Human P2Y14 receptor agonists: Truncation of the hexose moiety of uridine-5′-diphosphoglucose and its replacement with alkyl and aryl groups

Article abstract of DOI:10.1021/jm901432g

Uridine-5′-diphosphoglucose (UDPG) activates the P2Y₁₄ receptor, a neuroimmune system GPCR. P2Y₁₄ receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine 5′-diphosphate (UDP), a full agonist at the human P2Y₁₄ receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y₁₄ vs the P2Y₆ receptor, such as 2-thio-UDP 4 (EC₅₀=1.92 nMat P2Y₁₄, 224-fold selectivity vs P2Y₆) and its β-propyloxy ester 18. EC₅₀ values of the β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. β-tert-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y₁₄ receptor agonist activity. α,β-Methylene and difluoromethylene groups were well tolerated at the P2Y₁₄ receptor and are expected to provide enhanced stability in biological systems. α,β-Methylene-2-thio-UDP 11 (EC₅₀ = 0.92 nM) was 2160-fold selective versus P2Y₆. Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y₁₄ receptor.

Full text of DOI:10.1021/jm901432g

J. Med. Chem. 2010, 53, 471–480 471
DOI: 10.1021/jm901432g
Human P2Y14 Receptor Agonists: Truncation of the Hexose Moiety of Uridine-50-Diphosphoglucose
and Its Replacement with Alkyl and Aryl Groups
Arijit Das,† Hyojin Ko,† Lauren E. Burianek,‡ Matthew O. Barrett,‡ T. Kendall Harden,‡ and Kenneth A. Jacobson*,†
†Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National
Institutes of Health, Bethesda, Maryland 20892 and ‡Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill,
North Carolina 27599
Received September 25, 2009
Uridine-50-diphosphoglucose (UDPG) activates the P2Y14 receptor, a neuroimmune system GPCR.
P2Y14 receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine
50-diphosphate (UDP), a full agonist at the human P2Y14 receptor expressed in HEK-293 cells.
2-Thiouracil derivatives displayed selectivity for activation of the human P2Y14 vs the P2Y6 receptor,
such as 2-thio-UDP 4 (EC50 = 1.92 nM at P2Y14, 224-fold selectivity vs P2Y6) and its β-propyloxy ester
18. EC50 values of the β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively.
β-tert-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl
esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South
methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y14 receptor agonist activity. R,β-Methylene
and difluoromethylene groups were well tolerated at the P2Y14 receptor and are expected to provide
enhanced stability in biological systems. R,β-Methylene-2-thio-UDP 11 (EC50 = 0.92 nM) was 2160-
fold selective versus P2Y6. Thus, these nucleotides and their congeners may serve as important
pharmacological probes for the detection and characterization of the P2Y14 receptor.
Introduction
P2Y14 receptor initially was identified as a UDP-sugar-acti-
vated GPCR, we recently discovered that UDP is at least as
potent as UDP-glucose and other nucleotide sugars for
activation of this receptor.14 Thus, it is perhaps not surprising
that our recent detailed SAR (structure-activity relationship)
analyses with synthetic analogues of UDPG12,13 revealed that
the glucose moiety is the least restricted region of the structure
for substitutions that maintain P2Y14 receptor agonist po-
tency. This realization, together with conclusions made from
our recent SAR study of UDP analogues at the P2Y6 recep-
tor,15 suggests that β-phosphate substitution in a new series of
UDP analogues might favor activation of the P2Y14 receptor.
Therefore, with the ultimate goal of identifying highly selec-
tive ligands for the P2Y14 and P2Y6 receptors, we have
synthesized a newseries of β-phosphate-substitutedanalogues
of UDP and compared the potencies of these novel molecules
as well as a number of previously prepared uracil nucleotide
analogues at the human P2Y14 and P2Y6 receptors.15
Although the glucose moiety of UDPG was suggested to
interact with multiple H-bonding and/or charged resides with-
in the putative binding site of the P2Y14 receptor, its deletion or
substitution with smaller phosphoester groups was tolerated at
this receptor. Simple alkyl esters at this position and ana-
logues of UDP displayed highly potent agonist activity at the
P2Y14 receptor. The effects of these modifications to preserve
and enhance potency at the P2Y14 receptor were additive with
the previously identified potency enhancing effect of 2-thio
substitution of the uracil moiety, achieving in some cases
nanomolar and subnanomolar potency. Importantly, a num-
ber of these new UDP analogues exhibit high selectivity for
activation of the P2Y14 receptor over the P2Y6 receptor.
Purine and pyrimidine mononucleotides and dinucleotides
have a role as extracellular signaling molecules, in addition to
their diverse intracellular roles.1 TheP2Y family of G-protein-
coupled receptors (GPCRs) responds to various extracellular
nucleotides to induce intracellular signaling cascades.2-4
The P2Y14 receptor was identified initially as an orphan
GPCR activated by uridine 50-diphosphoglucose (UDPGa
1, Chart 1) and other endogenous UDP sugars. This Gi-
coupled receptor is expressed in the brain and in dendritic
cells,5 although no functional role has yet been clearly
assigned in these tissues. The P2Y14 receptor also is expressed
in the placenta, adipose tissue, stomach, intestine, brain,
spleen, thymus, lung, and heart.1 Thus, the P2Y14 receptor
is of potential therapeutic interest for modulation of the
immune system,7 as well as treatment of pain,8 diabetes,9
cystic fibrosis, and other pulmonary diseases.10,11
A current goal of our research is to identify and apply
potent, selective, and stable P2Y14 receptor ligands to define
the physiological role(s) of this receptor.12-14 Although the
*To whom correspondence should be addressed. Phone: 301-496-
9024. Fax: 301-480-8422. E-mail: kajacobs@helix.nih.gov.
a Abbreviations: cAMP, 30,50-cyclic adenosine monophosphate;
CHO Chinese hamster ovary; DCC, dicyclohexylcarbodiimide;
DMEM, Dulbecco’s modified Eagle medium; DMF, dimethylforma-
mide; HEK, human embryonic kidney; HEPES, N-2-hydroxyethylpi-
perazine-N0-2-ethanesulfonic acid; HPLC, high performance liquid
chromatography; PLC, phospholipase C; SAR, structure-activity re-
lationship; TBAP, tributylammonium phosphate; TEAA, triethylam-
monium acetate; UDP, uridine 50-diphosphate; UDPG, uridine 50-
diphosphoglucose.
r
2009 American Chemical Society
Published on Web 11/10/2009
pubs.acs.org/jmc

472 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Das et al.
