Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

Article abstract of DOI:10.1016/j.bmc.2008.05.013

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y₂, P2Y₄, and P2Y₆ receptors. The 2-thio modification, found previously to enhance P2Y₂ receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y₂ receptor, in the form of Up₄-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, α,β-methylene-UDP, a P2Y₆ receptor agonist; 30, Up₄-phenyl ester and 34, Up₄-[1]glucose, selective P2Y₂ receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y₂ receptor agonists; 43, the 2-thio analogue of INS37217 (P¹-(uridine-5′)-P⁴-(2′-deoxycytidine- 5′)tetraphosphate), a potent and selective P2Y₂ receptor agonist.

Full text of DOI:10.1016/j.bmc.2008.05.013

Bioorganic & Medicinal Chemistry 16 (2008) 6319–6332
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2
and P2Y6 receptor agonists
Hyojin Ko a, Rhonda L. Carter b, Liesbet Cosyn c, Riccardo Petrelli d, Sonia de Castro a, Pedro Besada a,
Yixing Zhou b, Loredana Cappellacci d, Palmarisa Franchetti d, Mario Grifantini d,
Serge Van Calenbergh c, T. Kendall Harden b, Kenneth A. Jacobson a,
*
a Molecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892-0810, USA
b Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, NC 27599-7365, USA
c Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences (FFW), Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium
d Department of Chemical Sciences, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 February 2008
Revised 2 May 2008
Accepted 5 May 2008
Available online 9 May 2008
The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of
agonist activity at the human P2Y2, P2Y4, and P2Y6 receptors. The 2-thio modification, found previously to
enhance P2Y2 receptor potency, could be combined with other favorable modifications to produce novel
molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges
introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency.
Truncation of dinucleotide agonists of the P2Y2 receptor, in the form of Up4-sugars, indicated that a ter-
minal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are
observed at this distal end of the molecule. Key compounds reported in this study include 9, a,b-meth-
ylene-UDP, a P2Y6 receptor agonist; 30, Up4-phenyl ester and 34, Up4-[1]glucose, selective P2Y2 receptor
agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y2 receptor agonists; 43, the
2-thio analogue of INS37217 (P1-(uridine-50)-P4-(20-deoxycytidine-50)tetraphosphate), a potent and
selective P2Y2 receptor agonist.
Keywords:
Uracil nucleotide
G protein-coupled receptor
Phospholipase C
UDP
UTP
Dinucleotide
Structure–activity relationships
Published by Elsevier Ltd.
1. Introduction
receptor agonists 3 and 4, which have progressed to clinical studies
for dry eye syndrome and pulmonary diseases, the potent and
The P2Y receptor family consists of at least eight human sub-
types that are activated by either or both adenine and uracil nucle-
otides.1,2 P2Y2 and P2Y4 nucleotide receptors respond to uridine 50-
triphosphate (UTP, 1) and its analogues, and the P2Y6 receptor re-
sponds to uridine 50-diphosphate (UDP, 2) and analogues.3 How-
ever, this delineation of agonist selectivities is not absolute. For
example, Müller and coworkers and Besada et al. reported that cer-
tain 50-triphosphate derivatives potently activate the P2Y6 recep-
tor.4,5 The conformational preference of the ribose moiety in
binding to uridine nucleotide-activated P2Y receptors has been ex-
plored through substitution with the sterically constrained metha-
nocarba (bicyclo[3.1.0]hexane) ring system. P2Y2 and P2Y4
receptors display a North conformational preference, while the
P2Y6 receptor prefers the South.6–8
selective P2Y2 receptor agonist 5,7 and the selective P2Y6 receptor
agonist 6 (Chart 1).12,13 Modification of the base moiety of UTP to
form C-linked nucleotides is possible in P2Y2 receptor agonists
and results in enhanced stability.10 Various dinucleotides tend to
activate P2Y2 and P2Y4 receptors (diuridine tetraphosphates) or
P2Y6 receptors (diuridine triphosphates) with moderate potency
and with greater stability than analogues of UTP and UDP.9 Also,
the pharmacological activity of diadenosine polyphosphates at
both P2Y and P2X receptor subtypes has been characterized.1,2
Diadenosine tetraphosphate is only 3-fold less potent than ATP at
the human P2Y2 receptor.
In the present study, we further investigated structure–activity
relationships at P2Y receptors through synthesis of molecules with
additional substitutions of the uracil, ribose, and phosphate moie-
ties and combinations thereof. These analogues of UDP, UTP, and
dinucleotides were assayed for capacity to promote P2Y2, P2Y4,
and P2Y6 receptor-mediated activation of phospholipase C (PLC).3
These novel derivatives incorporated groups such as 2-thio, found
previously to enhance receptor potency.4,7 Phosphonomethylene
bridges and nonphosphate linkages were introduced to enhance
Several recent studies have explored structure–activity rela-
tionships (SARs) at the P2Y2, P2Y4, and P2Y6 receptors.4–6,9–11 Key
pharmacological probes introduced include the nonselective P2Y2
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.05.013

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H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
NH2
N
O
N
O
N
O
N
HN
NH
HN
N
O
O
O
O
O
P
O
P
O
P
O
P
O
P
O
P
O
P
O
P
O
O
O
O
O
O
O
O
O
O
O
O
O
O
OH OH
OH OH
OH OH
OH OH
OH
HO OH
HO OH
HO OH
3 INS365
4 INS37217
O
N
O
O
N
NH
HN
HN
O
O
N
O
P
OH
O
P
O
P
S
O
P
O
P
O
P
O
O
O
O
HO
O
O
O
O
O
O
OH OH
OH
OH OH
HO OH
O O
HO NH2
6 INS48823
5 MRS2698
Chart 1. Agonists of the P2Y2 and P2Y6 receptors.
stability of molecules against the action of ectonucleotidases.14 We
also probed the effects of truncation of dinucleotide agonists of the
P2Y2 receptor by synthesizing and quantifying the activities of a
series of Up4-sugars.7
monophosphate18 were obtained as ammonium salts following
the Yoshikawa procedure19 starting from nucleosides 20-C-
methyl-uridine (49) and 30-C-methyl-uridine (50). The nucleo-
tides also were prepared by the one-pot method using a sequen-
tial reaction of 49 and 50 with phosphorous oxychloride and
pyrophosphoric acid tributylammonium salt, but the yields were
lower.
Most of the required nucleoside precursors were readily avail-
able, with several exceptions. 20-Ureido-20-deoxyuridine 46 was
prepared by a one-step method from 20-amino-20-deoxyuridine
57 (Scheme 6A).20 The synthesis of 2,4-dithiouridine 47 is depicted
2. Results and discussion
2.1. Chemical synthesis
The synthetic routes to the novel nucleotide derivatives (Tables
1–3) are shown in Schemes 1–6. The potencies of five known refer-
ence compounds are listed in Table 1 (P2Y6 agonist UDP 2), Table 2
(P2Y2 agonists UTP 1 and MRS2698 5), and Table 3 (P2Y2 agonists
Up4U 3 and INS37217 4). Types of modifications include UDP ana-
logues containing a methylene-bridged substitute for the diphos-
in Scheme 6B. The commercially available b-D-ribofuranose 1,2,3,5-
tetraacetate 58 was coupled with silylated 2-thiouracil under
SnCl4-catalyzed Vorbrüggen conditions.21 4-Thionation of the
resulting 20,30,50-tri-O-acetyl-2-thiouridine 59 was performed
using Lawesson’s reagent.22 Subsequent sugar deprotection of 60
afforded 2,4-dithiouridine 47 in 59% overall yield. The nucleoside
phate group (Scheme 1), UTP analogues containing
a b,c-
dihalomethylene-bridge in the triphosphate group4 (Scheme 2),
UTP analogues with modified uracil and ribose moieties7 (Scheme
3), a 5-iodo analogue of INS488235,13 (Scheme 4), analogues of uri-
dine 50-tetraphosphate7 (Scheme 5) in which the terminal phos-
phate moiety was condensed with various alcohols, including
sugars. Among the derivatives in Scheme 1, compounds 7 and 10
are new compounds, but 8 and 9 were previously reported15,16
(see Table 1).
1-(b-D-arabinofuranosyl)-2-thio(1H)pyrimidin-4-one 48 was ob-
tained via opening of 2,20-O-anhydrouridine 61with H2S and trieth-
ylamine in anhydrous DMF (Scheme 6C).23 The synthetic routes to
the nucleosides 20-C-methyl-uridine 49 and 30-C-methyl-uridine
50, synthesized using the strategy reported by Wolfe & Harry-
O’kuru24 and Mikhailov et al.25a with some modifications, are out-
lined in Scheme 6D.
The synthesis of UTP analogues 12 and 17–25 from the cor-
responding nucleosides was by standard methods of di-, tri-
phosphate formation.6 In each case the unprotected
nucleoside was first treated with phosphorous oxychloride
(Scheme 3). The reaction mixture was either treated immedi-
ately with bis(tri-n-butylammonium) pyrophosphate (phospho-
ric acid for 12) or the isolated 50-monophosphate was
activated with 1,10-carbonyldiimidazole (CDI) followed by the
pyrophosphate salt. An attempt to synthesize 2,4-dithio-UTP
led to isolation only of the 4-methylthio analogue 17. Identifi-
cation of nucleotide compounds was confirmed by NMR (1H
and 31P) and by high-resolution mass spectrometry (HRMS),
and purity was demonstrated with high-performance liquid
chromatography (HPLC).
2.2. Pharmacological activity
Activation of PLC by a range of concentrations of each nucleo-
tide derivative (7–44) was studied in [3H]inositol-labeled 1321N1
human astrocytoma cells stably expressing the human P2Y2,
P2Y4, or P2Y6 receptors (Tables 1–3) by methodology (see Section
3) we have described previously in detail.1,3,6,26
Table 1 illustrates UDP analogues that were designed for possi-
ble interaction with the P2Y6 receptor. Compounds 7 and 8 are
derivatives substituted with an anionic carboxylic acid or phospho-
nate acetyl ester moiety with the goal of approximating the charge
and electronic characteristics of the diphosphate moiety for inter-
action with cationic residues in the ligand binding pocket.8 These
molecules were inactive at the P2Y6 receptor. Introduction of an
a,b-methylene 9 substitution only slightly reduced potency at the
P2Y6 receptor (Fig. 1), while the a,b-difluoromethylene analogue
The preferred method of synthesis of 20-MeUTP (20) and 30-
MeUTP (21) was through isolation of the monophosphate. 20-C-
Methyl-uridine-50-monophosphate17 and 30-C-methyl-uridine-50-

