Synthesis and in vitro stability of nucleoside 5′-phosphonate derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.04.045

Nucleoside derivatives are largely synthesized and tested to investigate their influence on platelet aggregation. It's well known that P2Y receptors play an important role in the regulation of platelet function and, as consequence, in controlling atherothrombotic events. The research of compounds that antagonize P2Y₁ and, in particular, P2Y₁₂ receptors is of great interest in the aim to obtain platelet aggregation inhibitors that are effective in the prevention and treatment of arterial thrombosis. In this study we present the synthesis and in vitro metabolic stability in human blood and rat liver homogenate of a new class of nucleoside derivatives, in particular 5′-phosphonate adenosine, inosine, guanosine and thioadenosine analogues also modified at the ribose moiety. On the basis of the results obtained we can hypothesize compounds 4 and 18 to have in vivo a relatively high stability.

Full text of DOI:10.1016/j.ejmech.2012.04.045

European Journal of Medicinal Chemistry 54 (2012) 202e209
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro stability of nucleoside 50-phosphonate derivatives
,
a
b
b
Silvia Vertuani a , Anna Baldisserotto , Katia Varani , Pier Andrea Borea , Bonache De Marcos Maria
*
Cruz c, Luca Ferraro b, Stefano Manfredini a, Alessandro Dalpiaz a
a Department of Pharmaceutical Sciences, University of Ferrara, via Fossato di Mortara 17-19, 44121 Ferrara, Italy
b Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara, via Fossato di Mortara 17-19, 44121 Ferrara, Italy
c Instituto de Química Médica (C.S.I.C.), Juan de la Cierva 3, E-28006 Madrid, Spain
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< A class of nucleoside derivatives was
synthesized.
< Three derivatives were identified as
NH2
N
NH2
N
N
N
O
P
N
N
O
O
P
O
O
P
O
weak
inhibitors
of
platelets
X
Base
OH
X
N
O
O
O
aggregation.
R
R
N
EtO
OEt
O
R
R'
< Two derivatives showed high
stability in human plasma and in rat
liver homogenate.
HO
HO
OH
Base= Adenine, Guanine, Inosine; X= O, NH; R, R'= OH, OEt
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleoside derivatives are largely synthesized and tested to investigate their influence on platelet
aggregation. It’s well known that P2Y receptors play an important role in the regulation of platelet
function and, as consequence, in controlling atherothrombotic events. The research of compounds that
antagonize P2Y1 and, in particular, P2Y12 receptors is of great interest in the aim to obtain platelet
aggregation inhibitors that are effective in the prevention and treatment of arterial thrombosis. In this
study we present the synthesis and in vitro metabolic stability in human blood and rat liver homogenate
of a new class of nucleoside derivatives, in particular 50-phosphonate adenosine, inosine, guanosine and
thioadenosine analogues also modified at the ribose moiety. On the basis of the results obtained we can
hypothesize compounds 4 and 18 to have in vivo a relatively high stability.
Received 3 February 2012
Received in revised form
27 April 2012
Accepted 30 April 2012
Available online 15 May 2012
Keywords:
Nucleoside 50-phosphonate derivatives
Pharmacokinetics
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
currently known that ADP released from damaged vessels and red
blood cells is a key stimulus inducing platelets activation and aggre-
Platelets have a crucial role in the maintenance of normal hae-
mostasis, and perturbation of this system can lead to pathological
thrombus formation and vascular occlusion, resulting in stroke,
myocardial infarction and unstable angina [1] which are the most
common cause of morbidity and mortality in Western Word [2].
Intervention in the process of thrombus formation has long been
an attractive therapeutic target for the treatment or prophylaxis
of such events, and many approaches, centred on the various
components of the process, have been investigated [3e7]. It is
gationthroughits actionontwoGprotein-coupledreceptorsubtypes,
P2Y1 and P2Y12 [8].
Synergistic activation of both receptors is required to induce
ADP-mediated platelet aggregation [3e5]. Modulation of P2Y
receptor in platelets appears to be of primary importance in regu-
lating platelet function and, as consequence, in controlling throm-
botic disease [9]. Thus, both P2Y1 and P2Y12 receptors constitute
targets for antithrombotic therapy, antagonizing each subtype
separately has antithrombotic effects. P2Y12 is the target of platelet
aggregation inhibitors that are effective in the prevention and
treatment of arterial thrombosis [10]. Indeed many agents currently
on the market (such as ticlopidine, clopidogrel, prasugrel, ticagrelor)
target the P2Y12 receptor. The thienopyridines (ticlopidine,
* Corresponding author. Tel.: þ39 0532 455294; fax: þ39 0532 455378.
E-mail address: vrs@unife.it (S. Vertuani).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.045

S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
203
clopidogrel, and prasugrel) inactivatethe P2Y12 receptorirreversibly
via covalent binding of an active metabolite generated in the liver,
whereas ticagrelor acts as a competitive antagonist [11].
this receptor. This work led to the discovery of INS50589, an
adenosine monophosphate derivative with 20,30-cyclic acetal and
N6-urea (Fig. 1) [37,38].
Oral antiplatelet therapy with platelet P2Y12 antagonists,
primarily the thienopyridine clopidogrel, is a major strategy for
preventing cardiovascular events in patients with ACS and those
undergoing percutaneous coronary intervention [12e17]. Limita-
tions of clopidogrel include the requirement for metabolic
conversion to its active metabolite, and irreversible binding of the
active metabolite to the P2Y12 receptor [18e26]. The new thieno-
pyridine prasugrel is metabolized to its active form more efficiently
than clopidogrel and produces greater levels of platelet inhibition
[27,28]. Unlike thienopyridines, ticagrelor does not require meta-
bolic conversion to an active form; the parent compound and AR-
C124910XX (its main active metabolite) have a similar potency in
inhibiting the P2Y12 receptor [29,30].
Despite recent advances in the treatment of ACS, including dual
antiplatelet therapy with aspirin and a thienopyridine during the
acute phase and for secondary prevention, this condition remains
a leading cause of morbidity and mortality [31]. The limitations of
the currently available antiplatelet agents have triggered the
development of newer drugs.
Early structureeactivity studies on the effects of ADP analogues
on human platelets showed that the introduction of substituents,
such as alkylthio groups, at the 2-position of the adenine base
improved potency, while modifications of ribose sugar or diphos-
phate chain reduced it [32e34]. On the other hand di- and
triphosphates analogues of 2-phenylethynyladenosine appear able
to inhibit platelet aggregation; this behaviour may be associated to
the lacking of the 2-alkylthio moiety (PEAdo derivatives, Fig.1) [35].
Xu et al. have focused on a 2-chloro-N6-methyladenine-9-(2-
methylpropyl) scaffold (MRS2395 is the most representative of
the series, Fig. 1), characterized by the lack of the sugar portion
among the ATP analogues, for the design of P2Y receptors-based
inhibitors of platelet aggregation [36].
Other compounds have been reported to act as antagonist of
the P2Y12 receptor. In particular, it was reported that 6-amino-2-
mercapto-3H-pyrimidin-4-one can be used as lead for the synthesis
of ligands for P2Y12 receptors on platelets (Fig. 1). This nucleus
represents a simplified combination of the active metabolite of the
thienopyridines and the ATP derivatives [35].
Bases on the above observations about the SAR of the agonist
and antagonist of P2Y receptors, we have decided to investigate, in
the present study, a series of adenosine, inosine, guanosine and
thioadenosine nucleotide derivatives, selected from our previously
studies [39e44], and to synthesize new derivatives from these
remarks. In particular, we have explored the possibility to obtain
a new class of stable nucleotide analogues endowed with isosteric
substitution of the diphosphate group by substitution of the
diphosphate moiety of natural substrates with phosphonoacetic
acid ester and amide moieties. We have also explored the presence
of acyclic ribose equivalent and the absence of the ribose moiety.
Pharmacological data on their effect on human platelet aggre-
gation have shown that only compounds 4, 18 and 33 seem to be
able to inhibit the platelet aggregation (see below). We therefore
decided to focus our study on the in vitro stability of the three
compounds in human whole blood and rat liver homogenate to
evaluate their stability in physiological environments.
