Antiaggregatory activity in human platelets of potent antagonists of the P2Y1 receptor

Article abstract of DOI:10.1016/j.bcp.2004.06.026

Activation of the P2Y₁ nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500 (2-iodo-N⁶-methyl-(N)-methanocarba- 2′-deoxyadenosine-3′,5′-bi

Full text of DOI:10.1016/j.bcp.2004.06.026

Biochemical Pharmacology 68 (2004) 1995–2002
www.elsevier.com/locate/biochempharm
Antiaggregatory activity in human platelets of potent antagonists
of the P2Y1 receptor
Marco Cattaneoa,b, Anna Lecchib, Michihiro Ohnoc, Bhalchandra V. Joshic, Pedro Besadac,
Susanna Tchilibonc, Rossana Lombardib, Norbert Bischofbergerd, T. Kendall Hardene,
Kenneth A. Jacobsonc,
*
aHematology and Thrombosis Unit, Ospedale San Paolo, DMCO-University of Milano, Milan, Italy
bDepartment of Internal Medicine, Hemophilia and Thrombosis Center, IRCCS Ospedale Maggiore, University of Milano, Milan, Italy
cMolecular Recognition Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health,
Bldg. 8A, Rm. B1A-19, Bethesda, MD 20892-0810, USA
dGilead Sciences, 333 Lakeside Dr, Foster City, CA, USA
eDepartment of Pharmacology, University of North Carolina, School of Medicine,
Chapel Hill, NC 27599-7365, USA
Received 21 April 2004; accepted 21 June 2004
Abstract
Activation of the P2Y1 nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of
phospholipase C (PLC). Recently, MRS2500 (2-iodo-N6-methyl-(N)-methanocarba-20-deoxyadenosine-30,50-bisphosphate) was intro-
duced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and
compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative
MRS2496, which act as P2Y1 receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and
MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists
due to the non-hydrolyzable C–P bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC50 value of
0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC50 values of 62.8 nM and
1.5 mM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y1
receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca2+. No substantial antagonism of the
pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y1
receptor antagonists with the proaggregatory P2Y12 receptor, which is also activated by ADP. Thus, all three of the bisphosphate
derivatives are highly selective antagonists of the platelet P2Y1 receptor, and MRS2500 is the most potent such antagonist yet reported.
# 2004 Elsevier Inc. All rights reserved.
Keywords: P2Y1 nucleotide receptor; G protein-coupled receptor; Acyclic nucleotides; Purines; Platelet aggregation; Carbocyclic nucleotides
1. Introduction
P2Y receptors, which are G protein-coupled receptors
(GPCRs), and the P2X receptors, which are ligand-gated
ion channels (LGICs) [1,2]. The P2Y1,2,4,6,11 receptors
couple preferentially to the activation of phospholipase
C (PLC), via heterotrimeric G proteins of the Gq family
and therefore induce an increase in intracellular calcium.
The P2Y12,13,14 receptors are coupled preferentially to the
inhibition of adenylate cyclase via heterotrimeric G pro-
teins of the Gi family. The P2Y receptors are activated by
adenine and/or uracil nucleotides [3]. The P2Y2 receptor is
the only human subtype that is activated by both ATP and
UTP (but not the corresponding 50-diphosphates). The
pyrimidine-selective receptors include the P2Y4, P2Y6
The P2 receptors for extracellular nucleotides are
divided into two structurally-unrelated superfamilies: the
Abbreviations: MRS2279, 2-chloro-N6-methyl-(N)-methanocarba-20-
deoxyadenosine-30,50-bisphosphate; MRS2298, 2-[2-(2-chloro-6-methyla-
mino-purin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate);
MRS2496, [3-(2-chloro-6-methylamino-purin-9-yl)-2-phosphonomethyl-
propyl]-phosphonic acid ammonium salt; MRS2500, 2-iodo-N6-methyl-
(N)-methanocarba-20-deoxyadenosine-30,50-bisphosphate; PLC, phospholi-
pase C; Tris; Tris(hydroxymethyl)aminomethane.
* Corresponding author. Tel.: +1 301 496 9024; fax: +1 301 480 8422.