Chart 1. Structures of Two UDP-Sugars (1, 2) That Act as
P2Y14 Receptor Agonists, UDP 3, a Naturally Occurring
Ligand of the hP2Y14 Receptor, and Its Analogue 2-Thio-
UDP 4
commercially available alkyl monophosphates (41, 45). The
monophosphates were transformed to the correspon-
ding tributylammonium salts and were then activated with
1,10-carbonyldiimidazole in DMF for 5 h at room tempera-
ture followed by quenching of this reagent with methanol.
After removal of the solvent, the residue was dissolved in
DMF, and 2-thio or 4-thiouridine 50-monophosphate
tributylammonium salt was added to obtain compounds
15-18, 21, and 25 as shown in Scheme 2. All the nucleotide
analogues were characterized using HPLC, nuclear magnetic
resonance (1H NMR, 31P NMR), and high-resolution mass
spectrometry.
Quantification of Pharmacological Activity. Inhibition of
adenylyl cyclase was quantified in HEK-293 cells stably
expressing the hP2Y14 receptor. This cell line, as well as a
P2Y14 receptor-expressing Chinese hamster ovary (CHO)
cell line and a P2Y14 receptor-expressing C6 rat glioma cell
line that we recently developed, provides a physiologically
relevant test system, since receptor-promoted responses
mediated through a natively expressed heterotrimeric G
protein (Gi) and its natively expressed effector protein
(adenylyl cyclase) are quantified.14 We conclude that this
system is much preferable to assay methods, employed by us
and others, which utilized COS-7 cells transiently overex-
pressing the P2Y14 receptor with an engineered G protein,
GR-q/i protein (GRqi5), that allows coupling of Gi-coupled
receptors to activation of phospholipase C (PLC), resulting
in inositol lipid hydrolysis.22-24
Although UDPG 1 is usually thought of as a specific
agonist of the P2Y14 receptor, it also was found to activate
the P2Y2 receptor (EC50 ≈ 10 μM).13 We now report EC50
values for 1 of 0.40 (Figure 1A) and 16 μM at the P2Y14 and
P2Y6 receptor, respectively (Table 1). Therefore, although
selective for the P2Y14 receptor, UDPG also activates other
P2Y receptors. Thus, more potent and selective agonists,
preferably with a simplified chemical structure, are needed as
pharmacological probes of the P2Y14 receptor.
UDP 3 and its 4-thio derivative 5 were moderately potent
agonists at the hP2Y14 receptor. One of the most potent
P2Y14 receptor agonists in the current set of nucleotides was
the 2-thio derivative 4 of UDP, which displayed an EC50
value of 1.92 nM. Compound 4 was 83-fold more potent than
3 as a P2Y14 receptor agonist and 230-fold selective for the
P2Y14 receptor in comparison to the P2Y6 receptor. Replace-
ment of a terminal negatively charged oxygen of 3 with an
uncharged methyl group in the phosphonate derivative 6
resulted in a molecule with similarly weak potencies at the
two receptors. The N4-methyloxy cytidine derivative 7 was
20-fold less potent than 3 at the P2Y14 receptor and conse-
quently displayed selectivity for the P2Y6 receptor, at which
its high potency has been described.15 This finding is con-
sistent with the reported weak P2Y14 receptor activity of the
corresponding N4-methyloxy cytidine analogue of UDPG.13
Replacement of a ribose moiety of various P2Y receptor
agonists with a sterically constrained bicyclic ring has been
used to establish the receptor-preferred conformation.
Neither of the conformationally constrained methanocarba
analogues 8 (North, N) and 9 (South, S) of UDP 3 was active
at the P2Y14 receptor. Consistent with these findings with
analogues of 3, we have already established that the corre-
sponding (N)- and (S)-methanocarba analogues of UDPG
were both inactive at the P2Y14 receptor.13
Results
Chemical Synthesis. The principal objective of this study
was to structurally simplify the distal end of uracil nucleotide
sugars known to activate the hP2Y14 receptor and to com-
bine potency-enhancing modifications. By removal of the
glucose moiety of UDPG 1, this agonist is converted into
UDP 3, which we recently discovered is a highly potent
agonist of the hP2Y14 receptor.14 The activities of analogues
of 1 modified on the glucose moiety have been explored,
but intermediate structures and truncated analogues related
to UDP have not been systematically examined. We also
were searching for leads for UDP analogues that might
provide selectivity at the P2Y14 receptor over the P2Y6
receptor.
Simple ester and 50-diphosphate analogues of the hP2Y14
receptor agonist UDPG 1 and its 2-thio derivative 2 were
synthesized (Table 1). The only modifications of the nucleo-
base included were thiocarbonyl or methoxyamino substitu-
tion of carbonyl groups of the uracil moiety, which were
studied previously in P2Y14 receptor recognition.12 Several
of the analogues of UDP 3 examined here were reported in
studies of SAR at the P2Y6 receptor.15,17,18 All of the
nucleotide analogues were prepared in their ammonium or
triethylammonium salt form according to the methods
shown in Schemes 1-3 and tested in functional assays of
the hP2Y14 and hP2Y6 receptors (Table 1).
