6-Amino-2-mercapto-3H-pyrimidin-4-one derivatives as new candidates for the antagonism at the P2Y12 receptors

Article abstract of DOI:10.1016/j.bmc.2009.04.061

P2Y₁₂ plays an important role in platelet aggregation, which makes it an interesting target for antithrombotic agents. Compounds that antagonize P2Y₁₂ include the active metabolites of thienopyridines and molecules that are structurally related to ATP, which is an antagonist of P2Y₁₂. During the last few years, our group has been working on the development of P2Y₁₂ receptors antagonists that are based on an extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substituted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two known P2Y₁₂ antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light transmission aggregometry. None of the tested compounds induced platelet aggregation, while some of them, at concentration of 10^-4 M, partially inhibited platelet aggregation induced by ADP 10^-6 M. The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the forskolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y₁₂-receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of P2 receptors, and in particular of P2Y₁₂ and suggest that further development of this structurally new series of compounds as P2Y₁₂ receptors antagonists is recommended.

Full text of DOI:10.1016/j.bmc.2009.04.061

Bioorganic & Medicinal Chemistry 17 (2009) 4612–4621
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
6-Amino-2-mercapto-3H-pyrimidin-4-one derivatives as new candidates
for the antagonism at the P2Y12 receptors
a
b
e
Pamela Crepaldi a, Barbara Cacciari a, , Maria-Cruz Bonache , Giampiero Spalluto , Katia Varani ,
*
Pier Andrea Borea e, Ivar von Kügelgen c, Kristina Hoffmann c, Mariateresa Pugliano d,
Cristina Razzari d, Marco Cattaneo d,
*
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17/19, I-44100 Ferrara, Italy
b Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1, I-34127 Trieste, Italy
c Department of Pharmacology and Toxicology, University of Bonn, Reuterstrasse 2b, D-53113 Bonn, Germany
d Unità di Medicina III, Azienda Ospedaliera San Paolo, Dipartimento di Medicina Chirurgia e Odontoiatria, Università degli Studi di Milano, Via Di Rudinì 8, I-20142 Milano, Italy
e Dipartimento di Medicina Clinica e Sperimentale-Sezione di Farmacologia, Università di Ferrara, Via Fossato di Mortara 17/19, I-44100 Ferrara, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
P2Y12 plays an important role in platelet aggregation, which makes it an interesting target for antithrom-
botic agents. Compounds that antagonize P2Y12 include the active metabolites of thienopyridines and
molecules that are structurally related to ATP, which is an antagonist of P2Y12. During the last few years,
our group has been working on the development of P2Y12 receptors antagonists that are based on an
extremely simple chemical structure, the 6-amino-2-mercapto-3H-pyrimidin-4-one, variously substi-
tuted at the sulfur and oxygen functions. This nucleus represents the simplified combination of two
known P2Y12 antagonists: the active metabolite of the thienopyridines and ATP derivatives. The effects
of the synthesized compounds were tested on ADP-induced human platelet aggregation, using light
transmission aggregometry. None of the tested compounds induced platelet aggregation, while some
of them, at concentration of 10ꢀ4 M, partially inhibited platelet aggregation induced by ADP 10ꢀ6 M.
The most potent compound, 6b, antagonized the inhibitory effect of 2-methylthio-ADP on the for-
Received 24 April 2009
Accepted 28 April 2009
Available online 3 May 2009
Keywords:
P2Y12 receptors antagonists
6-Amino-2-mercapto-3H-pyrimidin-4-one
derivatives
Platelet aggregation
Recombinant human P2Y12-receptors
skolin-induced accumulation of cyclic-AMP in CHO FlpIN cells expressing recombinant human P2Y12
-
receptors. In addition, none of the tested compounds, including 6b, interfered with ligand binding to
P1 receptors. Our results suggest that some of the synthesized compounds are specific antagonists of
P2 receptors, and in particular of P2Y12 and suggest that further development of this structurally new ser-
ies of compounds as P2Y12 receptors antagonists is recommended.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
internal stores and mediates shape change and a slight and rapidly
reversible platelet aggregation, while the P2Y12 receptor mediates
In the last decade, many efforts have been made to characterize
a progressive and sustained aggregation not preceded by shape
the P2Y12 receptor in order to understand its physiological and
change.2
3
pathophysiological roles.1,2 The cloning of P2Y12
,
showed that it
In addition to its role in ADP-induced platelet aggregation,
P2Y12 also mediates (a) the potentiation of platelet secretion in-
duced by strong agonists, which is independent of the formation
of large aggregates and thromboxane A2 synthesis;6,7 (b) shear-in-
duced platelet aggregation8,9 (c) the stabilization of thrombin-in-
duced platelet aggregates,10,11 and (d) the potentiation of the
antiplatelet effects of the natural regulator of platelet function
prostacyclin, which reduces platelet reactivity to agonists by
increasing the platelet content of cyclic adenosine monophosphate
(cAMP), mediated by the stimulation of adenylyl cyclase.12
The selective tissue distribution of P2Y12 makes it an attractive
molecular target for therapeutic intervention. Indeed, P2Y12 is the
target of efficacious antithrombotic agents like the thienopyridines
ticlopidine, clopidogrel and prasugrel, which are already used in
belongs to the family of purinergic G-protein coupled P2Y recep-
tors, as previously suggested by functional studies on the response
to ADP of human platelets, and it is composed of 342 amino acids.3
The tissue distribution of P2Y12 is restricted to platelets, vascular
smooth muscle cells and some cells in the peripheral and central
nervous system.3–5 In cooperation with another P2Y receptor,
P2Y1, P2Y12 mediates platelet aggregation induced by ADP. The
P2Y1 receptor induces the mobilization of ionized calcium from
* Corresponding authors. Tel.: +39 0532455256; fax: +39 0532455953 (B.C.), tel.:
+39 0250323095/8184463; fax: +39 0250323090 (M.C.)
E-mail addresses: ccb@unife.it (B. Cacciari), marco.cattaneo@unimi.it (M.
Cattaneo).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.04.061

P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
4613
selective and stable, non-phosphate, competitive P2Y12 antago-
nist. Further refinement to increase the oral bio-availability re-
sulted in the discovery of the selective P2Y12 competitive
antagonist ticagrelor (AZD6140), which is currently being devel-
oped as oral antiplatelet agent. AR-C109318XX and ticagrelor
both belong to the new chemical class of cyclopentyl-triazolo-
pyrimidines (Fig. 2).13,21
O
Cl
F
R
H
N
N
O
S
S
O
clopidogrel R= COOCH3
ticlopidine R= H
prasugrel
During the last few years, our group has been working on the
development of P2Y12 receptors antagonists that are based on an
extremely simple chemical structure, the 6-amino-2-mercapto-
3H-pyrimidin-4-one, variously substituted at the sulfur and oxy-
gen functions. This nucleus represents a simplified combination
of known P2Y12 antagonists: the active metabolites of the thieno-
pyridines and the ATP derivatives. Our working hypothesis was
based on the observation that the active metabolites of thieno-
pyridines, which inhibit the P2Y12 receptor irreversibly, have a
simple monocyclic structure, which might imply that the pres-
ence of a bicyclic structure like that of a purine ring, is not an
absolute requirement for the affinity for the receptors. Moreover,
we maintained a free amino group and tried different substitu-
tions on the thio group in analogy with the ATP derivatives. The
oxygen was substituted preferentially with aryl sulfonyl groups
mimicking the aromatic, flat shape of the thienopyridines re-
ported in the literature (Fig. 3). The derivatives of 6-amino-2-
mercapto-3H-pyrimidin-4-one can be easily synthesized in few
steps with high yields.
