Structure-activity relationship of uridine 5′-diphosphoglucose analogues as agonists of the human P2Y14 receptor

Article abstract of DOI:10.1021/jm061222w

UDP-glucose (UDPG) and derivatives are naturally occurring agonists of the G_i protein-coupled P2Y₁₄ receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing novel ligands in conjunction with modeling. The recombinant human P2Y₁₄ receptor expressed in COS-7 cells was coupled to phospholipase C through an engineered Gα-q/i protein. Most modifications of the uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a 2-thiouracil modification in 15 (EC₅₀ 49 ± 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound 15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple H-bonding and charged residues and provides a fertile region for agonist modification.

Full text of DOI:10.1021/jm061222w

2030
J. Med. Chem. 2007, 50, 2030-2039
Structure-Activity Relationship of Uridine 5′-Diphosphoglucose Analogues as Agonists of the
Human P2Y14 Receptor
Hyojin Ko,† Ingrid Fricks,‡ Andrei A. Ivanov,† 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 October 18, 2006
UDP-glucose (UDPG) and derivatives are naturally occurring agonists of the Gi protein-coupled P2Y14
receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel
analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing
novel ligands in conjunction with modeling. The recombinant human P2Y14 receptor expressed in COS-7
cells was coupled to phospholipase C through an engineered GR-q/i protein. Most modifications of the
uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a
2-thiouracil modification in 15 (EC50 49 ( 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic
acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound
15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism
of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple
H-bonding and charged residues and provides a fertile region for agonist modification.
Chart 1. Structures of Three Naturally Occurring UDP-Sugars
(1, 2, and 4a) and Related Derivatives That Activate the P2Y14
Receptor
Introduction
The nucleotide-activated P2 receptors occur in two sub-
families of plasma membrane receptors. P2Y receptor sub-
types include eight members, the P2Y1, P2Y2, P2Y4, P2Y6,
P2Y11, P2Y12, P2Y13, and P2Y14 receptors, which couple to G
proteins. The seven subtypes of ligand-gated cation channels
that comprise the P2X receptor subfamily are denoted P2X1-
P2X7. All of these subtypes have been cloned and functionally
characterized.1-10 The family of P2Y receptors can be divided
into two subgroups. A P2Y1-like subgroup (P2Y1, P2Y2, P2Y4,
P2Y6, P2Y11) is Gq-coupled and stimulates phospholipase C
(PLCa), and a P2Y12-like subgroup (P2Y12, P2Y13, P2Y14) is
Gi coupled and inhibits adenylyl cyclase.11 P2Y receptors are
distributed in a broad range of tissues and are of interest as
therapeutic targets, including antithrombotic therapy, modulation
of the immune system and cardiovascular system, inflammation,
pain, diabetes, and treatment of cystic fibrosis and other
pulmonary diseases.12-15 P2X receptors are activated principally
by adenine nucleotides, and P2Y receptors are activated by
adenine and/or uracil nucleotides. For example, the P2Y2
receptor is activated by both uridine 5′-triphosphate (UTP) and
adenosine 5′-triphosphate (ATP), the P2Y4 receptor by UTP,
and the P2Y6 receptor by uridine 5′-diphosphate (UDP).16
The P2Y14 receptor is distributed in various tissues including
placenta, adipose, stomach, intestine, brain, spleen, thymus, lung,
and heart.3 Potent and selective ligands, currently lacking, are
needed for pharmacological studies. UDP-glucose (1, UDPG),
UDP-galactose (2), and UDP-N-acetylglucosamine (4a) are
naturally occurring agonists of the P2Y14 receptor (Chart 1).
The precise physiological function of the P2Y14 receptor has
not yet been clearly established, although it appears to be
involved in immune function.17 In contrast to other P2Y
receptors, the P2Y14 receptor is not activated by uridine 5′-di-
or triphosphates or by adenine nucleotides.21 Extracellular
release of UDPG has been demonstrated, and UDPG does not
activate any other P2Y receptor.17-23 The SAR (structure-
activity relationship) of synthetic nucleotides for activation of
the human P2Y14 receptor has not previously been probed in a
systematic fashion. Thus, we synthesized novel analogues of
UDPG and characterized their pharmacological activities at the
recombinant human P2Y14 receptor. The nucleobase, ribose, and
glucose moieties were modified in this study. Molecular
modeling was carried out to predict sites of interaction of the
ligands with the P2Y14 receptor and to provide recognition
hypotheses to be used in the design of additional analogues.
* To whom correspondence should be addressed. Phone: (301) 496-
3939. Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
† National Institutes of Health.
Results and Discussion
‡ University of North Carolina School of Medicine.
a Abbreviations: DCC, dicyclohexylcarbodiimide; DMF, dimethylform-
amide; MCMM, Monte Carlo multiple minimum; PLC, phospholipase C;
SAR, structure-activity relationship; UDPG, UDP-glucose; MD, molecular
dynamics.
Chemical Synthesis. Analogues of UDPG (1) with modifica-
tions in the ribose ring, uracil moiety, and sugar moiety, as well
as dinucleotides (Table 1), were synthesized. The ribose ring
10.1021/jm061222w CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/04/2007

Agonists of the Human P2Y14 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2031
Table 1. In Vitro Pharmacological Data for UDPG, 1, and Its Analogues in the Stimulation of PLC in COS-7 Cells Expressing Recombinant Human
P2Y14 Receptors
a COS-7 cells were transiently transfected with expression vectors for both the human P2Y14 receptor and an engineered GR-q/i protein that allows
coupling of Gi-coupled receptors to activation of PLC.35 Agonist potencies 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. N ) 3, unless noted. NE ) no effect at 100 µM. b 13, MRS2670; 15, MRS2690 (n ) 4). c Less than or equal to 50% of the maximal effect at 10
µM. d Exhibited a maximal effect somewhat less than 1 in some but not all experiments. e 100% of the maximal effect of 1 was achieved. f Compound 12 )

2032 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Ko et al.
Scheme 1. Synthesis of UDPG Analogues Substituted on the Ribose Moiety or the 2 Position of the Uracil Moietya
a Reagents and conditions: (a) (i) POCl3, proton sponge, PO(OMe)3, 0 °C; (ii) 0.2 M triethylammonium bicarbonate, rt; (b) CF3CO2Et, DIEA, DMF, rt;
(c) (i) 1,1′-carbonyldiimidazole, DMF, rt; (ii) Et3N 5% in H2O/MeOH 1/1, rt; (iii) glucose-1-monophosphate tributylammonium salt, DMF, rt; (d) H2, Pd/C,
MeOH, rt; (e) (i) morpholine, DCC, t-BuOH, H2O, reflux; (ii) glucose-1-monophosphate tributylammonium salt, DMF, rt.
