A general approach toward the synthesis of C-nucleoside pyrazolo[1,5-a]-1,3,5-triazines and their 3',5'-bisphosphate C-nucleotide analogues as the first reported in vivo stable P2Y(1)-receptor antagonists.

Article abstract of DOI:10.1021/jo026268l

In our effort to identify potent purinergic P2Y(1) receptor antagonists as potent platelet aggregation inhibitors with enhanced metabolic stability, we developed an efficient route for the large-scale preparation of 2'-deoxy-C-nucleosides of pyrazolo[1,5-a]-1,3,5-triazine. The key strategic elements of this novel synthetic approach involved the following: (i) the use of a novel activating group, the N-methyl-N-phenylamino group, which was easily generated in high yield by treatment of the pyrazolo[1,5-a]-1,3,5-triazin-4-one (5) with phosphorus oxychloride and dimethylaniline under high pressure, (ii) a regio- and stereospecific palladium-mediated coupling reaction of the readily available unprotected glycal 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b) and the 8-iodo derivative (16), and (iii) the stereoselective reduction of the ketone group of the furanosyl ring followed by the subsequent displacement of the N-methyl-N-phenylamino group upon treatment with methylamine. The beta configuration at the anomeric C-1' position of the glycal moieties was perfectly retained throughout this conversion. This procedure afforded 8-(2'-deoxy-beta-D-ribofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (21) and 8-(2'-deoxy-beta-D-xylofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-triazine (24) with an overall yield of 50% and 39%, respectively. Finally, the conversion of nucleosides 21 and 24 to the pyrazolotriazine C-nucleotides 3',5'-bisphosphate 2 and 3',5'-cyclophosphate 26 is also described herein and represents the first reported nucleotide derivatives within the pyrazolo[1,5-a]-1,3,5-triazine series. Preliminary biological testing has shown that compound 2 strongly inhibits ADP-induced human platelet aggregation and shape change and possesses significant efficacies 30 min after injection in rat, highlighting a strong P2Y(1)-receptor antagonist activity in vitro combined with a prolonged duration of action in vivo.

Full text of DOI:10.1021/jo026268l

A Gen er a l Ap p r oa ch tow a r d th e Syn th esis of C-Nu cleosid e
P yr a zolo[1,5-a ]-1,3,5-tr ia zin es a n d Th eir 3′,5′-Bisp h osp h a te
C-Nu cleotid e An a logu es a s th e F ir st Rep or ted in Vivo Sta ble
P 2Y1-Recep tor An ta gon ists
Pierre Raboisson,*,† Anthony Baurand,‡ J ean-Pierre Cazenave,‡ Christian Gachet,‡
Dominique Schultz,† Bernard Spiess,† and J ean-J acques Bourguignon†
Laboratoire de Pharmacochimie de la Communication Cellulaire, UMR 7081 du CNRS, Universite´ Louis
Pasteur, Faculte´ de Pharmacie, 74, route du Rhin, 67401 Illkirch Cedex, France, and Laboratoire de
Biologie et de Pharmacologie de l’He´mostase et de la Thrombose, Etablissement Franc¸ais du Sang-Alsace,
U.311 de l’INSERM, 10, rue Spielmann, BP 36, 67065 Strasbourg Cedex, France
pierreraboisson@aol.com
Received J uly 31, 2002
In our effort to identify potent purinergic P2Y1 receptor antagonists as potent platelet aggregation
inhibitors with enhanced metabolic stability, we developed an efficient route for the large-scale
preparation of 2′-deoxy-C-nucleosides of pyrazolo[1,5-a]-1,3,5-triazine. The key strategic elements
of this novel synthetic approach involved the following: (i) the use of a novel activating group, the
N-methyl-N-phenylamino group, which was easily generated in high yield by treatment of the
pyrazolo[1,5-a]-1,3,5-triazin-4-one (5) with phosphorus oxychloride and dimethylaniline under high
pressure, (ii) a regio- and stereospecific palladium-mediated coupling reaction of the readily available
unprotected glycal 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b) and the 8-iodo derivative (16),
and (iii) the stereoselective reduction of the ketone group of the furanosyl ring followed by the
subsequent displacement of the N-methyl-N-phenylamino group upon treatment with methylamine.
The â configuration at the anomeric C-1′ position of the glycal moieties was perfectly retained
throughout this conversion. This procedure afforded 8-(2′-deoxy-â-D-ribofuranosyl)-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (21) and 8-(2′-deoxy-â-D-xylofuranosyl)-2-methyl-4-(N-
methylamino)pyrazolo[1,5-a]-1,3,5-triazine (24) with an overall yield of 50% and 39%, respec-
tively. Finally, the conversion of nucleosides 21 and 24 to the pyrazolotriazine C-nucleotides
3′,5′-bisphosphate 2 and 3′,5′-cyclophosphate 26 is also described herein and represents the first
reported nucleotide derivatives within the pyrazolo[1,5-a]-1,3,5-triazine series. Preliminary biological
testing has shown that compound 2 strongly inhibits ADP-induced human platelet aggregation
and shape change and possesses significant efficacies 30 min after injection in rat, highlighting a
strong P2Y1-receptor antagonist activity in vitro combined with a prolonged duration of action in
vivo.
In tr od u ction
sented a very attractive challenge.2-8 In recent years a
number of modified 2′-deoxynucleoside analogues based
on structural modifications of the sugar have been
described.9-11 However, until now, no proof of better in
In our research program studying the synthesis and
biological evaluation of novel 2′-deoxyadenosine-3′,5′-
bisphosphates as P2Y1-receptor antagonists, we recently
reported the synthesis of 2,N6-dimethyl-2′-deoxyadeno-
sine-3′,5′-bisphosphate (1b, Figure 1), a potent and selec-
tive P2Y1-receptor antagonist.1 Preliminary in vivo test-
ing has shown that this compound possesses a very brief
activity as an anti-aggregating agent. Thus, the synthesis
of the corresponding isosteric compound 2 (Figure 1)
possessing significantly better in vivo activities repre-
(2) Ravi, R. G.; Kim, H. S.; Servos, J .; Zimmermann, H.; Lee, K.;
Maddileti, S.; Boyer, J . L.; Harden, T. K.; J acobson, K. A. J . Med. Chem.
2002, 45, 2090-2100.
(3) Gachet, C. Thromb. Haemost. 2001, 86, 222-232.
(4) Hechler, B.; Leon, C.; Vial, C.; Vigne, P.; Frelin, C.; Cazenave,
J . P.; Gachet, C. Blood 1998, 92, 152-159.
(5) Le´on, C.; Hechler, B.; Freund, M.; Eckly, A.; Vial, C.; Ohlmann,
P.; Dierich, A.; LeMeur, M.; Cazenave, J .-P.; Gachet, C. J . Clin. Invest.
1994, 104, 1731-1737.
(6) Fabre, J .-E.; Nguyen, M.; Latour, A.; Keifer, J . A.; Audoly, L.
P.; Coffman, T. M.; Koller B. H. Nat. Med. 1999, 5, 1199-1202.
(7) Le´on, C.; Freund, M.; Ravanat, C.; Baurand, A.; Cazenave, J .-
P.; Gachet, C. Circulation 2001, 103, 718-723.
* Author to whom correspondence should be addressed. Present
address: Department of Chemistry, 3-Dimensional Pharmaceuticals,
Inc., 665 Stockton Drive, Exton, Pennsylvania, 19341. E-mail:
PierreRaboisson@aol.com.
(8) Baurand, A.; Raboisson, P.; Freund, M.; Le´on, C.; Cazenave, J .
P.; Bourguignon, J . J .; Gachet, C. Eur. J . Pharmacol. 2001, 412, 213-
221.