The uridine-50-diphospho alkyl or aryl β-ester derivatives were
obtained by the following methods. The corresponding alkyl
or aryl monophosphate analogues were treated successively
with cation-exchange resin and tributylamine. To a solution
of the alkyl or aryl monophosphate tributylammonium salt
in DMF, commercially available uridine 50-monophosphate
morpholidate 4-morpholine-N,N-dicyclohexylcarboxami-
dine salt was added to form uridine-50-diphospho-β-ester
analogues (6, 14, 19, 20, 22-24, and 26-30) in a condensa-
tion reaction as shown in Scheme 1. 2-Thio UDP 4 and 4-thio
UDP 5 were synthesized by using the previously reported
procedure.17 (N)-Methanocarba-UDP 8 and (S)-methano-
carba-UDP 9 were synthesized using our previously reported
procedures.15,19,20 R,β-Methylene-UDP 10 and R,β-difluoro-
methylene-UDP 12 were also prepared using our recently
published procedures or modifications thereof. 2-Thio-R,
β-methylene-UDP 11 was prepared by the DCC coupling of
2-thiouridine with methylenediphosphonic acid (Scheme 3).18
Compounds 7 and 13 were synthesized by using a recently
developed procedure.15
For the preparation of the 2- or 4-thiocarbonyl β-alkyl
diphosphate esters (15-18, 21, and 25), we either synthe-
sized the alkyl monophosphate (42-44)21 or obtained the
The introduction of carbon-bridged substitutions of the
phosphate moieties of P2 agonists has led to greater stability

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 473
Table 1. In Vitro Pharmacological Data for UDPG 1, UDP 3, and Their Analogues (Non-Sugar β-Phosphoesters and Other Derivatives of UDP) in the
Inhibition of cAMP Formation at Recombinant hP2Y14 Receptors Expressed in HEK-293 Cells Stably Transfected with the hP2Y14 and in the
Stimulation of PLC at Recombinant hP2Y6 Receptors Stably Expressed in 1321N1 cellsf

474 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Table 1. Continued
Das et al.
a Agonist potencies reflect stimulation of phospholipase C, unless otherwise noted, and were calculated using a four-parameter logistic equation and the
GraphPad software package (GraphPad, San Diego, CA). EC50 values (mean ( standard error) represent the concentration at which 50% of the maximal effect
is achieved. Relative efficacies (%) were determined by comparison with the effect produced by a maximal effective concentration of reference agonist (UDP-
glucose, 1) in the same experiment. If no maximal effect is given, then 100% efficacy was achieved. N = 3. b Potency at P2Y6 receptor as reported by Maruoka et
al.,15 Besada et al.,17 and Ko et al.18 Potency at P2Y14 receptor as reported in Ko et al.12,13 c <50% effect at 10 μM. d Structure is given below.
e 12, MRS2802; 18, MRS2907.14 f Unless otherwise noted: X, Y = O, Z = H. NE: no effect at 10 μM.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 475
Scheme 1. Synthesis of UDPG Analogues Including UDP β-Estersa
a Reagents and conditions: (a) ROPO3H2, DMF, room temp.
Scheme 2. (A) Synthesis of Thio-UMP Derivatives 34 and 35 and (B) Synthesis of Thio-UDP Derivatives 15-18, 21, and 25a
a Reagents and conditions for part A: (a) (i) POCl3, proton sponge, PO(OMe)3, 0 °C; (ii) 0.2 M triethylammonium bicarbonate, room
temp. Reagents and conditions for part B: (b) H3PO4, pyridine, Et3N, Ac2O; (c) (i) CDI, DMF, room temp; (ii) 5% TEA in 1/1 MeOH/H2O;
(d) 2-thiouridine 50-monophosphate tributylammonium salt 34 or 4-thiouridine 50-monophosphate tributylammonium salt 35, DMF, room
temp.
Scheme 3. Synthesis of R,β-Methylene-2-thio-UDP 11a
receptor, resulting in high potency with EC50 values of 11 and
63 nM, respectively (Figure 1B). Indeed, the R,β-difluorometh-
ylene analogue 12 was >2000-fold selective for the P2Y14
receptor in comparison to the P2Y6 receptor, at which it was
inactive at the concentrations tested. Combining a carbon
bridge with a known potency-enhancing uracil modification
in 2-thio-R,β-methylene analogue 11 resulted in unprecedented
potency at the hP2Y14 receptor with an EC50 value of 0.92 nM.
The selectivity of 11 in comparison to the hP2Y6 receptor was
2160-fold. Combining the R,β-difluoromethylene and 5-iodo
modifications in 13 maintained equipotency to UDP 3 at the
P2Y14 receptor, which stands in contrast with the inactivity of
5-iodo-UDPG and suggests nonidentical modes of uracil
binding to the receptor between the two series.12 However,
13 was not selective for the P2Y14 receptor when compared to
the P2Y6 receptor.
a Reagents and conditions: (a) DCC, DMF, room temp.
in biological systems due to the inability of ectonucleotidases
to cleave these groups.1,27 Replacement of the bridging oxy-
gen of the diphosphate group of UDP with a methylene 10 or
difluoromethylene 12 group was well tolerated at the P2Y14

476 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Das et al.