Scheme 2. Synthesis of UDPG Analogues Substituted on the 4
Scheme 3. Synthesis of an (S)-Methanocarba Analogue of
2′-Deoxy-UDPGa
or 5 Position of the Uracil Moietya
a Reagents and conditions: (a) (i) 1,1′-carbonyldiimidazole, DMF, rt;
(ii) Et3N 5% in H2O/MeOH 1/1, rt; (iii) glucose-1-monophosphate tri-
butylammonium salt, DMF, rt.
a Reagents and conditions: (a) (i) 1,1′-carbonyldiimidazole, DMF, rt;
(ii) Et3N 5% in H2O/MeOH 1/1, rt; (iii) glucose-1-monophosphate tri-
butylammonium salt, DMF, rt; (b) (i) 0.25 M NaOH, H2O, MeOH, rt; (ii)
iodomethane, DMF, rt.
at room temperature, followed by reacting with an excess of
iodomethane at room temperature (Scheme 2).27 All synthesized
and commercially available nucleoside 5′-monophosphate ana-
logues were passed through cation-exchange resin and neutral-
ized with tributylamine. The salt form was changed to a
tributylammonium salt to allow the subsequent reaction to
proceed in an anhydrous organic solvent. The nucleoside 5′-
monophosphate was activated with 1,1′-carbonyldiimidazole in
DMF for 6 h at room temperature. After the reaction was
quenched and the intermediate containing a 2′-cyclic carbonyl
group was hydrolyzed with methanol and triethylamine, the
activated intermediate was directly condensed with a glucose
1-monophosphate tributylammonium salt in DMF to afford the
corresponding nucleoside-5′-diphosphoglucose derivative (5, 6,
8-14, 16, 19, 22, 24).28,29 Several intermediates, including
inositol-1-monophosphate, fructose-1-monophosphate, and man-
nose-1-monophosphate, were condensed with uridine-5′-mono-
phosphoimidazolide to afford the compounds 25-27.
The monophosphorylation of the 2′-amino-2′-deoxyuridine
32 using the above method provided only the undesired
3′-monophosphate derivative.27 As an alternate approach to
synthesize 2′-deoxy-2′-aminouridine-5′-diphosphoglucose (7),
the 2′-trifluoroacetylamino derivative 33, formed after protection
of the amino group in 32 with a trifluoroacetyl group, was
phosphorylated to give 38. However, the protecting group of
38 was removed during the reaction to activate the phosphate
with 1,1′-carbonyldiimidazole, and this reagent formed a side
product containing a cyclic carbonyl group between the 2′-amino
and 3′-oxygen. This intermediate was the precursor of compound
was modified by removal of the hydroxyl group at the 2′ position
5 and replacement of the hydroxyl at the 2′ position with various
functional groups, 6-8, 10, and 11. The hydroxyl group at the
3′ position was absent in compounds 9 and 10. Replacement of
the ribose ring with a 2′-deoxy (S)-methanocarba bicyclic ring24
(12) also was performed. The uracil ring was modified at the 2
and 4 positions with the synthesis of 4-thiouridine-5′-diphos-
phoglucose (13) and 2-thiouridine-5′-diphosphoglucose (15)25
and at the 5 position with iodide (16) and fluoride (24). Sugar-
modified compounds (25-27) were synthesized with com-
mercially available sugar monophosphates. Dinucleotides (29)
were obtained as byproducts during the synthesis of UDPG
analogues and of CDP-glucose 19. All 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 P2Y14 receptor (Table 1). The
nucleotide analogues were characterized using HPLC, nuclear
magnetic resonance (1H NMR, 31P NMR), and high-resolution
mass spectrometry.
The nucleoside-5′-diphosphoglucose derivatives were ob-
tained by the following methods. 5′-Monophosphorylation by
reaction of the nucleoside with phosphorus oxychloride provided
the nucleoside 5′-monophosphate analogues 36-40 (Scheme
1).26 4-Methylthiouridine-5′-monophosphate (42) was obtained
by the treatment of 41 with 0.25 M NaOH in methanol for 2 h

Agonists of the Human P2Y14 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2033
8, and it was necessary to prepare compound 7 by another route,
that is, by reduction of the 2′-deoxy-2′-azidouridine-5′-diphos-
phoglucose (6) with H2 and Pd/C.
1,1′-Carbonyldiimidazole activated not only the 5′-phosphate,
but also partially activated the 2′-hydroxy group of the
intermediate 3′-deoxyuridine-5′-monophosphate (39). The ac-
tivated 2′-(imidazole-1-carbonyl) group was transformed to a
methylcarbonate group when it was exposed to MeOH. After
these two transient intermediates were reacted with glucose
1-monophosphate, compounds 9 and 10 were readily separated.
When compound 36 was treated with 1,1′-carbonyldiimidazole,
a cyclic 3′,5′-monophosphate imidazolide was produced instead
of the desired 5′-monophosphate imidazolide. Alternatively,
compound 15 could be prepared via the nucleoside 5′-phos-
phoromorpholidate using morpholine and DCC,30 followed by
the same method of condensing with glucose 1-monophosphate
(Scheme 1).
Scheme 3 shows the synthesis of a sterically constrained
analogue of 2′-deoxy-UDPG, the (S)-methanocarba analogue
12. This analogue was prepared in an attempt to define the
preferred conformation of the ribose moiety in the receptor
binding site. The corresponding 2′-hydroxy analogue, which
perhaps would be more definitive, was not included in this study.
To test the ability to combine the potency enhancing 2-thio
modification with a substituted hexose moiety, 2-thio-UDP-
glucuronic acid 28, analogue was prepared. The method used
was an adaptation of a one-step selective enzymatic oxidation
starting from the corresponding glucose derivative 15, and the
product was purified using HPLC.41
Figure 1. Activation by potent agonists 13 (A) and 15 (B) of PLC in
COS-7 cells expressing both the human P2Y14 receptor and an
engineered GR-q/i protein that allows the Gi-coupled receptor to
stimulate inositol phosphate hydrolysis by PLC.
Quantification of Pharmacological Activity. Activation of
PLC was quantified in COS-7 cells transiently expressing the
human P2Y14 receptor and an engineered G protein, GR-q/i
protein (GRqi5) that allows coupling of Gi-coupled receptors to
activation of PLC.35 Thus, inositol lipid hydrolysis33,34 served
as a measure of agonist activity at the Gi-coupled P2Y14
receptor.
The high potency of analogues 2 and 3 suggested a flexibility
of structural modification at the glucose 4 and 6 positions. The
12-fold lower potency of 4a in comparison to 1 suggested a
limited tolerance for steric bulk at the glucose 2 position. Most
of the novel UDPG analogues modified on the uracil or ribose
moieties were inactive. Thus, the SAR of the P2Y14 receptor is
among the most restrictive of all of the P2Y receptors. For
example, at the P2Y6 receptor, most modifications of the uracil
or ribose moieties reduced, but did not entirely abolish activity.
At the P2Y2/P2Y4 receptors, some of the same ribose modifica-
tions introduced here (e.g., 2′-amino, 2′-azido) enhanced potency
or selectivity.36 Analogues with purine or pyrimidine base
substitutions 19-21 and the dinucleotide U[5′]p2[5′]U 29a were
inactive at the P2Y14 receptor. Also, UMP, UDP, UTP, and
U[5′]p4[5′]U failed to activate the human P2Y14 receptor (data
not shown).
The SAR of the uracil moiety indicated that substitution of
this entity was somewhat more permissive than that of the ribose
moiety. Among the novel analogues prepared, a 2-thiouracil
analogue 15 had a 7-fold higher potency (EC50 49 ( 2 nM)
than that of UDPG (Figure 1). A 4-thio analogue (13) was
equipotent to UDPG, but its S-methylation (14) was not tolerated
for receptor activation. Also, three modifications at the 5 position
of the uracil moiety (16-18) resulted in inactivity.
