Synthesis of Polyphosphorylated AZT Derivatives for the Development of Specific Enzyme Immunoassays

Article abstract of DOI:10.1021/jo982502p

The synthesis of a series of analogues of the different polyphosphorylated metabolites of AZT has been carried out. The compounds were designed in order to raise specific antibodies for the development of highly sensitive titration kits for the intracellular metabolites of AZT. The pyrophosphate moiety in AZT-DP and AZT-TP analogues is mimicked by the methylene bisphosphonate group to provide in vivo stability of the compounds. An ω-amino linker was introduced at the N³ position on the base to allow the further anchoring of the compounds to a carrier protein before immunization, tentitatively focusing the specificity of the immune response toward the phosphate mimic moiety and the nonnatural sugar.

Full text of DOI:10.1021/jo982502p

J . Org. Chem. 1999, 64, 5083-5090
5083
Syn th esis of P olyp h osp h or yla ted AZT Der iva tives for th e
Develop m en t of Sp ecific En zym e Im m u n oa ssa ys
Thierry Brossette,† Anne Le Faou,‡ Laure Goujon,‡ Alain Valleix,§ Christophe Cre´minon,‡
J acques Grassi,‡ Charles Mioskowski,*,†,§ and Luc Lebeau*,†
Universite´ Louis Pasteur de Strasbourg, Laboratoire de Synthe`se Bioorganique associe´ au
CNRS - Faculte´ de Pharmacie, 74, route du Rhin - BP 24 - 67 401 Illkirch Cedex - France,
CEA - Service de Pharmacologie et d’Immunologie, De´partement de Recherche Me´dicale, Baˆt. 136,
CEA-Saclay - 91 191 Gif sur Yvette - France, and CEA - Service des Mole´cules Marque´es,
De´partement de Biologie Mole´culaire et Cellulaire, Baˆt. 547, CEA-Saclay - 91 191 Gif sur Yvette - France
Received December 23, 1998
The synthesis of a series of analogues of the different polyphosphorylated metabolites of AZT has
been carried out. The compounds were designed in order to raise specific antibodies for the
development of highly sensitive titration kits for the intracellular metabolites of AZT. The
pyrophosphate moiety in AZT-DP and AZT-TP analogues is mimicked by the methylene bisphos-
phonate group to provide in vivo stability of the compounds. An ω-amino linker was introduced at
the N3 position on the base to allow the further anchoring of the compounds to a carrier protein
before immunization, tentitatively focusing the specificity of the immune response toward the
phosphate mimic moiety and the nonnatural sugar.
In tr od u ction
could interfere with the intracellular formation of d4T-
TP leading to a decrease in the treatment efficacy as seen
by insufficient lowering of viral load and poor restoration
of CD4+ lymphocytes. These results are supported by
previous observations indicating negative metabolic in-
terferences between antiviral nucleosides.5-7 This high-
lights the importance of the intracellular metabolism for
the activity of nucleoside inhibitors of HIV RT.
The efficacy of polytherapies in the treatments of AIDS
(combination of reverse transcriptase and protease in-
hibitors) essentially results from their ability to stop the
mechanisms of genetic resistance of the virus.1,2 Due to
the poor “fidelity” of the reverse transcriptase (RT) of
HIV, a large number of mutations spontaneously occur
during the replication process of the virus. That hyper-
variability of HIV acts in favor of the emergence of
variants that are resistant to the antiviral compounds
used. Whatever the antiviral drug administered in the
framework of monotherapies, resistance phenomena are
observed within a few weeks or months. The combination
of multiple drugs allows to prevent or delay the appear-
ance of these resistances as evidenced by the lowering
or even disappearance of the viral load for months or
years. In these conditions, the emergence of other causes
leading to therapy failure are observed. It is a common
estimation that more than 30% of the patients on
polytherapy are in check and this can be partly explained
on account of recent pharmacological results. In 1998 a
treatment escape mechanism related to the metabolism
of nucleoside derivatives has been reported.3,4 The au-
thors suggested that a combined use of AZT and d4T
Nucleoside inhibitors of HIV RT (AZT, ddI, ddC, d4T,
and 3TC) have been the first compounds used in large
scale to fight against AIDS and a number of new
nucleoside derivatives are currently under clinical evalu-
ation. All these compounds are 2′,3′-dideoxynucleosides
(ddNs) and are analogues of natural nucleosides. They
inhibit the virus replication by interfering with the RT
activity and by acting as chain terminators. They share
the common feature to require an intracellular transfor-
mation into their corresponding dideoxynucleoside tri-
phosphate (ddN-TP) that is the active species able to
inhibit RT. Thus the ddNs are prodrugs, and their
metabolism goes through the general metabolic pathway
of endogenous nucleosides and nucleotides.8 It takes place
in several steps: entry of the nucleosides into the cell
(passive process) and then 3 phosphorylation reactions
leading successively to the monophosphorylated (ddN-
MPs), diphosphorylated (ddN-DPs), and finally triphos-
phorylated derivatives (ddN-TPs). The nature and the
activity of the enzymes involved in these transformations
depend at one and the same time on the structure of the
nucleoside analogues, on the cell type, and on its activa-
tion state.9-13 Besides, the synthetic pathways to the
† Universite´ Louis Pasteur de Strasbourg.
‡ CEA - Service de Pharmacologie et d’Immunologie.
§ CEA - Service des Mole´cules Marque´es.
(1) Collier, A. C.; Coombs, R. W.; Schoenfeld, D. A.; Bassett, R. L.;
Timpone, J .; Baruch, A.; J ones, M.; Facey, K.; Whitacre, C.; McAuliffe,
V. J .; Merigan, R. C.; Reichman, R. C.; Hooper, C.; Corey, L. New Engl.
J . Med. 1996, 334, 1011-1018.
(2) Gulick, R. M.; Mellors, J . W.; Havlir, D.; Eron, J . J .; Gonzalez,
C.; McMahon, D.; Richman, D. D.; Valentine, F. T.; J onas, L.; Meibohm,
A.; Emini, E. A.; Chodakewitz, J . A. New Engl. J . Med. 1997, 337,
734-739.
(3) Havlir, D. Combination Zidovudine (ZDV) and Stavudine (d4T)
versus other nucleosides. Report of two randomized trials (ACTG 290
and 298). Presented at Vth Conference on Retrovirus and Opportunistic
Infections, Feb 1-5, 1998, Chicago, IL.
(4) Sommadossi, J . P. Impairment of Stavudine (d4T) phosphory-
lation in patient receiving a combination of Zidovudine (ZDV) and d4T
(ACTG 290). Presented at Vth Conference on Retrovirus and Op-
portunistic Infections, Feb 1-5, 1998, Chicago, IL.
(5) Wu, S.; Liu, X.; Solorzano, M. M.; Kwock, R.; Avramis, V. I. J .
Acquir. Immune Defic. Syndr. Hum. Retrovirol. 1995, 8, 1-9.
(6) Nyce, J .; Leonard, S.; Canupp, S.; Schulz, S.; Wong, S. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 2960-2964.
(7) Antonelli, G.; Turriziani, O.; Verri, A.; Narciso, P.; Ferri, F.;
D′Offizi, G.; Dianzani, F. AIDS Res. Hum. Retroviruses 1996, 12, 223-
228.
(8) Roy, B.; Fontecave, M. Bull. Inst. Pasteur 1993, 91, 89-100.
(9) Gao, W. Y.; Agbaria, R.; Driscoll, J . S.; Mitsuya, H. J . Biol. Chem.
1994, 259, 12633-12638.
