Bismethylene triphosphate nucleotides of uridine 4-phosphate analogues: A new class of anionic pyrimidine nucleotide analogues

Article abstract of DOI:10.1021/jo702249j

(Chemical Equation Presented) Cytidine-5′-triphosphate synthase (CTPS) catalyzes the formation of cytidine triphosphate (CTP) from glutamine, uridine 5′-triphosphate (UTP), and adenosine 5′-triphosphate (ATP). This reaction proceeds via formation of the high-energy intermediate UTP-4-phosphate (UTP-4-P). Stable analogues of UTP-4-P may be potent inhibitors of CTPS and useful as lead structures for the development of anticancer and antiviral agents. Several bismethylene triphosphate (BMT) nucleotides of uridine 4-phosphate (U-4-P) analogues have been prepared. A key step was the selective methanolysis, with the aid of a tin catalyst, of the 5′ ester moiety of 2′,3′,5′-tri-O-acetyl or tri-O-benzoyl U-4-P analogues. We believe this represents the first general approach to the selective cleavage of 5′ benzoyl esters in benzoylated nucleosides. Mitsunobu coupling of these 5′-deprotected U-4-P analogues to an unsymmetrical, protected BMT bearing a free phosphonic acid moiety at one of the terminal positions gave fully protected BMT-U-4-P analogues. Global deprotection of these species was achieved using TMSBr followed by treatment with NH₄OH-MeOH or NH ₄OH-pyridine. The resulting BMT nucleotides represent a new class of anionic pyrimidine nucleotide analogues.

Full text of DOI:10.1021/jo702249j

Bismethylene Triphosphate Nucleotides of Uridine 4-Phosphate
Analogues: A New Class of Anionic Pyrimidine Nucleotide
Analogues
Scott D. Taylor,*,† Farzad Mirzaei,† and Stephen L. Bearne‡
Department of Chemistry, UniVersity of Waterloo, 200 UniVersity AVenue West, Waterloo, ON, Canada,
and Department of Biochemistry and Molecular Biology, Dalhousie UniVersity,
Halifax, NS, B3H 1X5, Canada
s5taylor@sciborg.uwaterloo.ca
ReceiVed October 17, 2007
Cytidine-5′-triphosphate synthase (CTPS) catalyzes the formation of cytidine triphosphate (CTP) from
glutamine, uridine 5′-triphosphate (UTP), and adenosine 5′-triphosphate (ATP). This reaction proceeds
via formation of the high-energy intermediate UTP-4-phosphate (UTP-4-P). Stable analogues of UTP-
4-P may be potent inhibitors of CTPS and useful as lead structures for the development of anticancer
and antiviral agents. Several bismethylene triphosphate (BMT) nucleotides of uridine 4-phosphate (U-
4-P) analogues have been prepared. A key step was the selective methanolysis, with the aid of a tin
catalyst, of the 5′ ester moiety of 2′,3′,5′-tri-O-acetyl or tri-O-benzoyl U-4-P analogues. We believe this
represents the first general approach to the selective cleavage of 5′ benzoyl esters in benzoylated
nucleosides. Mitsunobu coupling of these 5′-deprotected U-4-P analogues to an unsymmetrical, protected
BMT bearing a free phosphonic acid moiety at one of the terminal positions gave fully protected BMT-
U-4-P analogues. Global deprotection of these species was achieved using TMSBr followed by treatment
with NH4OH-MeOH or NH4OH-pyridine. The resulting BMT nucleotides represent a new class of
anionic pyrimidine nucleotide analogues.
Introduction
tion of cytidine 5′-triphosphate (CTP) from uridine 5′-triphos-
phate (UTP) using either ammonia or L-glutamine as the nitrogen
source (Scheme 1). Numerous lines of evidence2a-h suggest that
CTPS performs this reaction by transferring the γ-phosphate
from ATP to the oxygen at position 4 of UTP to form UTP-
Nucleoside analogues have now been in use as antiviral and
anticancer agents for several decades.1a,b Consequently, their
synthesis continues to be an area of considerable importance to
the pharmaceutical industry. Our interest in nucleoside and
nucleotide analogues is a result of our desire to prepare inhibitors
of cytidine 5′-triphosphate synthase (CTPS, EC 6.3.4.2). CTPS
catalyzes the adenosine 5′-triphosphate (ATP)-dependent forma-
(2) (a) Long, C. W.; Levitzki, A.; Koshland, D. E., Jr. J. Biol. Chem.
1979, 245, 80. (b) Koshland, D. E., Jr.; Levitzki, A. Biochemistry 1971,
10, 3365. (c) Long, C. W.; Pardee, A. B. J. Biol. Chem. 1967, 242, 4715.
(d) von der Saal, W.; Villafranca, J. J.; Anderson, P. M. J. Am. Chem. Soc.
1985, 107, 703. (e) von der Saal, W.; Anderson, P. M.; Villafranca, J. J. J.
Biol. Chem. 1985, 260, 14993. (f) Levitzki, A.; Koshland, D. E., Jr.
Biochemistry 1972, 11, 241. (g) Levitzki, A.; Koshland, D. E., Jr.
Biochemistry 1971, 10, 3365. (h) Lewis, D. A.; Villafranca, J. J. Biochem-
istry 1989, 28, 8454.
† University of Waterloo.
‡ Dalhousie University.
(1) (a) Jordheim, L. P.; Galmarini, C. M.; Dumontet,C. Recent Patents
on Anti-Cancer Drug DiscoVery 2006, 1, 163. (b) Carroll, S. S.; Olen, D.
B. Infectious Disorders: Drug Targets 2006, 6, 17.
10.1021/jo702249j CCC: $40.75 © 2008 American Chemical Society
Published on Web 01/24/2008
J. Org. Chem. 2008, 73, 1403-1412
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Taylor et al.
SCHEME 1. Reaction Catalyzed by CTPS
4-phosphate (UTP-4-P) as an intermediate and ADP. Ammonia,
derived from the hydrolysis of glutamine or free in solution,
then displaces the phosphate at O-4 of UTP-4-P to give CTP.
CTPS activity is elevated in some forms of leukemia3a,b and
some solid tumors.4 Because CTP plays a central role in the
biosynthesis of phospholipids5 and nucleic acids,6 CTPS is a
recognized target for the development of antineoplastic agents.6,7
For example, the nucleoside derivative, 3-deazauridine (3-DaU),
has been shown to induce apoptosis in myeloid leukemia cells
in a dose-dependent manner.8 3-DaU, as well as another
nucleoside analogue, cyclopentenylcytosine (CPEC), enhances
the effects of various anticancer and antiviral agents suggesting
that they could be employed in combination drug therapy.9a-f
Studies suggest that 3-DaU and CPEC are triphosphorylated
intracellularly and it is most likely that it is the triphosphorylated
forms that are biologically active.10,11 In addition, CTPS is a
target for the development of antiviral7,12 and antiprotozoal
agents.13
nucleosides 1-6 (Figure 1), upon conversion to their respective
nucleotide triphosphates, would serve as structural and electronic
mimics of UTP-4-P and, consequently, be potent inhibitors of
CTPS (Figure 1a). Recently, we described the synthesis of
nucleosides 1-4.15
One method of generating potent inhibitors of enzymes is to
use analogues of either the transition state of the reaction or a
high-energy intermediate formed during catalysis.14a,b We,
therefore, anticipated that phosphonate and phosphoramidate
FIGURE 1. Structures of compounds 1-6.
Although there are a few reports of phosphonate16 and
phosphoramidate17 derivatives of the nucleosides uridine and
cytidine, to our knowledge, there is only a single report of a
nucleotide analogue bearing anionic groups on a pyrimidine.18
Conversion of nucleosides to their corresponding 5′-triphos-
phates is often difficult because of the low yields, difficulties
involved in large-scale purification, and the inherent instability
of the compounds to hydrolysis. This transformation is especially
challenging when a phosphorus-based substituent, such as a
phosphonate or phosphoramidate moiety, is already present in
the nucleoside precursor. However, nucleoside analogues are
often converted into hydrolytically stable nucleotides by the
attachment of nonhydrolyzable di- or triphosphate mimics for
in vitro characterization of some of their biological properties
and the methylene group has been widely used to replace the
labile pyrophosphate oxygens.19a-k Expanding on the pioneering
work of Mioskowski and co-workers,20a-c we recently reported
the synthesis of bismethylene triphosphate (BMT) analogues
of nucleosides by coupling compound 7 to the nucleoside
followed by global deprotection of the resulting protected BMT
nucleotide 8 (Scheme 2).21 This methodology was used to
(3) (a) Ellims, P. H.; Gan, T. E.; Medley, G. Cancer Res. 1983, 43, 1432.
(b) Verschuur, A. C.; van Gennip, A. H.; Muller, E. U. J.; Voute, P. A.;
van Kuilenburg, A. B. AdV. Exp. Med. Biol. 1998, 431, 667.