E-mail address: kajacobs@helix.nih.gov (K.A. Jacobson).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.06.026

1996
M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
and P2Y14 receptors, which are activated by UTP, UDP and
UDP-glucose, respectively [4]. The remaining subtypes
(P2Y1, P2Y11, P2Y12 and P2Y13 receptors) are activated by
adenine nucleotides.
chloro-N6-methyladenine-9-(2-methylpropyl) scaffold,
indicated that the ribose ring structure itself is not neces-
sary for receptor recognition but rather serves as a spacer.
A significant correlation was observed between the capa-
city of several of these P2Y1 receptor antagonists to inhibit
ADP-induced platelet aggregation in rat platelets [5,23]
and the inhibition of P2Y1 receptor-stimulated phospholi-
pase C activity previously determined in turkey erythro-
cytes [22]. The present study compares the activity of three
of these P2Y1 receptor antagonists on aggregatory proper-
ties and second messenger (cAMP and PLC) signaling in
human platelets.
ADP is released from platelets and endothelial cells to
induce aggregation of platelets and promote intravascular
coagulation by acting at the P2Y1 and P2Y12 receptors [5–
10]. Antagonists of either P2Y receptor subtype display
antiaggregatory properties. A mouse line lacking expression
of the P2Y1 receptor has been constructed [11,12], and the
absence of P2Y1 receptors in platelets of these mice inter-
fered with ADP-promoted aggregation and invivo thrombus
formation. A third nucleotide receptor, the P2X1 receptor, is
present on platelets, and its role in aggregation appears to be
most evident under conditions of high shear stress [13,31].
Fig. 1 illustrates the chemical structures of a variety of
recently-introduced antagonists of the P2Y1 receptor. The
structures differ markedly in the nature of the ribose-like
moiety to which the adenine nucleobase is attached.
MRS2179 (N6-methyl-20-deoxyadenosine 30,50-bispho-
sphate) 1 and its 2-chloro analogue MRS2216 2 (Fig. 1)
have been designed as nucleotide antagonists of the P2Y1
receptor [14–19]. In suspensions of washed human plate-
lets, MRS2179 inhibited ADP-induced platelet shape
change, aggregation and Ca2+ rise but had no effect on
ADP-induced inhibition of adenylate cyclase [20]. Binding
studies using 33P-labeled MRS2179 demonstrated the pre-
sence of an average of 134 binding sites per platelet.
MRS2179 inhibited both arterial and venous thrombosis,
further establishing this receptor as a potential target for
antithrombotic drugs [21]. Further structural modification
resulted in the highly potent P2Y1 receptor antagonists
MRS2279 3 and the 2-iodo analogue MRS2500 4 [19],
containing the rigid (N)-methanocarba ring system.
MRS2279 has been radiolabeled for use as a high affinity,
competitive radiotracer in binding experiments [18], We
previously characterized the SAR (structure activity rela-
tionship) as P2Y1 receptor antagonists of acyclic analogues
of adenine nucleotides, containing two phosphate groups
on a symmetrically-branched aliphatic chain, attached at
the 9-position of adenine [22,23]. The submicromolar
potency of the acyclic P2Y1 receptor antagonists
MRS2298 5 and MRS2496 6, both related to a 2-
2. Materials and methods
2.1. Reagents
myo-[3H]Inositol (20 Ci/mmol) was obtained from
American Radiolabeled Chemicals. Dowex AG 1-X8 resin
was purchased from Bio-Rad. DMEM and FBS were from
Life Technologies. All other reagents were purchased from
Sigma–Aldrich Chemical Co. HPLC mobile phases con-
sisted of System A: linear gradient solvent system:
CH3CN/triethyl ammonium acetate from 5/95 to 60/40
in 20 min, flow rate 1.0 mL/min; System B: linear gradient
solvent system: CH3CN/tetrabutyl ammonium phosphate
from 20/80 to 60/40 in 20 min, flow rate 1.0 mL/min.
Apyrase was a kind gift of R.L. Kinlough-Rathbone
(McMaster University, Hamilton, Ontario): the activity
of the compound was such that the enzyme (1 mL/mL)
converted 0.25 mmol/L ATP to adenosine monophosphate
within 120 s at 37 8C.
2.2. Chemical synthesis
The following synthetic methods are depicted schema-
tically in Fig. 2.