† Laboratoire de Pharmacochimie de la Communication Cellulaire.
‡ Laboratoire de Biologie et de Pharmacologie de l’He´mostase et de
la Thrombose.
(9) Nandanan, E.; J ang, S.-Y.; Moro, S.; Kim, H. O.; Siddiqui, M.
A.; Russ, P.; Marquez, V. E.; Busson, R.; Herdewijn, P.; Harden, T.
K.; Boyer, J . L.; J acobson, K. A. J . Med. Chem. 2000, 43, 829-842.
(1) Raboisson, P.; Baurand, A.; Cazenave, J .-P.; Gachet, C.; Retat,
M.; Spiess, B.; Bourguignon, J .-J . J . Med. Chem. 2002, 45, 962-972.
10.1021/jo026268l CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/16/2002
J . Org. Chem. 2002, 67, 8063-8071
8063

Raboisson et al.
F IGURE 1.
vivo activities was reported. Thus, C-nucleotide ana-
logues have become a focus of intense investigation with
the rationale that replacing the glycosidic carbon-ni-
trogen linkage by the carbon-carbon bond would in-
crease the metabolic stability of these derivatives toward
nucleosidase enzymes that cleave the link between the
base and the sugar moiety.12 To investigate the scope of
such an approach, we have now extended our investiga-
tion of the pyrazolo[1,5-a]-1,3,5-triazine-3′,5′-bisphos-
phate C-nucleotides and we report herein the details of
the synthetic studies which have successfully led to the
large-scale preparation of the 8-(2′-deoxy-â-D-ribofura-
nosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-a]-1,3,5-
triazine-3′,5′-bisphosphate (2). Subsequently, the P2Y1
receptor antagonist activities were also evaluated in vitro
and in vivo.
Two main synthetic approaches leading to pyrazolo-
[1,5-a]-1,3,5-triazine C-nucleosides were described in the
literature.13-17 The first started from a substituted pyra-
zole nucleus followed by triazine ring construction.13-15
More recently, Zhang and co-workers reported an inter-
esting two-step synthesis of the 8-(2′-deoxy-â-D-glycero-
pentofuran-3′-ulos-1′-yl)-4-(N-isobutyloxycarbonyl)ami-
nopyrazolo[1,5-a]-1,3,5-triazine 3 (Figure 1), which is
based upon a regio- and stereospecific Pd-mediated cross-
coupling reaction (PMCCR) of a 3-substituted tert-butyl-
diphenylsilyl glycal 4a 16 (Figure 1) with a protected
8-iodo-N4-diisobutyloxycarbonylamino derivative.17 It was
well-accepted that in this PMCCR of the furanoid glycal
4, the substituted hydroxyl groups exert a directing effect
on the attack of the organopalladium reagent on the enol
ether double bond and, thereby, determine the stereo-
chemistry of the resulting C-nucleoside product.18,19 When
a monoprotected glycal was used, PMCCR occurred from
the opposite face of the glycal ring that is substituted,
yielding a single C-nucleosidic product.17-19 However,
although the unique case of the PMCCR described in the
literature with the unprotected furanoide 4b21 and
mercuric acetate derivative had led to a poor R/â-C-
glycosides selectivity (a 3/4 ratio was encountered),18 no
other example with different aglycon and/or different
reaction conditions was previously reported.
Nevertheless, neither of these routes are readily ame-
nable to synthesis of N4-substituted derivatives because
the exclusively accessible 4-substituent obtained after
blocking and unblocking strategy would be the NH2
group.17 Additional steps would be necessary to introduce
other substituents in this position, resulting in a long
synthetic route, loss in yields, and a potential scrambing
of the anomeric center of the carbohydrate moiety.20
Moreover, the preparation of the ribosyl starting material
4a 16 is not conveniently accessible, and was obtained
through a 4-step synthesis and required a final depro-
tection step at the end of the reaction pathway.16,17 These
disadvantages limited the scope of this procedure in
C-nucleoside synthesis, in particular for their use in
parallel synthesis and multigram scale reactions.
With the aim of expanding the usefulness of this
approach, we decided to explore the possibility of intro-
ducing an activating group at position 4 that is stable
under the PMCCR and which could be displaced by
various amines at the final step of the synthesis. To avoid
the use of the hardly accessible glycal 4a ,16 we sought to
use the unsubstituted furanoside 1,4-anhydro-2-deoxy-
D-erythro-pent-1-enitol 4b,21 which is readily prepared in
2 steps from the cheap and commercially available
thymidine,22 as the starting material in the synthesis of
C-nucleoside pyrazolo[1,5-a]-1,3,5-triazine derivatives.
Resu lts a n d Discu ssion
1. Optim ized 4-Activatin g Gr ou p. The 8-iodo-4-meth-
oxy-2-methylpyrazolo[1,5-a]-1,3,5-triazine (9b, Scheme 1)
represented an interesting candidate for the explora-
tion of the coupling reaction with the unsubstituted
furanoid 4b,21 because of the potential to readily displace
(10) Kim, Y.-C.; Gallo-Rodriguez, C.; J ang, S.-Y.; Nandanan, E.;
Adams, M.; Harden T. K.; Boyer, J . L.; J acobson, K. A. J . Med. Chem.
2000, 43, 746-755.
(11) Kim, H. S.; Ravi, R. G.; Marquez, V. E.; Maddileti, S.; Wihlborg,
A.-K.; Erlinge, D.; Malmsjo¨, M.; Boyer, J . L.; Harden, T. K.; J acobson,
K. A. J . Med. Chem. 2002, 45, 208-218.
(12) Abusamhadneh, E.; McDonald, N. E.; Kline P. C. Plant Sci.
2000, 153, 25-32.
(13) Tam, S. Y.-K.; Hwang, J . S.; De Las Heras, F. G.; Klein, R. S.;
Fox, J . J . J . Heterocycl. Chem. 1976, 13, 1305-1308.
(14) Chu, C. K.; Watanabe, K. A.; Fox, J . J . J . Heterocycl. Chem.
1980, 17, 1435-1439.
(18) Hacksell, U.; Daves, G. D., J r. J . Org. Chem. 1983, 48, 2870-
2876.
(19) Cheng, J . C.-Y.; Hacksell, U.; Daves, G. D., J r. J . Org. Chem.
1986, 51, 3093-3098.
(15) Tam, S. Y.-K.; Klein, R. S.; Wempen, I.; Fox, J . J . J . Org. Chem.
1979, 44, 4547-4553.
(20) Zhang, H.-C.; Daves, G. D., J r. J . Org. Chem. 1992, 57, 4690-
4696.
(16) Farr, R. N.; Daves, G. D., J r. J . Carbohydr. Res. 1990, 9, 653-
(21) Cheng, J . C.-Y.; Hacksell, U.; Daves, G. D., J r. J . Org. Chem.
1985, 50, 2778-2780.
(22) Cameron, M. A.; Cush, S. B.; Hammer, R. P. J . Org. Chem.
1997, 62, 9065-9069.
660.
(17) Zhang, H.-C.; Brakta, M.; Daves, G. D., J r. Nucleosides Nucle-
otides 1995, 14, 105-116.
8064 J . Org. Chem., Vol. 67, No. 23, 2002

Synthesis of C-Nucleoside Pyrazolo[1,5-a]-1,3,5-triazines
SCHEME 1. Activa tion of 2-Meth ylp yr a zolo[1,5-a ]-1,3,5-tr ia zin -4-on e 5 w ith N,N-Dim eth yla n ilin e in
Reflu xin g P h osp h or u s Oxych lor id ea
a
Key: (a) POCl3, N,N-dimethylaniline, 87%; (b) MeOH, 54%; (c) NIS, 68%.
