Synthesis and biological activity of 2-alkylated deoxyadenosine bisphosphate derivatives as P2Y1 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2003.12.041

A previous study around adenine nucleotides afforded the reference N ⁶-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (1a, MRS 2179) as a selective human P2Y₁ receptor antagonist (pA ₂=6.55±0.05) with antithrombotic propertie

Full text of DOI:10.1016/j.bmc.2003.12.041

Bioorganic & Medicinal Chemistry 12 (2004) 1769–1779
Synthesis and biological activity of 2-alkylated deoxyadenosine
bisphosphate derivatives as P2Y1 receptor antagonists
Romain Mathieu,a Anthony Baurand,b Martine Schmitt,a Christian Gachetb
and Jean-Jacques Bourguignona,*
a
´
Laboratoire de Pharmacochimie de la Communication Cellulaire (CNRS, UMR 7081), Faculte de Pharmacie,
´
Universite Louis Pasteur, 74 route du Rhin, BP24, 67401 Illkirch cedex, France
b
´
Laboratoire de Biologie et de Pharmacologie de l’Hemostase et de la Thrombose (INSERM, U 311),
Etablissement Franc¸ais du Sang-Alsace, 10 rue Spielmann, BP36, 67065 Strasbourg cedex, France
Received 26 September 2003; accepted 22 December 2003
Abstract—A previous study around adenine nucleotides afforded the reference N6-methyl-20-deoxyadenosine-30,50-bisphosphate (1a,
MRS 2179) as a selective human P2Y1 receptor antagonist (pA2=6.55ꢀ0.05) with antithrombotic properties. In the present paper,
we have synthesized and tested in vitro various 2-substituted derivatives with the goal of exploring the 2-position binding region
and developing more potent P2Y1 receptor antagonists. Thus, we have adopted a novel and versatile chemical pathway using a
palladium-catalyzed cross-coupling reaction with the 2-iodinated derivative 7 as a common intermediate for a very efficient synth-
esis of the 2-alkyl-N6-methyl-20-deoxyadenosine-30,50-bisphosphate nucleotides 1e–i. The biological activity was evaluated through
the ability of compounds to inhibit ADP-induced platelet aggregation, intracellular calcium rise and to displace the specific binding
of [33P]2-MeSADP. 2-Ethyl and 2-propyl groups appeared to be tolerated, whereas a bulky group or a C3 linear substituent dra-
matically decreased potency of antagonists. The 2-ethynyl derivative 1h (pA2=7.54ꢀ0.10) was significantly more potent (10-fold)
as an antagonist when compared to the reference 1a, revealing a potential electronic interaction highly favorable between triple
bond orbitals and the P2Y1 receptor at this position.
# 2004 Published by Elsevier Ltd.
1. Introduction
tion through mobilization of calcium stores, whereas the
P2Y12 receptor coupled to adenylyl cyclase inhibition is
essential for a full aggregation response to ADP and the
stabilisation of aggregates.3,4 The latter has been shown to
be the target of the antiplatelet thienopyridine compounds
ticlopidine and clopidogrel5,6 and of the ATP analogues,
the Astra Zeneca compounds of the AR-C series.7ꢁ9 The
P2Y1 and P2Y12 receptors are both essential for normal
ADP-induced platelet aggregation to occur.10,11 Recent
studies with P2Y1-knockout mice or selective P2Y1
antagonists in experimental thrombosis models have
shown that the P2Y1 receptor is a promising potential
target for new antithrombotic agents.12ꢁ16 On the other
hand, the whole physiological role of this receptor is
unknown, and few pharmacological probes are available.
Thus, the development of potent and selective P2Y1
receptor antagonists is a critical need.
P2 receptors, which are activated by extracellular adenine
and uracil nucleotides, are widely distributed in many
different cell types and regulate a broad range of physio-
logical processes.1,2 These membrane-bound receptors,
collectively called purinergic receptors, are divided into
two structurally distinct families: G-protein-coupled
receptors (GPCR) termed P2Y, and ligand-gated cation
channels termed P2X.
Adenosine diphosphate (ADP, Fig. 1) plays a crucial
role in hemostasis and thrombosis, and its receptors are
potential targets for antithrombotic drugs. Two P2Y
receptors are involved in platelet aggregation: the P2Y1
receptor coupled to phospholipase C initiates aggrega-
Boyer et al. reported in 1996 that various endogenous
bisphosphates of adenosine (e.g., adenosine-30,50-
bisphosphate, A3P5P) behaved as competitive antagonists
of both turkey and human P2Y1 receptors with KB
Keywords: Platelet aggregation; P2Y1 receptor; Nucleotides; Palla-
dium-catalyzed cross-coupling reaction.
* Corresponding author. Tel.: +33-390-244-234; fax:+33-390-244-
310; e-mail: jjb@pharma.u-strasbg.fr
0968-0896/$ - see front matter # 2004 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2003.12.041

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R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
have not been described to our knowledge. These inter-
esting results encouraged us to synthesize and test in
vitro compounds 1e–i with the goal of better understand
the SAR of 2-substituted derivatives using homology
rules and introduction of unsaturation.
The biological activity at human P2Y1 receptor was
determined by measuring the ability to inhibit ADP-
induced platelet aggregation, shape change, intracellular
calcium rise and to displace the specific binding of
[33P]2-MeSADP in intact platelets. We also verified that
these compounds have no effect on P2Y12 receptor by
measuring adenylyl cyclase activity.
2. Results and discussion
2.1. Chemical synthesis
The recently reported synthesis of the 2-methyl derivative
1d was long and unsuitable to afford derivatives with
different substitutions at 2-position.22 In this goal, the
palladium-catalyzed cross-coupling reaction appeared
to be the best tool in C–C bond formation from a 2-
halogenated0 derivative. As shown in Schemes 1 and 2, all
N6-methyl-2 -deoxyadenosine-30,50-bisphosphate deriva-
tives 1e–i were synthesized by Sonogashira reaction of
the corresponding alkynes with the iodinated inter-
mediate 7.
Figure 1. Structures of nucleotides analogues acting on the P2Y1
receptor.
values in the micromolar range.17 These nucleotides also
displayed partial agonist activities at the turkey P2Y1
receptor.17 Starting from this observation of consider-
able interest, SAR (structure–activity relationship)
studies of derivatives of adenosine bisphosphates have
resulted in the reference N6-methyl-20-deoxyadenosine-
30,50-bisphosphate (1a, MRS 2179), a competitive
antagonist at human and turkey P2Y1 receptors, with a
KB value of 100 nM and an IC50 value of 0.33 mM in
blocking the effects of 10 nM 2-methylthioadenoine-50-
diphosphate (2-MeSADP).18,19 The presence of an N6-
The commercially available 20-deoxyadenosine (2) was
first protected as the 30,50-disilylated compound 3 in
good yield,23 followed by diazotization-chlorination24
with isoamyl nitrite in carbon tetrachloride to afford the
silylated 6-chloro-20-deoxynucleoside 4 in 41% yield.
Electron-withdrawing effects of the 6-chloro group
increased acidity at 2-position of the purine ring
according to a previously reported study.25 Treatment of
compound 4 with the bulky base lithium 2,2,6,6-tetra-
methyl piperidine (LTMP) and tributylstannyl chloride
as the electrophile allowed a selective stannylation at 2-
position against the 8-position of the purine moiety.26
To avoid protonolysis of the stannyl group on silica gel
during column chromatography, 2-tributylstannyl deri-
vative 5 was not purified and directly treated with iodine
to afford the 2-iodo-6-chloro-20-deoxynucleoside 6 in
55% yield. Nucleophilic displacement of the 6-chloro
groupwith methylamine led to the corresponding 2-
iodo-N6-methyl-20-deoxyadenosine 7 in good yield. The
key intermediate 7 was obtained in five steps and 18%
overall yield. This chemical pathway appeared to be
more efficient than classical strategies starting from
guanosine23 or 20-deoxyguanosine.20
0
methyl groupand the absence of a 2 -hydroxyl group
both enhanced affinity and turned the agonist character
into a pure antagonist at P2Y1 receptor.
Every substitution at the 8-position of the adenine moiety
considerably decreased affinity, whereas some substitu-
tion at the 2-position was known to be tolerated.20 The
corresponding 2-chloro derivative 1b was slightly more
potent than 1a with an IC50 of 0.20 mM and the 2-
methylthio derivative 1c was an antagonist as potent as
the reference 1a. On the other hand, major synthetic
modifications of the ribose moiety including replacement
with carbocyclics, smaller and larger rings, acyclics, and
conformationally constrained rings have been carried
out to increase activity and biological stability.21
Then, the intermediate 7 was engaged in a palladium-
catalyzed cross-coupling with (trimethylsilyl)acetylene,
propyne or phenylacetylene in presence of cuprous
iodide to afford 2-substituted derivatives 8a, b or c
respectively in good yields.23 Desilylation was achieved
with tetrabutylammonium fluoride (TBAF) in acetoni-
trile to give the desired compounds 9a–c, which were
finally phosphorylated using two different methods
(shown in Scheme 2).
