Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the identification of human P2Y6 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2015.07.004

P2Y₆ receptor (P2Y₆-R) is involved in various physiological and pathophysiological events. With a view to set rules for the design of UDP-based reversible P2Y₆-R antagonists as potential drugs, we established structure-activity relationship of UDP analogues, bearing modifications at the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-uridine-5′-α,β-methylene-diphosphonate, 16 and 23, or lack of 2′-OH, in 12-15, resulted in loss of both agonist and antagonist activity toward hP2Y₆-R. However, uridylyl phosphosulfate, 19, selectively inhibited hP2Y₆-R (IC₅₀ 112 μM) versus P2Y₂/₄-Rs. In summary, we have established a comprehensive SAR for hP2Y₆-R ligands towards the development of hP2Y₆-R antagonists.

Full text of DOI:10.1016/j.bmc.2015.07.004

Accepted Manuscript
Synthesis and structure-activity relationship of uracil nucleotide derivatives to-
wards the identification of human P2Y6 receptor antagonists
Diana Meltzer, Ofir Ben Yaacov, Guillaume Arguin, Yael Nadel, Ortal Danino,
Joanna Lecka, Jean Sévigny, Fernand-Pierre Gendron, Bilha Fischer
PII:
S0968-0896(15)00571-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.004
BMC 12433
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 June 2015
6 July 2015
7 July 2015
Please cite this article as: Meltzer, D., Yaacov, O.B., Arguin, G., Nadel, Y., Danino, O., Lecka, J., Sévigny, J.,
Gendron, F-P., Fischer, B., Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the
identification of human P2Y6 receptor antagonists, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
10.1016/j.bmc.2015.07.004
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Synthesis and structure-activity relationship of uracil nucleotide derivatives towards the
identification of human P2Y6 receptor antagonists
Diana Meltzera, Ofir Ben Yaacov a, Guillaume Arguinb, Yael Nadela, Ortal Daninoa, Joanna Leckac, Jean
Sévignyc, Fernand-Pierre Gendronb, and Bilha Fischera,*
a Department of Chemistry, Bar Ilan University, Ramat Gan, 52900, Israel
b Department of Anatomy and Cellular Biology, Université de Sherbrooke, rue Jean-Mignault, Sherbrooke,
3201, Quebec, Canada
c Centre de Recherche en Rhumatologie et Immunologie, Université Laval, Québec, QC, Canada

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis and structure-activity relationship of uracil nucleotide derivatives towards
the identification of human P2Y6 receptor antagonists
Diana Meltzera, Ofir Ben Yaacova, Guillaume Arguinb, Yael Nadela, Ortal Daninoa, Joanna Leckac, Jean
Sévignyc, Fernand-Pierre Gendronb, and Bilha Fischera,*
a Department of Chemistry, Bar Ilan University, Ramat Gan, 52900, Israel
b Department of Anatomy and Cellular Biology, Université de Sherbrooke, rue Jean-Mignault, Sherbrooke, 3201, Quebec, Canada
c Centre de Recherche en Rhumatologie et Immunologie, Université Laval, Québec, QC, Canada
ARTICLE INFO
ABSTRACT
Article history:
Received
P2Y6 receptor (P2Y6-R) is involved in various physiological and pathophysiological events.
With a view to set rules for the design of UDP-based reversible P2Y6-R antagonists as potential
drugs, we established structure–activity relationship of UDP analogues, bearing modifications at
the uracil ring, ribose moiety, and the phosphate chain. For instance, C5-phenyl- or 3-NMe-
uridine-5'-α,β-methylene-diphosphonate, 16 and 23, or lack of 2’-OH, in 12-15, resulted in loss
of both agonist and antagonist activity toward hP2Y6-R. However, uridylyl phosphosulfate, 19,
selectively inhibited hP2Y6-R (IC50 112 µM) vs. P2Y2/4-Rs. In summary, we have established a
comprehensive SAR for hP2Y6-R ligands towards the development of hP2Y6-R antagonists.
Received in revised form
Accepted
Available online
Keywords:
Human P2Y6 receptor
2009 Elsevier Ltd. All rights reserved
.
Antagonist
UDP
Structure-Activity Relationship (SAR)
1. Introduction
Pharmacological modulation of P2Y6-R has been proposed
to be useful for the treatment of various diseases, such as:
osteoporosis, neurodegeneration, gout, ocular hypertension,
glaucoma, inflammation, intestinal disorders, heart failure, and
diabetes.12-17
P2 receptors (P2-Rs) are membrane proteins that upon
activation by nucleoside di- and tri-phosphate, and in some
subtypes also by dinucleotides, lead to inhibitory and excitatory
effects in many tissues and organs under both normal and
pathophysiological conditions. The P2-Rs superfamily is divided
into two families: the ATP-selective P2X receptor (P2X-R)
family, which are ligand-gated ion channels, and the ATP (1; Fig.
1), ADP (2), UTP (3), UDP (4) and UPD-glucose sensitive
GPCR P2Y receptor (P2Y-R) family.1,2 P2-Rs are involved in the
modulation of vasoconstriction3, blood platelet aggregation,4
regulation of intraocular pressure5 and gastrointestinal disorders,6
and hence represent important targets for novel drugs
development.7
While P2Y6-R agonists are expected to be useful for the
treatment of diseases such as glaucoma, neurodegeneration, and
diabetes,18-20 the treatment of inflammatory bowel diseases, for
instance, requires the use of a P2Y6-R antagonist.21 Specifically,
inflammation of the mucosa of the colon up-regulates the
expression of P2Y6-R,6 and activation of the receptor by UDP
results in increased damage of the colonic mucosal tissue.
Blocking UDP activation of the over-expressed P2Y6-R on
epithelial cells, and consequently blocking the release of CXCL8,
has been suggested for the treatment of inflammatory bowel
diseases.22-24 This hypothesis is corroborated by the finding that a
P2Y6-R (irreversible) antagonist, MRS2578, 5, inhibited the
induction levels of CXCL8 in Caco-2/15 cells.21 Unlike P2Y6-R
agonists,25-27 currently no UDP-based antagonists of this receptor
are known.