(4) Kizaki, H.; Williams, J. C.; Morris, H. P.; Weber, G. Cancer Res.
1980, 40, 3921.
(5) Kennedy, E. P. In Lipids and membranes: Past, present and future;
Op den Kamp, J. A. F., Roelofsen, B., Wirtz, K. W. A., Eds.; Elsevier
Scientific Publishers: Amsterdam, The Netherlands, 1986; pp 171-206
(6) Hatse, S.; De Clercq; E.; Balzarini, J. Biochem. Pharmacol. 1999,
15, 58, 539.
(7) Kensler, T. W.; Cooney, D. A. In Design of Enzyme Inhibitors as
Drugs; Sandler, M., Smith, H. J., Eds.; Oxford University Press: New York,
1989; pp 379-401.
(8) Savickiene, J.; Gineitis, A. Int. J. Biochem. Cell Biol. 2003, 35, 1482.
(9) (a) Barlogie, B.; Plunkett, W.; Raber, M.; Latreille, J.; Keating, J.;
McCredie, K. B. Cancer Res. 1981, 41, 1227. (b) Mills-Yamamoto, C.;
Lauzon, G. J.; Peterson, A. R. P. Biochem. Pharmacol. 1978, 27, 181. (c)
Momparler, R, L.; Momparler, L. F. Cancer Chemother. Pharmacol. 1989,
25, 51. (d) Momparler, R. L; Bouffard, D. Y.; Momparler, L. F.; Marquet,
J.; Zittoun, J.; Marie, J-P.; Zittoun, R. Int. J. Cancer 1991, 49, 573. (e)
Momparler, R. L.; Vesely, J.; Momparler, L. F.; Rivard, G. E. Cancer Res.
1979, 39, 573. (f) Bouffard, D. Y.; Momparler, L. F.; Momparler, R. L.
Anticancer Drugs 1994, 5, 223.
(10) Kang, G. J.; Cooney, D. A.; Moyer, J. D.; Kelley, J. A.; Kim, H-Y.;
Marquez, V. E.; Johns, D. G. J. Biol. Chem. 1989, 264, 713.
(11) McPartland, R. P.; Wang, M. C.; Bloch, A.; Weinfeld, H. Cancer
Res. 1974, 34, 3107.
(12) (a) De Clercq, E.; Murase, J.; Marquez, V. E. Biochem. Pharmacol.
1991, 41, 1821. (b) Gao, W. Y.; Johns, D. G.; Mitsuya Nucleosides
Nucleotides Nucleic Acids 2000, 19, 371.
(13) (a) Hendriks, E. F.; O’Sullivan, W. J.; Stewart, T. S. Biochim.
Biophys. Acta 1998, 1399, 213. (b) Hofer, A.; Steverding, D.; Chabes, A.;
Brun, R.; Thelander, L. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 6412. (c)
Fijolek, A.; Hofer, A.; Thelander, L. J. Biol. Chem. 2007, 282, 11858.
(14) (a) Wolfenden, R. Acc. Chem. Res. 1972, 5, 10. (b) Wolfenden, R.
Ann. ReV. Biophys. Bioeng. 1976, 5, 271.
(15) Taylor, S. D.; Mirzaei, F.; Bearne, S. L. J. Org. Chem. 2006, 71,
9420.
(16) (a) Maguire, M. H.; Ralph, R. K.; Shaw, G. J. Chem. Soc. 1958,
2299. (b) Inoue, H.; Tomita, S.; Ueda, T. Chem. Pharm. Bull. 1975, 23,
2614. (c) Honjo, M.; Maruyama, T.; Horikawa, M.; Balzarini, J.; De Clercq,
E. Chem. Pharm. Bull. 1987, 35, 3227.
(17) Wada, T.; Moriguchi, T.; Sekine, M. J. Am. Chem. Soc. 1994, 116,
9901.
(18) Moriguchi, T.; Wada, T.; Sekine, M. Eur. J. Org. Chem. 2003, 2260.
1404 J. Org. Chem., Vol. 73, No. 4, 2008

Anionic Pyrimidine Nucleotide Analogues
SCHEME 2. General Approach to the Synthesis of BMT Nucleotides
SCHEME 3. Synthesis of Phosphoramidates 12-14
SCHEME 4. Selective 5′-Deacylation of 16 and 17 by Orita
et al.
prepare the first BMT analogues of UTP and CTP.21 Herein,
we report the first synthesis of phosphonate- and phosphora-
midate-pyrimidine BMT analogues of the UTP-4-P intermedi-
ate formed during catalysis by CTPS. We also describe what
we believe is the first general approach to achieve selective 5′-
O-debenzoylations of perbenzoylated pyrimidine nucleosides.
and 80% yields, respectively, using a similar approach starting
from compounds 10 and 11.22
We previously reported the synthesis of compounds 1-4 with
the phosphonate moiety protected as alkyl esters and the 2′, 3′,
and 5′ hydroxyl groups protected as acetyl or benzoyl esters.
To make the corresponding BMT nucleotides from these
protected nucleosides, as well as from nucleosides 12-14, it
was necessary to deprotect the 5′-position and then follow the
route outlined in Scheme 2. The selective cleavage of 5′-acetates
in acetylated nucleosides has been achieved by using enzymes
though this has yet to be shown to be of general utility.23
Relatively recently, Ren et al. reported selective cleavage of
5′-acetates in acetylated nucleosides in yields ranging from 55%
to 69% using 1% iodine in methanol at reflux.24 Orita et al.
described the selective deprotection of the 5′-acetate ester
moieties in 2′,3′,5′-tri-O-acetyl uridine (16) and 2′,3′,5′-O,N4-
tetraacetyl cytidine (17) in 89% and 77% yields, respectively,
by subjecting these compounds to methanol in the presence of
5 mol % tin catalyst 20 at 0-30 °C for 24 h (Scheme 4).25 The
good yields reported by Orita et al. prompted us to examine
this approach for the deacetylation of some of our protected
U-4-P analogues.
Results and Discussion
Previously, we described the synthesis of the U-4-P analogues
1-4 with their hydroxyl groups protected as either benzoate or
acetate esters and the phosphonate groups protected as methyl
or ethyl esters.15 We have also prepared 3-deaza phosporamidate
analogues of U-4-P in which 4-O is replaced with an NH (i.e.,
12-14). Phosphoramidate 12, in which the phosphoramidate
moiety is protected with a cyanoethyl (CNE) (Scheme 3), was
prepared by using a procedure similar to that developed by Wada
et al. for the synthesis of the O-benzoylated derivative.17 Thus,
2′,3′,5′-tri-O-acetyl cytidine (9) was reacted with reagent 15 in
the presence of tetrazole and the resulting nucleoside phos-
phoramidite was oxidized in situ to give phosphoramidate 12
in a 58% yield. The acetyl- and benzoyl-protected 3-deaza
derivatives of 12, compounds 13 and 14, were prepared in 68%
We first examined selective 5′-deacetylations starting with
model compound 21, which was readily obtained (Table 1, entry
1).26 Conducting the reaction in MeOH at room temperature
with 6.5 mol % catalyst, the reaction was complete within 18
h and compound 24 was obtained in a 75% yield. Applying
(19) For some examples: (a) Biller, S.; Forster, C. Tetrahedron, 1990,
46, 6645. (b) Blackburn, G. M.; Langston, S. P. Tetrahedron Lett. 1991,
32, 6425. (c) Morr, M.; Wray, V. Angew. Chem., Int. Ed. Engl. 1994, 33,
1394. (d) Yanachkov, I.; Pan, J. Y.; Wessling-Resnick, M.; Wright, G. E.
Mol. Pharmacol. 1997, 51, 47. (e) Ma, Q-F.; Kenyon, G. L.; Markham, G.
D. Biochemistry 1990, 29, 1412. (f) Dalton, A.; Hornby, D. P.; Langston,
S. P.; Blackburn, G. M. Biochem. J. 1992, 287, 871. (g) Labataille, P.;
Pelicano, H.; Maury, G.; Imbach, J.-L.; Gosselin, G. Biooorg. Med. Chem.
Lett. 1995, 5, 2315. (h) Spelta, V.; Mekhalfia, A.; Rejman, D.; Thompson,
M.; Blackburn, G. M.; North, R. A. Br. J. Pharmacol. 2003, 140, 1027. (i)
Towbridge, D. B.; Yamamoto, D. M.; Kenyon, G. L. J. Am. Chem. Soc.
1972, 94, 3816. (j) Stock, J. A. J. Org. Chem. 1979, 44, 3997. (k) Blackburn,
G. M.; Guo, M. J.; Langston, S. P.; Taylor, G. E. Tetrahedron Lett. 1990,
31, 5637.
(22) Cook, P. D.; Day, R. T.; Robins, R. K. J. Heterocycl. Chem. 1997,
14, 1295.