2. Results and discussion
2.1. Chemistry
Synthesis of 50-O-phosphonylacetyladenosine (4), 50-O-
[(diethyl)phosphonoacetyl]-adenosine (5), (Diethoxy-phosphoryl)-
acetic
acid
2-[1-(6-amino-purin-9-yl)-2-hydroxy-ethoxy]-3-
Douglass et al. have studied the structureeactivity relationships
of modified mono- and dinucleotides at P2Y12, and identified
lipophilic modifications to the ribose and base moieties that
imparted potent, selective, and reversible antagonist properties at
hydroxy-propyl ester (6), 50-deoxy-50-[(phosphonoacetyl)amino]-
adenosine (11), 50-deoxy-50-[[(diethoxyphosphinyl)acetyl]amino]-
adenosine (12), ({2-[1-(6-Amino-purin-9-yl)-2-hydroxy-ethoxy]-3-
hydroxy-propylcarbamoyl}-methyl)-phosphonic acid diethyl ester
NHCH3
NH2
N
N
N
N
O
N
O
O
N
N
Cl
N
R
O
R2
O
OH
O
HO
PeAdo derivatives R=C6H5
R2= diphosphate (IC50 60 µM)
MRS2395
(inhibits ADP-induced aggregation in both rat,
µ
KI 3.6 µM, and human platelets)
triphosphate (IC50 85 M)
NHEt
NH2
HN
O
N
N
N
N
NH2
TosO
N
S
N
O
4-Pyrimidinol, 6-amino-2-[(2-aminoethyl)thio]-,
4-(4-methylbenzenesulfonate)
O
NaO
NaO
O
(reduced the response to the agonist 2-methylthio-ADP
P
O
Ph
with an IC50 of 8.08 µM)
O
INS50589 (IC50 16 nM)
Fig. 1. Representative examples of P2Y12 antagonists.

204
S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
(13), (Diethoxy-phosphoryl)-acetic acid 2-(6-amino-purin-9-
ylmethoxy)-ethyl ester (17), (Ethoxy-hydroxy-phosphoryl)-acetic
acid 2-(6-amino-purin-9-ylmethoxy)-ethyl ester (18), and 50-O-
(diethyl)phosphonacetyl-8-methyltioadenosine (21) is shown in
Scheme 1. Adenosine was treated with perchloric acid in dryacetone
to obtain an intermediate with the hydroxyl group in 20 and 30
position protected. After protection of the N in position 6 with 4,40-
dimethoxytrityl chloride, the reaction with diethylphosphonoacetic
acid gave an intermediate that was treated with bromo-
trimethylsilane to give the expected compound 4 [45], and with
trifluoroacetic acid to give compound 5 [43].
2.2. Pharmacolocigal data
All the newly synthesized compounds were assayed in vitro
for their activity on the ADP induced aggregation of human
platelet-rich plasma (PRP) using light transmission aggregometry. All
the compounds were tested at a single concentration (10ꢀ4 M)
throughout the experiments. None of the compounds induced
aggregation of human platelets. Among the tested compounds 4, 18,
and 33 were found able to inhibit the platelet aggregation induced by
ADP up to 70% (Fig. 2).
Final treatment of 5 with sodium metaperiodate and sodium
triacetoxy borohydride gave the desired 6.
2.3. In vitro metabolism
The synthesis of the analogues 11 [42], 12 [42], and 13, started
by tosylation of the 50-hydroxyl group of Adenosine protected in
20 and 30 position and at N6 as described above, with tosyl
chloride to give an intermediate subsequently treated with NaN3
and then reduced with C/Pd under hydrogen atmosphere. The
intermediate obtained by reaction with diethylphosphonoacetic
acid, was treated with bromotrimethylsilane to give 11 and with
trifluoroacetic acid to give 12, that was treated with sodium
metaperiodate and subsequently with sodium triacetoxy boro-
hydride to give the compound 13.
The compounds 4, 18, and 33 are characterized by the pres-
ence of a 50-O-phosphono acetyl function that can potentially be
hydrolysed in physiologic environments [52]. The stability of
these compounds has been therefore tested in vitro, by evaluating
their degradation patterns in human whole blood and rat liver
homogenates. As reported in Fig. 3 the inhibitors 4 and 18 were
not significantly degraded in human blood during 6 h of incu-
bation at 37 ꢁC, whereas a relatively weak degradation (about
20%), following a first order kinetic, has been registered for
compound 33.
Adenosine treated with saturated bromine-water gave inter-
mediate 19 [46], subsequently reacted with sodium meth-
anethiolate [47] and finally with diethylphosphonacetic acid gave
the desired 21.
Fig. 4 evidences that this last compound is degraded also in rat
liver homogenates, following a first order kinetic, with a half-life
value of 2.33
ꢂ
0.25 h. In particular, the degradation of
compound 33 was about the 80% after 6 h; on the other hand any or
relatively weak degradation was registered for compounds 4 and
18, respectively.
The intermediated 15 [48] obtained by reaction of 14 [49]
with 6-chloropurine was treated with saturated methanolic
ammonia [50] and then with diethylphosphonacetic acid to give
the desired 17, then treated with bromotrimethylsilane to obtain
compound 18.
On the basis of these results we can hypothesize these last
compounds to have in vivo a relatively high stability. A lower
stability can be instead supposed for the derivative 33, that appears
also to be the weaker inhibitor among the three compounds ana-
lysed. We have hypothesized that the instability of the derivative 33
could be due to the hydrolysis of the 50-O-phosphono acetyl func-
tion, allowing to obtain guanosine as metabolite. As a consequence,
we have tested the stability of guanosine in rat liver homogenates
and founded that its degradation is very fast, following a first order
kinetic with a half-life value of 14.5 ꢂ 1.0 s (Fig. 4). This means that
guanosine, if derived by hydrolysis of 33, cannot be detected in our
Inosine derivatives 50-O-[(diethyl)phosphonoacetyl]-inosine
(24), 50-deoxy 50-N-phosphonylmethylinosine (29), 50-deoxy-50-N-
[(diethyl)phosphonoacetyl]-inosine (30), and Guanosine deriva-
tives 50-O-[(diethyl)phosphonoacetyl]-guanosine (33), 50-(phos-
phonoacetate)-guanosine
(34)
[51],
2-isobutylamide-50-O-
[(diethyl)phosphonoacetyl]-guanosine (38), and 2-isobutylamide-
50-O-phosphonylacetylguanosine (39) are shown in Scheme 2 and
were obtained according to the procedures described above.
i
1
ii
NH2
NH2
2
iii
NH2
N
3
iv or v
4: R=R'= H; X=O; Y=H;
vi,vii
6: R=R'= Et; X=O; Y=H;
N
N
N
N
5: R=R'= Et; X=O; Y=H;
N
N
N
N
O
P
O
P
O
O
Y
Y
X
X
N
N
O
N
O
i, ii, ia
RO
OR'
RO
OR'
HO
7
O
iia
8
iiia
11: R=R'= H; X=NH; Y=H;
12: R=R'= Et; X=NH; Y=H;
9
iii
vi,vii
10
iv or v
13: R=R'= Et; X=NH; Y=H;
HO
OH
6, 13
HO
OH
HO
OH
ib
4-5, 11-12, 21
19
iib
20
iiib
Adenosine
21: R=R'= Et; X=O; Y=SCH3;
Cl
N
N
NH2
N
H
N
N
N
N
O
P
O
O
6-chloropurine
ic
iv
15
iic
N
17: R=R'= Et; X=O; Y=SCH3;
16
+
iii
O
18: R=R'= Et; X=O; Y=SCH3.