Among P2Y-Rs, P2Y6-R is responsive to UDP, partially
responsive to UTP and ADP, and not responsive to ATP.8 P2Y6-
R is widely spread in the human body and has many
physiological roles.9-11
∗ Corresponding author. Tel.: +972-3-5318303; fax: +972-3-
6354907; e-mail: bilha.fischer@biu.ac.il
Identification of potent and selective P2Y6-R ligands has
been a challenge for researchers over the past decade. This
difficulty is exacerbated by the absence of a P2Y6-R crystal
structure. Hence, the structure–activity relationship (SAR) of
pyrimidine nucleotide agonists at P2Y6-R is only partially
known,28-30 and the mode of interaction between nucleotide

agonists and P2Y6-R is not fully understood.27 In this context, it
is thus not surprising that only one class of P2Y6-R antagonists
has been reported.7 The non-nucleotide di-isothiocyanate
derivative MRS2578, 56 (Fig. 1), is a potent and selective, yet
irreversible, antagonist of P2Y6-R. This isothiocyanate derivative
which likely binds covalently to P2Y6-R, is hydrophobic and has
limited stability in aqueous solution.7
2. Results
2.1. Synthesis of potential P2Y6-R antagonists
We attempted the identification of potential hP2Y6R antagonists
by modifying UDP-based hP2Y6R ligands, e.g. 5-OMe-UDP and
5-F-UDP scaffolds, which are a selective P2Y6-R agonist / partial
agonist, respectively,8,31 or 5-alkyl/aryl-UDP scaffold, where C5-
substituent is expected to improve the fit of the ligand to P2Y6-R
hydrophobic pocket.8,32
Specifically, UDP analogues 6-11 were obtained from
nucleosides 32 and 33. The latter were prepared by ribosylation
of uracil derivatives 24 and 25 (Scheme 1).33 Silylation of 24 and
25 was performed in HMDS in the presence of ammonium
sulfate followed by ribosylation with 1-acetate 2,3,5-tribenzoate
ribose. Compounds 30 and 31 were obtained by deprotection of
the benzoate groups, using NH3-MeOH for 30 and NaOMe for
31, respectively. Next, 2'- and 3'-hydroxyl groups of 30 and 31,
were protected by an acetonide group upon reaction with
dimethoxypropane and p-TsOH in acetone (Scheme 2).34
Hence, to allow controlled blocking of hP2Y6-R, we targeted
the identification of selective, reversible, stable and water-soluble
hP2Y6-R antagonists based on UDP scaffold.
Specifically, we describe here the synthesis of several series
of UDP analogues, 6-23, their SAR at hP2Y6-R, as well as the
metabolic stability of selected analogues.
Compound 35a was obtained by protection of commercially
available 5-methyluridine. Compound 34a was obtained by
Suzuki coupling of acetonide protected 5-iodo uridine and phenyl
boronic acid.35 To prepare compound 36a, first, Suzuki coupling
was performed between acetonide protected 5-iodo uridine and
isopropenylboronic acid pinacol ester,35 followed by
hydrogenation of the double bond over 10% Pd/C,36 to get the
desired isopropyl moiety (see Supplementary data for
experimental details).
Figure 1. Natural nucleotides active at P2Y-Rs and MRS2578, 5.
5'-Hydroxyl of 2',3'-acetonide-protected and 2'-deoxy
derivatives, 32a-36a, 41a and 37, was activated with p-TsCl in
pyridine. 5'-Tosyl-uridine analogues, 32-36, 38 and 41, were used
for the preparation of the corresponding diphosphate analogues
(Schemes 2-5). First, the nucleosides were treated with the
appropriate diphosphonate salt in DMF to produce the
diphosphonate products 6-14, 16-18 and 23. A high concentration
of the nucleosides, 0.5-1 M, was required for the reaction, since
5’-tosyl-uridine is of limited reactivity. The progress of the
reaction was monitored by 31P-NMR and/or 19F-NMR in 24 h
time intervals. The integration of the products' signals in 31P-
NMR vs. that of diphosphonate reagent signal helped
determining the completion of the reaction. The acetonide-
protecting group was then removed using 80% acetic acid at
95oC. Finally, the diphosphonate products were purified by
elution with 0.25-0.5 M ammonium bicarbonate on an ion-
exchange chromatography (DEAE-A25). The yields of products
6-14, 16-18 and 23 were 6-77%. The purity of most products,
determined on an analytical HPLC column in two solvents
systems, was >97%. Compound 15 was obtained from 5-fluoro-
2'-deoxyuridine, 37, in 25% yield, according to literature.37
Figure 2. Uracil nucleotide analogues designed as potential P2Y6-R
antagonists.
Scheme 1. Ribosylation of 5-F-uracil, 30, and 5-OMe-uracil, 31

Scheme 2. Synthesis of nucleotide analogues 6-11 and 16-18.
Scheme 4. Synthesis of nucleotide analogues 21-22.
Scheme 3. Synthesis of nucleotide analogues 12-15.
Scheme 5. Synthesis of nucleotide analogue 23.
Uridylyl phosphosulfate sodium salt, 19, was also prepared
according to literature.38 5-Fluoro-uridylyl phosphosulfate
sodium salt, 20, (see Supplementary data for experimental
details) was prepared similarly from 5-fluoro-5’-monophosphate-
uridine tri-n-octylammonium salt, tri-n-butyl-ammonium sulfate
and diphenyl chloro-phosphate, and was obtained in 12% yield
after HPLC separation. Analogues 21 and 22 were prepared
according to literature.39-42 Briefly, chloromethylphosphonic acid
was coupled to the appropriate acetonide protected uridine using
DCC in DMF. Then, sodium sulfite was added and after the
removal of acetonide protecting group, products 21 and 22 were
obtained. Compound 41 was obtained by methylation43 of
acetonide protected uridine followed by activation of 5'-OH with
a tosyl group. Finally, compound 23 was obtained by treating 41
with methylenediphosphonate tetrabutyl ammonium salt, as
described above.
2.2. Evaluation of uridine nucleotide analogues 6-23 as
hP2Y6-R ligands
Analogues 6-23 were first tested for their capacity to activate the
human recombinant P2Y6-R stably expressed in 1321N1 cells
(Figs. 3-4). P2Y6-R activation was determined by measuring
variations in intracellular calcium concentration (∆[Ca2+]i) as
previously described using UDP as control.44,45
Substitution of the 5-F-UDP and 5-OMe-UDP Pα, Pβ- bridging
oxygen atom by a CH2 group, in compounds 6 and 9, reduced
agonist potency at hP2Y6-R (Fig. 3A). The EC50 value for
compound 6 could not be determined because of a yellowish
background at concentrations above 10 µM. Although analogue 9
displayed some agonist activity, the elicited response at 10 µM
was 27% of the UDP response at the same concentration (Fig.
3A). Likewise, CCl2 and CF2-substituted compounds 7, 8, 10 and
11, were found to be poor P2Y6-R agonists (Fig. 3A).
The absence of 2'-hydroxyl group, as in analogues 12 to 15,
inhibited the ability of these molecules to stimulate P2Y6-R-
dependent changes of [Ca2+]i (Fig. 3B). Analogues 11, 12 and 15
showed some residual P2Y6-R activity at a concentration greater
than 100 µM, whereas analogues 13 and 14 did not activate
P2Y6-R at all.