10.1021/jo982502p CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/19/1999

5084 J . Org. Chem., Vol. 64, No. 14, 1999
Brossette et al.
different dideoxynucleotides (ddN-Ps) are not indepen-
dent one from each other and some modifications in one
of them can affect the others.13-17
There is a number of reports in the literature describ-
ing a great interindividual and longitudinal variability
in the production of the phosphorylated metabolites of
antiviral nucleosides in treated patients.18-21 Today it
seems obvious that the plasmatic ddNs levels do not
reflect the clinical efficacy of the treatment. As for all
bioprecursors, it is necessary to measure the intracellular
concentration of the active principles (ddN-TPs) as well
as that of their direct precursors (ddNs, ddN-MPs, and
ddN-DPs) produced in vivo. The monitoring of nucleoside
metabolites in treated patients requires highly sensitive
analytical methods able to detect minute amounts of
ddN-Ps (10-1000 fmol/106 cells, e.g., concentrations
between 2 × 10-11 M and 2 × 10-9 M). Most of the data
collected over the systemic metabolism of ddNs involved
chromatography techniques.22,23 HPLC coupled to clas-
sical detectors (UV or fluorescence spectrophotometers)
is not sensitive enough to allow the determination of
intracellular ddN-Ps levels. Thus the intracellular me-
tabolism has been mainly investigated on ex vivo sys-
tems, in cells cultured in the presence of radiolabeled
ddNs.11,12,18,24-26 The method is not adapted to intracel-
lular ddN-Ps monitoring in patients treated with ddNs.
Our efforts are directed toward the development of a
strategy for direct measurement of intracellular dideoxy-
nucleotides using specific enzyme immunoassays. The
objective is to establish competitive immunoassays based
on the use of specific rabbit polyclonal antibodies together
with a tracer (ddN-P covalently coupled to acetylcho-
linesterase).27 Immunoenzymological methods perfectly
fit the problem to be solved. As a matter of fact,
immunoassays are potentially very sensitive, and the
femtomolar concentration range is frequently attained.
Moreover these techniques present the valuable advan-
tage to be directly applicable to routine analysis of large
series of samples and protocols can be easily automated.
F igu r e 1.
Herein we describe the strategy we develop for the
production of specific antibodies directed against the most
widely used ddN in AIDS therapy, AZT, and its different
phosphorylated metabolites, AZT-MP, AZT-DP, and AZT-
TP (Figure 1). Our ultimate goal is the elaboration of the
corresponding enzyme immunoassays that will allow
specific monitoring of each compound in cells of treated
patients and that will be suited to the clinical environ-
ment requirements.
Resu lts a n d Discu ssion
The elaboration of enzyme immunoassays for monitor-
ing nucleosides and nucleotides comes up against two
specific difficulties. First, the molecules to assay (the
haptens) are low molecular weight compounds, and their
direct inoculation to rabbits would not give rise to any
immune response in the animals. Thus, it is necessary
to realize a covalent coupling of these haptens to an
antigenic carrier (bovine serum albumin, keyhole limpet
hemocyanin) prior to injections. The conjugates (the
immunogens) are then capable to trigger off an immune
reaction in the recipient animals susceptible to provoke
the synthesis of specific antibodies directed against the
haptens. The way the coupling to the antigenic carrier
is realized is of crucial importance and has to be
judiciously chosen to lead to the production of antibodies
exhibiting the higher specificity toward the haptens. In
our case, these antibodies will be required to specifically
recognize AZT or one of its three metabolites in the
complex pool of intracellular nucleosides and nucleotides,
among which are other AZT derivatives as well as
thymidine and its metabolites, structurally very close to
the hapten. In that respect, the coupling to the carrier
protein has been planned through the base to preserve
as much as possible the structural integrity of the sugar
and phosphorus-containing moieties in the different
molecules (Figure 2). Another difficulty results from both
the chemical and the enzymatic instability of pyrophos-
phate bridges, and AZT-DP and AZT-TP are rapidly
hydrolyzed in recipient animals.28 The methylene bis-
phosphonate group is stable under hydrolysis conditions
and has been widely used to mimic pyrophosphates in
nucleotides.29,30 The corresponding nucleotide analogues
are still recognized by much enzymes but are not
substrate for them anymore. Thus, the transposition of
that structural modification to AZT-DP and AZT-TP has
been reported31,32 and should yield haptens highly resis-
(10) Gao, W. Y.; Shirasaka, T.; J ohn, D. G.; Broder, S.; Mitsuya, H.
J . Clin. Invest. 1993, 71, 2326-2333.
(11) Balzarini, J .; Herdewijn, P.; De Clercq, E. J . Biol. Chem. 1989,
264, 6127-6133.
(12) Balzarini, J .; Pauwells, R.; Baba, M.; Herdewijn, P.; De Clercq,
E.; Broder, S.; J ohn, D. G. Biochem. Pharmacol. 1988, 37, 897-903.
(13) Sommadossi, J . P. Clin. Infect. Dis. 1993, 16, S7-S15, Suppl.
1.
(14) Gao, W. Y.; J ohns, D. G.; Chokekuchai, S.; Mitsuya, H. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 8333-8337.
(15) Lori, F.; Malykh, A.; Cara, A.; Sun, D.; Weinstein, J . N.;
Lisziewicz, J .; Gallo, R. C. Science 1994, 266, 801-805.
(16) Hartman, N. R.; Ahluwalia, G. S.; Cooney, D. A.; Mitsuya, H.;
Kageyama, S.; fridland, A.; Broder, S.; J ohns, D. G. Mol. Pharmacol.
1991, 40, 118-124.
(17) Bianchi, V.; Borella, S.; Calderazzo, F.; Ferraro, P.; Chieco
Bianchi, L.; Reichard, P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8403-
8407.
(18) Stretcher, B. N.; Pesce, A. J .; Murzai, J . A.; Hurtubise, P. E.;
Vine, W. H.; Frame, P. T. Ther. Drug Monit. 1991, 13, 325-331.
(19) Kamali, F. Clin. Investig. 1993, 71, 392-405.
(20) J ohnston, M. I.; Hoth, D. F. Science 1993, 260, 1286-1293.
(21) Richman, D. D.; Fischl, M. A.; Grieco, M. H.; Gottlieb, M. S.;
Volberding, P. A.; Laskin, O. L.; Leedom, J . M.; Groopman, J . E.;
Mildvan, D.; Hirsch, M. S. New Engl. J . Med. 1987, 317, 192-197.
(22) Molema, G.; J ansen, R. W.; Visser, J .; Meijer, D. K. J . Chro-
matogr. 1992, 579, 107-114.
(23) Toyoshima, T.; Kimura, S.; Maramatsu, S.; Takahagi, H.;
Shimada, K. Anal. Biochem. 1991, 196, 302-307.
(24) Stretcher, B. N.; Pesce, A. J .; Hurtubise, P. E.; Frame, P. T.
Ther. Drug Monit. 1992, 14, 281-285.
(26) Tornevik, Y.; J acobson, B.; Britton, S.; Eriksson, S. AIDS Res.
Hum. Retrov. 1991, 7, 751-759.
(27) Pradelles, P.; Grassi, J .; Chabardes, D.; Guiso, N. Anal. Chem.
1989, 61, 447-453.
(25) Furman, P. A.; Fyfe, J . A.; St. Clair, M. H.; Weinhold, K.;
Rideout, J . L.; Freeman, J . I. A.; Nusinhoff-Lehman, S.; Bolognesi, D.
P.; Broder, S.; Mitsuya, H.; Barry, D. Proc. Natl. Acad. Sci. U.S.A. 1986,
83, 8333-8337.
(28) Bredehorst, R.; Ferro, A.; Hilz, H. Eur. J . Biochem. 1978, 82,
105-113.
(29) Blackburn, G. M. Chem. Ind. 1981, 134-138.
(30) Engel, R. Chem. Rev. 1977, 77, 349-367.

Synthesis of Polyphosphorylated AZT Derivatives
J . Org. Chem., Vol. 64, No. 14, 1999 5085
Sch em e 2
F igu r e 2.
Sch em e 1
Mitsunobu reaction with 5-(2,2,2-trifluoroacetylamino)-
1-pentanol.34 The resulting compound 7 was treated with
potassium carbonate in methanol to remove the ester
protective group at the 5′ position, and treatment of the
subsequent compound 8 in anhydrous methanolic am-
monia allowed quantitative removal of the primary amine
protective group. Compound 1 was obtained in a 41%
overall yield.