Figure 1. (A) Activation of Gi-coupled P2Y14 receptor (left panel) was assessed by quantification of inhibition of forskolin-stimulated
[3H]cyclic AMP accumulation in HEK-293 cells stabling expressing the hP2Y14 receptor. Activation of the Gq-coupled P2Y6 receptor (right
panel) was assessed by quantification of [3H]inositol phosphate accumulation in 1321N1 human astrocytoma cells stably expressing the hP2Y6
receptor by agonists Up2-OMe 14, 2-thio-Up2-OMe 15, and its O-propyl ester 18. (B) Activation of Gi-coupled P2Y14 receptor (left panel) was
assessed by quantification of inhibition of forskolin-stimulated [3H]cyclic AMP accumulation in HEK-293 cells stabling expressing the hP2Y14
receptor. Activation of the Gq-coupled P2Y6 receptor (right panel) was assessed by quantification of [3H]inositol phosphate accumulation in
1321N1 human astrocytoma cells stably expressing the hP2Y6 receptor by methylene-bridged agonist 10, its 2-thio analogue 11, and the
difluoromethylene-bridged agonist 12.
The native agonist 1 is a β-ester of the diphosphate moiety,
which prompted us to explore the potency of structurally
simpler β-esters at the P2Y14 receptor. Since compound 14,
modified with a methyl ester group instead of glucose,
weakly activated the P2Y14 receptor and was inactive at
the P2Y6 receptor, this methyl ester was combined with other
modifications to probe the effects on biological activity. The
combination with the 2-thio modification in compound 15
increased potency by 42-fold. Thus, compound 15 was
>180-fold selective for the P2Y14 receptor. The 4-thio
modification in methyl ester 16 abolished and greatly re-
duced potency in comparison to 15 at the P2Y14 and P2Y6
receptors, respectively.
the P2Y14 receptor. Substitution of the β-methyl ester with a
tert-butyl ester group in 25 produced an equipotent agonist,
which was 8-fold more potent than the corresponding 2-oxo
analogue 24. The β-tert-butyloxy ester of 2-thio-UDP 25 was
11-fold more potent than UDPG. Introduction of a glyceryl
moiety esterified through the 2-hydroxyl group in 23 did not
diminish the potency of 3, but an isomeric glyceryl ester 22
was 10-fold less potent than 3.
Alkynyl ester derivatives 20 (2-oxo) or 21 (2-thio) were
either 2-fold less potent or 16-fold more potent, respectively,
than 3 at the P2Y14 receptor. Compound 21 was >900-fold
selective in comparison to the P2Y6 receptor and was de-
signed for coupling to azide-containing molecules by click
chemistry.
In the 2-thio series, homologation of the alkyl ester group
of 15 was tolerated at the P2Y14 receptor, with the ethyl 17
and propyl 18 derivatives both displaying EC50 values of ∼40
nM and >200-fold selectivity in comparison to the P2Y6
receptor. A 2-cyanoethyl group in 19 was poorly tolerated at
β-Aryloxy and cycloalkyloxy esters 26-30 of UDP were
found to only weakly activate the receptor in the order of
potency phenyl > 4-nitrophenyl > 3-chloro- and 4-methoxy-
phenyl > cyclohexyl.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 477
Discussion
expressing an endogenous P2Y14 receptor to aid in delineating
a role for this receptor.
In the present study, we have further expanded the range of
potent P2Y14 receptor ligands through a systematic explora-
tion of the SAR of structurally simple analogues at this
receptor, particularly with respect to substitution of the
glucose moiety with diverse alkyl moieties. We have found
that the introductionofR,β-methylene and difluoromethylene
groups is well tolerated at the P2Y14 receptor. These carbon-
bridged nucleotides, including R,β-methylene-UDP with an
EC50 value of 11 nM, are expected to display greater stability
in biological systems. Methylene-bridged nucleotides tend to
have increased stability by impeding breakdown by ecto-
nucleotidases present on the cell surface,27-29 and thus, these
carbon-bridged diphosphate analogues have good potential
for in vivo applications.
The 2-thio modification, but not the 4-thio modification of
theuracil ring, tended toincrease potencyand selectivity atthe
P2Y14 receptor in comparison to the P2Y6 receptor. The most
selective compounds (fold selectivity for P2Y14 in comparison
to P2Y6) in the present study were UDP analogues 4 (233), 11
(2160), 12 (>150), 15 (>180), 17 (>250), 18 (230), and 21
(>900). The most potent of these were 2-thio-UDP 4 and R,β-
methylene-2-thio-UDP 11. Compound 4 had a high potency
at the P2Y14 receptor, with an EC50 value of 1.92 nM, and the
corresponding R,β-methylene analogue 11 was even twice as
potent. Although it was a less potent P2Y14 receptor agonist,
difluoromethylene-UDP12did not activate the P2Y6 receptor
at the concentrations of analogue tested. Other compounds
that displayed lesser selectivity for the P2Y14 receptor were
(fold) 25 (64), 23 (59), 24 (43), and 10 (31). Thus, the 2-thio
derivatives 4, 11, and their congeners may serve as important
pharmacological probes for the detection and characteriza-
tion of the P2Y14 receptor.
Experimental Section
1
Chemical Synthesis. H NMR spectra were obtained with a
Varian Gemini 300 spectrometer using D2O as a solvent. The
chemical shifts are expressed as relative ppm from HOD (4.80
ppm). 31P NMR spectra were recorded at room temperature by
use of a Varian XL 300 spectrometer (121.42 MHz); orthophos-
phoric acid (85%) was used as an external standard.
Purity and extent of reaction of nucleotide derivatives was
checked using a Hewlett-Packard 1100 HPLC equipped with a
Zorbax Eclipse 5 μm XDB-C18 analytical column (250 mm ꢀ
4.6 mm; Agilent Technologies Inc., Palo Alto, CA), linear
gradient solvent system of 5 mM TBAP (tetrabutylammonium
dihydrogenphosphate)-CH3CN from 80:20 to 40:60 in 20 min
with a flow rate of 1 mL/min (system A), or Zorbax SB-Aq 5 μm
analytical column (50 mm ꢀ 4.6 mm; Agilent Technologies Inc.,
Palo Alto, CA). Mobile phase for linear gradient solvent system
was 5 mMTBAP(tetrabutylammonium dihydrogenphosphate)-
CH3CN from 80:20 to 40:60 in 13 min. The flow rate was
0.5 mL/min (system B). Peaks were detected by UV absorption
(254 nm) with a diode array detector.