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6321
Table 1
Relative potencies of UDP, 2, and UDP analogues for activation of the human P2Y6 receptor
O
R3
NH
N
O
R2
O
HO R1
Compound
Modification
Structure
EC50 (lM)
hP2Y6receptora
Nucleosides
O
HO C
O
C O
7
Uridine-50-malonate
NE
NE
R2 =
R2 =
O
HO P
O
C O
8
Uridine-50-phosphonoacetate
OH
Diphosphates
2
UDP
0.30 0.06
0.66 0.11
O
HO P
O
P O
9b
Up-CH2-p (a,b-Methylene UDP)
R2 =
R2 =
OH OH
F
O
HO P
O
P
10
Up-CF2-p (a,b-difluoromethylene UDP)
NE
O
F
OH OH
11
12
5-Amino-UDP
20-Deoxy-20-ureido-UDP
R
3 = NH2
0.61 0.17
4.70 0.44
R1 = NHCONH2
NE, no effect at 10 lM.
O
O
Unless noted: R1 = OH;
HO P O P
O
and R3 = H.
R2 =
OH OH
a
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 means standard error and are the average of three to six
different experiments with each molecule.
b
9, MRS2782.
10 was strikingly inactive. Nucleobase substitution was also exam-
ined in the UDP series. We previously reported that the higher
homologue of 11, 5-amino-UTP, displays increased potency rela-
tive to UTP as a P2Y6 receptor agonist.7 In contrast, the correspond-
ing diphosphate 11 was ꢀ50-fold less potent than UDP. The
introduction of a polar ureido group on the ribose moiety of aden-
osine derivatives provided tailored analogues for selective recogni-
tion by adenosine neoceptors.27 Therefore, we replaced the 20-
hydroxy group of UDP with a highly H-bonding ureido group. This
molecule exhibited a 360-fold reduction of potency at the P2Y6
receptor compared to UDP.
Table 2 illustrates composite data for UTP analogues designed
to activate the P2Y2 and P2Y4 receptors. We previously illustrated
that substitution of a 2-thio group enhances potency and/or selec-
tivity of UTP analogues (e.g., 5 and 14) at the P2Y2 receptor.7 The
combination of the 2-thio modification with a b,c-difluoromethyl-
ene 15 or b,c-dichloromethylene 16 substitution of the triphos-
phate moiety or 4-methylthio 17 group resulted in analogues
that were 50- to 100-fold weaker than 14. A report of another
UTP analogue,4 5-bromo-b,c-dichloromethylene-UTP, that dis-
played submicromolar P2Y2 receptor potency had suggested the
possibility of greater potency in 15 and 16 than we observed
experimentally in the current study. Nevertheless, the dichloro-
methylene group of 16 provides moderate selectivity for the P2Y2
receptor, while the equipotent difluoromethylene derivative 15
was only marginally selective. Replacement of the 20-hydroxy
group of UTP with a ureido group 18 resulted in a 36-fold reduction
of potency at the P2Y2 receptor and >100-fold reduction at the
P2Y4 receptor.
Methyl groups were placed on the ribose ring of UTP at the 20
and 30 positions in 20 and 21, respectively. Like the methanocarba
modification of ribose,6,28 this approach is a means of conforma-
tional control of the ribose ring that has proven effective for
achieving selectivity in A1 adenosine receptor agonists.29,30 Com-
pound 20 maintains a North conformation of the ribose ring and,
as is the case with UTP, activated the P2Y2 and P2Y4 receptors
nearly equipotently. However, a 20- to 30-fold decrease in potency
relative to UTP was observed. In contrast, 21, which maintains a
South conformation of the ribose ring, was inactive at both P2Y2
and P2Y4 receptors. These results are consistent with previously re-
ported conformational preferences of these receptors deduced
from our studies with methanocarba-derivatives of UTP.6
No previous studies have reported the potency of nucleoside 50-
tetraphosphates at the P2Y2 receptor, although adenosine 50-tetra-
phosphate (Ap4) was reported to activate a presumed P2X recep-
tor.31 Therefore, we synthesized and evaluated the activities of
50-tetraphosphate and pentaphosphate derivatives 22–25. The po-
tency of uridine 50-tetraphosphate 22 (Up4) was greatly reduced in

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H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
Table 2
Relative potencies of UTP, 1, and UTP analogues for activation of the human P2Y2, P2Y4 and P2Y6 receptors
X2
NH
N
X1
O
P
OH
O
P
OH
R2
X3
O
O
R3
R4
n
HO R1
a
Compound
Modification
Structure
EC50 (lM)
hP2Y2
hP2Y4
hP2Y6
Triphosphates (n = 2)
1b
(=UTP)
0.060 0.00
0.062 0.008
0.035 0.004
0.008 0.002
1.63 0.36
2.51 0.65
0.91 0.06
1.74 0.25
0.14 0.01
1.45 0.26
NE
0.090 0.01
1.2 0.3
0.35 0.10
2.4 0.8
8.11 0.69
NE
5.35 1.20
4.64 2.05
7.93 0.81
1.26 0.14
NE
>10d
NE
13b
14b
5b,c
15
20-Deoxy-20-amino-UTP
2-Thio-UTP
R1 = NH2
X1 = S
ꢀ1.5b
2-Thio-20-deoxy-20-amino-UTP
2-Thio-b,c-difluoromethylene-UTP
2-Thio-b,c-dichloromethylene-UTP
2-Thio-4-methylthio-UTP
20-Deoxy-20-ureido-UTP
2-Thio-arabino-UTP
X1 = S, R1 = NH2
X1 = S, X3 = CF2
X1 = S, X3 = CCl2
X1 = S, X2 = S CH3
R1 = NHCONH2
R1 = H, R3 = OH
R3 = CH3
NE
5.15 0.69
>10d
>10d
>10d
NE
16c
17
18
19
20
21
20-Methyl-UTP
30-Methyl-UTP
NE
NE
R4 = CH3
Tetra- (n = 3) and penta- (n = 4) phosphates
22
23
24
25
26
27
28
29
Up4
2-Thio-Up4
4-Thio-Up4
2-Thio-Up5
2.61 1.39
0.60 0.20
0.070 0.01
0.57 0.16
3.95 0.51
4.18 0.43
1.70 0.22
1.87 0.15
4.64 2.05
5.52 1.75
0.28 0.06
5.27 1.24
2.70 0.43
2.53 0.57
1.96 0.53
1.12 0.04
7.56 1.07
6.83 1.74
6.46 0.41
7.33 0.66
>10d
X1 = S
X2 = S
X1 = S, n = 4
R2 = OCH3
R2 = CH3
Up4-OMe
Up4-d-Me-phosphonate
Up4-O(CH2)2CN
Up4-OCH2CHOHCH2OH
8.16 0.74
R2 = O(CH2)2CN
>10d
R2 = OCH2CHOH-CH2OH
8.19 0.41
30c
31
Up4-OC6H5
Up4-OC6H11
1.89 1.07
5.86 0.33
NE
NE
O
R2 =
>10d
>10d
O
R2 =
Tetraphosphate sugars (n = 3)
R2 =
O
O
32
33
Up4-[5]ribose
HO
1.88 0.03
4.78 0.40
>10d
HO
OH
R2 =
HO
HO
HO
O
O
Up4-[6]fructose
3.33 0.42
6.30 0.75
>10d
OH
R2 =
OH
O
O
34c
Up4-[1]glucose
0.30 0.13
2.06 0.18
7.83 0.17
HO
OH
OH
R2 =
OH
O
O
35
Up4-[1]galactose
4.85 2.07
1.77 0.41
8.19 0.73
HO
OH
OH

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6323
Table 2 (continued)
a
Compound
Modification
Structure
EC50 (lM)
hP2Y2
hP2Y4
hP2Y6
R2 =
HO
HO
O
O
O
36
37
Up4-[6]mannose
>10d
>10d
>10d
OH
OH
R2 =
HO
O
Up4-[6]20-deoxyglucose
3.54 0.96
4.32 1.50
>10d
OH
OH
Unless noted: R1, R2 = OH; X = O; and R3, R4 = H.
NE, no effect at 10 lM.
a
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 GraphPad software package (GraphPad, San Diego, CA). The results are presented as means standard error and are the average of three
to six different experiments with each molecule.
b
Agonist potencies from Refs. 4 and 7.
5, MRS2698; 16, MRS2725; 30, MRS2768; 34, MRS2732.
650% effect at 10 lM.
c
d
Table 3
Relative potencies of dinucleotide derivatives for activation of the human P2Y2, P2Y4 and P2Y6 receptors
O
O
R1 O P
X
P O R2
OH
OH
a
Compound
Modification
Structure
EC50 (lM)
hP2Y2
hP2Y4
hP2Y6
Dinucleoside triphosphates (n = 1)
38b
Up3U
1.31 0.21
0.87 0.11
0.27 0.07
O
N
HN
O
N
I
O
NH
O
O
O
O
39c
5-I-Up3- (20,30-phenylethyl acetal)U
9.97 0.95
NE
5.49 0.68
O
O O
R1=
HO OH
R2=
Dinucleoside tetraphosphates (n = 2)
3
(=Up4U)
4-S-Up4(4-S-U)
0.21 0.03
0.030 0.010
2.27 1.39
7.77 1.39
0.14 0.04
0.08 0.03
0.14 0.04
0.13 0.01
0.08 0.01
>10d
1.16 0.42
2.03 0.18
>10d
40
41
42
4
43
44
R1 = R2 = 4-thio-uridine
X = CF2
Up2-CF2-p2-U (b,c-difluoromethylene Up4U)
Up2-CCl2-p2-U (b,c-dichloromethylene Up4U)
Up4-20-dC (INS37217)
2-Thio-Up4-20-dC
X = CCl2
NE
NE
R2 = 20-deoxy-cytidine
0.14 0.04
0.71 0.15
0.54 0.15
0.95 0.06
1.05 0.07
1.03 0.08
R1 = 2-thio uridine R2 = 20-deoxy-cytidine
R2 = 20-deoxy-guanosine
Up4-20-dG
Unless noted: R1, R2 = Uridine; and X = O.
NE, no effect at 10 lM.
a
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 GraphPad software package (GraphPad, San Diego, CA). The results are presented as means standard error and are the average of three
to six different experiments with each molecule.
b
Reported in Ref. 9.
39, MRS2752; 44, MRS2798; 43, MRS2657.
650% effect at 10 lM.
c
d