Haptens 2-4 were prepared similarly from the inter-
mediate compound 8 (Scheme 2). Phosphorylation and
phosphonylations of the 5′-hydroxyl group were carried
out via a Mitsunobu reaction. That procedure previously
proved to be highly efficient to achieve that kind of
transformation.35,36 Reaction with phosphoric acid diben-
zyl ester 937 quantitatively gave compound 12. Esterifi-
cation with phosphonic acids 10 and 1138,39 gave 13 and
14 in lower yields. This is due to the conjugation of the
reduced nucleophilicity of phosphonate anions (when
compared to phosphate anions) and of an increase in
steric hindrance in the close neighborhood of the nucleo-
philic center. Thus compound 13 was isolated as a
tant to hydrolytic enzymes. These considerations prompted
us to target compounds 1 to 4 (Figure 2).
(34) Hirschmann, R.; Nicolaou, K. C.; Pietranico, S.; Leahy, E. M.;
Salvino, J .; Arison, B.; Cichy, M. A.; Spoors, P. G.; Shakespear, W. C.;
Sprengeler, P. A.; Hamley, P.; Smith, A. B. I.; Reisine, T.; Raynor, K.;
Maechler, L.; Donaldson, C.; Vale, W.; Freidinger, R. M.; Cascieri, M.
R.; Strader, C. D. J . Am. Chem. Soc. 1993, 115, 12550-12568.
(35) Saady, M.; Lebeau, L.; Mioskowski, C. Tetrahedron Lett. 1995,
36, 2239-2242.
(36) Vincent, S.; Grenier, S.; Valleix, A.; Salesse, C.; Lebeau, L.;
Mioskowski, C. J . Org. Chem. 1998, 63, 7244-7257.
(37) Baddiley, J .; Clarck, V. M.; Michalski, J . J .; Todd, A. R. J . Chem.
Soc. 1949, 815-821.
Compound 1 was prepared from thymidine in five steps
(Scheme 1). Intermediate compounds 5 and 6 were
obtained following the procedure described by Czernecki
and Valery.33 The 5′-O-benzoyl AZT derivative 6 was
regioselectively alkylated at N3 in 88% yield via a
(31) Balzarini, J .; Herdewijn, P.; Pauwels, R.; Broder, S.; De Clercq,
E. Biochem. Pharmacol. 1988, 37, 2395-2403.
(38) Saady, M.; Lebeau, L.; Mioskowski, C. J . Org. Chem. 1995, 60,
2946-2947.
(39) Saady, M.; Lebeau, L.; Mioskowski, C. Helv. Chim. Acta 1995,
78, 670-678.
(32) Labataille, P.; Pe´licano, H.; Maury, G.; Imbach, J . L.; Gosselin,
G. Bioorg. Med. Chem. Lett. 1995, 5, 2315-2320.
(33) Czernecki, S.; Vale´ry, J . M. Synthesis 1991, 239-240.

5086 J . Org. Chem., Vol. 64, No. 14, 1999
Brossette et al.
Sch em e 3
F igu r e 3.
mixture of two diastereomers in 60% yield whereas
compound 14 was obtained as a mixture of four diaster-
eomers, together with compound 18 that could not be
separated by flash chromatography, in 47% yield (Figure
3). Compound 18 resulted from the coupling of nucleoside
8 to phosphinic acid 19 obtained as a nonseparable
byproduct during the preparation of 11. Removal of the
benzyl esters in this series of compounds could not be
achieved by hydrogenolysis due to the presence of the
3′-azido group. Direct saponification under basic condi-
tions was not much suitable, resulting in partial hydroly-
sis of the ester bond between the phosphorus containing
moiety and the deoxy sugar. We attempted to perform
debenzylations using trimethylsilyl bromide according to
a procedure described by McKenna and others.40-42 The
use of the experimental conditions described by these
authors resulted in partial depurination and produced
complex mixtures of compounds. The depurination side-
reaction could be avoided by quenching the reaction
mixture under slightly basic conditions. Thus compound
15 was quantitatively obtained by treating 12 with
TMSBr for 3 h at room temperature, the intermediately
formed bis-silyl phosphate being decomposed with diluted
aqueous ammonia. Final treatment of 15 with anhydrous
methanolic ammonia produced compound 2 in a high
yield. The debenzylation of phosphonylated compound 13
required a longer reaction time, and a second product
formed along with compound 16 that was not separated.
The treatment of the previous mixture with methanolic
ammonia led to a couple of compounds, and purification
was realized by reversed phase HPLC. The purified
compounds were analyzed by NMR (1H, 13C, and 31P) and
high-resolution mass spectrometry. The major component
was identified as compound 3 and the minor one as the
stereoisomer resulting from anomerization at the sugar.
This was confirmed by realizing the same chemical
transformations sequence in the nonnatural R-nucleoside
series (Scheme 3). Compound 6 was treated according to
the self-anomerization procedure described by Ward et
al.43,44 The separation of the resulting couple of diaster-
eomers 21 could not be achieved by chromatography over
silica gel. The two mixed isomers were then alkylated at
N3 to produce a nonseparable mixture of diastereomeric
compounds 22, and the benzoyl protective group was
subsequently removed under basic conditions. The two
resulting diastereomers 8 and 23 were satisfactorily
separated by chromatography over silica gel. The R-iso-
mer 23 was condensed with phosphonic acid 10, and
compound 24 was obtained as a mixture of two diaster-
eomers in 61% yield. Subsequent treatment with TMSBr
and aqueous ammonia produced 25. Final deprotection
in methanolic ammonia and purification by reversed
phase HPLC provided 26 in 55% yield (from 24) along
with compound 3 (24%).
Consistent with what was observed with compound 13
and 24, treatment of the mixture of compounds 14 and
18 with TMSBr for 12 h and subsequent hydrolysis
afforded 17 and 20 as two couples of diastereomers.
(40) Lazar, S.; Guillaumet, G. Synth. Commun. 1992, 22, 923-931.
(41) McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna, M. C.
Tetrahedron Lett. 1977, 155-158.
(42) McKenna, C. E.; Schmidhauser, J . J . Chem. Soc. Chem.
Commun. 1979, 739.
(43) Ward, I. D.; J effs, S. M.; Coe, P. L.; Walker, R. T. Tetrahedron
Lett. 1993, 34, 6779-6782.
(44) Larsen, E.; Abdel Aleem, A. A. H.; Pedersen, E. B. J . Heterocycl.
Chem. 1995, 32, 1645-1646.

Synthesis of Polyphosphorylated AZT Derivatives
J . Org. Chem., Vol. 64, No. 14, 1999 5087
Con clu sion
Functionalized analogues of AZT and of its three
intracellular metabolites were prepared from thymidine.
These compounds contain a spacer arm bearing a pri-
mary amine at the N3 position in the thymine moiety to
allow further coupling to a carrier protein using glut-
araldehyde as cross-linking reagent. The four title com-
pounds are actually under investigation for the produc-
tion of specific polyclonal antibodies, and initial results
were obtained very recently.50
Exp er im en ta l Section
Gen er a l. 1H-, 13C-, and 31P-NMR chemical shifts δ are
reported in ppm relative to an internal reference resulting from
incomplete deuteration of the NMR solvent (1H: CHCl3 at 7.27
ppm, HDO at 4.63 ppm, CD2HOD at 3.31 ppm; 13C: CDCl3 at
77.0 ppm, CD3OD at 49.0 ppm; 31P: H3PO4 at 0.00 ppm). IR
spectra were recorded in wavenumbers (cm-1). Mass spectra
(MS) were recorded at chemical ionization. High-resolution
mass spectra (HRMS) were recorded in the positive electro-
spray mode. Mass data are reported in mass units (m/z).
