Synthesis and P2Y receptor activity of a series of uridine dinucleoside 5′-polyphosphates

Article abstract of DOI:10.1016/S0960-894X(00)00612-0

A series of dinucleoside 5-polyphosphates Up_nU (n = 2-7) was synthesized. Their relative potencies as agonists at the G-protein-coupled receptors P2Y₁, P2Y₂, P2Y₄, and P2Y₆ were determined by intracellular calcium measurements using fluorescent imaging techniques. The correlation of phosphate chain length to activities at these receptors is discussed.

Full text of DOI:10.1016/S0960-894X(00)00612-0

Bioorganic & Medicinal Chemistry Letters 11 (2001) 157±160
Synthesis and P2Y Receptor Activity of a Series of Uridine
Dinucleoside 50-Polyphosphates
William Pendergast, Benjamin R. Yerxa, James G. Douglass, III, Sammy R. Shaver,Ã
Robert W. Dougherty, Catherine C. Redick, Ingrid F. Sims and Janet L. Rideout
Inspire Pharmaceuticals, Inc., 4222 Emperor Boulevard, Suite 470, Durham, NC 27703, USA
Received 5 September 2000; accepted 27 October 2000
AbstractÐA series of dinucleoside 5-polyphosphates UpnU (n=2±7) was synthesized. Their relative potencies as agonists at the G-
protein-coupled receptors P2Y1, P2Y2, P2Y4, and P2Y6 were determined by intracellular calcium measurements using ¯uorescent
imaging techniques. The correlation of phosphate chain length to activities at these receptors is discussed. # 2001 Elsevier Science
Ltd. All rights reserved.
Introduction
Synthesis
Diadenosine 50-polyphosphates (ApnA, n=2±6) have
received a great deal of attention over the last decade,
with their release from platelets and chroman cells
inspiring many studies on the potential biological activ-
ity of these molecules.1 Interest in uridine nucleotides as
extracellular signalling molecules increased with the
discovery that uridine 50-triphosphate (UTP) was an
agonist at the P2Y2 receptor, with a potency compar-
able to the natural purine agonist for the receptor,
adenosine 50-triphosphate (ATP). Stimulation of this
receptor results in activation of mucosal hydration and
mucociliary clearance mechanisms in the body.2 5 While
a variety of dipurine polyphosphates have been repor-
ted6 and the series ApnA (n=2±6) and GpnG (n=2±5)
are commercially available,7 several of the pyrimidine
dinucleotides UpnU (n=2±7) have not been described.8
Here we report syntheses of this latter series of com-
pounds of which three (n=5, 6, and 7) are new and
determine their relative potencies as agonists at the
P2Y1, P2Y2, P2Y4, and P2Y6 G-protein-coupled recep-
tors. One member of this family of dinucleotides, P1, P4-
di(uridine 50-)tetraphosphate (Up4U, INS365), exhibits
comparable potency with UTP as an agonist at the
P2Y2 and P2Y4 receptors and is currently in clinical
development for the treatment of dry eye9 and chronic
lung diseases, such as cystic ®brosis.10
As a result of our ongoing interest in dinucleoside
50-polyphosphates, we have developed a variety of coupl-
ing protocols to prepare them from the corresponding
nucleotides. In general, these procedures rely on con-
verting the water soluble salts of nucleotides into
organic soluble trialkylammonium salts, which allows
the phosphate couplings to be carried out under anhy-
drous conditions in DMF or DMSO. The present
method starts with the conversion of uridine monopho-
sphate (UMP, 1), uridine diphosphate (UDP, 3), or
UTP (5) from their sodium salts into their NBu3 salts by
standard procedures,11 followed by activation with
either CDI or DCC in DMF. The activated inter-
mediates were not isolated, but were directly condensed
with another nucleotide (also as the NBu3 salt).
Compound 1 was treated with CDI to give the phos-
phorimidazolidate 2 as the activated species,12 and this
was reacted with either 1 or 3 to give diuridine
50-diphosphate (Up2U, 7) or diuridine 50-triphosphate
(Up3U, 8), respectively (Scheme 1). Diuridine 50-tetra-
phosphate (9) was synthesized by activation of 5 with
DCC, which gave the known cyclic uridine 50-trimeta-
phosphate (6),13 followed by condensation with 1.
Alternately, 3 could be activated with CDI to give the
corresponding imidazolidate (4), which was condensed
with 3 to give 9. Diuridine 50-pentaphosphate (Up5U,
10) was prepared by conversion of 5 to 6 with DCC,
followed by ring opening of the cyclic 50-trimetaphos-
phate with 3. In the course of the synthesis of 10, diuridine
ÃCorresponding author. Tel.: +919-941-9777; fax: +919-941-9797;
e-mail: samray@inspirepharm.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00612-0