(23) (a) Singh, H. K.; Cote, G. L.; Sikorski, R. S. Tetrahedron Lett. 1993,
34, 5201. (b) Kuboki, A.; Ishihara, T.; Kobayashi, E.; Ohta, H.; Ishii, T.;
Inoue, A.; Mitsuda, S.; Miyazaki, T.; Kajhara, Y.; Sugai, T. Biosci.
Biotechnol. Biochem. 2000, 64, 363.
(20) (a) Klein, E.; Mons, S.; Valleix, A.; Mioskowski, M.; Lebeau, L. J.
Org. Chem. 2002, 67, 146. (b) Brossette, T.; Le Faou, A.; Goujon, L.;
Valleix, A.; Creminon, C.; Grassi, J.; Mioskowski, C.; Lebeau, L. J. Org.
Chem. 1999, 64, 5083. (c) Klein, E.; Nghiem, H.-O.; Valliex, A.;
Mioskowski, C.; Lebeau, L. Chem. Eur. J. 2002, 8, 4649.
(24) Ren, B.; Cai, L.; Zhang, L-R.; Yang, Z-J.; Zhang, L-H. Tetrahedron
Lett. 2005, 46, 8083.
(25) Orita, A.; Hamada, Y.; Nakano, T.; Toyoshima, S.; Otera, J. J. Chem.
Eur. J. 2001, 7, 3321.
(26) Matulic-Adamic, J.; Gonzalez, C.; Usman, N.; Beigelman, L. Bioorg.
Med. Chem. Lett. 1996, 373.
(21) Taylor, S. D.; Mirzaei, F.; Bearne, S. L. Org. Lett. 2006, 8, 4243.
J. Org. Chem, Vol. 73, No. 4, 2008 1405

Taylor et al.
TABLE 1. 5′-O-Deacetylation of Nucleosides 12, 13, and 21-23
that of the secondary benzoyl esters, they did report that
subjecting the benzoate ester of phenethyl alcohol to 5 mol %
catalyst 20 at 30 °C for 24 h resulted in 15% cleavage.25 This
suggested to us that selective 5′-O-debenzoylation of nucleosides
might be achieved by increasing the amount of catalyst, and/or
reaction time, and/or temperature. Unfortunately, debenzoylation
of 2′,3′,5′-tri-O-benzoyluridine (29) with 5 mol % 20 in refluxing
anhydrous methanol proceeded very slowly and increasing the
amount of catalyst to 17 mol % resulted in a complex mixture
of products. However, by using 17 mol % catalyst and by
maintaining the temperature at 66 °C, an 81% yield of the 5′-
O-debenzoylated product 34 was obtained after 48 h (Table 2,
entry 1). Applying these conditions to compound 30, we were
able to obtain an 84% yield of the debenzoylated product 35
(entry 2). Studies were conducted with nucleoside 30 to
determine if the amount of catalyst could be reduced and it was
found that compound 35 could also be obtained in good yield
by using 10 mol % catalyst though the reaction took 75 h (entry
3). Using 17 mol % catalyst at 66 °C, the 5′-O-benzoate esters
of compounds 3115 and 3215 were cleaved in excellent yields
(entries 4 and 5). Indeed, the yields for debenzoylation of 30-
32 (entries 2-5, Table 2) were superior to the yields obtained
for their acetylated counterparts (entries 1-3, Table 1). Subject-
ing compound 14 to 17 mol % catalyst in MeOH at 66 °C
resulted in cleavage of the acid-sensitive phosphoramidate group.
Nevertheless, the desired debenzoylated product 38 was obtained
in 56% yield by conducting the reaction in MeOH-THF (1:1),
increasing the amount of catalyst to 47 mol %, lowering the
temperature to 48 °C, and letting the reaction proceed for 72 h
(entry 6). Subjecting compound 3315 to the conditions employed
for compounds 29-32 or compound 14 did not yield the desired
product 39. The 1H NMR spectrum of the crude reaction mixture
suggested that depyrimidinylation occurred indicating that this
debenzolyation procedure may not be suitable when the pyri-
midine bears an electron-withdrawing group at the 4-position.28
We began our studies on the synthesis of the BMT derivatives
of the U-4-P analogues by coupling compound 7 to nucleoside
36 using a Mitsonobu reaction (Scheme 5). However, due to
the highly polar nature of the protected BMT product 40, it
proved quite challenging to purify the product and the yield
was only 15%. To decrease the polarity of the coupled products
and thereby facilitate their purification, we replaced the ethyl
groups in 7 with benzyl groups using the procedure outlined in
Scheme 6. Compound 4121 was treated with excess TMSBr for
30 h followed by MeOH to give the crude acid. Treatment of
this crude acid with excess tribenzylorthoformate at 150 °C for
2.5 h gave compound 42 in excellent yield (91%). Reaction of
42 with trimethylphosphite gave compound 43 in a 61% yield.
Treatment of compound 43 with KCN29 followed by exchange
of the potassium salt with H+ using a strong ion exchange matrix
gave acid 44 in 66% yield.
with Catalyst 20a
a All reactions were conducted in dry MeOH at room temperature with
6.5 mol % catalyst 20. b Percent yield of 5′-O-deacetylated product after
chromatography.
these conditions to U-4-P analogues 12, 13, 22, and 23, the
desired deacetylated products 25-28 were obtained in 81-87%
yield. It appears that the presence of the nitrogen at the
3-position of the base and the type of substituent at the
4-position, whether electron donating or withdrawing, have little
effect on the yield; however, the reaction proceeded faster with
compounds 12, 13, and 23 (6.5-9 h) compared to compounds
21 and 22, which required 18 h for the reaction to go to
completion.
Some of our U-4-P analogues were prepared with the OH
groups protected as benzoyl esters. The success we obtained
with the deacetylations prompted us to examine whether we
could also use Orita’s catalyst to perform selective debenzoy-
lations of these protected U-4-P analogues. To our knowledge,
only one report has appeared describing a selective cleavage of
the 5′-O-benzoyl ester of a benzoylated nucleoside. This was
achieved by Chavis et al., who subjected 2′,3′,5′-tri-O-benzoy-
luridine to NaBH4 in anhydrous EtOH for 3 h and obtained a
56% yield of the 5′-O-debenzoylated product.27 We also
performed this reaction using 2′,3′,5′-tri-O-benzoyluridine.
However, in our hands this reaction proceeded in only a 34%
yield and purification was difficult. Although Orita et al. did
not report the selective cleavage of the primary benzoates over
Compounds 26 and 35-37 were coupled to compound 44
using a Mitsunobu reaction in 50-72% yield and the products,
(28) We were unable to develop conditions that would allow us to
selectively deprotect 33. The fluorines in 33 are introduced by electrophilic
fluorination of 32 (see ref 15). Consequently, acetyl protection of the
hydroxyl groups was not an option. We also prepared compound 33, albeit
in low yield, by electrophilic fluorination of compound 32 except the 5′OH
was protected with a dimethoxytrityl (DMT) group. Surprisingly, all attempts
to remove the DMT group resulted in the formation of two inseparable
products in modest yield. 1H, 19F, 31P NMR suggested that one of these
compounds was compound 39 but we could not obtain it in pure form.
(29) Saady, M.; Lebeau, L.; Mioskowski, C. HelV. Chim. Acta 1995,
78, 670.
(27) Chavis, C.; Dumont, F.; Wightman, H.; Zeigler, H. C.; Imbach, J.
L. J. Org. Chem. 1982, 47, 202.
1406 J. Org. Chem., Vol. 73, No. 4, 2008

Anionic Pyrimidine Nucleotide Analogues
TABLE 2. 5′-O-Debenzoylation of Nucleosides 14 and 29-33 with Catalyst 20
a Percent yield of 5′-O-debenzoylated product after chromatography. b Reaction performed in anhydrous MeOH. c Reaction performed in anhydrous MeOH-
THF (1:1).
SCHEME 5. Coupling of 7 to Nucleoside 36
SCHEME 6. Synthesis of Compound 44
45-48, were readily purified by column chromatography
(Scheme 7). Global deprotection of compounds 45-47 was
accomplished by treatment with 6-9 equiv of TMSBr for 8 h
followed by ammonium hydroxide in methanol. The resulting
ammonium salts were purified using HPLC and then converted
to their corresponding sodium salts 49-51. BMT’s 49 and 50
were obtained cleanly in a 60% yield. We previously reported
that nucleoside 1 undergoes a slow dephosphonylation reaction
to give 1-(â-D-ribofuranosyl)-4-methyl-2-pyrimidinone and we
were unable to obtain 1 in pure form even after HPLC.15
Similarly, we were unable to obtain 51 in pure form and the
dephosphonylated product 52 consisted of about 15% of the
total product (∼54% overall yield). Due to the presence of the
ethyl groups in fluorophosphonate 48, 12 equiv of TMSBr and
5 days were required for complete phosphonate deprotection.