Analytical HPLC studies were carried out in the isocratic mode
using a reversed phase column (Zorbax SB C18, 250 × 4.6 mm,
5 µm; flow rate 1 mL/min at 25 °C) and a photodiode array
detector (LKB 2410, detection at at 267 nm). Reversed phase
preparative HPLC was carried out in the isocratic mode
(Hypersil BDS C18, 300 × 50 mm, 12 µm; Zorbax SB C18, 250
× 20 mm, 5 µm).
F igu r e 4.
Removal of the trifluoroacetamide protective group in
methanolic ammonia and purification by reversed phase
HPLC allowed isolation of the four major compounds 4
(17%), 27 (17%), 28 (13%), and 29 (17%) (Figure 4).
The self-anomerization of nucleosides and nucleotides
catalyzed by TMSBr has not been described yet and
appears to be a very interesting alternative to the
previously reported procedures.43-49 The transformation
occurs in very mild conditions that can be paralleled to
those reported by Kiss et al.45 and Yamaguchi et al.46 The
first authors mentioned anomerization of a 5′-deoxy-5-
fluorouridine analogue catalyzed by trimethylsilyl tri-
fluoromethanesulfonate (TMS-OTf). The transformation
however is described to be extremely slow at room
temperature, and only a minute amount of the R-anomer
(2%) was formed over a week. In these conditions the
major product obtained (10%) resulted from deglycosy-
lation, and, besides, the authors indicate that the ano-
merization reaction is not reversible, as only the â-nu-
cleoside anomerizes. At higher temperature (70 °C) and
in the presence of stoichiometric bis(trimethylsilyl)-
acetamide (BSA), anomerization of deoxycytidine and
deoxythymidine derivatives proceeds satisfactorily within
a few hours and is fully reversible.46 The authors propose
the transformation to occur in two steps as an intermo-
lecular reaction. First the trimethylsilylated base is
released from the nucleoside, and then the liberated
active sugar carbonium cation at the C1 position is
attacked again by the nucleophilic N1 position of the
pyrimidine base. An alternative pathway, however, goes
through a ring-opening reaction at the sugar43 and can
account for these results as well as for ours. We actually
do not have any experimental evidence for consideration
of one mechanism over the other, but additional study is
currently underway.
3′-Azid o-3′-d eoxy-N3-[1-(5-a m in op en tyl)]th ym id in e (1).
Compound 8 (90 mg, 0.20 mmol) is stirred for 24 h at room
temperature in saturated methanolic ammonia (5 mL). Solvent
is removed in vacuo, and the residue is purified by chroma-
tography over silica gel (AcOEt/MeOH: 100/0 to 70/30) to yield
1 (68 mg, 97%) as a white powder. 1H NMR (CD3OD, 300 MHz)
δ 1.28-1.40 (m, 2H); 1.48-1.62 (m, 4H); 1.90 (s, 3H); 2.40 (t,
2H, J ) 6.2 Hz); 2.66 (t, 2H, J ) 7.2 Hz); 3.80 (AB part of
ABX syst, 2H, J AB ) 12.1 Hz, J AX ) 3.0 Hz, J BX ) 3.0 Hz);
3.89-4.36 (m, 3H); 4.33 (pseudo-q, 1H, J ) 5.7 Hz); 6.19 (t,
1H, J ) 6.2 Hz); 7.83 (s, 1H). 13C NMR (CD3OD, 50 MHz) δ
13.2; 25.0; 28.1; 29.4; 38.4; 40.5; 42.1; 61.5; 62.3; 86.2; 86.9;
110.7; 136.4; 152.2; 165.3. MS (NH3) m/z 353 [M + H]+; 370
[M + NH4]+. IR (neat) ν 1470; 2105; 3307 (br). Anal. Calcd for
C15H24N6O4 (352.41): C 51.12, H 6.87, N 23.85. Found: C 50.96,
H 6.91, N 23.61.
3′-Azid o-3′-d eoxy-5′-O-(d ih yd r oxyp h osp h or yl)-N3-[1-(5-
a m in op en tyl)] th ym id in e (2). Compound 2 is obtained from
15 following the same procedure as for 1. Purification is
achieved by flash chromatography over reversed phase silica
gel (Merck, RP-18, 40-63 µm, CH3CN/H2O: 0/100 to 15/85),
and compound 2 is obtained as a white hygroscopic solid (34
mg, 87%). 1H NMR (D2O, 200 MHz) δ 1.10-1.30 (m, 2H); 1.35-
1.55 (m, 4H); 1.74 (s, 3H); 2.29 (t, 2H, J ) 6.9 Hz); 2.79 (t, 2H,
J ) 7.1 Hz); 3.71 (t, 2H, J ) 6.8 Hz); 3.80-4.05 (m, 3H); 4.30
(m, 1H); 6.08 (pseudo-t, 1H, J ) 6.9 Hz); 7.58 (s, 1H). 13C NMR
(D2O, 50 MHz) δ 13.4; 24.0; 27.2; 27.4; 37.5; 40.3; 42.3; 61.8;
65.5; 84.3; 87.0; 112.1; 136.7; 152.8; 166.8. 31P NMR (D2O,
121.5 MHz) δ 4.90 (s). MS (NH3) m/z 308 [M - H2PO4 - N2 +
H]+; 325 [M - H2PO4 - N2 + NH4]+. IR (Kbr) ν 1107; 1467;
2098; 3214 (br). Anal. HPLC (H2O/CH3CN/TFA 90/10/0.1) tR
16.2 min.
3′-Azid o-3′-d eoxy-5′-O-[(d ih yd r oxyp h osp h or ylm eth yl)-
h yd r oxyp h osp h or yl]-N3-1-(5-a m in op en tyl)th ym id in e (3).
Trimethylsilyl bromide (67 µL, 0.55 mmol) is added dropwise
to compound 13 (96 mg, 0.11 mmol) in anhydrous methylene
chloride (5 mL). The reaction mixture is stirred for 6 h at room
temperature before 5% aqueous ammonia (0.5 mL) is added.
The solution is evaporated to dryness, and the crude residue
(45) Kiss, J .; D’Souza, R.; van Koeveringe, J . A.; Arnold, W. Helv.
Chim. Acta 1982, 65, 1522-1537.
(46) Yamaguchi, I.; Saneyoshi, M. Chem. Pharm. Bull. 1984, 32,
1441-1450.
(47) Matulic-Adamic, J .; Pavela-Vrancic, M. P.; Skaric, V. J . Chem.
Soc., Perkin Trans. 1 1988, 2681-2686.
(48) Seela, F.; Menkhoff, S.; Behrendt, S. J . Chem. Soc., Perkin
Trans. 2 1986, 525-530.
(50) Goujon, L.; Brossette, T.; Dereudre-Bosquet, N.; Creminon, C.;
Clayette, P.; Dormont, D.; Mioskowski, C.; Grassi, J . J . Immunol.
Methods 1998, 218, 19-30.
(49) Sakthivel, K.; Pathak, T.; Suresh, C. G. Tetrahedron 1996, 52,
1767-1772.

5088 J . Org. Chem., Vol. 64, No. 14, 1999
Brossette et al.
is stirred for 24 h at room temperature in saturated methanolic
ammonia (5 mL). Solvent is removed in vacuo, and purification
is achieved by reversed phase HPLC (Hypersil BDS C18, 12
µm, 300 × 50 mm; isocratic mode: ammonium carbonate 0.5
M/acetonitrile 94:6, pH 8.8; flow rate: 30 mL/min). The bis-
ammonium salt of compound 3 is obtained as a white hygro-
scopic solid (retention time: 45 min; 38 mg, 58%). 1H NMR
(D2O, 300 MHz) δ 1.21-1.29 (m, 2H); 1.45-1.60 (m, 4H); 1.80
(s, 3H); 2.01-2.23 (m, 2H); 2.37 (m, 2H); 2.84 (t, 2H, J ) 7.1
Hz); 3.78 (t, 2H, J ) 7.1 Hz); 3.95-4.06 (m, 3H); 4.39 (m, 1H);
6.12 (pseudo-t, 1H, J ) 6.0 Hz); 7.58 (s, 1H). 13C NMR (D2O,
75 MHz) δ 12.3; 22.9; 26.1; 26.3; 36.1; 39.2; 41.2; 60.6; 64.0;
83.2; 86.0; 110.9; 135.6; 151.7; 165.7. 31P NMR (D2O, 121.5
MHz) δ 16.93 (m, 1P); 19.01 (m, 1P). HRMS: calcd for
H]+; 466 [M + NH4]+. IR (neat) ν 1472; 2105; 2944; 3307 (br).