2.2.1. (10R,20S,40R,50S)-4-(tert-Butyl-diphenyl-
silanyloxy)-1-hydroxymethyl-bicyclo[3.1.0]hexan-2-ol (8)
To a solution of acetic acid 1-acetoxymethyl-4-hydroxy-
bicyclo [3.1.0]hex-2-yl ester 7 ([24], 750 mg, 3.29 mmol)
Fig. 1. Structures of nucleotide (bisphosphate) derivatives that have been shown to act as inhibitors of P2Y1 receptors. The nucleotide analogues differ mainly in
the nature of the ribose-like moiety and include 9-ribosides (1, 2), the (N)-methanocarba ring system, which is sterically-constrained in the North 20-exo
conformation favored by the P2Y1 receptor (3, 4), and acyclic analogues (5, 6).

M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
1997
Fig. 2. Improved synthetic routes for the preparation of the (N)-methanocarba antagonist MRS2500 4 and the acyclic antagonist MRS2496 6. The route applied
to MRS2500 involves phosphorylation of the [3.1.0]bicyclohexane system in 8, condensation with the nucleobase using a Mitsunobu coupling to yield 11,
followed by deprotection of the phosphate groups and functional group replacement on the adenine moiety. The single step applied to the preparation of
MRS2500 involves hydroboration to form the intermediate 14.
in CH2C12 (10 mL) was added 4-N,N-dimethylaminopyr-
idine (50 mg, 0.41 mmol), Triethylamine (1.00 mL,
7.17 mmol) and t-butyl-diphenylsilyl chloride (TBDPS,
1.85 mL, 7.11 mol) and the reaction mixture was stirred
at rt for 23 h. The reaction mixture was diluted with ethyl
acetate (EtOAc) and was washed sequentially with 5%
citric acid, brine, sat NaHCO3, and brine. The organic layer
was dried over Na2SO4 and the solvent evaporated. The
residue was dissolved in MeOH (50 mL) and K2CO3
(166 mg, 1.20 mmol) was added. The reaction mixture
was stirred at rt for 3 days and evaporated. The residue
was dissolved with AcOEt and washed with 5% citric acid,
sat NaHCO3, and brine, and dried over Na2SO4 and the
solvent evaporated. The residue obtained was purified by
column chromatography (silica gel, eluent: AcOEt/petro-
(t, 1H, J = 7.7 Hz); MS (m/e) (positive-FAB) 381 (M ꢀ H)+,
405 (M + Na)+.
2.2.2. (10R,20S,40R,50S)-Phosphoric acid di-tert-butyl
ester 4-(tert-butyl-diphenyl-silanyloxy)-1-(di-tert-butoxy-
phosphoryloxymethyl)-bicyclo[3.1.0]hex-2-yl ester (9)
To a stirred solution of 8 (82 mg, 0.21 mmol) and 1H-
tetrazole (79 mg, 1.13 mmol) in anhydrous tetrahydrofuran
(THF, 2.0 mL) was added di-t-butyl diethylphosphorami-
dite (0.400 mL, 1.44 mmol), and the reaction mixture was
stirred for 3 h at room temperature. The reaction mixture
was cooled to ꢀ78 8C and treated with a solution of m-
chloroperbenzoic acid (70% max, 530 mg) in CH2Cl2
(5.0 mL). The resulting mixture was warmed to room
temperature and stirred for 30 min. 5% NaHSO3
(2.0 mL) was added to the reaction mixture, and was stirred
for another 10 min at room temperature. The reaction
mixture was extracted with AcOEt and the organic phase
was subsequently washed with saturated aqueous NaHCO3
and brine and dried over Na2SO4 and filtered. The solvent
1
leum ether = 1/2 then 1/0), to give 8 (1.24 g, 99%). H
NMR (CDCl3) d 7.72–7.62 (m, 4H), 7.43–7.34 (m, 6H),
4.36–4.23 (m, 2H), 3.72 (dd, 1H, J = 5.1, 11.1 Hz), 3.50
(dd, 1H, J = 5.7, 11.1 Hz), 2.17–2.06 (m, 1H), 1.83 (d, 1H,
J = 5.1 Hz), 1.57 (m, 1H), 1.32 (m, 2H), 1.04 (s, 9H), 0.55

1998
M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
was removed under reduced pressure. The obtained residue
was purified by silica gel column chromatography (AcOEt/
petroleum ether = 1/2 to 1/0), to give 9 (152 mg, 93%). 1H
NMR (CDC13) d 7.70–7.60 (m, 4H), 7.43–7.31 (m, 6H),
4.72 (m, 1H), 4.32 (m, 2H), 3.61 (dd, 1H, J = 6.9, 11.4 Hz),
2.27 (dt, 1H, J = 7.5, 13.5 Hz), 1.72 (m, 1H), 1.52 (s, 9H),
1.46 (s, 18H), 1.39 (s, 9H), 1.33 (s, 9H), 1.26 (m, 1H),
1.07(m, 1H), 0.67(m, 1H); MS (m/e) (positive-FAB) 767
(M + H)+.