Surprisingly, after treatment of the crude product with am-
monium hydroxide, HPLC purification, and ion exchange
chromatography, the yield of BMT 53 (53%) was similar to
that for 49-51 indicating that, even after 5 days of being
J. Org. Chem, Vol. 73, No. 4, 2008 1407

Taylor et al.
SCHEME 7. Synthesis of Compounds 49-51 and 53
subjected to excess TMSBr, loss of product due to cleavage at
C-5′ was not a significant problem.
Mitsunobu reaction in THF.33 They found that formation of this
byproduct could be minimized by conducting the reaction in
dry pyridine.33 We found that Mitsunobu coupling of 27 and
44 in dry pyridine led to the formation of the desired compound
56 in 55% yield (Scheme 8). However, even when pyridine was
used as solvent, Mitsunobu coupling of 28 and 44 still gave
mainly compound 55. We also attempted this reaction using
the modified Mitsunobu reaction conditions that we described
previously for the synthesis of the BMT derivatives of uridine
and cytidine;21 however, this also proved unsuccessful. Clearly,
the cyclization of 28 upon activation of its 5′-OH occurs very
rapidly, much faster than it does for the corresponding aza
analogue 27.
Coupling of phosphoramidates 27 and 28 to 44 using the same
conditions as described above for the synthesis of 45-48 led
almost exclusively to the formation of byproducts whose
structures were assigned as 2-5′ anhydro compounds 54 and
55 (Figure 2).30 Only small amounts of the desired BMT
nucleotides were formed and these were inseparable from the
byproducts by silica gel chromatography.
This result is not unprecedented in that Shibuya and co-
workers,32a Kimura and co-workers,32b and Li and Miller32c have
shown that 2-5′ anhydro pyrimidine nucleosides can be readily
formed from the corresponding pyrimidine nucleosides using
triphenylphosphine and DIAD in dioxane or THF. Saady and
co-workers reported the formation of N3-5′ anhydro purine
nucleosides when attempting to couple phosphoric or phospho-
nic acid derivatives to the 5′-OH of purine nucleosides via the
At present, the reason for this difference in reactivity is not
clear. So far, we have been unable to couple acid 44 to
nucleoside 28 using a Mitsunobu reaction or any other reaction
conditions.34 Global deprotection of compound 56 was achieved
by using 6 equiv of TMSBr followed by concd NH4OH-
pyridine.17 After RP-HPLC and conversion to the sodium salt,
the desired product 57 was obtained in approximately 40% yield.
We found that some dephosphorylation occurred during this
process and about 3-4% of the product consisted of compound
(30) The structures of 54 and 55 were assigned based on the 31P NMR,
1H NMR, and mass spectral data of partially purified material. For example,
the LREIMS of the crude product that was isolated from the attempted
Mitsunobu reaction of phosphoramidate 28 and 44 exhibited a m/z of 494,
which is consistent with compound 55. In the 31P NMR this product
exhibited a chemical shift of 8.1 ppm, which is a downfield shift of
approximately 6.5 ppm compared to compound 28. This is consistent with
a phosphoramidate to phosphoryl imine conversion (see ref 31). The 1H
NMR of the product that was isolated from the attempted Mitsunobu reaction
of phosphoramidate 28 and 44 exhibited significant changes in comparison
to the 1H NMR of compound 28. These include the following: (a) the
doublet corresponding to phosphoramidate N-H proton in 28 is no longer
evident, (b) downfield shifts with increased geminal coupling for the two
diastereotopic protons attached to C-5′, (c) an upfield shift of about 0.4
ppm for the H-1′ proton, and (d) a downfield shift of approximately 0.5
ppm for the H-5 proton. Similar differences were also found between
compound 27 and the product that was isolated from the attempted
Mitsunobu reaction of phosphoramidate 27 and 44. All of these changes
are consistent with the conversion of a nucleoside into a 1,5-anhydro
nucleoside (see refs 32a-c).
1
58 (by H NMR), which we were unable to remove.35
Conclusions
Several bismethylene triphosphate (BMT) nucleotides of
uridine 4-phosphate (U-4-P) analogues were prepared. A key
step in this process was the use of the tin catalyst of Orita et al.
to effect the selective cleavage of both 5′-acetyl and 5′-benzoyl
esters in 2′,3′,5′-tri-O-acetyl or tri-O-benzoyl U-4-P analogues.
To the best of our knowledge, this represents the first general
approach to the selective cleavage of 5′-benzoyl esters from
benzoyl-protected nucleosides. Further studies on the generality
of this methodology, such as its application to selective
debenzoylations of benzoylated carbohydrates, are in progress.
(31) Napierzaj, A.; Zaeadzki, S.; Zwierzak, A. Tetrahedron 2000, 56,
6299.
(32) (a) Shibuya, S.; Kuninaka, A.; Hiroshi, Y. Chem. Pharm. Bull. 1974,
22, 719. (b) Kimura, J.; Fujisawa, Y.; Sawada, T. Chem. Lett. 1974, 691.
(c) Li., H.; Miller, M. J. J. Org. Chem. 1999, 64, 9289.
(33) Saady, M.; Lebeau, L.; Mioskowski, C. Tetrahedron Lett. 1995,
36, 2239.
(34) We have also attempted to convert phosphonic acid 44 to the
corresponding phosphonyl chloride in situ and then react the crude
phosphonyl chloride with 28. However, only trace amounts of the coupled
product were obtained using this procedure.
(35) Wada et al. reported the synthesis of unprotected 12 and found the
compound to be stable under basic conditions (0.1 M NaOH, concd
ammonia) unstable under acidic conditions (t1/2 ) 8 h in 0.1 N HCl) and
have a t1/2 of 60 min at 70 °C in 0.1 M ammonium acetate, pH 7.0. No
studies were performed under neutral conditions at room temperature (see
refs 17 and 18). We have found that its BMT nucleotide, compound 55,
undergoes very slow dephosphorylation in D2O at room temperature. The
dephosphorylation is presumed to proceed by a metaphosphate intermediate.
FIGURE 2. Structures of byproducts 54 and 55.
1408 J. Org. Chem., Vol. 73, No. 4, 2008

Anionic Pyrimidine Nucleotide Analogues
SCHEME 8. Synthesis of Compound 57
(d, J ) 4.8 Hz, 1H), 5.49 (overlapping dd, J ) 4.9 Hz, 1H), 5.42
(overlapping dd, J ) 4. 9 Hz, 1H), 4.14-4.26 (m, 4 H), 3.92 (d, J
) 11.8 Hz, 1H), 3.76 (d, J ) 12.3 Hz, 1H), 2.05 (s, 3H), 2.03 (s,
3H), 1.31 (t, J ) 7.3 Hz, 6 H); 13C NMR (75 MHz) δ 169.8, 169.3,
161.4, 144.5 (dt, J ) 22.3, 14.0 Hz), 134.5, 118.3 (dt, J ) 6.5, 3.1
Hz), 116.2 (dt, J ) 264, 214 Hz), 103.2, (t, J ) 4.2 Hz), 87.8,
83.3, 74.0, 70.5, 65.3 (d, J ) 5.6 Hz), 60.9, 20.4, 20.2, 16.1 (d, J
) 5.5 Hz); 31P NMR (121 MHz) δ 6.1 (t, J ) 108 Hz); 19F NMR
(282 MHz) δ -112.61 (d, J ) 110 Hz), -112.63 (d, J ) 110 Hz);
LREIMS m/z (rel intensity) 497.1 (M+, 9), 321.0 (46), 282.1 (33),
217.1 (100), 127.1 (44); HREIMS m/z calculated for C19H26F2NO10P
497.1262, found 497.1273
Several of these 5′-deprotected U-4-P analogues were converted
into their fully protected BMT nucleotides by coupling an
unsymmetrical, protected BMT analogue bearing a free phos-
phonic acid moiety at one of the terminal positions using a
Mitsunobu reaction. Global deprotection of these species was
achieved by using TMSBr followed by treatment with NH4-
OH-MeOH or NH4OH-pyridine, which afforded the UTP-
4-P intermediate analogues. These analogues are currently being
examined as inhibitors of purified CTPS and the results of these
studies will be reported in due course.
2′,3′-Di-O-acetylcytidine 4-N-[O,O-Bis(2-cyanoethyl)phospho-
ramidate] (27). Compound 27 was prepared from compound 12
as a white foam in 84% yield (5% MeOH-95% EtOAc) by using
the general procedure described above. 1H NMR (300 MHz) δ 10.25
(br s, 1H), 7.94 (d, J ) 8.0 Hz, 1H), 6.29 (d, J ) 7.9 Hz), 6.01 (s,
1H), 5.39 (s, 1H), 4.15-4.31 (m, 5H), 3.85 (d, J ) 11.3 Hz, 1H),
3.74 (d, J ) 11.8 Hz, 1H), 2.75 (t, J ) 5.9 Hz, 4H), 2.06 (s, 3H),
2.01 (s, 3H); 13C NMR (75 MHz) δ 170.0, 169.8, 160.8, 149.3,
141.3, 117.3, 101.53 (d, J ) 11.9 Hz), 87.3, 83.8, 73.4, 71.2, 61.5,
61.41 (d, J ) 20.9 Hz), 20.6, 20.4, 19.6 (d, J ) 6.6 Hz); 31P NMR
(121 MHz) δ 6.5; LR+ESIMS m/z (rel intensity) 514.1 (M + 1,
100); HR+ESIMS m/z calculated for C19H25N5O10P (M + 1)
514.1339, found 514.1346.