Anal. Calcd for C17H23F3N6O5 (448.42): C 45.53, H 5.17, N
18.74. Found: C 45.71, H 5.25, N 19.01.
3′-Azid o-3′-d eoxy-5′-O-(b is-b en zyloxy-p h osp h or yl)N3-
{1-[5-(2,2,2-tr iflu or o-acetylam in o)pen tyl]}th ym idin e (12).
A mixture of triphenylphosphine (146 mg, 0.56 mmol) and
diidopropyl azodicarboxylate (110 µL, 0.56 mmol) in anhydrous
THF (1.5 mL) is added dropwise to compound 8 (100 mg, 0.22
mmol) and phosphoric acid dibenzyl ester 9 (77 mg, 0.27 mmol)
in THF (4.5 mL). The resulting solution is stirred for 1 h at
room temperature, and the solvent is removed in vacuo. The
crude residue is purified by chromatography over silica gel
(Et2O/CHCl3/CH3CN: 80/15/5 to 57/28/15) to yield compound
12 as a glassy solid (160 mg, 99%). 1H NMR (CDCl3, 200 MHz)
δ 1.32-1.43 (m, 2H); 1.60-1.70 (m, 4H); 1.86 (s, 3H); 2.03-
2.40 (m, 2H); 3.36 (dt, 2H, J ) 6.0, 6.6 Hz); 3.90-4.21 (m, 6H);
5.07 (AB part of ABX syst, 4H, J AB ) 16.0 Hz, J AX ) 9.7 Hz,
J BX ) 9.3 Hz, ∆ν ) 0.03); 6.19 (pseudo-t, 1H, J ) 6.5 Hz); 6.74
(m, 1H); 7.31-7.37 (m, 11H).13C NMR (CDCl3, 50 MHz) δ 13.1;
23.7; 27.0; 27.8; 37.5; 40.0; 40.5; 60.1; 66.3 (d, J ) 5.6 Hz);
69.9 (m); 82.1 (d, J ) 7.7 Hz); 85.5; 110.8; 128.3; 128.8; 129.0;
C
16H28N6O9NaP2 [M + Na]+ 533.1291, found 533.1304; calcd
for C16H28N6O9P2K [M + K]+ 549.1031, found 549.1039. IR
(KBr) ν 1197; 1467; 2109; 3200 (br).
3′-Azid o-3′-d eoxy-5′-O-{[(d ih yd r oxy-p h osp h or ylm eth -
yl)h yd r oxyp h osp h or ylm eth yl]h yd r oxyp h osp h or yl}-N3-
[1-(5-a m in op en tyl)]th ym id in e (4). Compound 4 is obtained
as its tris-ammonium salt from 14 following the same proce-
dure as for 3. Purification is achieved by reversed phase HPLC
(Zorbax SB C18, 250 × 20 mm; isocratic mode: ammonium
carbonate 0.5 M/acetonitrile 94:6, pH 8.8; flow rate: 17 mL/
min), and compound 4 is obtained as a white hygroscopic solid
(retention time: 37 min; 8 mg, 17%). 1H NMR (D2O, 200 MHz)
δ 1.28-1.38 (m, 2H); 1.53-1.70 (m, 4H); 189 (s, 3H); 2.15-
2.35 (m, 4H); 2.45 (m, 2H); 2.92 (t, 2H, J ) 7.0 Hz); 3.87 (t,
2H, J ) 7.0 Hz); 4.07-4.15 (m, 3H); 4.46 (m, 1H); 6.20 (pseudo-
t, 1H, J ) 6.1 Hz); 7.66 (m, 1H). 31P NMR (D2O, 121.5 MHz)
δ 15.40 (m, 1P); 19.74 (m, 1P); 31.17 (m, 1P). HRMS: calcd
for C17H31N6O11NaP3 [M + Na]+ 611.1161, found 611.1165;
calcd for C17H31N6O11Na2P3 [M - H+2Na]+ 633.0981, found
633.0953; calcd for C17H31N6O11KNaP3 [M - H + Na + K]+
649.0720, found 649.0740. IR (neat) ν 1096; 1188; 1484; 2112;
3381 (br).
135.3 (d, J ) 5.7 Hz); 150.8; 152.2 (q, J ) 36.8 Hz); 163.5.31
P
NMR (CDCl3, 121.5 MHz) δ 0.67 (s). MS (NH3) m/z 709 [M +
H]+; 726 [M + NH4]+. IR (neat) ν 1467; 2108; 2943; 3307.
3′-Azid o-3′-d e oxy-5′-O-[(b is-b e n zyloxyp h osp h or yl-
m e t h y l)b e n z y lo x y p h o s p h o r y l)]-N 3 -{1-[5-(2,2,2-t r i -
flu or o-a cetyla m in o)p en tyl]}th ym id in e (13). A mixture of
triphenylphosphine (524 mg, 2.00 mmol) and diidopropyl
azodicarboxylate (315 µL, 0.56 mmol) in anhydrous THF (2
mL) is added dropwise to compound 8 (200 mg, 0.45 mmol)
and phosphonic acid 10 (219 mg, 0.49 mmol) in THF (10 mL).
The resulting solution is stirred for 4 h at room temperature,
and the solvent is removed in vacuo. The crude residue is
purified by chromatography over silica gel (Et2O/AcOEt/EtOH:
100/0/0 to 0/85/15) to yield compound 13 as a colorless syrup
(mixture of two diastereomers, 235 mg, 60%). 1H NMR (CDCl3,
200 MHz) δ 1.19-1.29 (m, 2H); 1.51-1.59 (m, 4H); 1.86 (s,
1.5H); 1.92 (s, 1.5H); 2.21-2.40 (m, 2H); 2.48 (t, 1H, J ) 21.1
Hz); 2.50 (t, 1H, J ) 21.1 Hz); 3.23 (dt, 2H, J ) 6.6, 6.2 Hz);
3.94 (t, 2H, J ) 6.2 Hz); 4.02-4.25 (m, 4H); 4.90-5.12 (m, 6H);
6.09-6.15 (m, 1H); 6.84 (m, 1H); 7.31-7.37 (m, 16H).13C NMR
(CDCl3, 75 MHz) δ 12.9; 23.6; 25.7 (t, J ) 138.1 Hz); 26.8; 27.5;
38.0; 39.7; 40.3; 52.9; 64.9; 68.0-68.2 (m); 84.5-85.5 (m); 86.3;
109.5; 115.9 (q, J ) 289.2 Hz); 127.8-128.9 (m); 132.8; 135.6
(m); 150.4; 157.2 (q, J ) 36.3 Hz); 163.4.31P NMR (CDCl3, 121.5
MHz) δ 20.75 (d, 0.5P, J ) 5.8 Hz); 20.97 (d, 0.5P, J ) 5.4
Hz); 21.05 (d, 0.5P, J ) 5.4 Hz); 21.44 (d, 0.5P, J ) 5.8 Hz).
MS (NH3) m/z 894 [M + NH4]+. IR (neat) ν 1437; 2108; 2919;
3237; 3495. Anal. HPLC (H2O/CH3CN/TFA 40/60/0.1) tR 9.7
min.