We have recently reported the synthesis and the phar-
macological properties of the 2-methyl derivative 1d.22
This compound was a full antagonist at the human
P2Y1 receptor with a pA2 value of 7.11ꢀ0.11 and was
found to be 4-fold more potent than the reference 1a
(pA2=6.55ꢀ0.05). However the effects of longer alkyl
groups or aryl groups at 2-position of the adenine moiety

R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
1771
The tetrabenzyl pyrophosphate (TBPP) procedure27
which needs hydrogenolysis to remove the benzyl groups
was first displayed with the goal to obtain saturated and
flexible alkyl chains at 2-position. Then, transformation of
compounds 9a–c in bis(dibenzyl)phosphates 10a–c fol-
lowed by catalytic hydrogenation led to nucleotides 1e–g
in good yields. Alternatively, phosphorylation of nucleo-
sides 9a,b was carried out using a phosphoramidite
method28 to keepunsaturations and rigidity of 2-sub-
stituents. The bis(di-tert-butyl)phosphate intermediates
11a,b were deprotected under acidic conditions to afford
2-alkynyl derivatives 1h,i in moderate yields.
Scheme 1. Reagents and conditions: (a) TBDMS-Cl, imidazole, DMF, 35 ꢂC, 16 h; (b) isoamyl nitrite, CCl4, 50 ꢂC, 3 h; (c) LTMP, Bu3SnCl, THF,
ꢁ78 ꢂC, 30 min; (d) I2, THF, rt, 4 h; (e) MeNH2, EtOH, rt, 16 h; (f) HCꢃC-R, (PPh3)2PdCl2, CuI, Et3N, rt, 3 h; (g) TBAF, CH3CN, rt, 1 h.
Scheme 2. Reagents and conditions: (a) tBuOK, TBPP, THF, ꢁ40 ꢂC, 30 min; (b) H2, Pd/C, MeOH, 60 psi, rt, 48 h; (c) Et2NP(OtBu)2, tetrazole,
THF, rt, 2 h, then MCPBA, ꢁ78 ꢂC, 30 min; (d) TFA, CH2Cl2, rt, 30 min.

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R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
Figure 2. Inhibition of ADP-induced aggregation of washed human platelets by the compound 1h. (A) Platelet aggregation was evaluated quanti-
tatively using a turbidimetric method across a suspension of washed platelets. Aggregation and shape change induced by 5 mM ADP (control) were
inhibited by 10 mM 1h. Traces are from one representative experiment. (B) The pA2 value corresponding to the potency of inhibition, was deter-
mined by generation a series of concentration-response curves for ADP, in the presence of the indicated increasing concentrations of the compound
1h added 30 s before ADP. The following Schild regression analysis allows us to determine the pA2 value. Curves represent the mean of three
independent experiments and give a pA2 of 7.54ꢀ0.10.
2.2. Biological activity
affected by these molecules in human platelets, whereas
AR-C66096,30 a selective P2Y12 receptor antagonist,
totally reversed the inhibitory effect of ADP (Fig. 3B).
In conclusion, the potential inhibitory effect of 1e–i on
ADP-induced platelet aggregation is entirely due to the
antagonistic activity on the P2Y1 receptor.
2.2.1. Platelet aggregation. The new derivatives 1e–i
prepared in the present study were first tested as
antagonists in a platelet aggregation assay induced by
ADP using platelet aggregometry. Traces representing
the aggregation as a function of time following exposure
to 5 mM ADP were shown in Figure 2.
2.2.3. Binding assays. The affinity of ligands for the
P2Y1 receptor could be evaluated quantifying their
potency to displace a radioligand. Studies of the binding
of ADP to its platelet receptors are currently performed
using [33P]2-MeSADP, a radioligand which binds to
both P2Y1 and P2Y12 receptors.5,29,31 In order to verify
that 1e–i displaced [33P]2-MeSADP only from P2Y1
receptors, we compared its effects to those of reference
1a, a well known P2Y1 receptor antagonist.29 The spe-
cific binding of [33P]2-MeSADP to washed human pla-
telets was competitively and partially displaced by 1a
(Ki=110 nM) at approximately 20% of [33P]2-MeSADP
sites while the 2-MeSADP totally displaced the radi-
olabeled 2-MeSADP (Fig. 4, filled square). Similarly, we
confirmed that the compounds 1e–i displaced the radi-
oligand from the P2Y1 binding sites as shown for
Addition of compounds 1e–i (100 mM) to washed
human platelets 30 s before ADP (5 mM) inhibited pla-
telet aggregation and shape change as shown in the
presence of 10 mM 1h (Fig. 2A), while did not induce
shape change or aggregation by themselves even at high
concentrations (upto 100 mM) (data not shown). As
seen with the reference 1a no agonist activity was
observed.29 The potency of inhibition was determined
by generating a series of concentration-response curves
for ADP in the presence of different concentrations of
each compound 1e–i. The compounds caused a parallel
shift to the right of the concentration-response curve,
but high concentrations of ADP could completely
override high concentrations of compounds 1e–i, as
illustrated with the compound 1h (Fig. 2B). Schild analysis
of the inhibition gave pA2 values summarized in the
Table 1 (n=3).
Table 1. In vitro pharmacological data for inhibition of ADP-
induced aggregation and displacement of the radiolabeled ligand
[
33P]2-MeSADP to washed human platelets
2.2.2. Calcium measurements and inhibition of adenylyl
cyclase. The aggregation process involves the activation
of two receptors (P2Y1 and P2Y12 receptors) and their
transduction machinery. In platelets, as previously
described, ADP induced simultaneous mobilization of
intracellular Ca2+ stores and inhibition of adenylyl
cyclase, through activation of the P2Y1 and P2Y12
receptors, respectively. We have verified that compounds
1e–i act selectively on the P2Y1 receptor. The intracel-
lular Ca2+ rise induced in washed human platelets by
ADP could be inhibited by the different derivatives, in
the presence or absence of 2 mM external Ca2+ (data
not shown). As an illustration, the compound 1h (10
mM) totally inhibited ADP (1 mM)-induced calcium rise
(Fig. 3A). Conversely, 100 mM of the compounds 1e–i
had no influence on basal levels of cAMP in human
platelets, or on the cAMP levels induced by 10 mM
prostaglandin E1 (PGE1). The ability of ADP to inhibit
PGE1-stimulated cAMP accumulation was likewise not
a
Compd
R
pA2
Ki (nM)b
1a
1d
1e
1f
1g
1h
1i
H
CH3
6.55ꢀ0.05
7.11ꢀ0.11
6.59ꢀ0.14
6.75ꢀ0.34
6.03ꢀ0.18
7.54ꢀ0.10
4.7
110
60
15
140
4500
10
CH2CH3
CH2CH2CH3
CH2CH2Ph
CꢃCH
CꢃC–CH3
600
a The inhibition of ADP-induced platelet aggregation by molecules
was measured using aggregometry. The pA2 value corresponds to the
potency of inhibition and was determined by generating a series of
concentration-response curves for ADP in the presence of different
concentrations of each compound 1a, 1d–i. Means of three indepen-
dent experiments were shown.
b The affinity of each compound was evaluated by a competition
experiment using the radioligand [33P]2-MeSADP. Displacement
experiments were performed using a single concentration of radi-
olabeled [33P]2-MeSADP in the presence of increasing concentrations
of the appropriate unlabeled ligand (1a, 1d–i) and the Ki determined
(n=1).

R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
1773
Figure 3. Selective inhibition of the P2Y1 receptor transduction pathway. (A) The antagonistic properties of each compound were evaluated on
ADP-induced calcium rise through the P2Y1 receptor in washed human platelets. As an example, the compound 1h (10 mM) totally abolished the
intracellular calcium rise induced by 1 mM ADP in the presence of 2 mM external Ca2+. Graphs are from one experiment representative of three
independent experiments. (B) The selectivity of each compound between the platelet P2Y1 and P2Y12 receptors was determined by cAMP mea-
surements. The cAMP inhibition induced by ADP through the P2Y12 receptor (gray bars) after prestimulation by the prostaglandin PGE1 (black
bars) was not modified by the derivative 1h as well as each compound. However, a selective P2Y12 receptor antagonist (AR-C66096) reversed the
inhibition of cAMP induced by ADP (striped bars).
example with the compound 1h (Fig. 4, open square).