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H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
comparison to UTP 1. The high P2Y2 receptor potency of 2-thio-
O
N
O
N
UTP 14 was remarkably preserved in the corresponding 4-thio-
50-tetraphosphate analogue 24; in contrast, the potency of the 2-
thio analogue 23 was reduced. Nevertheless, these results illustrate
that 2- or 4-thio substitution of Up4 results in marked increases of
potency in tetraphosphate molecules since both 23 and 24 were
more potent at the P2Y2 receptor than Up4 22. Homologation of
23 to the pentaphosphate 25 had no effect on potency at the
P2Y2 receptor.
Although dinucleoside tetraphosphates, such as 3 and 4, are
known to activate P2Y2 and P2Y4 receptors with moderate po-
tency,9 the SAR of the terminal nucleoside moiety mainly has been
explored for uridine and other nucleoside units. Therefore, we
investigated the effects of substitution of the terminal nucleoside
moiety with small organic moieties 26–31 or to sugars alone 32–
37. Most of these modifications led to P2Y2 receptor potencies in
the micromolar range. A terminal methyl phosphoester 26 and
the corresponding phosphonate 27 were identical in potency as
relatively weak P2Y2 receptor agonists, with EC50 values of 4 lM.
A terminal cyclohexyl ester 31 was significantly less potent than
the corresponding terminal phenyl ester 30, suggesting that aro-
matic or hydrophilic groups are more favored than simple hydro-
phobic groups in this region. Curiously, substitution of an acyclic
alkyl phosphate or phosphonate at the terminal position (26–29)
did not favor selective interaction with the P2Y2 versus P2Y4 recep-
tors, while an aryl phosphate ester (30) substitution resulted in
high selectivity for the P2Y2 receptor (Fig. 2).
HN
HN
O
O
a
R
O
HO
O
O
HO OH
HO OH
Uridine
7, R = COCH2CO2H
8, R =COCH2PO3H
9, R = PO2CH2PO3H
10, R = PO2CF2PO3H
Scheme 1. Synthesis of UDP analogues containing a methylene-bridged substitute
for the diphosphate group. Reagent and condition: (a) DCC, ROH, DMF, rt.
O
N
O
N
HN
HN
S
S
a
O
P
OH
O
P
X
O
P
O
P
O
N
O
HO
O
O
O
O
OHX OH OH
HO OH
HO OH
15, X = F
16, X = Cl
45
Scheme 2. Synthesis of UTP analogues containing a b,c-dihalomethylene-bridge in
the triphosphate group. Reagent and condition: (a) PO(OH)2CX2PO(OH)2, DMF, rt.
X2
X2
HN
HN
X1
N
X1
N
a or b or c
O
P
O
P
HO
R3
HO R1
HO
O
O
O
O
OH OH
R2
R
R2
3
n
HO R1
46, X1 = O, X2 = O, R1 = NHCONH2, R2 = H, R3 = H
47, X1 = S, X2 = S, R1 = OH, R2 = H, R3 = H
48, X1 = S, X2 = O, R1 = H, R2 = OH, R3 = H
49, X1 = O, X2 = O, R1 = OH, R2 = CH3, R3 = H
50, X1 = O, X2 = O, R1 = OH, R2 = H, R3 = CH3
51, X1 = O, X2 = S, R1 = OH, R2 = H, R3 = H
52, X1 = S, X2 = O, R1 = OH, R2 = H, R3 = H
12, X1 = O, X2 = O, R1 = NHCONH2, R2 = H, R3 = H, n =1
17, X1 = S, X2 = SCH3, R1 = OH, R2 = H, R3 = H, n = 2
18, X1 = O, X2 = O, R1 = NHCONH2, R2 = H, R3 = H, n = 2
19, X1 = S, X2 = O, R1 = H, R2 = OH, R3 = H, n = 2
20, X1 = O, X2 = O, R1 = OH, R2 = CH3, R3 = H, n = 2
21, X1 = O, X2 = O, R1 = OH, R2 = H, R3 = CH3, n = 2
23, X1 = S, X2 = O, R1 = OH, R2 = H, R3 = H, n = 3
24, X1 = O, X2 = S, R1 = OH, R2 = H, R3 = H, n = 3
25, X1 = S, X2 = O, R1 = OH, R2 = H, R3 = H, n = 4
Scheme 3. Synthesis of UTP analogues with modified uracil and ribose moieties. Reagents and conditions: (a) i—POCl3, proton sponge, PO(OMe)3, 0 °C; ii—(Bu3NH)2H2P2 O7,
Bu3N, DMF, 0 °C; iii—TEAB 0.2 M rt; (b) i—POCl3, PO(OMe)3, 4 h, 0 °C; ii—concd NH4OH; iii—Bu3N, CDI, DMF, 48; iv—(Bu3NH)2H2P2 O7; TEAB 1 M for 19; (c) i—POCl3, PO(OMe)3,
49 or 50, 0 °C; ii—concd NH4OH; iii—CDI, DMF, rt; iv—(Bu3NH)2H2P2 O7, DMF, rt for 20 or 21. Note that the UDP analogue 6 (not shown in scheme) was prepared by a similar
method to (a), except for use of a phosphoric acid salt in the second step: (ii) (Bu3NH)2H2PO4, Bu3N, DMF, 0 °C.
O
N
O
N
O
N
O
N
I
HN
HN
NH
HN
O
O
O
O
a
b
O
O
O
O
O
HO P
O
HO P
O
O P O P O P
O
O
O
O
O
OH
OH
OH
OH OH
54
39
HO OH
O O
HO OH
O O
53
Scheme 4. Synthesis of a 5-iodo analogue of INS48823. Reagents and conditions: (a) phenylacetaldehyde dimethylacetal, TFA, rt; (b) 5-iodo-uridine-50-diphosphoimidazol-
idate, DMF, rt.

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6325
O
O
N
HN
X1
HN
X1
N
O
P
O
P
O
P
O
P
O
P
O
P
O
a
HO
X2
O
O
R
O
X2
O P O
O
O
OH OH OH
OH OH
OH OH
HO OH
HO OH
1, X1 = O, X2 = O,
26, X1 = O, X2 = O, R = OCH3
14, X1 = S, X2 = O,
55, X1 = O, X2 = CCl2
56, X1 = O, X2 = CF2
27, X1 = O, X2 = O, R = CH3
28, X1 = O, X2 = O, R = O(CH2)2CN
29, X1 = O, X2 = O, R = OCH2CHOHCH2OH
30, X1 = O, X2 = O, R = OC6H5
31, X1 = O, X2 = O, R = OC6H11
32, X1 = O, X2 = O, R = [5]ribose
33, X1 = O, X2 = O, R = [6]fructose
34, X1 = O, X2 = O, R = [1]glucose
35, X1 = O, X2 = O, R = [1]galactose
36, X1 = O, X2 = O, R = [6]mannose
37, X1 = O, X2 = O, R = [6]2'-deoxyglucose
41, X1 = O, X2 = CF2, R = uridine
42, X1 = O, X2 = CCl2, R = uridine
43, X1 = S, X2 = O, R = 2'-deoxy-cytidine
Scheme 5. Synthesis of 50-tetraphosphate analogues, including Up4-sugars and dinucleotides. The 20-deoxyguanosine derivative 44 was prepared in the same manner as 43.
Reagents and conditions: (a) i—DCC, DMF, rt; ii—RPO(OH)2, DMF, rt.
Among tetraphosphate sugar derivatives, Up4-[1] glucose 34
was the most potent with an EC50 of 0.3 lM (Fig. 3). The P2Y2
receptor selectivity of 34 in comparison to P2Y4 and P2Y6 receptors
was 7- and 26-fold, respectively. Inversion of the chirality of the 40-
hydroxyl group in the sugar, that is, galactose rather than glucose,
in 35 reduced potency at the P2Y2 receptor (16-fold), but not at the
selectivity for the P2Y4 receptor subtype over the other uridine
nucleotide activated receptors.
In conclusion, we have synthesized novel uracil nucleotide
derivatives that are directed toward activation of the P2Y2 or
P2Y6 receptor. Key compounds reported in this study include 34,
Up4-[1]glucose, which displayed submicromolar potency at the
P2Y2 receptor; 16 and 41, dihalomethylene phosphonate analogues
which were selective P2Y2 receptor agonists; and 43, the 2-thio
analogue of INS37217, which was a potent and selective P2Y2
receptor agonist. Thus, the 2-thio modification, found previously
to enhance P2Y2 receptor potency, could be combined with other
favorable modifications to produce novel molecules that exhibit
high potencies and receptor selectivities. Phosphonomethylene
bridges introduced for stability in analogues of UDP, UTP, and ura-
cil dinucleotides markedly reduced potency. Truncation of dinucle-
otide agonists of the P2Y2 receptor, in the form of Up4-sugars,
indicated that a terminal uracil ring is not essential for moderate
potency at this receptor and that specific SAR patterns are ob-
served at this distal end of the molecule.
6
P2Y4 receptor. The mannose adduct 36 of Up4 was essentially
inactive at the P2Y2 receptor. Removal of a single hydroxyl group
of 36 to form 37 increased the potency at both P2Y2 and P2Y4
receptors. These results are consistent with our recent P2Y2 recep-
tor modeling study, which proposes specific interactions in the re-
gion of the distally-binding uridine moiety of Up4U.7
The SAR of dinucleotides at the P2Y2 and P2Y6 receptors also was
further explored (Table 3). An analogue of a known P2Y6 receptor
agonist,9 Up3U 38, was designed to combine multiple reported
favorable modifications of uracil nucleotides directed toward acti-
vation of the P2Y6 receptor based on the agonist INS48823 6.13
The resulting hybrid compound 39, containing 5-iodo substitution
and a 20,30-phenylmethylacetyl group on opposite uridine moieties
of Up3U, was a nonselective agonist of the P2Y6 receptor.
Several new dinucleoside tetraphosphate derivatives 41–44
were synthesized, and their activities were compared to previously
studied compounds 3, 4.9,12 Compound 40 was reported by Shaver
et al.9 The inclusion of a b,c-difluoromethylene 41 or dichloro-
methylene 42 bridge in the tetraphosphate moiety resulted in ana-
logues that were at least an order of magnitude weaker at the P2Y2
receptor than Up4U 3. The difluoro analogue 41, which exhibited
an EC50 of ꢀ2 lM at the P2Y2 receptor, apparently provides a better
mimic of the phosphate ester group than does the dichloro substi-
tution in 42. These results are consistent with electronic effects of
the difluoromethylene group noted for other phosphonates.32 The
P2Y2 receptor agonist INS37217 4 also was prepared for compari-
son.12 Substitution of a 2-thio group in 43 resulted in enhanced po-
tency (EC50 ꢀ80 nM) and selectivity for the P2Y2 receptor (9-fold in
comparison to P2Y4). A 20-deoxyguanosine analogue 44 also exhib-
ited moderate potency at the P2Y2 receptor, as reported.12
3. Experimental
3.1. Chemical synthesis
1H NMR spectra were obtained with a Varian Gemini 300 or
Varian Mercury 400 spectrometers 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 rt (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 due to high dilution.
Purity of compounds was checked using a Hewlett–Packard
1100 HPLC equipped with a Zorbax Eclipse 5 lm XDB-C18 analyt-
ical column (250 ꢁ 4.6 mm; Agilent Technologies Inc., Palo Alto,
CA). Mobile phase: linear gradient solvent system: 5 mM TBAP
(tetrabutylammonium dihydrogenphosphate)-CH3CN from 80:20
to 40:60 in 20 min; the flow rate was 1 mL/min. Peaks were de-
tected by UV absorption with a diode array detector at 254, 275,
Many of the analogues synthesized and studied including Up4-
sugars retained moderate potency at the P2Y4 receptor. However,
our work to date has not revealed molecules that exhibit notable