R
N
HOOC
HS
active metabolite of thienopyridines
Figure 1.
clinical practice either alone or in combination with other anti-
thrombotic drugs.13 Thienopyridines are pro-drugs that are inac-
tive in vitro and need to be metabolized in vivo by the hepatic
cytochrome P-450 enzymatic pathway to active metabolites.13
Their metabolites expose a SH group, which interacts with cysteine
residues in the P2Y12 receptor, forming a disulfide bond, which
irreversibly inactivates the receptor (Fig. 1).14–16
Other compounds have been reported to act as antagonists of
the P2Y12 receptor. Among them, cangrelor, [N6-(2-methylthio-
ethyl)-2-(3,3,3-trifluoropropylthio)-50-adenylic acid, monoanhy-
dride with dichloromethylenbis (phosphinic acid)], previously
known as AR-C69931MX, belongs to a family of analogs of ATP
that are relatively resistant to breakdown by ectonucleotidases
and display high affinity for the P2Y12 receptor (Fig. 2).17,18 These
ATP analogs are characterized by the presence of substituents in
the 2-position of the adenine ring, which increase the affinity
2. Chemistry
The 6-amino-2-mercapto-3H-pyrimidin-4-one was synthesized
by reacting thiourea and ethyl cyanoacetate in the presence of so-
dium ethylate, following the well established procedure.22 The thio
group was alkylated under basic conditions in the presence of the
appropriate alkyl halide, followed by the reaction with the various
aryl sulfonyl chlorides in order to synthesize compounds 5a–n
(Schemes 1 and 2).
The BOC-protected derivatives 5b,c were reacted with trifluoro
acetic acid to give the compounds 6b,c by standard procedure
(Scheme 3).
properties of the molecule, and of b,c-methylene substitutions
in the tri-phosphate part of the molecule, which increase the
metabolic stability. Among the ATP analogs, PEAdo and HeAdo in-
hibit human platelet aggregation: they are substituted at 2-posi-
tion but lack the alkyl-thio moiety, whereas, MRS 2395 lacks also
the sugar portion.19,20 Efforts to find compounds for oral adminis-
tration led first to the discovery of AR-C109318XX, the first oral,
SCH
3
HN
N
HN
N
N
F
N
N
N
N
Cl
Cl
N
F
CF
O
P
O
P
O
P
3
S
HO
O
O
N
SC H
3 7
O
O
HO
OH OH OH
OH
OH
HO
AR-C69931MX, cangrelor
HO
AZD6140, ticagrelor
NH
NHCH
2
3
N
N
N
N
N
O
N
N
R
R
O
O
N
Cl
2
O
O
O
OH
HO
HeAdo R=nC H
4
9
MRS2395
R = mono-, di, triphosphate
2
PEAdo R=C H
6
5
Figure 2.

4614
P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
concentration (10ꢀ4 M) throughout the experiments, induced
NH2
R2
O
aggregation of human platelets, indicating that they are not ago-
nists at the platelet P2Y receptors. The majority of the tested com-
pounds did not inhibit platelet aggregation induced by ADP
10ꢀ5 M, with only a few (4b, 4g, 5m, and 14) causing partial inhi-
bition (about 10–20%) (Tables 1 and 2). When ADP was used at
lower concentration (10ꢀ6 M), the number of inhibitory com-
pounds and the extent of inhibition of platelet aggregation that
they caused increased: 4b, 4f, 4g, 5d, 5n, 6b, and 14 inhibited
platelet aggregation by 40–60% (Tables 1 and 2).
Our results indicate that a simple monocyclic structure can
be used as a core for the synthesis of a series of analogs en-
dowed with antagonistic activity against the P2Y receptors on
platelets.
N
N
N
N
N
OH
HS
R1
R
Purinic structures
Active metabolite
NH2
N
N
R1O
SR
Despite the limited number of the synthesized compounds, it is
possible to observe that the presence of a free amino group is
important for activity, since compounds 16 and 9 lost their activity
in comparison to the analog 4e. The substituent on the thio group
seems critical for the activity. In analogy with the substituent in
the ticlopidine derivative, we tested the 2-chloro-benzyl moiety
which did not have effects in this series of compounds. Sterical hin-
drance might have a key role: the small methyl group in 5a or the
highly hampered trytil function in 4h, are detrimental for activity.
The pentyl chain on the sulfur gave better results (4f and 5n),
whereas, the presence of alkyl functions bearing hydrogen bond
donors, such as the 2-amino- or 2-hydroxy-ethyl chains, seems
important for the activity compared to the hydrogen bond acceptor
2,2,2-trifluoro ethyl substituent, which lose completely the
activity.
The presence of an aryl sulfonyl group appears to highly influ-
ence the activity of the compounds. The tosyl group is preferred re-
spect to a more hampered group such as the dansyl moiety or the
electron-withdrawing 2-chloro-benzensulfonyl group. In fact, its
presence increases the activity as in the analog 5j respect to the
unsubstituted analog 4e. Preferentially, we insert these substitu-
ents on the oxygen at the 4- position, but the tosyl group seems
to increase the activity independently of its position, as shown
by the activity of 4g. Furthermore, we tried to substitute the oxy-
6-Amino-2-Substituted mercapto-4Substitued-pyrimidinine
Figure 3.
In a similar way, compound 9 was synthesized by alkylation of
compound 8 obtained by reacting thiourea and diethyl malonate
under the same reaction conditions.
The reaction of thiourea with malononitrile provided compound
11, which, after analogous steps of reactions, gave compound 13.
Alternatively, compound 4e was alkylated with iodomethane
and 2-chloro-benzyl chloride in the presence of anhydrous potas-
sium carbonate in DMF, respectively, to give compounds 14 and
15. Probably, the different positions of the substituent in the two
compounds were due to the steric hindrance of the alkyl halide.
Reaction of 4e with phenyl isocyanate gave the urea derivative
16 (Schemes 4–6).
3. Results and discussion
All compounds were screened for their effects on human plate-
let aggregation induced by ADP using light transmission aggre-
gometry. None of the compounds, which were tested at a single
NH2
NH
NH2
N
S
Alkyl Halide
NaOH 0.1M
S
EtONa
reflux, 3h
CN
CO Et
+
H N NH
2
2
N
H
3
N
H
4 a-h
2
O
O
SR
CH OH
3
1
2
Compound
R
4a
4b
4c
4d
4e
4f
CH3
CH2CH2NHBOC
CH2CH2OH
CH2CF3
2-Chlorobenzyl
C5H11
4g
4h
CH2CH2NHTos
Ph3C
Scheme 1.

P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
4615
NH
NH
2
2
N
N
ArSO Cl
2
r.t.
O
N
SR
R O
N
SR
1
H
4 a-h
5 a-n
Compound
5a
R
Compound
5h
R
R1
R1
Cl
CH3
CH2CF3
O2S
O2S
CH3
O2S
CH2CH2NHBOC
5b
5c
5d
5e
5f
5i
5j
CH2CF3
O2S
CH3
CH3
N
CH3
CH2CH2NHBOC O2S
CH3
2-Cl-Benzyl
2-Cl-Benzyl
O2S
CH3
N
CH3
Cl
CH2CH2OH
5k
5l
O2S
CH3
O2S
Cl
CH2CH2OH
CH2CH2OH
CH2CF3
2-Cl-Benzyl O2S
CH3
N
O2S
CH3
O2S
CH3
5m
5n
C5H11
C5H11
O2S
CH3
N
CH3
Cl
5g
O2S
CH3
O2S
Scheme 2.