Based on the potency of compounds 2-4 and on the modeling
results (see below) indicating as predominance of hydrophilic,
H-bonding residues in the region of the hexose moiety, we
proposed that the glucose moiety of UDPG should be amenable
to extensive structural modification. Consequently, we synthe-
sized UDP-fructose, UDP-mannose, and UDP-inositol and tested
these derivatives as agonists of the P2Y14 receptor. Indeed, such
variation of the glucose moiety of UDPG in the form of inositol
25, fructose 26, and mannose 27 preserved the agonist potency
at the P2Y14 receptor.
UDP-galactose 2, UDP-glucuronic acid 3, and UDP-N-
acetylglucosamine 4a also were agonists with potencies similar
to that of UDPG 1 at the P2Y14 receptor. The maximal effect
observed with 2, 3, and 4a was somewhat variable. Maximal
effects identical to that of UDPG were observed in some
experiments, whereas the maximal effects observed with these
three molecules were somewhat less than that of UDPG in other
experiments. 2-Thio-UDP-glucuronic acid 28 acted as an agonist
at the P2Y14 receptor and was equipotent to compounds 1 and
3. Thus, the favorable 2-thio modification preserved but did
not enhance the potency of a hexose sugar-modified analogue.
Molecular Modeling. We recently reported a rhodopsin-
based homology model of the human P2Y14 receptor inserted
into a phospholipid bilayer and refined by molecular dynamics
(MD) simulation.31 The model was utilized to study the binding
mode of UDPG at the P2Y14 receptor. Ligand docking modes
were based on automatic molecular docking protocols combined
with the Monte Carlo multiple minimum (MCMM) calculations.
Despite being based on the ground-state of rhodopsin, the model
seems to be applicable to study ligand-receptor interactions
for agonists. For example, similar approaches to study the
interactions of agonists with P2Y1 and P2Y6 receptors were
recently reported.24,37 The diphosphate moiety was found to
interact with only one cationic residue in the P2Y14 receptor,
namely, Lys171 of EL2, while in other P2Y receptor subtypes
three positively charged residues interact with the phosphate
chain. It should be noted that in nature the phosphate groups of
UDPG could be also coordinated by a metal cation. The model
of the P2Y1 receptor complex with UTP-Mg2+ was recently

2034 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Ko et al.
Figure 2. Possible conformations of the hexose ring of UDPG.
Figure 4. Molecular model of the P2Y14-compound 15 complex, with
a hexose conformation corresponding to Figure 2A. A rhodopsin-based
molecular model of the human P2Y14 receptor11,31 was refined using
20 ns MD simulation in the phospholipid bilayer. The complex obtained
after the automatic docking of 15 was used as a starting point for a
MCMM conformational search analysis of the ligand inside the putative
binding site of the P2Y14 receptor.
and an additional hydrogen bond with the hydroxyl group at
the 5 position of the hexose ring. Also, this hydroxyl group of
UDPG can interact with the backbone oxygen atom of a
conserved Cys172 (EL2). The â-phosphate group formed a
H-bond with the hydroxyl group of Thr280 (7.38), while Ser284
(7.42) appeared in proximity to the R-phosphate group of
UDPG. K277 (7.35) interacted with hydroxyl groups at the
hexose 2 and 3 positions, while Arg253 (6.55) was found near
the hydroxyl group at position 3. The glutamate residue Glu174
(EL2) can interact with hydroxyl groups at the 3 and 4 positions
of the hexose ring. The second glutamate residue Glu166 (EL2)
appeared near hydroxyl groups at the 2 and 3 positions and in
proximity to Lys277 (7.35). Similarly, hexose binding regions
in protein crystallographic structures typically contain charged
amino acid side chains, which H-bond to the hydroxyl groups.39
The hydroxyl groups of the ribose moiety of UDPG were
found to be H-bonded with two asparagine residues. In
particular, it was observed that the 2′-hydroxyl group can interact
with the side chain amino group of Asn104 (3.35) and the side
chain CdO group of Asn287 (7.45). The 3′-hydrogen group of
UDPG seemed to be H-bonded with the side chain amino group
of Asn287 (7.45). These results are in a good agreement with
the experimental values of EC50 measured for the ribose-
modified UDPG derivatives, suggesting the importance of both
2′- and 3′-hydroxyl groups.
Similar to our previous model,31 the ribose ring oxygen atom
was not involved in hydrogen bonding with the receptor. This
observation makes it possible to propose that replacement of
this oxygen atom of UDPG by a carbon atom will not decrease
the potency of a ligand. Several examples of such modifications
of the ribose ring were already published for other subtypes of
P2Y receptors.
As shown in Table 1, replacement of the oxygen atom at the
uracil 2 position of UDPG by a sulfur atom increases agonist
potency. This finding is in good agreement with the receptor
docking models obtained for UDPG and 2-thio-UDPG 15
(Figure 4), in which there is a mainly hydrophobic pocket
surrounding the 2 position of the uracil ring. The oxygen atom
at the 2 position of UDPG was not involved in H-bonding with
the receptor, implying that this oxygen is not critical for ligand
recognition. The more hydrophobic sulfur atom of larger radius
at the 2 position of 15 would be expected to favorably occupy
Figure 3. The molecular model of the human P2Y14 receptor obtained
after 20 ns of MD simulation. A centroid of Lys171, Arg253, and
Lys277 (red sphere) was used as a center of the box for molecular
docking of UDPG.
reported by Major et al.38 The distal hexose moiety appeared
to be H-bonded by other conserved cationic residues, namely,
Arg253 (6.55) and Lys277 (7.35). To facilitate the comparison
among receptors, throughout this paper we use the GPCR
residue indexing system, as explained in detail elsewhere.32 To
refine the binding mode of UDPG, especially the binding mode
of the hexose ring, the MCMM calculations were continued as
described in the Experimental Section. The results of MCMM
calculations suggested that two conformations of the hexose ring
are possible at the receptor binding site (Figure 2). However,
values of total energy of the entire ligand-receptor complex
calculated for both models demonstrated that conformation A
is more favorable. This is in a good agreement with the hexose
conformation in X-ray data published for complexes of UDPG
with various proteins.39 On the other hand, conformation B also
seems to be possible due to the interactions between Lys277
(7.35) and hydroxyl groups of the hexose ring. These interactions
can lock the hexose ring in its otherwise less favorable
conformation.
The UDPG binding mode A was proved by independently
performed automatic docking of the ligand with the Glide
program of MacroModel.40 As mentioned in the Experimental
Section, a box with a side of 46 Å around the centroid of three
conserved cationic residues was used for the docking study
covering more than half of the entire receptor (Figure 3). The
binding mode of UDPG obtained after the Glide-aided molecular
docking was very similar to the binding mode of UDPG obtained
after MCMM calculations.
It was observed in the docking model obtained for UDPG
(Figure 4) that, in the case of conformation A, Lys171 (EL2)
can form an electrostatic interaction with the â-phosphate group

Agonists of the Human P2Y14 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2035
moiety was also found to be H-bonded with Glu174 (EL2) and
with Lys277.
The results of molecular docking performed for ligands
containing various other sugar moieties 25-27 indicated that
all these structures display similar binding modes at the P2Y14
receptor. In general, the sugar moieties of these ligands were
involved in similar interactions with the receptor as UDPG. For
this reason, compounds 25-27 were proposed as potential
agonists of the P2Y14 receptor and subsequently synthesized
and tested biologically (see above).