6326
H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
O
O
N
A
B
HN
HN
O
N
a
O
HO
HO
O
O
HO NHCONH2
HO NH2
57
46
O
N
S
N
S
N
HN
HN
HN
OAc
O
S
S
a
c
S
b
AcO
AcO
HO
AcO
O
O
O
AcO OAc
58
AcO OAc
HO OH
AcO OAc
59
60
47
O
O
N
C
HN
HN
S
N
a
O
HO
O
HO
O
OH
HO
HO
48
61
O
N
OBz
D
BzO
O
O
HN
Me
HN
O
BzO OBz
R'''O
R'
O
N
H
O
62
R
a
or
R''O OR''
BzO
Me
O
OAc
64 R = CH3, R' = H, R'',R''' = Bz
65 R = H, R' = CH3, R'' = Ac, R''' = Bz
49 R = CH3, R',R'',R''' = H
b
b
AcO OAc
50 R = H, R' = CH3, R'',R''' = H
63
Scheme 6. Synthesis of nucleoside intermediates. Reagents and conditions: A, benzotriazole-1-carboxamide, DMF; B, (a) silylated 2-thiouracil, SnCl4, C2H4Cl2; (b) Lawesson’s
reagent, toluene, 80 °C, overnight; (c) NaOMe, MeOH, reflux, 4 h; C, (a) H2S, Et3N, DMF, 200 psi, 2 days; D, (a) i—1,2-dichloroethane, hexamethyldisilazane, trimethylsilyl-
chloride, 80 °C, 4 h; ii—SnCl4, rt, 4 h; (b) NH3/CH3OH, rt, overnight.
and 280 nm. All derivatives tested for biological activity showed
>99% purity by HPLC analysis (detection at 254 nm).
uridine 61 was obtained from Wako Chemicals. 5-Amino-UDP was
purchased from ALT, Inc. (Lexington, KY). Other reagents and sol-
vents were purchased from Sigma–Aldrich (St. Louis, MO).
High-resolution mass measurements were performed on Micro-
mass/Waters LCT Premier Electrospray Time of Flight (TOF) mass
spectrometer coupled with a Waters HPLC system, unless 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 bicarbon-
ate as the mobile phase. Compounds 16, 20, 21, 39, and 54 were
purified by Sephadex alone (and isolated in the ammonium salt
form), and all other compounds were additionally purified by
HPLC with a Luna 5 l RP-C18(2) semipreparative column (250 ꢁ
10.0 mm; Phenomenex, Torrance, CA) and using the following con-
ditions: flow rate of 2 mL/min; 10 mM triethylammonium acetate
(TEAA)-CH3CN from 100:0 to 95:5 (or up to 99:1 to 92:8) in 30 min
(and isolated in the triethylammonium salt form).
3.1.1. General procedure for the preparation of nucleoside
triphosphates (e.g., 14 and 17), tetraphosphates (22–24),
pentaphosphate (25). Procedure A
To a solution of nucleoside (0.054 mmol) and Proton Sponge
(23 mg, 0.11 mmol) in trimethyl phosphate (1 mL) was added
phosphorous oxychloride (0.01 mL, 0.11 mmol) at 0 °C. The reac-
tion mixture was stirred for 2 h at 0 °C and tributylamine
(0.03 mL, 0.12 mmol) was added. Tributylammonium pyrophos-
phate (1.6 moles C12H27N per mole 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 (2 mL)
was added, and the clear solution was stirred at rt for 1 h. The lat-
ter was lyophilized overnight, and the resulting residue was puri-
fied by ion-exchange column chromatography using a Sephadex-
All reagents were of analytical grade. 20-Amino-20-deoxyuridine
was from Metkinen Chemistry (Kuusisto, Finland). 2,20-O-Anhydro-

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6327
Figure 1. Activity of compounds 2 (the native agonist, UDP) and 9 (a,b-methylene-
UDP) at the P2Y6 receptor as indicated by activation of PLC in stably transfected
astrocytoma cells.
Figure 3. Activity of compounds 34–37 (uridine 50-tetraphosphate d-sugars) at the
P2Y2 receptor as indicated by activation of PLC in stably transfected astrocytoma
cells.
the reaction mixture at rt for 1 h, a solution of the corresponding
monophosphate tributylammonium salt (0.109 mmol) in DMF
(1 mL) was added. The reaction mixture was stirred at rt for
48 h. After removal of the solvent, the residue was purified by
ion-exchange column chromatography with a Sephadex-DEAE A-
25 resin, followed by a semipreparative HPLC purification as de-
scribed above. Free nucleoside 50-tetra- and pentaphosphates
were not stable upon prolonged storage at 4 °C, but were stable
at ꢂ20 °C for at least 3 weeks. Dinucleoside tetraphosphates were
stable for several months at ꢂ20 °C and later showed signs of
gradual decomposition.
3.1.3. General procedure for the preparation of compounds (7–
10). Procedure C
To a solution of uridine (25 mg, 0.1 mmol) and DCC (62 mg,
0.3 mmol) in DMF (1.5 mL) was added the appropriate carboxylic
acid or phosphonic acid (0.15 mmol): malonic acid for 7, phospho-
noacetic acid for 8, methylene diphosphonic acid for 9, difluorom-
ethylene diphosphonic acid for 10. After stirring the reaction
mixture at rt for 24–48 h, the solvent was removed. The residue
was purified by ion-exchange column chromatography with a
Sephadex-DEAE A-25 resin, followed by a semipreparative HPLC
purification as described above.
Figure 2. Activity of compound 30 (uridine 50-tetraphosphate d-phenyl ester) at
P2Y2, P2Y4, and P2Y6 receptors as indicated by activation of PLC in stably transfe-
cted astrocytoma cells. The effect of UTP corresponds to 100%.
DEAE A-25 resin with a linear gradient (0.01–0.5 M) of 0.5 M
ammonium bicarbonate as the mobile phase to obtain the corre-
sponding nucleotides as the ammonium salts. The collected por-
tions were purified by HPLC again as described above.
3.1.4. Uridine-50-phosphonoacetate triethylammonium salt (8).
Procedure C
Compound 8 (13.4 mg, 36%) was obtained as a white solid from
uridine (25 mg, 0.1 mmol). 1H NMR (D2O) d 7.81 (d, J = 8.1 Hz, 1H),
5.94 (d, J = 8.1 Hz, 1H), 5.91 (d, J = 4.5 Hz, 1H), 4.41 (m, 3H), 4.33
(m, 2H), 2.89 (d, J = 20.4 Hz, 2H); 31P NMR (D2O) d 12.23; 13C
NMR (D2O) d (169.27, 169.19), 164.92, 150.34, 140.53, 101.18,
88.21, 80.14, 72.06, 68.06, 62.87, (36.28, 34.73); HRMS-EI found
367.0658 (MꢂH)ꢂ. C11H16N2O10P requires 367.0543; purity >99%
by HPLC (retention time: 5.9 min).
3.1.2. General procedure for the preparation of nucleosides
tetraphosphate (26–31), nucleoside tetraphosphate sugars (32–
37), and dinucleoside tetraphosphates (40–44). Procedure B
Uridine triphosphate trisodium salt (15 mg, 0.027 mmol or UTP
analogues for compounds 40–44) and the corresponding mono-
phosphate (0.109 mmol) were converted to the tributylammoni-
um salts by treatment with ion-exchange resin (DOWEX
50WX2-200 (H)) and tributylamine. After removal of the water,
the obtained tributylammonium salts were dried under high-vac-
uum overnight. To a solution of uridine triphosphate tributylam-
monium salt (0.027 mmol) in DMF (2 mL) was added N,N0-
dicyclohexylcarbodiimide (DCC, 14 mg, 0.07 mmol). After stirring
3.1.5. Uridine-50-a,b-methylene-diphosphate triethyl-
ammonium salt (9). Procedure C
Compound 9 (14 mg, 33%) was obtained as a white solid from
uridine (25 mg, 0.1 mmol). 1H NMR (D2O) d 8.03 (d, J = 8.1 Hz,