General Procedure for the Debenzoylation of 29-32 To Give
Nucleosides 34-37. Catalyst 20 (17 mol %) was added to a solution
of the nucleoside (29-32) in dry MeOH (0.133 mmol of nucleoside/
mL of dry MeOH) and the resulting solution was stirred for 48 h
at 66 °C (oil bath). The solution was concentrated and the product
purified by flash chromatography (34, 36, and 37) or recrystalization
(35).
Experimental Section
2′,3′,5′-Tri-O-acetylcytidine 4-N-[O,O-Bis(2-cyanoethyl)phos-
phoramidate] (12). To a solution of 2′,3′,5′-tri-O-acetylcytidine36
(1.5 g, 4.06 mmol) and tetrazole (0.569 g, 8.12 mmol) in dry THF
(20 mL) was added a solution of phosphoramidite 1537 (2.20 g,
8.12 mmol) in dry THF (10 mL). After 20 h, the solution was cooled
to -10 °C and tert-butyl hydroperoxide (2.43 mL, 24.36 mmol)
was added and the solution stirred for 1 h. The reaction was diluted
with EtOAc and washed with sat. NaHCO3 and the aqueous layer
was back extracted with EtOAc. The combined organic layers were
dried and concentrated and the residue purified by flash chroma-
tography (2% MeOH-98% EtOAc to 4% MeOH-96% EtOAc)
1
to give 12 as a white foam (1.298 g, 58%). H NMR (300 MHz)
δ 10.25 (br s, 1H), 7.43 (d, J ) 7.7 Hz, 1H), 6.13 (d, J ) 8.5 Hz,
1H), 5.94 (d, J ) 4.9 Hz, 1H), 5.39 (m, 2H), 4.20-4.34 (m, 7H),
2.75 (t, J ) 6.1 Hz, 4H), 2.07-2.11 (three overlapping singlets,
9H); 13C NMR (75 MHz) δ 170.2, 169.6, 160.4, 148.9, 140.1, 117.2,
101.9 (d, J ) 14.8 Hz), 88.4, 79.9, 72.9, 69.9, 62.9, 61.47 (d, J )
5.4 Hz), 20.74, 20.42, 20.39, 19.64 (d, J ) 7.2 Hz); 31P NMR (121
MHz) δ 6.53; LR+ESIMS m/z (rel intensity) 556.1 (M + 1, 100),
298.1 (52); HR+ESIMS m/z calculated for C21H27N5O11P (M + 1)
556.1453, found 556.1445.
General Procedure for the Deacetylation of 12, 13, and 21-
23 To Give Nucleosides 24-28. To a solution of the nucleoside
(12, 13, 21-23) in dry MeOH (0.133 mmol of nucleoside/mL of
MeOH) was added catalyst 2038 (6.5 mol %). This solution was
stirred for 6.5-18 h (see Table 1 for reaction times) followed by
concentration and purification of the product using flash chroma-
tography.
{1-[3,4-Bis(benzoyloxy)-5-hydroxymethyltetrahydrofuran-2-
yl]-2-oxo-1,2-dihydropyridin-4-ylmethyl}phosphonic Acid Dim-
ethyl Ester (36). Compound 36 was prepared from compound 3115
as a white foam in 90% yield (95% EtOAc-5% EtOH then 90%
EtOAc-10% EtOH) by using the general procedure described
1
above. H NMR (300 MHz) δ 7.90 (d, J ) 7.9 Hz, 4H), 7.77 (d,
J ) 7.3 Hz, 1H), 7.45-7.52 (m, 2H), 7.28-7.35 (m, 4H), 6.40 (s,
1H), 6.39 (d overlapping with s, J ) 5.1 Hz, 1H), 6.28 (d, J ) 7.3
Hz, 1H), 5.84-5.93 (m, 2H), 4.45 (s, 1H), 3.91-4.09 (m, 3H), l
3.71 (d, J ) 10.9 Hz, 6H), 2.96 (d, J ) 22.4 Hz, 2H); 13C NMR
(75 MHz) δ 165.5, 165.0, 162.0, 145.5 (d, J ) 8.6 Hz), 134.0,
133.42, 133.36, 129.73, 129.66, 128.86, 128.76, 128.34, 128.30,
121.3 (d, J ) 8.7 Hz), 108.62 (d, J ) 3.2 Hz), 89.8, 83.9, 74.5,
71.4, 61.5, 53.0 (d, J ) 4.1 Hz), 32.3 (d, J ) 136.3 Hz); 31P NMR
(121 MHz) δ 27.9; LR+ESIMS m/z (rel intensity) 558.1 (M + 1,
100), 341.1 (32); HR+ESIMS m/z calculated for C27H29NO10P
558.1529, found 558.1534.
[Difluoro{1-[3,4-bis(acetoxy)-5-hydroxymethyltetrahydrofu-
ran-2-yl]-2-oxo-1,2-dihydropyridin-4-yl}methyl]phosphonic Acid
Diethyl Ester (26). Compound 26 was prepared from compound
238 as a white foam in 81% yield (80% EtOAc-20% hexane) by
using the general procedure described above. 1H NMR (300 MHz)
δ 7.90 (d, J ) 7.4 Hz, 1H), 6.67 (s, 1H), 6.38 (d, J ) 7.2 Hz), 6.21
(36) Nowak, I.; Conda-Sheridan, M.; Robins, M. J. J. Org. Chem. 2005,
70, 7455.
(37) Gaffney, P. R.; Reese, C. B. J. Chem. Soc. Perkin 2001, 1, 192.
(38) (a) Chu, C. V.; Murray, J. D. J. Chem. Soc. A 1971, 360. (b) Puff,
H.; Hevendehl, H.; Hoffer, K.; Reuter, H.; Schuh, W. Organomet. Chem.
1985, 287, 163.
{1-[3,4-Bis(benzoyloxy)-5-hydroxymethyltetrahydrofuran-2-
yl]-2-oxo-2,3-dihydro-1H-pyrimidin-4-ylidenemethyl}-
phosphonic Acid Dimethyl Ester (37). Compound 37 was prepared
from compound 3215 as a white foam in 93% yield (90% EtOAc-
1
10% EtOH) by using the general procedure described above. H
J. Org. Chem, Vol. 73, No. 4, 2008 1409

Taylor et al.
NMR (300 MHz) δ 10.40 (s, 1H), 7.93 (d, J ) 7.3 Hz, 4H), 7.49-
7.56 (m, 2H), 7.23-7.39 (m, 4H), 7.01 (d, J ) 6.8 Hz, 1H), 6.20
(d, J ) 4.7 Hz, 1H), 5.75-5.80 (m, 2H), 5.62 (d, J ) 6.7 Hz, 1H),
4.38 (s, 1H), 3.85-3.40 (m, 3H), 3.67 (d, J ) 11.3 Hz, 6H), 2.68
(s, 1H); 13C NMR (75 MHz) δ 165.5, 165.2, 152.3 (d, J ) 22.7
Hz), 148.5, 133.4, 131.9, 129.7, 129.6, 128.9, 128.5, 128.4, 128.3,
104.2 (d, J ) 22.7 Hz), 86.5, 83.5, 73.4 (d, J ) 196.5 Hz), 61.9,
52.1 (dd, J ) 4.0, 1.8 Hz); 31P NMR (121 MHz) δ 27.7; LR+-
ESIMS m/z (rel intensity) 559.2 (M + 1, 100), 341.1 (17), 219.1
(28); HR+ESIMS m/z calculated for C26H28N2O10P (M + 1)
559.1482, found 559.1494.
5.4 Hz), 66.9 (d, J ) 6.6 Hz), 51.7 (d, J ) 6.1 Hz), 27.3 (dd, J )
133.2, 89.5 Hz); 31P NMR (121 MHz, CD3OD) δ 41.9, 22.5 (d, J
) 7.6 Hz), 20.1; LR+ESIMS m/z (rel intensity) 539.0 (M + 1,
100); HR+ESIMS m/z calculated for C24H30O8P3 539.1154, found
539.1159.