The affinity of each compound was evaluated by a
competition experiment and gave Ki values summarized
in Table 1 (n=1).
affinity. On the other hand, compound 1h (pA2=
7.54ꢀ0.10) was significantly more potent (10-fold) as an
antagonist when compared to the reference 1a
(pA2=6.55ꢀ0.05),29 reflecting the potency-enhancing
effect of the 2-ethynyl group. Moreover, this compound
exhibited a better affinity (Ki=10 nM) for the human
P2Y1 receptor than the reference 1a (10-fold) and the
other derivatives. Thus, an electronic interaction
between triple bond orbitals and the receptor at this
position seems to be greatly favorable.
The 2-ethyl (1e) and 2-propyl (1f) derivatives showed pA2
values similar to the reference 1a (Table 1). Moreover,
the presence of an ethyl group (1e) at the 2-position of
the adenine moiety enhanced the affinity of the ligand
for the P2Y1 receptor (Ki=15 nM) when compared to
the reference 1a (about 8-fold). Short and medium alkyl
groups are well tolerated at this position revealing a
relatively open area of the P2Y1 receptor around this
position. However, the 2-phenylethyl derivative 1g or
the 2-propynyl derivative 1i displayed low potency as an
antagonist, with higher affinity constant (Ki=4500 and
600 nM respectively) than the unsubstituted compound
1a. A bulky groupor a C 3 linear substituent dramatically
decreased activity and highlighted some steric hindrance
at this position. This topological exploration showed a
medium and weakly sterically restricted hydrophobic
pocket at the 2-position of the human P2Y1 receptor,
which allows various modifications of N6-methyl-20-
deoxyadenosine-30,50-bisphosphate nucleotides to improve
3. Conclusion
In summary, we have synthesized and evaluated the 2-
N6-methyl-20-deoxyadenosine-30,50-bispho-
alkylated
sphate derivatives 1e–i. The chosen chemical pathway
allowed us to explore in a very efficient way different
substitutions at 2-position by means of the corresponding
alkynes and the 2-iodinated derivative as a common
intermediate leading to possible topological exploration
at this position. The 2-alkylated derivatives 1e–i were
tested as inhibitors of platelet function in comparison to
the unsubstituted reference 1a. 2-Alkyl groups from
methyl to propyl appeared to be completely tolerated,
whereas a bulky groupor a C
linear substituent
3
dramatically decreased potency of antagonists. These
results reflect a medium hydrophobic pocket at the 2-
position of the human P2Y1 receptor. The 2-ethynyl
derivative 1h (pA2=7.54ꢀ0.10) was found more
potent (10-fold) as an antagonist when compared to the
reference 1a and exhibited high affinity (Ki=10 nM) for
its receptor, revealing a potential electronic interaction
highly favorable between triple bond orbitals and the
human P2Y1 receptor at this position. Furthermore, this
2-ethynyl derivative seems to be the most potent P2Y1
receptor antagonist with a N6-methyl-20-deoxyadenosine-
30,50-bisphosphate scaffold reported to date to our
knowledge. In addition, as the 2-ethyl derivative 1e pre-
sented the same potent affinity, the ethynyl derivative 1h
may serve for easy and efficient introduction of tritium
in its structure by means of catalytic hydrogenation with
tritium gas. This novel radioligand may constitute a new
pharmacological tool for further binding experiments.
Figure 4. Affinity for the P2Y1 receptor. Competition experiments
were performed using a single concentration of the radiolabeled [33P]2-
MeSADP on washed human platelets in the presence of increasing
concentrations of the each unlabeled compound. The compound 1h as
well as the other compounds (1e–i) displaced approximately 20% of
[
33P]2-MeSADP sites corresponding to the P2Y1 binding sites on pla-
telets, while the remaining binding sites were the P2Y12 receptors.
Curves are from one experiment.

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R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
4. Experimental
3.4 Hz, 1H, 50-HA), 3.88 (dd, J=11.2, 4.4 Hz, 1H, 50-
HB), 4.00–4.05 (m, 1H, 40-H), 4.59–4.65 (m, 1H, 30-H),
5.69 (bs, 2H, NH2), 6.46 (t, J=6.5 Hz, 1H, 10-H), 8.14
(s, 1H, 8-H), 8.36 (s, 1H, 2-H). 13C NMR (75 MHz,
CDCl3): d ꢁ5.50, ꢁ5.40, ꢁ4.82, ꢁ4,68 (2ꢄSi(CH3)2),
18.0, 18.4 (2ꢄSiC), 25.9, 26.0 (2ꢄSiC(CH3)3), 41.3 (20-
C), 62.6 (50-C), 71.8 (30-C), 84.3 (10-C), 87.9 (40-C), 120.0
(5-C), 140.0 (8-C), 149.6, 153.0 (4-C+6-C), 155.4 (2-C).
MS: m/z 480 (M+H)+, 502 (M+Na)+. HRMS for
C22H41N5O3Si2 (M+H)+ calcd: 480.2826, found:
480.2836.
4.1. Chemical synthesis
Reagents used for the synthesis were purchased from
Sigma-Aldrich (Isle d’Abeau Chesnes, France) and
Lancaster (Bischheim-Strasbourg, France). All solvents
were obtained from commercial suppliers and used
without further purification with the exception of tetra-
hydrofuran (THF) which was freshly distilled from
sodium benzophenone ketyl. Flash chromatography
was performed on Geduran silica gel Si 60 (40–63 mm,
Merck). Thin-layer chromatography was carried out
using plates of silica gel 60 F254 (Merck). The spots were
visualized under UV light (l=254 nm). All chemical
yields are unoptimized and generally represent the result
4.1.2. 9-[3,5-bis-O-(tert-Butyldimethylsilyl)-2-deoxy-ꢀ-D-
erythro-pentofuranosyl]-6-chloropurine (4). Argon was
bubbled through a solution of 3 (8.60 g, 17.9 mmol) in
CCl4 (150 mL) and this solution was cooled to 0 ꢂC.
Isoamyl nitrite (7.24 mL, 53.8 mmol) was added and the
mixture was stirred at 50 ꢂC for 3 h. The solvent was
removed under reduced pressure and the crude product
was purified by silica gel column chromatography
(AcOEt/hexane 1/4 then 1/2) to give 4 (3.71 g, 41%) as a
yellow syrup. Rf 0.85 (AcOEt/hexane 1/1). 1H NMR
(300 MHz, CDCl3): d 0.10, 0.12 (s, 12H, 2ꢄSi(CH3)2),
0.91, 0.93 (s, 18H, 2ꢄSiC(CH3)3), 2.45–2.53 (m, 1H, 20-
HA), 2.60–2.68 (m, 1H, 20-HB), 3.79 (dd, J=11.2, 2.8
Hz, 1H, 50-HA), 3.90 (dd, J=11.2, 3.8 Hz, 1H, 50-HB),
4.04–4.08 (m, 1H, 40-H), 4.61–4.66 (m, 1H, 30-H), 6.53
(t, J=6.5 Hz, 1H, 10-H), 8.50 (s, 1H, 8-H), 8.75 (s, 1H,
2-H). 13C NMR (75 MHz, CDCl3): d ꢁ5.51, ꢁ5.41,
ꢁ4.84, ꢁ4.69 (2ꢄSi(CH3)2), 18.0, 18.4 (2ꢄSiC), 25.7,
25.9 (2ꢄSiC(CH3)3), 41.6 (20-C), 62.7 (50-C), 71.8 (30-C),
84.9 (10-C), 88.2 (40-C), 132.1 (5-C), 143.8 (8-C), 151.0,
151.1 (4-C+6-C), 151.8 (2-C). MS: m/z 500 (35Cl-
M+H)+, 502 (37Cl-M+H)+.
1
of a single experiment. H, 13C and 31P NMR spectra
were recorded on a Bruker AC 200 or a Bruker DPX
300 spectrometer at room temperature; orthopho-
sphoric acid (85%) was used as an external standard for
31P NMR calibration. The chemical shifts (d) are
expressed as relative ppm downfield from CHCl3
(dH=7.27, dC=77.0) or DMSO (dH=2.52, dC=40.0),
coupling constants (J) are given in hertz (Hz), and sig-
nals are designated as follows: s, singlet; d, doublet; t,
triplet; q, quadruplet; m, multiplet; bs, broad singlet.