6328
H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
1H), 5.98 (m, 2H), 4.41 (m, 2H), 4.28 (m, 1H), 4.19 (m, 2H), 2.18 (t,
J = 19.5 Hz, 2H); 31P NMR (D2O) d 19.37 (m), 14.44 (m); HRMS-EI
found 403.0245 (M+H)ꢂ. C10H17N2 O11P2 requires 403.0308; purity
>99% by HPLC (retention time: 12.6 min).
3.1.10. 2-Thio-4-methylthio-uridine-50-triphosphate
triethylammonium salt (17). Procedure A
Compound 17 (3.2 mg, 6.4%) was obtained as a white solid from
2,4-dithio-uridine, 47 (15 mg, 0.054 mmol). 1H NMR (D2O) d 8.51
(d, J = 7.5 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 6.53 (br s, 1H), 4.45 (m,
2H), 4.38 (m, 3H), 2.60 (s, 3H); 31P NMR (D2O) d ꢂ11.00 (d,
J = 19.2 Hz), ꢂ21.02 (m); HRMS-EI found 528.9315 (MꢂH)ꢂ.
3.1.6. Uridine-50-a,b-difluoromethylenediphosphate triethyl-
ammonium salt (10). Procedure C
Compound 10 (14 mg, 33%) was obtained as a white solid from
uridine (25 mg, 0.1 mmol). 1H NMR (D2O) d 8.02 (d, J = 8.1 Hz, 1H),
5.98 (m, 2H), 4.40 (m, 2H), 4.26 (m, 1H), 4.19 (m, 2H); 31P NMR
(D2O) d 4.69 (m), 3.48 (m); HRMS-EI found 436.9977 (MꢂH)ꢂ.
C10H16N2O13 P3S2 requires 528.9307; purity >99% by HPLC (reten-
tion time: 17.1 min).
3.1.11. 20-Deoxy-20-ureido uridine-50-triphosphate
triethylammonium salt (18). Procedure A
C10H13N2O11 F2P2 requires 436.9963; purity >99% by HPLC (reten-
tion time: 14.1 min).
A solution of the compound 46 (38 mg, 0.13 mmol) and Pro-
ton Sponge (43 mg, 0.20 mmol) in trimethyl phosphate (1 mL)
was stirred for 10 min at 0 °C. Phosphorous oxychloride (25 lL,
0.27 mmol) was then added dropwise, and the reaction mixture
was stirred for 2 h at 0 °C. A solution of tributylammonium pyro-
phosphate (377 mg, 0.80 mmol) and tributylamine (0.13 mL,
0.53 mmol) in DMF (1 mL) was added and stirring was continued
at 0 °C for additional 10 min. Triethylammonium bicarbonate
solution (TEAB, 3 mL of 0.2 M) was added, and the reaction mix-
ture was stirred at rt for 30 min. The latter was lyophilized over-
night. The residue was purified by Sephadex-DEAE A-25 resin
ion-exchange column chromatography, followed by semiprepar-
ative HPLC as described above to obtain 18 (9 mg, 7%) as a white
solid. 1H NMR (D2O) d 7.95 (d, J = 8.4 Hz, 1H), 6.05 (d, J = 7.8 Hz,
1H), 6.01 (d, J = 8.4 Hz, 1H), 4.51 (m, 1H), 4.35 (m, 2H), 4.25 (m,
2H); 31P NMR (D2O) d ꢂ10.15, ꢂ12.15 (d, J = 19.5 Hz), ꢂ23.6;
HRMS-EI found 524.9812 (MꢂH)ꢂ. C10H16N4O15 P3 requires
524.9825; purity >99% by HPLC (retention time: 16.6 min).
3.1.7. 20-Deoxy-20-ureido-uridine-50-diphosphate triethyl-
ammonium salt (12)
A solution of the compound 46 (38 mg, 0.13 mmol) and Proton
Sponge (43 mg, 0.20 mmol) in trimethyl phosphate (1 mL) was
stirred for 10 min at 0 °C. Then phosphorous oxychloride (25 lL,
0.27 mmol) was added dropwise, and the reaction mixture was
stirred for 2 h at 0 °C. A mixture of tributylamine (0.25 mL,
1.05 mmol) and a solution 0.35 M of bis(tributylammonium) salt
of phosphoric acid in DMF (2.28 mL) was added at once. This salt
was prepared by mixing tributylamine (0.4 mL, 1.65 mmol) and
phosphoric acid (85 mg, 0.87 mmol) in DMF (2.5 mL). After
10 min, 0.2 M triethylammonium bicarbonate (TEAB) solution
(3 mL) was added, and the clear solution was stirred at rt for
30 min. The latter was lyophilized overnight. The residue
was purified by Sephadex-DEAE A-25 resin ion-exchange column
chromatography, followed by semipreparative HPLC as described
above to obtain 12 (8 mg, 8%) as a white solid. 1H NMR (D2O) d
7.98 (d, J = 8.1 Hz, 1 H), 6.02 (m, 2H), 4.51 (m, 1H), 4.34 (m,
3.1.12. 1-(b-D
-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one 50-
triphosphate triethyl ammonium salt (19)
2H), 4.20 (m, 2H); 31P NMR (D2O)
d
ꢂ7.24, ꢂ10.14 (d,
J = 22.0 Hz); HRMS-EI found 445.0172 (MꢂH)ꢂ. C10H15N4O12 P2
requires 445.0162; purity >99% by HPLC (retention time:
12.7 min).
Solution of 48 (150 mg, 0.58 mmol) in trimethyl phosphate
(5.8 mL) was cooled to 0 °C, POCl3 (342 lL, 3.8 mmol) was added
dropwise and the mixture was stirred for 4 h at 0 °C and for
30 min at rt. The mixture was poured into ice-water (10 mL), neu-
tralized with concentrated NH4OH, and evaporated to dryness. The
resulting residue was purified by column chromatography
(i-PrOH:NH4OH:H2O 60:30:5). After lyophilization of the collected
pure fractions, the 50-monophosphate of 48 was obtained as a
white solid (124 mg, 60%). 1H NMR (D2O) d 7.97 (d, J = 8.2 Hz,
1H), 6.78 (d, J = 5.0 Hz, 1H), 6.07 (d, J = 8.2 Hz, 1H), 4.48 (app t,
J = 4.8 Hz, 1H), 4.12 (app t, J = 4.8 Hz, 1H), 3.90–4.01 (m, 3H); 31P
NMR (D2O) d 3.42; HRMS-EI found 363.0271 [M+Na]+. C9H12N2O8
P1S1Na requires 363.0281. To a solution of 50-monophosphate of
48 (32 mg, 0.088 mmol) and tributylamine (21 lL, 0.088 mmol)
in DMF (3.2 mL) was added CDI (71 mg, 0.44 mmol). After being
stirred for 3 h at rt, the reaction mixture was quenched by addition
of methanol (14 lL). Bis(tri-n-butylammonium)pyrophosphate
(228 mg, 0.51 mmol) was added, and the mixture was stirred and
subsequently concentrated under reduced pressure. The resulting
residue was stirred in 1 M triethylammonium bicarbonate (TEAB)
buffer (6 mL, pH 7.4) for 30 min, lyophilized and purified on a
3.1.8. 2-Thio-uridine-50-b,c-difluoromethylene-triphosphate
triethylammonium salt (15)
To a solution of 2-thio-uridine 50-monophosphate morpholidate
4-morpholine-N,N dicyclohexylcarboxamidine salt, 4533 (10 mg,
0.014 mmol) in DMF (2 mL), difluoromethylene diphosphonate
tributylammonium salt (20 mg, 0.021 mmol) was added. After
being stirred for 3 days at rt, the reaction mixture was evaporated
to remove the solvent and purified by Sephadex-DEAE A-25 resin
followed by HPLC purification to give 15(4 mg, 31%). 1H NMR
(D2O) d 8.18 (d, J = 8.1 Hz, 1H), 6.69 (d, J = 3.0 Hz, 1H), 6.28 (d,
J = 8.1 Hz, 1H), 4.31–4.47 (m, 5H); 31P NMR (D2O) d 3.47, ꢂ3.80
(m), ꢂ10.69 (d, J = 31.2 Hz); HRMS-EI found 532.9375 (MꢂH)ꢂ.
C10H14N2O13 F2P3S requires 532.9398; purity >99% by HPLC (reten-
tion time: 18.1 min).
3.1.9. 2-Thio-uridine-50-b,c-dichloromethylene-triphosphate
ammonium salt (16)
To a solution of 2-thio-uridine 50-monophosphate morpholidate
4-morpholine-N,N dicyclohexylcarboxamidine salt, 4533 (7 mg,
0.01 mmol) in DMF (2 mL), dichloromethylene diphosphonate trib-
utylammonium salt (25 mg, 0.025 mmol) was added. After being
stirred for 3 days at rt, the reaction mixture was evaporated to re-
move the solvent and purified by Sephadex-DEAE A-25 resin to
give 16 (5 mg, 80%). 1H NMR (D2O) d 8.20 (d, J = 8.1 Hz, 1H), 6.69
(d, J = 2.7 Hz, 1H), 6.29 (d, J = 8.4 Hz, 1H), 4.45 (m, 2H), 4.39 (m,
2H), 4.34 (m, 1H); 31P NMR (D2O) d 8.08 (d, J = 17.7 Hz), 1.17 (dd,
J = 17.7, 31.2 Hz), ꢂ10.60 (d, J = 31.2 Hz); HRMS-EI found
564.8817 (MꢂH)ꢂ. C10H14N2O13 Cl2P3S requires 564.8807; purity
>99% by HPLC (retention time: 19.5 min).
preparative HPLC apparatus equipped with a source 15 Q
column (100% water ? 100% 1 M TEAB/water in 45 min) to yield
10 lmol (11%) of compound 19 after lyophilizing the appropriate
fractions. 1H NMR (D2O) d 7.90 (d, J = 8.1 Hz, 1H), 6.80 (d,
J = 5.2 Hz, 1H), 6.08 (d, J = 8.2 Hz, 1H), 4.48 (app t, J = 5.0 Hz, 1H),
4.16 (m, 3H), 4.03 (m, 1H); 31P NMR (D2O)
d
ꢂ9.54 (d,
J = 19.6 Hz), ꢂ10.35 (d, J = 19.6 Hz), ꢂ22.19 (t, J = 19.6 Hz), HRMS-
EI found 498.9099 [MꢂH]ꢂ. C9H14N2O14 P3S requires 498.9384.
Structural assignment was confirmed with COSY. All signals as-
signed to hydroxyl groups were exchangeable with D2O. Exact
mass measurements were performed on a quadrupole/orthogo-
nal-acceleration time-of-flight (Q/oaTOF) tandem mass spectrome-