3′-Azid o-3′-d e oxy-5′-O-(4-m e t h oxyb e n zoyl)-N 3-{1-[5-
(2,2,2-t r iflu or oa cet yla m in o)p en t yl]}t h ym id in e (7). Tri-
phenylphosphine (1.63 g, 6.2 mmol) in anhydrous THF (8 mL)
is added dropwise to a mixture of 6 (1.00 g, 2.5 mmol), 5-(2,2,2-
trifluoroacetylamino)-1-pentanol (0.59 g, 3.0 mmol), and di-
isopropyl azodicarboxylate (1.23 mL, 6.2 mmol) in THF (5 mL)
at room temperature. The reaction mixture is stirred for 30
min, solvent is removed in vacuo, and the residue is purified
by chromatography over silica gel (n-C6H14/Et2O: 25/75 to 20/
1
80) to yield compound 7 as a white powder (1.27 g, 88%). H
NMR (CDCl3, 200 MHz) δ 1.25-1.36 (m, 2H); 1.50-1.63 (m,
7H); 2.28-2.49 (m, 2H); 3.27 (dt, 2H, J ) 6.6, 5.8 Hz); 3.80 (s,
3H); 3.84 (t, 2H, J ) 6.9 Hz); 4.15 (dt, 1H, J ) 4.7, 3.7 Hz);
4.32 (dt, 1H, J ) 7.3, 4.7 Hz); 4.53 (AB part of ABX syst, 2H,
J AB ) 12.4 Hz, J AX ) 3.3 Hz, J BX ) 3.7 Hz, ∆ν ) 0.07); 6.14
(pseudo-t, 1H, J ) 4.7 Hz); 6.87 (m, 2H); 7.17 (s, 1H); 7.33 (m,
1H); 7.91 (m, 2H). 13C NMR (CDCl3, 50 MHz) δ 12.7; 23.6; 26.7;
27.8; 37.6; 39.6; 40.5; 55.3; 60.3; 63.1; 81.8; 85.6; 110.2; 113.7;
115.7 (q, J ) 285.9 Hz); 121.1; 131.4; 133.0; 150.4; 157.1 (q, J
) 36.3 Hz); 163.1; 163.7; 167.6. MS (NH3) m/z 583 [M + H]+;
600 [M + NH4]+ IR (neat) ν 1461; 2108; 2943; 3319. Anal. Calcd
for C25H29F3N6O7 (582.55): C 51.54, H 5.02, N 14.43. Found:
C 51.63, H 5.08, N 14.31.
3′-Azid o-3′-d e oxy-5′-O-{[(b is-b e n zyloxyp h osp h or yl-
m e t h yl)b e n zyloxyp h osp h or ylm e t h yl]b e n zyloxyp h os-
p h or yl}-N3-{1-[5-(2,2,2-t r iflu or oa cet yla m in o)p en t yl]}-
th ym id in e (14). A mixture of triphenylphosphine (128 mg,
0.45 mmol) and diidopropyl azodicarboxylate (76 µL, 0.45
mmol) in anhydrous THF (3 mL) is added dropwise to
compound 8 (49 mg, 0.11 mmol) and phosphonic acid 11 (80
mg, 0.13 mmol) in THF (4 mL). The resulting solution is stirred
for 4 h at room temperature, and the solvent is removed in
vacuo. The crude residue is purified by chromatography over
silica gel (Et2O/AcOEt/EtOH: 100/0/0 to 0/85/15) to yield
compound 14 as a colorless syrup (mixture of 4 diastereomers,
54 mg, 47%). 1H NMR (CDCl3, 300 MHz) δ 1.30-1.45 (m, 2H);
1.61-1.68 (m, 4H); 1.84-1.94 (m, 3H); 2.15-2.45 (m, 2H);
2.67-3.15 (m, 4H); 3.34 (dt, 2H, J ) 6.4, 6.2 Hz); 3.93 (t, 2H,
J ) 7.0 Hz); 4.14-4.36 (m, 4H); 4.91-5.20 (m, 8H); 6.08-6.20
(m, 1H); 6.90 (m, 1H); 7.28-7.40 (m, 21H).13C NMR (CDCl3,
75 MHz) δ 12.9; 23.6; 26.9-30.2 (m); 37.0; 39.7; 40.6; 59.8-
60.1 (m); 62.6-65.4 (m); 66.9-68.5 (m); 82.0; 85.1-85.7 (m);
110.4; 115.8 (q, J ) 291.8 Hz); 127.8-128.7 (m); 133.6-134.1
3′-Azid o-3′-d eoxy-N3-{1-[5-(2,2,2-tr iflu or oa cetyla m in o)-
p en tyl]} th ym id in e (8). Compound 7 (3.39 g, 5.8 mmol) and
potassium carbonate (0.48 g, 3.5 mmol) are stirred in anhy-
drous methanol (40 mL) for 90 min at room temperature.
Methanol is evaporated under reduced pressure, and the
residue is dissolved in chloroform and washed with brine. The
organic layer is dried over sodium sulfate and filtered. The
filtrate is reduced in vacuo and purified by chromatography
over silica gel (Et2O) to yield 8 as a white solid (1.80 g, 72%).
1H NMR (CDCl3, 200 MHz) δ 1. 23-1.41 (m, 2H); 1.56-1.71
(m, 4H); 1.94 (s, 3H); 2.25-2.57 (m, 2H); 3.28-3.37 (m, 3H);
3.70-3.98 (m, 5H); 4.34-4.43 (m, 1H); 6.08 (pseudo-t, J ) 6.6
Hz, 1H); 7.20 (m, 1H); 7.49 (s, 1H). 13C NMR (CDCl3, 75 MHz)
δ 13.1; 23.7; 26.9; 27.9; 37.4; 39.8; 40.6; 59.9; 61.8; 84.5; 86.9;
110.2; 115.8 (q, J ) 287.7 Hz); 134.9; 150.8; 157.4 (q, J ) 36.3
Hz); 163.5. MS (NH3) m/z 424 [M - N3 + NH4]+; 449 [M +
(m); 135.4-135.7 (m); 150.6; 157.2 (q, J ) 36.3 Hz); 163.3. 31
P
NMR (CDCl3, 121.5 MHz) δ 20.35-22.74 (m, 1P); 30.21 (s,
0.5P); 31.63 (s, 0.5P); 38.19-40.47 (m, 1P). IR (neat) ν 1461;
2108; 2355; 2943; 3237.

Synthesis of Polyphosphorylated AZT Derivatives
J . Org. Chem., Vol. 64, No. 14, 1999 5089
3′-Azid o-3′-d eoxy-5′-O-(d ih yd r oxyp h osp h or yl)-N3-{1-[5-
(2,2,2-tr iflu or oa cetyla m in o)p en tyl]}th ym id in e (15). Tri-
methylsilyl bromide (60 µL, 0.50 mmol) is added dropwise to
compound 12 (63 mg, 0.10 mmol) in anhydrous methylene
chloride (5 mL). The reaction mixture is stirred for 3 h at room
temperature before 5% aqueous ammonia (0.5 mL) is added.
The solution is evaporated to dryness, and the bis-ammonium
salt of compound 15 is obtained as a white cristalline pow-
300 MHz) δ 1.25-1.39 (m, 2H); 1.54-1.69 (m, 4H); 1.91 (s,
3H); 2.13 (m, 1H); 2.82 (m, 1H); 3.30 (m, 2H); 3.63-3.79 (m,
2H); 3.88 (t, 2H, J ) 7.0 Hz); 4.27-4.33 (m, 2H); 6.18 (dd, 1H,
J ) 7.0, 3.7 Hz); 7.30-7.34 (m, 2H). 13C NMR (CDCl3, 50 MHz)
δ 13.2; 23.6; 26.8; 27.8; 38.1; 39.7; 40.6; 60.7; 62.5; 86.1; 86.8;
109.8; 115.8 (q, J ) 287.7 Hz); 133.5; 150.8; 157.3 (q, J ) 36.3
Hz); 163.6. MS (NH3) m/z 421 [M - N2 + H]+; 449 [M + H]+;
466 [M + NH4]+. IR (neat) ν 1467; 2097; 2943; 3307; 3448.