Conclusions
We have identified various modifications that deselect for
this receptor subtype in relation to other uracil nucleotide-
responsive receptors and one modification (i.e., 2-thio) that
provides higher potency at the P2Y14 receptor. The 4-thio
analogue of UDPG was equipotent to the parent nucleotide.
Most other modifications of the uracil or ribose moieties led to
inactivity. Thus, the P2Y14 receptor is among the least permis-
sive P2Y receptors with respect to SAR. The molecular
modeling studies allowed us to propose that the glucose moiety
can be replaced by various six-membered or five-membered
sugar rings. This prediction was tested experimentally and it
was shown that variation of the glucose moiety of UDPG in
the form of inositol, fructose, and mannose preserved the
potency. In the models, all these sugar moieties were able to
interact with various hydrophilic and H-bonding side chains
located in the vicinity. Therefore, the hexose moiety provides
a fertile region for chemical modification of P2Y14 receptor
agonists.
Figure 5. The fructose moiety of 26 docked within the binding site
of the human P2Y14 receptor.
more free space inside this pocket and result in improved
potency of the ligand. The sulfur atom at the 2 position of the
uracil ring occupied a space between several residues, namely,
Val32 (1.42), Met70 (2.53), Ala285 (7.43), and Val288 (7.46).
The binding modes of several UDP derivatives with various
sugar moieties were examined with molecular docking studies.
In particular, binding modes of UDP-mannose 27 and UDP-
inositol 25 were studied. In general, the position and conforma-
tion of these two ligands were found to be very similar to
UDPG. In the case of 27, the 2-OH group was located closer to
Glu166 than to Lys277 (7.35), which formed a H-bond with
the 3-OH group. Also, hydroxyl groups at the 3 and 4 positions
of 27 formed H-bonds with Glu174 (EL2). The hydroxyl group
at the 5 position can form a H-bond with Lys171 (EL2), while
the oxygen atom of the mannose ring of 27 was not involved
in H-bonding with the receptor.
The molecular model of the P2Y14 receptor with UDP-inositol
25 obtained after MCMM calculations indicated that similar to
UDPG and 27, the ligand 25 can interact with Lys277 (7.35)
and Glu174 (EL2) due to H-bonding between these residues
and the hydroxyl groups at the 3 and 4 positions of the inositol
moiety. In addition, the 3-OH group of the inositol moiety can
interact with Arg253 (6.55). In contrast, Glu166 (EL2) appeared
far from the hydroxyl groups of 25 and did not form H-bonds
with the ligand. The hydroxyl group at the 6 position of the
inositol ring was found near Lys171 (EL2). We propose that
introduction of negatively charged groups at 2, 3, and 6 positions
of the inositol ring could provide more favorable interactions
with cationic residues.
Among UDP derivatives is a five-membered sugar ring
derivative, UDP-fructose 26. The docking complex of 26 in the
P2Y14 receptor indicated that the ligand can form several
H-bonds with the P2Y14 receptor (Figure 5). In particular, similar
to UDPG, the oxygen atom of the hydroxyl group at the 5
position of 26 can be H-bonded to Lys171 (EL2). In addition,
Lys171 can interact with the ring oxygen atom of the fructose
moiety. The H-bonds between the ligand and Glu166 (EL2) were
not observed in the model obtained after MCMM calculations.
However, the hydroxyl group at the 4 position was H-bonded
to Glu174 (EL2) and could interact with the backbone oxygen
atom of Cys172 (EL2). The 3-hydroxyl group of the fructose
Experimental Section
Chemical Synthesis. 2-Thiouridine (30) was purchased from
Berry and Associates, Inc. (Dexter, MI). 2′-Azido-2′-deoxyuridine
(31) and 2′-amino-2′-deoxyuridine (32) were purchased from CMS
Chemicals Ltd. (Oxfordshire, U.K.). 3′-Deoxyuridine (34) was
purchased from T.R.C., Inc. (North York, Ontario, Canada). 2′-
Ara-fluoro-2′-deoxyuridine (35) was purchased from R.I. Chemical,
Inc. (Orange, CA). 5-Azidouridine diphosphoglucose (17) and
5-aminouridine diphosphoglucose (18) were purchased from A.L.T.,
Inc. (Lexington, KY). Compounds 1-4, 20, 21, 23, and all reagents
and solvents were purchased from Sigma-Aldrich (St. Louis, MO).
Compounds 5, 19, 22, 29a, and 29b were synthesized as reported.28
1H 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 Varian XL 300
spectrometer (121.42 MHz); orthophosphoric acid (85%) was used
as an external standard. Purity of compounds was checked using a
Hewlett-Packard 1100 HPLC equipped with a Zorbax Eclipse
5 µ XDB-C18 analytical column (250 × 4.6 mm; Agilent
Technologies, Palo Alto, CA). System A: linear gradient solvent
system; 5 mM TBAP (tetrabutylammonium dihydrogenphosphate)-
CH3CN from 80:20 to 40:60 in 20 min, then isocratic for 2 min;
the flow rate was 1 mL/min. System B: linear gradient solvent
system; 10 mM TEAA (triethylammonium acetate)-CH3CN from
100:0 to 85:15 in 20 min, then isocratic for 2 min; the flow rate
was 1 mL/min. Peaks were detected by UV absorption with a diode
array detector. All derivatives tested for biological activity showed
>98% purity in the HPLC systems.
High-resolution mass measurements were performed on Micro-
mass/Waters LCT Premier electrospray time-of-flight mass spec-
tometer coupled with a Waters HPLC system. Purification of the
nucleotide analogues for biological testing was carried out on
(diethylamino)ethyl (DEAE)-A25 Sephadex columns as described
below. Compounds 6, 7, 9, 10, 12, 16, and 25-27 were additionally
purified by HPLC using system C. System C: gradient solvent
system; 10 mM TEAA-CH3CN from 100:0 to 90:10 in 30 min,

2036 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Ko et al.
then isocratic for 2 min; the flow rate was 2 mL/min with a Luna
5 µ RP-C18(2) semipreparative column (250 × 10.0 mm; Phe-
nomenex, Torrance, CA).
((2S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-
hydroxytetrahydrofuran-2-yl)methyl Phosphate, Ammonium
Salt (39). Procedure A. Compound 39 (9.2 mg, 41%) was obtained
from 34 (15 mg, 0.066 mmol). 1H NMR (D2O) δ 8.09 (d, J ) 8.1
Hz, 1H), 5.88 (d, J ) 7.8 Hz, 1H), 5.79 (d, J ) 1.2 Hz, 1H), 4.59
(m, 1H), 4.48 (m, 1H), 4.09 (ddd, J ) 11.7, 4.5, 2.7 Hz, 1H), 3.86
(ddd, J ) 11.7, 4.5, 4.5 Hz, 1H), 2.14 (ddd, J ) 14.1, 9.6, 5.7 Hz,
1H), 2.00 (ddd, J ) 13.8, 6.3, 2.7 Hz, 1H); 31P NMR (D2O) δ 2.35
(s); HRMS m/z calcd for C9H12N2O8P (M - H+)-, 307.0331; found,
307.0309.