SCHEME 2
the methoxy group at the end of the reaction sequence.
Attempts to drive this reaction using a previously de-
scribed experimental procedure17 were not successful and
led to a complex mixture of unseparable and unidentified
products. However, LC-MS analysis showed a signal at
374, highlighting the susceptibility of the methoxy com-
pound 9b (Scheme 1) to a displacement by the free
hydroxyl groups of glycal 4b (Figure 1). Therefore, it
was of great interest to develop a new and efficient ac-
tivating group for the nucleophilic replacement of C-
nucleoside that would offer the necessary stability under
the PMCCR of those previously reported.
The reaction of 2-methylpyrazolo[1,5-a]-1,3,5-triazin-
4-one (5, Scheme 1)23 with N,N-dimethylaniline in re-
fluxing phosphorus oxychloride afforded the desired
4-chloro derivative 6 as the major product in 87% yield,
together with traces of the two byproducts 7 and 8 which
were obtained in small amounts (<5%, Scheme 1). These
two unexpected products were purified and analyzed and
their structures were unambiguously assigned by el-
emental analysis and mass and NMR spectroscopy. The
1H NMR spectra of the first byproduct exhibited an NCH3
signal at δ 3.76 ppm and a phenyl group at δ 7.17-7.44
ppm. The mass spectrum and the microanalysis data
were in perfect accordance with the structure of 7.
Moreover, the structure of compound 7 was identical in
all respects with the product prepared through the
reaction of iminochloride 6 and N-methylaniline.
elimination sequence of the dimethylaniline as depicted
in Scheme 2. The first step likely involves formation of a
dimethylphenylammonium intermediate (10, Scheme 2),
generated by the N-attack of the dimethylaniline followed
by a demethylation and a subsequent rearomatization
of the triazine ring, leading to the thermodynamically
more stable N-methyl-N-phenylamino adduct 7. A distant
analogy to the second step of the proposed mechanism is
the reported heteroatom-demethylation with trimethyl-
silyl chloride.24 Similar N-methyl-N-phenylamino adducts
have been previously observed in the literature.25,26
The other side product obtained during the preparation
of the iminochloride 6 exhibited an N(CH3)2 signal at δ
3.16 ppm and an AB system at δ 7.87 ppm (∆δ ) 1.37,
J AB ) 9.5 Hz). The mass as well as the microanalysis data
supported the structure of the 4-(N,N-dimethylamino)-
phenyl derivative 8 (Schemes 1 and 3). This product is
not stable under strong aqueous acidic conditions and led
to two identified hydrolysis products: the 3-aminopyra-
zole 14 and the N-acetyl-4-(N,N-dimethylamino)benz-
amide (15, Scheme 3). We suggest that this addition of
dimethylaniline under the iminochloride 6 leading to a
C-C bond formation proceeds as shown in Scheme 3,
through an aromatic nucleophilic substitution. Interest-
ingly, this example is the first reported C-C coupling
reaction between a halogenated aromatic ring and di-
methylaniline.
To support the two proposed mechanisms depicted in
Schemes 2 and 3 involving a nucleophilic attack of the
We postulate that the reaction leading to 7 occurs
through a thermal promoted nucleophilic addition/
(24) Olah, G. A.; Narang, S. C.; Balaram Gupta, B. G.; Malhorta,
R. J . Org. Chem. 1979, 44, 1247-1251.
(25) Robins, R. K. J . Am. Chem. Soc. 1956, 78, 784-790.
(26) Hull, R. J . Chem. Soc. 1959, 481-484.
(23) Robins, R. K.; Revankar, G. R.; O’Brien, D. E.; Springer, R. H.;
Novinson, T.; Albert, A.; Senga, K.; Miller, J . P.; Streeter, D. G. J .
Heterocycl. Chem. 1985, 22, 601-634.
J . Org. Chem, Vol. 67, No. 23, 2002 8065

Raboisson et al.
SCHEME 3
TABLE 1. Syn th esis of Com p ou n d s 7 a n d 8 u n d er
Differ en t Exp er im en ta l Con d ition s
SCHEME 4. Nu cleop h ilic Disp la cem en t of th e
N-Meth yl-N-p h en yla m in o Gr ou p by Va r iou s
Am in esa
yield (%)b
conditionsa
7
8
a
b
c
d
<5
10
35
92
<5
7
21
<5
a
Conditions: (a) 5, POCl3, N,N-dimethylaniline, reflux, 4 h; (b)
6, N,N-dimethylaniline, CHCl3, reflux, 24 h; (c) 5, POCl3, N,N-
dimethylaniline, sealed tube, 125 °C, 4 h; (d) (1) 5, POCl3, N,N-
dimethylaniline, reflux, 2 h, (2) N-methylaniline, triethylamine,
b
CH2Cl2, 0 °C to room temperature. Yields of isolated products.
dimethylaniline on the 4-chloro derivative 6, a pure
sample of 6 was heated under refluxing chloroform with
freshly distilled dimethylaniline. Under these condi-
tions, the chloro derivative 6 slowly disappears and un-
dergoes a coupling reaction with the excess of dimeth-
ylaniline present leading to byproducts 7 and 8 (condi-
tions b, Table 1).
a
Key: (a) NIS, 87%; (b) HNR6R6′, 53-98%.
TABLE 2. Disp la cem en t of th e 4-(N-Meth yl-N-p h en yl-
a m in o) Gr ou p w ith Va r iou s Am in esa
compd
R6
R6′
R8
yield (%)b
17a
17b
17c
17d
CH3
H
Bn
H
H
H
H
I
I
78
53
98
92
However, since dimethylaniline is a very weak nucleo-
phile, an increase in the temperature and pressure of the
reaction was necessary to accelerate the rate of the
reaction. Thus, as shown in Table 1, when 2-methylpyra-
zolo[1,5-a]-1,3,5-triazin-4-one (5)23 was treated with an
excess of N,N-dimethylaniline and phosphorus oxychlo-
ride in a sealed tube at 125 °C over 4 h (conditions c),
the starting material was completely consumed and the
two stable target dimethylaniline adducts 7 and 8 were
obtained respectively in 35% and 21% yields. Finally, the
N-methyl-N-phenylamino derivative was obtained in
excellent yield (92%) when the quite unstable iminochlo-
ride 6 was directly treated in situ without purification
with N-methylaniline at 0 °C (conditions d). Moreover,
the pKa of compound 7 (close to 3.0) and its high stability
in the presence of water or refluxing ethanol make the
N-methyl-N-phenylamino group a suitable candidate for
nucleophilic addition-elimination displacements. To our
satisfaction, treatment of 7 with methylamine as a typical
nucleophile amine led to the desired N-methylamino
derivative 17a (Scheme 4, Table 2) in 78% isolated
yield. Although this sequence required extended reac-
tion times at room temperature presenting a draw-
back for an expeditious preparation of 4-substited deriva-
-(CH2)4-
I
a
b
Conditions: R6R6′NH, EtOH, 100 °C, sealed tube, 1 h. Yields
of isolated products.
tives (after 2 days, only 10% of the desired product was
obtained), the reaction times could be shortened to 1 h
by running the reaction in a sealed tube. As shown in
Table 2, the facile displacement of the N-methyl-N-
phenylamino group with various amines should be useful
as it opens up access to a wide range of N-4 substituted
compounds.
2. Ap p lica tion to C-Nu cleosid e P r ep a r a tion . Hav-
ing demonstrated the viability of this approach employing
the N-methyl-N-phenylamino group as an activating
group with derivatives 17a -d (Table 2), its application
to the preparation of C-nucleosides was also assessed.