no agonist activity at hP2Y6-R. On the other hand, analogue 19
significantly inhibited hP2Y6-R at 100 µM (Fig. 6A).
Concentration-response curve showed inhibition of P2Y6-R
activity with IC50 112 µM (Fig. 6B). Hence, compound 19 may
be a promising lead molecule for the design of UDP-based P2Y6-
R antagonists.
This result should be taken with care as it could be due to
agonist-induced receptor desensitization and internalization,46
and/or calcium pool depletion following the weak activation of
the receptor by analogue 19 (Fig. 4).
A
250
200
150
100
50
UDP
6
7
8
9
10
11
Compound 19 was evaluated for its selectivity to P2Y6-R vs.
hP2Y2- and hP2Y4-Rs and showed no agonist activity at the latter
receptors (data not shown).
0
0.01
0.1
1 10
[analogues] (µM)
100
1000
B
250
200
150
100
50
250
UDP
12
13
14
15
UDP
17
200
19
20
150
100
50
0
0
0.01
0.1
1 10
[analogues] (µM)
100
1000
0.01
0.1
1
10
100
1000
[analogues] (µM)
Figure 3. P2Y6-R agonist activity of analogues 6-15 vs. UDP was determined
by measuring intracellular calcium variation (∆[Ca2+]i) in 1321N1
astrocytoma cell line stably expressing recombinant human P2Y6-R (hP2Y6-
R). A) Activity of analogues 6-11. B) Activity of analogues 12-15. The
results of two experiments performed in duplicate are presented.
Figure 4. Activity of analogues 17, 19 and 20 vs. UDP at hP2Y6-R. Potential
P2Y6-R agonist activity was determined by measuring intracellular calcium
variation (∆[Ca2+]i) in 1321N1 astrocytoma cell line stably expressing
recombinant hP2Y6-R. The results of the two experiments performed in
duplicate are presented.
Among 5-alkyl/aryl-α,β-CH2-UDP, 16-18, analogues 16 and
18 could not activate the receptor (data not shown), however,
compound 17 displayed significant P2Y6-R activity with a
response corresponding to 60% of the response induced by UDP
at 100 µM (Fig. 4). 5-Fluoro-uridylyl phosphosulfate, 20, also
elicited a calcium response similar to the one produced by 17,
albeit less potent. However, the parent compound, uridylyl
phosphosulfate, 19, displayed only weak P2Y6-R activation at
concentration of 100 µM and higher. The related uridine-
phosphono-methylene-sulfonate, 21, and 5-OMe-uridine-
phosphono-methylene-sulfonate, 22, did not induce a calcium
response even at 100 µM (data not shown). Finally, substitution
of the hydrogen atom on N3 for a methyl group completely
abolished the capacity of analogue 23 to induce ∆[Ca2+]i by
P2Y6-R (data not shown).
Since most of these analogues displayed only a partial agonist
activity or even no activity at all, we tested these molecules for
their ability to block changes of [Ca2+]i induced by 1 µM UDP in
1321N1 cells stably expressing the recombinant hP2Y6-R (Figs.
5-6). Compounds 9, 17 and 20 were not tested as antagonists due
to their significant agonist activity as compared to UDP (Figs. 3A
and 4). Compound 6, tested at 10 µM, could not be analyzed due
to the formation of an interfering yellowish color.
A
200
150
**
*
100
50
0
30 100
7
30 100
30 100 (µM)
8
Analogues
10
B
200
150
100
50
***
0
30 100 30 100 30 100 30 100 30 100 (µM)
11 12 13 14 15
Analogues
Figure 5. hP2Y6-R antagonist activity of compounds 7-15. The tested
analogues were added prior to the addition of 1 µM UDP and ∆[Ca2+]i was
measured. Horizontal bar showed the calcium response to 1 µM UDP. A)
P2Y6-R antagonist activity of compounds 7-10. B) P2Y6-R antagonist activity
Analogues 7, 8 and 10-15 tested at 30 µM failed to block UDP-
induced P2Y6-R activation, whereas at 100 µM analogues 8, 10
and 11 significantly, but weakly, blocked P2Y6-R stimulation by
1 µM UDP (Fig. 5).
of compounds 11-15. Results are presented as the mean
SEM of two
Compounds 16, 21, 22 and 23 were found to be neither P2Y6-R
agonists nor P2Y6-R antagonists (Fig. 6). Compound 18
exhibited limited P2Y6-R antagonist activity reducing UDP-
induced calcium variation by 24% at 100 µM (the observed
reduction is not statistically significant (p = 0.07)), and showing
experiments performed in duplicate. Statistical significance was determined
by one-way ANOVA with multiple comparisons post-test: *P < 0.05, **P <
0.01 and ***P < 0.001 compared to control vehicle (dashed line).

Table 1. Hydrolysis of analogues 19-22 by human
ectonucleotidases, hNPP1/3.
A
400
300
200
100
0
relative hydrolysis (% SD of UDP hydrolysis)a
human
19
20
21
22
**
ectonucleotidase
hNPP1
27.9 1.0b
28.3 0.1b
18.9 1.0b
44.4 1.9b
9.5 2.5b
13.5 4.8b
21.6 3.8b
11.0 5.0b
vehicle 16 18 19 21 22 23
hNPP3
1 µM UDP
B
400
300
200
100
0
a After incubation at 37 °C in buffer (1 mM CaCl2, 200 mM NaCl, 10 mM
KCl and 100 mM Tris, pH 8.5) for 2 h with NPP1/3.
b Values represent mean S.D. of three experiments.
**
***
Table 2. Inhibition of UDP hydrolysis by hNPP1/3 in the
presence of analogues 21 and 22.
0
10
30
100
300
[compound 19] (µM)
relative inhibition (% SD of UDP hydrolysis)a
Figure 6. Antagonist activity of analogues 16, 18, 19 and 21-23 at human
recombinant P2Y6-R. A) Antagonists were tested at 100 µM upon 1 µM UDP
stimulation of recombinant hP2Y6-R stably expressed in 1321N1 cells. B)
Concentration-dependent inhibitory effect of compound 19 on 1 µM UDP-
dependent activation of hP2Y6-R. Results are presented as the mean SEM
of two experiments performed in duplicate. Statistical significance was
determined by one-way ANOVA with multiple comparisons post-test: **P <
0.01 and ***P < 0.001 compared to control vehicle (A) or 0 µM (B).
human ectonucleotidase
NPP1
21
22
26.7 3.6b
48.4 2.9b
NPP3
12.2 2.6b
10.5 3.1b
a
Analogue 21 or 22 was incubated at 37 ºC in buffer (1 mM CaCl2, 140 mM
NaCl, 5 mM KCl and 50 mM Tris, pH 8.5) for 2 h with hNPP1/3 and UDP.
b Values represent mean S.D. of three experiments.