linear gradient (0.01–0.7 M) of 0.5 M NH4HCO3 as the
mobile phase. After lyophilization, 12 (16.3 mg, 62%) was
1
obtained as a white solid. H NMR(D2O) d 8.83 (s, 1H),
5.30–5.20 (m, 1H), 5.16 (d, 1H, J = 6.3 Hz), 4.60–4.50 (m,
1H), 3.75–3.65 (m, 1H), 2.40–2.20 (m, 1H), 2.10–1.95 (m,
1H), 1.95–1.90 (m, 1H), 1.25–1.20 (m, 1H), 1.05–1.00 (m,
1H); 31P NMR (D2O) d 2.02, 1.40 (2s, 30-P, 50-P); MS (m/e)
(negative-FAB) 565, 567 (peak height ratio = 3:1) (M ꢀ
H)+; HPLC 9.8 min (98%) in solvent system A, 16.0 min
(98%) in solvent system B.
2.2.3. (10R,20S,40R,50S)-Phosphoric acid di-tert-butyl
ester 1-(di-tert-butoxy-phosphoryloxymethyl)-4-hydroxy-
bicyclo[3.1.0]hex-2-yl ester (10)
2.2.6. (10R,20S,40S,50S)-4-(2-iodo-6-methylamino-
purin-9-yl)-l-[(phosphato)-methyl]-2-(phosphato)-
bicyclo[3.1.0]hexane (4)
To a stirred solution of 9 (2.16 g, 2.81 mmol) in 6.0 mL
of anhydrous THF was added 1.0 M tetrabutyl ammonium
fluoride THF solution (4.3 mL, 4.3 mmol) and the reaction
mixture was stirred at room temperature for 24 h. The
solvent was removed under reduced pressure. The residue
obtained was purified by silica gel column chromatography
(MeOH/CHCl3 = 1/10), which furnished 10 (1.34 g, 90%).
1H NMR (CDCl3) 4.89 (q, 1H, J = 7.4 Hz), 4.41 (m, 2H),
3.69 (dd, 1H, J = 6.9, 10.8 Hz), 2.46 (dt, 1H, J = 8.0,
13.5 Hz), 1.80 (m, 1H), 1.49 (s, 18H), 1.48(s, 9H), 1.47 (s,
9H), 1.23 (m, 1H), 1.09 (m, 1H), 0.69 (m, 1H); MS (m/e)
(positive-FAB) 529 (M + H)+.
To a solution of 12 (10.9 mg, 0.017 mmol) in water
(5.00 mL) was added 40% MeNH2 in water (1.0 mL) and
the reaction mixture was stirred for 2 h at room tempera-
ture. The reaction was monitored by HPLC. The reaction
mixture was subsequently frozen and lyophilized. Purifi-
cation of the residue obtained was performed on an ion-
exchange column packed with Sephadex-DEAE A-25
resin. A linear gradient (0.01–0.7 M) of 0.5 M ammonium
bicarbonate was applied as the mobile phase, and UV and
HPLC were used to monitor the elution, which furnished 4
(10.2 mg, 95%). 1H NMR (D2O) d 8.54 (bs, 1H), 5.19 (m,
1H), 5.01 (d, 1H, J = 6.9 Hz), 4.58 (dd, 1H, J = 4.7,
11.3 Hz), 3.73 (dd, 1H, J = 4.4, 11.0 Hz), 3.07 (bs, 3H),
2.28 (dd, 1H, J = 7.7, 14.6 Hz), 1.92–2.09 (m, 2H), 1.26
(dd, 1H, J = 4.1, 6.1 Hz), 1.06 (dd, 1H, J = 9.7, 16.3 Hz).