158
W. Pendergast et al. / Bioorg. Med. Chem. Lett. 11 (2001) 157±160
Scheme 1. Reagents and conditions: (i) CDI, NBu3, DMF; (ii) 1, DMF, 50 ꢀC, 63%; (iii) 3, DMF, 50 ꢀC, 50%; (iv) DCC, NBu3, DMF, rt, quan-
tative; (v) 3, DMF, 40 ꢀC, 8.5% as compound 10; (vi) 1, DMF, 40 ꢀC, 32%; (vii) 3, DMF, 50 ꢀC, 25%; (viii) triethylammoniumpyrophosphate,
pyridine, 30 ꢀC.
Table 1. Correlation of P2Y agonist activity with chain length in
50-hexaphosphate (Up6U, 11) and diuridine 50-hepta-
UpnU derivatives versus UTP and UDP
phosphate (Up7U, 12) were generated as minor bypro-
ducts. Compound 11 arose from the condensation of 6
with 5, as a result of a small amount of hydrolysis of 6
in the reaction mixture. Compound 12 was likely gen-
erated from the reaction between 6 and uridine 50-tetra-
phosphate (Up4), which is present as a minor impurity
in 5. All compounds were puri®ed14 using either gel ®l-
tration chromatography or ion exchange HPLC, and
were characterized by 1H NMR, 31P NMR, and
HRMS.15
Biological Activity
EC50 (mM)
P2Y2 P2Y4
The functional activities of test compounds were asses-
sed by measuring changes in the levels of cytosolic cal-
cium in 1321N astrocytoma cells infected with a
retrovirus encoding the human P2Y1, P2Y2, P2Y4, and
P2Y6 receptors. For assay, the cells were plated in black
wall/clear bottom cell culture plates (cat. # 3904, Corn-
ing Inc., Corning, NY) and grown to con¯uence. On the
day of assay, the growth medium was aspirated and
replaced with a solution of Fluo-3, AM (2.5 mM ®nal
concentration (Molecular Probes, Eugene, OR)) in an
Compound
m
p
P2Y1
P2Y6
>10
0.1
>30
0.2
20.0
>30
0
0
1
1
1
1
1
1
2
1
1
2
3
4
5
6
iaa
ia
ia
ia
ia
ia
ia
ia
0.03
>10
ia
22.0
0.1
ia
ia
.
5 (UTP 3Na)
.
3 (UDP 2Na)
.
7 (Up2U 2NH4)
.
8 (Up3U 3NH4)
>100
.
9 (Up4U 4Na)
0.10
0.4
.
10 (Up5U 5NH4)
5.6
9.8
8.4
>20
>30
>30
.
11 (Up6U 6NH4)
>30
>100
.
12 (Up7U 7NH4)
aia, inactive at 100 mM.