Electrospray mass spectra (ESMS) and high resolution
mass spectra (HRMS) were obtained on a Perseptive-
Biosystem Mariner mass spectrometer. Melting points
were determined with a Mettler FP62 or a Bibby SMP3
apparatus and are uncorrected (all final nucleotides 1e–i
decomposed between 130–150 ꢂC). The determination of
purity was performed with a Waters 600HPLC system
using a XTerra MS C18 5 mm analytical column (4.6
mmꢄ250 mm) in the linear gradient solvent system
0.1 M triethylammonium acetate buffer+50 mM tetra-
butylammonium bromide/CH3CN in ratios of 95/5 to
40/60 for 30 min with flow rate 1 mL/min. Peaks were
detected by UV absorption using a Waters 996 photo-
diode array detector. All final compounds showed more
than 95% purity as determined using HPLC.
4.1.3. 9-[3,5-bis-O-(tert-Butyldimethylsilyl)-2-deoxy-ꢀ-D-
erythro-pentofuranosyl]-6-chloro-2-iodopurine (6). 1.6 M
BuLi solution in hexane (8.50 mL, 13.6 mmol) was
added dropwise to
a
solution of 2,2,6,6-tetra-
methylpiperidine (2.87 mL, 17.0 mmol) in anhydrous
THF (20 mL) at ꢁ78 ꢂC under an argon atmosphere.
After stirring for 10 min, a solution of 4 (1.70 g, 3.40
mmol) in anhydrous THF (15 mL) was added dropwise
to the mixture at ꢁ78 ꢂC. After stirring for 2 min,
Bu3SnCl (2.77 mL, 10.2 mmol) was added to the lithi-
ated mixture and stirring was continued for 30 min at
ꢁ78 ꢂC. The reaction was quenched by adding saturated
aqueous NH4Cl (10 mL) and the mixture was warmed
to room temperature. The mixture was partitioned
between ethyl acetate (100 mL) and saturated aqueous
NaHCO3 (100 mL) and the aqueous layer was re-
extracted with ethyl acetate (100 mL). The combined
organic layer was dried over Na2SO4, filtered and con-
centrated under reduced pressure. The residue was dis-
solved in THF (25 mL) and iodine (1.30 g, 5.11 mmol)
was added to the solution. After stirring for 4 h at room
temperature, the reaction mixture was diluted with 10%
aqueous Na2S2O3 (50 mL) and extracted with ethyl
acetate (2ꢄ100 mL). The organic layer was washed with
brine (20 mL) and evaporated under reduced pressure.
The residue was dissolved in ethyl acetate (20 mL) and
treated with 15% aqueous KF (10 mL) for 15 min
without stirring. The resulting suspension was filtered
4.1.1. 30,50-bis-O-(tert-Butyldimethylsilyl)-20-deoxyadeno-
sine (3). 20-Deoxyadenosine monohydrate (5.10 g, 18.9
mmol) was suspended in pyridine (20 mL) and evapo-
rated to dryness. This operation was repeated twice, and
then, the residue was dissolved in dry DMF (30 mL)
under an argon atmosphere. Imidazole (3.87 g, 56.8
mmol) and tert-butyldimethylsilyl chloride (6.28 g, 41.7
mmol) were added and the resulting solution was stirred
at 35 ꢂC for 16 h. The solvent was removed under
reduced pressure, the residue was partitioned between
ethyl acetate (100 mL) and water (100 mL) and the
aqueous layer was re-extracted with ethyl acetate (100
mL). The combined organic layer was dried over
Na2SO4, filtered and concentrated under reduced pres-
sure. The crude product was purified by silica gel col-
umn chromatography (AcOEt/CH2Cl2 1/1 then AcOEt/
CH2Cl2/EtOH 4/5/1) to give 3 (8.77 g, 96%) as a white
solid which may be recrystallized from EtOH and Et2O;
mp131–132 ꢂC. Rf 0.61 (AcOEt/CH2Cl2/EtOH 4/5/1).
1H NMR (300 MHz, CDCl3): d 0.10, 0.11 (s, 12H, 2ꢄ
Si(CH3)2), 0.92 (s, 18H, 2ꢄSiC(CH3)3), 2.39–2.47 (m,
1H, 20-HA), 2.59–2.69 (m, 1H, 20-HB), 3.78 (dd, J=11.2,

R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
1775
and washed with ethyl acetate. The aqueous layer from
filtrate was extracted with ethyl acetate (2ꢄ50 mL) and
the combined organic layer was concentrated under
reduced pressure. This KF treatment was carried out
once again to remove Bu3SnCl excess and then, the
organic layer was dried over Na2SO4, filtered and con-
centrated under reduced pressure. The crude product
was purified by silica gel column chromatography (hexane
then AcOEt/hexane gradient from 1/50 to 1/4) to give 6
(1.17 g, 55%) as a yellow syrup. Rf 0.45 (AcOEt/hexane
and concentrated under reduced pressure. The crude
product was purified by silica gel column chromato-
graphy (AcOEt/hexane 1/2 then 1/1) to give 8a (240 mg,
90%) as a yellow syrup. Rf 0.67 (AcOEt/hexane 1/1). 1H
NMR (300 MHz, CDCl3): d 0.09, 0.10 (s, 12H,
2ꢄSi(CH3)2), 0.30 (s, 9H, Si(CH3)3), 0.92 (s, 18H,
2ꢄSiC(CH3)3), 2.37–2.47 (m, 1H, 20-HA), 2.51–2.62 (m,
1H, 20-HB), 3.23 (bs, 3H, NCH3), 3.77 (dd, J=11.3, 3.4
Hz, 1H, 50-HA), 3.86 (dd, J=11.3, 3.8 Hz, 1H, 50-HB),
3.94–3.99 (m, 1H, 40-H), 4.58–4.65 (m, 1H, 30-H), 5.80
(bs, 1H, NH), 6.49 (t, J=6.4 Hz, 1H, 10-H), 8.11 (s, 1H,
8-H). 13C NMR (50 MHz, CDCl3): d ꢁ5.51, ꢁ5.38,
ꢁ4.82, ꢁ4.66 (2ꢄSi(CH3)2), ꢁ0.21 (Si(CH3)3), 18.0, 18.4
(2ꢄSiC), 25.8, 25.9 (2ꢄSiC(CH3)3), 41.6 (20-C), 62.8 (50-
C), 71.8 (30-C), 83.9 (10-C), 87.8 (40-C), 90.1, 103.9
(CꢃC-TMS), 119.7 (5-C), 139.1 (8-C), 146.0 (2-C),
155.1 (4-C). MS: m/z 590 (M+H)+, 612 (M+Na)+.
HRMS for C28H51N5O3Si3 (M+H)+ calcd: 590.3378,
found: 590.3379.
1
1/4). H NMR (300 MHz, CDCl3): d 0.10, 0.12 (s, 12H,
2ꢄSi(CH3)2), 0.92, 0.93 (s, 18H, 2ꢄSiC(CH3)3), 2.43–
2.52 (m, 1H, 20-HA), 2.59–2.68 (m, 1H, 20-HB), 3.79 (dd,
J=11.2, 3.1 Hz, 1H, 50-HA), 3.91 (dd, J=11.2, 3.8 Hz,
1H, 50-HB), 4.02–4.06 (m, 1H, 40-H), 4.61–4.67 (m, 1H,
30-H), 6.45 (t, J=6.2 Hz, 1H, 10-H), 8.40 (s, 1H, 8-H).
13C NMR (75 MHz, CDCl3): d ꢁ5.45, ꢁ5.37, ꢁ4.80,
ꢁ4.64 (2ꢄSi(CH3)2), 18.0, 18.4 (2ꢄSiC), 25.8, 26.0
(2ꢄSiC(CH3)3), 41.5 (20-C), 62.6 (50-C), 71.7 (30-C), 85.1
(10-C), 88.3 (40-C), 116.4 (2-C), 132.2 (5-C), 144.0 (8-C),
150.4, 151.8 (4-C+6-C). MS: m/z 625 (35Cl-M+H)+, 627
(37Cl-M+H)+, 647 (35Cl-M+Na)+, 649 (37Cl-M+Na)+.
4.1.6. N6-Methyl-2-(prop-1-ynyl)-30,50-bis-O-(tert-butyl-
dimethylsilyl)-20-deoxyadenosine (8b). Propyne (1 mL,
17 mmol) was condensed in a sealed tube at ꢁ78 ꢂC and
Et3N (8 mL) was added dropwise. (PPh3)2PdCl2 (39 mg,
0.055 mmol), CuI (10 mg, 0.055 mmol) and 7 (340 mg,
0.549 mmol) were added to this solution. The reaction
mixture was warmed to room temperature under stir-
ring for 3 h. The solvent was removed under reduced
pressure, the residue was taken up in ethyl acetate (50
mL) and successively washed with 10% aqueous
Na2EDTA (2ꢄ25 mL) and brine (25 mL). The organic
layer was dried over Na2SO4, filtered and concentrated
under reduced pressure. The crude product was purified
by silica gel column chromatography (AcOEt/hexane 1/
2 then 1/1) to give 8b (230 mg, 79%) as a yellow syrup.