General Procedure for the Preparation of Compounds 45-
48. To a solution of the nucleoside (26, 35-37), compound 44
(1.2 equiv), and DIAD (3 equiv) in dry THF (0.10 mmol of
nucleoside/mL of THF) was added a solution of triphenylphosphine
(3 equiv) in dry THF (8 mL, 0.17 mmol of triphenylphosphine/mL
of THF) over a period of 1.5-2 h by syringe pump. The reaction
was stirred for 3.5 h then the solution was concentrated and the
residue purified with use of flash chromatography.
(Bromomethylbenzyloxyphosphinoylmethyl)phosphonic Acid
Dibenzyl Ester (42). Trimethylsilyl bromide (2.94 mL, 22.3 mmol)
was added to a solution of compound 4121 (1.5 g, 4.46 mmol) in
dry dichloromethane (11 mL) and the solution was then stirred for
30 h. After removal of the solvent, the resulting residue was
subjected to high vacuum for several hours. Dry MeOH (15 mL)
was added and the solution stirred for 30 min. The solution was
then concentrated and this process was repeated two more times
and the final residue subjected to high vacuum overnight. Triben-
zylorthoformate (8.88 g, 26.8 mmol) was added and the mixture
heated to 150 °C for 2.5 h. The mixture was subjected to high
vacuum rotary evaporation to remove the benzyl alcohol and the
resulting residue was purified with use of flash chromatography
(50% EtOAC-50% hexane then 70% EtOAC-30% hexane) to give
compound 42 as a pale yellow oil that solidified as a white solid
after being subjected to high vacuum overnight (2.12 g, 91%). Mp
{1-[3,4-Bis(benzoyloxy)-5-(bis[(benzyloxyphosphorylmethyl)-
benzyloxyphosphorylmethyl]methyloxyphosphoryl) tetrahydro-
furan-2-yl]-2-oxo-1,2-dihydropyridin-4ylmethyl}phosphonic Acid
Dimethyl Ester (46). Compound 46 was prepared from compound
36 as a white foam in 50% yield (100% EtOAc then 5% EtOH-
95% EtOAc then 10% EtOH-90% EtOAc) by using the general
procedure described above. 1H NMR (300 MHz) δ 7.82-7.89 (m,
4H), 7.61-7.69 (m, 1H), 7.42-7.46 (m, 2H), 7.15-7.37 (m, 17H),
6.52-6.64 (m, 1H), 6.35 (s, 1H), 6.22 (overlapping dd, J ) 7.2
Hz, 1H), 5.90 (overlapping dd, J ) 5.3 Hz, 0.5 H), 5.77
(overlapping dd, J ) 5.3 Hz, 0.5 H), 5.63-5.70 (m, 1H), 4.85-
5.15 (m, 6H), 4.40-4.69 (m, 3H), 3.63-3.79 (m, 9H), 2.63-3.17
(m, 6H); 13C NMR (75 MHz) δ 165.3, 165.23, 165.17, 165.10,
161.79, 161.76, 161.75, 161.73, 145.54, 145.49, 145.42, 145.39,
145.32, 135.89, 135.83, 135.81, 135.79, 135.76, 135.74, 133.61,
133.59, 133.52, 133.06, 133.00, 132.8, 129.88, 129.87, 129.79,
129.78, 128.79, 128.73, 128.71, 128.68, 128.67, 128.61, 128.60,
128.56, 128.51, 128.49, 128.48, 128.44, 128.42, 128.20, 128.17,
128.12, 128.08, 128.02, 128.00, 120.91, 120.89, 120.84, 120.82,
120.78, 108.81, 108.78, 108.76, 108.62, 108.59, 87.6, 87.2, 86.9,
81.23, 81.17, 87.14, 81.09, 74.66, 74.61, 74.58, 70.8, 70.7, 70.6,
687.23, 68.18, 68.13, 68.11, 68.06, 68.02, 67.97, 67.21, 67.14,
67.12, 67.09, 67.08, 65.66, 65.62, 65.34, 65.30, 65.25, 53.68, 53.62,
53.41, 53.37, 53.10, 53.08, 53.05, 53.03, 32.5 (d, J ) 134.5 Hz),
26.93-29.61 (m); 31P NMR (121 MHz) δ 39.78-39.84 (m),
39.36-39.54 (m), 27.74, 27.72, 23.3-23.9 (m), 21.7-21.9 (m);
LR+ESIMS m/z (rel intensity) 1078 (M + 1, 100); HR+ESIMS
m/z calculated for C51H56N1O17P4 1078.2499, found 1078.2528.
{1-[3,4-Bis(benzoyloxy)-5-(bis[(benzyloxyphosphorylmethyl)-
benzyloxyphosphorylmethyl]methyloxyphosphoryl)tetra-
hydrofuran-2-yl]-2-oxo-2,3-dihydro-1H-pyrimidin-4-ylidenemethyl}-
phosphonic Acid Dimethyl Ester (47). Compound 47 was prepared
from compound 37 as a white foam in 72% yield (95% EtOAc-
1
50-51 °C; H NMR (300 MHz) δ 7.20-7.40 (m, 15 H), 4.93-
5.13 (m, 6H), 3.32-3.50 (m, H), 2.49-2.77 (m, 2H); 13C NMR
(75 MHz) δ 135.7, 135.62, 135.58, 135.54, 135.45, 128.64, 128.58,
128.15, 128.13, 128.06, 68.3 (d, J ) 6.8 Hz), 68.0 (d, J ) 6.4 Hz),
67.5 (d, J ) 6.2 Hz), 25.9 (dd, J ) 144.8, 90.0 Hz), 21.6 (d, J )
101.9 Hz); 31P NMR (121 MHz) δ 40.1 (d, J ) 2.3 Hz), 21.1 (d,
J ) 2.3 Hz); LREIMS m/z (rel intensity) 433.1 (M+ - 91, 5), 327.0
(22), 91.1 (100); HREIMS m/z calculated for C16H18BrO5P2 (M+
- 91) 432.9790, found 432.9783.
[(Dibenzyloxyphosphorylmethyl)benzyloxyphosphinoylmethyl]-
phosphonic Acid Dimethyl Ester (43). A solution of compound
42 (0.300 g, 0.575 mmol) in freshly distilled trimethylphosphite
(2.1 mL, 33 equiv) was heated to 120 °C for 12 h. The mixture
was subjected to high vacuum rotary evaporation to remove the
trimethylphosphite and dimethyl methylphosphonate. The resulting
residue was purified with flash chromatography (5% MeOH-95%
EtOAc) to give compound 43 as a pale yellow oil (0.203 g, 61%).
1H NMR (300 MHz) δ δ7.20-7.40 (m, 15H), 4.95-5.17 (m, 6H),
3.75 (d, J ) 9.5 Hz, 3H), 3.67 (d, J ) 9.5 Hz, 3H), 2.60-2.90 (m,
4H); 13C NMR (75 MHz) δ 135.8, 135.7, 135.6, 128.5, 128.44,
128.37, 128.01, 127.94, 127.91, 68.0 (d, J ) 5.5 Hz), 67.8 (d, J )
5.3 Hz), 66.9 (d, J ) 6.5 Hz), 53.0 (d, J ) 6.5 Hz), 52.8 (d, J )
6.5 Hz), 28.5 (dd, J ) 133.0, 89.0 Hz), 27.4 (dd, J ) 134.0, 88.9
Hz); 31P NMR (121 MHz) δ δ 39.8 (t, J ) 4.7 Hz), 23.6 (d, J )
4.0 Hz), 21,9 (d, J ) 3.6 Hz); LREIMS m/z (rel intensity) 552.1
(M+, 4%), 355.0 (100), 279.0 (20), 91.0 (100); HREIMS m/z
calculated for C25H31O8P 552.1232, found 552.1237.
1
5% MeOH) by using the general procedure described above. H
NMR (300 MHz) δ 10.37 (s, 1H), 7.83-7.92 (m, 4H), 7.49-7.59
(m, 2H), 7.20-7.39 (m, 19H), 7.01-7.15 (m, 1H), 6.40-6.53 (m,
1H), 5.50-5.92 (m, 3H), 4.90-5.21 (m, 6H), 4.30-4.60 (m, 3 H),
3.61-3.89 (m, 9H), 2.65-3.23 (m, 4H); 13C NMR (75 MHz) δ
165.30, 165.25, 165.18, 165.11, 152.14, 152.01, 151.96, 148.42,
148.33, 148.29, 135.80, 135.71, 135.64, 135.58, 135.55, 133.54,
133.51, 133.46, 131.28, 131.08, 129.77, 129.67, 128.70, 128.61,
128.53, 128.50, 128.47, 128.43, 128.40, 128.36, 104.65, 104.48,
104.35, 104.18, 85.42, 85.25, 85.17, 85.09, 80.91, 80.81, 80.76,
80.66, 75.47, 75.30, 73.00, 72.88, 72.71, 72.42, 72.37, 72.30, 71.00,
70.85, 68.17, 68.13, 68.06, 67.96, 67.87, 67.80, 67.26, 67.18, 67.08,
67.06, 67.00, 66.97, 66.88, 65.80, 65.73, 65.42, 65.34, 53.75, 53.66,
53.49, 53.42, 53.36, 53.29, 53.11, 53.06, 52.97, 52.06, 52.03, 52.00,
51.97, 26.22-30.02 (m); 31P NMR (121 MHz) δ 39.20-39.84 (m),
27.95, 27.83, 27.81, 27.75, 23.28-24.16 (m), 21.66-21.90 (m);
LR+ESIMS m/z (rel intensity) 1079.2 (M + 1, 100), 540.1 (30),
219.1 (10); HR+ESIMS m/z calculated for C50H55N2O17P4 (M +
1) 1079.2451, found 1079.2461.