NH2
NH2
N
N
N
CF3COOH
r.t., 45 min
NH2
NHBOC
N
S
S
R1O
R1O
5 b,c
6 b,c
Scheme 3.
Cl
OH
NH
OH
Cl
S
1
EtONa
reflux, 3h
CO2Et
CO2Et
N
Cl
+
H2N
NH2
NaOH 0.1M
CH3OH
N
H
O
N
H
S
S
O
7
8
9
Scheme 4.
gen at 4-position with a nitrogen in order to synthesize the isoster
of the more interesting compound of this series 6b. The derivative
13 did not show activity, confirming the influence of this position
in the interaction with the receptors.
In order to test whether our compounds with inhibitory activity
towards ADP-induced platelet aggregation antagonize the P2Y12
receptor, we tested only compound 6b, which displayed the high-
est inhibitory effect and representative of the series, on CHO FlpIn

4616
P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
NH2
NH
S
1
CN
CN
EtONa
reflux, 3h
+
H2N
NH2
N
S
H2N
10
11
BrCH2CH2NHBOC
NaOH 0.1M
CH3OH
NH2
N
NH2
1) TosCl
N
N
2) CF COOH
3
NH2
NHBOC
TosHN
S
N
S
H2N
13
12
Scheme 5.
O
NH2
N
N
H
NH2
N
HN
NCO
N
CH3I
N
H
SR
N
CH3
O
O
O
SR
N
H
SR
K2CO3
DMF
4 e
16
14
Cl
K2CO3
DMF
Cl
NH2
N
Cl
N
O
SR
15
Scheme 6.
cells expressing the recombinant human P2Y12-receptor. 2-
Methylthio-ADP (10 nM), a poorly hydrolysable ADP analog, was
used to activate the receptors. Receptor activation caused inhibi-
tion of the forskolin-induced cyclic AMP response element (CRE)-
directed luciferase expression. Compound 6b reduced the response
to the agonist 2-methylthio-ADP in a concentration-dependent
ments of ligand binding to the adenosine receptors A1, A2A, A2B,
and A3. As shown in Table 3, none of our tested compounds dis-
played activity for P1-receptors, proving to be selective for the
P2Y receptors family.
4. Conclusions
manner (Fig. A), with an IC50 of 8.08 lM, proving to be a possible
candidate as lead compound to develop a new series of P2Y12
In conclusion, it is possible to assume that 6-amino-2-mer-
capto-3H-pyrimidin-4-one can be used as a lead for the synthesis
of ligands for P2Y12 receptors on platelets. They behave as antago-
nists, inhibiting the aggregation induced by ADP. In particular, the
activity can be attributed to an interaction with the P2Y12 recep-
tors as shown by the results of our experiments performed on
cloned receptors expressed by FlpIn cells. These new compounds
can be synthesized easily in few steps, in contrast to many of the
P2Y12 antagonists reported in the literature, which are usually
ATP congeners. The base structure is promptly available and suit-
antagonists.
The other compounds showed such a poor activity, that trying
higher concentrations in the biological experiments could be
meaningless.
Recently, some derivatives of the 6-amino-2-mercapto-3H-pyr-
imidin-4-one, variously substituted at the sulfur and oxygen moi-
eties, structurally related to our series, were reported to be
antagonists of the human A3 adenosine receptors, which belong
to the P1-receptors family.23 For this reason, we tested some se-
lected compounds of our series (4b,g, 5d,f,n,and 6b), in experi-

P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
4617
Table 1
5. Experimental
Effect of different compounds on platelet aggregation induced by two different
concentrations of ADP
5.1. General
R
1
Reactions were routinely monitored by thin-layer chromatogra-
phy (TLC) on silica gel (precoated F254 Merck plates) and products
visualized with iodine or potassium permanganate solution. Infra-
red spectra (IR) were measured on a Perkin–Elmer 257 instrument.
1H NMR were determined in CDCl3 or DMSO-d6 solutions with a
Bruker AC 200 spectrometer, peak positions are given in parts
per million (d) downfield from tetramethylsilane as internal stan-
dard, and J values are given in hertz. Light petroleum ether refers
to the fractions boiling at 40–60 °C. Melting points were deter-
mined on a Buchi-Tottoli instrument and are uncorrected. Chroma-
tographies were performed using Merck 60–200 mesh silica gel. All
reported products showed IR and 1H NMR spectra in agreement
with the assigned structures. Organic solutions were dried over
anhydrous magnesium sulfate. Elemental analyses were performed
by the microanalytical laboratory of Dipartimento di Chimica, Uni-
versity of Trieste, and they were within 0.4% of the theoretical val-
ues for C, H, and N.
N
O
N
SR
R
2
4b-h, 9, 14, 16
Platelet aggregation (% of control)*
Compound
R
R1
R2
ADP 10–5
ADP 10ꢀ6
4b
4c
4e
4f
CH2CH2NHBOC
CH2CH2OH
2-Cl-Benzyl
C5H11
NH2
NH2
NH2
NH2
NH2
NH2
OH
NH2
H
H
H
H
H
H
H
CH3
H
76
99
101
100
89
100
96
88
40
114
88
63
51
81
97
61
91
4g
4h
9
14
16
CH2CH2NHTos
Ph3C
2-Cl-Benzyl
2-Cl-Benzyl
2-Cl-Benzyl
NHCONHPh
94
* Platelet aggregation observed in the presence of each compound was expressed as
% of platelet aggregation that was measured in the presence of the vehicle (mean
values of at least 2 experiments).
5.2. Synthesis of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidin-
4-one (3)
The thiourea (1) (1 g, 0.013 mol) and ethyl cyanoacetate (2)
(1.38 mL, 0.013 mol) were added to a freshly prepared solution of
sodium ethylate (Na 0.3 g, 0.013 mol, in EtOH, 20 mL).
The mixture was refluxed for 3 h, then the precipitate was fil-
tered off. The solid was dissolved in water (15 mL), the pH adjusted
around 7–8, and the resulted precipitate was filtered.
White solid (78% yield, mp >300 °C); 1H NMR (DMSO-d6) d: 4.69
(s, 1H); 6.37 (br s, 2H); 11.52 (br s, 1H); 11.63 (br s, 1H); IR (KBr):
3450, 3220, 1690, 1320 cmꢀ1. Anal. Calcd for C4H5N3OS: C, 33.56;
H, 3.52; N, 29.35; S, 22.40. Found: C, 33.54; H, 3.54; N, 29.34; S,
22.40.
Table 2
Effect of different compounds on platelet aggregation induced by two different
concentrations of ADP
NH2
N
N
SR
R1
5a-n, 6b,c,13,15
Compound
R
R1
ADP 10ꢀ5
ADP 10ꢀ6
Platelet aggregation (% of control)*
5.3. General procedure for the preparation of compounds 4a–h
5a
5b
5d
5e
5f
5g
5h
5i
CH3
OTos
OTos
OTos
96
97
99
100
100
101
102
104
100
96
101
88
95
96
99
120
75
57
110
93
105
97
115
68
92
96
96
CH2CH2NHBOC
CH2CH2OH
CH2CH2OH
CH2CH2OH
CH2CF3
To a solution of compound 3 (0.5 g, 3.49 mmol) in 35 mL of
NaOH 0.1 M in CH3OH, the opportune alkyl halide (3.49 mmol)
was added, and the solution was stirred for 5 h at room tempera-
ture. Then the solvent was concentrated to 4–5 mL under reduced
pressure and the residue left in the fridge for 24 h. The residue was
washed with water (10–15 mL) and filtered to furnish the right
compound.