Rf 0.34 (AcOEt/hexane 1/2). 1H NMR (300 MHz,
CDCl3): d 0.09, 0.10 (s, 12H, 2ꢄSi(CH3)2), 0.91, 0.93 (s,
18H, 2ꢄSiC(CH3)3), 2.10 (s, 3H, CꢃC–CH3), 2.38–2.47
(m, 1H, 20-HA), 2.49–2.58 (m, 1H, 20-HB), 3.22 (bs, 3H,
NCH3), 3.77 (dd, J=11.3, 2.8 Hz, 1H, 50-HA), 3.88 (dd,
J=11.3, 3.8 Hz, 1H, 50-HB), 3.96–4.02 (m, 1H, 40-H),
4.57–4.64 (m, 1H, 30-H), 5.78 (bs, 1H, NH), 6.50 (t,
J=6.4 Hz, 1H, 10-H), 8.11 (s, 1H, 8-H). 13C NMR
(50 MHz, CDCl3): d ꢁ5.53, ꢁ5.41, ꢁ4.84, ꢁ4.66
(2ꢄSi(CH3)2), 4.57 (CꢃC–CH3), 14.2 (NCH3), 18.0,
18.4 (2ꢄSiC), 25.8, 26.0 (2ꢄSiC(CH3)3), 41.7 (20-C),
62.8 (50-C), 71.8 (30-C), 84.0 (10-C), 87.8 (40-C), 138.8 (8-
C), 146.8 (2-C), 155.2 (4-C), 160.4 (6-C). MS: m/z 532
(M+H)+, 554 (M+Na)+. HRMS for C26H45N5O3Si2
(M+H)+ calcd: 532.3139, found: 532.3152.
4.1.4. 2-Iodo-N6-methyl-30,50-bis-O-(tert-butyldimethyl-
silyl)-20-deoxyadenosine (7). 2 M MeNH2 solution in
THF (3.0 mL, 6.00 mmol) was added to a solution of 6
(380 mg, 0.608 mmol) in EtOH (10 mL) and the result-
ing mixture was stirred at room temperature for 16 h.
The solvent was removed under reduced pressure, the
residue was dissolved in ethyl acetate (50 mL) and
washed with brine (25 mL). The organic layer was dried
over Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel
column chromatography (AcOEt/hexane 1/3 then 1/2)
to give 7 (316 mg, 84%) as a colorless syrup. Rf 0.39
(AcOEt/hexane 1/3). 1H NMR (300 MHz, CDCl3): d
0.09, 0.11 (s, 12H, 2ꢄSi(CH3)2), 0.91 (s, 18H,
2ꢄSiC(CH3)3), 2.33–2.43 (m, 1H, 20-HA), 2.61–2.70 (m,
1H, 20-HB), 3.15 (bs, 3H, NCH3), 3.76 (dd, J=11.2, 3.4
Hz, 1H, 50-HA), 3.87 (dd, J=11.2, 4.4 Hz, 1H, 50-HB),
3.95–4.00 (m, 1H, 40-H), 4.60–4.65 (m, 1H, 30-H), 5.94
(bs, 1H, NH), 6.34 (t, J=6,5 Hz, 1H, 10-H), 7.92 (s, 1H,
8-H). 13C NMR (75 MHz, CDCl3): d ꢁ5.47, ꢁ5.37,
ꢁ4.80, ꢁ4.66 (2ꢄSi(CH3)2), 18.0, 18.4 (2ꢄSiC), 25.8, 26.0
(2ꢄSiC(CH3)3), 40.9 (20-C), 62.8 (50-C), 71.9 (30-C), 84.5
(10-C), 87.9 (40-C), 120.1, 120.5 (2-C+5-C), 138.3 (8-C),
154.9 (4-C), 171.1 (6-C). MS: m/z 620 (M+H)+, 642
(M+Na)+. HRMS for C23H42IN5O3Si2 (M+H)+
calcd: 620.1949, found: 620.1971.
4.1.5. N6-methyl-2-(2-trimethylsilylethynyl)-30,50-bis-O-
(tert-butyldimethylsilyl)-20-deoxy adenosine (8a). Argon
was bubbled through a solution of 7 (280 mg, 0.452
mmol) in Et3N (6 mL) and then, (PPh3)2PdCl2 (32 mg,
0.045 mmol) and CuI (8.6 mg, 0.045 mmol) were added
to this solution. (Trimethylsilyl)acetylene (0,12 mL,
0.904 mmol, 2 equiv) was subsequently added dropwise
to the reaction mixture, which was then stirred under
argon at room temperature for 3 h. The solvent was
removed under reduced pressure, the residue was taken
upin ethyl acetate (50 mL) and successively washed
with 10% aqueous Na2EDTA (2ꢄ25 mL) and brine (25
mL). The organic layer was dried over Na2SO4, filtered
4.1.7. N6-Methyl-2-(2-phenylethynyl)-30,50-bis-O-(tert-butyl-
dimethylsilyl)-20-deoxyadenosine (8c). This compound
was prepared from 7 (430 mg, 0.694 mmol) and pheny-
lacetylene (0.11 mL, 1.04 mmol) by the procedure
described for the preparation of 8a. Purification by
silica gel column chromatography (AcOEt/hexane 1/2
then 1/1) afforded 8c (350 mg, 85%) as a yellow syrup.
Rf 0.44 (AcOEt/hexane 1/1). 1H NMR (200 MHz,
CDCl3): d 0.10, 0.11 (s, 12H, 2ꢄSi(CH3)2), 0.92 (s, 18H,
2ꢄSiC(CH3)3), 2.40–2.52 (m, 1H, 20-HA), 2.55–2.68 (m,
1H, 20-HB), 3.27 (bs, 3H, NCH3), 3.78 (dd, J=11.2, 3.2
Hz, 1H, 50-HA), 3.84 (dd, J=11.2, 3.9 Hz, 1H, 50-HB),

1776
R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
3.97–4.04 (m, 1H, 40-H), 4.58–4.66 (m, 1H, 30-H), 5.86
(bs, 1H, NH), 6.52 (t, J=6.5 Hz, 1H, 10-H), 7.32–7.41,
7.62–7.73 (m, 5H, Ph), 8.13 (s, 1H, 8-H). 13C NMR
(50 MHz, CDCl3): d ꢁ5.51, ꢁ5.39, ꢁ4.82, ꢁ4.66
(2ꢄSi(CH3)2), 14.1 (NCH3), 18.0, 18.4 (2ꢄSiC), 25.8,
25.9 (2ꢄSiC(CH3)3), 41.5 (20-C), 62.8 (50-C), 71.8 (30-
C), 84.1 (10-C), 87.8 (40-C), 122.2 (5-C), 128.2, 139.0,
131.9, 132.5 (Ph), 139.1 (8-C), 155.2 (4-C). MS: m/z 594
(M+H)+, 616 (M+Na)+. HRMS for C31H47N5O3Si2
(M+Na)+ calcd: 616.3115, found: 616.3108.
3.81–3.91 (m, 1H, 50-HA), 4.02–4.09 (m, 1H, 50-HB),
4.23–4.26 (m, 1H, 40-H), 4.80–4.85 (m, 1H, 30-H), 6.23–
6.32 (m, 1H, 10-H), 6.35 (bs, 3H, NH+2ꢄOH), 7.37–
7.42, 7.68–7.74 (m, 5H, Ph), 7.75 (s, 1H, 8-H). 13C
NMR (75 MHz, CDCl3): d 40.9 (20-C), 63.6 (50-C), 73.5
(30-C), 87.7 (10-C), 89.6 (40-C), 121.7 (5-C), 128.4, 129.4,
132.7 (Ph), 139.9 (8-C), 155.5 (4-C). MS: m/z 366
(M+H)+, 388 (M+Na)+. HRMS for C19H19N5O3
(M+H)+ calcd: 366.1566, found: 366.1556.