RO
OR'
O
Br
O
O
14
Scheme 1. Synthesis of the adenosine derivatives 4, 5, 6, 11, 12, 13, 17, 18 and 21. Reagents and conditions: i) HClO4 60%, dry acetone; ii) TMSCl, 4,40-dimethoxytrityl chloride,
pyridine, r.t.; iii) Diethylphosphonacetic acid, DMAP, DCC, CH2Cl2 or DMF; iv) TMSBr, CH2Cl2; v) TFA 50%; vi) NaIO4, MeOH, H2O, r.t.; vii) Na(AcO)3BH, AcOH, r.t; ia) Tosyl chloride,
DMAP, CH2Cl2, r.t.; iia) NaN3, DMF; iiia) H2, C/Pd, MeOH; ib) Saturated bromine-water, NaOAc buffer pH4, 47 h, r.t.; iib) NaSCH3, DMF, 12 h, r.t.; ic) DMF, TEA, 0 ꢁC for 2 h, r.t. for 12 h;
iic) NH3/MeOH, 150 ꢁC.

S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
205
O
N
O
N
ia
N
N
NH
22
O
P
NH
O
ii
23
iii
X
N
24: R=R'= Et; X=O;
N
O
RO
OR'
HO
O
ia
22
iva
25
v
HO
OH
26 vi
27
29: R=R'= H; X=NH;
30: R=R'= Et; X=NH;
ii
HO
OH
28
iii or ivb
24, 29, 30
Inosine
O
O
N
N
ib
N
31
NH
O
P
ii
NH
O
32
iii or ivb
X
33: R=R'= Et; X=O;Y=H;
34: R=R'= H; X=O; Y=H;
N
NH-Y
N
O
N
NH2
RO
OR'
HO
O
ic
HO
OH
35
ia
36
ii
HO
OH
38: R=R'= Et; X=O; Y=COCH(CH3)2;
39: R=R'= H; X=O; Y=COCH(CH3)2.
37 iii or ivb
33, 34 38, 39
Guanosine
Scheme 2. Synthesis of the derivatives 24, 29, 30, 33, 34, 38, and 39. Reagents and conditions: ia) Dimethxypropane, p-toluenesulfonic acid monohydrate, DMF; ib) HClO4 60%, dry
acetone; ic) 1) TMSCl, pyridine, 2) Isobutyryl chloride, 3) NH4OH conc.; ii) Diethylphosphonacetic acid, DMAP, DCC, CH2Cl2 or DMF; iii) TFA 50%; iva) Tosyl chloride, DMAP, pyridine,
40 ꢁC; ivb) TMSBr, CH2Cl2; v) NaN3, DMF; vi) H2, C/Pd, MeOH; ivb) TMSBr, CH2Cl2.
100
A
100
75
75
4
50
18
33
50
25
0
25
0
Control
4
18
33
0
60
120
180
240
300
360
Fig. 2. Effect of compound 4, 18, and 33 on platelet aggregation induced by ADP.
Time (min)
B
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
100
2.1
4
75
2.0
1.9
18
33
50
25
0
4
1.8
1.7
1.6
18
33
0
60
120
180
240
300
360
Time (min)
0
60
120
180
240
300
360
Time (min)
0
60
120
180
240
300
360
Time (min)
Fig. 4. Degradation time-courses of compounds 4, 18, and 33 in rat liver homogenates
(A). Compound 33 showed a significant degradation following a first order kinetic,
confirmed by the semilogarithmic plot (B) (n ¼ 11, r ¼ 0.983, P < 0.0001) and its half-
life was calculated to be 140 ꢂ 15 min. The compound 4 showed a weak but significant
degradation (about 20% within 6 h) following a first order kinetic, confirmed by the
semilogarithmic plot (B) (n ¼ 11, r ¼ 0.904, P < 0.001). Compound 18 was not
Fig. 3. Degradation time-courses of compounds 4, 18, and 33 in human whole blood.
Only compound 33 showed a significant degradation (about 20% within 6 h) following
a first order kinetic, confirmed by the semilogarithmic plot reported in the inset (n ¼ 9,
r ¼ 0.904, P < 0.001). The compounds 4 and 18 were not significantly degraded within
6
h, accordingly the linear regressions of their semilogarithmic plots were not
significantly degraded within 6 h, accordingly the linear regression of its semi-
significant (n ¼ 10, r < 0.61, P > 0.061). Data are expressed as the mean ꢂ SD of three
logarithmic plot (B) was not significant (n ¼ 11, r ¼ 0.027, P ¼ 0.94). Data are expressed
independent experiments.
as the mean ꢂ SD of three independent experiments.

206
S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
coated plates of silica gel MachereyeNagel durasil-25. Nuclear
100
75
50
25
0
magnetic resonance spectra were determined in DMSO-d6, and D2O
and solution with a Varian VXR-200 MHz spectrometer or Varian
MERCURYplus 400 MHz and chemical shifts are presented in ppm
from internal tetramethylsilane as a standard. HPLC analysis was
performed using an Agilent1100 Series HPLC System equipped with
a G1315A DAD and with an Hydro RP18 Sinergi 80A column
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(4.6 ꢃ 150 mm, 4
mm) from Phenomenex. Male Wistar rats were
purchased from Harlan SRC (Milan, Italy).
All other reagents were of analytical grade and obtained from
commercial sources.
0
10
20
30
40
50
60
Time (sec)
4.2. Compounds synthesis
0
30
60
90
Time (sec)
120
150
180
4.2.1. N6-(4,40-Dimethoxytrityl) -20,30-isopropylidene-adenosine (2)
To a solution of 1 (1.10 g, 3.57 mmol) in pyridine (32 mL) was
added trimethylchlorosilane (TMSCl) (0.762 mL, 5.35 mmol). After
1 h, 4,4-dimethoxytrityl chloride (1.68 g, 5.03 mmol) was added
and the reaction mixture was stirred for 1.5 h. After this time,
8 mL of NH3 were added and the solution stirred for others
45 min. Then the reaction was evaporated to dryness and the
residue obtained was dissolved in CH2Cl2 and washed with
NaHCO3 at 5%. The organic phase was dried, filtered and evapo-
rated to give a crude residue purified by chromatography over
silica gel (eluent: CH2Cl2/MeOH, 98:2/95:5, v/v) to afford
desiderated compound 2 (yield 62%). 1H NMR (400 MHz, DMSO-
Fig. 5. Degradation time-course of guanosine in rat liver homogenates. This compound
showed a relatively fast degradation following a first order kinetic, confirmed by the
semilogarithmic plot in the inset (n ¼ 4, r ¼ 0.993, P < 0.01) and its half-life was
calculated to be 14.5 ꢂ 1.0 s. Data are expressed as the mean ꢂ SD of three independent
experiments.
experimental conditions. We have therefore verified that guano-
sine was degraded to guanine in rat liver homogenates, being the
guanine amounts increased during guanosine degradation (data
not shown). Compound 33 was the only one, among those ana-
lysed, able to induce the guanine formation in rat liver homoge-
nates during its degradation. To support our data concerning the
hydrolysis of 33 in rat liver homogenates, we have observed that
this compound differs from 4 and 18 adenosine derivatives by the
presence of an ethyl-radical at all the phosphonic hydroxyl groups.
Thus, at physiologic pH values the compounds 4 and 18 are nega-
tively charged on the phosphonic group, whereas the derivative 33
is not. It has been reported that some hydrolase activities can be
sensibly hindered by the presence of negative charges on the
substrates, as demonstrated, for instance, in the case of butyr-
ylcholinesterase [53,54]. Also in our case the hydrolysis of the 50-O-
phosphono acetyl function could be possible only in the absence of
negative charges. We have recently observed a similar phenom-
enon also with a novel conjugated molecule between dopamine
and an A2A adenosine receptor antagonist, were the presence of
negative charges inhibited hydrolysis phenomena in physiologic
environments [55] (Fig. 5).
d6) d
: 1.30, 1.52 (6H, s, (CH3)2C), 3.45e3.50 (2H, m, H-50), 3.72 (6H,
s, 2OCH3), 4.20 (1H, m, H-40), 4.98 (1H, dd, H-30), 5.12 (1H, t,
J ¼ 2.8 Hz, OH-50), 5.38 (1H, dd, J ¼ 4.0 Hz, H-20), 6.10 (1H, d,
J ¼ 2.8 Hz, H-10), 6.83e7.30 (15H, m, Ph), 7.94 (1H, s, H-2), 8.42
(1H, s, H-8). ESI MS: m/z 612.8 Da [M þ H]þ, C34H37N5O6 Mol. Wt.
