introduction of the iodo-
substituent at the 5-position of the uracil ring (see compounds 1
and 3) is consistent with this reasoning and supports the orienta-
tion of this moiety of the ligand toward the extracellular opening
of the helical bundle of the receptor as suggested by our models.
A molecular model of N4-methoxy-CDP 12 docked in the
P2Y6 receptor showed similar coordination to that of UDP;
i.e., S291(7.43) coordinates the base, and four cationic
residues coordinate the diphosphate moiety (Figure 2B).
The methyl group at the 4 position seems to fit well in a
sterically restricted hydrophobic pocket, which consisted of
V32(1.38), Y33(1.39), I83(2.61), and P288(7.40). These hydro-
phobic interactions may be the reason for the increased
activity of 12 (Table 2).
Discussion
The present set of analogues provides new insights into the
structural requirements for activation of the P2Y6 receptor at
each of the three regions modified: ribose, nucleobase, and
terminal phosphate. In addition to studying the SAR of
pyrimidine nucleotides in receptor activation, we have also
confirmed that blockade of the terminal phosphate with an
alkyl or aryl group or other groups may increase its stability.
Using sterically constrained carbocyclics, we have extended
to the ribo (20-hydroxy) series the prediction that the (S)-
conformation of a ribose-like moiety is preferred for recogni-
tion at the human P2Y6 receptor, consistent with previous
activity of the 20-deoxy analogues. The first synthesis of
enantiomerically pure (S)-methanocarbauridine enabled the
preparation of nucleotide derivatives in the 20-hydroxy series
(7, 18, and 28). These (S)-methanocarba nucleotides exhibited
enhanced potency over the corresponding natural ribose
analogue in each case.
In homology-based modeling of the P2Y6 receptor and
ligand docking, an intramolecular hydrogen bond was detected
between the 20-hydroxy group and the uracil 2-oxo group of the
conformationally constrained, potent analogue of UDP 7. We
suggest that this interaction might further restrain 7 in its
bioactive conformation and thus contribute to the greater
potency of 7 over the corresponding 20-deoxy analogue 6.
One of the striking findings of this study was that the
4-methoxyimino and 4-benzyloxyimino modifications of the
pyrimidine ring in the appropriate nucleotide derivative
served to enhance potency at the P2Y6 receptor. This is one
of the few modifications of the uracil ring that is tolerated for
activation of the P2Y6 receptor. CDP itself is nearly inactive at
the humanP2Y6 receptor, and its EC50 atthe P2Y14 receptor is
>10 μM.17,30 The 4-methoxyimino 50-diphosphate analogue
12 displayed a selectivity of 51- and 47-fold versus the P2Y2
and P2Y14 receptors, respectively, and a substituent as large as
O-benzyl 15 was tolerated. In the 50-triphosphate analogue 20,
the 4-methoxyimino modification also preserved potency at
P2Y2 and P2Y4 receptors and abolished potency at the P2Y14
receptor.27 In Schemes 1 and 2, the imino group of N4-meth-
oxycytidine is depicted as the iminooxo tautomer, based on
an early study of the poly-C derivative after treatment with
Figure 1. (A) Activity of agonists 7, 12, 15, and 23 at the human
P2Y6 receptor as indicated by activation of PLC in stably trans-
fected 1321N1 human astrocytoma cells. The effect of UDP corres-
ponds to 100%. (B) Mobilization of intracellular calcium in astro-
cytoma cells expressing the human P2Y6 receptor (EC50 values in
nM) by nucleotide agonists 3 (50), 21 (370), 23 (83), and 24 (630).
The EC50 value of 1 was 200 nM.
Table 4. Half-Lives of Nucleotide (50-Triphosphate) Derivatives When Incubated at 37ꢀC in Acidic Aqueous Solution or in the Presence of Mammalian
(1321N1 Human Astrocytoma) Cell Membranes, Determined by HPLC
t1/2, h
(pH 1.2)
t1/2, h
(membranes)
compd
pharmacological properties at hP2Y6 receptor
12, N4-MeO-CDP
15, N4-BnO-CDP
17, UTP
116
126
23
55
26
31
42
62
23
62
43
29
19
selective, potent agonist
selective, potent agonist
∼inactive
21, Up3U
218
34
moderately potent agonist, only 4- to 5-fold selective
weak agonist, selective
selective, potent agonist
weak agonist, nonselective vs P2Y2 receptor
weak agonist, selective
∼inactive
22, R,β-me-Up3U
23, N4-MeO-Cp3U
25, Up3-O-cyclohexyl
26, Up3-O-Ph
120
193
164
5.1
2.8
27, Up3-gluc
31, R,β-Me-Up3-gluc
∼inactive

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4495
Figure 2. Molecular modeling of the human P2Y6 receptor. (A) Details of the nucleotide binding site of the receptor complexed with the
agonist 7 as obtained after a fully flexible Monte Carlo conformational search. The ligand is represented as balls and sticks, colored by element.
The binding pocket is represented as a van der Waals surface colored according to the electrostatic potential, with positive charges in blue and
negative charges in red. For clarity, the residues located in front of the ligand are represented as sticks, colored by element. Labels indicate all of
the residues represented as sticks (adjacent), and the key residues are represented as van der Waals surfaces (trough arrows). A schematic
representation of the receptor-ligand complex is given in the lower left inset. In the tube representations the receptor is colored according to
residue positions, with colors that range from red (N-terminus) to purple (C-terminus): TM1 is in orange, TM2 in ochre, TM3 in yellow, TM4 in
green, TM5 in cyan, TM6 in blue, TM7 in purple. (B) Docking of the potent agonist N4-methoxycytidine 50-diphosphate 12. A hydrophobic
4
˚
binding pocket for the N -alkyloxy substituent is defined in this docking model. Distances in A from the methoxy carbon atom to neighboring
amino acid residues are shown.
methoxyamine.31 This predominant form, which resembles the
major lactam (keto) tautomer of UDP, was used in the
molecular modeling. However, the amino tautomeric form,
which is found in native cytosine in DNA, might also be present.
The 4-methoxyimino group in a γ-[1]glucose triphosphate ester
32 also enhanced potency. However, the γ-[1]glucose triphos-
phate in another analogue 27 suffered from low stability, and
therefore, 32 is not a likely candidate for generation of a potent
and stable agonist.
The introduction of a phenyl or cyclohexyl ring at the
terminal phosphate of UTP in compounds 25 and 26, respec-
tively, enhanced stability in both acidic solution and in the
presence of membranes to at least the same extent as Up3U.