Anal. Calcd for C17H23F3N6O5 (448.42): C 45.53, H 5.17, N
18.74. Found: C 45.89, H 5.22, N 18.86.
1
der without further purification (47 mg, 99%). H NMR (D2O,
300 MHz) δ 1.08-1.19 (m, 2H); 1.41-1.45 (m, 4H); 1.76 (d,
3H, J ) 0.9 Hz); 2.30-2.34 (m, 2H); 3.15 (t, 2H, J ) 7.2 Hz);
374 (t, 2H, J ) 7.2 Hz); 3.81-4.04 (m, 3H); 4.32 (m, 1H); 6.12
(pseudo-t, 1H, J ) 6.4 Hz); 7.59 (d, 1H, J ) 0.9 Hz). 13C NMR
(D2O/CD3OD: 95/5, 75 MHz) δ 13.3; 24.2; 27.2; 28.4; 37.5; 40.4;
42.4; 61.5; 65.5; 84.1; 86.8; 111.9; 136.4; 152.7; 166.7. 31P NMR
(D2O/CD3OD, 121.5 MHz) δ 1.50 (s). MS (NH3) m/z 449 [M -
COCF3 + NH4]+; 519 [M - N2 + NH4]+. IR (neat) ν 1402; 1467;
2108; 3119 (br). Anal. HPLC (H2O/CH3CN/TFA 70/30/0.1) tR
7.3 min.
3′-Azid o-3′-d e oxy-5′-O-[(b is-b e n zyloxyp h osp h or yl-
m et h yl)b en zyloxyp h osp h or yl]-N3-{1-[5-(2,2,2-t r iflu or o-
a cetyla m in o)p en tyl]}-r-th ym id in e (24). Compound 24 is
prepared starting from phosphonic acid 10 and nucleoside 23
following the same procedure as for 13 and is obtained as a
1
mixture of two diastereomers (59 mg, 61%). H NMR (CDCl3,
300 MHz) δ 1.30-1.40 (m, 2H); 1.60-1.70 (m, 4H); 1.93 (s,
3H); 1.94-2.04 (m, 1.5H); 2.51 (t, 1H, J ) 21.1 Hz); 2.52 (t,
1H, J ) 21.1 Hz); 2.60-2.75 (m, 0.5H); 3.34 (pseudo-q, 2H, J
) 6.4 Hz); 3.93 (t, 2H, J ) 6.8 Hz); 3.96-4.30 (m, 4H); 4.95-
5.20 (m, 6H); 6.08-6.10 (m, 1H); 7.19 (s, 0.5H); 7.20 (s, 0.5H);
7.32-7.45 (m, 15H). 13C NMR (CDCl3, 50 MHz) δ 14.2; 23.7;
25.9 (t, J ) 136.6 Hz); 26.9; 27.9; 37.5 and 37.7; 39.8; 40.5;
60.5 and 60.6; 65.1-65.4 (m); 67.6-68.5 (m); 83.6-83.9 (m);
87.0; 109.9; 115.9 (q, J ) 287.7 Hz); 128.1-128.8 (m); 133.3;
135.4-136.0 (m); 150.7; 157.2 (q, J ) 36.3 Hz); 163.6. 31P NMR
(CDCl3, 121.5 MHz) δ 20.71 (d, 0.5P, J ) 6.6 Hz); 20.73 (d,
0.5P, J ) 6.6 Hz); 22.06 (d, 0.5P, J ) 6.6 Hz); 22.11 (d, 0.5P,
J ) 6.6 Hz). MS (NH3) m/z 877 [M + H]+, 894 [M + NH4]+. IR
(neat) ν 1466; 2107; 2954; 3063; 3241.
3′-Azid o-3′-d eoxy-5′-O-[(d ih yd r oxyp h osp h or ylm eth yl)-
h yd r oxyp h osp h or yl]-N 3-[1-(5-a m in op e n t yl)]-r-t h ym i-
d in e (26). Compound 26 is obtained as its bis-ammonium salt
starting from 24 following the same procedure as for 3
(retention time: 37 min; 16 mg, 55%). 1H NMR (D2O, 300 MHz)
δ 1.22-1.29 (m, 2H); 1.45-1.60 (m, 4H); 1.79 (s, 3H); 2.07 (t,
2H, J ) 20.3 Hz); 2.13-2.18 (m, 1H); 2.77 (m, 1H); 2.84 (t,
2H, J ) 7.5 Hz); 3.78 (t, 2H, J ) 7.5 Hz); 3.89 (m, 2H); 4.37
(m, 1H); 4.46 (m, 1H); 6.06 (dd, 1H, J ) 6.4, J ) 2.3 Hz); 7.52
(s, 1H). 13C NMR (D2O, 75 MHz) δ 12.3; 22.9; 25.3 (t, J ) 142.5
Hz; 26.2; 26.3; 37.0; 39.3; 41.0; 60.8; 64.4; 85.3; 88.2; 111.0;
135.7; 151.5; 165.9. 31P NMR (D2O, 121.5 MHz) δ 16.57 (m,
1P); 19.16 (m, 1P). HRMS: calcd for C16H28N6O9NaP2 [M +
Na]+ 533.1291, found 533.1298; calcd for C16H28N6O9P2K [M
+ K]+ 549.1031, found 549.1037. IR (neat) ν 1455; 1922; 2108;
29.31; 3369.
An om er iza tion of Com p ou n d 6 in to 21. To compound 6
(1.05 g, 2.6 mmol) in CH2Cl2 (2 mL) is added CH3CN/CH2Cl2/
Ac2O/H2SO4: v/v/v/v: 20/20/4/1 (11.5 mL). The resulting mixture
is stirred at room temperature for 1 h before saturated aqueous
NaHCO3 (40 mL) is added. Solvents are removed under
reduced pressure, and the residue is extracted with CH2Cl2.
The organic layer is dried over Na2SO4, reduced in vacuo, and
purified by chromatography over silica gel (Et2O) to yield a
mixture of the two anomers 21 (371 mg, 35%, R/â: 63/37)
that could not be separated by flash chromatography. 1H
NMR (CDCl3, 300 MHz) δ 1.72 (s, 3Hâ); 1.97 (s, 3HR); 2.14-
2.59 (m, 2Hâ, 1HR); 2.87 (m, 1HR); 3.88 (s, 3HR and 3Hâ); 4.20-
4.69 (m, 4HR and 4Hâ); 6.19 (pseudo-t, 1Hâ, J ) 6.8 Hz); 6.27
(dd, 1HR, J ) 6.8, 3.8 Hz); 6.92-6.98 (m, 2HR and 2Hâ);
7.22 (s, 1Hâ); 7.33 (s, 1HR); 7.97-8.01 (m, 2HR and 2Hâ); 8.43
(m, 1HR and 1Hâ). 13C NMR (CDCl3, 50 MHz) δ 12.0â; 12.4R;
37.4â; 37.7R; 55.2Râ; 60.4â; 61.2R; 63.2â; 63.7R; 81.7â; 83.2R; 84.9â;
85.9R; 110.6R; 111.0â; 113.6Râ; 121.2Râ; 131.5Râ; 134.9Râ; 150.2â;
150.4R; 163.5R; 163.6â; 163.9â; 164.1R; 165.5Râ. MS (NH3) m/z
374 [M - N2 + H]+; 391 [M - N2 + NH4]+; 419 [M + NH4]+.
IR (neat) ν 1467; 2097; 2931; 3049; 3178. Anal. HPLC (H2O/
CH3CN/TFA 70/30/0.1) tR 36.8 min (â anomer), 40.8 min (R
anomer).
3′-Azid o-3′-d e oxy-5′-O-(4-m e t h oxyb e n zoyl)-N 3-{1-[5-
(2,2,2-tr iflu or oa cetyla m in o)p en tyl]}th ym id in e (m ixtu r e
of r a n d â a n om er s, 22). Compound 22 is obtained from 21
following the same procedure as for 7. The two anomers are
not separated by chromatography over silica gel (502 mg, 93%,
3′-Azid o-3′-d eoxy-5′-O-{[(d ih yd r oxyp h osp h or ylm et h -
yl)h yd r oxyp h osp h or ylm eth yl]h yd r oxyp h osp h or yl}-N3-
[1-(5-a m in op en tyl)]-r-th ym id in e (27). Compound 27 is
produced together with compound 4 from 14 and is purified
by HPLC (Zorbax SB C18, 250 × 20 mm; isocratic mode:
ammonium carbonate 0.5 M/acetonitrile 94:6, pH 8.8; flow
rate: 17 mL/min). Its tris-ammonium salt is obtained as a
white hygroscopic solid (retention time: 28 min; 8 mg, 17%).