O-2-Cl-Benzensulfonyl
ODansyl
OTos
O-2-Cl-Benzensulfonyl
ODansyl
OTos
O-2-Cl-Benzensulfonyl
ODansyl
OTos
O-2-Cl-Benzensulfonyl
OTos
ODansyl
CH2CF3
CH2CF3
5j
5k
5l
5m
5n
6b
6c
13
15
2-Cl-Benzyl
2-Cl-Benzyl
2-Cl-Benzyl
C5H11
5.3.1. 6-Amino-2-methylsulfanyl-3H-pyrimidin-4-one (4a)
Yield: 81%, mp 275–277 °C; 1H NMR (DMSO-d6) d: 2.37 (s, 3H);
4.90 (s,1H); 5.87 (br s,2H); 7.97 (br s,1H); IR (KBr): 3465, 3218,
1685 cmꢀ1. Anal. Calcd for C5H7N3OS: C, 38.20; H, 4.49; N, 26.73;
S, 20.40. Found: C, 38.18; H, 4.49; N, 26.75; S, 20.39.
C5H11
58
37
83
91
CH2CH2NH2
CH2CH2NH2
CH2CH2NH2
2-Cl-Benzyl
NHTos
O-2-Cl-Benzyl
91
95
86
*
Platelet aggregation observed in the presence of each compound was expressed as
percentage of platelet aggregation that was measured in the presence of the vehicle
(mean values of at least two experiments).
5.3.2. [2-(4-Amino-6-oxo-1,6-dihydro-pyrimidin-2-ylsulfanyl)-
ethyl]- carbamic acid tert-butyl ester (4b)
Yield: 76%, mp183–185 °C; 1H NMR (DMSO-d6) d: 1.37 (s,1H);
3.04 (t, 2H, J = 3.4 Hz); 3.16 (q, 2H, J = 2 Hz); 4.86 (s, 1H); 6.10 (br
s, 2H); 7.01 (br s, 1H); 11.20 (br s,1H); IR (KBr): 3450, 3220,
1720, 1690 cmꢀ1. Anal. Calcd for C11H18N4O3S: C, 46.14; H, 6.34;
N, 19.57; S, 11.20. Found: C, 46.12; H, 6.36; N, 19.56; S, 11.20.
able for various substitutions in different positions with the pur-
pose of increasing the activity, which is poor, till now. In ongoing
studies, the structure–response relationship is being studied in
more detail, in order to understand the requirements for the inter-
action of the compounds with the receptors. This work will allow
us to increase the affinity of this series of compounds for the P2
receptors of the platelets. Moreover, we will investigate the affinity
and selectivity for the cloned P2Y12 receptor.
5.3.3. 6-Amino-2-(2-hydroxy-ethylsulfanyl)-3H-pyrimidin-4-
one (4c)
Yield: 81%, mp: >270 °C; 1H NMR (DMSO-d6) d: 3.15 (t, 2H,
J = 4 Hz); 3.38 (br s, 1H); 3.58 (t, 2H, J = 6 Hz); 4.88 (s, 1H); 6.40

4618
P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
Table 3
Inhibition of binding and effects of examined compounds to A1, A2A, A2B and A3 adenosine receptors
Compound
[3H] DPCPX binding hA1CHO
cells % of displacement
[3H]ZM241385 binding hA2ACHO
cells % of displacement
cAMP assay in hA2BCHO
cells % of inhibition
[
125I]ABMECA binding hA3CHO
cells % of displacement
4b
4g
5d
5f
7
6
2
1% (10
1% (10
1% (10
l
l
l
M)
M)
M)
7
1
1
1
1
2% (10
1% (10
1% (10
1% (10
1% (10
l
l
l
l
l
M)
M)
M)
M)
M)
8
3
3
5
4
2
5
7
1% (10
1% (10
1% (10
1% (10
1% (10
1% (10
1% (10
1% (10
l
l
l
l
l
l
l
l
M)a
M)b
M)a
M)b
M)a
M)b
M)a
M)b
21 2% (10
44 4% (10
46 4% (10
47 5% (10
23 2% (10
36 3% (10
lM)
lM)
lM)
lM)
lM)
lM)
48 4% (10 lM)
5n
6b
4
3
1% (10
1% (10
l
M)
M)
10 1% (10
8
7
l
M)a
1% (10
1% (10 l
l
M)b
l
18 2% (10
l
M)
M)a
10 1% (10
l
M)b
Data are expressed as means SEM of four independent experiments performed in triplicate.
Percent displacement of the specific binding or of cAMP inhibition in the absencea and in the presenceb of NECA 200 nM is indicated. The tested concentration of examined
compounds is reported within brackets.
(br s, 2H); 10.80 (br s,1H); IR (KBr): 3450, 3280, 3220, 1690 cmꢀ1
Anal. Calcd for C6H9N3O2S: C, 38.49; H, 4.85; N, 22.44; S, 17.13.
Found: C, 38.50; H, 4.84; N, 22.41; S, 17.13.
.
temperature for 5–7 h, then the solvent was removed under re-
duced pressure. The residue was taken up with water (25 mL)
and extracted with ethyl acetate (20 mL ꢁ 3). The organic phase,
dried with anhydrous Na2SO4, evaporated, and purified by flash
chromatography (ethyl acetate/petroleum ether 20%), afforded
the right compounds.
5.3.4. 6-Amino-2-(2,2,2-trifluoro-ethylsulfanyl)-3H-pyrimidin-
4-one (4d)
Yield: 72%, mp: 215–217 °C; 1H NMR (CDCl3) d: 3.94 (q, 2H,
J = 10 Hz); 4.95 (br s, 2H); 5.28 (s, 1H); 10.10 (br s, 1H); IR (KBr):
3400, 3223, 1690 cmꢀ1. Anal. Calcd for C6H6F3N3OS: C, 32.00; H,
2.69; N, 18.66; S, 14.24. Found: C, 32.02; H, 2.67; N, 18.65; S, 14.21.
5.4.1. Toluene-4-sulfonic acid 6-amino-2-methylsulfanyl-
pyrimidin-4-yl ester (5a)
Yield: 68%, mp: >270 °C; 1H NMR (DMSO-d6) d: 2.16 (s, 3H);
2.42 (s, 3H); 5.83 (s, 1H); 7.33 (br s, 2H); 7.50 (d, 2H, J = 8), 7.86
5.3.5. 6-Amino-2-(2-chloro benzylsulfanyl)-3H-pyrimidin-4-
one (4e)
(d, 2H, J = 8), 11.49 (br s, 1H); IR (KBr): 3385, 3320, 1715 cmꢀ1
.
Anal. Calcd for C12H13N3O3S2: C, 46.29; H, 4.21; N, 13.49; S,
20.60. Found: C, 46.30; H, 4.20; N, 13.49; S, 20.58.
Yield: 76%, mp: >270 °C; 1H NMR (DMSO-d6) d: 4.40 (s, 2H);
4.98 (br s, 1H); 6.61 (br s, 2H); 7.28–7.31(m, 2H); 7.45–7.47(m,
1H); 7.68–7.71 (m, 1H); 11.45 (br s, 1H); IR (KBr): 3380, 3234,
1692 cmꢀ1. Anal. Calcd for C11H10ClN3OS: C, 49.35; H, 3.76; N,
15.69; S, 11.98. Found: C, 49.34; H, 3.77; N, 15.67; S, 11.98.