611.69.
4.2.2. General synthetic procedures for the preparation of
compounds 3, 17, 21, 23, 28, 32, and 37
To a solution of 2 (1.00 g, 1.63 mmol), 16 [50] (0.35 g, 1.67 mmol),
20 [47] (1.6 g, 5.1 mmol), 22 [56] (0.2 g, 0.64 mmol), 27 (0.2 g,
0.65 mmol), 31 [57] (0.3 g, 0.92 mmol), or 36 [58] (0.38 g,
0.95 mmol) in CH2Cl2 under Ar or dry DMF, were added dieth-
ylphosphonoacetic acid (PPA) and 4-(dimethylamino)pyridine
(DMAP). At 0 ꢁC N,N0-dicyclohexylcarbodiimide (DCC) was added
and then the reaction mixture was stirred at r.t. for 12 h. After
filtration of dicyclohexyl urea (DCU), the solvent was evaporated to
dryness.
3. Conclusion
4.2.2.1. N6-(4,40-Dimethoxytrityl)-20,30-isopropylidene-50-O-[(diethyl)
phosphonoacetyl]-adenosine (3). The crude residue was purified by
chromatography over silica gel (eluent: CH2Cl2/MeOH, 98:2/95:5,
v/v) to afford the desiderated compound 3 (yield 54%). 1H NMR
The synthesis of a series of adenosine, inosine, guanosine and
thioadenosine nucleoside derivatives obtained by substitution of
the diphosphate moiety of native compounds with phosphono-
acetic acid ester led to the identification of the derivatives 4, 18, and
33 identified as weak inhibitors of platelets aggregation induced by
ADP. Compounds 4 and 18 showed high stability in human plasma
and in rat liver homogenate while compound 33 undergoes
degradation in human plasma and rat liver homogenates with
different rates. Thus 4 and 18 may represent interesting lead
candidate for the development of inhibitors of platelets
aggregation.
(200 MHz, DMSO-d6) d: 1.15e1.25 (6H, m, 2CH3CH2O),1.35,1.53 (6H,
s, (CH3)2C), 2.72, 2.88 (2H, s, J ¼ 21.2 Hz, CH2P), 3.71 (6H, s, 2OCH3),
4.26e4.39 (3H, m, H-40, 2H-50), 5.20 (1H, dd, H-30), 5.40 (1H, dd,
J ¼ 3.8 Hz, H-20), 6.10 (1H, d, J ¼ 6.0 Hz, H-10), 6.81e7.27 (15H, m, Ph),
7.95 (1H, s, H-2), 8.39 (1H, s, H-8). ESI MS: m/z 790.7 Da [M þ H]þ,
C40H48N5O10P Mol. Wt. 789.81.
4.2.2.2. (Diethoxy-phosphoryl)-acetic acid 2-(6-amino-purin-9-
ylmethoxy)-ethyl ester (17). The resulting residue was purified by
silica gel column chromatography (eluent: CH2Cl2/MeOH 95/5 v/v)
to afford desiderated compound 17 as white foam (yield 75%). 1H
4. Experimental
4.1. General
NMR (200 MHz, DMSO-d6)
d
: 1.19 (6H, t, J ¼ 8.0 Hz, 2OCH2CH3), 3.08
Chemicals and solvents were purchased from SigmaeAldrich
and Carlo Erba reagenti (Italia). The molecular weights of the
compounds were determined by ESI (MICROMASS ZMD 2000), and
the values are expressed as [MH]þ. TLC was performed on pre-
(2H, d, J ¼ 20.0 Hz, CH2P), 3.75 (2H, m, CH2), 4.00 (4H, m,
2OCH2CH3), 4.18 (m, 2H, CH2O), 5.57 (s, 1H, OCH2N); 7.30 (2H, br s,
NH2); 8.17 (1H, s, H-2); 8.28 (1H, s, H-8). ESI MS: m/z 388.5 Da
[M þ H]þ, C14H22N5O6P Mol. Wt. 387.3.

S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
207
4.2.2.3. 50-O-(diethyl)phosphonacetyl-8-methylthioadenosine (21). The
resulting residue was purified by silica gel column chromatography
(eluent: CH2Cl2/MeOH 95/5, v/v) to afford desiderated compound
4.2.3.1. (Diethoxy-phosphoryl)-acetic acid 2-[1-(6-aminopurin-9-yl)-
2-hydroxy-ethoxy]-3-hydroxypropyl ester (6). The crude material
was purified by silica gel column chromatography (eluent: CH2Cl2/
MeOH 98/2/90/10, v/v) to afford desiderated compound 6 as
21 (yield 10%).1H NMR (200 MHz, DMSO-d6)
d: 1.2 (6H, t, 2OCH2CH3),
2.70 (3H, s, SCH3), 3.08 (2H, d, J ¼ 21.4 Hz, CH2P), 3.95e4.08 (2H, m,
H-50, H-500), 4.2e4.3 (6H, m, 2OCH2CH3, H-30, H-40), 5.1 (1H, m, H-20),
5.4 (1H, br t, OH30), 5.58 (1H, br t, OH20), 5.85 (1H, J ¼ 6.8 Hz, H-10),
7.25 (2H, br s, NH2), 8.12 (1H, s, H-2). ESI MS: m/z 492.6 Da [M þ H]þ,
C17H26N5O8PS Mol. Wt. 491.46.
white foam (yield 60%). 1H NMR (200 MHz, DMSO-d6)
d: 1.16 (6H, t,
2OCH2CH3), 2.76 (2H, d, J ¼ 20.6 Hz, CH2P), 3.63e3.54 (7H, m,
2OCH2CH3, H20, H30, H40), 3.97e3.95 (2H, m, H50, H500), 4.94 (1H, br
t, OH30), 5.19 (1H, br t, OH20), 5.85 (1H, t, J ¼ 6.4 Hz, H10), 7.25 (2H, br
s, NH2), 8.14 (1H, s, H2), 8.29 (1H, s, H8). ESI MS: m/z 450.3 Da
[M þ H]þ, C16H26N5O8P Mol. Wt. 449.40.
4.2.2.4. 20,30-Isopropylidene-50-O-[(diethyl)phosphonoacetyl]-inosine
(23). The resulting residue was purified by silica gel column
chromatography (eluent: CH2Cl2/MeOH 98/2/95/5, v/v) to afford
desiderated compound 23 (yield 70%). 1H NMR (200 MHz, DMSO-
4.2.3.2. ({2-[1-(6-Aminopurin-9-yl)-2-hydroxy-ethoxy]-3-hydroxy-
propylcarbamoyl}-methyl)-phosphonic acid diethyl ester (13). The
obtained residue, solubilized in water, was applied to a carbon
column and the column was eluted with water. The product was
then eluted with 3% concentrated NH4OH in 1/1 water/ethanol.
Selected fractions were concentrated to give 13 as brown solid
d6) d: 1.15e1.22 (6H, m, 2CH3CH2O), 1.32, 1.54 (6H, s, (CH3)2C), 3.06,
3.17 (2H, s, J ¼ 22 Hz, CH2P), 4.37e4.39 (4H, m, 2OCH2CH3), 4.96
(1H, m, H-30), 5.39 (1H, dd, J ¼ 3.7 Hz, H-20), 6.16 (1H, d, J ¼ 2.0 Hz,
H-10), 8.09 (1H, s, H-2), 8.29 (1H, s, H-8), 12.44 (1H, br s, NH-3). ESI
MS: m/z 487.3 Da [M þ H]þ, C19H27N4O9P Mol. Wt. 486.41.