Initial studies demonstrated that the direction of attack
by the organopalladium reagent on the furanoid enol
ether double bond is highly dependent on the relative
steric bulks of the C3 and C4 substituents of glycals 4.18,19
Unexpectedly, the palladium-mediated coupling of the
8-iodo derivative 16 and the readily available unprotected
glycal 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b),21
8066 J . Org. Chem., Vol. 67, No. 23, 2002

Synthesis of C-Nucleoside Pyrazolo[1,5-a]-1,3,5-triazines
SCHEME 5a
a
Key: (a) 4b, bis(dibenzylideneacetone)Pd(0), Ph3As, TEA, 75%; (b) sodium triacetoxyborohydride, 92%; (c) NH2CH3, 67-73%; (d)
t-BuOK, TBPP, 63-66%; (e) H2, Pd/C, 91-92%; (f) K-selectride, 78%.
SCHEME 6. P r op osed Mech a n ism for th e P MCCR
using the conditions depicted in the literature,17 led
exclusively to an adduct in 75% isolated yield that could
be assigned as the â-C-nucleoside 19 (Scheme 5). The
coupling reaction was accompanied by aglycon deiodina-
tion leading to 7 (15%). Convincing evidence for the
â-anomeric configuration assigned to nucleoside 19 was
face bearing the 3-hydroxyl group. Therefore, in the
opposite of the example reported in the literature using
less bulky ligands,19 the PMCCR selectively proceeds via
a syn-addition on the sterically most open face of 4b (the
face R) leading to adduct 28, which subsequently under-
goes a syn-elimination of the hydridopalladium (Scheme
6). Finally, a tautomerism equilibrum would afford the
single target compound 19.
Stereospecific reductions of the 3′-keto group of inter-
mediate 19 with sodium triacetoxyborohydride16,27 yielded
the 2′-deoxyribofuranosyl C-nucleoside 20 in 92% yield
while K-selectride permitted reduction by the less steri-
cally hindered face of the glycal ring16 leading to 23 (78%
isolated yield). Finally, the N-methyl-N-phenylamino
group was easily displaced with methylamine, with
retention of stereochemistry of the 2′-deoxyfuranosyl
moiety. Thus, an efficient preparation of the C-nucleoside
analogues 21 and 24 (Scheme 5) was accomplished.
3. C-Nu cleotid e P r ep a r a tion . Phosphorylation of 21
with tetrabenzylpyrophosphate (TBPP),28,29 followed by
1
obtained from the comparison of their H NMR coupling
constants J 1′2′a, J 1′2′b (6.6 and 10.6 Hz, respectively), which
are very close to those of the known â- nucleosides.17
Moreover, the 1H spectrum exhibited signals for the
anomeric hydrogen (H-1′) at δ 5.39 ppm assigned to the
â anomer.20 To the best of our knowledge, this is the only
instance in which a product resulting from organopalla-
dium attack on a single face of unsubstituted glycal 4b
has been isolated. However, the conditions used in the
present study were different from those reported in the
literature, employing glycal 4b, mercuric acetate salts,
and palladium II.19 Thus, we postulated that the more
bulky Palladium ligands (triphenylarsine), which were
used in the present study, make the organopalladium
reagent 27 (Scheme 6), which was obtained via an initial
transmetalation, a very hindered complex that is prob-
ably not able to attack the olefinic glycal 4b through the
(27) Gribble, G. W.; Ferguson, D. C. J . Chem. Soc., Chem. Commun.
1975, 535-536.
J . Org. Chem, Vol. 67, No. 23, 2002 8067

Raboisson et al.
catalytic hydrogenolysis, afforded the expected 2-methyl-
C-nucleotide derivative 2 in 91% yield (Scheme 5).
Attempts to apply this same methodology to the prepara-
tion of the xylofuranosyl derivative failed and the cyclic
compound 25 was obtained as the major product, high-
lighting the proximity of the two hydroxy groups in the
cis configuration which facilitate the cyclization. As such,
hydrogenolysis of compound 25 led to the corresponding
cyclic phosphate derivative 26 in 92% yield.
4. Biologica l Eva lu a tion . The new derivatives 2
(Figure 1) and 26 (Scheme 5) prepared in the present
study were first tested as antagonists in a platelet
aggregation assay as described earlier.1 Even though 2
exhibited strong antiaggregating properties (pA2 ) 6.5
( 0.3) identical with those of the P2Y1 receptor reference
antagonist MRS-2179 (pA2 ) 6.55 ( 0.05),29 the cyclic
derivative 26 was found to be inactive. As seen with
compounds 1 (Figure 1) no agonist activity was observed.1
Moreover, the intracellular Ca2+ rise induced in washed
human platelets by 5 µM ADP could be totally inhibited
by 100 µM 2, in the presence or absence of 2 mM external
Ca2+. Finally, studies in rat showed that ADP-induced
platelet aggregation was still inhibited 30 min after
injection (38% inhibition of aggregation to ADP 1 µM),
revealing that the in vitro potency of compound 2 was
sustained in vivo.
onstrated. Preliminary biological evaluations have also
revealed that compound 2 inhibits ADP-induced platelet
aggregation (pA2 ) 6.5 ( 0.3) and shape change in vitro
and possesses significant efficacies 30 min after injection
in rat. Thus, compound 2 appears to be the most stable
and one of the more potent P2Y1 receptor antagonists
known to date and represents a very promising pharma-
cological tool to obtain a better understanding of physiol-
ogy and physiopathology involving ADP-promoted ag-
gregation through the P2Y1 receptor pathway.
Exp er im en ta l Section
Ch em ica l Syn th esis. With the exception of THF and Et2O,
all solvents were obtained from commercial suppliers and used
without further purification. These two solvents were freshly
distilled from sodium benzophenone ketyl. Flash chromatog-
raphy was performed on Si 60 (40-63 µm) silica gel. Thin-
layer chromatography was carried out using Silica gel 60 F254
plates. The spots were visualized either under UV light (λ )
254 nm) or by spraying with molybdate reagent (H2O/
concentrated H2SO4/(NH4)6Mo7O24‚4H2O/(NH4)2‚Ce(SO4)4‚2H2O,
90/10/25/1, v/v/w/w) and charring at 140 °C for a few minutes.
All chemical yields are unoptimized and generally represent
the result of a single experiment. 1H NMR were recorded on a
200- or 300-MHz spectrophotometer at room temperature.
Chemical shifts are given in ppm (δ), coupling constants (J )
are in hertz (Hz), and signals are designated as follows: s,
singlet; d, doublet; t, triplet; q, quadruplet; quint, quintuplet;
m, multiplet; br s, broad singlet. Melting points are uncor-
rected. Elemental analyses were performed by the CNRS
department of microanalysis (CNRS, Vernaison, France) and
are indicated only by the elemental symbols within (0.4% of
the theoretical values unless otherwise noted.