((2R,3R,4S,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl Phosphate, Am-
monium Salt (40). Procedure A. Compound 40 (9.5 mg, 33%)
was obtained from 35 (20 mg, 0.08 mmol). 1H NMR (D2O) δ 7.90
(dd, J ) 8.1, 1.2 Hz, 1H), 6.31 (dd, J ) 15.9, 4.2 Hz, 1H), 5.89
(dd, J ) 8.1, 1.2 Hz, 1H), 5.21 (d, J ) 51.5, 3.0 Hz, 1H), 4.52 (d,
J ) 19.2, 4.2 Hz, 1H), 4.18 (m, 3H); 31P NMR (D2O) δ 4.75 (s);
HRMS m/z calcd for C9H11N2O8FP (M - H+)-, 325.0237; found,
325.0240.
((2R,3S,4R,5R)-3,4-Dihydroxy-5-(4-(methylthio)-2-oxopyrimi-
din-1(2H)-yl)tetrahydrofuran-2-yl)methyl Phosphate, Ammo-
nium Salt (42). To a suspension of 4-thiouridine 5′-monophosphate,
41 (sodium salt, 10 mg, 0.03 mmol) in MeOH/H2O (1.0 mL/0.5
mL) was added 0.25 M NaOH (0.5 mL). The reaction mixture was
stirred at room temperature for 2 h, and then the solvent was
removed under high vacuum. The resulting residue was dissolved
in dry DMF (2.0 mL), and iodomethane (0.09 mL, 1.5 mmol) was
added. The reaction mixture was stirred at room temperature for 1
h, and an additional 0.09 mL of iodomethane was added. After 2
h, the solvent was removed under reduced pressure. The crude
compound was purified by the same method in procedure A to give
42 (7 mg, 62%). 1H NMR (D2O) δ 8.40 (d, J ) 6.9 Hz, 1H), 6.78
(d, J ) 7.5 Hz, 1H), 5.97 (d, J ) 2.4 Hz, 1H), 4.34-4.37 (m, 2H),
4.30 (m, 1H), 4.10-4.17 (m, 1H), 3.96-4.02 (m, 1H); 31P NMR
(D2O) δ 2.09 (s); HRMS m/z calcd for C10H14N2O8SP (M - H+)-,
325.0209; found, 353.0238.
General Procedure for the Preparation of Nucleoside 5′-
Monophosphates. Procedure A. A solution of the corresponding
nucleoside (0.04-0.19 mmol) and proton sponge (1.5 equiv) in
trimethyl phosphate (2 mL) was stirred for 10 min at 0 °C. Then
phosphorus oxychloride (2 equiv) was added dropwise, and the
reaction mixture was stirred for 2 h at 0 °C. Triethylammonium
bicarbonate solution (0.2 M, 2 mL) was added to the reaction
mixture, and the clear solution was stirred at room temperature for
1 h. The latter was lyophilized overnight. 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. The portions of desired compounds
were collected, frozen, and lyophilized to give the corresponding
nucleoside 5′-monophosphates as the ammonium salts.
General Procedure for the Preparation of Nucleoside 5′-
Diphosphoglucose Procedure B. An ammonium salt or sodium
salt of the nucleoside 5′-monophosphate (0.01-0.02 mmol) was
treated with ion-exchange resin (Dowex 50WX2-200 (H)), fol-
lowed by neutralization with tributylamine. The mixture was
evaporated and lyophilized to afford the nucleoside 5′-monophos-
phate tributylamine salt. To a solution of the dried corresponding
nucleoside 5′-monophosphates tributylamine salt in DMF (1.5-
2.0 mL) was added 1,1′-carbonyldiimidazole (3 equiv). The reaction
mixture was stirred at room temperature for 6 h. Then 5%
triethylamine solution in 1/1 water/methanol (2 mL) was added and
stirring was continued at room temperature for an additional 2 h.
After removal of the solvent, the residue was dried in high vacuum
and dissolved in DMF (1 mL). Glucose-1-monophosphate tri-
butylammonium salt (1.5 equiv, inositol-1-monophsphate for
compound 25, fructose-1-monophosphate for compound 26, and
mannose-1-monophosphate for compound 27 were used) was added
to a solution of the nucleotide imidazolide in DMF. The reaction
mixture was stirred at room temperature for 2 days. After removal
of the solvent, 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. The portion of desired compounds were collected,
frozen, and lyophilized to give the corresponding nucleoside 5′-
diphosphoglucoses as the ammonium salts. Some of the products
were additionally purified by HPLC using system C.
((2R,3S,4R,5R)-3,4-Dihydroxy-5-(4-oxo-2-thioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl Phosphate, Am-
monium Salt (36). Procedure A. Compound 36 (8 mg, 35%) was
obtained from 30 (15 mg, 0.06 mmol). 1H NMR (D2O) δ 8.21 (d,
J ) 8.1 Hz, 1H), 6.64 (d, J ) 2.7 Hz, 1H), 6.22 (d, J ) 8.1 Hz,
1H), 4.23 (dd, J ) 4.5, 3.0 Hz, 1H), 4.31 (br d, J ) 3.0 Hz, 1H),
4.26 (dd, J ) 12.3, 3.9 Hz, 1H), 4.11 (dd, J ) 12.0, 5.0 Hz, 1H);
31P NMR (D2O) δ 0.81 (s); HRMS m/z calcd for C9H12N2O8P2S
(M - H+)-, 339.0052; found, 339.0056.
((2R,3S,4R,5R)-4-Azido-5-(2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)-3-hydroxytetrahydrofuran-2-yl)methyl Phosphate, Am-
monium Salt (37). Procedure A. Compound 37 (7 mg, 41%) was
obtained from 31 (12 mg, 0.045 mmol). 1H NMR (D2O) δ 7.91 (d,
J ) 8.1 Hz, 1H), 5.98 (d, J ) 5.1 Hz, 1H), 5.91 (d, J ) 8.1 Hz,
1H), 4.52 (t, J ) 5.4 Hz, 1H) 4.32 (t, J ) 5.4 Hz, 1H), 4.23 (m,
1H), 4.18 (ddd, J ) 12.0, 4.5, 2.7 Hz, 1H), 4.08 (ddd, J ) 12.0,
5.1, 2.7 Hz, 1H); 31P NMR (D2O) δ -3.52 (s); HRMS m/z calcd
for C9H11N5O8P (M - H+)-, 348.0345; found, 348.0366.