Both 25 and 26 containing terminal hydrophobic groups
showed higher potency than UTP 17 at the P2Y6 receptor,
and the more hydrophilic Up3-[1]glucose 27 was nearly inac-
tive. Thus, the nature of the terminal group can greatly affect
agonist activity. Since Up4-[1]glucose is a potent agonist of the

4496 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
P2Y2 receptor and since Up2-[1]glucose is a native agonist of
the P2Y14 receptor, we speculated that the intermediate triphos-
phate homologue 27 might be an effective agonist of the P2Y6
receptor. However, this receptor is not as tolerant of a terminal
glucose moiety as are the P2Y2 and P2Y14 receptors. Never-
theless, the introduction of a rigid (S)-methanocarba ring
system in 18 and 28 resulted in agonists that exceeded the
P2Y6 potency of both UTP and Up3-[1]glucose.
The combination of two modifications in the present set
of compounds either produced an additive effect on P2Y6
receptor potency, as in γ-[1]glucose triphosphate ester 32, or
failed to increase potency, as in the γ-phenyl ester 33 and the
R,β-methylene analogue 16. The inconsistent effects of com-
bination of various structural modifications on receptor
recognition suggested multiple modes of binding of these
nucleotide series.
The homology modeling and docking, interpreted in light
of the biological activity, indicate conformational cross-talk
between the nucleobase and the terminal phosphate substitu-
ent. Although the molecular model of N4-methoxy-CDP 12
docked in the P2Y6 receptor showed coordination similar to
that of UDP, Up3-O-phenyl 26 docked a different mode (not
shown). The base of 26 did not coordinate with Y33(1.39) and
S291(7.43) because the position of the base had shifted within
the binding site. Also, the ribose ring showed a planar rather
than a (S)-conformation. The terminal phenyl group of 26
seemed to fit in a hydrophobic region in the model, which
might be the reason for the dislocation of the nucleobase, i.e.,
by withdrawal of the entire molecule. On the other hand, the
docked model of N4-methoxy-Cp3-O-phenyl 33 in the recep-
tor showed a position and coordination similar to N4-meth-
oxy-CDP 12, but the phenyl ring did not interact with the
distal hydrophobic region occupied by 26. The strong inter-
action of the methyl group of 33 in a hydrophobic pocket near
the nucleobase may cause the phenyl group to exit its large
hydrophobic binding region. This interpretation is consistent
with the lack of enhanced activity of compound 33 in com-
parison to Up3-O-phenyl 26, despite the presence of methoxy-
imino moiety at the 4 position.
possibly neurodegeneration, and several compounds such as
23 from the present SAR analysis could provide useful
pharmacological tools.
Experimental Section
Synthesis. 1H NMR spectra were obtained with a Varian
Gemini 300 or Varian Mercury 400 spectrometer using D2O,
CDCl3, or DMSO-d6 as a solvent. The chemical shifts are
expressed as relative ppm from HOD (4.80 ppm). 31P NMR
spectra were recorded at room temperature (rt) by use of Varian
XL 300 (121.42 MHz) or Varian Mercury 400 (162.10 MHz)
spectrometers; orthophosphoric acid (85%) was used as an
external standard. In several cases the signal of the terminal
phosphate moiety was not visible because of high dilution.
Purity of compounds was checked using a Hewlett-Packard
1100 HPLC instrument equipped with a Zorbax Eclipse 5 μm
XDB-C18 analytical column (250 mm ꢀ 4.6 mm; Agilent
Technologies Inc., Palo Alto, CA). For system A, the mobile
phase was a linear gradient solvent system of 5 mM TBAP-
CH3CN from 80:20 to 40:60 in 20 min; the flow rate was 1 mL/
min or Zorbax SB-Aq 5 μm analytical column (50 mm ꢀ 4.6 mm;
Agilent Technologies Inc., Palo Alto, CA). For system B, the
mobile phase was a linear gradient solvent system of 5 mM
TBAP-CH3CN from 80:20 to 40:60 in 13 min; the flow rate was
0.5 mL/min. Peaks were detected by UV absorption with a diode
array detector at 254, 275, and 280 nm. All derivatives tested for
biological activity showed >99% purity by HPLC analysis
(detection at 254 nm).
High-resolution mass measurements were performed on a
Micromass/Waters LCT Premier electrospray time of flight
(TOF) mass spectrometer coupled with a Waters HPLC system
unless otherwise noted. Purification of the nucleotide analogues
for biological testing was carried out on (diethylamino)ethyl
(DEAE)-A25 Sephadex columns with a linear gradient (0.01-
0.5 M) of 0.5 M ammonium bicarbonate as the mobile phase.
Compounds 7 were purified by Sephadex alone (and isolated in
the ammonium salt form), and all other compounds were
purified by HPLC with a Luna 5 μm RP-C18(2) semipreparative
column (250 mm ꢀ 10.0 mm; Phenomenex, Torrance, CA) and
using the following conditions: flow rate of 2 mL/min; 10 mM
triethylammonium acetate (TEAA)-CH3CN from 100:0 to
95:5 (or up to 100:0 to 75:25) in 20-40 min (and isolated in
the triethylammonium salt form).
The modeling and receptor docking also predicted that
various water molecules are located in proximity of the 5
position of the ligand. The enhanced affinity of the 5-iodo-
substituted analogues could be a result of the fact that the
halogen displaced some of the structured water molecules
present in the binding site, with a consequent entropic gain.
All reagents were of analytical grade. 3-Phenacyl-UDP 5 was
purchased from Tocris. 5-Iodouridine 34 was purchased from
Berry Associates (Ann Arbor, MI). Compound 36 was synthe-
sized as reported.19,25 Other reagents and solvents were pur-
chased from Sigma-Aldrich (St. Louis, MO).
(S)-Methanocarbauridine 50-Diphosphate Triethylammo-
nium Salt (7). A solution of the (S)-methanocarbauridine 40
(1-[(1S,2S,5S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)bicyclo-
[3.1.0]hexyl]-1,3-dihydropyrimidine-2,4-dione, 10mg, 0.04mmol,
ref 13) and Proton Sponge (17 mg, 0.08 mmol) in trimethyl
phosphate (1 mL) was stirred for 10 min at 0 ꢀC. Then phosphorus
oxychloride (0.008 mL, 0.08 mmol) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 ꢀC. A mixture of tributy-
lamine (0.8 mL, 0.35 mmol) and a solution 0.35 M of the bis-
(tributylammonium) salt of phosphoric acid in DMF (0.7 mL) was
added at once. After 10 min, 0.2 M triethylammonium bicarbonate
solution (1.5 mL) was added, and the clear solution was stirred at rt
for 1 h. After removal of solvents, the residue was purified using the
same method as the general procedure using Sephadex-DEAE A-25
resin and HPLC to get compound 7 (1.3 mg, 45%) as a white solid.