1H NMR (D2O, 200 MHz) δ 1.27-1.38 (m, 2H); 1.54-1.68 (m,
4H); 1.87 (s, 3H); 2.15-2.28 (m, 5H); 2.83 (m, 1H); 2.92 (t, 2H,
J ) 7.2 Hz); 3.87 (t, 2H, J ) 7.0 Hz); 3.97 (m, 2H); 4.45 (m,
1H); 4.55 (m, 1H); 6.15 (m, 1H); 7.61 (m, 1H). 31P NMR (D2O,
121.5 MHz) δ 15.53 (m, 1P); 19.73 (m, 1P); 29.25 (m, 1P).
HRMS: calcd for C17H31N6O11NaP3 [M + Na]+ 611.1161, found
1
R/â: 63/37). H NMR (CDCl3, 200 MHz) δ 1.21-1.40 (m, 2HR
and 2Hâ); 1.50-1.70 (m, 4HR and 7HR); 1.63 (s, 3Hâ); 1.89 (s,
3HR); 2.15-2.55 (m, 1HR and 2Hâ); 2.74-2.89 (m, 1HR); 3.27
(pseudo-q, 2HR and 2Hâ, J ) 6.2 Hz); 3.79 (s, 3HR and 3Hâ);
3.86 (t, 2HR and 2Hâ, J ) 6.6 Hz); 4.15 (dt, 1Hâ, J ) 4.7, 3.7
Hz); 4.23-4.63 (m, 4HR and 3Hâ); 6.14 (pseudo-t, 1Hâ, J ) 6.6
Hz); 6.19 (dd, 1HR, J ) 6.9, 3.7 Hz); 6.87 (d, 2HR and 2Hâ, J )
8.8 Hz); 7.17 (s, 1Hâ); 7.28 (s, 1HR); 7.33 (m, 1HR and 1Hâ);
7.91 (d, 2HR and 2HR, J ) 8.8 Hz). 13C NMR (CDCl3, 50 MHz)
δ 12.9R; 13.1R; 23.6Râ; 26.8Râ; 27.8Râ; 37.5â; 37.8R; 39.6Râ; 40.4R;
40.5â; 55.2Râ; 60.3â; 61.5R; 63.1â; 63.6R; 81.8â; 83.4R; 85.6â; 109.7R;
110.2â; 113.6R; 113.7â; 115.7Râ (q, J ) 287.7 Hz); 121.1Râ; 131.4R;
131.8â; 133.0â; 133.1R; 150.4â; 150.6R; 157.1Râ (q, J ) 36.3 Hz);
163.1â; 163.3R; 163.6R; 164.0â; 165.6Râ. MS (NH3) m/z 555 [MH
- N2]+; 572 [M - N2 + NH4]+; 600 [M + NH4]+. IR (neat) ν
1261;1467;2108;2355;2943;3307. Anal. Calcd for C25H29F3N6O7
(582.55): C 51.54, H 5.02, N 14.43. Found: C 51.75, H 5.15, N
14.26.
611.1122; calcd for
C
17H30N6O11Na2P3 [M - H + 2Na]+
633.0981, found 633.0973; calcd for C17H32N6O11KNaP3 [M -
H + Na + K]+ 649.0720, found 649.0687. IR (neat) ν 1471;
2110; 2955; 3377 (br).
3′-Azid o-3′-d eoxy-5′-O-[bis(d ih yd r oxyp h osp h or ylm eth -
yl)ph osph or yl]-N3-[1-(5-am in open tyl)]th ym idin e (28). Com-
pound 28 is produced together with compound 4 from 14 and
is purified by HPLC (Zorbax SB C18, 250 × 20 mm; isocratic
mode: ammonium carbonate 0.5 M/acetonitrile 94:6, pH 8.8;
flow rate: 17 mL/min). Its tris-ammonium salt is obtained as
a white hygroscopic solid (retention time: 33 min; 6 mg, 13%).
1H NMR (D2O, 200 MHz) δ 1.27-1.35 (m, 2H); 1.50-1.68 (m,
4H); 1.89 (s, 3H); 2.47-2.56 (m, 6H); 2.92 (t, 2H, J ) 7.5 Hz);
3.87 (t, 2H, J ) 7.0 Hz); 4.17-4.27 (m, 3H); 4.49 (m, 1H); 6.17
(pseudo-t, 1H, J ) 6.4 Hz); 7.53 (m, 1H).31P NMR (D2O, 121.5
3′-Azid o-3′-d eoxy-N3-{1-[5-(2,2,2-tr iflu or oa cetyla m in o)-
p en tyl]}r-th ym id in e (23). Compound 22 (490 mg, 0.84
mmol) is stirred with potassium carbonate (70 mg, 0.51 mmol)
in anhydrous methanol (10 mL) for 2 h at room temperature.
Solvent is removed in vacuo before saturated aqueous NaHCO3
(15 mL) is added. The solution is extracted with CH2Cl2, and
the organic layer is dried over Na2SO4, filtered, and reduced
in vacuo. The crude residue is purified by chromatography over
silica gel (Et2O) and yields separately compound 8 (98 mg,
26%) and its R-anomer 23 (166 mg, 44%). 1H NMR (CDCl3,

5090 J . Org. Chem., Vol. 64, No. 14, 1999
Brossette et al.
MHz) δ 9.42 (m, 2P); 59.11 (m, 1P). HRMS: calcd for
1.52-1.67 (m, 4H); 1.86 (s, 3H); 2.23-2.27 (m, 1H); 2.51
(pseudo-t, 4H, J ) 19.2 Hz); 2.86 (m, 1H); 2.91 (t, 2H, J ) 7.3
Hz); 3.85 (t, 2H, J ) 7.0 Hz); 4.15-4.25 (m, 3H; 4.48 (m, 1H);
6.15 (dd, 1H, J ) 6.8, J ) 3.1 Hz); 7.59 (m, 1H). 31P NMR
(D2O, 121.5 MHz) δ 9.64 (m, 2P); 59.39 (m, 1P). HRMS: calcd
for C17H32N6O11P3 [M + H]+ 589.1342, found 589.1345; calcd
for C17H31N6O11NaP3 [M + Na]+ 611.1161, found 611.1149.
C
17H31N6O11NaP3 [M + Na]+ 611.1161, found 611.1171; calcd
for C17H31N6O11KP3 [M + K]+ 627.0901, found 627.0917;
calcd for C17H30N6O11Na2P3 [M - H + 2Na]+ 633.0981, found
633.0979; calcd for C17H32N6O11KNaP3 [M - H + Na +
K]+ 649.0720, found 649.0719. IR (neat) ν 1458; 2106; 2928;
3364 (br).
3′-Azid o-3′-d eoxy-5′-O-[bis(d ih yd r oxyp h osp h or ylm eth -
yl)p h osp h or yl]-N3-[1-(5-a m in op en tyl)]-r-th ym id in e (29).
Compound 29 is produced together with compound 4 from 14
and is purified by HPLC (Zorbax SB C18, 250 × 20 mm;
isocratic mode: ammonium carbonate 0.5 M/acetonitrile 94:
6, pH 8.8; flow rate: 17 mL/min). Its tris-ammonium salt is
obtained as a white hygroscopic solid (retention time: 21 min;
8 mg, 17%). 1H NMR (D2O, 200 MHz) δ 1.24-1.44 (m, 2H);
Ack n ow led gm en t. This work was supported by a
grant from ANRS (Agence Nationale de Recherche sur
le Sida, France). The authors thank Eric Le Gallo for
analytical work.
J O982502P