4.1.11. 2-Ethynyl-N6 -methyl-20 -deoxyadenosine-30,50-
bis(dibenzylphosphate) (10a). 1.0 M potassium tert-but-
oxide solution in THF (0.72 mL, 0.720 mmol) was
added dropwise to a stirred solution of 9a (95 mg, 0.328
mmol) in anhydrous THF (10 mL) at ꢁ40 ꢂC. After 5
min, tetrabenzyl pyrophosphate (390 mg, 0.720 mmol)
was added and the resulting mixture was stirred at
ꢁ40 ꢂC for 30 min. The reaction was quenched by add-
ing saturated aqueous NH4Cl (10 mL) and the mixture
was allowed to warm to room temperature. The mixture
was diluted with water (100 mL) and extracted with
ethyl acetate (2ꢄ100 mL). The combined organic layer
was dried over Na2SO4, filtered and concentrated under
reduced pressure. The crude product was purified by
silica gel column chromatography (AcOEt/CH2Cl2/
EtOH 4/5/1) to give 10a (200 mg, 75%) as a yellow
4.1.8. 2-Ethynyl-N6-methyl-20-deoxyadenosine (9a). 1 M
TBAF solution in THF (1.12 mL, 1.12 mmol) was
added to a solution of 8a (220 mg, 0.373 mmol) in
CH3CN (10 mL) and the resulting mixture was stirred
at room temperature for 1 h. The solvent was removed
under reduced pressure and the residue was purified by
silica gel column chromatography (CH2Cl2/MeOH 9/1)
to give 9a (100 mg, 92%) as a white solid which may be
recrystallized from EtOH and Et2O; mp83–84 ꢂC. Rf
1
0.39 (CHCl3/MeOH 9/1). H NMR (300 MHz, CDCl3):
d 2.26–2.36 (m, 1H, 20-HA), 3.04 (s, 1H, CꢃCH), 3.07–
3.18 (m, 1H, 20-HB), 3.21 (bs, 3H, NCH3), 3.77–3.88 (m,
1H, 50-HA), 3.98–4.07 (m, 1H, 50-HB), 4.20–4.25 (m, 1H,
40-H), 4.81–4.85 (m, 1H, 30-H), 5.99 (bs, 1H, NH), 6.18
(bs, 2H, 2ꢄOH), 6.31 (dd, J=5.4, 9.6 Hz, 1H, 10-H),
7.82 (s, 1H, 8-H). 13C NMR (50 MHz, CDCl3): d 40.8
(20-C), 63.6 (50-C), 73.5, 73.8 (30-C+CꢃCH), 87.7 (10-
C), 89.8 (40-C), 140.3 (8-C). MS: m/z 290 (M+H)+, 312
(M+Na)+. HRMS for C13H15N5O3 (M+H)+ calcd:
290.1253, found: 290.1247.
1
syrup. Rf 0.59 (AcOEt/CH2Cl2/EtOH 4/5/1). H NMR
(300 MHz, CDCl3): d 2.40–2.48 (m, 2H, 20-H), 2.95 (s,
1H, CꢃCH), 3.22 (bs, 3H, NCH3), 4.02–4.12 (m, 2H, 50-
H), 4.17–4.23 (m, 1H, 40-H), 5.00, 5.06 (d, 2ꢄ4H,
4ꢄCH2), 5.02–5.10 (m, 1H, 30-H), 5.88 (bs, 1H, NH),
6.35 (t, J=7.2 Hz, 1H, 10-H), 7.29, 7.31, 7.36 (s, 20H,
4ꢄPh ), 7.97 (s, 1H, 8-H). 13C NMR (50 MHz, CDCl3):
d 38.6 (20-C), 66.4 (50-C), 69.5, 69.6, 69.7, 69.9 (4ꢄCH2),
72.7 (30-C), 83.0, 83.7 (10-C+40-C), 128.0, 128.1, 128.2,
128.6, 128.7, 128.8 (4ꢄPh), 135.4 (CꢃCH), 138.7 (8-C),
145.5 (2-C), 155.2 (4-C). MS: m/z 810 (M+H)+, 832
(M+Na)+. HRMS for C41H41N5O9P2 (M+H)+ calcd:
810.2458, found: 810.2459.
4.1.9. N6-Methyl-2-(prop-1-ynyl)-20-deoxyadenosine (9b).
This compound was prepared from 8b (220 mg, 0.414
mmol) and 1 M TBAF solution in THF (0.83 mL, 0.830
mmol) by the procedure described for the preparation
of 9a. Purification by silica gel column chromatography
(CH2Cl2/MeOH 9/1) afforded 9b (125 mg, 99%) as a
white solid which may be recrystallized from EtOH and
Et2O; mp91–92 ꢂC. Rf 0.34 (CH2Cl2/MeOH 9/1). H
1
NMR (300 MHz, CDCl3): d 2.10 (s, 3H, CꢃC–CH3),
2.26–2.35 (m, 1H, 20-HA), 3.04–3.15 (m, 1H, 20-HB),
3.21 (bs, 3H, NCH3), 3.77–3.88 (m, 1H, 50-HA), 4.00
(dd, J=12.8, 1.3 Hz, 1H, 50-HB), 4.20–4.24 (m, 1H, 40-
H), 4.78–4.84 (m, 1H, 30-H), 5.92 (bs, 1H, NH), 6.23 (bs,
2H, 2ꢄOH), 6.31 (dd, J=9.4, 5.3 Hz, 1H, 10-H), 7.81 (s,
1H, 8-H). 13C NMR (75 MHz, CDCl3): d 4.35 (CH3),
13.5 (NCH3), 40.7 (20-C), 63.0 (50-C), 72.3 (30-C), 79.3,
83.6 (CꢃC), 86.7 (10-C), 89.1 (40-C), 120.0 (5-C), 139.6
(8-C), 146.3 (2-C), 155.2 (4-C), 171.2 (6-C). MS: m/z 304
(M+H)+, 326 (M+Na)+. HRMS for C14H17N5O3
(M+H)+ calcd: 304.1410, found: 304.1445.
4.1.12. N6-Methyl-2-(prop-1-ynyl)-20-deoxyadenosine-30,50-
bis(dibenzylphosphate) (10b). This compound was pre-
pared from 9b (60 mg, 0.198 mmol) by the procedure
described for the preparation of 10a. Purification by
silica gel column chromatography (AcOEt/CH2Cl2/
EtOH 4/5/1) afforded 10b (138 mg, 85%) as a colorless
1
syrup. Rf 0.57 (AcOEt/CH2Cl2/EtOH 4/5/1). H NMR
(300 MHz, CDCl3): d 2.05 (s, 3H, CꢃC–CH3), 2.34–2.47
(m, 2H, 20-H), 3.19 (bs, 3H, NCH3), 4.01–4.11 (m, 2H,
50-H), 4.16–4.20 (m, 1H, 40-H), 4.98, 5.06 (d, 2ꢄ4H,
4ꢄCH2), 5.03–5.08 (m, 1H, 30-H), 6.13 (bs, 1H, NH),
6.39 (dd, J=8.1, 5.9 Hz, 1H, 10-H), 7.27, 7.29, 7.34 (s,
20H, 4ꢄPh ), 7.95 (s, 1H, 8-H). 13C NMR (50 MHz,
CDCl3): d 4.60 (CH3), 39.6 (20-C), 66.4 (50-C), 69.5,
69.6, 69.7, 69.8 (4ꢄCH2), 72.7 (30-C), 82.8, 83.5 (CꢃC),
87.6 (10-C), 90.2 (40-C), 127.8, 127.9, 128.0, 128.4, 128.5
(4ꢄPh), 138.8 (8-C), 146.5 (2-C), 155.1 (4-C). MS: m/z
824 (M+H)+, 846 (M+Na)+. HRMS for C42H43N5O9P2
(M+H)+ calcd: 824.2614, found: 824.2592.
4.1.10. N6-Methyl-2-(2-phenylethynyl)-20-deoxyadenosine
(9c). This compound was prepared from 8c (350 mg,
0.589 mmol) and 1 M TBAF solution in THF (1.18 mL,
1.18 mmol) by the procedure described for the prepara-
tion of 9a. Purification by silica gel column chromato-
graphy (CH2Cl2/MeOH 9/1) yielded 9c (203 mg, 94%)
as a white solid which may be recrystallized from EtOH
ꢂ
and Et2O; mp126–128 C. Rf 0.41 (CH2Cl2/MeOH 9/1).
4.1.13. N6-Methyl-2-(2-phenylethynyl)-20-deoxyadenosine-
30,50-bis(dibenzylphosphate) (10c). This compound was
prepared from 9c (198 mg, 0.542 mmol) by the proce-
1H NMR (300 MHz, CDCl3): d 2.31–2.40 (m, 1H, 20-
HA), 3.01–3.13 (m, 1H, 20-HB), 3.28 (bs, 3H, NCH3),

R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
1777
dure described for the preparation of 10a. Purification
by silica gel column chromatography (AcOEt/CH2Cl2/
EtOH 4/5/1) yielded 10c (456 mg, 95%) as a colorless
(m, 1H, 30-H), 6.40 (t, 1H, 10-H), 7.23–7.35 (m, 5H, Ph),
7.41 (bs, 1H, NH), 8.30 (s, 1H, 8-H). 13C NMR
(75 MHz, DMSO-d6): d 34.1 (20-C), 41.5 (CH2), 49.3
(CH2), 67.6 (50-C), 76.2 (30-C), 83.4 (10-C), 83.7 (40-C),
127.7, 127.8, 128.2, 128.5 (Ph), 137.6 (8-C), 142.0 (2-C).