1HNMR(D2O) δ7.79 (d, J= 7.8 Hz, 1H), 5.86 (d, J= 8.1 Hz, 1H),
4.69 (m, 1H), 4.15 (m, 3H), 2.39 (t, J = 6.3 Hz, 1H), 1.87 (m, 1H),
Conclusions
We have identified novel nucleotides, such as N4-alkoxycy-
tidine derivatives, that are more potent, selective, and poten-
tially more stable in vivo than both the native agonist and
previously reported synthetic agonists of the P2Y6 receptor.
Thus, N4-methoxycytidine nucleotides 12, 15, and 23 were
relatively potent and selective agonists, and moreover, com-
pound 23 displayed favorable stability. Thus, in future SAR
studies (S)-methanocarba-(R,β-CH2)-N4-OMe-CDP could be
a suitable combination to maximize the potency while retain-
ing selectivity and stability.
The P2Y6 receptor is implicated in neurodegeneration,
osteoporosis, ischemic effects in skeletal muscle, and possibly
diabetes.4,32,33 Therefore, new potent and selective ligands are
of interest in the search for new treatments for these condi-
tions. Agonists of the P2Y6 receptor are predicted to produce
beneficial responses in, for example, muscle wasting and
1.71 (t, J = 6.0 Hz, 1H), 1.30 (ddd, J = 2.1, 6.3, 8.4 Hz, 1H); 31
P
NMR (D2O) δ -5.87 (d, J = 22.0 Hz), -10.10 (d, J = 23.2 Hz);
HRMS-EI found 413.0170 (M - Hþ)-. C11H15N2O11P2 requires
413.01511; purity >99% by HPLC (system A, 11.1 min).

Article
5-Iodouridine 50-r,β-Methylenediphosphate Triethylammo-
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4497
1H), 4.27-4.17(m, 2H), 3.95(dd, 1H, J =6.1, 3.0Hz), 3.80-3.68
(m, 2H), 1.26 (s, 9H); HRMS-EI found 316.1509 (M þ Hþ)-.
C13H22N3O6 requires 316.1516.
nium Salt (11). Methylene diphosphonic acid (14 mg, 0.08 mmol)
and DCC (25 mg, 0.121 mmol) were added to a solution of
5-iodouridine 34 (15 mg, 0.040 mmol) in DMF (1.0 mL). The
mixture was stirred for 2 days at rt under a nitrogen atmosphere.
Solvent was evaporated and the residue was purified onSephadex-
DEAE A-25 resin and HPLC using the same method as the
general procedure, to give the compound 11 (2 mg, 11%) as a
white powder. 1H NMR (D2O) δ 8.26 (s, 1H), 5.98 (d, J = 6.1 Hz,
1H), 4.39-4.46 (m, 2H), 4.29-4.34 (m, 1H), 4.19-4.24 (m, 2H),
2.23 (t, J=19.1Hz, 2H);31PNMR(D2O) δ19.02 (d, J=8.34Hz),
15.04 (d, J = 8.34 Hz); HRMS-EI found 526.9108 (M - H).
C10H15IN2O11P2 requires 527.9109.
A solution of the N4-tert-butyloxycytidine 38 (39 mg, 0.12 mmol)
and Proton Sponge (40 mg, 0.19 mmol) in trimethyl phosphate
(1.2 mL) was stirred for 10 min at 0 ꢀC. Then phosphorus
oxychloride (0.023 mL, 0.25 mmol) was added dropwise, and
the reaction mixture was stirred for 2 h at 0 ꢀC. A mixture of
tributylamine (0.24 mL, 1.0 mmol) and a 0.35 M solution of the
bis(tributylammonium) salt of phosphoric acid in DMF (2.8 mL)
was added at once. After 10 min 0.2 M triethylammonium
bicarbonate solution (3 mL) was added, and the clear solution
was stirred at rt for 1 h. After removal of solvents, the residue was
purified using the same method as the general procedure using
Sephadex-DEAE A-25 resin and HPLC to obtain compound 14
N4-Methoxycytidine 50-Diphosphate Ammonium Salt (12). A
solution of the N4-methoxycytidine 36 (10 mg, 0.037 mmol) and
Proton Sponge (17 mg, 0.08 mmol) in trimethyl phosphate (1 mL)
was stirred for 10 min at 0 ꢀC. Then phosphorus oxychloride
(0.008 mL, 0.08 mmol) was added dropwise, and the reaction
mixture was stirred for 2 h at 0 ꢀC. A mixture of tributylamine
(0.8 mL, 0.35 mmol) and a solution 0.35 M of the bis(tributyl-
ammonium) salt of phosphoric acid in DMF (0.7 mL) was added
at once. After 10 min 0.2 M triethylammonium bicarbonate
solution (1.5 mL) was added, and the clear solution was stirred
at rt for 1 h. After removal of solvents, the residue was purified
using the same method as the general procedure using Sephadex-
DEAE A-25 resin and HPLC to get compound 12 (4.4 mg, 25%)
as a white solid. 1H NMR (D2O) δ 7.23 (d, J = 8.1 Hz, 1H), 5.92
(d, J = 5.7 Hz, 1H), 5.79 (d, J = 7.8 Hz, 1H), 4.35 (m, 2H), 4.21
(m, 1H), 4.15 (m, 2H), 3.78 (s, 3H); 31P NMR (D2O) δ -7.74 (d,
J = 21.4 Hz), -10.6 (d, J = 21.4 Hz); HRMS-EI found 432.0207
(M - Hþ)-. C10H16N3O12P2 requires 432.0209; purity >98% by
HPLC (system A, 13.6 min).
1
(12.5 mg, 21%) as a white amorphous solid. H NMR (D2O)
δ 7.12 (d, J = 8.3 Hz, 1H), 5.84 (d, J = 6.0 Hz, 1H), 5.74 (d, J =
8.3 Hz, 1H), 4.32-4.23 (m, 2H), 4.13-4.03 (m, 2H), 3.08 (dd, J =
14.7, 7.4 Hz, 12H), 1.18 (s, 9H), 1.16 (t, J =7.3Hz, 18H); HRMS-
EI found 474.0679 (M - Hþ)-. C13H22N3O12P2 requires
474.0669; purity >98% by HPLC (system B, 8.06 min).
N4-Benzyloxycytidine 50-Diphosphate Ammonium Salt (15). A
solution of the cytidine 35 (200 mg, 0.82 mmol) and O-benzyl-
hydroxylamine hydrochloride (263 mg, 1.64 mmol) in pyridine
(2 mL) was stirred overnight at 100 ꢀC. The reaction mixture was
concentrated, and the residue was purified on a silica gel column
(CHCl3/MeOH = 10/1) to obtain compound 39 (288 mg, 100%)
1
as a pale-yellow amorphous product. H NMR (acetone-d6) δ
7.40-7.26 (m, 5H), 7.23 (d, J = 8.2 Hz, 1H), 5.85 (d, J = 5.6 Hz,
1H), 5.52 (d, J = 8.2 Hz, 1H), 5.00 (s, 2H), 4.27-4.18 (m, 2H),
3.96 (dd, J = 6.1, 3.0 Hz, 1H), 3.82-3.69 (m, 2H); HRMS-EI
found 350.1352 (M þ Hþ)-. C16H19N3O6 requires 350.1356.