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′′-Deoxy-
2′′-azido)uridin-5′′-yl) Ester, Triethylammonium Salt (6). Pro-
cedure B. Compound 6 (2.5 mg, 63%) was obtained from 37
(ammonium salt, 2 mg, 0.005 mmol). 1H NMR (D2O) δ 7.94 (d, J
) 8.1 Hz, 1H), 6.01 (d, J ) 5.4 Hz, 1H), 5.96 (d, J ) 7.8 Hz, 1H),
5.58 (dd, J ) 7.2, 3.6 Hz, 1H), 4.80 (m, 1H), 4.36 (t, J ) 5.4 Hz,
1H), 4.19-4.25 (m, 3H), 3.82-3.89 (m, 2H), 3.72-3.78 (m, 2H),
3.51 (dd, J ) 9.9, 3.0 Hz, 1H), 3.44 (t, J ) 9.6 Hz, 1H); 31P NMR
(D2O) δ -10.89 (d, J ) 20.8 Hz), -12.47 (d, J ) 20.8 Hz); HRMS
m/z calcd for C15H22N5O16P2 (M - H+)-; found, 590.0531. HPLC
(System A) 13.7 min (98%), (System B) 7.2 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′′-Deoxy-
2′′-amino)uridin-5′′-yl) Ester, Triethylammonium Salt (7). H2
at atmospheric pressure was applied to a mixture of 6 (1.5 mg,
0.002 mmol) and Pd/C 10% (0.3 mg) in MeOH (0.5 mL) for 2 h
at room temperature. The reaction mixture was filtered, and the
filtrate was evaporated under the reduced pressure. The residue was
1
purified by HPLC using system C to afford 7 (1.0 mg, 69%). H
NMR (D2O) δ 7.96 (d, J ) 7.8 Hz, 1H), 6.02 (d, J ) 7.8 Hz, 1H),
5.98 (d, J ) 8.1 Hz, 1H), 5.62 (dd, J ) 8.1, 3.6 Hz, 1H), 4.38 (m,
1H), 4.33 (m, 1H), 4.20 (m, 2H), 3.77-3.91 (m, 4H), 3.46-3.64
(m, 3H); 31P NMR (D2O) δ -10.83 (d, J ) 20.8 Hz), -12.49 (d,
J ) 20.8 Hz); HRMS m/z calcd for C15H24N3O16P2 (M -
H+)-,564.0632; found, 564.0600. HPLC (System A) 5.7 min (98%),
(System B) 5.4 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′′-Deoxy-
2′′-aminocarbonyl-3′′-O)uridin-5′′-yl) Ester, Ammonium Salts
(8). Procedure B. Compound 8 (2.0 mg, 48%) was obtained from
38 (ammonium salt, 3.0 mg, 0.007 mmol). 1H NMR (D2O) δ 7.84
(d, J ) 8.4 Hz, 1H), 5.91 (m, 2H), 5.59 (dd, J ) 7.5, 3.6 Hz, 1H),
5.40 (dd, J ) 8.4, 3.3 Hz, 1H), 4.67-4.75 (m, 2H), 4.30 (m, 2H),
3.75-3.92 (m, 4H), 3.43-3.55 (m, 2H); 31P NMR (D2O) δ -11.25
(br s), -12.52 (br s); HRMS m/z calcd for C16H22N3O17P2 (M -
H+)-, 590.0424; found, 590.0449. HPLC (System A) 11.7 min
(98%), (System B) 5.5 min (98%).
((2R,3S,4R,5R)-5-(2,4-Dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
-3-hydroxy-4-(2,2,2-trifluoroacetamido)tetrahydrofuran-2-yl)-
methyl Phosphate, Ammonium Salt (38). Procedure A. Com-
pound 38 (3.2 mg, 45%) was obtained from 33 (5.8 mg, 0.017
mmol). 1H NMR (D2O) δ 8.09 (dd, J ) 8.4, 1.8 Hz, 1H), 6.05 (d,
J ) 8.1 Hz, 1H), 6.00 (dd, J ) 7.8, 1.5 Hz, 1H), 4.41 (m, 1H),
4.31 (m, 1H), 3.96 (m, 2H), 3.80 (m, 1H); 31P NMR (D2O) δ 3.85
(s); HRMS m/z calcd for C11H12N3O9F3P1 (M - H+)-, 418.0263;
found, 418.0251.

Agonists of the Human P2Y14 Receptor
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9 2037
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((3′′-Deoxy)-
uridin-5′′-yl) Ester, Triethylammonium Salt (9) and Diphos-
phoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′′,3′′-Dideoxy-2′′-
acetyloxy)uridin-5′′-yl) Ester, Triethylammonium Salt (10).
Procedure B. Compounds 9 (1.3 mg, 7%) and 10 (3.4 mg, 16%)
were obtained from 39 (ammonium salts, 9 mg, 0.026 mmol).
Compound 9 1H NMR (D2O) δ 8.02 (d, J ) 8.1 Hz, 1H), 5.95 (d,
J ) 8.1 Hz, 1H), 5.85 (d, J ) 2.1 Hz, 1H), 5.62 (dd, J ) 7.2, 3.3
Hz, 1H), 4.71 (m, 1H), 4.54 (m, 1H), 4.34 (m, 1H), 4.15 (m, 1H),
3.77-3.93 (m, 4H), 3.44-3.57 (m, 2H), 2.07-2.24 (m, 2H); 31P
NMR (D2O) δ -10.66 (d, J ) 21.5 Hz), -12.48 (d, J ) 20.8 Hz);
HRMS m/z calcd for C15H23N2O16P2 (M - H+)-, 549.0523; found,
549.0543. HPLC (System A) 12.9 min (98%), (System B) 8.5 min
(98%). Compound 10 1H NMR (D2O) δ 7.96 (d, J ) 8.1 Hz, 1H),
6.02 (d, J ) 1.5 Hz, 1H), 5.96 (d, J ) 7.8 Hz, 1H), 5.61 (dd, J )
7.2, 3.6 Hz, 1H), 5.37 (m, 1H), 4.65 (m, 1H), 4.35 (ddd, J ) 11.7,
5.1, 2.4 Hz, 1H), 4.14 (ddd, J ) 11.7, 6.3, 4.2 Hz, 1H), 3.92 (m,
1H), 3.84 (s, 3H), 3.76-3.86 (m, 3H), 3.54 (dt, J ) 9.9, 3.3 Hz,
1H), 3.47 (t, J ) 9.3 Hz, 1H), 2.44 (m, 1H), 2.31 (ddd, J ) 14.1,
6.0, 2.1 Hz, 1H); 31P NMR (D2O) δ -10.72 (d, J ) 20.8 Hz),
-12.47 (d, J ) 20.8 Hz); HRMS m/z calcd for C17H25N2O18P2 (M
- H+)-, 607.0578; found, 607.0568. HPLC (System A) 13.1 min
(98%), (System B) 8.0 min (98%).
for 4 h and cooled to room temperature. After removal of solvent,
the reaction was diluted with H2O and washed with diethyl ether
twice. The aqueous layer was concentrated and passed through H2O
and ion-exchange resin (Dowex 50WX2-200 (H)), followed by
treatment with tributylamine. After removal of all solvents, the
residue was dried in high vacuum and dissolved in DMF (1 mL).
Glucose-1-monophosphate tributylammonium salt was added to a
solution of the nucleotide morpholidate in DMF. The reaction
mixture was stirred for 2 days, followed by the same purification
procedure B to give 15 (5 mg, 77%). 1H NMR (D2O) δ 8.15 (d, J
) 8.4 Hz, 1H), 6.68 (d, J ) 3.0 Hz, 1H), 6.26 (d, J ) 8.1 Hz, 1H),
5.61 (dd, J ) 6.9, 3.3 Hz, 1H), 4.20-4.45 (m, 5H), 3.70-3.95 (m,
4H), 3.54 (dt, J ) 9.0, 2.7 Hz, 1H), 3.47 (t, J ) 9.3 Hz, 1H); 31P
NMR (D2O) δ -10.23 (d, J ) 20.8 Hz), -11.83 (d, J ) 20.8 Hz);
HRMS m/z calcd for C15H23N2O16P2S (M - H+)-, 581.0244; found,
581.0248. HPLC (System A) 12.6 min (98%), (System B) 5.0 min
(98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((5′-Iodo)-
uridin-5′′-yl)ester, Triethylammonium Salt (16). Procedure B.