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 ammo-
nium bicarbonate as the mobile phase. Some of the compounds
were additionally purified by HPLC with a Luna 5 μm RP-
C18(2) semipreparative column (250 mm ꢀ 10.0 mm; Phenom-
enex, Torrance, CA) and using a linear gradient solvent system
of 10 mM TEAA (triethylammonium acetate)-CH3CN from
100:0 to 95:5 (or up to 99:1 to 90:10) in 30 min with a flow rate of
2 mL/min. The tested nucleotide derivatives were confirmed by
HPLC to possess a g95% purity. Compounds purified by
HPLC were isolated as the triethylammonium salts. Com-
pounds 41 and 45, other reagents, and solvents were purchased
from Sigma-Aldrich (St. Louis, MO).
It is now clear that the glucose moiety of 1 is not required for
activation of the P2Y14 receptor, although when it is present,
there is a SAR pattern related to interaction of specific func-
tionality of this hexose moiety with the receptor.13,26 We
conclude here that UDP and its analogues are potent full
agonists of the human P2Y14 receptor, consistent with our
recent pharmacological studies of this receptor stably expressed
in three different cell lines that utilized native Gi proteins for
signal transduction.14 This finding contradicts the original
work of Fricks et al. who reported that UDP is a partial
agonist/competitive antagonist of the human P2Y14 receptor
while acting as a full agonist at the rat P2Y14 receptor.16
However, the latter study quantified activity of a recombinant
P2Y14 receptor transiently coexpressed with an unnatural
chimeric G protein, GRq/i, that couples Gi-activating recep-
tors to activation of PLC-β isozymes. It was suggested that this
system favors “a conformation of the P2Y14-R..., which results
in ligand binding selectivities and agonist activities that are not
altogether consistent with activities of the receptor obtained
when coupled to its cognate heterotrimeric G protein”.14 Thus,
the efficacy of UDP appears to be a function of the G protein
signaling system activated by the receptor.
General Procedure for the Preparation of Uridine 50-Diphos-
phoglucose Analogues. The appropriate alkyl or aryl monophos-
phate was first converted to its tributylammonium salt form by
treatment with ion-exchange resin (DOWEX 50WX2-200 (H)),
which upon acidification of the supernatant was followed by
addition of tributylamine until a basic pH was reached. The
aqueous solvent was removed by lyophilization, and the residue
was used without further purification. A solution in DMF
(1.5 mL) of uridine 50-monophosphate morpholidate 4-morpho-
line-N,N-dicyclohexylcarboxamidine salt (20 mg, 0.029 mmol)
and the corresponding monophosphate (0.035 mmol, tributyl-
ammonium salt, was stirred at room temperature for 2 days.
After that the solvent was removed and the residue was purified
by ion-exchange column chromatography using a Sephadex-
DEAE A-25 resin (with a linear gradient (0.01-0.5 M) of 0.5 M
ammonium bicarbonate as the mobile phase) to provide the
corresponding nucleotide as the ammonium salt. Some of the
uridine 50-diphosphoglucose analogues were additionally puri-
fied by HPLC as described above.
Diphosphoric Acid 1-β-Methyl (C-P) 2-(Uridine-50-yl)ester,
Triethylammonium Salt (6). Compound 6 (4.0 mg, 23%) was
obtained as a white solid following the general procedure. H
1
NMR (D2O) δ 7.96 (d, J = 8.1 Hz, 1H), 6.00 (d, J = 4.8 Hz, 1H),
5.97 (d, J = 7.8 Hz, 1H), 4.38 (m, 2H), 4.29 (m, 1H), 4.21 (m,
2H), 1.46 (d, J = 16.8 Hz, 3H); 31P NMR (D2O) δ 17.82 (d, J =
23.9 Hz), -11.06 (d, J = 23.8 Hz); HRMS-EI found 401.0158
(M - Hþ)-. C10H15N2O11P2 requires 401.0151; purity >98%
by HPLC (system A, 13.3 min).
Diphosphoric Acid 1-β-Methyl Ester 2-(Uridine-50-yl)ester,
Ammonium Salt (14). Compound 14 (3.9 mg, 30%) was obtained
as a white solid following the general procedure. 1H NMR
(D2O) δ 7.97 (d, J = 8.1 Hz, 1H), 6.01 (dd, J = 3.6, 1.2 Hz,
In conclusion, we have identified new analogues of UDP
and its simple esters that display enhanced potency and
selectivity for the P2Y14 receptor and that promise to be useful
as pharmacological tools to distinguish the effects of uracil
nucleotides acting at P2Y14 versus P2Y6 receptors. The na-
nomolar potency achieved in this series is until now unprece-
dented for small molecular ligands of the P2Y14 receptor. It is
now possible to examine these potent agonists on human cells

478 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Das et al.
1H), 5.97 (d, J = 8.1 Hz, 1H), 4.38 (m, 2H), 4.29 (m, 1H), 4.20
(m, 2H), 3.66 (d, J = 11.4 Hz, 3H); 31P NMR (D2O) δ -9.22 (d,
J = 21.4 Hz), -11.03 (d, J = 21.4 Hz); HRMS-EI found
417.0087 (M - Hþ)-. C10H15N2O12P2 requires 417.0100; purity
>98% by HPLC (system A, 11.3 min).
(M - Hþ)-. C15H16N3O14P2 requires 524.0108; purity >98%
by HPLC (system A, 13.7 min).