A solution of the N4-benzyloxycytidine 39 (80 mg, 0.23 mmol)
and Proton Sponge (74 mg, 0.34 mmol) in trimethyl phosphate
(2.4 mL) was stirred for 10 min at 0 ꢀC. Then phosphorus
oxychloride (0.042 mL, 0.46 mmol) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 ꢀC. A mixture of
tributylamine (0.43 mL, 1.83 mmol) and a 0.35 M solution of
the bis(tributylammonium) salt of phosphoric acid in DMF
(5.1 mL) was added at once. After 10 min 0.2 M triethylammo-
nium bicarbonate solution (1.5 mL) was added, and the clear
solution was stirred at rt for 1 h. After removal of solvents, the
residue was purified using the same method as the general
procedure using Sephadex-DEAE A-25 resin and HPLC to
obtain compound 15 (54 mg, 47%) as a white amorphous solid.
1H NMR (D2O) δ 7.50-7.39 (m, 5H), 7.26 (d, J = 8.4 Hz, 1H),
5.95 (d, J = 5.9 Hz, 1H), 5.77 (d, J = 7.8 Hz, 1H), 5.07 (s, 2H),
4.46-4.43 (m, 1H), 4.37 (t, J = 5.7 Hz, 1H), 4.27-4.11 (m, 3H);
31P NMR (D2O) δ -7.33 (s), -11.04 (s), -11.18(s); HRMS-EI
found 508.0522 (M - Hþ)-. C16H20N3O12P2 requires 508.0507;
purity >98% by HPLC (system B, 9.45 min).
N4-Ethoxycytidine 50-Diphosphate Ammonium Salt (13). A
solution of the cytidine 35 (200 mg, 0.82 mmol) and O-ethylhy-
droxylamine hydrochloride (160 mg, 1.64 mmol) in pyridine
(2 mL) was stirred overnight at 100 ꢀC. The reaction mixture was
concentrated, and the residue was purified on a silica gel column
(CHCl3/MeOH = 10/1) to obtain compound 37 (230 mg, 97%)
1
as a yellow amorphous solid. H NMR (acetone-d6) δ 7.32 (d,
J = 8.2 Hz, 1H), 5.86 (d, J = 5.1 Hz, 1H), 5.62 (d, J = 8.2 Hz,
1H), 4.27-4.21 (m, 2H), 4.07-3.95 (m, 3H), 3.79 (dd, J = 12.0,
2.8 Hz, 1H), 3.73 (dd, J = 12.0, 2.8 Hz, 1H), 1.22 (t, J = 7.0 Hz,
3H); HRMS-EI found 288.1196 (M þ Hþ)-. C11H18N3O6
requires 288.1185
A solution of the N4-ethoxycytidine 37 (85 mg, 0.30 mmol)
and Proton Sponge (95 mg, 0.44 mmol) in trimethyl phosphate
(2.5 mL) was stirred for 10 min at 0 ꢀC. Then phosphorus
oxychloride (0.054 mL, 0.59 mmol) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 ꢀC. A mixture of tri-
butylamine (0.56 mL, 2.4 mmol) and a 0.35 M solution of the
bis(tributylammonium) salt of phosphoric acid in DMF (6.6 mL)
was added at once. After 10 min 0.2 M triethylammonium
bicarbonate solution (6.9 mL) was added, and the clear solution
was stirred at rt for 1 h. After removal of solvents, the residue was
purified using the same method as the general procedure using
Sephadex-DEAE A-25 resin and HPLC to obtain compound 13
N4-Methoxycytidine 50-r,β-Methylenediphosphate Triethy-
lammonium Salt (16). Methylene diphosphonic acid (13 mg,
0.07 mmol) and DCC (23 mg, 0.11 mmol) were added to a
solution of N4-methoxycytidine 36 (10 mg, 0.037 mmol) in DMF
(1.0 mL). The mixture was stirred for 2 days at rt under a
nitrogen atmosphere. Solvent was evaporated and the residue
was purified on Sephadex-DEAE A-25 resin and HPLC using
the same method as the general procedure, to give the compound
16 (7.5 mg, 48%) as a white powder. 1H NMR (D2O) δ 7.30 (d,
J = 8.1 Hz, 1H), 5.97 (br, 1H), 5.84 (d, J = 8.1 Hz, 1H),
4.42-4.39 (m, 2H), 4.28-4.23 (m, 1H), 4.17-4.12 (m, 2H), 3.83
(s, 3H), 2.20 (t, J = 19.5 Hz, 2H); 31P NMR (D2O) δ 18.88 (br),
14.75 (br); HRMS-EI found 430.0425 (M - Hþ)-. C11H18-
N3O11P2 requires 430.0417; purity >98% by HPLC (system B,
6.93 min).
1
(21.3 mg, 14%) as a white amorphous solid. H NMR (D2O)
δ 7.24 (d, J = 8.2 Hz, 1H), 5.96 (d, J = 5.6 Hz, 1H), 5.82 (d, J =
8.2 Hz, 1H), 4.42-4.33 (m, 2H), 4.26-4.14 (m, 3H), 4.05(dd, J =
14.1, 7.0 Hz, 2H); HRMS-EI found 446.0366 (M - Hþ)-.
C10H16N3O12P2 requires 446.0373; purity >98% by HPLC
(system B, 7.54 min).