31P NMR (81 MHz, DMSO-d6): d ꢁ1.28 (30-OPO3H2),
ꢁ0.52 (50-OPO3H2). MS: m/z no (M+H)+, 254 (ade-
nine fragment). HPLC: RT=27.24 min.
1
syrup. Rf 0.69 (AcOEt/CH2Cl2/EtOH 4/5/1). H NMR
(300 MHz, CDCl3): d 2.42–2.49 (m, 2H, 20-H), 3.27 (bs,
3H, NCH3), 4.05–4.13 (m, 2H, 50-H), 4.18–4.25 (m, 1H,
40-H), 5.00, 5.06 (d, 2ꢄ4H, 4ꢄCH2), 5.04–5.10 (m, 1H,
30-H), 5.94 (bs, 1H, NH), 6.40 (t, J=7.2 Hz, 1H, 10-H),
7.28-7.39, 7.65–7.68 (m, 25H, 5ꢄPh), 70.98 (s, 1H, 8-H).
13C NMR (75 MHz, CDCl3): d 38.7 (2 -C), 66.4 (50-C),
69.5, 69.6, 69.8 (4ꢄCH2), 77.9 (30-C), 83.6 (10-C), 84.8
(40-C), 128.0, 128.1, 128.2, 128.6, 128.7, 128.8, 129.0
(5ꢄPh), 138.4 (8-C), 146.9 (2-C), 155.2 (4-C). MS: m/z 886
(M+H)+, 908 (M+Na)+. HRMS for C47H45N5O9P2
(M+Na)+ calcd: 908.2590, found: 908.2606.
4.1.17. 2-Ethynyl-N6-methyl-20-deoxyadenosine-30,50-bis-
(di-tert-butylphosphate) (11a). Di-tert-butyl N,N-die-
thylphosphoramidite (0.29 mL, 1.04 mmol) and tetra-
zole (145 mg, 2.07 mmol) were added to a solution of 9a
(100 mg, 0.346 mmol) in anhydrous THF (7 mL). After
the solution was stirred at room temperature for 2 h, the
reaction mixture was cooled to ꢁ78 ꢂC and MCPBA
(60–77%, 298 mg, 1.04 mmol) was rapidly added. The
reaction mixture was stirred at ꢁ78 ꢂC for 30 min and
warmed to room temperature for 1 h under stirring.
Excess of MCPBA was reduced by 10% aqueous
Na2SO3 (25 mL) and the mixture was extracted with
ethyl acetate (2ꢄ50 mL). The combined organic layer
was dried over Na2SO4, filtered and concentrated under
reduced pressure. The crude product was purified by
silica gel column chromatography (AcOEt/CH2Cl2/
EtOH 4/5/1) to give 11a (100 mg, 43%) as a colorless
4.1.14. 2-Ethyl-N6-methyl-20-deoxyadenosine-30,50-bispho-
sphate (1e). A mixture of 10a (180 mg, 0.222 mmol)
and 10% Pd/C (50 mg) in MeOH (100mL) was shaken
in a hydrogenation apparatus under 60 psi pressure at
room temperature for 24 h. The catalyst was removed
by filtration and washed with MeOH and water. The
filtrate was concentrated to dryness and the product was
purified by recrystallization from MeOH and Et2O to
furnish 1e (60 mg, 60%) as a colorless solid; decomp.
ꢂ
1
>130 C. H NMR (300 MHz, D2O): d 1.29 (t, J=7.2
Hz, 3H, CH3), 2.68–2.77 (m, 1H, 20-HA), 2.82–2.98 (m,
3H, 20-HB+CH2), 3.16 (bs, 3H, NCH3), 4.03–4.11 (m,
2H, 50-H), 4.37–4.44 (m, 1H, 40-H), 4.97–5.05 (m, 1H, 30-
H), 6.51 (t, J=5.9 Hz, 1H, 10-H), 8.41 (s, 1H, 8-H). 13C
NMR (75 MHz, DMSO-d6): d 13.1 (CH3), 31.5 (CH2),
32.3 (20-C), 67.8 (50-C), 76.2 (30-C), 83.2 (10-C), 83.8 (40-
C), 139.9 (8-C). 31P NMR (81 MHz, DMSO-d6): d-1.37
(30-OPO3H2), 0.20 (50-OPO3H2). MS: m/z no (M+H)+,
178 (adenine fragment). HPLC: RT=20.75 min.
1
syrup. Rf 0.54 (AcOEt/CH2Cl2/EtOH 4/5/1). H NMR
(200 MHz, CDCl3):
d 1.43, 1.45, 1.48 (s, 36H,
4ꢄC(CH3)3), 2.67–2.77 (m, 2H, 20-H), 2.95 (s, 1H,
CꢃCH), 3.18 (bs, 3H, NCH3), 4.11–4.19 (m, 2H, 50-H),
4.37–4.45 (m, 1H, 40-H), 5.02–5.09 (m, 1H, 30-H), 6.17
(bs, 1H, NH), 6.52 (t, J=7.1 Hz, 1H, 10-H), 8.13 (s, 1H,
8-H). 13C NMR (50 MHz, CDCl3): d 14.2 (NCH3), 29.8,
29.9 (C(CH3)3), 40.6 (20-C), 66.1 (50-C), 71.3 (30-C), 72.7,
77.3 (CꢃCH), 83.8 (10-C), 85.0 (40-C), 119.9 (5-C), 139.0
(8-C), 145.4 (2-C), 155.1 (4-C). MS: m/z 674 (M+H)+,
696 (M+Na)+. HRMS for C29H49N5O9P2 (M+H)+
calcd: 674.3084, found: 674.3071.
4.1.15. N6-methyl-2-propyl-20-deoxyadenosine-30,50-bis-
phosphate (1f). This compound was prepared from 10b
(125 mg, 0.152 mmol) by the procedure described for
the preparation of 1e. Recrystallization from MeOH
afforded 1f (70 mg, 99%) as a white solid; decomp.
>130 ꢂC. 1H NMR (200 MHz, DMSO-d6): d 0.94 (t,
J=7.1 Hz, 3H, CH3), 1.08–1.29 (m, 2H, CH2), 1.66–
1.87 (m, 2H, CH2), 2.61–2.80 (m, 2H, 20-H), 2.98 (bs,
3H, NCH3), 3.93–4.18 (m, 2H, 50-H), 4.25–4.36 (m, 1H,
40-H), 4.96-5.12 (m, 1H, 30-H), 6.38 (t, 1H, 10-H), 7.71
(bs, 1H, NH), 8.27 (s, 1H, 8-H). 13C NMR (75 MHz,
DMSO-d6): d 13.6 (CH3), 15.7 (CH2), 32.1 (CH2), 32.8
(20-C), 67.8 (50-C), 76.4 (30-C), 83.1 (10-C), 83.9 (40-C),
138.9 (8-C), 141.5 (2-C). 31P NMR (81 MHz, DMSO-
d6): d ꢁ1.55 (30-OPO3H2), ꢁ0.56 (50-OPO3H2). MS: m/z
no (M+H)+, 192 (adenine fragment). HPLC:
RT=23.13 min.
4.1.18. N6-Methyl-2-(prop-1-ynyl)-20-deoxyadenosine-30,50-
bis(di-tert-butylphosphate) (11b). This compound was
prepared from 9b (60 mg, 0.198 mmol) by the procedure
described for the preparation of 11a. Purification by
silica gel column chromatography (AcOEt/CH2Cl2/
EtOH 4/5/1) afforded 11b (97 mg, 71%) as a colorless
1
syrup. Rf 0.49 (AcOEt/CH2Cl2/EtOH 4/5/1). H NMR
(300 MHz, CDCl3):
d 1.48, 1.50, 1.52 (s, 36H,
4ꢄC(CH3)3), 2.10 (s, 3H, CꢃC–CH3), 2.66–2.75 (m, 2H,
20-H), 3.22 (bs, 3H, NCH3), 4.16–4.22 (m, 2H, 50-H),
4.41–4.47 (m, 1H, 40-H), 5.03–5.11 (m, 1H, 30-H), 5.80
(bs, 1H, NH), 6.59 (t, J=7.2 Hz, 1H, 10-H), 8.15 (s, 1H,
8-H). 13C NMR (75 MHz, CDCl3): d 4.57 (CH3), 29.8,
29.9, 30.0 (C(CH3)3), 39.6 (20-C), 66.3 (50-C), 83.2, 83.6
(CꢃC), 88.4 (10-C), 91.7 (40-C), 119.4 (5-C), 138.6 (8-C),
146.9 (2-C), 155.2 (4-C). MS: m/z 688 (M+H)+, 710
(M+Na)+. HRMS for C30H51N5O9P2 (M+H)+ calcd:
688.3240, found: 688.3243.