31P NMR (D2O) 0.651 (s). High-resolution MS (negative-
ion FAB) calcd for C13H17N5O8P2I 559.9597, found
559.9604, HPLC 9.8 min (99%) in solvent system A,
15.4 min (99%) in system B.
2.2.4. (10R,20S,40S,50S)-Phosphoric acid di-tert-butyl
ester 1-(di-tert-butoxy-phosphoryloxymethyl)-4-(6-chloro-
2-iodo-purin-9-yl)-bicyclo[3.1.0]hex-2-yl ester (11)
To
a
solution of triphenylphosphine (101 mg,
0.385 mmol) in anhydrous THF (1.00 mL) was added
diisopropyl azodicarboxylate (0.075 mL, 0.38 mmol) at
rt with stirring for 1.5 h. Compound 10 (102 mg,
0.194 mmol) and 6-chloro-2-iodopurine ([19], 70 mg,
0.25 mmol) in THF (2.20 mL) were added to the reaction
mixture, and it was stirred at room temperature for 23 h.
The solvent was removed under vacuum and the residue
obtained was purified by preparative thin-layer chromato-
graphy (AcOEt), which furnished 11 (81.3 mg, 53%). 1H-
NMR (CDC13) d 8.44 (s, 1H), 5.34 (dd, 1H, J = 8.1,
15.0 Hz), 5.16 (d, 1H, J = 6.9 Hz), 4.69 (dd, 1H, J = 5.1,
11.4 Hz), 3.94 (dd, 1H, J = 6.6, 11.4 Hz), 2.40–2.30 (m,
1H), 2.22–2.10 (m, 1H), 1.85–1.80 (m, 1H), 1.50 (s, 9H),
1.49 (s, 18H), 1.48 (s, 9H), 1.18–1.14 (m, 1H), 1.09–
1.03(m, 1H); MS (m/e) (positive-FAB) 791, 793 (peak
height ratio 3:1) (M + H)+.
2.2.7. Tetraethyl 2-hydroxymethyl-1,3-
propanebisphosphonate (14)
Compound 13 (3.28 g, 10 mmol) was dissolved in THF
(25 mL) and BH3-THF complex (34 mL of 1 M in THF,
34 mmol) was added at 0–5 8C, and the reaction mixture
was allowed to warm to room temperature. After stirring
for an additional 8 h, the mixture was cooled to 0 8C and
treated with solid K2CO3 (10 g) and 30% hydrogen per-
oxide (20 mL), added slowly. The resulting reaction mix-
ture was stirred at room temperature for 2 h. It was then
extracted twice with EtOAc (100 mL X 2), and the com-
bined organic layer was dried over sodium sulfate and
concentrated in vacuo. The final purification was effected
by silica-gel column chromatography (EtOAc:MeOH,
75:25) to afford 14 (2.49 g, 72%) as an oil. The spectral
data of 14 were found to be comparable with a genuine
sample prepared previously [21].
2.2.5. (10R,20S,40S,50S)-4-(6-chloro-2-iodo-9H-purin-9-
yl)-1-[(phosphato)-methyl]-2-(phosphato)-
bicyclo[3.1.0]hexane tetrakis ammonium salt (12)
A mixture of 11 (33.0 mg, 0.042 mmol) in CH2C12
(3 mL) was treated with trifluoroacetic acid (TFA,
0.100 mL) and the reaction mixture was stirred at room
temperature for 3 h. After removal of the solvent, the crude
12 was purified with ion-exchange column chromatogra-
phy with the use of Sephadex-DEAE-A-25 resin with a
2.3. Binding assay
The affinities of bisphosphate analogues for the human
P2Y1 receptor were directly determined by using

M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
1999
[3H]MRS2279 in a radioligand binding assay, as we
recently described in detail [18]. Briefly, the human
P2Y1 receptor was expressed in 1321N1 astrocytoma cells.
Membranes prepared from these cells were incubated for
30 min at 4 8C in the presence of ꢁ20 nM [3H]MRS2279
and a wide range of concentrations of the nucleotide
analogues. Binding reactions were terminated by the addi-
tion of ice-cold tris(hydroxymethyl)aminomethane (Tris)
wash buffer (10 mM Tris, pH 7.5, and 145 mM NaCl) to
the samples followed by rapid filtration over GF/A glass-
fiber filters. Each filter was washed with an additional 4 mL
of wash buffer, and radioactivity retained by the filters was
quantified by liquid scintillation spectrometry. All assays
were carried out in triplicate, and competition curves for all
molecules were generated in three separate experiments.