5.4.2. Toluene-4-sulfonic acid 6-amino-2-(2-tert-
butoxycarbonylamino-ethylsulfanyl)-pyrimidin-4-yl ester (5b)
Yield: 71%, mp: 228–230 °C; 1H NMR (CDCl3) d: 1.42 (s, 9H);
2.46 (s, 3H); 2.96 (t, 2H, J = 4 Hz); 3.27 (q, 2H, J = 4 Hz); 4.99 (br
s, 1H); 5.12 (br s, 2H); 5.85 (s, 1H); 5.36 (d, 2H, J = 4 Hz); 7.87 (d,
2H, J = 4 Hz); IR (KBr): 3450, 3220, 1720, 1362, 1180 cmꢀ1. Anal.
Calcd for C18H24N4O5S2: C, 49.07; H, 5.49; N, 12.72; S, 14.56. Found:
C, 49.05; H, 5.51; N, 12.72; S, 14.57.
5.3.6. 6-Amino-2-pentylsulfanyl-3H-pyrimidin-4-one (4f)
Yield: 68%, mp: >270 °C; 1H NMR (DMSO-d6) d: 0.86 (t, 3H,
J = 3 Hz); 1.30–1.33 (m, 4H); 1.58–1.61 (m, 2H); 3.04 (t, 2H,
J = 4); 5.75 (s, 1H); 6.40 (br s, 2H); 11.55 (br s, 1H); IR (KBr):
3378, 3241, 1690 cmꢀ1. Anal. Calcd for C9H15N3OS: C, 50.68; H,
7.09; N, 19.70; S, 15.03. Found: C, 50.70; H, 7.07; N, 19.67; S, 15.03.
5.4.3. 5-Dimethylamino-naphtalene-1-sulfonic acid-6-amino-
2-(2-tert-butoxycarbonylamino-ethylsulfanyl)-pyrimidin-4-yl
ester (5c)
5.3.7. N-[2-(4-Amino-6-oxo-1,6-dihydro-pyrimidin-2-
ylsulfanyl)-ethyl]-4-methyl-benzenesulfonamide (4g)
Yield: 67%, foam; 1H NMR (CDCl3) d: 1.37 (s, 9H); 2.61 (t, 2H,
J = 6 Hz); 2.85 (s, 6H); 2.96–3.11 (m, 2H); 4.83 (t, 1H, J = 6 Hz), 5.56
(br s, 2H); 5.84 (s, 1H); 7.18 (d, 2H, J = 8 Hz), 7.51–7.62 (m, 4H);
8.22–8.31 (m, 4H); 8.60 (d, 2H, J = (Hz); IR (KBr): 3455, 3230, 1717,
1358, 1180 cmꢀ1. Anal. Calcd for C23H29N5O5S2: C, 53.16; H, 5.63;
N, 13.48; S, 12.34. Found: C, 53.15; H, 5.64; N, 13.48; S, 12.33.
Yield:65%, colorlessoil; 1H NMR (CDCl3) d: 2.42 (s, 3H); 3.09–3.12
(m, 2H); 3.26 (s, 2H); 3.39 (t, 2H, J = 6); 4.92 (s, 1H); 7.30 (d, 2H, J = 6);
7.61 (br s, 1H); 7.74 (d, 2H, J = 6); 11.41 (br s, 1H); IR (KBr): 3380,
3292, 3250, 1690 cmꢀ1. Anal. Calcd for C13H16N4O3S2: C, 45.87; H,
4.74; N, 16.46; S, 18.84. Found: C, 45.87; H, 4.75; N, 16.45; S, 18.82.
5.3.8. 6-Amino-2-tritylsulfanyl-3H-pyrimidin-4-one (4h)
Yield: 52%, mp: >270 °C; 1H NMR (DMSO-d6) d: 6.25 (s, 1H);
6.49 (br s, 2H); 7.22–7.44 (m, 15H); 11.49 (br s, 1H); IR (KBr):
3380, 3312, 1690 cmꢀ1. Anal. Calcd for C23H19N3OS: C, 71.66; H,
4.97; N, 10.90; S, 8.32. Found: C, 71.65; H, 4.98; N, 10.92; S, 8.32.
5.4.4. Toluene-4-sulfonic acid 6-amino-2-(2-hydroxy-
ethylsulfanyl)-pyrimidin-4-yl ester (5d)
Yield: 75%, foam; 1H NMR (DMSO-d6) d: 2.42 (s, 3H); 2.85 (t, 2H,
J = 6 Hz); 3.45 (t, 2H, J = 6 Hz); 4,75 (br s, 1H); 5.83 (s, 1H); 7.33 (br
s, 2H); 7.49 (d, 2H, J = 8 Hz); 7.86 (d, 2H, J = 8 Hz); IR (KBr): 3385,
1721, 1358, 1180 cmꢀ1. Anal. Calcd for C13H15N3O4S2: C, 45.73;
H, 4.43; N, 12.31; S, 18.78. Found: C, 45.73; H, 4.45; N, 12.29; S,
18.79.
5.4. General procedure for the synthesis of aryl sulfonyl
derivatives 5a–n
To a solution of the 6-amino-2-substituted-3H-pyrimidin-4-
ones (4a–f) (1.74 mmol) in dioxane (10 mL) and DMF (10 mL), tri-
ethyl amine (1.74 mmol, 0.24 mL) and the opportune aryl sulfonyl
chloride (1.74 mmol) were added. The mixture was stirred at room
5.4.5. 2-Chloro-benzensulfonic acid 6-amino-2-(2-hydroxy-
ethylsulfanyl)-pyrimidin-4-yl ester (5e)
Colorless oil, 54% yield; 1H NMR (CDCl3) d: 2.86 (br s, 1H); 2.90 (t,
2H, J = 6 Hz); 3.67 (t, 2H, J = 6 Hz), 5.58 (br s, 2H); 5.86 (s, 1H); 7.45–

P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
4619
7.49 (m, 1H); /.57-/.60 (m, 2H); 8.08–8.12 (m, 1H); IR (KBr): 3398,
1720, 1360, 1180 cmꢀ1. Anal. Calcd for C12H12ClN3O4S2: C, 39.83;
H, 3.34; N, 11.61; S, 17.72. Found: C, 39.85; H, 3.32; N, 11.61; S, 17.71.
(s, 1H); 7.33 (d, 2H, J = 8 Hz); 7.88 (d, 2H, J = 8 Hz); IR (KBr): 3450,
3215, 1350, 1172 cmꢀ1. Anal. Calcd for C16H21N3O3S2: C, 52.29; H,
5.76; N, 11.43; S, 17.45. Found: C, 52.30; H, 5.76; N, 11.42; S, 17.43.
5.4.6. 5-Dimethylamino-naphtalene-1-sulfonic 6-amino-2-(2-
hydroxy-ethylsulfanyl)-pyrimidin-4-yl ester (5f)
5.4.14. 2-Chloro-benzensulfonic acid 6-amino-2-pentylpyrimidin-
4-yl ester (5n)
Yellow oil, 77% yield; 1H NMR (CDCl3) d: 2.83 (br s, 1H); 2.73 (t,
2H, J = 6 Hz); 2.85 (s, 6H); 3.48 (t, 2H, J = 6 Hz), 5.49 (br s, 2H); 5.81
(s, 1H); 7.18 (d, 2H, J = 8 Hz); 7.48–7.63 (m, 4H); 8.24–8.30 (m,
4H); 8.60 (d, 2H, J = 8 Hz); IR (KBr): 3456, 3230, 1720, 1356,
1180 cmꢀ1. Anal. Calcd for C18H20N4O4S2: C, 51.41; H, 4.79; N,
13.32; S, 15.25. Found: C, 51.44; H, 4.76; N, 13.32; S, 15.26.