2.3. Resistance of phosphosulfate and phosphono-methylene-
sulfonate analogues 19-22 to hydrolysis by NPP1/3
3. Discussion
To further evaluate the potential application of 19 as a P2Y6-R
antagonist, we studied its resistance to hydrolysis by nucleotide
pyrophosphatase/phosphodiesterases (NPP1/3) vs. the hydrolysis
of UDP and phosphosulfate and phosphonosulfonate analogues
20-22. NPP1/3 are some of the major enzymes responsible for
the hydrolysis of extracellular nucleotides.47 The hydrolysis rate
of analogues 19-22 by each enzyme was determined after
incubation at 37 ºC in the appropriate buffer for 2 or 3 h (for
NPP1 and NPP3, respectively), as compared to 100% hydrolysis
of UDP. The enzymatic reaction was stopped by adding the
reaction mixture into ice-cold perchloric acid. The stability of
UDP and analogues 19-22 to hydrolysis by NPPs was determined
by measuring the change in the integration of the HPLC peaks
for each analogue vs. control, to take into consideration the
degradation of the compounds due to the addition of acid to stop
the enzymatic reaction. Percentages of hydrolysis of UDP and
compounds 19-22 by NPP1 and 3 under the above conditions are
shown in Table 1. Analogues 19 and 20 were partially
metabolized by NPP1/3 vs. UDP. Surprisingly, analogues 21 and
22 were also hydrolyzed by NPP1/3, although to a lesser extent.
Currently, no reversible nucleotide-based P2Y6-R antagonists are
known. Likewise, structure-activity relationships (SAR) for
P2Y6-R antagonists are practically unknown. In the present study
we have established SAR of several series of UDP derivatives at
recombinant hP2Y6-R stably expressed in 1321N1 cells. Pα,Pß–
CH2 modification48 of P2Y6-R agonist 5-OMe-UDP,8 i.e.
analogue 9, resulted in reduced agonist activity.
CH2 and CF2 groups are considered as isosteres of the bridging
oxygen atom in PPi.49 Hence, in addition to Pα,Pß–CH2 UDP
analogues 6 and 9, Pα,Pß–CF2 and Pα,Pß–CCl2 modified UDP
analogues were synthesized, 10-11 and 13-14, and found to be
poor hP2Y6-R agonists or no agonists at all (analogues 13 and
14). Compound 11, containing a bridging CF2 group and 5-OMe
substitution, reduced calcium release by 40% at 100 µM, but was
not active at lower concentrations. At 100 µM analogue 10
weakly blocked P2Y6-R stimulation by UDP. The minor or lack
of P2Y6-R agonist and antagonist activity of 10 and 7, containing
a bridging CCl2 group (vs. 11 and 9, Fig. 5) may be related to the
large size of dichloro methylene bisphosphonate, vs. difluoro
methylene bisphosphonate or methylene bisphosphonate anion,50
which may not be compatible with the binding pocket of the
receptor.
Compounds 21 and 22, the enzymatic hydrolysis of which
was relatively limited, were also tested as inhibitors of hNPP1/3
(Table 2). These compounds were found to be poor and non-
selective hNPP1/3 inhibitors.
Reduced activation of P2Y6-R by 2'-dUDP analogues has been
described before.26 2'-Deoxy analogues 12-14, showed neither
agonist nor antagonist activity at P2Y6-R. Likewise, 5-fluoro-2'-
deoxyuridine 3',5'-bis-phosphate, 15, was inactive as either an
agonist or antagonist at P2Y6-R.
However, C5-alkyl/aryl substitution in compounds 16-18 which
was expected to improve the fit to the hydrophobic pocket of
P2Y6-R,32 resulted in loss of any activity at P2Y6-R or in poor
agonist activity. These findings are in accordance with a report

on 5-substituted UTP derivatives as agonists of the related P2Y2-
R, showing that alkyl substituents at UTP C5-position are not
tolerated by P2Y2-R either.51
inhibition of P2Y6-R at 100 µM. Yet, the corresponding 5-F-UDP
analogues 6, 8, and 14 proved to be poor P2Y6-R ligands.
Analogue 19 is thus proposed as a lead structure for the
development of P2Y6-R antagonists. Furthermore, this compound
was a P2Y6-R selective ligand showing no activity at hP2Y2- and
hP2Y4-Rs.
The SAR of the new UDP derivatives presented here and
summarized in Fig. 7, may contribute to future design of
selective ligands for P2Y6-R and other uracil nucleotide-sensitive
P2Y-Rs.
The loss of activity of compound 23, bearing a methyl moiety on
N3, at hP2Y6-R may be due to a loss of a significant H-bond
present in 98% of the complexes of uracil-nucleotides and
proteins, between the uracil N3-H and a protein H-bond
acceptor.8,32
At 100 µM, compounds 21 and 22, bearing a phosphono-
methylene-sulfonate moiety, neither activated P2Y6-R nor
inhibited its activity.
Analogue 19, bearing a phosphosulfate moiety, although partially
hydrolysable by NPP1/3, proved to be a very poor P2Y6-R
agonist and caused an apparent inhibition of P2Y6-R with an IC50
value of 112 µM (Fig. 6). Furthermore it showed selectivity vs.
P2Y2-R and P2Y4-R.
5. Experimental Section
5.1. Chemistry
Hence, analogue 19 may be used as a scaffold to develop the next
generation of potential UDP analogues displaying antagonist
activity at human P2Y6-R.
5.1.1. General
All air and moisture sensitive reactions were carried out in flame-
dried, argon-flushed, two-neck flasks sealed with rubber septa,
and the reagents were introduced by syringe. The separation on
the automatic column was carried out using an HPFC automated
flash purification system (Biotage SP1 separation system (RP)).
Compounds were characterized by NMR using Bruker AC-200,
DPX-300, or DMX-600 spectrometers. 1H NMR spectra were
recorded at 200, 300, or 600 MHz. High resolution mass spectra
were recorded on an AutoSpec Premier (Waters, UK)
spectrometer by chemical ionization. Nucleotides were analyzed
under ESI (electron spray ionization) conditions on a Q-TOF
micro instrument (Waters, UK). Primary purification of the
nucleotides was achieved on a LC (Isco UA-6) system using a
Sephadex DEAE-A25 column, swollen in 1M NaHCO3 at room
temperature for 1 day. The resin was washed with deionized
water before use. LC separation was monitored by UV detection
at 280 nm. A buffer gradient of NH4HCO3 was applied as
detailed below. Final purification of the nucleotides was achieved
on an HPLC (Merck-Hitachi) system, using a semipreparative
reverse-phase column (Gemini 5u C-18 110A, 250 X 10.00 mm,
5 µm, Phenomenex, Torrance, CA). The purity of the nucleotides
was evaluated on an analytical reverse-phase column system
(Gemini 5u, C-18, 110A, 150 X 4.60 mm, 5 µm, Phenomenex,
Torrance, CA), in two solvent systems as described below.