IC50 values were determined from each competition curve,
and a Ki value was calculated for each analogue according
to the relationship Ki = IC50/1 + [[3H]MRS2279]/Kd of
[3H]MRS2279, where the Kd of [3H]MRS2279 determined
in separate experiments was 8 nM.
ously before and after stimulation with ADP, using a
spectrofluorimeter (LS50B, Perkin-Elmer Co.). The exci-
tation wavelength was alternatively fixed at 340 and
380 nm, and fluorescence emission was determined at
510 nm.
2.7. Measurement of cyclic AMP (cAMP) in platelets
Platelet cyclic AMP levels were measured by a radio-
isotopic assay, using a commercially available kit (Amer-
sham). Duplicate samples of 1 mL washed platelets
containing theophylline (1 mM) were incubated with: (a)
Tyrode’s buffer and PGE1 (1 mmol/L), (b) Tyrode’s buffer,
PGE1 and ADP (10 mM) or (c) Tyrode’s buffer alone. After
incubation at 37 8C (2 min), 1 mL of 5% trichloroacetic
acid was added, and the samples were snap-frozen in dry
ice and methanol, thawed at room temperature (22–25 8C),
and then shaken at 4 8C for 45 min. After centrifugation at
4 8C for 30 min, the supernatant was extracted three times
with 5 mL of water-saturated ether, dried under a stream of
nitrogen at 60 8C, and stored at ꢀ20 8C. Before assay, the
samples were reconstituted with 50 mM Tris buffer con-
taining 4 mM EDTA.
2.4. Preparation of washed platelet suspensions
Six volumes of blood were drawn into one volume of
acid-citrate-dextrose anticoagulant. Twice washed platelet
suspensions were prepared according to the method
described by Mustard et al. [25], with the exception that
500 nmol/L PGI2 was added during the first and second
wash. The number of platelets was adjusted to 4 ꢂ 1011/L
in the final suspension, which contained Tyrode’s buffer,
0.35% bovine serum albumin, 0.1% dextrose and 1 mL/mL
apyrase.
2.8. Statistical analysis
Pharmacological parameters were analyzed by Graph-
PAD Prism software. Data were expressed as mean ꢃ
standard error.
3. Results
2.5. Studies of platelet aggregation and shape change
3.1. Chemistry
The inhibition of ADP-induced activation of human
platelets was measured using aggregometry using washed
platelet suspensions. Samples of washed platelet suspen-
sions (0.45 mL) were incubated with vehicle or test com-
pounds at 37 8C for 30 s and stirred at 1000 rpm in an
aggregometer (Lumi-Aggregometer, Chrono-log Corp.).
After incubation, 10 mL of ADP (final concentration,
5 mmol/L) was added and the aggregation tracings were
recorded for 3 min. The initial rapid decrease in oscilla-
tions of the basal tracings and increase in optical density
were interpreted as being caused by a platelet shape
change. Concentration response curves for antagonists
were constructed, and the IC50 values determined (n = 3).
An improved synthetic method for the preparation of
MRS2500 4 is shown in Fig. 2. The previous route of Kim
et al. [19] featured a condensation of the N6-methyl-2-
iodoadenine with the prephosphorylated and protected (N)-
methanocarba ring system 10. In contrast, the present
method began with the Mitsunobu coupling of 6-chloro-
2-iodoadenine with the same methanocarba ring moiety 10
to provide 11. Following deprotection of the phosphate
esters of 11 to yield 12, the 6-chloro was substituted with
methylamine resulting in MRS2500 4. This synthetic route
also would be amenable to tritium labeling upon treatment
of 12 with labeled methylamine, similar to the route used
for labeling MRS2279 [18].