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6329
ter (qToF 2, Micromass, Manchester, UK) equipped with a standard
electrospray ionization (ESI) interface. Samples were infused in a i-
PrOH/water (1:1) mixture at 3 lL/min.
4.43 (m, 2H), 4.32 (m, 3H); 31P NMR (D2O)
d
ꢂ11.22 (d,
J = 18.3 Hz), ꢂ22.78 (m); HRMS-EI found 658.8782 (MꢂH)ꢂ.
C9H16N2O20 P5S requires 658.8705; purity >99% by HPLC (retention
time: 19.9 min).
3.1.13. General procedure for the preparation of the nucleoside
50-triphosphates 20-MeUTP (20) and 30-MeUTP (21)
3.1.19. Uridine-50-methyl-tetraphosphate triethylammonium
salt (26). Procedure B
To a solution of 20-17 or 30-C-methyl-UMP18 (0.15 mmol) dis-
solved in dry DMF (1.5 mL) was added tri-n-butylamine
(0.15 mmol) and the solution was stirred for 20 min at rt. After
evaporation under anhydrous condition, the residue was sus-
pended in 1.4 mL of dry DMF and CDI (122 mg, 0.75 mmol)
was added and the mixture was stirred for 6 h at rt. Methanol
(49 ll, 1.2 mmol) was added and the mixture was stirred for
30 min. Then 6 mL (3 mmol) of a 0.5 M solution of bis(tri-n-
butylammonium) pyrophosphate in dry DMF was added. The
mixture was stirred for 24 h at rt, and the solvent was removed
under high vacuum at rt. The mixture dissolved in water was
purified by Sephadex DEAE A-25 resin ion exchange column
chromatography with a linear gradient (0.01–0.5 M) of 0.5 M
ammonium bicarbonate. Compounds 20 and 21 were isolated
as ammonium salts (yield 32 and 34%, respectively). Mass spec-
troscopy was carried out on an HP 1100 series instrument in the
negative ion mode using atmospheric pressure electrospray ion-
ization (API-ESI).
Compound 26 (7.8 mg, 33%) was obtained as a white solid using
uridine triphosphate (20 mg, 0.036 mmol) and methylphosphate
(45 mg, 0.15 mmol). 1H NMR (D2O) d 8.00 (d, J = 8.1 Hz, 1H), 6.03
(d, J = 5.7 Hz, 1H), 6.00 (d, J = 8.1 Hz, 1H), 4.44 (m, 2H), 4.31 (m,
1H), 4.26 (m, 2H), 3.69 (d,J = 11.7 Hz, 3H); 31P NMR (D2O) d
ꢂ8.70 (d, J = 17.7 Hz), ꢂ10.59 (d, J = 18.9 Hz), ꢂ22.46; HRMS-EI
found 576.9375 (MꢂH)ꢂ. C10H17N2O18P4 requires 576.9427; purity
>99% by HPLC (retention time: 18.8 min).
3.1.20. Uridine-50-methyl(C-P)-tetraphosphate
triethylammonium salt (27). Procedure B
Compound 27 (8.6 mg, 34%) was obtained as a white solid using
uridine triphosphate (22 mg, 0.04 mmol) and methylphosphoric
acid (22 mg, 0.23 mmol). 1H NMR (D2O) d 7.98 (d, J = 8.4 Hz, 1H),
6.02 (d, J = 5.4 Hz, 1H), 5.99 (d, J = 8.4 Hz, 1H), 4.43 (m, 2H), 4.28
(m, 1H), 4.25 (m, 2H), 1.49 (d, J = 17.1 Hz, 3H); 31P NMR (D2O) d
19.05, ꢂ10.57 (d, J = 18.3 Hz), ꢂ22.34 (d, J = 18.9 Hz); HRMS-EI
found 560.9476 (MꢂH)ꢂ. C10H17N2O17 P4 requires 560.9478; purity
>99% by HPLC (retention time: 19.2 min).
3.1.14. 20-C-Methyl-uridine-50-triphosphate ammonium salt
(20)
1H NMR (D2O) d 7.76 (d, J = 8.1 Hz, 1H), 5.86 (s, 1H), 5.75 (d,
J = 8.1 Hz, 1H), 3.85–3.90 (m, 2H), 3.75 (d, J = 9.0 Hz, 1H), 3.68
(dd, J = 4.3, 13.7 Hz, 1H), 1.05 (s, 3H); 31P NMR (D2O) d ꢂ4.75 (br
s), ꢂ19.34 (br s), ꢂ20.59 (m); MS m/z 497.10 [MꢂH]ꢂ.
3.1.21. Uridine-50-(2-cyanoethyl)-tetraphosphate
triethylammonium salt (28). Procedure B
Compound 28 (2.5 mg, 13%) was obtained as a white solid using
uridine triphosphate (15 mg, 0.027 mmol) and 2-cyanoethyl phos-
phate (35 mg, 0.11 mmol). 1H NMR (D2O) d 7.98 (d, J = 8.1 Hz, 1H),
5.99 (m, 2H), 4.41 (m, 2H), 4.29 (m, 1H), 4.24 (m, 2H), 4.18 (t,
J = 6.2 Hz, 2H), 2.88 (t, J = 6.2 Hz, 2H); 31P NMR (D2O) d ꢂ10.54
(d, J = 17.7 Hz), ꢂ10.83 (d, J = 18.2 Hz), ꢂ22.39; HRMS-EI found
639.9489 (M+Na–H)ꢂ. C12H19N3O18 P4Na requires 639.9512; purity
>99% by HPLC (retention time: 19.4 min).
3.1.15. 30-C-Methyl-uridine-50-triphosphate ammonium salt
(21)
1H NMR (D2O) d 7.78 (d, J = 8.1 Hz, 1H), 5.84 (d, J = 7.7 Hz, 1H),
5.75 (d, J = 8.1 Hz, 1H), 4.03 (d, J = 7.7 Hz, 1H), 3.94 (dd, J = 3.4, 5.1
Hz, 1H), 3.65 (dd, J = 3.4, 12.8 Hz, 1H), 3.58 (dd, J = 4.9, 12.6,
1H),1.20 (s, 3H); 31P NMR (D2O) d ꢂ4.83 (d, J = 15.9 Hz), ꢂ19.25
(t, J = 15.3 Hz), ꢂ20.66 (t, J = 15.9 Hz); MS m/z 497.10 [MꢂH]ꢂ.
3.1.22. Uridine-50-a-glycerol-tetraphosphate
triethylammonium salt (29). Procedure B
3.1.16. 2-Thio-uridine-50-tetraphosphate triethylammonium
salt, 2-thio-UP4 (23). Procedure A
Compound 29 (2.1 mg, 11%) was obtained as a white solid using
uridine triphosphate (15 mg, 0.027 mmol) and D-L-a-glycerol
Compound 23 (1.5 mg, 4.5%) was obtained as a white solid from
2-thio-uridine (10 mg, 0.038 mmol). 1H NMR (D2O) d 8.17 (d,
J = 8.4 Hz, 1H), 6.73 (d, J = 3.0 Hz, 1H), 6.27 (d, J = 8.1 Hz, 1H),
phosphate (35 mg, 0.11 mmol). 1H NMR (D2O)
d 7.99 (d,
J = 7.8 Hz, 1H), 6.01 (m, 2H), 4.44 (m, 2H), 4.30 (m, 1H), 4.26 (m,
2H), 4.01 (m, 3H), 3.67 (m, 2H); 31P NMR (D2O) d ꢂ9.83 (d,
J = 17.7 Hz), ꢂ10.55 (d, J = 18.3 Hz), ꢂ22.40; HRMS-EI found
636.9638 (MꢂH)ꢂ. C12H21N2O20 P4 requires 636.9638; purity
>99% by HPLC (retention time: 18.9 min).
4.46 (m, 2H), 4.33 (m, 3H); 31P NMR (D2O)
d
ꢂ11.18 (d,
J = 18.9 Hz), ꢂ22.55 (m); HRMS-EI found 578.9042 (MꢂH)ꢂ.
C9H15N2O17 P4S requires 578.9042; purity >99% by HPLC (retention
time: 19.8 min).
3.1.23. Uridine-50-phenyl-tetraphosphate triethylammonium
salt (30). Procedure B
3.1.17. 4-Thio-uridine-50-tetraphosphate triethylammonium
salt (24). Procedure A
Compound 30 (1.1 mg, 6%) was obtained as a white solid
using uridine triphosphate (15 mg, 0.027 mmol) and phenyl
Compound 24 (5.2 mg, 7.6%) was obtained as a white solid from
4-thio-uridine (20 mg, 0.077 mmol). 1H NMR (D2O) d 7.88 (d,
J = 7.8 Hz, 1H), 6.66 (d, J = 7.8 Hz, 1H), 5.95 (d, J = 4.5 Hz, 1H),
4.47 (m, 1H), 4.40 (m, 1H), 4.28 (m, 3H); 31P NMR (D2O) d
ꢂ10.91 (d, J = 20.2 Hz), ꢂ21.97 (m); HRMS-EI found 578.8890
(MꢂH)ꢂ. C9H15N2O17 P4S requires 578.9042; purity >99% by HPLC
(retention time: 19.2 min).
phosphate (28 mg, 0.11 mmol). 1H NMR (D2O)
d 7.92 (d,
J = 7.8 Hz, 1H), 7.37 (t, J = 7.2 Hz, 2H), 7.25 (d, J = 7.8 Hz, 2H),
7.17 (t, J = 7.5 Hz, 1H), 5.93 (m, 2H), 4.37 (m, 1H), 4.31 (m,
1H), 4.21 (m, 3H); 31P NMR (D2O) d ꢂ10.51 (d, J = 17.7 Hz),
ꢂ14.80 (d, J = 17.1 Hz), ꢂ22.43; HRMS-EI found 638.9577
(MꢂH)ꢂ. C15H19N2O18 P4 requires 638.9583; purity >99% by HPLC
(retention time: 18.6 min).
3.1.18. 2-Thio-uridine-50-pentaphosphate triethylammonium
salt (25). Procedure A
Compound 25 (1.1 mg, 3%) was obtained as a white solid from
2-thio-uridine 52 (10 mg, 0.038 mmol). 1H NMR (D2O) d 8.15 (d,
J = 7.8 Hz, 1H), 6.70 (d, J = 3.6 Hz, 1H), 6.26 (d, J = 8.1 Hz, 1H),
3.1.24. Uridine-50-cyclohexane-tetraphosphate
triethylammonium salt (31). Procedure B
Compound 31 (6.7 mg, 20%) was obtained as a white solid using
uridine triphosphate (20 mg, 0.036 mmol) and cyclohexene mono-