W. Pendergast et al. / Bioorg. Med. Chem. Lett. 11 (2001) 157±160
159
assay bu€er consisting of (mM): KCl (10.0), NaCl (118),
CaCl2 (2.5), MgCl2 (1.0), HEPES (20), glucose (10) pH
7.4. After a 60 min incubation with Fluo-3, AM at
25 ꢀC, cells were washed free of dye (Columbus Plate
Washer, TECAN U.S., Inc., Research Triangle Park,
NC). Receptor/cell activation was measured by mon-
itoring changes in ¯uorescence intensity (an indicator of
cytosolic calcium mobilization) using the FLIPRTM
(Molecular Devices Corp., Sunnyvale, CA). Data (rela-
tive ¯uorescence units) were modeled using PRISMTM
(San Diego, CA). EC50 values (Table 1) were estimated
from the ®tted curve functions.
also dependent upon phosphate chain length. In the
dinucleotide series, when the phosphate chain was
extended by one, the activity paralleled that of the nat-
ural nucleotide agonist for a given receptor. Thus,
compound 8 mirrored the activity of UDP on P2Y2 and
P2Y6, whereas compound 9 mimicked that of UTP on
P2Y2 and P2Y4. Compounds 10, 11, and 12 showed
some selectivity for P2Y2, with 10 being the most potent
of the three. However, this apparent increase in potency
might be attributable to the small amount of 9 that was
present in 10.
In conclusion, we have described the synthesis and bio-
logical activities of a series of diuridine polyphosphate
compounds. No activity was observed at the P2Y1
receptor with any of the compounds. The P2Y2 and
P2Y4 receptors exhibited a marked preference for 9
whereas the P2Y6 receptor exhibited a marked pref-
erence for 8.
Results and Discussion
While seemingly simple and relatively mild, the phos-
phate coupling reactions depicted in Scheme 1 often
give rise to byproducts that are dicult to remove.
Consequently, we found that multiple chromatographic
puri®cations on either Sephadex DEAE A-25 or PRP-
X100 HPLC were required to obtain compounds with
adequate purity for biological testing.14 In general, the
yields were inversely proportional to phosphate chain
length, in keeping with the larger number of unwanted
side reactions possible between the starting materials,
their degradation products and any excess of coupling
reagents in the more complex reaction mixtures. For
example, the reaction between 2 and 1 or 3 proceeded
with greater than 90% HPLC conversion to 7 or 8, and
gave isolated yields of 63 and 50%, respectively. At the
other extreme, compound 10 was obtained in only 20%
crude chromatographic purity, contaminated with the
closely-eluting byproducts 9, 11, and 12. Following
chromatography on Sephadex DEAE A-25, an 8.5%
yield of 10 was obtained, with a purity of 87%
(remainder 0.7% 9 and 12% 11). Compounds 11 and 12
were isolated from the same reaction by HPLC, with a
®nal purity of 94% (remainder 2.5% 10 and 3.3% 12)
and 91% (remainder 1.4% 10 and 7.2% 11), respec-
tively. Compound 9 was prepared by several routes,
with the method employing DCC giving the greatest
conversion by HPLC (>50%) and highest isolated yield
(32%). Use of CDI to couple 3 gave a somewhat lower
isolated yield (25%), while the coupling of two mol-
ecules of intermediate 2 with inorganic pyrophosphate16
gave a low yield of 9 which was tainted with Up4
following puri®cation on Sephadex. Of the six diuridine
5-polyphosphates tested, only 7, 8, and 9 were obtained
with purity greater than 99%.
Acknowledgements
High-resolution mass spectra (HRMS-FAB) were
obtained at the Mass Spectrometry Laboratory for
Biotechnology (North Carolina State University,
Raleigh). Partial funding for the facility was obtained
from the North Carolina Biotechnology Center and the
National Science Foundation.
References and Notes
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None of the compounds tested exhibited activity at the
P2Y1 receptor. The activities at the P2Y2, P2Y4, and
P2Y6 receptors were dependent upon the length of the
phosphate chains. The rank-order of potency at the
P2Y2 and P2Y4 receptors were similar with 9 being the
most potent compound. The potencies of all compounds
in this series were approximately 4-fold lower at the
P2Y4 receptor as compared with the P2Y2 receptor.
Only 8 exhibited signi®cant activity at the P2Y6 recep-
tor. These results are consistent with the activities
observed with UTP and UDP, where the nucleotides'
activities at the P2Y2, P2Y4, and P2Y6 receptors were
7. Sigma, St. Louis, MO.
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from the sodium salts by treatment in water with a cationic
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15. Selected spectroscopic data: Compound 9 (4Na salt), H
NMR (300 MHz, D2O) d 7.81 (d, J=8.1 Hz, 1H), 5.86 (d,
J=5.4 Hz, 1H), 5.84 (d, J=8.1 Hz, 1H), 4.27 (m, 1H), 4.25 (m,
1H), 4.14 (m, 1H), 4.11 (m, 2H); 31P NMR (D2O, H3PO4 std)
d
22.32 (m, 2P), 10.75 (m, 2P). HRMS (FAB) m/z
C18H22N4O23P4Na4 (M Na) ; calcd: 854.9318; found:
854.9268. Compound 7 (NH4 salt): 31P NMR (D2O) d 10.10
(s), HRMS (FAB) m/z C18H23N4O17P2 (M H) ; calcd:
629.0533; found: 629.0552. Compound 8 (NH4 salt): 31P NMR
(D2O) d 10.31 (d, J=19.2 Hz, 2P), 21.87 (t, J=18.95 Hz,
1P). HRMS (FAB) m/z C18H24N4O20P3 (M H) ; calcd:
709.0197; found: 709.0191. Compound 10 (NH4 salt): 31P
NMR (D2O) d 10.30 (d, 2P), 22.14 (m, 3P), HRMS (FAB)
m/z C18H26N4O26P5 (M H) ; calcd: 868.9523; found:
868.9561. Compound 11 (NH4 salt): 31P NMR (D2O) d
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gradient from water to 1 M NH4HCO3; HPLC: Hamilton
PRP-X100 column, 250 4.1 mm, 10 mm, gradient from water
to 90% 1 M NH4HCO3/10% ACN over 30 min, 2 mL/min,
monitor at 260 nm. All reported yields in Scheme 1 are for
isolated compounds.
10.12 (d, 2P),
21.76 (m, 4P). HRMS (FAB) m/z
C18H27N4O29P6 (M H) ; calcd: 948.9187; found: 948.9199.
Compound 12 (Na salt): 31P NMR (D2O) d 10.18 (d, 2P),
21.72 (m, 5P). HRMS (FAB) m/z C18H29N4O32P7
(M 2H+Na) ; calcd: 1050.8670; found: 1050.8629.
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