N4-tert-Butyloxycytidine 50-Diphosphate Ammonium Salt
(14). A solution of the cytidine 35 (200 mg, 0.82 mmol) and
O-tert-butylhydroxylamine hydrochloride (207 mg, 1.64 mmol)
in pyridine (2 mL) was stirred overnight at 100 ꢀC. The reaction
mixture was concentrated, and the residue was purified on a
silica gel column (CHCl3/MeOH = 10/1) to obtain compound
38 (143 mg, 55%) as a white solid. 1H NMR (acetone-d6) δ 7.18
(d, J = 8.2 Hz, 1H), 5.85 (d, J = 5.7 Hz, 1H), 5.56 (d, J = 8.2 Hz,
(S)-Methanocarbauridine 50-Triphosphate Triethylammonium
Salt (18). A solution of (S)-methanocarbauridine 41 (10 mg, 0.04
mmol) and Proton Sponge (17 mg, 0.08 mmol) in trimethyl phos-
phate (1 mL) was stirred for 10 min at 0 ꢀC. Then phosphorus

4498 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
oxychloride (0.008 mL, 0.08 mmol) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 ꢀC. Tributylammonium
pyrophosphate (1.6 mol of C12H27N per mol of H4P2O7, 110 mg,
0.23 mmol) in DMF (0.3 mL) was added at once to the reaction
mixture. After 10 min, 0.2 M triethylammonium bicarbonate
solution (1.5 mL) was added, and the clear solution was stirred at
rt for 1 h. After removal of solvents, the residue was purified using
the same methodas the generalprocedureusing Sephadex-DEAE
A-25 resin and HPLC to get compound 18 (2.0 mg, 56%) as a
white solid. 1H NMR (D2O) δ 7.78 (d, J = 7.8 Hz, 1H), 5.85 (d,
J = 8.1 Hz, 1H), 4.67 (m, 1H), 4.16 (m, 3H), 2.38 (t, J = 6.3 Hz,
1H), 1.86 (m, 1H), 1.71 (t, J = 6.0 Hz, 1H), 1.30 (ddd, J = 2.1,
6.3, 8.4 Hz, 1H); 31P NMR (D2O) δ -6.76 (m), -10.7 (d, J =
20.2 Hz), -22.63 (app t); HRMS-EI found 492.9847 (M - Hþ)-.
C11H16N2O14P3 requires 492.9814; purity >98% by HPLC
(system A, 17.5 min).
(d, J=19.9 Hz), -23.07 (t, J = 19.1 Hz); HRMS-EI found
558.9928 (M - Hþ)-. C15H18N2O15P3 requires 558.9920; purity
>99% by HPLC (system B, 8.9 min).
P1-Uridine 50-P3-Phenyltriphosphate Triethylammonium Salt
(26). Compound 1 (4 mg, 0.010 mmol) and sodium phenylpho-
sphate dibasic dihydrate (5.0 mg, 0.020 mmol) were cation-
exchanged to the tributylammonium salts. A solution of com-
pound 1, tributylammonium salt, and DIC (4.6 μL, 0.030 mmol)
in DMF (0.08 mL) was stirred for 3 h at rt. Then a solution of
phenylphosphate tributylammonium salt in DMF (0.08 mL) and
a solutionof MgCl2 (2.8 mg, 0.030mmol)inDMF(0.04 mL) were
added. After the reaction mixture was stirred at rt for 16 h, the
solvent was removedandwater (0.4 mL) was added. Theresulting
solid was removed by filtration, and the filtrate was purified by
semipreparative HPLC as described above to obtain 25 (1.24 mg,
16%) as a white solid. 1H NMR (D2O) δ 8.02 (d, J = 8.7 Hz, 1H),
6.07-6.00 (m, 2H), 4.49-4.40 (m, 2H), 4.35-4.15 (m, 4H),
4.01-3.77 (m, 4H), 2.06-1.96 (m, 2H), 1.79-1.69 (m, 2H),
1.59-1.14 (m, 6H); 31P NMR (D2O) δ -11.5 (br, 2H), -23.1
(br, 1H);HRMS-EI found 565.0405 (M - Hþ)-. C15H24N2O15P3
requires 565.0390; purity >99% by HPLC (system B, 9.8 min).
P1-Uridine 50-P3-[1]Glucose-10-Triphosphate Triethylammo-
nium Salt (27). Compound 1 (10 mg, 0.025 mmol) and R-D-
glucose-1-phosphate disodium salt hydrate (10 mg, 0.038 mmol)
were exchanged to the tributylammonium salts. A solution of
compound 1, tributylammonium salt, and DIC (12 μL, 0.075
mmol) in DMF (0.10 mL) was stirred for 3 h at rt. Then a solution
of the R-D-glucose-1-phosphate tributylammonium salt in DMF
(0.10 mL) and a solution of MgCl2 (7 mg, 0.075 mmol) in DMF
(0.05 μL) were added. After the reaction mixture was stirred at rt
for 16 h, the solvent was removed and water (0.4 mL) was added.
The resulting solid was removed by filtration, and the filtrate was
purified by semipreparative HPLC as described above to obtain
27 (1.8 mg, 9%) as a white solid. 1H NMR (D2O) δ 7.97 (d, J =
7.8 Hz, 1H), 6.01 (d, J = 4.5 Hz, 1H), 5.99 (d, J = 7.8 Hz, 1H),
5.63 (dd, J = 7.2, 3.6 Hz, 1H), 4.45-4.37 (m, 2H), 4.32-4.22 (m,
3H), 3.97-3.73 (m, 4H), 3.52 (dt, J = 9.9, 3.3 Hz, 1H), 3.45 (t,
J = 9.6 Hz); 31P NMR (D2O) δ -11.27 (d, J = 19.8 Hz), -12.65
(d, J = 19.8 Hz), -22.70 (d, J = 19.8 Hz); HRMS-EI found
645.0100 (M - Hþ)-. C15H24N2O20P3 requires 645.0135; purity
>99% by HPLC (system B, 8.8 min).
N4-Methoxycytidine 50-Triphosphate Ammonium Salt (20). A
solution of N4-methoxycytidine 36 (10 mg, 0.037 mmol) and Proton
Sponge (17 mg, 0.08 mmol) in trimethyl phosphate (1 mL) was
stirredfor 10 minat0ꢀC. Then phosphorus oxychloride (0.008 mL,
0.08 mmol) was added dropwise, and the reaction mixture was
stirred for 2 h at 0 ꢀC. Tributylammonium pyrophosphate (1.6 mol
of C12H27N per mol of H4P2O7, 110 mg, 0.23 mmol) in DMF
(0.3 mL) was added at once to the reaction mixture. After 10 min,
0.2 M triethylammonium bicarbonate solution (1.5 mL) was
added, and the clear solution was stirred at rt for 1 h. After removal
of solvents, the residue was purified using the same method as the
general procedure using Sephadex-DEAE A-25 resin and HPLC to
get compound 20 (3.9 mg, 31%) as a white solid. 1H NMR (D2O)
δ 7.25 (d, J = 8.1 Hz, 1H), 5.97 (d, J = 6.3 Hz, 1H), 5.83 (d, J =
8.4 Hz, 1H), 4.40 (m, 2H), 4.22 (m, 3H), 3.81 (s, 3H); 31P NMR
(D2O) δ -8.82 (d, J = 20.2 Hz), -11.0 (d, J = 20.2 Hz), -22.40 (t,
J = 19.5 Hz); HRMS-EI found 511.9873 (M - Hþ)-. C10H17-
N3O15P3 requires 511.9857; purity >98% by HPLC (system A,
17.2 min).