6330
H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
phosphate tributylammonium salt (20 mg, 0.037 mmol). 1H NMR
(D2O) d 8.02 (d,J = 8.1 Hz, 1H), 6.03 (m, 2H), 4.42–4.50 (m, 2H),
4.22–4.34 (m, 4H), 2.01 (m, 2H), 1.74 (m, 2H), 1.33 (m, 6H); 31P
NMR (D2O) d ꢂ11.26 (m), ꢂ23.10 (m); HRMS-EI found 645.0037
(MꢂH)ꢂ. C15H25N2O18 P4 requires 645.0053; purity >99% by HPLC
(retention time: 19.9 min).
3.1.30. (2-Benzyl-1,3-dioxolo-4-yl)uridine 50-monophosphate
ammonium salt (54)
To a solution of uridine 50-monophosphate (100 mg, 0.27 mmol)
in TFA (1 mL) was added phenylacetaldehyde dimethylacetal
(0.3 mL, 1.81 mmol). The reaction mixture was stirred at rt for
4 h. After removal of the solvent, the residue was treated with
1 M NaHCO3 (4 mL) and AcOEt (2 mL). Aqueous phase was sepa-
rated, evaporated, and purified by Sephadex-DEAE A-25 resin as
described above to obtain 54 (100 mg, 86%) as a white solid. 1H
NMR (D2O) d 7.92 (d, J = 8.3 Hz, 1H), 7.36 (m, 5H), 5.90 (d,
J = 8.3 Hz, 1H), 5.63 (m, 1H), 5.47 (m, 1H), 4.95 (m, 1H), 4.90 (m,
1H), 4.42 (m, 1H), 3.89 (m, 2H), 3.16 (m, 2H); 31P NMR (D2O) d
2.71; HRMS-EI found 425.0748 (MꢂH)ꢂ. C17H18N2O9P requires
425.0750; purity >99% by HPLC (retention time: 12.9 min).
3.1.25. Uridine-50-fructose-60-tetraphosphate
triethylammonium salt (33). Procedure B
Compound 33 (4.2 mg, 19%) was obtained as a white solid
using uridine triphosphate (15 mg, 0.027 mmol) and D-fructose-
6-phosphate (51 mg, 0.12 mmol). 1H NMR (D2O)
d 7.98 (d,
J = 8.3 Hz, 1H), 6.02 (d, J = 5.4 Hz, 1H), 5.99 (d, J = 8.3 Hz, 1H),
4.43 (m, 2H), 4.32–4.06 (m, 7H), 3.95 (m, 1H), 3.63 (m, 1H),
3.56 (m, 1H); 31P NMR (D2O) d ꢂ10.08 (d, J = 15.3 Hz), ꢂ10.54
(d, J = 18.3 Hz), ꢂ22.27; HRMS-EI found 724.9796 (MꢂH)ꢂ.
C15H25N2O23 P4 requires 724.9799; purity >99% by HPLC (reten-
tion time: 19.5 min).
3.1.31. P1-((2-benzyl-1,3-dioxolo-4-yl)uridine 50)P3-(5-iodouridine
50) triphosphate ammonium salt (39)
To a solution of 5-iodouridine 50-diphosphate[5] (10 mg, 0.017
mmol) in DMF (1 mL) was added CDI (7 mg, 0.04 mmol). The reac-
tion mixture was stirred at rt for 6 h. Methanol (1 mL) was then
added, and stirring was continued at rt for an additional 1 h. After
removal of the solvent, the residue was dried in high vacuum over-
night, was dissolved in DMF (2 mL), and was added compound 54
(11 mg, 0.025 mmol). The reaction mixture was stirred at rt over-
night. After removal of the solvent, the residue was purified by
Sephadex-DEAE A-25 resin as described above to obtain 39
(2.5 mg, 15%) as a white solid. 1H NMR (D2O) d 8.17 (s, 1H), 7.77
(d, J = 8.1 Hz, 1H), 7.35 (m, 5H), 5.88 (m, 2H), 5.53 (m, 1H), 5.44
(m, 1H), 4.92 (m, 2H), 4.45 (m, 1H), 4.31 (m, 2H), 4.19 (m, 5H),
3.13 (m, 2H); 31P NMR (D2O) d ꢂ10.68 (d, J = 17.7 Hz), ꢂ10.97 (d,
J = 20.2 Hz), ꢂ22.2; HRMS-EI found 936.9611 (MꢂH)ꢂ.
3.1.26. Uridine-50-glucose-10-tetraphosphate triethylammonium
salt (34). Procedure B
Compound 34 (10 mg, 28%) was obtained as a white solid using
uridine triphosphate (25 mg, 0.045 mmol) and D-glucose-1-phos-
phate (68 mg, 0.18 mmol). 1H NMR (D2O) d 8.01 (d, J = 8.4 Hz,
1H), 6.02 (m, 2H), 5.66 (m, 1H), 4.45 (m, 2H), 4.32 (m, 1H), 4.28
(m, 2H), 4.01–3.77 (m, 4H), 3.55 (m, 1H), 3.46 (m, 1H); 31P NMR
(D2O) d ꢂ10.53 (d, J = 15.9 Hz), ꢂ11.90 (d, J = 16.5 Hz), ꢂ22.27;
HRMS-EI found 724.9800 (MꢂH)ꢂ. C15H25N2O23 P4 requires
724.9799; purity >99% by HPLC (retention time: 18.9 min).
3.1.27. Uridine-50-galactose-10-tetraphosphate triethylammonium
salt (35). Procedure B
C26H29N4O20 P3I requires 936.9633; purity >99% by HPLC (reten-
tion time: 18.5 min).
Compound 35 (4.5 mg, 16%) was obtained as a white solid using
uridine triphosphate (20 mg, 0.036 mmol) and D-galactose-1-
3.1.32. P1,P4-di(uridine 50-)b,c-difluoromethylenetetra-
phosphate triethylammonium salt (41). Procedure B
Compound 41 (1.4 mg, 21%) was obtained as
phosphate (51 mg, 0.12 mmol). 1H NMR (D2O)
d 7.99 (d,
J = 8.3 Hz, 1H), 6.03 (d, J = 5.7 Hz, 1H), 6.00 (d, J = 8.3 Hz, 1H),
5.67 (m, 1H), 4.44 (m, 2H), 4.26 (m, 4H), 4.05 (m, 1H), 3.98 (m,
1H), 3.77 (m, 3H); 31P NMR (D2O) d ꢂ10.54 (d, J = 18.3 Hz),
ꢂ11.74 (d, J = 18.2 Hz), ꢂ22.19; HRMS-EI found 724.9781 (MꢂH)ꢂ.
a
white
solid from uridine-50-b,c-difluoromethylenetriphosphate (3.2 mg,
0.0055 mmol) and uridine-50-monophosphate (3.2 mg, 0.01 mmol).
1H NMR (D2O) d 7.97 (br d, J = 6.1 Hz, 2H), 6.01 (m, 4H), 4.41 (m, 4H),
4.28 (m, 6H); 31P NMR (D 2O) d ꢂ6.28 (m), ꢂ10.96 (m); HRMS-EI
found 822.9910 (MꢂH)ꢂ. C19H25N4O22 F2P4 requires 822.9879; pur-
ity >99% by HPLC (retention time: 19.8 min).
C15H25N2O23 P4 requires 724.9799; purity >99% by HPLC (retention
time: 19.0 min).
3.1.28. Uridine-50-mannose-60-tetraphosphate triethylammonium
salt (36). Procedure B
3.1.33. P1,P4-di(uridine 50-)b,c-dichloromethlyenetetraphosphate
triethylammonium salt (42). Procedure B
Compound 36 (2.4 mg, 11%) was obtained as a white solid using
uridine triphosphate (15 mg, 0.027 mmol) and D-mannose-6-
Compound 42 (2.2 mg, 19%) was obtained as a white solid from
uridine-50-b,c-dichloromethylenetriphosphate (5 mg, 0.009 mmol)
and uridine-50-monophosphate (6.4 mg, 0.019 mmol). 1H NMR
(D2O) d 7.99 (d, J = 8.1 Hz, 2H), 5.98 (m, 4H), 4.45 (m, 2H), 4.40
(m, 2H), 4.29 (m, 6H); 31P NMR (D2O) d ꢂ1.61 (m), ꢂ10.94 (m);
HRMS-EI found 854.9203 (MꢂH)ꢂ. C19H25N4O25 Cl2P4 requires
854.9288; purity >99% by HPLC (retention time: 20.0 min).
phosphate (23 mg, 0.08 mmol). 1H NMR (D2O)
d 7.97 (d,
J = 8.4 Hz, 1H), 5.99 (m, 2H), 5.18 (m, 3/5H), 4.92 (m, 2/5H), 4.43
(m, 2H), 4.24 (m, 5H), 3.95–3.74 (m, 3H), 3.68 (m, 3/5H), 3.52 (m,
2/5H); 31P NMR (D2O) d ꢂ9.92 (m), ꢂ10.46 (m), ꢂ22.20 (m);
HRMS-EI found 724.9814 (MꢂH)ꢂ. C15H25N2O23 P4 requires
724.9799; purity >99% by HPLC (retention time: 18.6 min).
3.1.34. P1-(2-thiouridine 50-)-P4-(20-deoxycytidine 50-)tetra-
phosphate triethylammonium salt (43). Procedure B
3.1.29. Uridine-50-(20-deoxy-glucose)-60-tetraphosphate
triethylammonium salt (37). Procedure B
Compound 37 (4.2 mg, 20%) was obtained as a white solid using
uridine triphosphate (15 mg, 0.027 mmol) and 20-deoxy-D-glu-
cose-6-phosphate (20 mg, 0.08 mmol). 1H NMR (D2O) d 7.99 (d,
J = 7.8 Hz, 1H), 6.01 (m, 2H), 5.38 (m, 1/2H), 4.96 (m, 1/2H), 4.44
(m, 2H), 4.33–4.09 (m, 6H), 3.93 (m, 1/2H), 3.73 (m, 1/2H), 3.51
(m, 1H), 2.23 (m, 1/2H), 2.13 (m, 1/2H), 1.74 (m, 1/2H), 1.53 (m,
1/2H); 31P NMR (D2O) d ꢂ11.60 (d, J = 14.7 Hz), ꢂ12.24 (d,
J = 18.9 Hz), ꢂ23.58, -24.11 (t, J = 12.2 Hz); HRMS-EI found
708.9827 (MꢂH)ꢂ. C15H25N2O22 P4 requires 708.9849; purity
>99% by HPLC (retention time: 18.5 min).
Compound 43 (1.1 mg, 7.1%) was obtained as a white solid from
2-thio-uridine triphosphate tributylammonium salt, (14 mg,
0.013 mmol) and 20-deoxycytidine mono phosphate tributylam-
monium salt (27 mg, 0.055 mmol). 1H NMR (D2O) d 8.16 (d,
J = 8.4 Hz, 1H), 7.97 (d, J = 7.8 Hz, 1H), 6.63 (d, J = 3.0 Hz, 1H),
6.31 (t, J = 6.6 Hz, 1H), 6.26 (d, J = 8.4 Hz, 1H), 6.15 (d, J = 7.2 Hz,
1H), 4.62 (m, 1H), 4.36 (m, 5H), 4.20 (m, 3H), 2.42 (m, 1H), 2.28
(m, 1H); 31P NMR (D2O) d ꢂ11.10 (m), ꢂ22.94 (m); HRMS-EI found
787.9837 (MꢂH)ꢂ. C18H26N5O20 P4S requires 787.9842; purity
>99% by HPLC (retention time: 19.5 min).