(yield 60%). 1H NMR (200 MHz, DMSO-d6)
d: 1.18 (6H, m,
2OCH2CH3), 2.60e2.80 (2H, m, CH2P), 3.90e3.20 (2H, m, H-50, H-
500), 3.40e3.60 (3H, m, H-30, H-40), 3.80e4.00 (6H, m, 2OCH2CH3, H-
20), 4.80 (1H, br s, OH30), 5.20 (1H, br s, OH20), 5.85 (1H, t, J ¼ 5.6 Hz,
H-10), 7.25 (2H, br s, NH2), 7.80 (1H, br t, NH), 8.13 (1H, s, H-2), 8.27
(1H, s, H-8). ESI MS: m/z 447.2 Da [M þ H]þ, 469.4 Da [M þ Na]þ,
C16H27N6O7P Mol. Wt. 446.4.
4.2.2.5. 20,30-Isopropylidene-50-deoxy-50-N-[(diethyl)phosphonoace-
tyl]-inosine (28). The resulting residue was purified by silica gel
column chromatography (eluent: CH2Cl2/MeOH 98/2/95/5, v/v)
to afford desiderated compound 28 (yield 94%). 1H NMR (400 MHz,
DMSO-d6)
d: 1.18e1.22 (6H, m, 2CH3CH2O), 1.23, 1.52 (6H, m,
(CH3)2C), 2.85e2.93 (2H, m, CH2P), 3.93 (2H, m, H-50), 3.97e4.04
(4H, m, 2OCH2CH3), 4.16e4.17 (1H, m, H-40),4.87 (1H, dd,
J ¼ 3.2 Hz, H-30), 5.34 (1H, dd, H-20), 6.07 (1H, d, J ¼ 2.8 Hz, H-10),
8.10 (1H, s, H-2), 8.21 (1H, t, NH-50), 8.35 (1H, s, H-8), 12.47 (1H, br s,
NH-3). ESI MS: m/z 486.3 Da [M þ H]þ, C19H28N5O8P Mol. Wt.
485.43.
4.2.4. General synthetic procedures for the preparation of
compounds 18, 29 and 39
To a solution of 17 (0.125 g, 0.32 mmol), 28 (0.13 g, 0.26 mmol) or
37 (0.09 g, 0.16 mmol) in CH2Cl2 (5 mL) was added TMSBr (10 eq).
The reaction mixture was stirred at r.t. for 5 h and the solvent was
then evaporated in vacuo.
4.2.2.6. 20,30-Isopropylidene-50-O-[(diethyl)phosphonoacetyl]-guano-
sine (32). The resulting residue was purified by silica gel column
chromatography (eluent: CH2Cl2/MeOH 95/5, v/v) to afford desid-
4.2.4.1. (Ethoxy-hydroxy-phosphoryl)-acetic acid 2-(6-aminopurin-
9-ylmethoxy)-ethyl ester (18). The residue obtained was purified by
reversed-phase chromatography over silica gel (eluent: H2O) to
erated compound 32 (yield 63%). 1H NMR (400 MHz, DMSO-d6)
d:
afford 18 (yield 60%). 1H NMR (200 MHz, D2O)
d: 1.02 (3H, t,
1.16e1.20 (6H, m, 2CH3CH2O), 1.24e1.51 (6H, m, (CH3)2C), 3.10e3.16
(2H, s, J ¼ 24 Hz, CH2P), 3.98e4.02 (4H, m, 2OCH2CH3), 4.03e4.32
(3H, m, H-40, 2H-50), 5.11 (1H, dd, J ¼ 3.2 Hz, H-30), 5.24 (1H, dd,
J ¼ 3.8 Hz, H-20), 6.01 (1H, d, J ¼ 2.0 Hz, H-10), 6.50 (2H, br s, NH2),
7.89 (1H, s, H-8), 10.60 (1H, br s, NH-3). ESI MS: m/z 502.3 Da
[M þ H]þ, C19H28N5O9P Mol. Wt. 501.43.
J ¼ 7.2 Hz, OCH2CH3), 2.59 (2H, d, J ¼ 20.6 Hz, CH2P), 3.74 (4H, m,
CH2, OCH2CH3), 4.09(2H, m, CH2), 5.61 (2H, s, OCH2N), 8.30 (1H, s,
H-2), 8.33 (1H, s, H-8). ESI MS: m/z 360.1 Da [M þ H]þ, C12H18N5O6P
Mol. Wt. 359.28.
4.2.4.2. 50-Deoxy 50-N-phosphonylmethylinosine (29). The residue
obtained was purified by reversed-phase chromatography over
silica gel (eluent: H2O) to afford 29 (yield 40%). 1H NMR (400 MHz,
4.2.2.7. 20,30-Isopropylidene-2-isobutylamide-50-O-[(diethyl)phospho-
noacetyl]-guanosine (37). The resulting residue was purified by silica
gel column chromatography (eluent: CH2Cl2/MeOH 95/5, v/v) to
afford desiderated compound 37 (yield 86%) as white foam. 1H NMR
D2O)
d
: 2.58, 2.63 (2H, s, J ¼ 20 Hz, CH2P), 3.37 (1H, dd, H-50a), 3.54
(1H, dd, J ¼ 4.8 Hz, J ¼ 14.8 Hz, H-50b), 4.12e4.16 (3H, m, H-20, H-30,
H-40), 5.88 (1H, d, J ¼ 5.6 Hz, H-10), 8.1 (1H, s, H-2), 8.3 (1H, s, H-8).
ESI MS: m/z 390.2 Da [M þ H]þ, C12H16N5O8P Mol. Wt. 389.26.
(200 MHz, DMSO-d6) d: 1.12e1.22 (6H, m, 2CH3CH2O),1.26, 1.53 (6H,
s, (CH3)2C), 3.08, 3.18 (2H, s, CH2P), 3.96e4.07 (4H, m, OCH2CH3),
4.28e4.32 (3H, m, H-40, 2H-50), 5.03e5.16 (1H, m, H-30), 5.40 (1H,
dd, H-20), 6.10 (1H, d, J ¼ 2.0 Hz, H-10), 8.22 (1H, s, H-8), 11.50 (1H, br
s, NHeCO), 12.12 (1H, br s, NH-3). ESI MS: m/z 572.4 Da [M þ H]þ,
C23H34N5O10P Mol. Wt. 571.52.
4.2.4.3. 2-Isobutylamide-50-O-phosphonylacetylguanosine (39). To
a solution of 37 (0.09 g, 0.16 mmol) in CH2Cl2 (2.5 mL) was added
TMSBr (0.210 mL, 1.58 mmol). The reaction mixture was stirred at
r.t. for 5 h and the solvent was then evaporated in vacuo. The
residue obtained was purified by reversed-phase chromatography
over silica gel (eluent: H2O) to afford 39 (yield 40%) as white foam.
4.2.3. General synthetic procedures for the preparation of
compounds 6 and 13
1H NMR (400 MHz, D2O)
d: 1.18e1.20 (6H, s, (CH3)2C), 2.71e2.77
A solution of 5 [43] or 12 [42] (2.2 mmol) and sodium
metaperiodate (0.48 g, 2.2 mmol) in a mixture of methanol
(14.5 mL) and water (4.4 mL) was stirred at room temperature
for 1 h. The iodate salts were removed by filtration, and the
obtained filtrate was evaporated in vacuo. Sodium triacetoxy
borohydride or sodium borohydride was slowly added to a stir-
red solution of the dialdehyde in acetic acid or ethanol. After
stirred for 12 h at room temperature the mixture was evapo-
rated to dryness in vacuo.
(1H, m, CH), 2.85, 2.90 (2H, s, J ¼ 24.1 Hz, CH2P), 4.44e4.47 (4H,
m, H-30, H-40, 2H-50), 4.73 (1H, t, H-20), 6.00 (1H, d, J ¼ 6.0 Hz, H-10),
8.53 (1H, s, H-8). ESI MS: m/z 476.3 Da [M þ H]þ, C16H22N5O10P Mol.
Wt. 475.35.