Con clu sion s
In summary, we have disclosed herein a very efficient
and particularly attractive route toward the large-scale
preparation of 2′-deoxy-C-nucleosides of the pyrazolo[1,5-
a]-1,3,5-triazine and their C-nucleotide derivatives. The
impetus for this work was the easy access to the 4-(N-
methyl-N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine de-
rivatives (e.g. 7), obtained by treatment of the corre-
sponding pyrazolo[1,5-a]-1,3,5-triazin-4-one (5) with
phosphorus oxychloride and dimethylaniline, and our
observation of the facile nucleophilic displacement of this
optimized N-methyl-N-phenylamino activating group
with amines. Another main advantage of this approach
is the use of the readily available 1,4-anhydro-2-deoxy-
D-erythro-pent-1-enitol (4b)21 as a source of glycal in a
regio- and stereoselective palladium-mediated cross-
coupling reaction with the 8-iodo-2-methyl-4-(N-methyl-
N-phenylamino)pyrazolo[1,5-a]-1,3,5-triazine (16) per-
mitting a multigram scale synthesis of C-furanosyl-4-one
compounds 19. Moreover, displacement of the N-methyl-
N-phenylamino group at the end of the reaction repre-
sents a more concise and general route for the introduc-
tion of substitutions and functionalities at the 4-position,
thus avoiding additional protection/deprotection steps
which can compromise the integrity of the anomeric
center. The conversion of so-formed C-nucleoside 8-(2′-
deoxy-â-D-ribofuranosyl)-2-methyl-4-(N-methylamino)-
pyrazolo[1,5-a]-1,3,5-triazine (21) and 8-(2′-deoxy-â-D-
xylofuranosyl)-2-methyl-4-(N-methylamino)pyrazolo[1,5-
a]-1,3,5-triazine (24) to corresponding C-nucleotide 3′,5′-
bisphosphate 2 and 3′,5′-cyclophosphate 26 with use of
the tetrabenzylpyrophosphate procedure was also dem-
2-Meth yl-4-(N-m eth yl-N-p h en yla m in o)p yr a zolo[1,5-a ]-
1,3,5-tr ia zin e (7) a n d 2-Meth yl-4-[4-(N,N-d im eth yla m i-
n o)p h en yl]p yr a zolo[1,5-a ]-1,3,5-tr ia zin e (8). Meth od A.
A solution of 512 (1.0 g, 6.66 mmol), N,N-dimethylaniline (3.0
mL, 23.7 mmol), and phosphorus oxychloride (8 mL, 107 mmol)
was stirred at 125 °C in a sealed tube for 4 h. After the solution
was cooled to room temperature, the excess of phosphorus
oxychloride was removed under vacuum. The mixture was
taken up with ice-cooled water (100 mL) and the resulting
solution was neutralized with saturated NaHCO3, then ex-
tracted with ethyl acetate (3 × 100 mL). The organic layer
was dried (Na2SO4) and concentrated to dryness under reduced
pressure. Chromatography on silica (EtOAc/hexanes, 1:1)
afforded the pure compounds 7 (555 mg, 35%) and 8 (355 mg,
21%). Compound 7: colorless solid; mp 116 °C (Et2O/hexane).
1H NMR (300 MHz, CDCl3) δ 2.58 (s, 3H), 3.76 (s, 3H), 6.26
(d, J ) 1.9, 1H), 7.17-7.44 (m, 5H), 7.71 (d, J ) 1.9, 1H); 13C
NMR (CDCl3, 75 MHz) δ 26.0, 42.5, 94.5, 126.3, 126.4, 127.4,
129.4, 145.2, 149.6, 151.9, 163.0. FABMS m/z 239 (M)+. Anal.
Calcd for C13H13N5: C, 65.26; H, 5.48; N, 29.27. Found: C,
65.35; H, 5.50; N, 29.23. Compound 8: colorless solid, mp 123
1
°C (Et2O/hexane). H NMR (300 MHz, CDCl3) δ 2.75 (s, 3H),
3.16 (s, 6H), 6.52 (d, J ) 2.2, 1H), 7.87 (AB system, ∆δ ) 1.37,
J AB ) 9.5, 4H), 8.20 (d, J ) 2.2, 1H); 13C NMR (CDCl3, 75 MHz)
δ 26.2, 40.4, 95.1, 111.3, 117.0, 133.7, 146.7, 151.5, 153.7, 153.8,
162.3. FABMS m/z 254 (M + H)+. Anal. Calcd for C14H15N5:
C, 66.38; H, 5.97; N, 27.65. Found: C, 66.27; H, 5.99; N, 27.51.
Meth od B. A solution of 512 (1.0 g, 6.66 mmol), N,N-
dimethylaniline (3.0 mL, 23.7 mmol), and phosphorus oxy-
chloride (8 mL, 107 mmol) was refluxed for 2 h. After the
solution was cooled to room temperature, the excess of
phosphorus oxychloride was removed under reduced pressure.
Then, the residue was redissolved in CH2Cl2 (40 mL), and
N-methylaniline (1.5 mL, 11.8 mmol) was added dropwise with
stirring at 0 °C under argon. After 10 min, the reaction mixture
was allowed to warm to room temperature. After evaporation
of the solvent, the residue was chromatographed on silica
(28) Vacca, J . P.; deSolms, S. J .; Huff, J . R. J . Am. Chem. Soc. 1987,
109, 3478-3479.
(29) Yu, K.-L.; Fraser-Reid, B. Tetrahedron Lett. 1988, 29, 979-
982.
8068 J . Org. Chem., Vol. 67, No. 23, 2002

Synthesis of C-Nucleoside Pyrazolo[1,5-a]-1,3,5-triazines
(EtOAc/hexanes, 1:1) to give 7 (1.46 g, 92%), which was
identical in all respects with the compound that was obtained
through Method A.
ammonium hydroxide (2.0 mL, 15.9 mmol) in ethanol (10 mL)
was stirred at 110 °C in a sealed tube for 14 h. After the
solution was cooled to room temperature, the solvent was
evaporated under reduced pressure. The crude reaction prod-
uct was purified by column chromatography on silica gel
(EtOAc/CH2Cl2/EtOH, 4:5:1). Recrystallization from ethanol
and diethyl ether yielded compound 17b (138 mg, 53%) as
4-Meth oxy-2-m eth ylp yr a zolo[1,5-a ]-1,3,5-tr ia zin e (9a ).
A solution of 512 (2.70 g, 18.0 mmol), phosphorus oxychloride
(30 mL, 401 mmol), and N,N-dimethylaniline (8 mL, 63.2
mmol) was refluxed for 1.5 h. The excess of phosphorus
oxychloride was then removed under vacuum. The residue was
poured onto ice-water (30 mL), then the solution was ex-
tracted with EtOAc (3 × 40 mL), dried (Na2SO4), and concen-
trated to dryness under reduced pressure. The residue was
directely put into MeOH (40 mL). After 12 h of stirring at 20
°C, the solvent was evaporated to dryness and the residue was
dissolved in ice-cold EtOAc (50 mL). This solution was suc-
cessively washed with cold, saturated NaHCO3 (50 mL), brine
(40 mL), and finally water (30 mL). The organic layer was
dried (Na2SO4) and concentrated under reduced pressure.
Recrystallization from Et2O/hexanes yielded compound 9a
(1.60 g, 54%) as a yellow solid. 13C NMR (CDCl3, 75 MHz) δ
25.8, 56.5, 96.3, 147.2, 151.4, 153.6, 163.5. EIMS m/z 165 (M
+ H)+. Anal. Calcd for C7H8N4O: C, 51.21; H, 4.91; N, 34.13.
Found: C, 51.28; H, 4.95; N, 34.42.
8-Iod o-4-m e t h oxy-2-m e t h ylp yr a zolo[1,5-a ]-1,3,5-t r i-
a zin e (9b). A solution of 9a (300 mg, 1.83 mmol) and
N-iodosuccinimide (493 mg, 2.19 mmol) in chloroform (20 mL)
was stirred under argon for 3 h at reflux. After evaporation of
the solvent, the residue was diluted with EtOAc (30 mL),
washed with saturated sodium bisulfite (10 mL) and cold water
(20 mL), dried (Na2SO4), then concentrated to dryness under
reduced pressure. Chromatography on silica (EtOAc/hexanes,
1:1) afforded compound 9b (361 mg, 68%) as a colorless solid.