Yield: 67%, foam: ; 1H NMR (DMSO-d6)d:0.84(t, 3H, J = 6 Hz); 1.18–
1.38 (m, 6H); 2.87–2-89 (m, 2H); 5.87 (s, 1H), 7.37 (br s, 2H); 7.7.58–
7.63 (m, 2H); 7.80–7.82 (m, 2H); 8.15–8.17 (m, 1H); IR (KBr): 3432,
3278, 1358, 1180 cmꢀ1. Anal. Calcd for C15H18ClN3O3S2: C, 46.44; H,
4.68; N, 10.83; S, 16.53. Found: C, 46.42; H, 4.70; N, 10.83; S, 16.53.
5.5. General procedure for the synthesis of 6b,c
5.4.7. Toluene-4-sulfonic acid 6-amino-2-(2,2,2-trifluoro-
ethylsulfanyl)-pyrimidin-4-yl ester (5g)
A solution of compounds 5b,c (1.25 mmol) in CF3COOH (3 mL)
was stirred at room temperature for 40 min. Then, the solvent
was removed and the residue was dissolved in water (5 mL) neu-
tralized with saturated NaHCO3 solution, and extracted with ethyl
acetate (10 mL ꢁ 3). The organic layer, dried with Na2SO4, was
evaporated to furnish the compounds 6b,c.
Yield: 71%, mp: 133–136 °C; 1H NMR (CDCl3) d: 2.45 (s, 3H);
3.57 (q, 2H, J = 10 Hz); 5.23 (br s, 2H); 5.95 (s, 1H); 7.35 (d, 2H,
J = 8 Hz); 7.87 (d, 2H, J = 8 Hz); IR (KBr): 3450, 3220, 1360,
1170 cmꢀ1. Anal. Calcd for C13H12F3N3O3S2: C, 41.16; H, 3.19; N,
11.08; S, 16.90. Found: C, 41.15; H, 3.20; N, 11.08; S, 16.87.
5.4.8. 2-Chloro-benzensulfonic acid 6-amino-2-(2,2,2-trifluoro-
ethylsulfanyl)-pyrimidin-4-yl ester (5h)
5.5.1. Toluene-4-sulfonic acid 6-amino-2-(2-amino-
ethylsulfanyl)-pyrimidin-4-yl ester (6b)
Yield: 62%, mp: 119–121 °C; 1H NMR (CDCl3) d: 3.42 (q, 2H,
J = 10 Hz); 5.26 (br s, 2H); 5.98 (s, 1H); 7.54–7.57 (m, 1H); 7.60–
7.62 (m, 2H); 8.09–8.13 (m, 1H); IR (KBr): 3400, 3230, 1360,
1180 cmꢀ1. Anal. Calcd for C12H9ClF3N3O3S2: C, 36.05; H, 2.27; N,
10.51; S, 16.04. Found: C, 36.02; H, 2.30; N, 10.51; S, 16.05.
Yield: 87%, mp: 104–106 °C 1H NMR (DMSO-d6) d: 2.43 (s, 3H);
2.63 (t, 2H, J = 8 Hz); 2.78 (t, 2H, J = 8 Hz); 3.35 (br s,2H); 5.82
(s,1H); 7.31 (br s,2H); 7.50 (d, 2H,J = 8 Hz); 7.86 (d, 2H, J = 8 Hz);
IR (KBr): 3560, 3450, 3220, 1360, 1183 cmꢀ1. Anal. Calcd for
C13H16N4O3S2: C, 45.87; H, 4.74; N, 16.46; S, 18.84. Found: C,
45.86; H, 4.74; N, 16.47; S, 18.81.
5.4.9. 5-Dimethylamino-naphtalene-1-sulfonic acid 6-amino-2-
(2,2,2-trifluoro-ethylsulfanyl)-pyrimidin-4-yl ester (5i)
Yield: 65%, mp: 163–165 °C; 1H NMR (CDCl3) d: 2.90 (s, 6H);
3.34 (q, 2H, J = 10 Hz); 5.10 (br s, 2H); 5.96 (s, 1H); 7.24 (d, 1H,
J = 7 Hz); 7.52–7.67 (m, 2H); 8.28–8.35 (m, 2H); 8.66 (d, 1H, J =
7 Hz); IR (KBr): 3450, 3225, 1350, 1170 cmꢀ1. Anal. Calcd for
C18H17F3N4O3S2: C, 47.15; H, 3.74; N, 12.22; S, 13.99. Found: C,
47.15; H, 3.75; N, 12.24; S, 13.99.
5.5.2. 5-Dimethylamino-naphtalene-1-sulfonic acid 6-amino-2-
(2-amino-ethylsulfanyl)-pyrimidin-4-yl ester (6c)
Yield: 86%, mp: 144–146 °C; 1H NMR (CDCl3) d: 2.80 (s, 6H);
2.81–2.98 (m, 4H); 5.18 (br s, 2H); 5.69 (s, 1H); 5.85 (br s, 2H);
7.12 (d, 1H, J = 8 Hz); 7.42–7.57 (m, 2H); 8.21 (d, 2H, J = 8 Hz);
8.54 (d, 2H, J = 8 Hz); IR (KBr): 3564, 3449, 3220, 1365,
1178 cmꢀ1. Anal. Calcd for C18H21N5O3S2: C, 51.53; H, 5.05; N,
16.69; S, 15.29. Found: C, 51.52; H, 5.06; N, 16.69; S, 15.27.
5.4.10. Toluene-4-sulfonic acid 6-amino-2-(2-chloro
benzylsulfanyl)-pyrimidin-4-yl ester (5j)
5.6. Synthesis of 6-hydroxy-2-mercapto-3H-pyrimidin-4-one (8)
Yield: 68%, foam; 1H NMR (DMSO-d6) d: 2.35 (s, 3H); 4,10 (s,
2H); 5.87 (s, 1H); 7.25–7.30 (m, 2H); 7.42–7.49 (m, 6H); 7.86 (d,
2H, J = 8 Hz); IR (KBr): 3430, 3280, 1358, 1180 cmꢀ1. Anal. Calcd
for C18H16ClN3O3S2: C, 51.24; H, 3.82; N, 9.96; S, 15.20. Found: C,
51.25; H, 3.81; N, 9.98; S, 15.20.
The thiourea (1) (1 g, 0.013 mol) and diethyl malonate (7)
(1.97 mL, 0.013 mol) were added to a freshly prepared solution of
sodium ethylate (Na 0.3 g, 0.013 mol, in EtOH, 20 mL).
The mixture was refluxed for 3 h, then the precipitate was fil-
tered off. The solid was dissolved in water (10 mL), the pH adjusted
around 7–8, and the resulted precipitate was filtered.
5.4.11. 2-Chloro-benzensulfonic acid 6-amino-2-(2-chloro
benzylsulfanyl)-pyrimidin-4-yl ester (5k)
White solid (67% yield, mp >300 °C); 1H NMR (DMSO-d6) d: 3.34
(s, 1H); 4.10 (s, 1H); 10.46 (br s, 2H); IR (KBr): 3390, 3334, 1690,
1320 cmꢀ1. Anal. Calcd for C4H4N2O2S: C, 33.33; H, 2.80; N,
19.43; S, 22.24. Found: C, 33.36; H, 2.80; N, 19.40; S, 22.23.