Aqueous solutions of the products were passed through a sodium
form Dowex 50WX8-200 or CM Sephadex ion-exchange resin
column and the products were eluted with deionized water to
obtain the corresponding sodium salts after freeze-drying.
Figure 7. SAR of the new UDP derivatives presented herein.
4. Conclusions
5.1.2.
5-Fluoro-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 6, was obtained from 32 (150 mg, 0.33 mmol) in a
14% yield (23 mg) after HPLC separation. Final separation was
achieved by applying an isocratic TEAA/CH3CN 97:3 in 13 min
(5 mL/min): tR 8.00 min. Purity data was obtained on an
analytical column: tR 2.58 min (100% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.34
min (99.9% purity) using solvent system II (100:0 PBS/CH3CN
Here, we synthesized several series of UDP analogues, and
established their SAR as hP2Y6-R ligands (Fig. 7). Analogues
lacking 2’-OH (compounds 12-15) had no agonist or antagonist
activity at hP2Y6-R, namely, 2’-OH is crucial for any activity at
the receptor. Furthermore, 3-NMe and C5-alkyl/aryl
modifications of Pα,Pß–CH2-UDP, compounds 16-18 and 23,
were not tolerated by hP2Y6-R, as well. They showed poor
agonist activity towards hP2Y6-R with the exception of
compounds 16 and 23 that did not activate the receptor at all.
Compounds 16 and 23 also did not inhibit P2Y6-R. The
replacement of β-phosphate in UDP by sulfate group, compounds
19-22, reduced agonist activity, and even resulted in antagonist
activity of compound 19. Pα,Pß–CCl2 modification of 5-F- and 5-
OMe-UDP decreased agonist activity at hP2Y6-R (compounds 7,
10, and 13), however, did not result in antagonist activity, while
5-OMe substitution of UDP-Pα,Pß–CF2, 11, resulted in 40%
1
over 10 min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.15 (d, J
= 6.2 Hz, H-6, 1H), 5.95 (d, J = 1.8 Hz, H-1', 1H), 4.43-4.32 (m,
2H), 4.31-4.23 (m, 1H), 4.23-4.08 (m, 2H), 2.19 (t, J = 20 Hz,
PCH2P, 2H) ppm. 31P NMR (D2O, 81 MHz): δ 19.03 (m, Pɑ),
15.19 (m, Pß) ppm. 19F NMR (D2O, 188 MHz): δ -165.28 (d, J =
6.25 Hz, F-5) ppm. HR MALDI (negative): calcd for
C10H14F1N2O11P2, 419.0102; found, 419.0081.
5.1.3. 5-Fluoro-uridine-5'-α,β-dichloromethylene-
diphosphonate sodium salt, 7, was obtained from 32 (150 mg,
0.33 mmol) in a 8% yield (14.3 mg) after HPLC separation. Final

separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 7.60 min. Purity data was obtained
on an analytical column: tR 8.66 min (99.9% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 4.40
min (99.7% purity) using solvent system II (100:0 PBS/CH3CN
separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 7.70 min. Purity data was obtained
on an analytical column: tR 3.91 min (99.5% purity) using solvent
system I (96:4 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.31
min (97.8% purity) using solvent system II (98:2 PBS/CH3CN
over 10 min, 1 mL/min). 1H NMR (D2O, 200 MHz): δ 7.32 (s, H-
6, 1H), 6.00 (d, J = 4.8 Hz, H-1', 1H), 4.48-4.18 (m, 5H), 3.79 (s,
OMe, 3H) ppm. 31P NMR (D2O, 81 MHz): δ 8.70-5.60 (m, Pα),
5.53-2.67 (m, Pß) ppm. 19F NMR (D2O, 188 MHz): δ -116.6 (m,
CF2, 2F) ppm. HR MALDI (negative): calcd for
C11H15F2N2O12P2, 467.0071; found, 467.0062.
1
over 10 min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.14 (d, J
= 6.2 Hz, H-6, 1H), 5.94 (d, J = 3.0 Hz, H-1', 1H), 4.40-4.28 (m,
4H), 4.27-4.18 (m, 1H) ppm. 31P NMR (D2O, 81 MHz): δ 8.90
(m, Pɑ), 8.20 (m, Pß) ppm. 19F NMR (D2O, 188 MHz): δ -165.19
(d, J = 6.2 Hz, F-5) ppm. HR MALDI (negative): calcd for
C10H12Cl2F1N2O11P2, 486.9272; found, 486.9274.
5.1.4.
5-Fluoro-uridine-5'-α,β-difluoromethylene-
5.1.8.
5-Fluoro-uridine-2'-deoxy-5'-α,β-methylene-
diphosphonate sodium salt, 8, was obtained from 32 (45 mg,
0.10 mmol) in a 16% yield (8.5 mg) after HPLC separation. Final
separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 9.00 min. Purity data was obtained
on an analytical column: tR 5.67 min (100% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.36
min (99.8% purity) using solvent system II (100:0 PBS/CH3CN
diphosphonate sodium salt, 12, was obtained from 38 (70 mg,
0.18 mmol) in a 17% yield (14 mg) after HPLC separation. Final
separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 7.00 min. Purity data was obtained
on an analytical column: tR 3.11 min (99.5% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.98
min (99.1% purity) using solvent system II (100:0 PBS/CH3CN
1
1
over 10 min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.08 (d, J
over 10 min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.08 (d, J
= 6.2 Hz, H-6, 1H), 5.94 (d, J = 3.0 Hz, H-1', 1H), 4.38-4.17 (m,
5H) ppm. 31P NMR (D2O, 81 MHz): δ 8.70-5.60 (m, Pɑ), 5.53-
2.67 (m, Pß) ppm. 19F NMR (D2O, 188 MHz): δ -116.03 to -
117.10 (m, CF2, 2F), -165.42 (d, J = 6.2 Hz, F-5) ppm. HR
MALDI (negative): calcd for C10H12F3N2O11P2, 454.9902; found,
454.9861.
= 6.3 Hz, H-6, 1H), 6.24 (t, J = 6.4 Hz, H-1', 1H), 4.60-4.50 (m,
1H), 4.19-4.00 (m, 3H), 2.33 (t, J = 5.7 Hz, 2H), 2.12 (t, J = 20.0
Hz, PCH2P, 2H) ppm. 31P NMR (D2O, 81 MHz): δ 19.78 (d, J =
9.5 Hz, Pα), 14.71 (d, J = 9.5 Hz, Pß) ppm. 19F NMR (D2O, 188
MHz): δ -165.26 (d, J = 6.3 Hz, F-5) ppm. HR MALDI
(negative): calcd for C10H14FN2O10P2, 403.0110; found,
403.0082.