1H NMR (200 MHz, CDCl3) δ 2.67 (s, 3H), 4.32 (s, 3H), 8.07
(s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.1, 49.0, 57.1, 150.3,
151.1, 153.5, 165.2. EIMS m/z 291 (M + H)+. Anal. Calcd for
C7H7IN4O: C, 28.99; H, 2.43; N, 19.32. Found: C, 29.03; H,
2.54; N, 19.21.
colorless crystals: mp 220 °C. 1H NMR (300 MHz, CDCl3
+
DMSO-d6) δ 2.36 (s, 3H), 7.00 (br s, 2H), 7.78 (s, 1H); 13C NMR
(75 MHz, CDCl3) δ 26.1, 48.6, 149.5, 150.0, 151.2, 165.2. EIMS
m/z 276 (M + H)+. Anal. Calcd for C6H6IN5: C, 26.20; H, 2.20;
N, 25.47. Found: C, 25.94; H, 2.21; N, 25.46.
4-(N-Ben zylam in o)-8-iodo-2-m eth ylpyr azolo[1,5-a ]-1,3,5-
tr ia zin e (17c) was prepared from 16 and benzylamine as
described for 17b: a colorless solid (98%); mp 142 °C. 1H NMR
(300 MHz, CDCl3) 2.62 (s, 3H), 4.85 (d, J ) 5.9, 2H), 6.76 (br
s, 1H), 7.35-7.42 (m, 5H), 7.92 (s, 1H); 13C NMR (75 MHz,
CDCl3) δ 26.6, 45.1, 47.9, 128.3, 128.5, 129.3, 137.0, 148.9,
149.3, 149.4, 165.8. EIMS m/z 366 (M + H)+. Anal. Calcd for
C
13H12IN5: C, 42.75; H, 3.31; N, 19.18. Found: C, 42.86; H,
3.36; N, 19.07.
8-Iod o-2-m eth yl-4-(1-p yr r olid in yl)p yr a zolo[1,5-a ]-1,3,5-
tr ia zin e (17d ) was prepared from 16 and pyrrolidine as
1
described for 17b: colorless solid (92%); mp 127 °C. H NMR
(200 MHz, CDCl3) δ 1.95-2.10 (m, 4H), 2.54 (s, 3H), 3.70-
4.60 (m, 4H), 7.88 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.2,
46.6, 50.3, 50.8, 148.5, 148.6, 151.5, 165.5. EIMS m/z 330 (M
+ H)+. Anal. Calcd for C10H12IN5: C, 36.49; H, 3.67; N, 21.28.
Found: C, 36.34; H, 3.64; N, 20.75.
2-Meth yl-4-(N-m eth yl-N-p h en yla m in o)-8-(â-D-glycer o-
pen tofu r an -3′-u los-1′-yl)pyr azolo[1,5-a ]-1,3,5-tr iazin e (19).
Bis(dibenzylideneacetone)palladium(0) (310 mg, 0.53 mmol)
and triphenylarsine (330 mg, 1.08 mmol) were mixed in
acetonitrile (50 mL) and stirred under nitrogen at 25 °C for
15 min. The complex was then transferred by syringe to a
solution of 1,4-anhydro-2-deoxy-D-erythro-pent-1-enitol (4b;
800 mg, 6.89 mmol), 8-iodo-2-methyl-4-(N-methyl-N-phenyl-
amino)pyrazolo[1,5-a]-1,3,5-triazine (16; 2.47 g, 6.76 mmol),
and tri-n-butylamine (1.89 mL, 7.92 mmol) in acetonitrile (150
mL). The reaction mixture was stirred under nitrogen at 60
°C for 18 h. After the solution was cooled to room temperature,
the solvent was evaporated under reduced pressure. The crude
reaction product was purified by column chromatography on
silica gel (EtOAc/hexanes, 1:1). Recrystallization from ethyl
acetate and hexanes yielded compound 19 (1.80 g, 75%) as
colorless crystals: mp 70 °C. 1H NMR (200 MHz, CDCl3) δ
2.55 (s, 3H), 2.85 (ddd, J ) 6.6, J ) 10.6, J ) 17.8, 2H), 3.74
(s, 3H), 3.86-4.11 (m, 3H), 5.39 (dd, J ) 6.6, J ) 10.6, 1H),
6.06 (dd, J ) 10.3, J ) 2.7, 1H), 7.14-7.45 (m, 2H), 7.31-7.45
(m, 3H), 7.66 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 25.7, 42.7,
45.1, 63.5, 71.3, 82.7, 107.7, 126.6, 127.8, 129.4, 143.9, 144.8,
148.9, 149.5, 163.6, 214.9. EIMS m/z 354 (M + H)+. Anal. Calcd
for C18H19N5O3: C, 61.18; H, 5.42; N, 19.82. Found: C, 61.41;
H, 5.46; N, 20.11.
8-(2′-Deoxy-â-D-r ib ofu r a n osyl)-2-m et h yl-4-(N-m et h yl-
N-p h en yla m in o)p yr a zolo[1,5-a ]-1,3,5-t r ia zin e (20). So-
dium triacetoxyborohydride (4.50 g, 21.2 mmol) was added to
a stirred solution of 19 (1.50 g, 4.24 mmol) in acetonitrile (150
mL). The reaction mixture was allowed to stir for 20 min at
room temperature and then quenched with water (250 mL)
and extracted with dichloromethane (3 × 150 mL), dried
(Na2SO4), and evaporated under reduced pressure. Purification
by column chromatography (EtOAc/EtOH, 9:1) followed by
recrystallization from ethyl alcohol and diethyl ether yielded
compound 20 (1.71 g, 92%) as colorless crystals: mp 153 °C.
1H NMR (200 MHz, CDCl3) δ 1.78 (br s, 1H), 2.08 (dd, J )
5.2, J ) 11.3, 1H), 2.54-2.68 (m, 4H), 3.72-3.81 (m, 4H), 3.98
(dd, J ) 2.0, J ) 12.5, 1H), 4.12 (br s, 1H), 4.67 (br d, J ) 4.7,
1H), 5.33 (dd, J ) 5.2, J ) 11.3, 1H), 6.28 (d, J ) 11.5, 1H),
7.11-7.17 (m, 2H), 7.32-7.43 (m, 3H), 7.62 (s, 1H); 13C NMR
(75 MHz, CDCl3) δ 25.6, 42.6, 43.9, 64.6, 74.4, 76.3, 89.2, 108.0,
126.5, 127.6, 129.4, 144.4, 144.9, 148.5, 149.6, 162.9. EIMS
8-Iodo-2-m eth yl-4-(N-m eth yl-N-ph en ylam in o)pyr azolo-
[1,5-a ]-1,3,5-tr ia zin e (16). To a solution of 7 (1.00 g, 4.18
mmol) in dry chloroform (100 mL) was added N-iodosuccin-
imide (1,33 g, 5.91 mmol), and the mixture was refluxed for
0.5 h. After the mixture was cooled at room temperature, the
solvent was evaporated in vacuo. Then, the residue was
partitioned between dichloromethane (100 mL) and water (70
mL). The aqueous layer was extracted twice with additional
dichloromethane (2 × 50 mL). The combined organic phases
was washed with 10% sodium bisulfite (70 mL). The organic
layer was dried (Na2SO4) and concentrated under reduced
pressure. The crude material was purified by column chroma-
tography on silica (EtOAc/CH2Cl2/hexanes, 2:3:5). Recrystal-
lization from ethanol yielded compound 16 (1.33 g, 87%) as
colorless crystals: mp 192 °C. 1H NMR (200 MHz, CDCl3) δ
2.66 (s, 3H), 3.76 (s, 3H), 7.16-7.44 (m, 5H), 7.71 (s, 1H); 13
C
NMR (50 MHz, CDCl3) δ 26.3, 42.7, 47.4, 126.5, 127.6, 129.4,
144.9, 148.9, 149.5, 164.7. EIMS m/z 366 (M + H)+. Anal. Calcd
for C13H12IN5: C, 42.76; H, 3.31; N, 19.18. Found: C, 42.54;
H, 3.38; N, 19.04.