Diphosphoric Acid 1-β-(3-Chlorophenyl) Ester 2-(Uridine-50-
yl)ester, Ammonium Salt (29). Compound 29 (3.7 mg, 25%) was
obtained as a white solid following the general procedure. H
1
Diphosphoric Acid 1-β-(2-Cyanoethyl) Ester 2-(Uridine-50-
yl)ester, Ammonium Salt (19). Compound 19 (3.7 mg, 26%)
was obtained as a white solid following the general procedure.
1H NMR (D2O) δ 7.99 (d, J = 8.4 Hz, 1H), 5.99 (m, 2H),
4.89 (m, 2H), 4.26 (m, 3H), 4.16 (dd, J = 12.9, 6.0 Hz, 2H), 2.87
(t, J = 6.07 Hz, 2H); 31P NMR (D2O) δ -11.18 (dd, J = 42.8,
21.4 Hz); HRMS-EI found 456.0212 (M - Hþ)-. C12H16N3-
O12P2 requires 456.0209; purity >98% by HPLC (system A,
12.8 min).
NMR (D2O) δ 7.85(d, J = 8.1 Hz, 1H), 7.33 (m, 3H), 7.17 (m,
1H), 5.95 (d, J = 4.2 Hz, 1H), 5.76 (d, J = 8.1 Hz, 1H), 4.29 (m,
4H), 4.18 (m, 1H); 31P NMR (D2O) δ -11.04 (d, J = 21.3 Hz),
-16.18 (d, J = 21.3 Hz); HRMS-EI found 512.9867 (M - Hþ)-.
C15H16N2O12ClP2 requires 512.9854; purity >98% by HPLC
(system B, 8.6 min).
Diphosphoric Acid 1-β-(4-Methoxyphenyl) Ester 2-(Uridine-
50-yl)ester, Ammonium Salt (30). Compound 30 (3.3 mg, 21%)
was obtained as a white solid following the general procedure.
1H NMR (D2O) δ 7.82 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 8.7 Hz,
2H), 6.93 (d, J = 8.7 Hz, 2H), 5.95 (d, J = 4.8 Hz, 1H), 5.77 (d,
J = 8.1 Hz, 1H), 4.29 (m, 4H), 4.16 (m, 1H); 31P NMR (D2O)
δ -11.04 (d, J = 21.3 Hz), -15.37 (d, J = 21.3 Hz); HRMS-EI
found 509.0362 (M - Hþ)-. C16H19N2O13P2 requires 509.0353;
purity >98% by HPLC (system B, 8.84 min).
Diphosphoric Acid 1-β-(3-Butynyl) Ester 2-(Uridine-50-yl)-
ester, Triethylammonium Salt (20). Compound 20 (5.31 mg,
28%) was obtained as a white solid following the general
1
procedure. H NMR (D2O) δ 7.88 (d, J = 8.1 Hz, 1H), 5.98
(m, 2H), 4.41 (m, 1H), 4.21 (m, 3H), 4.08 (m, 1H), 3.83 (m, 2H),
2.53 (m, 3H); 31P NMR (D2O) δ -9.61 (d, J = 16.5 Hz), -11.2
(m); HRMS-EI found 455.0257 (M - Hþ)-. C13H17N2 O12P2
requires 455.0265; purity >97% by HPLC (system B, 6.89 min).
Diphosphoric Acid 1-r-Glycerol Ester 2-(Uridine-50-yl)ester,
Ammonium Salt (22). Compound 22 (2.2 mg, 15%) was obtained
as a white solid following the general procedure. 1H NMR
(D2O) δ 7.96 (d, J = 8.4 Hz, 1H), 5.60 (m, 2H), 4.40 (m, 2H),
4.26 (m, 3H), 3.99 (m, 3H), 3.68 (m, 2H); 31P NMR (D2O)
δ -10.35 (d, J = 21.4 Hz), -10.97 (d, J = 20.8 Hz); HRMS-EI
found 477.0315 (M - Hþ)-. C12H19N2O14P2 requires 477.0312;
purity >98% by HPLC (system A, 13.6 min).
General Procedure for the Preparation of Diphosphoric Acid 1-
Alkyl Ester 2-(Thiouridine-50-yl)ester (15-18, 21, and 25).
Monophosphates (42-44) were prepared from the correspond-
ing alcohols (37-39) using a published procedure,21 and the
spectral data were consistent with the assigned structures.
Each of the prepared monophosphates (42-44)21 or commer-
cially available monophosphates (41, 45) was transformed to its
tributylammonium salt by treatment with ion-exchange resin fol-
lowed by the addition of tributylamine. 1,10-Carbonyldiimidazole
(10 mg, 0.06 mmol) was added to the alkyl monophosphate
tributylammonium salts (0.017 mmol) (41-45) in DMF (1 mL).
The reaction mixture was stirred at room temperature for 5 h.
Methanol (1 mL) was added, and stirring was continued at room
temperature for an additional 1 h. After removal of the solvent, the
residue was dried under high vacuum and dissolved in DMF
(1.5 mL). 2-Thiouridine 50-monophosphate tributylammonium salt
(34)12 (5.7 mg, 0.008 mmol) for 15,17,18,21,and25 or 4-thiouridine
50-monophosphate tributylammonium salt (35)12 (5.7 mg, 0.008
mmol) for 16 was added to the reaction mixture, and it was stirred at
room temperature for 2 days. After removal of the solvent, the
residue was purified by the same method as the general procedure
using Sephadex-DEAE A-25 resin. Compounds 16 and 21 were
further purified by HPLC to provide homogeneous products.