4.1.16. N6-Methyl-2-(2-phenylethyl)-20-deoxyadenosine-
30,50-bisphosphate (1g). This compound was prepared
from 10c (455 mg, 0.514 mmol) by the procedure
described for the preparation of 1e. Recrystallization
from MeOH yielded 1g (120 mg, 44%) as a white solid;
4.1.19. 2-Ethynyl-N6-methyl-20-deoxyadenosine-30,50-bis-
phosphate (1h). Trifluoroacetic acid (0.15 mL) was
added to a solution of 11a (100 mg, 0.148 mmol) in
CH2Cl2/MeOH (3 mL/1 mL) and the reaction mixture
was stirred at room temperature for 30 min. The solvent
decomp. >130 ꢂC. H NMR (300 MHz, DMSO-d6): d
1
2.93–3.02 (m, 2H, 20-H), 3.07 (bs, 3H, NCH3), 3.98–4.16
(m, 2H, 50-H), 4.26–4.34 (m, 1H, 40-H), 4.88 (t, J=6.8
Hz, 2H, CH2), 4.99 (t, J=6.8 Hz, 2H, CH2), 5.08–5.18

1778
R. Mathieu et al. / Bioorg. Med. Chem. 12 (2004) 1769–1779
was removed under reduced pressure and the residue
was triturated in EtOH. The precipitate was filtered
and recrystallized from MeOH and Et2O to give 1g
4.2.2. [Ca2+
]
i
Measurements. Fura-2/AM-loaded
human platelets were prepared as previously described10
and resuspended in Tyrode’s buffer with 2 mM CaCl2.
Aliquots of fura-2-loaded platelets were transferred to a
10ꢄ10 mm quartz cuvette maintained at 37 ꢂC and
fluorescence measurements were performed under con-
tinuous stirring, in a PTI Deltascan spectrofluorimeter
(Photon Technology International Inc., Princetown, NJ,
USA).10 The excitation wavelength was alternately fixed
at 340 or 380 nm, fluorescence emission was determined
at 510 nm and results were calculated as the fluores-
cence ratio (340/380) in arbitrary units.
(33 mg, 50%) as a white solid; decomp. >130 ꢂC. H
1
NMR (300 MHz, DMSO-d6): d 2.58–2.72 (m, 2H, 20-H),
2.96 (bs, 3H, NCH3), 3.27 (s, 1H, CꢃCH), 3.94–4.07
(m, 2H, 50-H), 4.25–4.36 (m, 1H, 40-H), 4.91–5.02
(m, 1H, 30-H), 6.38 (t, 1H, 10-H), 7.82 (bs, 1H, NH), 8.24
(s, 1H, 8-H). 13C NMR (75 MHz, DMSO-d6): d 28.1 (20-C),
66.2 (50-C), 75.9 (CꢃCH), 76.3 (30-C), 83.9 (10-C), 84.7
(40-C), 140.5 (8-C), 141.3 (2-C). 31P NMR (81 MHz,
DMSO-d6): d-0.49 (30-OPO3H2), ꢁ0.19 (50-OPO3H2).
MS: m/z no (M+H)+, 174 (adenine fragment). HPLC:
RT=14.12 min.
4.2.3. Measurement of adenylyl cyclase activity. A 450
mL aliquot of washed platelets resuspended in Tyrode’s
buffer containing 2 mM Ca2+ and 1 mM Mg2+ was
stirred at 1100 rpm in an aggregometer cuvette and the
following reagents were added at 30 s intervals: (i) 10
mM PGE1, (ii) 1 mM AR-C66096MX or different con-
centrations of compounds 1e–i, and (iii) 5 mM ADP or
vehicle (Tyrode’s buffer containing no Ca2+ or Mg2+).
The reaction was stopped 1 min later by addition of
50 mL of ice-cold 6.6 N perchloric acid. Perchloric acid
extracts were centrifuged at 11,000 g for 5 min to
eliminate protein precipitate, and cAMP was isolated
from the supernatants using a mixture of trioctylamine
and Freon (28/22, vol/vol). The upper aqueous phase
was lyophilized and the dry residue dissolved in the
buffer provided with the commercial radioimmunoassay
kit for cAMP measurement.10
4.1.20. N6-Methyl-2-(prop-1-ynyl)-20-deoxyadenosine-30,50-
bisphosphate (1i). This compound was prepared from
11b (89 mg, 0.129 mmol) by the procedure described for
the preparation of 1g. Recrystallization from MeOH
and Et2O afforded 1h (35 mg, 58%) as a white solid;
decomp. >130 ꢂC. H NMR (300 MHz, DMSO-d6): d
1
2.06 (s, 3H, CꢃC–CH3), 2.58–2.70 (m, 2H, 20-H), 2.95
(bs, 3H, NCH3), 3.96–4.17 (m, 2H, 50-H), 4.26–4.35 (m,
1H, 40-H), 4.93–5.15 (m, 1H, 30-H), 6.37 (t, J=7.1 Hz,
1H, 10-H), 7.86 (bs, 1H, NH), 8.21 (s, 1H, 8-H). 13C
NMR (75 MHz, DMSO-d6): d 4.82 (CH3), 30.9 (20-C),
67.1 (50-C), 76.1 (30-C), 81.2, 83.4 (CꢃC), 84.3 (10-C),
84.9 (40-C), 139.7 (8-C), 142.9 (2-C). 31P NMR
(81 MHz, DMSO-d6): d ꢁ0.62 (30-OPO3H2), ꢁ0.08 (50-
OPO3H2). MS: m/z no (M+H)+, 188 (adenine frag-
ment). HPLC: RT=16.30 min.
4.2.4. Binding studies. Competitive binding of [33P]2-
MeSADP (850 Ci/mmol) to washed platelets at 37 ꢂC
for 5 min was determined as described in earlier work.31
Displacement experiments were performed using a sin-
gle concentration of radiolabeled ligand [33P]2-
MeSADP (0.2 nM, 200.000 dpm), in the presence of
increasing concentrations of the appropriate unlabeled
ligand. The reactions were terminated by addition of ice
cold Tyrode’s buffer and rapid filtration through What-
man GF/C glass fiber filters under vacuum, after which
the tubes and filters were rinsed twice. Radioactivity
bound to the platelets on the filters was measured by
scintillation counting (Wallac 1409 b-counter, count
rate (DPM/CPMꢀS.E.M.): 1.070ꢀ0.0018; Turku, Fin-
land). The inhibition constants for the drugs (Ki) were
calculated using the GraphPad software package
(GraphPad, San Diego, CA).
4.2. Biological tests
2-Methylthioadenosine-50-diphosphate
(2-MeSADP),
adenosine-50-diphosphate (ADP), prostaglandin E1
(PGE1) were from Sigma (Saint Quentin-Fallavier,
France). Human fibrinogen was from Kabi (Stockholm,
Sweden), fura-2/acetoxymethyl ester (fura-2/AM) from
Calbiochem (Meudon, France) and the cyclic adenosine-
30,50-monophosphate (cAMP) assay kit from Amersham
(Les Ulis, France). Apyrase was purified from potatoes
as previously described.32 [33P]2-MeSADP was provided
by Du Pont NEN (Le Blanc Mesnil, France). AR-
C66096MX was a generous gift from AstraZeneca
(Charnwood, UK).
4.2.1. Washed human platelet aggregation. Washed
human platelets were prepared as previously described32
and resuspended at 3ꢄ105 platelets/mL in Tyrode’s
buffer containing 2 mM CaCl2, in the presence of 0.02
U/mL of the ADP scavenger apyrase (adenosine-50-
triphosphate diphosphorylase, EC 3.6.1.5), a concen-
tration sufficient to prevent the desensitization of plate-
let ADP receptors during storage.33 Platelets were kept
at 37 ꢂC throughout all experiments and aggregation
was measured by standard methods.10,32 Briefly, a 450
mL aliquot of platelet suspension was stirred at 1100
rpm and activated by addition of agonists and for
human platelets of human fibrinogen (0.8 mg/mL), in a
final volume of 500 mL. The extent of aggregation was
estimated quantitatively by measuring the maximum
curve height above the baseline.
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