[(Dibenzyloxyphosphorylmethyl)benzyloxyphosphinoylmethyl]-
phosphonic Acid Monomethyl Ester (44). KCN (147 mg, 2.26
mmol) was added to a solution of compound 43 (1.00 g, 1.81 mmol)
in dry DMF (20 mL). After the solution was heated to 75 °C over
5 h, the solution was concentrated and the product was purified by
using flash chromatography (MeOH-CH2Cl2-NH4OH, 2:10:0.5).
The purified material was dissolved in MeOH-H2O (7:3, 150 mL)
and mixed with Dowex 50W×8 H+ ion-exchange resin and stirred
for 4 h. The mixture was filtered and the filtrate concentrated to
1
give compound 44 as a pale yellow oil (644 mg, 66%). H NMR
(300 MHz, CD3OD) δ 7.34-7.41 (m, 15H), 4.90-5.36 (m, 6H),
3.65 (d, J ) 11.4 Hz), 3.03 (overlapping dd, J ) 20.2 Hz), 2.82
(overlapping dd, J ) 19.5 Hz); 13C NMR (75 MHz, CD3OD) δ
136.0, 135.88, 135.78, 135.74, 135.70, 135.66, 128.18, 128.13,
128.0, 127.78, 127.75, 127.69, 68.0 (d, J ) 5.5 Hz), 67.95 (d, J )
[Difluoro{1-[3,4-bis(acetoxy)-5-(bis[(benzyloxyphosphorylm-
ethyl)benzyloxyphosphorylmethyl]methyloxyphosphoryl)-
tetrahydrofuran-2-yl]-2-oxo-1,2-dihydropyridin-4-yl}methyl]-
phosphonic Acid Diethyl Ester (48). Compound 48 was prepared
1410 J. Org. Chem., Vol. 73, No. 4, 2008

Anionic Pyrimidine Nucleotide Analogues
from compound 26 as a white foam in 68% yield (95% EtOAc-
used for preparative HPLC: solvent A, 100 mM TEAA, pH 8.8;
solvent B, CH3CN. The following elution profile was used: 0-13
min, 99% A-1% B; 13-40 min, linear gradient of 99% A-1% B
to 94% A-6% B. Flow rate ) 8 mL/min. tr ) 27.36 min.
Conversion of the compound to the hexasodium salt by using a
Dowex 50W×8 Na+ ion exchange column gave nucleotide 51 as
a white powder (37.0 mg, 54%). Analysis of the NMR spectra
revealed that the powder contained approximately 15% of com-
1
5% MeOH) by using the general procedure described above. H
NMR (300 MHz) δ 7.71-7.79 (m, 1H), 7.16-7.38 (m, 15H), 6.69
(s, 1H), 6.28-6.46 (m, 2H), 5.31-5.55 (m, 2H), 4.80-5.15 (m,
6H), 4.13-4.61 (m, 7H), 3.65-3.78 (m, 3H), 2.59-3.14 (m, 4H),
2.04 (s, 6H), 1.31 (t, J ) 6.9 Hz); 13C NMR (125 MHz) δ 169.43,
169.38, 169.18, 161.0, 144.1-147.8 (m), 135.70, 135.61, 135.53,
133.55, 133.47, 133.32, 128.49, 128.07, 128.00, 129.97, 127.90,
118.7, 116.0 (dt, J ) 258, 208 Hz), 113.14-103.35 (m), 87.20,
87.09, 86.73, 80.52, 80.43, 80.33, 73.69-73.89 (m), 69.37, 67.82-
68.10 (m), 66.91-67.26 (m), 64.67-65.16 (m), 52.77-53.50 (m),
26.08-30.30 (m), 20.44, 20.34, 16.26, 16.19; 31P NMR (121 MHz)
δ 39.27-40.06 (m), 23.50-24.14 (m), 21.63-21.77 (m), 6.26 (t,
J ) 108.1 Hz), 6.22 (108.1 Hz); 19F NMR (283 MHz) δ -112.43
(d, J ) 108.0 Hz), -112.44 (d, J ) 107.4 Hz), -112.47 (d, J )
108.6 Hz), -112.53 (d, J ) 107.5 Hz); LR+ESIMS m/z (rel
intensity) 1018.2 (M + 1, 100), 550.6 (28); HR+ESIMS m/z
calculated for C43H54NO17F2P4 (M + 1) 1018.2310, found 1018.2324.
General Procedure for the Deprotection of 45-48. TMSBr
(6 equiv for 45, 9 equiv for 46 and 47, 12 equiv for 48) was added
to a solution of 45-48 in dry CH2Cl2 (0.040 mmol/mL of CH2-
Cl2). The reaction mixture was stirred for 8 h (for 45-47) or 5
days (for 48). The solution was then concentrated, dry CH2Cl2 was
added, and the mixture was concentrated again, then this was
repeated and the residue was subjected to high vacuum for 1 h. A
solution of NH4OH-MeOH (5 mL, 3:2) was then added to the
residue and the resulting solution was stirred for 24 h. The solution
was concentrated and the resulting white residue was dissolved in
water and lyophilized. The resulting white powder was dissolved
in 100 mM triethylammonium acetate (TEAA) (pH 7.0 for crude
49, 50, and 53) or pH 8.8 (for crude 51) and purified by using
semipreparative reversed-phase HPLC with 100 mM TEAA-CH3-
CN as the eluent. Compounds obtained after HPLC purification
were dissolved in water and repeatedly lyophilized until all of the
TEAA was removed (by 1H NMR). The resulting triethylammonium
salts were converted into their penta- (compound 49) or hexa-
(compounds 50, 51, and 53) sodium salts, using a Dowex 50W×8
Na+ form ion exchange column.
{1-[3,4-Bis(hydroxy)-5-{[(dihydroxyphosphorylmethyl)-
hydroxyphosphorylmethyl]hydroxyphosphoryl}tetrahydrofuran-
2-yl]-2-oxo-1,2-dihydropyridin-4-ylmethyl}phosphonic Acid, Hex-
asodium Salt (50). Compound 50 was prepared from compound
46 (0.108 g, 0.0928 mmol) by using the general procedure described
above. The following solvent systems were used for preparative
HPLC: solvent A, 100 mM TEAA, pH 7.0; solvent B, CH3CN.
The following elution profile was used: 0-15 min, 99% A-1%
B; 15-50 min, linear gradient of 99% A-1% B to 94% A-6% B.
Flow rate ) 8 mL/min. tr ) 30.04 min. Conversion of the compound
to the hexasodium salt by using a Dowex 50W×8 Na+ ion exchange
column gave nucleotide 50 as a white powder (38.4 mg, 60%). 1H
NMR (300 MHz, D2O) δ 7.85 (d, J ) 5.5 Hz, 1H), 6.64 (d, J )
5.5 Hz, 1H), 6.43 (s, 1H), 6.13 (s, 1H), 4.12-4.30 (m, 5H), 2.86
(d, J ) 21.3 Hz, 2H), 2.137-2.34 (m, 4H); 13C NMR (75 MHz,
D2O) δ 163.2, 152.6 (d, J ) 8.4 Hz), 132.3, 118.0 (d, J ) 6.6 Hz),
111.9 (d, J ) 2.7 Hz), 83.1, 82.8 (d, J ) 7.5 Hz), 74.8, 69.1, 63.0
(d, J ) 3.6 Hz), 36.4 (d, J ) 121.9 Hz), 29.7-33.3 (m); 31P NMR
(121 MHz, D2O) δ 27.2 (Pâ), 18.3 (PR), 16.8, 15.8 (Pγ); LR-ESIMS
m/z (rel intensity) 556 (M - 6Na + 5H+, 100). HR-ESIMS m/z
calculated for C13H22NO15P4 (M - 6Na + 5H+) 555.9940, found
555.9953. The analytical HPLC chromatogram of 50, using the
elution profile described above for the purification of 50, indicated
it to be greater than 98.8% pure (flow rate 1 mL/min, tr ) 12.94
min).