P1,P3-Di(uridine 50-)r,β-MethylenetriphosphateTriethylammo-
nium Salt (22). Compound 10 (3 mg, 0.006 mmol) and UMP
(1.6 mg, 0.006 mmol) were converted to the corresponding tri-
butylammonium salts by ion exchange. A solution of compound
10, tributylammonium salt, and N,N0-diisopropylcarbodiimide
(DIC, 2.8 μL, 0.018 mmol) in DMF (0.06 mL) was stirred for 3 h
at rt. Then a solution of the UMP tributylammonium salt in DMF
(0.04 mL) and a solution of MgCl2 (1.7 mg, 0.018 mmol) in DMF
(0.02 μL) were added. After the reaction mixture was stirred at rt
for 16 h, the solvent was removed and water (0.6 mL) was added.
The resulting solid was removed by filtration, and the filtrate was
purified by semipreparative HPLC as described above to obtain 22
(S)-Methanocarbauridine 30,50-Cyclic-diphosphate Triethyla-
mmonium Salt (9) and (S)-Methanocarbauridine 50-Glucose-10-
triphosphate Triethylammonium Salt (28). To a solution of
compound 7 (2.6 mg, 0.0036 mmol) in DMF (2 mL), 1,10-
carbonyldiimidazole (3.0 mg, 0.018 mmol) was added. The
reaction mixture was stirred at rt for 5 h. Then 5% triethylamine
solution in 1/1 water/methanol (1 mL) was added and stirring
was continued at rt for an additional 2 h. After removal of the
solvent, the residue was dried under high vacuum and dissolved
in DMF (2 mL). Glucose-1-monophosphate tributylammonium
salt (4 mg, 0.006 mmol) in DMF (0.2 mL) was added to this
mixture. The reaction mixture was stirred at rt for 2 days. After
removal of the solvent, the residue was purified using the same
method as the general procedure using Sephadex-DEAE A-25
resinandHPLC togetcompound 28 (0.8 mg, 23%) and9 (1.1 mg,
51%) additionally. Compound 28: 1H NMR(D2O) δ 7.79 (d, J =
8.1 Hz, 1H), 5.86 (d, J = 8.1 Hz, 1H), 5.64 (dd, J = 3.3, 7.2 Hz,
1H), 4.62 (m, 1H), 4.15 (t, J = 6.3 Hz, 2H), 3.86 (m, 4H), 3.50 (m,
3H), 2.39 (t, J = 6.3 Hz, 1H), 1.88 (dd, J = 4.8, 9.9 Hz, 1H), 1.72
(t, J = 5.7 Hz, 1H), 1.39 (m, 1H); 31P NMR (D2O) δ -10.87 (d,
J = 21.4 Hz), -12.58 (d, J = 20.76 Hz); HRMS-EI found
655.0326 (M - Hþ)-. C17H26N2O19P3 requires 655.0343; purity
>98% by HPLC (system A, 16.9 min). Compound 9: 1H NMR
(D2O) δ 7.81 (d, J = 8.1 Hz, 1H), 5.84 (d, J = 8.1 Hz, 1H), 5.30
(m, 1H), 4.08 (m, 3H), 2.63 (t, J = 6.3 Hz, 1H), 2.03 (m, 1H), 1.71
(t, J = 6.0Hz, 1H), 1.50 (ddd, J = 2.1, 6.3, 8.4Hz, 1H); 31P NMR
(D2O) δ -9.55 (app d), -22.66 (app t); HRMS-EI found
395.0044 (M - Hþ)-. C11H13N2O10P2 requires 395.0045; purity
>98% by HPLC (system A, 12.0 min).
1
(1.2 mg, 27%) as a white solid. H NMR (D2O) δ 8.03 (d, J =
8.1 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 6.02-5.94 (m, 4H), 4.43-4
.35 (m, 2H), 4.32-4.17 (m, 3H), 2.37 (t, J = 20.1 Hz, 1H); 31P
NMR (D2O) δ 17.35 (s), 7.42 (d, J = 27.6 Hz), -10.93 (d, J = 27.6
Hz; HRMS-EI found 751.0009 (M þ 2Naþ - 3Hþ)-. C19H24-
N4Na2O19P3 requires 751.0043; purity >99% by HPLC (system B,
9.3 min).
P1-Uridine 50- P3-Cyclohexyltriphosphate Triethylammonium
Salt (25). Compound 1 (4 mg, 0.010 mmol) was exchanged to the
tributylammonium salt form. A solution of compound 1, tribu-
tylammonium salt, and DIC (4.6 μL, 0.030 mmol) in DMF
(0.08 mL) was stirred for 3 h at rt. Then a solution of cyclohexyl-
phosphate tributylammonium salt in DMF (0.08 mL) and a
solution of MgCl2 (2.8 mg, 0.030 mmol) in DMF (0.04 mL) were
added. After the reaction mixture was stirred at rt for 16 h, the
solvent was removed and water (0.4 mL) was added. The
resulting solid was removed by filtration, and the filtrate was
purified by semipreparative HPLC as described above to obtain
26 (1.35 mg, 18%) as a white solid. 1H NMR (D2O) δ 7.92 (d,
J = 8.1 Hz, 1H), 7.39 (dd, J = 8.2, 8.7 Hz, 2H), 7.26 (d, J =
8.7 Hz, 2H), 7.19 (t, J = 8.2 Hz, 1H), 5.94 (d, J = 4.5 Hz,
1H), 5.92 (d, J = 8.7 Hz, 1H), 4.38-4.32 (m, 1H), 4.30-4.18
(m, 4H); 31P NMR (D2O) δ -11.22 (d, J = 19.1 Hz), -15.71

Article
5-Iodouridine 50-Glucose-10-triphosphate Triethylammonium
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11 4499
resulting solid was removed by filtration, and the filtrate was
purified by semipreparative HPLC as described above to obtain
32 (0.62 mg, 10%) as a white solid. 1H NMR (D2O) δ 7.28 (d, J =
8.4 Hz, 1H), 5.99 (d, J = 5.4 Hz, 1H), 5.86 (d, J = 8.4 Hz, 1H),
5.65 (dd, J = 3.6, 7.2 Hz, 1H), 4.45-4.36 (m, 2H), 4.30-4.15 (m,
3H), 3.97-3.77 (m, 4H), 3.82 (s, 3H), 3.57-3.50 (m, 1H), 3.47 (t,
J = 9.3 Hz, 1H); 31P NMR (D2O) δ -11.27 (d, J = 19.1 Hz),
-12.65 (d, J = 19.1 Hz), -22.71 (t, J = 19.1 Hz); HRMS-EI
found 674.0387 (M - Hþ)-. C16H27N3 O20P3 requires 674.0401;
purity >99% by HPLC (system B, 9.3 min).