5.1.5.
5-OMe-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 9, was obtained from 33 (100 mg, 0.22 mmol) in a
30% yield (33.5 mg) after HPLC separation. Final separation was
achieved by applying an isocratic TEAA/CH3CN 97:3 in 13 min
(5 mL/min): tR 7.80 min. Purity data was obtained on an
analytical column: tR 2.77 min (99.5% purity) using solvent
system I (96:4 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.62
min (99.0% purity) using solvent system II (98:2 PBS/CH3CN
over 10 min, 1 mL/min). 1H NMR (D2O, 200 MHz): δ 7.30 (s, H-
6, 1H), 5.95 (d, J = 5.3 Hz, H-1', 1H), 4.43-4.32 (m, 2H), 4.26-
4.05 (m, 3H), 2.11 (t, J = 20 Hz, PCH2P, 2H) ppm. 31P NMR
(D2O, 81 MHz): δ 18.49 (m, Pɑ), 15.19 (m, Pß) ppm. 13C-NMR
(D2O, 100 MHz): δ 163.8, 152.2, 137.6, 120.3, 89.2, 84.1 (d, J =
7.9 Hz, C-4'), 73.9, 70.3, 64.1 (d, J = 4.6 Hz, C-5'), 58.3, 28.6 (t,
J = 120.6 Hz, PCH2P) ppm. HR MALDI (negative): calcd for
C11H17N2O12P2, 431.0260; found, 431.0252.
5.1.9.
5-Fluoro-uridine-2'-deoxy-5'-α,β-dichloromethylene-
diphosphonate sodium Salt, 13, was obtained from 38 (135 mg,
0.30 mmol) in a 6% yield (9.1 mg) after HPLC separation. Final
separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 6.80 min. Purity data was obtained
on an analytical column: tR 3.63 min (98.7% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 3.20
min (98.0% purity) using solvent system II (100:0 PBS over 10
1
min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.10 (d, J = 6.1
Hz, H-6, 1H), 6.30 (t, J = 6.6 Hz, H-1', 1H), 4.71-4.60 (m, 1H),
4.45-4.10 (m, 3H), 2.38 (t, J = 6.1 Hz, 2H) ppm. 31P NMR (D2O,
81 MHz): δ 11.02 (d, J = 16.0 Hz, Pα), 8.58 (d, J = 16.0 Hz, Pß)
ppm. 19F NMR (D2O, 188 MHz): δ -165.08 (d, J = 6.1 Hz, F-5)
ppm. HR MALDI (negative): calcd for C10H12Cl2FN2O10P2,
470.9330; found, 470.9321.
5.1.6.
5-OMe-uridine-5'-α,β-dichloromethylene-
5.1.10. 5-Fluoro-uridine-2'-deoxy-5'-α,β-difluoromethylene-
diphosphonate sodium salt, 14, was obtained from 38 (71 mg,
0.18 mmol) in a 23% yield (21 mg) after HPLC separation. Final
separation was achieved by applying an isocratic TEAA/CH3CN
97:3 in 13 min (5 mL/min): tR 7.20 min. Purity data was obtained
on an analytical column: tR 3.25 min (99.9% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 2.56
min (99.3% purity) using solvent system II (100:0 PBS over 10
diphosphonate sodium salt, 10, was obtained from 33 (50 mg,
0.10 mmol) in a 28% yield (15.7 mg) after HPLC separation.
Final separation was achieved by applying an isocratic
TEAA/CH3CN 97:3 in 13 min (5 mL/min): tR 8.30 min. Purity
data was obtained on an analytical column: tR 5.31 min (99.9%
purity) using solvent system I (96:4 TEAA/CH3CN over 10 min,
1 mL/min); tR 3.49 min (99.8% purity) using solvent system II
1
1
(98:2 PBS/CH3CN over 10 min, 1 mL/min). H NMR (D2O, 200
min, 1 mL/min). H NMR (D2O, 200 MHz): δ 8.07 (d, J = 6.5
MHz): δ 7.32 (s, H-6, 1H), 5.97 (d, J = 5.1 Hz, H-1', 1H), 4.51-
4.17 (m, 5H), 3.80 (s, OMe, 3H) ppm. 31P NMR (D2O, 81 MHz):
δ 11.06 (d, J = 16.0 Hz, Pα), 8.62 (d, J = 16.0 Hz, Pß) ppm. 13C-
NMR (D2O, 151 MHz): δ 163.5, 151.9, 137.6, 120.8, 88.9, 84.3
(d, J = 6.8 Hz, C-4'), 80.9 (dd, J = 118.5 Hz, J = 135.7 Hz,
PCH2P), 73.8, 70.3, 67.1 (d, J = 5.3 Hz, C-5'), 58.8 ppm. HR
MALDI (negative): calcd for C11H15Cl2N2O12P2, 498.9480;
found, 498.9470.
Hz, H-6, 1H), 6.29 (t, J = 6.6 Hz, H-1', 1H), 4.68-4.54 (m, 1H),
4.38-4.06 (m, 3H), 2.44-2.30 (m, 2H) ppm. 31P NMR (D2O, 81
MHz): δ 8.70-5.60 (m, Pα), 5.53-2.67 (m, Pß) ppm. 19F NMR
(D2O, 188 MHz): δ -117.38 (dd, J = 88.0 Hz, 2F), -165.42 (d, J =
6.5 Hz, F-5) ppm. HR MALDI (negative): calcd for
C10H12F3N2O10P2, 438.9921; found, 438.9901.
5.1.11. 5-Fluoro-2'-deoxyuridine 3',5'-bisphosphate disodium
salt, 15. Starting from 40 mg (0.16 mmol) of 5-fluoro-2'-
deoxyuridine, 37, and following literature procedure,37 20 mg
(25% yield) of 15 were obtained after HPLC separation. Final
separation was achieved by applying an isocratic TEAA/CH3CN
5.1.7. 5-OMe-uridine-5'-α,β-difluoromethylene-
diphosphonate sodium salt, 11, was obtained from 33 (46 mg,
0.10 mmol) in a 9% yield (4.6 mg) after HPLC separation. Final

97:3 in 13 min (5 mL/min): tR 6.20 min. Purity data was obtained
on an analytical column: tR 4.16 min (97.0% purity) using solvent
system I (98:2 TEAA/CH3CN over 10 min, 1 mL/min); tR 3.81
min (96.5% purity) using solvent system II (100:0 PBS over 10
7.8 Hz, C-4'), 73.1, 69.7, 63.5 (d, J = 4.8 Hz, C-5'), 27.8 (t, J =
124.1 Hz, PCH2P), 25.8, 20.6, 20.4 ppm. HR MALDI (negative):
calcd for C13H21N2O11P2, 443.0626; found, 443.0615.