Yield: 43%, foam; 1H NMR (DMSO-d6) d: 3.92 (s, 2H); 5.91 (s,
1H); 7.23–7.81 (m, 8H); 8.08–8.12 (m, 2H); IR (KBr): 3435, 3282,
1358, 1180 cmꢀ1. Anal. Calcd for C17H13Cl2N3O3S2: C, 46.16; H,
2.96; N, 9.50; S, 14.50. Found: C, 46.15; H, 2.97; N, 9.50; S, 14.47.
5.7. Synthesis of 6-hydroxy-2-(2-chloro benzylsulfanyl)-3H-
pyrimidin-4-one (9)
5.4.12. 5-Dimethylamino-naphtalene-1-sulfonic acid 6-amino-
2-(2-chloro benzylsulfanyl)-pyrimidin-4-yl ester (5l)
Yield: 51%, foam; 1H NMR (CDCl3) d: 2.76 (s, 6H); 3.75 (s, 2H);
5.09 (br s, 2H); 5.89 (s, 1H); 6.90–6.92 (m, 2H); 7.03–7.27 (m,
3H); 7.48–7.62 (m, 2H); 8.27–8.34 (m, 2H); 8.56–8.59 (m, 1H); IR
To a solution of compound 8 (0.3 g, 2 mmol) in 20 mL of NaOH
0.1 M in CH3OH, the 2-chlorobenzyl chloride (0.66 mL, 5 mmol)
was added, and the solution was stirred for 2.5 h at room temper-
ature. Then the solvent was removed and the residue was washed
with water (10–15 mL) and filtered to furnish the right compound.
Yield: 68% mp >300 °C; 1H NMR (DMSO-d6) d: 3.35 (br s, 2H);
4.21 (s, 1H); 4.32 (s, 2H); 7.25–7.27 (m, 2H); 7.41–7.43 (m, 1H);
7.46–7.60 (m, 1H); IR (KBr): 3390, 3334, 1690, 1320 cmꢀ1. Anal.
Calcd for C11H9ClN2O2S: C, 49.17; H, 3.38; N, 10.42; S, 11.93.
Found: C, 49.15; H, 3.40; N, 10.42; S, 11.93.
(KBr): 3445, 3220, 1350, 1170 cmꢀ1
.
Anal. Calcd for
C23H21ClN4O3S2: C, 55.14; H, 4.22; N, 11.18; S, 12.80. Found: C,
55.13; H, 4.23; N, 11.18; S, 12.80.
5.4.13. Toluene-4-sulfonic acid 6-amino-2-pentyl-pyrimidin-4-
yl ester (5m)
Yield: 71%, foam; 1H NMR (CDCl3) d: 0.87 (t, 3H, J = 6 Hz); 1.19–
1.42 (m, 6H); 2.44 (s, 3H); 2.82 (t, 2H, J = 8 Hz)); 5.09 (br s, 2H); 5.87

4620
P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
5.8. Synthesis of 4,6-diamino-pyrimidine-2-thiol (11)
5.12. Synthesis of 6-(2-chloro-benzyloxy)-2-(2-chloro-
benzylsulfanyl)-pyrimidin-4-yl amine (15)
The thiourea (1) (1 g, 0.013 mol) and malononitrile (10)
(860 mg, 0.013 mol) were added to a freshly prepared solution of
sodium ethylate (Na 0.3 g, 0.013 mol, in EtOH, 20 mL).
To a solution of compound 4e (0.05 g, 0.19 mmol) and K2CO3
(0.025 g, 0.19 mmol) in dry DMF (3 mL), 2-chloro-benzyl chloride
(0.048 mL, 0.38 mmol) was added. The mixture was stirred at room
temperature for 12 h, then the solvent was removed under reduced
pressure. The residue was dissolved in water (10 mL), neutralized
with aqHCl1 M, and extractedwith ethyl acetate (10 mL ꢁ 3). The or-
ganic layer was dried and evaporated to furnish the right compound.
Yield: 82%, mp >270 °C; 1H NMR (DMSO-d6) d: 4.37 (s, 2H); 5.37
(s, 2H); 5.52 (s, 1H); 6.86 (br s, 2H); 7.21–7.53 (m, 8H); IR (KBr):
3356, 1558 cmꢀ1. Anal. Calcd for C18H15Cl2N3OS: C, 55.11; H,
3.85; N, 10.71; S, 8.17. Found: C, 55.10; H, 3.86; N, 10.73; S, 8.17.
The mixture was refluxed for 3 h, then the precipitate was fil-
tered off. The solid was dissolved in water (10 mL), the pH adjusted
around 7–8, and the resulted precipitate was filtered.
White solid; 52% yield, mp >300 °C; 1H NMR (DMSO-d6) d: 5.01
(s, 1H); 6.56 (br s, 4H); 10.97 (br s, 1H); IR (KBr): 3434, 3350, 1692,
1320 cmꢀ1. Anal. Calcd for C4H6N4S: C, 33.79; H, 4.25; N, 39.40; S,
22.55. Found: C, 33.77; H, 4.27; N, 39.41; S, 22.54.
5.9. Synthesis of [2-(4,6-diamino-pyrimidin-2-ylsulfanyl)-
ethyl]-carbamic acid tert-butyl ester (12)
5.13. Synthesis of 1-[2-(2-chloro-benzylsulfanyl)-6-oxo-1,6-
dihydro-pyrimidin-4-yl]-3-phenyl-urea (16)
To a solution of compound 11 (0.1 g, 0.7 mmol) in 7 mL of NaOH
0.1 M in CH3OH, the (2-bromo-ethyl)-carbamic acid tert-butyl ester
(0.4 g, 1.75 mmol) was added, and the solution was stirred for 18 h
at room temperature. Then the solvent was removed and the resi-
due was purified by flash chromatography (ethyl acetate 100%) to
afford the right compound 12.
To a solution of compound 4e (0.05 g, 0.19 mmol) in dioxane
(10 mL), phenyl isocyanate (0.03 mL, 0.26 mmol) was added and
the mixture was refluxed for 18 h. The solvent was removed under
reduced pressure and the residue was purified by flash chromatog-
raphy (EtOAc 100%).
Yield: 72% (colorless oil); 1H NMR (CDCl3) d: 1.42 (s, 9H); 3.14 (t,
2H, J = 4 Hz); 3.40–3.48 (m, 2H); 4.73 (br s, 4H); 5.26 (s, 1H); 5.75
(br s, 1H); 7.12 (d, 1H, J = 8 Hz); 7.42–7.57 (m, 2H); 8.21 (d, 2H,
J = 8 Hz); 8.54 (d, 2H, J = 8 Hz); IR (neat): 3450, 3220, 1720,
1690 cmꢀ1. Anal. Calcd for C11H19N5O2S: C, 46.30; H, 6.71; N,
24.54; S, 11.24. Found: C, 46.32; H, 6.71; N, 24.52; S, 11.21.
Yield: 43%, mp >270 °C; 1H NMR (DMSO-d6) d: 4.49 (s, 2H); 5.81
(s, 1H); 6.99 (br s, 1H); 7.06–7.70 (m, 9H); 8.42 (br s, 1H); 9.27 (br
s, 1H); IR (KBr): 3425, 3340, 1678, 1558 cmꢀ1. Anal. Calcd for
C18H15ClN4O2S: C, 55.88; H, 3.91; N, 14.48; S, 8.29. Found: C,
55.87; H, 3.90; N, 14.48; S, 8.31.