1
pound 52. H NMR (300 MHz, D2O) δ 8.37, 8.34 (overlapping
singlets, 1H), 6.74, 6.66 (overlapping singlets, 1H), 5.87 (s, 1H),
4.42 (br s, 4H), 4.12 (s, 1H), 3.01 (d, J ) 16.5 Hz, 0.4 H, exchange
with solvent deuterons), 2.10-2.34 (m, 4H); 31P NMR (121 MHz,
D2O) δ 27.6 (Pâ), 18.3 (PR), 15.8 (Pγ), 14.0, 1.05; LR-ESIMS m/z
(rel intensity) 557.0 (M - 6Na + 5H+, 100), 477.0 (85).
HR-ESIMS m/z calculated for C12H21N2O15P4 (M - 6Na + 5H+)
556.9892, found 556.9896.
[Difluoro{1-[3,4-bis(hydroxy)-5-(bis{[(dihydroxyphosphory-
lmethyl)hydroxylphosphorylmethyl]hydroxyphosphoryl})tetra-
hydrofuran-2-yl]-2-oxo-1,2-dihydropyridin-4-yl}methyl]phosphonic
Acid, Hexasodium Salt (53). Compound 53 was prepared from
compound 48 (0.110 g, 0.108 mmol) by using the general procedure
described above. The following solvents were used for preparative
HPLC: solvent A, 100 mM TEAA, pH 7.0; solvent B, CH3CN.
The following elution profile was used: linear gradient of 99%
A-1% B to 93% A-7% B over 40 min. Flow rate ) 8 mL/min.
tr ) 28.24 min. Conversion of the compound to the hexasodium
salt by using a Dowex 50W×8 Na+ ion exchange column gave
1
nucleotide 53 as a white powder (41.0 mg, 53%). H NMR (300
MHz, D2O) δ 7.95 (d, J ) 6.1 Hz, 1H), 6.72 (s, 2H), 6.14 (s, 1H),
4.42-4.26 (m, 5H), 2.14-2.29 (m, 4H); 13C NMR (75 MHz, D2O)
δ 163.4, 150.8 (dt, J ) 22.7, 11.0 Hz), 133.1, 120.2 (dt, J ) 264,
176 Hz), 116.2 (t, J ) 7.0 Hz), 106.6 (t, J ) 4.5 Hz), 89.6, 82.7
(d, J ) 7.2 Hz), 74.9, 69.1, 63.0 (d, J ) 4.2 Hz), 29.8-33.4 (m);
31P NMR (121 MHz, D2O) δ 27.7 (Pâ), 18.3 (PR), 15.9 (Pγ), 4.0 (t,
J ) 84.4 Hz); 19F NMR (282 MHz, D2O) δ -110.7 (d, J ) 85.3
Hz); LR-ESIMS m/z (rel intensity) 591.9 (M - 6Na + 5H+, 100);
HR-ESIMS m/z calculated for C13H21F2NO15P4 (M - 6Na + 5H+)
591.9752, found 591.9742. The analytical HPLC chromatogram of
53, obtained by using the elution profile described above for the
purification of 53, indicated that the product was greater than 99%
pure (flow rate 1 mL/min, tr ) 17.42 min).
2′,3′-Di-O-acetyl-5′-(bis[(benzyloxyphosphorylmethyl)-
benzyloxyphosphorylmethyl]methyloxyphosphoryl)cytidine 4-N-
[O,O-Bis(2-cyanoethyl)phosphoramidate] (56). To a solution of
nucleoside 27 (0.051 g, 0.10 mmol), compound 44 (0.087 g, 0.15
mmol), and DIAD (0.051 mL, 0.26 mmol) in dry pyridine (1 mL)
was added a solution of triphenylphosphine (0.65 mg, 0.25 mmol)
in dry pyridine (1 mL) over a period of 1.5 h by syringe pump.
The reaction was stirred for 7 h then concentrated and the residue
was purified with FC (10% MeOH-90% EtOAc) to give compound
1
56 as a white foam (0.057 g, 55%). H NMR (300 MHz) δ 10.40
(br s, 1H), 7.68-7.81 (m, 1H), 7.21-7.38 (m, 15H), 6.08 (m, 2H),
5.30-5.57 (m, 2H), 4.83-5.39 (m, 6H), 4.11-4.59 (m, 7H), 3.66-
3.79 (m, 3H), 2.55-3.18 (m, 8H), 2.02-2.08 (m, 6H); 13C NMR
(75 MHz) δ 169.7, 169.6, 169.5, 160.0, 148.4, 140.9, 140.4, 135.8,
135.7, 135.6, 128.6, 128.1, 128.07, 128.01, 116.8, 103.4, 103.2,
102.9, 86.7, 86.5, 86.3, 81.1, 80.98, 80.87, 72.8, 72.5, 70.1, 70.0,
68.24, 68.17, 68.08, 68.00, 67.5, 67.4, 67.2, 67.1, 65.22, 65.16,
65.0, 64.9, 64.8, 61.34, 61.26, 53.6, 53.5, 53.23, 53.16, 25.5-30.1
(m), 20.5, 20.4, 19.7, 19.6; 31P NMR (121 MHz) δ 39.0-39.9 (m),
23.6-24.3 (m), 21.5-21.8 (m), 6.9; LR+ESIMS m/z (rel intensity)
1034.2 (M + 1, 100), 944.1 (50), 536.6 (86); HR+ESIMS m/z
calculated for C43H52N5O17P4 (M + 1) 1034.2309, found 1034.2280.
5′-(Bis[(dihydroxyphosphorylmethyl)hydroxyphosphor-
ylmethyl]hydroxyphosphoryl)cytidine 4-N-Phosphoramidic Acid,
Hexasodium Salt (57). TMSBr (0.063 mL, 0.52 mmol) was added
to a solution of nucleotide 56 (0.90 mg, 0.087 mmol) in 2 mL of
dry CH2Cl2 and the solution was stirred for 8 h. The solution was
concentrated and the resulting residue was dissolved in dry CH2-
{1-[3,4-Bis(hydroxy)-5-{[(dihydroxyphosphorylmethyl)-
hydroxyphosphorylmethyl]hydroxyphosphoryl}tetrahydrofuran-
2-yl]-2-oxo-2,3-dihydro-1H-pyrimidin-4-ylidenemethyl}-
phosphonic Acid, Hexasodium Salt (51). Compound 51 was
prepared from compound 47 (0.108 g, 0.100 mmol) by using the
general procedure described above. The following solvents were
J. Org. Chem, Vol. 73, No. 4, 2008 1411

Taylor et al.
Cl2 and then concentrated again. This process was repeated and
the resulting residue was subjected to high vacuum for 12 h. A
solution of NH4OH-pyridine (9:1, 5 mL) was added, followed by
stirring for 36 h. The mixture was concentrated and the resulting
residue was dissolved in water and lyophilized to give a white
powder. The product was then purified by using semipreparative
RP-HPLC with the following solvents: solvent A, 100 mM TEAA,
pH 9.0; solvent B, CH3CN. The following elution profile was
used: 0-35 min, 100% A; 35-55 min, linear gradient of 100% A
to 90% A-10% B. Flow rate ) 8 mL/min. tr ) 49.2 min.
Conversion of the compound to the hexasodium salt by using a
Dowex 50W×8 Na+ ion exchange column gave nucleotide 57 as
a white powder (24.0 mg, 40%). 1H NMR (300 MHz, D2O) δ 7.94
(br s, 1H), 6.40 (br s, 0.6H), 5.86 (s, 1H), 4.00-4.26 (m, 5H),
2.07-3.30 (m, 4H); 31P NMR (121 MHz, D2O) δ 28.7 (Pâ), 19.43
(PR), 16.9 (Pγ), -2.3; LR-ESIMS m/z (rel intensity) 557.9 (M -
6Na + 5H+, 22), 478.0 (100); HR-ESIMS m/z calculated for
C11H20N3O15P4 (M - 6Na + 5H+) 557.9845, found 557.9835. We
did not attempt to obtain a 13C NMR due to a spontaneous
dephosphorylation that occurs when in D2O. The analytical HPLC
chromatogram of 57, using 100% solvent A as eluent, indicated it
to be greater than 98.8% pure (flow rate 1 mL/min, tr ) 14.78
min) with compound 58 (tr ) 9.76 min) constituting about 1% of
the product. In contrast, 1H NMR indicated that approximately
3-4% of compound 58 was present.
Acknowledgment. This work was supported by a Natural
Sciences and Engineering Research Council (NSERC) of
Canada Discovery Grant to S.D.T., and an NSERC Collaborative
Health Research Project Grant to S.L.B. and S.D.T.
Supporting Information Available: Characterization data
for compounds 13, 14, 22, 24, 25, 28, 34, 35, 38, 45, and 49,
and NMR spectra [(1H, 13C, 31P (when applicable), 19F (when
applicable)] and HPLC chromatograms (when applicable) for
compounds 12-14, 22, 24-28, 34-38, 42-51, 53, 56, and 57.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO702249J
1412 J. Org. Chem., Vol. 73, No. 4, 2008