Compound 16 (1.4 mg, 11%) was obtained from 43 (sodium salt,
1
7 mg, 0.014 mmol). H NMR (D2O) δ 8.27 (s, 1H), 5.95 (d, J )
4.8 Hz, 1H), 5.63 (dd, J ) 6.9, 4.5 Hz, 1H), 4.23-4.42 (m, 5H),
3.77-4.01 (m, 4H), 3.54 (dt, J)9.0, 3.0 Hz, 1H), 3.47 (t, J ) 9.6
Hz, 1H); 31P NMR (D2O) δ -10.40 (d, J ) 20.8 Hz), -11.87 (d,
J ) 20.8 Hz); HRMS m/z calcd for C15H22N2O17IP2 (M - H+)-,
690.9439; found, 690.9444. HPLC (System A) 12.8 min (98%),
(System B) 6.9 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′-Fluoro-
2′′-deoxyara)uridin-5′′-yl)ester, Ammonium Salts (11). Proce-
dure B. Compound 11 (ammonium salt, 2.4 mg, 24%) was obtained
1
from 40 (ammonium salt, 6 mg, 0.017 mmol). H NMR (D2O) δ
7.92 (dd, J ) 8.1, 1.2 Hz, 1H), 6.33 (dd, J ) 15.9, 3.9 Hz, 1H),
5.94 (d, J ) 8.4 Hz, 1H), 5.61 (dd, J ) 7.2, 3.6 Hz, 1H), 5.23 (dd,
J ) 51.3, 4.2 Hz, 1H), 4.57 (dd, J ) 18.9, 3.3 Hz, 1H), 4.25 (m,
3H), 3.76-3.92 (m, 4H), 3.44-3.56 (m, 2H); 31P NMR (D2O) δ
-10.73 (d, J ) 20.8 Hz), -12.45 (d, J ) 20.8 Hz); HRMS m/z
calcd for C15H22N2O16FP2 (M - H+)-, 567.0429; found, 567.0427.
HPLC (System A) 11.7 min (98%), (System B) 8.4 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-(((S)-
Methanocarba-2′′-deoxy)uridin-5′′-yl)ester, Triethylammonium
Salt (12). Procedure B. Compound 12 (1.4 mg, 59%) was obtained
from 4524 (ammonium salt, 1.1 mg, 0.003 mmol). 1H NMR (D2O)
δ 7.81 (d, J ) 8.1 Hz, 1H), 5.80 (d, J ) 7.8 Hz, 1H), 5.59 (dd, J
) 6.9, 3.3 Hz, 1H), 4.37 (d, J ) 6.6 Hz, 1H), 4.09 (m, 2H), 3.74-
3.87 (m, 4H), 3.42-3.53 (m, 2H), 2.40 (m, 1H), 2.19 (m, 2H),
1.86 (m, 1H), 1.63 (m, 1H), 1.37 (m, 1H); 31P NMR (D2O) δ
-10.48 (d, J ) 20.8 Hz), -12.53 (d, J ) 20.8 Hz); HRMS m/z
calcd for C17H25N2O15P2 (M - H+)-, 559.0730; found, 559.0735.
HPLC (System A) 11.6 min (98%), (System B) 7.9 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((4′-Thio)-
uridin-5′′-yl)ester, Ammonium Salts (13). Procedure B. Com-
pound 13 (1.9 mg, 18%) was obtained from 41 (12 mg, 0.017
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′′-Deoxy-
5′-flouro)uridin-5′′-yl)ester, Ammonium Salts (24). Procedure
B. Compound 24 (1.8 mg, 21%) was obtained from 44 (tributyl-
1
ammonium salt, 10 mg, 0.014 mmol). H NMR (D2O) δ 8.04 (d,
J ) 5.7 Hz, 1H), 6.30 (t, J ) 6.9 Hz, 1H), 5.59 (dd, J ) 7.5, 3.2
Hz, 1H), 4.18 (m, 2H), 3.74-3.91 (m, 4H), 3.42-3.53 (m, 2H),
3.10-3.21 (m, 2H), 2.34-2.38 (m, 2H); 31P NMR (D2O) δ -10.89
(d, J ) 20.8 Hz), -12.51 (d, J ) 20.8 Hz); HRMS m/z calcd for
C9H13N2O11P2 (M - H+)-, 386.9995; found, 386.9995. HPLC
(System A) 14.5 min (98%), (System B) 4.8 min (98%).
UDP-Inositol, Triethylammonium Salt (25). Procedure B.
Compound 25 (3.2 mg, 30%) was obtained from uridine-5′-
1
monophosphate (tributylammonium salt, 10 mg, 0.014 mmol). H
NMR (D2O) δ 7.97 (d, J ) 7.8 Hz, 1H), 5.99 (m, 2H), 4.72 (m,
1H), 4.40 (m, 2H), 4.31 (m, 1H), 4.26 (m, 2H), 3.74 (t, J ) 9.9
Hz, 2H), 3.56 (m, 2H), 3.30 (t, J ) 9.3 Hz, 1H); 31P NMR (D2O)
δ -9.95 (d, J ) 21.4 Hz), -10.56 (d, J ) 21.4 Hz); HRMS m/z
calcd for C15H23N2O17P2 (M - H+)-, 565.0472; found, 565.0463.
HPLC (System A) 12.3 min (98%), (System B) 9.2 min (98%).
UDP-Fructose, Triethylammonium Salt (26). Compound 26
(4.0 mg, 37%) was obtained from uridine-5′-monophosphate
1
1
mmol). H NMR (D2O) δ 7.86 (d, J ) 7.8 Hz, 1H), 6.68 (d, J )
(tributylammonium salt, 10 mg, 0.014 mmol). H NMR (D2O) δ
7.5 Hz, 1H), 5.96 (d, J ) 3.6 Hz, 1H), 5.62 (dd, J ) 7.5, 3.6 Hz,
1H), 4.23-4.39 (m, 5H), 3.83-3.90 (m, 2H), 3.76-3.84 (m, 2H),
7.98 (d, J ) 8.1 Hz, 1H), 6.00 (m, 2H), 4.40 (m, 2H), 4.31 (m,
1H), 4.26 (m, 2H), 4.04-4.11 (m, 2H), 3.94-4.01 (m, 2H), 3.91
(m, 1H), 3.85 (m, 1H), 3.72 (m, 1H); 31P NMR (D2O) δ -10.82
(d, J ) 4.2 Hz), -10.96 (s); HRMS m/z calcd for C15H23N2O17P2
(M - H+)-, 565.0472; found, 565.0468. HPLC (System A) 12.4
min (98%), (System B) 8.7 min (98%).
3.54 (dt, J ) 9.0, 3.0 Hz, 1H), 3.48 (dd, J ) 9.9, 9.3 Hz, 1H); 31
P
NMR (D2O) δ -10.33 (d, J ) 20.8 Hz), -12.46 (d, J ) 20.8 Hz);
HRMS m/z calcd for C15H23N2O16P2S (M - H+)-, 581.0224; found,
581.0249. HPLC (System A) 13.7 min (98%), (System B) 6.6 min
(98%).
UDP-Mannose, Triethylammonium Salt (27). Procedure B.
Compound 27 (2.8 mg, 26%) was obtained from uridine-5′-
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((4′-Methyl-
thio)uridin-5′′-yl)ester, Ammonium Salts (14). Procedure B.