1
min, 1 mL/min). H NMR (D2O, 200 MHz) δ 2.38 (m, CH2-2',
5.1.15. 5-Fluoro-uridylyl phosphosulfate sodium salt, 20, was
synthesized according to literature,38 and obtained in a 12% yield
1H), 2.55 (m, CH2-2', 1H), 4.00 (m, CH2-5', 2H), 4.32 (m, H-4',
1H), 4.89 (m, H-3', 1H), 6.34 (t, J = 7.0 Hz, H-1', 1H), 8.20 (d, J
= 6.5 Hz, H-5, 1H). 31P NMR (D2O, 81 MHz): δ 3.32 (s, 5'-P),
2.99 (s, 3'-P). 19F NMR (D2O, 188 MHz): δ -165.36 (d, J = 6.5
Hz, F-5). HR MALDI (negative): calcd for C9H12FN2O11P2,
404.9922; found, 404.9902.
1
(22 mg) after HPLC separation. H NMR (D2O, 600 MHz): δ
8.02 (d, J = 6.4 Hz, 1H, H-6), 6.01 (dd, J = 4.9, 1.6 Hz, 1H, H-1'),
4.20-4.40 (m, 5H, H-2',H-3',H-4', H-5', H-5'') ppm. 31P NMR
(D2O, 81 MHz): δ -10.11 (s, 1P) ppm. 19F NMR (D2O, 188
MHz): -164.62 (d, J = 5.6 Hz) ppm. HR MALDI (negative):
calcd for C9H11FN2O12PS, 420.9760; found, 420.9749. Purity
data was obtained on an analytical column: tR 7.65 min (88.0%
purity) using solvent system I (99:1 TEAA/CH3CN over 14 min,
1 mL/min); tR 2.01 min (85.0% purity) using solvent system II
(99:1 PBS/CH3CN over 8 min, 1 mL/min).
5.1.12.
5-Phenyl-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 16, was obtained from 34 (90 mg, 0.175 mmol) in a
20% yield (16 mg) after HPLC separation. Final separation was
achieved by applying an isocratic TEAA/CH3CN 90:10 in 10 min
(4 mL/min): tR 6.25 min. Purity data was obtained on an
analytical column: tR 5.31 min (99.5% purity) using solvent
system I (92:8 TEAA/CH3CN over 15 min, 1 mL/min); tR 4.28
min (99.4% purity) using solvent system II (94:4 PBS/CH3CN
5.1.16.
Uridine-phosphono-methylene-sulfonate
triethylammonium salt, 21, was synthesized according to
literature,42 and obtained in a 17% yield (20 mg) after LC
separation. 1H NMR (D2O, 600 MHz): δ 8.02 (d, J = 8.4 Hz, 1H,
H-6), 5.98 (d, J = 5.4 Hz, 1H, H-1'), 5.95 (d, J = 8.4 Hz, 1H, H-
5), 4.36-4.40 (m, 2H, H-2',H-3'), 4.26-4.27 (m, 1H, H-4'), 4.17-
4.20 (m, 2H, H-5', H-5''), 3.43 (d, J = 15.6 Hz, 2H, PCH2S) ppm.
31P NMR (D2O, 162 MHz): δ 11.38 (s, 1P) ppm. 13C NMR (D2O,
150 MHz): δ 167.1, 152.7, 142.6, 103.3, 88.8, 84.3 (d, J = 8.0
Hz, C-4'), 74.4, 70.5, 64.6 (d, J = 5.3 Hz, C-5'), 49.6 (d, J 130.1 =
Hz, PCH2S) ppm. HR MALDI (negative): calcd for
C10H14N2O11PS, 401.0050; found, 401.0030. Purity data was
obtained on an analytical column: tR 4.90 min (99.8% purity)
using solvent system I (99:1 TEAA/CH3CN over 15 min, 1
mL/min); tR 2.51 min (99.9% purity) using solvent system II
(99.8:0.2 PBS/CH3CN over 10 min, 1 mL/min).
1
over 13 min, 1 mL/min). H NMR (D2O, 200 MHz): δ 7.87 (s,
1H, H-6), 7.42-7.52 (m, 5H, Ph), 6.01 (d, J = 5.4 Hz, 1H, H-1'),
4.48 (t, J = 5.4 Hz, 1H, H-2'), 4.37 (t, J = 4.6 Hz, 1H, H-3'), 4.2-
4.3 (m, 1H, H-4'), 4.15-4.05 (m, 2H, H-5', H-5''), 1.98 (t, J = 19.8
Hz, 2H, PCH2P) ppm. 31P NMR (D2O, 81 MHz): δ 19.10 (d, J =
8.9 Hz, Pα), 15.22 (d, J = 8.9 Hz, Pβ) ppm. 13C NMR (D2O, 150
MHz): δ 164.8, 151.5, 138.7, 131.8, 128.8, 128.5, 116.2, 88.8,
83.6 (d, J = 7.8 Hz, C-4'), 73.4, 69.8, 63.4 (d, J = 5.0 Hz, C-5'),
27.5 (t, J = 123.2 Hz, PCH2P). HR MALDI (negative): calcd for
C16H19N2O11P2, 477.0459; found, 477.0464.
5.1.13.
5-Methyl-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 17, was obtained from 35 (90 mg, 0.20 mmol) in a
33% yield (27 mg) after HPLC separation. Final separation was
achieved by applying an isocratic TEAA/CH3CN 98:2 in 11 min
(4 mL/min): tR 6.80 min. Purity data was obtained on an
analytical column: tR 4.56 min (99.9% purity) using solvent
system I (99:1 TEAA/CH3CN over 9 min, 1 mL/min); tR 2.53
min (99.7% purity) using solvent system II (95.5:0.5
PBS/CH3CN over 10 min, 1 mL/min). 1H NMR (D2O, 200
MHz): δ 7.77 (s, 1H, H-6), 5.97 (d, J = 4.2 Hz, 1H, H-1'), 4.43-
4.32 (m, 2H, H-2', H-3'), 4.31-4.23 (m, 1H, H-4'), 4.23-4.08 (m,
2H, H-5', H-5''), 2.03 (t, J = 19.6 Hz, 2H, PCH2P), 1.94 (s, 3H,
CH3) ppm. 31P NMR (D2O, 81 MHz): δ 21.07 (d, J = 8.4 Hz, Pα),
13.33 (d, J = 8.4 Hz, Pβ) ppm. 13C NMR (D20, 150 MHz): δ
166.7, 152.1, 137.2, 111.7, 88.3, 83.3 (d, J = 7.3 Hz, C-4'), 73.5,
69.5, 63.3 (d, J = 4.6 Hz, C-5'), 28.1 (t, J = 120.8 Hz, PCH2P),
11.66 ppm. HR MALDI (negative): calcd for C11H17N2O11P2,
415.0313; found, 415.0302.