We devised an alternate approach to the synthesis of
intermediate 10. The previous study used a 10-acetoxy
protection scheme in the phosphorylation step. The present
method used the orthogonal protection scheme of transient
acetylation of the 30-and 50-hydroxyl groups 7, followed by
silylation of the 10-hydroxyl and removal of the esters using
potassium carbonate, to provide intermediate 8. Phosphor-
ylation was carried out using the phosphoramidite method
2.6. Measurement of intracellular ionized calcium
([Ca2+]i)in platelets
Platelets were loaded with 2 mmol/L fura-2/AM at 37 8C
for 45 min as previously described [26]. Aliquots of fura-2/
AM-loaded platelets were transferred to quartz cuvettes
maintained at 37 8C. Fluorescence was monitored continu-

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M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
as described [19], and subsequent desilylation provided 10
in high yield.
All three antagonists also inhibited ADP-promoted
shape change with an order of potency identical to that
observed for aggregation and Ca2+ responses. In contrast
to platelet aggregation, the inhibition of ADP (10 mM)-
induced platelet shape change was complete at concen-
trations of the compounds that completely inhibited the
ADP-induced intracellular Ca2+ rise (Fig. 3).
The acyclic phosphonate MRS2496 was prepared by the
method reported by Xu et al. [23], except that an improve-
ment was made in the preparation of the intermediate
tetraethyl 2-hydroxymethyl-1,3 -propane bisphosphonate
14 from diethyl 2-(diethylphosphonomethyl)-allylpho-
sphonate 13. Use of borane-tetrahydrofuran complex in
this hydroboration step proved superior to 9-borabicy-
clo[3.3.1]nonane (9-BBN) in giving higher yields as well
as a simpler product isolation procedure.
The cAMP inhibitory pathway in platelets [26] is asso-
ciated with activation of P2Y12 receptors. We examined
whether any of these three antagonists for interaction with
the P2Y12 receptor-promoted pathway (Table 1 and Fig. 3).
No significant effect of compound 4 was observed on the
capacity of 10 mM ADP to inhibit PGE1-stimulated cAMP
accumulation. Compounds 4 and 6 at the highest concen-
tration tested (1 mM) caused approximately 40% inhibi-
tion of the ADP (10 mM)-induced effects on cAMP.
Compounds 4, 5, and 6 alone had no effect on basal cAMP
levels (not shown).
3.2. Inhibition of radioligand binding at the
human P2Y1 receptor
The affinity of the three nucleotide bisphosphate analo-
gues at the human P2Y1 receptor expressed in 1321N1
astrocytoma cells was determined in a binding assay using
the recently introduced antagonist radioligand [3H]MRS2279
[18] and Ki values were calclulated (Table 1). The order of
affinity based on Ki values was 4 ꢄ 5 > 6. Thus, the
conformationally constrained analogue 4was demonstrated
to have extremely high receptor affinity, while the two
acyclic compounds were of intermediate affinity.
4. Discussion
We have compared the effects of three antagonists of
the P2Y1 receptor on ADP-induced responses in human
platelets and at recombinant human P2Y1 receptors. All
compounds exhibited selective and potent antagonism of
the P2Y1-dependent responses of platelets to ADP. The
potency order was consistently 4 > 5 > 6 by a variety of
criteria for antagonism of P2Y1 receptors (Table 1). The
same order of potency was observed for all three antago-
nists for the inhibition of ADP-promoted Ca2+ mobiliza-
tion, shape change, and aggregation. Lower concentrations
of antagonists were necessary to inhibit completely platelet
aggregation than shape change and Ca2+ mobilization,
apparently because platelet aggregation requires a higher
degree of receptor occupancy by ADP than shape change
and Ca2+ mobilization [27]. In a previous study [23] it was
demonstrated the 5 and 6 inhibited the aggregation of
rat platetets induced by 3.3 mM ADP in platelet-rich
plasma with IC50 values of 300 and 680 nM, respectively.
Compound 4 was reported to inhibit the activation of PLC
induced by 30 nM 2-methylthio-ADP with an IC50 value of
8.4 nM [19]. Thus, the order of potency is consistent with
previous findings.