Salt (29). Compound 3 (2 mg, 0.003 mmol) and R-D-glucose-1-
phosphate disodium salt hydrate (2 mg, 0.005 mmol) were
exchanged to the tributylammonium salts. A solution of com-
pound 3, tributylammonium salt, and DIC (1.3 μL, 0.008 mmol)
in DMF (0.04 mL) was stirred for 3 h at rt. Then a solution of
the R-D-glucose-1-phosphate tributylammonium salt in DMF
(0.04 mL) and a solution of MgCl2 (0.8 mg, 0.008 mmol) in
DMF (0.02 μL) were added. After the reaction mixture was
stirred at rt for 16 h, the solvent was removed and water (0.2 mL)
was added. The resulting solid was removed by filtration, and
the filtrate was purified by semipreparative HPLC as described
N4-Methoxycytidine 50-Phenyltriphosphate Triethylammonium
Salt (33). Compound 12 (6.9 mg, 0.011 mmol) and sodium
phenylphosphate dibasic dihydrate (8.3 mg, 0.033 mmol) were
exchanged to the tributylammonium salts. A solution of com-
pound 12 tributylammonium salt and DIC (5.0 μL, 0.033 mmol)
in DMF (0.07 mL) was stirred for 3 h at rt. Then a solution of the
phenylphosphate tributylammonium salt in DMF (0.07 mL) and
a solutionof MgCl2 (3.1 mg, 0.033mmol)inDMF(0.07 mL) were
added. After the reaction mixture was stirred at rt for 16 h, the
solvent was removed and water (1 mL) was added. The resulting
solid was removed by filtration, and the filtrate was purified by
semipreparative HPLC as described above to obtain 33 (0.9 mg,
14%) as a white amorphous solid. 1H NMR (D2O) δ 7.44-7.36
(m, 2H), 7.31-7.17(m, 4H), 5.92 (d, J = 6.0Hz, 1H), 5.77 (d, J =
8.4 Hz, 1H), 4.38-4.28 (m, 2H), 4.24-4.15 (m, 3H); HRMS-EI
found 588.0186 (M - Hþ)-. C16H22N3 O15P3 requires 588.0199;
purity >99% by HPLC (system B, 10.3 min).
1
above to obtain 29 (0.35 mg, 13%) as a white solid. H NMR
(D2O) δ 8.26 (s, 1H), 6.01 (d, J = 4.8 Hz, 1H), 5.68 (dd, J = 3.0,
6.9 Hz, 1H), 4.49-4.40 (m, 2H), 4.36-4.25 (m, 3H), 4.03-3.80
(m, 4H), 3.62-3.47 (m, 2H); 31P NMR (D2O) δ -11.46 (d, J =
19.1 Hz), -12.60 (d, J = 19.1 Hz), -22.60 (t, J = 19.1 Hz);
HRMS-EI found 770.9131 (M - Hþ)-. C15H23IN2O20P3 requires
770.9102; purity >99% by HPLC (system B, 9.7 min).
3-Phenacyluridine 50-Glucose-10-triphosphate Triethylammo-
nium Salt (30). Compound 5 (2 mg, 0.005 mmol) and R-D-
glucose-1-phosphate disodium salt hydrate (2 mg, 0.010 mmol)
were exchanged to the tributylammonium salts. A solution of
compound 5, tributylammonium salt, and DIC (2 μL, 0.015
mmol) in DMF (0.04 mL) was stirred for 3 h at rt. Then a
solution of the R-D-glucose-1-phosphate tributylammonium salt
in DMF (0.04 mL) and a solution of MgCl2 (1 mg, 0.015 mmol)
in DMF (0.02 μL) were added. After the reaction mixture was
stirred at rt for 16 h, the solvent was removed and water (0.2 mL)
was added. The resulting solid was removed by filtration, and
the filtrate was purified by semipreparative HPLC as described
Pharmacological Analyses. UDP was purchased from Sigma
(St. Louis, MO). myo-[3H]Inositol (20 Ci/mmol) was obtained
from American Radiolabeled Chemicals (St. Louis, MO). Mea-
surement of intracellular calcium ion concentration in response
to nucleotides was performed as described.4
1
above to obtain 30 (1.0 mg, 29%) as a white solid. H NMR
Assay of PLC Activity Stimulated by P2Y2, P2Y4, and P2Y6
Receptors. Stable cell lines expressing the human P2Y2, P2Y4, or
P2Y6 receptor in 1321N1 human astrocytoma cells were gener-
ated as described.26 Agonist-induced [3H]inositol phosphate
production was measured in 1321N1 cells plated to 20 000
cells/well on 96-well plates 2 days prior to assay. Sixteen hours
before the assay, the inositol lipid pool of the cells was radi-
olabeled by incubation in 100 μL of serum-free inositol-free
Dulbecco’s modified Eagle’s medium, containing 1.0 μCi of
myo-[3H]inositol. No changes of medium were made subsequent
to the addition of [3H]inositol. On the day of the assay, the cells
were challenged with 25 μL of a 5-fold concentrated solution
of receptor agonists in 200 mM HEPES (N-(2-hydroxyethyl)-
piperazine-N0-2-ethanesulfonic acid), pH 7.3 in HBSS, contain-
ing 50 mM LiCl for 30 min at 37 ꢀC. Incubations were terminated
by aspiration of the drug-containing medium and addition of
450 μL of ice-cold 50 mM formic acid. [3H]Inositol phosphate
accumulation was quantified using scintillation proximity assay
methodology as previously described in detail.26
Data Analysis. Agonist potencies (EC50 values) were deter-
mined from concentration-response curves by nonlinear re-
gression analysis using the GraphPad software package Prism
(GraphPad, San Diego, CA). All experiments examining the
activity of newly synthesized molecules also included full con-
centration effect curves for the cognate agonist of the target
receptor: UTP for the P2Y2 receptor, UTP for the P2Y4
receptor, and UDP for the P2Y6 receptor. Each concentration
of drug was tested in triplicate assays, and concentration effect
curves for each test drug were repeated in at least three separate
experiments with freshly diluted molecule. The results are pre-
sented as the mean ( SEM from multiple experiments or in the
case of concentration effect curves from a single experiment
carried out with triplicate assays that were representative of
results from multiple experiments.