5.1.17. 5-OMe-uridine-phosphono-methylene-sulfonate
triethylammonium salt, 22, was synthesized according to
literature,42 and obtained in a 40% yield (50 mg) after LC
1
separation. H NMR (D2O, 600 MHz): δ 7.33 (s, 1H, H-5), 5.97
(d, J = 6.0 Hz, 1H, H-1'), 4.29-4.36 (m, 2H, H-2',H-3'), 4.17-4.19
(m, 1H, H-4'), 4.11-4.12 (m, 2H, H-5', H-5''), 3.69 (s, 3H, 5-
OMe), 3.36 (d, J = 16.4 Hz, 2H, PCH2S) ppm. 31P NMR (D2O,
162 MHz): δ 11.02 (s, 1P) ppm. 13C NMR (D2O, 150 MHz): δ
163.8, 152.4, 138.5, 121.1, 89.3, 85.4 (d, J = 7.8 Hz, C-4'), 74.6,
71.6, 65.9 (d, J = 5.1 Hz, C-5'), 49.6 (d, J 130.1 = Hz, PCH2S)
ppm. HR MALDI (negative): calcd for C11H16N2O12PS,
431.0156; found, 431.0170. Purity data was obtained on an
analytical column: tR 5.32 min (99.5% purity) using solvent
system I (98:2 TEAA/CH3CN over 15 min, 1 mL/min); tR 3.16
min (99.4% purity) using solvent system II (99.5:0.5
PBS/CH3CN over 10 min, 1 mL/min).
5.1.14. 5-Isopropyl-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 18, was obtained from 36 (100 mg, 0.2 mmol) in a
30% yield (27 mg) after HPLC separation. Final separation was
achieved by applying an isocratic TEAA/CH3CN 99:9 in 25 min
(4 mL/min): tR 18.40 min. Purity data was obtained on an
analytical column: tR 6.82 min (98.9% purity) using solvent
system I (95:5 TEAA/CH3CN over 22 min, 1 mL/min); tR 3.61
min (99.9% purity) using solvent system II (98:2 PBS/CH3CN
5.1.18.
N3-Me-uridine-5'-α,β-methylene-diphosphonate
sodium salt, 23, was obtained from 41 (100 mg, 0.22 mmol) in a
77% yield (75 mg) after HPLC separation. Final separation was
achieved by applying a linear gradient of TEAA/CH3CN 94:6 to
93:7 in 10 min (4 mL/min): tR 5.5 min. Purity data was obtained
on an analytical column: tR 3.03 min (99.9% purity) using solvent
system I (96:4 to 90:10 TEAA/CH3CN over 10 min, 1 mL/min);
tR 1.90 min (100% purity) using solvent system II (99:1 to 93:17
of 0.01M KH2PO4 (pH=4.6)/ CH3CN over 10 min, 1 mL/min). 1H
NMR (D2O, 600 MHz): δ 7.99 (d, J = 8.4 Hz, 1H, H-6), 6.01 (d, J
= 8.4 Hz, 1H, H-5), 5.96 (d, J = 3.6 Hz, 1H, H-1'), 4.35-4.40 (m,
2H, H-2', H-3'), 4.23-4.28 (m, 1H, H-4'), 4.12-4.21 (m, 2H, H-5',
H-5''), 3.28 (s, 3H, N-CH3), 2.12 (t, J = 21.0 Hz, 2H, PCH2P)
ppm. 31P NMR (D2O, 162 MHz): δ 20.66 (d, J = 9.1 Hz, Pα),
14.03 (d, J = 9.1 Hz, Pβ) ppm. 13C NMR (D2O, 150 MHz): δ
1
over 10 min, 1 mL/min). H NMR (D2O, 600 MHz): δ 7.48 (s,
1H, H-6), 5.93 (d, J = 5.4 Hz, 1H, H-1'), 4.43 (t, J = 4.6 Hz, 1H,
H-2'), 4.37 (dd, J = 5.4, 4.2 Hz, 1H, H-3'), 4.2-4.3 (m, 1H, H-4'),
4.10-4.18 (m, 2H, H-5', H-5''), 2.82 (septet, J = 7.2 Hz, 1H, CH),
2.12 (t, J = 19.2 Hz, 2H, PCH2P), 1.16 (d, J = 1.8 Hz, 3H, CH3),
1.15 (d, J = 1.8 Hz, 3H, CH'3) ppm. 31P NMR (D2O, 81 MHz): δ
20.29 (d, J = 8.3 Hz, Pα), 14.08 (d, J = 8.3 Hz, Pβ) ppm. 13C NMR
(D2O, 150 MHz): δ 165.6, 151.6, 135.7, 121.7, 88.7, 83.3 (d, J =

165.5, 152.1, 139.5, 101.6, 89.6, 83.0 (d, J = 8.0 Hz, C-4'), 73.8,
69.0, 62.8 (d, J = 4.8 Hz, C-5'), 27.8 (t, J = 124.1 Hz, PCH2P),
27.6 ppm. HRMS (ESI): calcd for C11H17N2O11P2, 415.0312
found, 415.0311.
Triton X-100 and 50ꢀmM EDTA to cell suspensions. The
following equation was used to relate the fluorescence intensity
to Ca2+ levels: [Ca2+]ꢀ=ꢀKd(Fꢀ−ꢀFmin)/(Fmaxꢀ−ꢀF). Kd is the Ca2+
dissociation constant of Fluo-4 (345ꢀnM).52
5.2. Evaluation of the resistance of analogues 19-22 to
hydrolysis by hNPP1/3
Acknowledgments
This research was supported by Crohn’s and Colitis
Foundation of Canada Grant in Aid of Research (2009–2012) and
a CIHR operating grant (MOP-286567) to F.P.G.
100 µM analogues 19-22 was incubated with hNPP1 and hNPP3
at 37 °C in 487 µl of incubation buffer (1 mM CaCl2, 200 mM
NaCl, 10 mM KCl and 100 mM Tris, pH 8.5). After 2 h for
hNPP1 and 3 h for hNPP3 the reaction was quenched by the
addition of 350 µl of 1M perchloric acid. The samples were
centrifuged for 1 min at 10,000 g and subsequently neutralized
with 2M KOH until pH 7 and then centrifuged for 1 min at
10,000 g. Substance hydrolysis was analyzed by HPLC (on a
Gemini analytical column (5u, C-18, 110A; 150 × 4.60 mm)
using isocratic elution with TEAA (pH 7)/CH3CN, at a flow rate
of 1 mL/min). The percentage of degradation was calculated
from the area under the curve of the nucleoside monophosphate
peak, after subtraction of the control.
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