Diphosphoric Acid 1-β-Methyl Ester 2-(2-Thiouridine-50-yl)-
ester, Ammonium Salt (15). Compound 15 (0.82 mg, 22%) was
Diphosphoric Acid 1-β-Glycerol Ester 2-(Uridine-50-yl)ester,
Triethylammonium Salt (23). Compound 23 (3.1 mg, 16%) was
obtained as a white solid following the general procedure. H
1
NMR (D2O) δ 7.97 (d, J = 7.8 Hz, 1H), 6.01 (m, 1H), 5.98 (d,
J = 7.8 Hz, 1H), 4.39 (m, 2H), 4.30 (m, 2H), 4.24 (m, 2H), 3.77
(m, 4H); 31P NMR (D2O) δ -10.89 (dd, J = 37.9, 21.4 Hz);
HRMS-EI found 477.0302 (M - Hþ)-. C12H19N2O14P2 requires
477.0312; purity >98% by HPLC (system A, 11.2 min).
Diphosphoric Acid 1-β-tert-Butyl Ester 2-(Uridine-50-yl)ester,
Ammonium Salt (24). Compound 24 (4.4 mg, 31%) was obtained
as a white solid following the general procedure. 1H NMR
(D2O) δ 7.99 (d, J = 7.8 Hz, 1H), 5.99 (m, 2H), 4.39 (m, 2H),
4.29 (m, 1H), 4.24 (m, 2H), 1.44 (d, J = 0.3 Hz, 9H); 31P NMR
(D2O) δ -11.51 (d, J = 21.4 Hz), -14.73 (d, J = 21.4 Hz);
HRMS-EI found 459.0565 (M - Hþ)-. C13H21N2O12P2 re-
quires 459.0570; purity >98% by HPLC (system A, 13.6 min).
Diphosphoric Acid 1-β-Cyclohexyl Ester 2-(Uridine-50-yl)e-
ster, Ammonium Salt (26). Compound 26 (6.2 mg, 41%) was
1
obtained as a white solid following the general procedure. H
NMR (D2O) δ 8.15 (d, J = 8.1 Hz, 1H), 6.70 (d, J = 3.6 Hz, 1H),
6.24 (d, J = 8.1 Hz, 1H), 4.43 (m, 1H), 3.34 (m, 3H), 4.24 (m,
1H), 3.66 (d, J = 11.7 Hz, 3H); 31P NMR (D2O) δ -10.68 (dd,
J = 21.5, 6.6 Hz), -11.05 (d, J = 21.4 Hz); HRMS-EI found
432.9886 (M - Hþ)-. C10H15N2O11P2S requires 432.9872;
purity >98% by HPLC (system A, 12.9 min).
1
obtained as a white solid following the general procedure. H
NMR (D2O) δ 8.00 (d, J = 8.4 Hz, 1H), 6.00 (m, 2H), 4.40 (m,
2H), 4.30 (m, 1H), 4.24 (m, 2H), 4.20 (m, 1H), 1.97 (m, 2H), 1.72
(m, 2H), 1.50-1.10 (m, 6H); 31P NMR (D2O) δ -11.26; HRMS-
EI found 485.0719 (M - Hþ)-. C15H23N2O12P2 requires
485.0726; purity >98% by HPLC (system A, 11.8 min).
Diphosphoric Acid 1-β-Phenyl Ester 2-(Uridine-50-yl)ester,
Ammonium Salt (27). Compound 27 (4.7 mg, 32%) was obtained
as a white solid following the general procedure. 1H NMR
(D2O) δ 7.87 (d, J = 8.4 Hz, 1H), 7.36 (m, 2H), 7.19 (m, 3H),
5.93 (d, J = 5.1 Hz, 1H), 5.81 (d, J = 8.4 Hz, 1H), 4.30 (m, 1H),
4.25 (m, 2H), 4.20 (m, 1H), 4.15 (m, 1H); 31P NMR (D2O) δ
-11.11 (d, J = 21.5 Hz), -15.74 (d, J = 20.8 Hz); HRMS-EI
found 479.0247 (M - Hþ)-. C15H17N2O12P2 requires 479.0257;
purity >98% by HPLC (system A, 13.1 min).
Diphosphoric Acid 1-β-(4-Nitrophenyl) Ester 2-(Uridine-50-
yl)ester, Ammonium Salt (28). Compound 28 (6.1 mg, 38%)
was obtained as a white solid following the general procedure.
1H NMR (D2O) δ 8.21 (d, J = 9.3 Hz, 2H), 7.76 (d, J = 7.83 Hz,
1H), 7.37 (d, J = 9.0 Hz, 2H), 5.92 (m, 1H), 5.69 (d, J = 7.5 Hz,
1H), 4.25 (m, 4H), 4.14 (m, 1H); 31P NMR (D2O) δ -11.15 (d,
J = 20.8Hz), -17.12 (d, J= 20.8 Hz); HRMS-EI found 524.0110
Diphosphoric Acid 1-β-Methyl Ester 2-(4-Thiouridine-50-yl)-
ester, Triethylammonium Salt (16). Compound 16 (0.91 mg, 18%)
was obtained as a white solid following the general procedure. 1H
NMR (D2O) δ 7.9 (d, J = 7.7 Hz, 1H), 6.69 (d, J = 4.3 Hz, 1H),
5.99(d, J = 7.5 Hz, 1H), 4.40 (m, 3H), 4.30 (m, 2H), 3.69 (m, 3H);
31P NMR (D2O) δ -10.83 (d, J = 21.3, Hz), -11.12 (d, J = 21.3
Hz); HRMS-EI found 432.9884 (M - Hþ)-. C10H15N2O11P2S
requires 432.9872; purity >98% by HPLC (system B, 7.2 min in
negative absorbance).
Diphosphoric Acid 1-β-Ethyl Ester 2-(2-Thiouridine-50-yl)-
ester, Ammonium Salt (17). Compound 17 (7.7 mg, 20%) was
obtained as a white solid following the general procedure. H
1
NMR (D2O) δ 8.22 (d, J = 8.1 Hz, 1H), 6.72 (d, J = 3.0 Hz, 1H),
6.32 (d, J = 8.4 Hz, 1H), 4.49 (m, 1H), 3.39 (m, 3H), 4.29 (m,
1H), 4.09 (m, 2H), 1.33 (m, 3H); 31P NMR (D2O) δ -10.39 (t,
J = 21.3, 42.7 Hz), -11.08 (d, J = 21.3 Hz); HRMS-EI found
447.0022 (M - Hþ)-. C11H17N2O11P2S requires 447.0028;
purity >98% by HPLC (system B, 7.09 min).