Compound 14 (5.1 mg, 40%) was obtained from 42 (ammonium
salt, 6.2 mg, 0.016 mmol). 1H NMR (D2O) δ 8.20 (d, J ) 7.5 Hz,
1H), 6.76 (d, J ) 7.2 Hz, 1H), 5.94 (d, J ) 2.4 Hz, 1H), 5.59 (dd,
J ) 6.9, 3.0 Hz, 1H), 4.18-4.35 (m, 5H), 3.73-3.90 (m, 4H),
3.20-3.53 (m, 2H), 2.53 (s, 3H); 31P NMR (D2O) δ -10.80 (d, J
) 20.8 Hz), -12.46 (d, J ) 20.8 Hz); HRMS m/z calcd for
C16H25N2O16P2S (M - H+)-, 595.0400; found, 595.0415. HPLC
(System A) 14.2 min (98%), (System B) 8.0 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosyl Ester 2-((2′-Thio)-
uridin-5′′-yl)ester, Ammonium Salts (15). To a suspension of 36
(tributylammonium salt, 8 mg, 0.01 mmol) in t-BuOH/H2O (0.5
mL/0.5 mL) were added morpholine (0.006 mL, 0.06 mmol) and
DCC (14 mg, 0.06 mmol). The reaction mixture was heated to reflux
1
monophosphate (tributylammonium salt, 10 mg, 0.014 mmol). H
NMR (D2O) δ 7.99 (d, J ) 8.4 Hz, 1H), 6.00 (m, 2H), 5.53 (dd,
J ) 7.2, 2.3 Hz, 1H), 4.40 (m, 2H), 4.31 (m, 1H), 4.25 (m, 2H),
4.07 (m, 1H), 3.67-3.97 (m, 5H); 31P NMR (D2O) δ -11.17 (d, J
) 20.8 Hz), -13.46 (d, J ) 20.9 Hz); HRMS m/z calcd for
C15H23N2O17P2 (M - H+)-, 565.0472; found, 565.0471. HPLC
(System A) 12.5 min (98%), (System B) 7.8 min (98%).
Diphosphoric Acid 1-R-D-Glucopyranosuronic Acid 2-((2′-
Thio)uridin-5′′-yl)ester, Triethylammonium Salts (28). Com-
pound 15 (ammonium salt, 0.7 mg, 0.001 mmol) was dissolved in
0.7 mL of stock solution (stock solution: Tris buffer (pH 7.4, 10
mL), NAD (17 mg, 0.026 mmol), sodium pyruvate (177 mg, 1.6
mmol), and lactate dehydrogenase (67 U, EC 1.1.1.27, from rabbit
muscle, Sigma). The pH of this solution was adjusted to 8.7 with

2038 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 9
Ko et al.
0.05 NaOH). Uridine-5′-diphosphoglucose dehydrogenase (0.3 U,
Sigma, from bovine liver) was added to the mixture.41 The reaction
was monitored by HPLC (System A). After 20 h at room
temperature, an additional 0.1 U of UDPG dehydrogenase was
added. After 5 h, the reaction mixture was lyophilized and purified
by HPLC (gradient solvent system: 10 mM TEAA-CH3CN from
100:0 to 97:3 in 40 min, then isocratic for 5 min; the flow rate was
2 mL/min with a Luna 5 µ RP-C18(2) semipreparative column (250
× 10.0 mm; Phenomenex, Torrance, CA)). The portion of desired
compound was collected, frozen, and lyophilized to give 28 (0.5
mg, 0.56 µmol, 56%, starting material 15 (0.3 mg, 0.38 µmol, 38%)
20 ns of MD simulation was utilized for automatic docking of
UDPG. The receptor grid generation was performed for the box
with a side of 46 Å with a center in the centroid of Lys171, Arg253,
and Lys277. Extra precision (XP) of docking was used. The
flexibility of a ligand was allowed. The conformations of the
sidechains of residues located within 6 Å from UDPG were refined
by 100 steps of MCMM calculations with an energy window for
saving structures of 100 kJ‚mol-1. The model obtained after this
refinement was used for a Glide-aided automatic docking of all
other ligands.
Acknowledgment. Mass spectral measurements were carried
out by Dr. John Lloyd and NMR by Wesley White (NIDDK).
We thank Dr. Victor Marquez and Dr. Stefano Costanzi
(NIDDK) for helpful discussion and Savitri Maddileti for
technical assistance. This research was supported in part by the
Intramural Research Program of the NIH, National Institute of
Diabetes and Digestive and Kidney Diseases. This work was
supported by National Institutes of Health Grants GM38213 and
HL34322 to T.K.H. This work was partly supported by the
Korea Research Foundation Grant (E00076) to H.K.
1
was recovered). H NMR (D2O) δ 8.17 (d, J ) 8.1 Hz, 1H), 6.72
(d, J ) 3.3 Hz, 1H), 6.24 (d, J ) 7.5 Hz, 1H), 5.63 (dd, J ) 7.2,
3.0 Hz, 1H), 4.43 (m, 1H), 4.33 (m, 2H), 4.24 (m, 1H), 4.10-4.16
(m, 2H), 3.78 (t, J ) 9.6 Hz, 1H), 3.57 (m, 1H), 3.51 (t, J ) 9.6
Hz, 1H); 31P NMR (D2O) δ -10.81 (br s), -12.45 (br s); HRMS
m/z calcd for C15H21N2O17P2S (M - H+)-, 595.0036; found,
595.0032. HPLC (System A) 17.1 min (98%), (System B) 12.4
min (98%).
Assay of P2Y14 Receptor-Stimulated PLC Activity. COS-7
cells were transiently transfected with the human P2Y14 receptor
and GRqi5.
35 Twenty-four hours after transfection, the inositol lipid
pool of the cells was radiolabeled by incubation in 200 µL of serum-
free inositol-free Dulbecco’s modified Eagle’s medium, containing
0.4 µCi of myo-[3H]inositol. No changes of medium were made
subsequent to the addition of [3H]inositol. Forty-eight hours after
transfection, cells were challenged with 50 µL of the 5-fold
concentrated solution of receptor agonists in 200 mM N-(2-
hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid, pH 7.3, contain-
ing 50 mM LiCl for 20 min at 37 °C. Incubations were terminated
by aspiration of the drug-containing medium and addition of 450
µL of ice-cold 50 mM formic acid. After 15 min at 4 °C, samples
were neutralized with 150 µL of 150 mM NH4OH. [3H]Inositol
phosphates were isolated by ion exchange chromatography on
Dowex AG 1-X8 columns as previously described.34
Data Analysis. Agonist potencies (EC50 values) were obtained
from concentration-response curves by nonlinear regression analy-
sis 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 mean
( SEM from multiple experiments or, in the case of concentration
effect curves, from a single experiment carried out with triplicate
assays that were representative of results from multiple experiments.
Molecular Modeling. Conformational Search. The recently
reported complex of the P2Y14 receptor with UDPG was utilized
as a starting point for further calculations.31 The conformational
analysis of the UDP-sugars in the putative binding site of the P2Y14
receptor was performed with MCMM implemented in MacroModel
9.0 software.40
Supporting Information Available: Graphical views of the
P2Y14-compound 15 complex (but not including those of the
phospholipid bilayer and aqueous medium) are included. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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