4.2.5. General synthetic procedures for the preparation of
compounds 24, 30, 33 and 38
6 mL of TFA 50% in water were added to 23 (0.22 g, 0.43 mmol),
28 (0.2 g, 0.41 mmol), 32 (0.200 g, 0.40 mmol) or 37 (0.200 g,

208
S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
0.38 mmol). The reaction mixture was stirred at r.t. for 5 h and then
4.2.7. 50-Azido-20,30-isopropylidene-inosine (26)
the solvent was evaporated in vacuo to give an oil.
To a solution of 25 (0.487 g, 1.15 mmol) in DMF (20 mL) was
added NaN3 (0.3 g, 4.61 mmol). The reaction mixture was stirred at
80 ꢁC for 3 h. After evaporation of the solvent, the residue was
dissolved in CH2Cl2 and washed with brine. The organic phase was
dried, filtered and evaporated to give a crude residue purified by
chromatography over silica gel (eluent: CH2Cl2/MeOH 98/2/95/5,
v/v) to afford the desired products 26 (yield 45%). 1H NMR
4.2.5.1. 50-O-[(Diethyl)phosphonoacetyl]-inosine (24). The residue
obtained was purified by chromatography over silica gel (eluent:
CH2Cl2/MeOH, 95:5/90:1, v/v) to afford the desiderated
compound 24 (yield 92%). 1H NMR (400 MHz, DMSO-d6)
d: 1.18 (6H,
m, 2CH3CH2O), 3.11, 3.22 (2H, s, J ¼ 22 Hz, CH2P), 3.97e4.18 (8H, m,
H-30, H-40, 2H-50, 2OCH2CH3), 4.60 (1H, m, H-20), 5.40 (1H, d,
J ¼ 2.6 Hz, OH-30), 5.60 (1H, d, J ¼ 3.0 Hz, OH-20), 5.88 (1H, d,
J ¼ 2.8 Hz, H-10), 8.07 (1H, s, H-2), 8.34 (1H, s, H-8), 12.29 (1H, br s,
NH-3). ESI MS: m/z 447.2 Da [M þ H]þ, C16H23N4O9P Mol. Wt.
446.35.
(200 MHz, DMSO-d6) d: 1.32, 1.53 (6H, s, (CH3)2C), 3.57 (2H, d,
J ¼ 5.8 Hz, H-50), 4.29e4.31 (1H, m, H-40), 4.30 (1H, dd, H-30), 4.95
(1H, dd, J ¼ 3.2 Hz, H-20), 6.17 (1H, d, J ¼ 2.8 Hz, H-10), 8.11 (1H, s, H-
2), 8.30 (1H, s, H-8), 12.45 (1H, br s, NH-3). ESI MS: m/z 334.1 Da
[M þ H]þ, C13H15N7O4 Mol. Wt. 333.30.
4.2.5.2. 50-Deoxy-50-N-[(diethyl)phosphonoacetyl]-inosine (30). The
residue obtained was purified by chromatography over silica gel
(eluent: CH2Cl2/MeOH, 95:5/90:1, v/v) to afford the desiderated
5. Pharmacological data
5.1. Platelet function studies
compound 30 (yield 77%). 1H NMR (400 MHz, DMSO-d6)
d:
1.18e1.22 (6H, m, 2CH3CH2O), 2.86, 2.92 (2H, s, J ¼ 24 Hz, CH2P),
3.37e3.41 (2H, m, H-50), 3.90e3.98 (1H, m, H-40), 4.00e4.05 (5H, m,
H-30, 2OCH2CH3), 4.57 (1H, t, J ¼ 5.6 Hz, H-20), 5.83 (1H, d, J ¼ 6.4 Hz,
H-10), 8.09 (1H, s, H-2), 8.12 (1H, t, NH-50), 8.37 (1H, s, H-8), 12.42
(1H, br s, NH-3). ESI MS: m/z 446.2 Da [M þ H]þ, C16H24N5O8P Mol.
Wt. 445.36.
The effects of the compounds on platelets were studied using
platelet-rich plasma (PRP) obtained from whole blood collected in
sodium citrate 10.9 mmol (9:1, v/v) from healthy volunteers, 2 h
later the intake of a tablet of acetyl salicylic acid and upon
centrifugation at 150g at 21 ꢁC for 10 min. 10ꢀ6 M ADP, was added
to PRP samples, which had been pre-warmed 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 [59].
4.2.5.3. 50-O-[(Diethyl)phosphonoacetyl]-guanosine (33). The residue
obtained was purified by chromatography over silica gel (eluent:
CH2Cl2/MeOH, 95:5, v/v) to afford the desiderated compound 33
(yield 76%) as white foam. 1H NMR (400 MHz, DMSO-d6)
d: 1.17e1.22
6. Metabolic stability studies
(6H, m, 2CH3CH2O), 2.86, 2.92 (2H, s, J ¼ 24 Hz, CH2P), 3.99e4.02
(4H, m, 2OCH2CH3), 4.11 (1H, dd, H-40), 4.19 (1H, dd, H-50a),
5.53 (1H, d, J ¼ 6.0 Hz, OH-20), 4.27 (1H, dd, J ¼ 3.6 Hz, J ¼ 12.0 Hz, H-
50b), 4.46 (1H, dd, H-30), 5.32 (1H, d, J ¼ 5.2 Hz, OH-30), 5.70 (1H,
d, J ¼ 5.6 Hz, H-10), 6.48 (2H, br s, NH2), 7.95 (1H, s, H-8),10.64 (1H, br
s, NH-3). ESI MS: m/z 462.1 Da [M þ H]þ, C16H24N5O9P Mol. Wt.
461.36.
6.1. HPLC methods
The quantification of 4, 18, 33, Guanosine and Guanine in all
samples generated from the experimental procedures was per-
formed by HPLC. The detector was set at 259 nm for 4 and 18
analyses and at 254 nm for 33 Guanosine and Guanine analyses.
For 4 and 18 analyses the mobile phase consisted of water
(0.01 M H3PO4) (solvent A) and acetonitrile (0.01 M H3PO4) (solvent
B) with a ratio of 98:2 (v/v). The flow rate was of 1 mL/min and the
retention times of 4 and 18 were 5.8 and 11.8 min, respectively.
For 33 analyses the mobile phase consisted of water/acetonitrile
(95:5, 0.01 M H3PO4) (solvent A) and acetonitrile/water (95:5,
0.01 M H3PO4) (solvent B) with a ratio of 95:5 (v/v). The flow rate
was of 1.2 mL/min and the retention times of 33 were 11.4 min.
For Guanosine analyses the mobile phase consisted of water
(0.01 M H3PO4) (solvent A) and acetonitrile (0.01 M H3PO4) (solvent
B) with a ratio of 98:2 (v/v). The flow rate was of 1.2 mL/min and the
retention times of Guanosine was 6 min.
4.2.5.4. 2-Isobutylamide-50-O-[(diethyl)phosphonoacetyl]-guanosine
(38). The residue obtained was purified by chromatography over
silica gel (eluent: CH2Cl2/MeOH, 95:5, v/v) to afford the desider-
ated compound 38 (yield 90%) as white foam. 1H NMR (400 MHz,
DMSO-d6) d: 1.11e1.19 (6H, m, (CH3)2CH), 2.90e2.96 (1H, m, CH),
3.16, 3.21 (2H, s, J ¼ 20 Hz, CH2P), 3.97e4.03 (4H, m, 2OCH2CH3),
4.06e4.20 (4H, m, H-30, H-40, 2H-50), 4.54 (1H, t, H-20), 5.40 (1H,
d, J ¼ 3.9 Hz, OH-30), 5.60 (1H, d, J ¼ 4.3 Hz, OH-20), 5.90 (1H, d,
J ¼ 5.6 Hz, H-10), 8.28 (1H, s, H-8),11.67 (1H, br s, NHeCO),12.09
(1H, br s, NH-3). ESI MS: m/z 532.3 Da [M þ H]þ, C20H30N5O10
P
Mol. Wt. 531.45.
For Guanine analyses the mobile phase consisted of water
(0.01 M H3PO4) (solvent A) and acetonitrile (0.01 M H3PO4) (solvent
B) with a ratio of 99:1 (v/v). The flow rate was of 0.8 mL/min and the
retention times of Guanine was 4.1 min.