Article
Diphosphoric Acid 1-β-Propyl Ester 2-(2-Thiouridine-50-yl)-
ester, Ammonium Salt (18). Compound 18 (0.91 mg, 23%) was
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 479
serum-free DMEM for at least 2 h prior to assay. Assays were
initiated by addition of HEPES-buffered, serum-free DMEM
containing 200 μM 3-isobutyl-1-methylxanthine (IBMX) and
30 μM forskolin, with or without drugs, and incubation con-
tinued for 15 min at 37 °C. Incubations were terminated by
aspiration of the medium and addition of 450 mL of ice-cold 5%
trichloroacetic acid. [3H]cAMP was isolated by sequential Dowex
and alumina chromatography and quantified by liquid scintillation
counting14,16 as previously described by Harden et al.30
1
obtained as a white solid following the general procedure. H
NMR 1H NMR (D2O) δ 8.21 (d, J = 8.4 Hz, 1H), 6.69 (d, J =
3.1 Hz, 1H), 6.28 (d, J = 8.4 Hz, 1H), 4.47 (m, 1H), 3.41 (m, 3H),
4.26 (m, 1H), 3.95 (m, 2H), 1.65 (m, 2H); 0.96 (m, 3H); 31P NMR
(D2O) δ -10.68 (d, J = 21.5, 4.), -11.10 (d, J = 21.3 Hz);
HRMS-EI found 461.0145 (M - Hþ)-. C12H19N2O11P2S re-
quires 461.0151; purity >98% by HPLC (system B, 7.4 min).
Diphosphoric Acid 1-β-(3-Butynyl) Ester 2-(2-Thiouridine-
50-yl)ester, Triethylammonium Salt (21). Compound 21(1.18
mg, 22%) was obtained as a white solid following the general
procedure. 1H NMR (D2O) δ 8.12 (d, J = 8.1 Hz, 1H), 6.79 (d,
J = 3.1 Hz, 1H), 6.23 (d, J = 8.1 Hz, 1H), 4.43 (m, 1H), 4.35
(m, 1H), 4.10 (m, 3H), 3.89 (m, 2H), 2.54 (m, 2H), 2.41 (m,
1H); 31P NMR (D2O) δ -9.63 (d, J = 21.3), -11.12 (d, J =
21.3 Hz); HRMS-EI found 471.0022 (M - Hþ)-. C13H17N2-
O11P2S requires 471.0035; purity >97% by HPLC (system B,
6.9 min).
Data Analysis. Agonist potencies (EC50 values) were obtained
from concentration-response curves by nonlinear regression
analysis using the GraphPad software package Prism
(GraphPad, San Diego, CA). All experiments were performed
in triplicate assays and repeated at least three times. The results
are presented as the mean ( SEM from multiple experiments or
in the case of concentration-effect curves from a single experi-
ment carried out with triplicate assays that were representative
of results from multiple experiments.
Diphosphoric Acid 1-β-tert-Butyl Ester 2-(2-Thiouridine-50-
yl)ester, Ammonium Salt (25). Compound 25 (0.83 mg, 21%)
was obtained as a white solid following the general procedure.
1H NMR (D2O) δ 8.26 (d, J = 8.4 Hz, 1H), 6.68 (d, J = 3.0 Hz,
1H), 6.26 (d, J = 8.1 Hz, 1H), 4.41 (m, 1H), 3.36 (m, 3H), 4.27
(m, 1H), 1.47 (s, 9H); 31P NMR (D2O) δ -11.00 (d, J = 21.3,
Hz), -11.53 (d, J = 21.3 Hz); HRMS-EI found 475.0341 (M -
Hþ)-. C13H21N2O11P2S requires 475.0341; purity >98% by
HPLC (system B, 7.16 min).
Acknowledgment. Mass spectral measurements were car-
ried out by Dr. John Lloyd and Dr. Noel Whittaker
(NIDDK). We thank Dr. Andrei A. Ivanov and Dr. Stefano
Costanzi (NIDDK) for helpful discussion and Dr. Sonia de
Castro (NIDDK) for technical assistance. This research was
supported in part by the Intramural Research Program of the
NIH, National Institute of Diabetes and Digestive and Kid-
ney Diseases. This work was supported by National Institutes
of Health Grant GM38213 to T.K.H.
Preparation of 2-Thiouridine-50-r,β-methylene Diphosphate,
Triethylammonium Salt (11). A solution of 2-thiouridine (13 mg,
0.05 mmol) and DCC (30 mg 0.15 mmol) was stirred in DMF
under a nitrogen atmosphere. Methylenediphosphonic acid (13
mg, 0.075 mmol) was added, and the stirring was continued for
48 h. The solvent was removed, and the product was purified by
ion exchange column chromatography with a Sephadex-DEAE
A-25 resin, followed by semipreparative HPLC as described
above. Compound 11 (11.8 mg, 33%) was obtained as a white
solid. 1H NMR (D2O) δ 8.22 (d, J = 7.8 Hz, 1H), 6.65 (d, J =
3.2 Hz, 1H), 6.28 (d, J = 7.8 Hz, 1H), 4.47 (m, 2H), 4.37 (m, 2H),
4.26 (m, 1H), 1.47 (t, J = 19.2 Hz, 2H); 31P NMR (D2O) δ 16.70,
Supporting Information Available: NMR spectral data and
HPLC traces of selected nucleotide derivatives. This material is
available free of charge via the Internet at http://pubs.acs.org.
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