2-Met h yl-4-(N-m et h yla m in o)p yr a zolo[1,5-a ]-1,3,5-t r i-
a zin e (17a ). A solution of 7 (300 mg, 1.26 mmol) and
methylamine (2 M, 2.0 mL, 4.0 mmol) in ethanol (10 mL) was
stirred at 100 °C in a sealed tube for 12 h. After the mixture
was cooled the solvent was evaporated under reduced pressure.
The crude reaction product was purified by column chroma-
tography on silica gel (EtOAc/CH2Cl2/EtOH, 4:5:1). Recrystal-
lization from ethanol and diethyl ether yielded compound 17a
(160 mg, 78%) as colorless crystals: mp: 165 °C. 1H NMR (200
MHz, CDCl3) δ 5.44 (s, 3H), 5.70 (d, J ) 1.9, 3H), 6.89 (d, J )
0.9, 1H), 6.98 (br s, 1H), 7.52 (d, J ) 0.9, 1H); 13C NMR (50
MHz, CDCl3) δ 26.3, 27.6, 95.5, 145.4, 149.5, 149.6, 164.1.
EIMS m/z 164 (M + H)+. Anal. Calcd for C7H9N5: C, 51.52;
H, 5.56; N, 42.92. Found: C, 51.20; H, 5.61; N, 42.90.
4-Am in o -8-io d o -2-m e t h y lp y r a zo lo [1,5-a ]-1,3,5-t r i-
a zin e (17b). A solution of 16 (350 g, 0.95 mmol) and 28%
J . Org. Chem, Vol. 67, No. 23, 2002 8069

Raboisson et al.
m/z 356 (M + H)+. Anal. Calcd for C18H21N5O3‚1.5H2O: C,
8-(2′-De oxy-â-D-xylofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-tr ia zin e-3′,5′-cyclic p h os-
p h a te ben zyl ester (25) was prepared from 24 as described
56.53; H, 6.32; N, 18.31. Found: C, 56.72; H, 6.01; N, 18.06.
8-(2′-De oxy-â-D-r ib ofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-tr ia zin e (21) was prepared
from 20 as described for 17a : colorless solid (73%); mp 156
°C. 1H NMR (300 MHz, CDCl3) δ 1.90 (d, J ) 3.1, 1H), 2.11
(dd, J ) 5.4, J ) 11.2, 1H), 2.53-2.65 (m, 4H), 3.23 (d, J )
5.0, 3H), 3.72-3.80 (m, 1H), 3.95-4.00 (dd, J ) 1.9, J ) 12.5,
1H), 4.13 (br s, 1H), 4.69 (br t, J ) 3.9, 1H), 5.40 (dd, J ) 5.4,
J ) 11.2, 1H), 6.28 (dd, J ) 11.8, J ) 1.9, 1H), 6.56 (br d, J )
4.4, 1H), 7.86 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 25.8, 27.7,
44.2, 64.5, 74.3, 76.0, 89.1, 109.8, 144.5, 146.3, 149.6, 164.1.
EIMS m/z 280 (M + H)+. Anal. Calcd for C12H17N5O3‚0.1H2O:
C, 51.27; H, 6.17; N, 24.91. Found: C, 51.56; H, 6.06; N, 24.79.
8-(2′-De oxy-â-D-r ib ofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-t r ia zin e -3′,5′-b is(d ib e n -
zyl p h osp h a te) (22). Potassium tert-butoxide (1.0 M in THF,
5.5 mL) was slowly added, at - 40°C, to a stirred solution of
21 (700 mg, 2.5 mmol) in anhydrous THF (100 mL). After 5
min, tetrabenzyl pyrophosphate (2.59 g, 5.5 mmol) was added
and stirring was continued for 15 min at -40 °C. The reaction
mixture was allowed warm to 0 °C, then quenched with acetic
acid (250 µL). The mixture was diluted with ethyl acetate (200
mL), washed with ice-cold water (100 mL), dried (Na2SO4), and
concentrated to dryness under reduced pressure. Chromatog-
raphy on silica (EtOAc/CH2Cl2/EtOH, 40:50:10) followed by
recrystallization from ethanol and diethyl ether yielded com-
pound 22 (2.9 g, 66%) as colorless crystals: mp 120 °C. 1H
NMR (300 MHz, CDCl3) δ 2.35-2.41 (m, 2H), 2.52 (s, 3H), 3.20
(d, J ) 4.9, 3H), 4.08 (t, J ) 4.5, 2H), 4.19-4.24 (m, 1H), 4.99-
5.12 (m, 9H), 5.39 (t, J ) 8.1, 1H), 6.43 (q, J ) 5.2, 1H), 7.28-
7.37 (m, 20H), 7.90 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.4,
27.6, 39.8, 67.4, 69.8, 72.1, 80.3, 83.7, 107.7, 128.4, 129.1, 136.1,
138.5, 144.3, 147.1, 149.4, 164.2. EIMS m/z 800 (M + H)+.
8-(2′-De oxy-â-D-r ib ofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-t r ia zin e -3′,5′-b isp h os-
p h a te (2). A mixture of 22 (1.2 g, 1.5 mmol) and 10% Pd/C
(1.0 g) in absolute methanol (200 mL) was shaken in a
hydrogenation apparatus under 60 psi pressure at room
temperature for 1.4 h. The catalyst was removed by filtration
and washed with water, and the filtrate was concentrated to
dryness. Recrystallization from ethanol and diethyl ether
yielded compound 2 (600 mg, 91%) as colorless crystals: mp
145 °C. 1H NMR (300 MHz, DMSO-d6) δ 2.26-2.41 (m, 5H,
2′-H), 3.00 (d, J ) 4.7, 3H), 3.85-3.98 (m, 2H), 4.10-4.18 (m,
1H), 4.79-4.84 (m, 1H), 5.25 (dd, J ) 5.6, J ) 10.0, 1H), 8.14
(s, 1H), 8.61 (q, J ) 4.7, 1H); 13C NMR (75 MHz, CDCl3) δ
26.5, 27.9, 41.5, 66.0, 71.5, 77.8, 84.3, 108.3, 144.7, 147.1, 149.6,
163.6; 33P NMR (300 MHz, D2O, 80°C) δ 0.46, 1.21. Anal. Calcd
for C12H19N5O9P2‚2 H2O: C, 30.32; H, 4.87; N, 14.74. Found:
C, 30.38; H, 4.76; N, 14.61.
1
for 22: colorless solid (63%); mp 182 °C. H NMR (300 MHz,
CDCl3) δ 2.43 (dd, J ) 5.3, J ) 14.4, 1H), 2.54 (s, 3H), 2.70-
2.78 (m, 1H), 3.22 (d, J ) 5.0, 3H), 3.82-3.85 (m, 1H), 4.39-
4.62 (m, 2H), 4.92-4.94 (m, 1H), 5.16 (dd, J ) 0.6, J ) 8.1,
2H), 5.46 (dd, J ) 5.6, J ) 9.4, 1H), 6.54 (q, J ) 5.0, 1H), 7.37-
7.45 (m, 5H), 8.17 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 26.4,
27.6, 41.4, 67.9, 69.5, 70.6, 75.1, 81.7, 109.4, 128.6, 129.1, 129.2,
136.0, 144.6, 146.7, 149.4, 164.3. EIMS m/z 432 (M + H)+.