(D2O) δ 8.08-8.04 (m, 3H), 7.80 (t, J = 7.2 Hz, 1H), 7.65 (t, J =
7.2 Hz, 2H), 6.17 (d, J = 7.8 Hz, 1H), 6.04 (d, J = 4.5 Hz, 1H),
5.66 (dd, J = 3.6, 6.9 Hz), 5.55 (s, 2H), 4.49-4.43 (br, 2H),
4.37-4.29 (br, 3H), 4.01-3.77 (m, 4H), 3.60-3.50 (br, 1H), 3.48
(t, J = 9.1 Hz, 1H); 31P NMR (D2O) δ -11.24 (d, J = 25.4 Hz),
-12.63 (d, J = 25.4 Hz), -22.65 (d, J = 25.8 Hz); HRMS-EI
found 763.0554 (M - Hþ)-. C23H30N2O21P3 requires 763.0589;
purity >99% by HPLC (system B, 9.9 min).
Uridine 50-Glucose-10-r,β-methylenetriphosphate Triethylam-
monium Salt (31). Compound 10 (2.4 mg, 0.004 mmol) and R-D-
glucose-1-phosphate disodium salt hydrate (3.6 mg, 0.012 mmol)
were exchanged to the tributylammonium salts. A solution of
compound 10, tributylammonium salt, and DIC (1.8 μL, 0.012
mmol) in DMF (0.04 mL) was stirred for 3 h at rt. Then a solution
of the R-D-glucose-1-phosphate tributylammonium salt in DMF
(0.04 mL) and a solution of MgCl2 (2.6 mg, 0.028 mmol) in DMF
(0.02 μL) were added. After the reaction mixture was stirred at rt
for 16 h, the solvent was removed and water (0.4 mL) was added.
The resulting solid was removed by filtration, and the filtrate was
purified by semipreparative HPLC as described above to obtain
31 (2.7 mg, 80%) as a white solid. 1H NMR (D2O) δ 8.07 (d, J =
7.8 Hz, 1H), 6.06-5.99 (m, 2H), 5.70-5.63 (m, 1H), 4.49-4.43
(m, 2H), 4.35-4.27 (m, 1H), 4.27-4.21 (m, 2H), 4.00-3.77 (m,
4H), 3.60-3.43 (m, 2H), 2.43 (t, J = 19.8 Hz, 2H); 31P NMR
(D2O) δ 17.32 (d, J = 9.1 Hz), 8.27 (dd, J = 9.1, 26.7 Hz), -12.23
(d, J = 26.7 Hz); HRMS-EI found 643.0348(M - Hþ)-. C16H26-
N2O19P3 requires 643.0343; purity >99% by HPLC (system B,
9.2 min).
N4-Methoxycytidine 50-Glucose-10-triphosphate Triethylammo-
nium Salt (32). Compound 12 (4.9 mg, 0.009 mmol) and R-D-
glucose-1-phosphate disodium salt hydrate (8.4 mg, 0.028 mmol)
were exchanged to the tributylammonium salts. A solution of
compound 12, tributylammonium salt, and DIC (4.3 μL, 0.028
mmol) in DMF (0.1 mL) was stirred for 3 h at rt. Then a solution
of the R-D-glucose-1-phosphate tributylammonium salt in DMF
(0.1 mL) and a solution of MgCl2 (2.6 mg, 0.028 mmol) in DMF
(50μL) wereadded. After the reactionmixturewas stirred at rtfor
16 h, the solvent was removed and water (1 mL) was added. The
Stability of Nucleotide Derivatives. UTP was purchased from
Sigma (St. Louis, MO). Up3U 19 was synthesized by the
procedure previously reported.21 HCl buffer (pH 1.2) solution
was prepared as follows. Concentrated HCl (7 mL, 12 N) was

4500 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 11
Maruoka et al.
added to 2.0 g of sodium chloride and diluted with water to a
volume of 1000 mL. Membranes were prepared as follows. P2Y1
receptor-expressing astrocytoma cells were grown to 80% con-
fluence and then harvested. The cells were homogenized, and the
nuclear fraction was removed by centrifuging at 100g for 5 min.
The pellet was resuspended in 50 mM tris(hydroxymethyl)-
aminomethane (Tris) hydrochloride buffer (pH 7.4). The suspen-
sion was homogenized witha Polytronhomogenizer (Brinkmann)
for 10 s and was ultracentrifuged at 20000g for 20 min at 4 ꢀC.
The resultant pellets were resuspended in Tris buffer (pH 7.4),
and the membrane was stored at -80 ꢀC until the experiments.
The protein concentration of the membrane preparation was
measured by Bradford assay.34 The membrane preparation
was diluted with 4 mL of Dulbecco’s phosphate buffered saline
(pH 7.4) and homogenized before use for the stability check.
The final protein concentration was 14.9 μg/mL.
An amount of 10 μL of a 2 mg/mL aqueous solution of each
nucleotide derivative was mixed with 90 μL of HCl buffer
(pH 1.2) or the membrane suspension and incubated at 37 ꢀC.
At regular intervals, a 6 μL aliquot of the above mixture was
mixed with 54 μL of water or 5 mM TBAP. Then 50 μL of the
mixure was injected into the HPLC.
AUC of compounds were measured using a Hewlett-Packard
1100 HPLC equipped with a Zorbax Eclipse 5 μm XDB-C18
analytical column (250 mm ꢀ 4.6 mm; Agilent Technologies
Inc., Palo Alto, CA). The mobile phase was a linear gradient
solvent system of 5 mM TBAP-CH3CN from 80:20 to 40:60 in
6.5 min, and the flow rate was 1 mL/min (system C) or system B.
Peaks were detected by UV absorption with a diode array
detectorat254, 275, and280nm, andAUCvalueswerecalculated
based on the peak at 254 nm.
the OPLS_2001 force field.36 The minimizations were based on
the Polak-Ribier conjugate gradient algorithm and were per-
formed with a threshold on the gradient of 0.05 kJ/mol. The
conformational search was based on 100 steps of extended
Monte Carlo multiple minimum (MCMM) torsional sampling.
The sampling regarded the torsional angles of residues and
ligand, as well as the rotation and translation of the ligand.
Residue Indexing. To facilitate the comparison among recep-
tors, residues are identified through the GPCR residue indexing
system. In each TM, the index X.50, where X is the TM number,
is assigned to the position hosting the most conserved residue
among class A GPCRs. All other positions in the TM are
numbered relative to the X.50 position.37
Acknowledgment. Mass spectral measurements were per-
formed by Dr. Noel Whittaker and NMR measurements by
Wesley White and Dr. Herman Yeh (NIDDK). We thank Dr.
Andrei A. Ivanov (NIDDK) for helpful discussions and Rhonda
Carter for technical assistance. This research was supported in
part by the Intramural Research Program of the NIH, NIDDK,
and NIH extramural Grant GM38213 from the National
Institute of General Medical Sciences.
Supporting Information Available: Time course of decom-
position/hydrolysis of selected pyrimidine nucleotide deriva-
tives in acidic solution and in the presence of cell membranes;
stick representation of Figure 2A. This material is available free
of charge via the Internet at http://pubs.acs.org.
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