Quantification was performed by integration of peak areas using
external standardization.
4.2.6. 50-O-Tosyl-20,30-isopropylidene-inosine (25)
To a solution of 22 (0.15 g, 0.49 mmol) in pyridine (10 mL) was
added DMAP (0.035 g, 0.2 mmol). At 0 ꢁC tosyl chloride (0.122 g,
0.63 mmol) was added and the reaction mixture was stirred at
40 ꢁC overnight. The reaction mixture was washed with HCl 1 N,
sat.d aq. NaHCO3 solution and brine. The organic phase was dried,
filtered and evaporated to give a crude residue purified by
chromatography over silica gel (eluent: CH2Cl2/MeOH 98/2/95/
5/90/10, v/v) to afford 25 (yield 53%). 1H NMR (200 MHz,
6.2. Preparation of rat liver homogenates
The livers of male Wistar rats were isolated from the rats imme-
diately after their decapitation. The excided tissue was washed in ice-
cold saline solution (0.9% sodium chloride), weighted, cut into small
pieces and 4 mL of ice-cold incubation buffer (50 mM TriseHCl, pH 7.4)
wasadded per1 gof tissue. Thetissuewas then homogenizedwiththe
employment of a PottereElvehjem apparatus. The supernatant
obtained after centrifugation (2000ꢃ g for 10 min, at 4 ꢁC) was
DMSO-d6)
d: 1.28, 1.50 (6H, s, (CH3)2C), 2.35 (3H, s, CH3-tos),
4.16e4.35 (3H, m, H-40, 2H-50), 4.93 (1H, m, H-30), 5.25 (1H, dd,
J ¼ 3.0 Hz, H-20), 6.14 (1H, d, J ¼ 2.0 Hz, H-10), 7.27 (2H, d, Ph),
7.60 (2H, d, J ¼ 8.2 Hz, Ph), 7.96 (1H, s, H-2), 8.15 (1H, s, H-8),
12.43 (1H, br s, NH-3). ESI MS: m/z 463.4 Da [M
þ
H]þ,
C20H22N4O7S Mol. Wt. 462.48.

S. Vertuani et al. / European Journal of Medicinal Chemistry 54 (2012) 202e209
209
decanted off and stored at ꢀ80 ꢁC before its employment for degra-
[20] V.L. Serebruany, S.R. Steinhubl, P.B. Berger, A.I. Malinin, D.L. Bhatt, E.J. Topol,
J. Am. Coll. Cardiol. 45 (2005) 246.
[21] T. Geisler, H. Langer, M. Wydymus, K. Göhring, C. Zürn, B. Bigalke, K. Stellos,
A.E. May, M. Gawaz, Eur. Heart J. 27 (2006) 2420.
[22] P.A. Gurbel, U.S. Tantry, Nat. Clin. Pract. Cardiovasc. Med. 3 (2006) 387.
[23] S. Heptinstall, J. Thromb. Haemost. 4 (2006) 539.
dation studies. The total protein concentration in the tissue homoge-
nate was determined using the Lowry procedure [60] (29.4 ꢂ 1.1
mg
protein/ L).
m
[24] D.J. Angiolillo, A. Fernandez-Ortiz, E. Bernardo, F. Alfonso, C. Macaya, T.A. Bass,
M.A. Costa, J. Am. Coll. Cardiol. 49 (2007) 1505.
[25] K.P. Bliden, J. DiChiara, U.S. Tantry, A.K. Bassi, S.K. Chaganti, P.A. Gurbel, J. Am.
Coll. Cardiol. 49 (2007) 657.
6.3. Stability studies in human plasma and rat liver homogenates
An aliquot of 15
L solution of each compounds (10ꢀ2 M in
m
[26] P. Buonamici, R. Marcucci, A. Migliorini, G.F. Gensini, A. Santini, R. Paniccia,
G. Moschi, A.M. Gori, R. Abbate, D. Antoniucci, J. Am. Coll. Cardiol. 49 (2007) 2312.
[27] J.T. Brandt, C.D. Payne, S.D. Wiviott, G. Weerakkody, N.A. Farid, D.S. Small,
J.A. Jakubowski, H. Naganuma, K.J. Winters, Am. Heart J. 153 (66) (2007) e9.
[28] T. Jernberg, C.D. Payne, K.J. Winters, C. Darstein, J.T. Brandt, J.A. Jakubowski,
H. Naganuma, A. Siegbahn, L. Wallentin, Eur. Heart J. 27 (2006) 1166.
[29] J.J.J. van Giezen, R.G. Humphries, Semin. Thromb. Hemost. 31 (2005) 195.
[30] B. Springthorpe, A. Bailey, P. Barton, T.N. Birkinshaw, R.V. Bonnert, R.C. Brown,
D. Chapman, J. Dixon, S.D. Guile, R.G. Humphries, S.F. Hunt, F. Ince, A.H. Ingall,
I.P. Kirk, P.D. Leeson, P. Leff, R.J. Lewis, B.P. Martin, D.F. McGinnity,
M.P. Mortimore, S.W. Paine, G. Pairaudeau, A. Patel, A.J. Rigby, R.J. Riley,
B.J. Teobald, W. Tomlinson, P.J. Webborn, P.A. Willis, Bioorg. Med. Chem. Lett.
17 (2007) 6013.
DMSO) was incubated (at a final concentration of 50 M) in rat liver
m
homogenate or human blood obtained from healthy subjects
(3 mL). Incubation of the aliquots was carried out at 37 ꢁC for
various periods up to 360 min. At different incubations times, an
aliquot of the solution (100
degradation was blocked by addition of ice water (100
mixing the solution, 50 L of SSA 10% and 50 L of internal standard
(50 M solution) were subsequently added. After centrifugation
(10 min at 14,000g), the supernatant was evaporated to dryness by
N2 flow. The residue was dissolved in 400 L of water and after
L) of supernatant
mL) was removed and compound
mL). After
m
m
m
m
[31] K. Balasubramaniam, G.N. Viswanathan, S.M. Marshall, A.G. Zaman, Cardiol.
Res. Pract. (2012) ID 909154.
[32] G. Cristalli, D.C. Mills, Biochem. J. 291 (1993) 875.
centrifugation (10 min at 19,800g) an aliquot (20
m
was injected into RP-HPLC.
[33] D.C. Mills, Blood 76 (1996) 835.
[34] N.J. Cusack, S.M.O. Hourani, J. Auton. Nerv. Syst. 81 (2000) 37.
[35] P. Crepaldi, B. Cacciari, M.C. Bonache, G. Spalluto, K. Varani, P.A. Borea,
I. Kügelgen, K. Hoffmann, M. Pugliano, C. Razzari, M. Cattaneo, Bioorg. Med.
Chem. 17 (2009) 4612.
[36] B. Xu, A. Stephens, G. Kirschenheuter, A.F. Greslin, X. Cheng, J. Sennelo,
M. Cattaneo, M.L. Zighetti, A. Chen, S.A. Kim, H.S. Kim, N. Bischofberger,
G. Cook, K.A. Jacobson, J. Med. Chem. 45 (2002) 5694.
[37] J.G. Douglass, R.I. Patel, B.R. Yerxa, S.S. Shaver, P.S. Watson, K. Bednarski,
R. Plourde, C.C. Redick, K. Brubaker, A.C. Jones, J.L. Boyer, J. Med. Chem. 51
(2008) 1007.
[38] J.G. Douglass, J.B. deCamp, E.H. Fulcher, W. Jones, S. Mahanty, A. Morgan,
D. Smirnov, A.C. Jones, J.L. Boyer, P.S. Watson, Bioorg. Med. Chem. Lett. 18
(2008) 2167.
Acknowledgements
Authors wish to thank Alberto Casolari and Elisa Durini for
careful NMR experiments. This work was supported by Ministry of
Education, University and Research (grant FIRB RBNE03YA3L_008).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2012.04.045.