3.3. Inhibition of ADP-induced platelet responses
Three nucleotide bisphosphate analogues were exam-
ined as inhibitors of ADP-induced aggregation of human
platelets, using platelet aggregometry [23,26]. Compound
4, the (N)-methanocarba derivative, was highly potent in
inhibiting ADP (10 mM)-induced platelet aggregation with
an IC50 value of 0.95 nM. Compound 5, the acyclic bispho-
sphate antagonist MRS2298, and compound 6, the bispho-
sphonate acyclic nucleotide, also inhibited ADP (10 mM)-
induced platelet aggregation in a concentration-dependent
manner (Table 1) and with IC50 values of 62.8 nM and
1.5 mM, respectively. The effect of these three P2Y1
receptor antagonists in ADP-promoted Ca2+ mobilization
also was examined in human platelets. All three antago-
nists inhibited ADP (10 mM)-induced increases in intra-
cellular calcium, and the order of potency for inhibition
(4 > 5 > 6) was the same as observed for inhibition of
ADP-promoted aggregation (Table 1).
Table 1
Activities of the adenine nucleotide derivatives as antagonists of functions induced by 10 mM ADP in human platelets and in a competitive binding assay at
heterologously expressed human P2Y1 receptors in 1321 astrocytoma cells (n = 3)
IC50 or Ki (nM) (mean ꢃ S.E.M.)
Compound
Inhibition of P2Y1
receptor bindinga (Ki)
Inhibition of platelet
aggregationb (IC50
Inhibition of Ca2+
rise in plateletsb IC50
Inhibition of cAMP
effects in plateletsb (IC50
)
)
4 MRS2500
5 MRS2298
6 MRS2496
a
0.78 ꢃ 0.08
29.6 ꢃ 3.3
76 ꢃ 10
0.95 ꢃ 0.16
62.8 ꢃ 15.6
1500 ꢃ 250
49.1 ꢃ 6.7
810 ꢃ 207
>100,000
>100,000
>100,000
16,100 ꢃ 4600
Using [3H]MRS2279 as radioligand [18].
In washed platelet suspensions.
b

M. Cattaneo et al. / Biochemical Pharmacology 68 (2004) 1995–2002
2001
P2Y1-receptor mediated platelet function at micromolar
concentrations.
No substantial antagonism of the pathway linked to the
inhibition of cyclic AMP was observed with these three
P2Y1 receptor antagonists, indicating no interaction with
the proaggregatory P2Y12 receptor, which is also activated
by ADP. Thus, we have demonstrated the functional
selectivity of the derivatives for the P2Y1 receptor in
human platelets. It will be important to examine the
activity of these compounds in isolated organs and in vivo.
This would further clarify the feasibility of using P2Y1
receptor antagonists as antithrombotic agents, which has
already been established experimentally. A previously
reported P2Y1 receptor antagonist, the riboside derivative
MRS2179, was studied in the isolated guinea pig heart
[28], to demonstrate that vasodilation induced by endo-
genous ADP occurs principally through the P2Y1 receptor
rather than the adenosine A2A receptor (as a result of the
breakdown of ADP to adenosine). These compounds will
also be useful in determining the relative importance of the
two metabotropic ADP receptors in platelet activation in
vivo [29]. Since there are other known sites of the P2Y1
receptor in adult and developing mammals [30], both in the
brain and in the periphery (i.e. endothelial cells, bone cells,
skeletal muscle), it would be important to assess possible
side effects of such proposed therapeutic use using potent
and relatively stable analogues. In summary, these selec-
tive antagonists potentially are valuable as pharmacologi-
cal tools for defining the role of P2Y1 receptors in platelet
function and in other physiological processes.
Acknowledgements
B.V. Joshi thanks Gilead Sciences (Foster City, CA) for
financial support. We also thank Toray Industries, Inc.,
Pharmaceutical Research Laboratories (Kamakura, Japan)
for financial support. We thank Dr. G. Cook and G.
Kirschenheuter (formerly of Gilead Sciences) and Heng
T. Duong (NIDDK) for helpful discussion and Savitri
Maddileti (University of North Carolina, Chapel Hill,
NC) for technical assistance.
Fig. 3. Concentration-response curve for inhibition of ADP-induced pro-
cesses in human platelets by MRS2500 4 (upper panel), the acyclic bispho-
sphate derivative 5 (middle panel), and the acyclic bisphosphonate
derivative 6 (lower panel). Aggregation was induced in the presence of
10 mM ADP. The antagonism of the following parameters is expressed as
percent (mean ꢃ S.D.) of ADP-induced control in the presence of 1%
DMSO: aggregation, increase in Ca2+, and the inhibition of adenylate
cyclase. An upper bar indicates whether the initial shape change in the
platelets was observed at each concentration of the antagonist.
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