H. Ko et al. / Bioorg. Med. Chem. 16 (2008) 6319–6332
6331
3.1.35. 20-Deoxy-20-ureidouridine (46)
(CH2Cl2/MeOH 96:4), yielding compound 48 (1.6 g, 70%). 1H NMR
(DMSO-d6)d 12.59 (br s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 6.73 (d,
J = 3.9 Hz, 1H), 5.94 (d, J = 8.4 Hz, 1H), 5.59 (d, J = 5.4 Hz, 1H),
5.48 (d, J = 3.9 Hz, 1H), 5.04 (t, J = 5.4 Hz, 1H), 4.18 (m, 1H,), 3.91
(m, 1H), 3.84 (m, 1H), 3.62 (app t, J = 5.1 Hz, 2H); HRMS-EI found
261.0540 (M+H)+. C9H13N2O5 S1 requires 261.0545.
Benzotriazole-1-carboxamide20 (20 mg, 0.12 mmol) was added
to a solution of 20-amino-20-deoxyuridine 57 (20 mg, 0.08 mmol)
in DMF (4 mL). The reaction mixture was stirred at rt for 6 h. After
removal of the solvent, the residue was purified by preparative
thin-layer chromatography (CH2Cl2-MeOH, 8:2) to obtain 46
(21 mg, 89%) as a white solid. 1H NMR (CD3OD) d 7.99 (d,
J = 8.3 Hz, 1H), 5.98 (d, J = 8.1 Hz, 1H), 5.71 (d, J = 8.3 Hz, 1H),
4.41 (m, 1H), 4.20 (m, 1H), 4.03 (m, 1H), 3.75 (m, 2H); HRMS-EI
found 285.0859 (MꢂH)+. C10H13N4O6 requires 285.0835.
3.1.40. General procedure for synthesis of 20-C-methyl-uridine
(49) and 30-C-methyl-uridine (50)
To dry uracil (0.45 g, 4 mmol) in dry 1,2-dichloroethane (20 mL)
were added HMDS (0.68 mL, 0.8 equiv) and trimethylsilyl chloride
(TMSCl, 0.3 mL, 0.8 equiv). The reaction mixture was heated at
80 °C for 4 h in the absence of moisture. After cooling to rt, 1,2,
3.1.36. 20,30,50-Tri-O-acetyl-2-thiouridine (59)
A suspension of 2-thiouracil (2 g, 15.6 mmol) and trimethylsilyl
chloride (1.8 mL) in hexamethyldisilazane (80 mL) was treated
with a few crystals of ammonium sulfate and refluxed overnight.
3,5-tetra-O-benzoyl-2-C-methyl-b-
3-tri-O-acetyl-5-O-benzoyl-3-C-methyl-b-
D
-ribofuranose (62)24 or 1,2,
-ribofuranose (63)25b
D
The clear greenish solution was evaporated and a solution of b-
D
-
(1 equiv) in 1,2-dichloroethane (20 mL) was added followed by
SnCl4 (0.93 mL, 2 equiv) dropwise. The mixture was stirred at rt for
4 h and quenchedby NaHCO3 saturated watersolutionand extracted
with CHCl3 (3ꢁ 10 mL). The organic layers were dried (MgSO4), fil-
tered, and concentrated under reduced pressure to give compounds
64 or 65, which were purified by chromatography on a silica gel col-
umn eluting with CHCl3. Compounds 64 and 65 (1 mmol) were trea-
ted with methanol (20 mL) saturated with ammonia at 0 °C stirring
at rt overnight. Evaporation of the solvent gave the desired com-
pounds 49 and 50, which were purified by chromatography on a sil-
ica gel column.
ribofuranose 1,2,3,5-tetraacetate (5.5 g, 17.3 mmol) in 20 mL dry
dichloroethane was added. After a few minutes, stannic chloride
(2.4 mL, 20.8 mmol) was added and after 1 h, the mixture was
poured into a saturated aq NaHCO3 solution under vigorous stir-
ring and then allowed to stand for 1 h. The suspension was filtered
over a silica gel pad, which was washed with CH2Cl2. The organic
layer was separated, dried over MgSO4, and evaporated to dryness.
Silica gel chromatography (CH2Cl2:MeOH 99:1) yielded 4.75 g
(79%) of compound 59. 1H NMR (DMSO-d6) d 10.64 (br s, 1H),
7.49 (d, J = 7.9 Hz, 1H), 6.68 (d, J = 3.8 Hz, 1H), 6.62 (dd, J = 2.3,
7.9 Hz, 1H), 5.45 (dd, J = 3.8, 5.6 Hz, 1H), 5.20 (app t, J = 5.7 Hz,
1H), 4.41–4.47 (m, 2H), 4.31–4.37 (dd, J = 3.2, 13.5 Hz, 1H), 2.09
(s, 6H), 2.06 (s, 3H); HRMS-EI found 387.08604 (M+H)+ꢃC15H19N2O8
S1 requires 387.08620.
3.1.41. 1-(2,3,5-Tri-O-benzoyl-2-C-methyl-b-D-ribofuranosyl)
uracil (64)
The title compound was obtained as a white foam (67% yield).
1H NMR (CDCl3) d 9.35 (br s, 1H), 8.06 (d, J = 7.7 Hz, 4H), 7.86 (d,
J = 7.7 Hz, 2H), 7.4–7.6 (m, 10H), 6.48 (s, 1H), 5.76 (d, J = 5.7 Hz,
1H), 5.66 (d, J = 7.7 Hz, 1H), 4.82 (dd, J = 4.7, 6.2 Hz, 2H), 4.62 (m,
1H), 1.72 (s, 3H).
3.1.37. 20,30,50-Tri-O-acetyl-2,4- dithiouridine (60)
To a solution of 59 (290 mg, 0.75 mmol) in dry toluene (10 mL),
Lawesson’s reagent (304 mg, 0.75 mmol) was added. After heating
the reaction mixture at 80 °C overnight, insoluble materials were
filtered off and the filtrate was purified by silica gel chromatogra-
phy (Hex:EtOAc 65:35), yielding compound 60 as a yellow foam
(250 mg, 83%). 1H NMR (DMSO-d6) d 9.97, (br s, 1H), 7.65 (d,
J = 8.2 Hz, 1H), 6.76 (d, J = 4.1 Hz, 1H), 5.98 (d, J = 8.2 Hz, 1H),
5.37 (dd, J = 4.1, 5.6 Hz, 1H), 5.16 (app t, J = 5.7 Hz, 1H), 4.34–4.40
(m, 2H), 4.25–4.30 (dd, J = 3.5,13.5 Hz, 1H), 2.09 (s, 3H), 2.07 (s,
3H), 2.06 (s, 3H); HRMS-EI found 403.06338 (M+H)+ꢃ C15H19N2O7
S2. requires 403.06335.
3.1.42. 1-(2-C-methyl-b-D-ribofuranosyl)uracil (49)
The title compound was obtained as a white foam after chroma-
tography on a silica gel column eluting with CHCl3/MeOH (86:14)
(80% yield). 1H NMR (DMSO-d6) d 11.35 (br s, 1H), 8.05 (d,
J = 8.1 Hz, 1H), 5.78 (s, 1H), 5.58 (d, J = 8.1 Hz, 1H), 5.15 (br s,
2H), 5.10 (s, 1H), 3.40–3.80 (m, 4H), 1.0 (s, 3H).
3.1.43. 1-(2,3-Di-O-acetyl-5-O-benzoyl-3-C-methyl-b-D-
ribofuranosyl)uracil (65)
3.1.38. 2,4-Dithiouridine (47)
The title compound was obtained as a white foam (68% yield).
1H NMR (CDCl3): d 8.70 (br s, 1H), 8.05 (d, J = 7.3 Hz, 2H), 7.62 (t,
J = 7.7 Hz, 1H), 7.48 (t, J = 7.9 Hz, 2H), 7.42 (d, J = 8.1 Hz, 1H) 6.22
(d, J = 7.7 Hz, 1H), 5.45 (d, J = 8.1 Hz, 1H), 5.40 (d, J = 7.7 Hz, 1H),
4.90 (t, J = 3.4 Hz, 1H), 4.72 (dd, J = 3.3, 12.6 Hz, 1H), 4.52 (dd,
J = 3.8, 12.8, 1H), 2.16 (s, 6H), 1.70 (s, 3H).
Compound 60 (240 mg, 0.60 mmol) was dissolved in dry meth-
anol (15 mL) and the warmed solution was treated with 130 lL of
a solution of NaOMe (30% w/w) in methanol. The reaction was re-
fluxed for 4 h and then treated with diluted acetic acid to pH 5,
evaporated to dryness, and purified on
a silica gel column
(CH2Cl2:MeOH 93:7) to obtain compound 47 as a yellow foam
(150 mg, 91%).1H NMR (DMSO-d6) d 13.80 (br s, 1H), 8.07 (d,
J = 7.7 Hz, 1H), 6.63 (d, J = 7.7 Hz, 1H), 6.40 (d, J = 2.7 Hz, 1H),
5.53 (d, J = 5.3 Hz, 1H), 5.29 (t, J = 5.0 Hz, 1H), 5.11 (d, J = 5.9 Hz,
1H), 4.08 (m, 1H), 3.93 (m, 2H), 3.72–3.78 (m, 1H), 3.58–3.64 (m,
1H); HRMS-EI found 275.01637 [MꢂH]ꢂ. C9H11N2O4 S2 requires
275.01602.
3.1.44. 1-(3-C-methyl-b-D-ribofuranosyl)uracil (50)
The title compound was obtained as a white foam after chroma-
tography eluting with CHCl3/MeOH (88:12) (82% yield). 1H NMR
(DMSO-d6) d 11.3 (br s, 1H), 8.0 (d, J = 8.1 Hz, 1H), 5.85 (d,
J = 8.1 Hz, 1H), 5.65 (d, J = 7.7 Hz, 1H), 5.33 (d, J = 6.6 Hz, 1H),
5.10 (t, J = 4.9 Hz, 1H), 4.72 (s, 1H), 3.85 (dd, J = 6.6, 7.7 Hz, 1H),
3.75 (pseudo t, 1H), 3.55 (m, 2H), 1.20 (s, 3H).
3.1.39. 1-b-D-Arabinofuranosyl)-2-thio(1H)pyrimidin-4-one
(48)
3.1.45. Assay of PLC activity stimulated by P2Y2, P2Y4, and P2Y6
receptors
In a parr apparatus 2,20-O-anhydrouridine (61, 2 g, 8.8 mmol)
was dissolved in dry DMF (40 mL) and triethylamine (6 mL). The
solution was saturated with H2S at ꢂ40 °C and allowed to warm
to rt resulting in a pressure of 200 psi. After stirring for two days,
the remaining H2S was released and the solvent evaporated to dry-
ness. The brown residue was purified on a silica gel column
Stable cell lines expressing the human P2Y2, P2Y4, or P2Y6 recep-
tor in 1321N1 human astrocytoma cells were generated as de-
scribed.3 Agonist-induced [3H]inositol phosphate production was
measured in 1321N1 cells plated to 20,000 cells/well on 96-well
plates two days prior to assay. Sixteen h before the assay, the inositol

6332
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Acknowledgments
Mass spectral measurements were performed by Dr. Noel Whit-
taker and NMR by Wesley White (NIDDK). We thank Dr. Andrei A.
Ivanov (NIDDK) for helpful discussions. This research was sup-
ported in part by research grant GM38213, by the Intramural Re-
search Programs of NIDDK, National Institutes of Health, and by
MIUR (Italian Ministry for the Research and University) (PRIN 2006).
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Supplementary data
NMR spectra and HPLC traces for selected derivatives are avail-
able. Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.bmc.2008.05.013.
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