[39] S. Manfredini, P.G. Baraldi, R. Bazzanini, M. Marangoni, D. Simoni, J. Balzarini,
E. De Clercq, J. Med. Chem. 38 (1995) 199.
[40] S. Manfredini, P.G. Baraldi, E. Durini, S. Vertuani, J. Balzarini, E. De Clercq,
A. Karlsson, V. Buzzoni, L. Thelander, J. Med. Chem. 42 (1999) 3243.
[41] S. Manfredini, P.G. Baraldi, R. Bazzanini, E. Durini, S. Vertuani, A. Pani,
T. Marceddu, F. Demontis, L. Vargiu, P. La Colla, Nucleosides Nucleotides
Nucleic Acids 19 (2000) 705.
[42] S. Manfredini, N. Solaroli, A. Angusti, F. Nalin, E. Durini, S. Vertuani, S. Pricl,
M. Ferrone, S. Spadari, F. Focher, A. Verri, E. De Clercq, J. Balzarini, Antivir.
Chem. Chemother. 14 (2003) 183.
[43] S. Manfredini, A. Angusti, C.A. Veronese, E. Durini, S. Vertuani, F. Nalin,
N. Solaroli, S. Pricl, M. Ferrone, M. Mura, M.A. Piano, B. Poddesu, A. Cadeddu,
P. La Colla, R. Loddo, Pure Appl. Chem. 76 (2004) 1007.
[44] A. Angusti, S. Manfredini, E. Durini, N. Ciliberti, S. Vertuani, N. Solaroli, S. Pricl,
M. Ferrone, M. Fermeglia, R. Loddo, B. Secci, A. Visioli, T. Sanna, G. Collu,
M. Pezzullo, P. La Colla, Chem. Pharm. Bull. (Tokyo) 56 (2008) 423.
[45] Y. Chen, S. Morera, C. Pasti, A. Angusti, N. Solaroli, M. Veron, J. Janin,
S. Manfredini, D. Deville-Bonne, Med. Chem. 1 (2005) 529.
[46] N. Kohyama, T. Katashima, Y. Yamamoto, Synthesis 17 (2004) 2799.
[47] G.S. Buenger, V. Nair, Synthesis 10 (1990) 962.
References
[1] G. Hollopeter, H.M. Jantzen, D. Vincent, G. Li, L. England, V. Ramakrishnan,
R.B. Yang, P. Nurden, A. Nurden, D. Julius, P.B. Conley, Nature 409 (2001) 202.
[2] E. ten Boekel, P. Bartels, Pathophysiol. Haemost. Thromb. 32 (2002) 137.
[3] J. Jin, J.L. Daniel, S.P. Kunapuli, J. Biol. Med. 273 (1998) 2030.
[4] G.E. Jarvis, R.G. Humphries, M.J. Robertson, P. Leff, Br. J. Pharmacol. 129 (2000)
275.
[5] M.P. Abbracchio, C. Verderio, Novartis Found. Symp. 276 (2006) 91.
[6] C. Léon, C. Vial, C. Gachet, P. Ohlmann, B. Hechler, J.P. Cazenave, A. Lecchi,
M. Cattaneo, Thromb. Haemost. 81 (1999) 775.
[7] E.A. Barnard, J. Simon, Trends Pharmacol. Sci. 22 (2001) 388.
[8] F. Marteau, E. Le Poul, D. Communi, C. Labouret, P. Savi, J.M. Boeynaems,
N.S. Gonzalez, Mol. Pharmacol. 64 (2003) 104.
[9] M. Cattaneo, C. Gachet, Arterioscler. Thromb. Vasc. Biol. 19 (1999) 2281.
[10] P. Savi, J.M. Pereillio, M.F. Uzabiaga, J. Combalbert, C. Picard, J.P. Maffrand,
M. Pascal, J.M. Herbert, Thromb. Haemost. 84 (2000) 891.
[11] J.M. Boeynaems, H. van Giezen, P. Savi, J.M. Herbert, Curr. Opin. Invest. Drugs 6
(2005) 275.
[12] S. Yusuf, F. Zhao, S.R. Mehta, S. Chrolavicius, G. Tognoni, K.K. Fox, N. Engl. J.
Med. 345 (2001) 494.
[13] S.R. Steinhubl, P.B. Berger, J.T. Mann III, E.T. Fry, A. DeLago, C. Wilmer,
E.J. Topol, JAMA 288 (2002) 2411 (published correction appears in JAMA
289:987 (2003)).
[14] M.S. Sabatine, C.P. Cannon, C.M. Gibson, J.L. López-Sendón, G. Montalescot,
P. Theroux, M.J. Claeys, F. Cools, K.A. Hill, A.M. Skene, C.H. McCabe,
E. Braunwald, N. Engl. J. Med. 352 (2005) 1179.
[48] A. Lanver, H.G. Schmalz, Molecules 10 (2005) 508.
[49] M.J. Robins, P.W. Hatfield, Can. J. Chem. 60 (1982) 547.
[50] C.J. Marasco Jr., D.L. Kramer, J. Miller, C.W. Porter, C.J. Bacchi, D. Rattendi,
L. Kucera, N. Iyer, R. Bernacki, P. Pera, J.R. Sufrin, J. Med. Chem. 45 (2002)
5112.
[51] S.P. Vincent, S. Grenier, C. Mioskowski, C. Salesse, L. Lebeau, Bioorg. Med.
Chem. Lett. 11 (2001) 1185.
[52] B. Pavan, A. Dalpiaz, N. Ciliberti, C. Biondi, S. Manfredini, S. Vertuani, Mole-
cules 13 (2008) 1035.
N.M. Nielsen, H. Bundgaard, J. Med. Chem. 32 (1989) 727.
[53]
[54] P. Masson, M.T. Froment, P.L. Fortier, J.E. Visicchio, C.F. Bartels, O. Lockridge,
Biochim. Biophys. Acta 1387 (1998) 41.
[15] M.S. Sabatine, C.P. Cannon, C.M. Gibson, J.L. López-Sendón, G. Montalescot,
P. Theroux, B.S. Lewis, S.A. Murphy, C.H. McCabe, E. Braunwald, JAMA 294
(2005) 1224.
[16] J.L. Anderson, C.D. Adams, E.M. Antman, C.R. Bridges, R.M. Califf, D.E. Casey Jr.,
W.E. Chavey 2nd, F.M. Fesmire, J.S. Hochman, T.N. Levin, A.M. Lincoff,
E.D. Peterson, P. Theroux, N.K. Wenger, R.S. Wright, Circulation 116 (2007) 803.
[17] J.P. Bassand, C.W. Hamm, D. Ardissino, E. Boersma, A. Budaj, F. Fernández-
Avilés, K.A. Fox, D. Hasdai, E.M. Ohman, L. Wallentin, W. Wijns, Eur. Heart J. 28
(2007) 1598.
[55] A. Dalpiaz, B. Cacciari, C.B. Vicentini, F. Bortolotti, G. Spalluto, S. Federico,
B. Pavan, F. Vincenzi, P.A. Borea, K. Varani, Mol. Pharm. 9 (2012) 591.
[56] A. Hampton, J. Am. Chem. Soc. 83 (1961) 3640.
[57] Y. Xu, H. Jin, Z. Yang, L. Zhang, L. Zhang, Tetrahedron 65 (2009) 5228.
[58] J. Howarth, P. Kenny, S. McDonnell, A. O’Connor, Bioorg. Med. Chem. Lett. 13
(2003) 2693.
[59] S. Dionisotti, S. Ferrara, C. Molta, C. Zocchi, E. Ongini, J. Pharmacol. Exp. Ther.
298 (1996) 726.
[60] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951)
265.
[18] P.A. Gurbel, K.P. Bliden, Am. J. Cardiol. 91 (2003) 1123.
[19] S. Matetzky, B. Shenkman, V. Guetta, M. Shechter, R. Bienart, I. Goldenberg,
I. Novikov, H. Pres, N. Savion, D. Varon, H. Hod, Circulation 109 (2004) 3171.