Anal. Calcd for C19H22N5O5P: C, 52.90; H, 5.14; N, 16.23.
Found: C, 53.01; H, 5.15; N, 16.18.
8-(2′-De oxy-â-D-xylofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-tr ia zin e-3′,5′-cyclic p h os-
p h a te (26) was prepared from 25 as described for 2: colorless
solid (92%); mp 156 °C. 1H NMR (300 MHz, CD3OD) δ 2.28
(dd, J ) 4.1, J ) 14.7, 1H), 2.69 (s, 3H), 2.70-2.87 (m, 1H),
3.28 (s, 3H), 3.94-3.97 (m, 1H), 4.41-4.63 (m, 2H), 4.98-5.01
(m, 1H), 5.39 (dd, J ) 4.1, J ) 9.4, 1H), 8.21 (s, 1H); 13C NMR
(75 MHz, CDCl3) δ 26.4, 28.0, 41.5, 67.6, 70.2, 75.3, 80.8, 110.1,
144.8, 146.4, 149.6, 163.6. EIMS m/z 342 (M + H)+. Anal. Calcd
for C12H16N5O5P: C, 42.23; H, 4.73; N, 20.52. Found: C, 42.09;
H, 5.03; N, 20.48.
Biologica l Tests: Wa sh ed Hu m a n P la telet Aggr ega -
tion . Washed human platelets were prepared as previously
described1 and resuspended at 3 × 105 platelets/µL in Tyrode’s
buffer containing 2 mM CaCl2, in the presence of 0.02 U/mL
of the ADP scavenger apyrase (adenosine 5′-triphosphate
diphosphorylase, EC 3.6.1.5). Platelets were kept at 37 °C
throughout all experiments and aggregation was measured by
standard methods.1
Briefly, a 450-µL aliquot of platelet suspension was stirred
at 1100 rpm and activated by addition of agonists and of
human fibrinogen (0.8 mg/mL), in a final volume of 500
µL. The extent of aggregation was estimated quantitatively
by measuring the maximum curve height above the base-
line.
[Ca 2+]i Mea su r em en ts. Fura-2/AM loaded human platelets
were prepared as previously described1 and resuspended in
Tyrode’s buffer with 2 mM CaCl2. Aliquots of fura-2-loaded
platelets were transferred to a 10 × 10 mm2 quartz cuvette
maintained at 37 °C and fluorescence measurements were
performed under continuous stirring, in a spectrofluorimeter.1
The excitation wavelength was alternately fixed at 340 or 380
nm, fluorescence emission was determined at 510 nm, and
results were calculated as the fluorescence ratio (340/380) in
arbitrary units.
In vivo stu d ies. Male Wistar rats weighing 300 g (Iffa-
Credo, l’Arbresle, France) were anesthetized by intraperitoneal
injection of 200 µL of xylazine base (0.2 mg/kg) and ketamine
(1 mg/kg). At time zero, 2 (30 mg/kg) or vehicle was injected
into the penis vein. Blood (6.3 mL) was drawn (1 and 30 min)
later from the abdominal aorta into syringes containing 0.7
mL of 3.15% sodium citrate and immediately centrifuged (70
s at 1570g) at room temperature. Citrated platelet-rich plasma
(cPRP) was removed and platelets were adjusted to 5 × 105/
µL with platelet-poor plasma (PPP). Platelet aggregation was
measured in citrated platelet-rich plasma from control and
compound 2-treated rats.
8-(2′-Deoxy-â-D-xylofu r a n osyl)-2-m et h yl-4-(N-m et h yl-
N-p h en yla m in o)p yr a zolo[1,5-a ]-1,3,5-t r ia zin e (23) was
prepared from 19 and K-selectride as described for 20: color-
less solid (78%). 1H NMR (200 MHz, CDCl3) δ 2.33 (dd, J )
5.6, J ) 14.7, 1H), 2.59-2.79 (m, 4H), 3.76 (s, 3H), 4.02-4.14
(m, 2H), 4.23 (dd, J ) 1.5, J ) 12.9, 1H), 4.57-4.62 (m, 1H),
5.42 (dd, J ) 5.9, J ) 9.3, 1H), 7.14-7.19 (m, 2H), 7.34-7.43
(m, 3H), 7.80 (s, 1H). EIMS m/z 356 (M + H)+. Anal. Calcd for
C
18H21N5O3: C, 60.83; H, 5.96; N, 19.71. Found: C, 60.93; H,
Deter m in a tion of th e p Ka . To solutions of compound 7 at
a concentration of ca. 3 × 10-3 mol dm-3 was added 1.2 equiv
of HCl before the solution was titrated with potassium
hydroxide. Due to the poor solubility of the compound in
aqueous medium, the titration was performed in CH3OH/H2O
50:50 at 20 °C in the presence of 0.2 M KCl in order to hold
the ionic strength constant. The pH measurements were made
6.01; N, 19.84.
8-(2′-De oxy-â-D-xylofu r a n osyl)-2-m e t h yl-4-(N -m e t h -
yla m in o)p yr a zolo[1,5-a ]-1,3,5-tr ia zin e (24) was prepared
from 23 as described for 17a : colorless solid (67%); mp 145
1
°C. H NMR (300 MHz, CDCl3) δ 2.28-2.36 (m, 1H), 2.54 (s,
3H), 2.67-2.73 (m, 1H), 2.80 (d, J ) 5.0, 1H), 3.23 (d, J ) 5.3,
3H), 3.95-4.08 (m, 3H), 4.40-4.51 (m, 1H), 5.11 (dd, J ) 6.8,
J ) 8.7, 1H), 5.82 (d, J ) 10.6, 1H), 6.62 (br s, 1H), 7.87 (s,
1H); 13C NMR (50 MHz, CDCl3) δ 25.9, 27.7, 43.2, 63.2, 71.4,
74.2, 82.4, 110.4, 144.8, 145.8, 149.6, 164.2. EIMS m/z 280 (M
+ H)+. Anal. Calcd for C12H17N5O3: C, 51.60; H, 6.13; N, 25.07.
Found: C, 51.63; H, 6.27; N, 25.11.
(30) Boyer, J . L.; Mohanram, A.; Gamaioni, E.; J acobson, K. A.;
Harden, T. K. Br. J . Pharmacol. 1998, 124, 1-3.
(31) Gans, P.; Sabatini, A.; Vacca, A. Investigation of equilibria in
solution. Determination of equilibrium constants with the HYPER-
QUAD suite programs. Talanta 1996, 43, 1739-1753.
8070 J . Org. Chem., Vol. 67, No. 23, 2002

Synthesis of C-Nucleoside Pyrazolo[1,5-a]-1,3,5-triazines
in 2 cm3 solutions with a combined semimicroglass electrode
connected to a pH meter. The electrode was calibrated in terms
of concentrations at pH 2 and its slope was checked by a
previous acid-base titration. The titration curves allowed the
calculation of the total acid (CH), whereas the concentration
of the ligands (CL) was obtained by weight. Since the pKa
values appeared to be very low, the average degree of proto-
nation pj versus pH was calculated according to the following
equation: pj ) (CH - [H+] + [OH-])/CL, to provide an initial
constant for the refinement by HYPERQUAD.31 Compound 7
remains soluble at a millimolar concentration over all the
studied pH range (2.4 to 10.5) in the CH3OH/H2O 50:50
medium. The pj reaches 0.75 and its pKa value was calculated
to be 3.02 ( 0.04. Addition of 1.2 equiv of H+ to 7 in aqueous
medium led to its solubilization by protonating it at 80%
according to the pj ) f(pH) curve.
J O026268L
J . Org. Chem, Vol. 67, No. 23, 2002 8071