5.10. Synthesis of N-[6-amino-2-(2-amino-ethyl)-pyrimidin-4-
yl]-4-methyl-benzenesulfonamide (13)
6. Pharmacological data
6.1. Platelet function studies
To a solution of compound 12 (0.29 g, 1 mmol) in pyridine (5 mL),
tosyl chloride (0.23 g, 1.2 mmol) was added. The mixture was stirred
at room temperature for 12 h, then the solvent was removed under
reduced pressure and, the residue was purified by flash chromatog-
raphy (ethyl acetate/petroleum ether 40%). Then, the compound
Boc-protected was immediately dissolved in CF3COOH (3 mL) and
the solution was stirred at room temperature for 45 min. The solvent
was removed and the residue was dissolved in water (5 mL) neutral-
ized with saturated NaHCO3 solution, and extracted with ethyl ace-
tate (10 mL ꢁ 3). The organic layer, dried with Na2SO4, was
evaporated to furnish the compounds 13.
The effects of the compounds on platelets was studied using
platelet-rich plasma (PRP) obtained from whole blood collected
in sodium citrate 10.9 mmol (9:1, v:v) from healthy volunteers,
upon centrifugation at 150g at 21 °C for 10 min. ADP (10ꢀ5 M or
10ꢀ6 M, final concentration) was added to PRP samples, which
had been prewarmed at 37 °C, in the presence of the compounds
at study or of their vehicles, in a light transmission aggregometer
(Macia Brunelli, Italy). Platelet shape change and aggregation were
monitored for 3 min after the addition of ADP.
Yield: 42%, mp: 282–284 °C ; 1H NMR (DMSO-d6) d: 2.30 (s,3H);
2.81–2.84 (m, 2H); 2.96–3.03 (m, 2H); 4.98 (br s, 2H); 5.49 (s, 1H);
5.81 (br s, 2H); 7.18 (d, 2H, J = 8 Hz); 7.63 (d, 2H, J = 8 Hz); 10.97
(br s, 1H); IR (KBr): 3455, 3230, 1690 cmꢀ1. Anal. Calcd for
C13H17N5O2S2: C, 46.00; H, 5.05; N, 20.63; S, 18.89. Found: C,
46.02; H, 5.03; N, 2.,63; S, 18.88.
6.2. Recombinant human P2Y12-receptors studies
The effect of 6b on the 2-methylthio-ADP mediated inhibition of
forskolin-induced cAMP response element (CRE)-directed lucifer-
ase expression were tested in CHO FlpIN cells expressing the re-
combinant human P2Y12-receptor. Cells were transiently
transfected with the pCRE-luc vector (Stratagene, Amsterdam,
Netherlands). 24 h later the medium was replaced by physiological
5.11. Synthesis of 6-amino-2-(2-chloro-benzylsulfanyl)-3-
methyl-3H-pyrimidin-4-one (14)
salt solution and the cells were treated with forskolin 1 lM and,
when used, 2-methylthio-ADP 10 nM and the antagonists indi-
cated for 3.5 h at 37 °C. Luciferase expression was quantified by
measuring luminescence after lysis of cells by Bright-Glo assay
solution (Promega, Mannheim). Compound 6b was solved in
dimethylsulfoxide (DMSO, Sigma, Deisenhofen, Germany). Value
is expressed as percent response to 2-methylthio-ADP in the ab-
sence of antagonist (presence of the solvent DMSO only).
To a solution of compound 4e (0.05 g, 0.19 mmol) and K2CO3
(0.025 g, 0.19 mmol) in dry DMF (3 mL), CH3I (0.034 mL,
0.55 mmol) was added. The mixture was stirred at room tempera-
ture for 12 h, then the solvent was removed under reduced pres-
sure. The residue was dissolved in water (10 mL), neutralized
with aq HCl 1 M, and extracted with ethyl acetate (10 mL ꢁ 3).
The organic layer was dried and evaporated to furnish the right
compound.
6.3. P1 Binding assays
Yield: 87%, mp >270 °C; 1H NMR (DMSO-d6) d: 3.21 (s,3H); 4.50
(s, 2H); 4.93 (s, 1H); 6.57 (br s, 2H); 7.30–7.34 (m, 2H); 7.46–7.51
(m, 1H); 7.74–7.78 (m, 1H); IR (KBr): 3440, 3220, 1685 cmꢀ1. Anal.
Calcd for C12H12ClN3OS: C, 51.15; H, 4.29; N, 14.91; S, 11.38.
Found: C, 51.13; H, 4.29; N, 14.91; S, 11.36.
6.3.1. Human cloned A1, A2A and A3 adenosine receptor binding
assay
All synthesized compounds were tested for evaluating their
affinity to human A1, A2A and A3 adenosine receptors. Displace-

P. Crepaldi et al. / Bioorg. Med. Chem. 17 (2009) 4612–4621
4621
of cyclic AMP levels. The reaction was terminated by the addition
of cold 6% trichloroacetic acid (TCA). The TCA suspension was cen-
trifuged at 2000g for 10 min at 4 °C and the supernatant was ex-
tracted four times with water saturated diethyl ether. The final
aqueous solution was tested for cyclic AMP levels by a competition
protein binding assay. Samples of cyclic AMP standard (0–10 pmol)
were added to each test tube containing [3H] cyclic AMP and the
incubation buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2-
mercaptoethanol 6.0 mM, pH 7.4). The binding protein prepared
from beef adrenals, was added to the samples previously incubated
at 4 °C for 150 min, and after the addition of charcoal were centri-
fuged at 2000g for 10 min.28
Acknowledgment
Figure A. The effect of 6b on the 2-methylthio-ADP mediated inhibition of
forskolin-induced cAMP response element (CRE)-directed luciferase expression
was tested in CHO FlpIN cells expressing the recombinant human P2Y12-receptor.
After transient transfection with the pCRE-luc vector, the cells were incubated at
The investigation was supported by research Grant FIRB-
RNBE03YA3L-008 from MIUR, Italy.
37 °C with forskolin 1 lM in the presence or absence of 2-methylthio-ADP (10 nM)
and different concentrations of 6b for 3.5 h. Luciferase expression was quantified by
measuring luminescence after lysis of the cells. Values are expressed as percentage
of the responses to 2-methylthio-ADP in the presence of the vehicle (DMSO),
instead of 6b. Means SEM of 4–8 experiments. *p < 0.05, compared with control (t-
test modified according to Bonferroni). Antagonism of the response to 2-methyl-
thio-ADP by compound 6b in CHO FlpIN cells expressing recombinant human
P2Y12-receptors.
References and notes
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6.3.2. Measurement of cyclic AMP levels in CHO cells
transfected with hA2B adenosine receptors
CHO cells transfected with hAR subtypes were washed with
phosphate-buffered saline, diluted trypsin and centrifuged for
10 min at 200g. The pellet containing the CHO cells
(1 ꢁ 106 cells/assay) was suspended in 0.5 mL of incubation mix-
ture (mM): NaCl 15, KCl 0.27, NaH2PO4 0.037, MgSO4 0.1, CaCl2
0.1, Hepes 0.01, MgCl2 1, glucose 0.5, pH 7.4 at 37 °C, 2 IU/mL aden-
osine deaminase and 4-(3-butoxy-4-methoxybenzyl)-2-imidazo-
lidinone (Ro 20–1724) as phosphodiesterase inhibitor and
preincubated for 10 min in a shaking bath at 37 °C. The potency
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