Synthesis of haptens for the development of immunoassays for the monitoring of intracellular anti-HIV nucleosides and nucleotides

Article abstract of DOI:10.1016/S0040-4020(01)00800-6

A series of nine modified dideoxynucleosides and dideoxynucleotides has been synthesized for preparing antigenic conjugates with keyhole lympet haemocyanin in order to produce specific antibodies, and develop immunoassays. Derivatives of ddI, ddA, d4T, 3TC, and the corresponding 5′-O-monophosphates were designed incorporating an amino spacer at the base for conjugation with the proteinic antigenic carrier.

Full text of DOI:10.1016/S0040-4020(01)00800-6

TETRAHEDRON
Pergamon
Tetrahedron 57 92001) 8129±8143
Synthesis of haptens for the development of immunoassays for the
monitoring of intracellular anti-HIV nucleosides and nucleotides
a
a
b
b
Â
Thierry Brossette, Emmanuel Klein, Christophe Creminon, Jacques Grassi,
Charles Mioskowskia,p and Luc Lebeaua,p
a
Á Â Â Â
Laboratoire de Synthese Bioorganique associe au CNRS, Faculte de Pharmacie, Universite Louis Pasteur de Strasbourg,
74 route du Rhin, BP 24 - 67 401 Illkirch, France
 Â
Departement de Recherche Medicale, CEA, Service de Pharmacologie et d'Immunologie,
b
Ã
Bat. 136, CEA Saclay - 91 191 Gif sur Yvette, France
Received 21 May 2001; revised 19 June 2001; accepted 1 August 2001
AbstractÐA series of nine modi®ed dideoxynucleosides and dideoxynucleotides has been synthesized for preparing antigenic conjugates
with keyhole lympet haemocyanin in order to produce speci®c antibodies, and develop immunoassays. Derivatives of ddI, ddA, d4T, 3TC,
and the corresponding 50-O-monophosphates were designed incorporating an amino spacer at the base for conjugation with the proteinic
antigenic carrier. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
of anti-HIV agents has shown to prevent or delay the
appearance of these resistances as evidenced by the lower-
ing or even disappearance of the viral load for months or
years.6±9 In these conditions however, the emergence of
other causes leading to therapy failure are observed.10±12
They are essentially related to the complex metabolism of
the drugs interfering with the general metabolic pathway of
nucleosides and nucleotides. For example it has been shown
that long-term exposure to AZT affects in vitro and in vivo
the ef®ciency of phosphorylation of thymidine kinase,13±15
or that intracellular accumulation of AZT monophosphate
9AZTMP) may decrease the conversion of the host cell
thymidine monophosphate to thymidine diphosphate and
thus lead to cellular toxicity.16,17 More recently, results
were reported suggesting that a combined use of AZT and
d4T could interfere with the intracellular formation of d4T
triphosphate leading to a decrease in the treatment
ef®cacy.18,19 These results and others highlight the impor-
tance of the intracellular metabolism for the activity of
nucleoside inhibitors of HIV RT.20±24 In addition, a number
of reports in the literature describe a great variability in the
production of the phosphorylated metabolites of antiviral
nucleosides in treated patients.25±28 That interindividual
variability considerably complicates the interpretation of
clinical data on treated patients in such a manner that treat-
ments and posologies cannot be ef®ciently optimized from
one patient to the other. This prompted us to develop
competitive enzyme immunoassays 9EIA) to monitor the
intracellular concentration of anti-HIV nucleosides and
their phosphorylated metabolites.29±32 The EIA technique
is fast and ef®cient, and is perfectly adapted to monitoring
of intracellular nucleotides as it may allow speci®c detec-
The ®rst compounds to demonstrate anti-HIV 9Human
Immunode®ciency Virus) activity were dideoxynucleosides
9ddNs). At present, ®ve ddNs are approved by the US Food
and Drug Administration and are currently used for treating
HIV-infected patients 9AZT, ddI, ddC, d4T, and 3TC). They
will be followed by a number of new nucleoside compounds
that are currently under clinical evaluation. It has been well
established that these compounds do not have intrinsic anti-
viral activity.1 They penetrate cells through a passive
process and are successively metabolized into their
monophosphate, diphosphate, and triphosphate by
intracellular kinases and nucleotidases. The resulting
triphosphates can then inhibit competitively the ADN
polymerase of the virus, called reverse transcriptase 9RT),
because they all lack a 30-hydroxyl group and work as chain
terminators.
Due to the poor ®delity of RT, a large number of mutations
spontaneously occur during the replication process.2,3 That
hypervariability of the virus acts in favor of the emergence
of variants that are resistant to the antiviral compound used
and severely limits the long-term effectiveness of the
drug.4,5 Whatever the antiviral drug administered, in the
framework of monotherapies, resistance phenomena are
observed within a few weeks or months. The combination
Keywords: dideoxynucleoside; HIV; enzyme immunoassay; mono-
phosphate; ddI; d4T; 3TC.
Corresponding authors. Tel.: 133-390244298; fax: 133-390244306;
e-mail: mioskowski@bioorga.u-strasbg.fr; lebeau@aspirine.u-strasbg.fr
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020901)00800-6

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T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
against ddI, d4T, 3TC, and their monophosphate 9MP)
metabolites.
2. Results and discussion
The speci®c antibodies needed to build up an immunoassay
are produced in animal hosts injected with the hapten or a
derivative. In order to trigger off an immune response in a
recipient animal, low molecular weight antigens 9such as the
nucleosides and nucleotides we are interested in) have to be
covalently coupled to a macromolecular carrier, i.e. a
protein 9bovine serum albumin, keyhole limpet hemo-
cyanin), a liposome, `a polymer'. The coupling reaction
generally requires a prior chemical modi®cation of the
antigen in order to introduce a functionalized spacer arm
able to react with the carrier. Furthermore the antigen modi-
®cation should not hamper the recognition speci®city of the
hapten by the elicited antibodies. In the case of the non-
natural nucleoside and nucleotide antigens we are
concerned with, we opted for anchoring the spacer at the
nucleobase to point out the ribose moiety and the phosphate
group, when present, as major antigenic determinant9s)
9Fig. 1). Compounds 2, 8, and 12 were designed to
immunize rabbits against ddI 1, d4T 7, and 3TC 11, respec-
tively. An amino spacer was introduced in the O6-position in
ddI, at N3 in d4T, and at N4 in 3TC. This will allow the
further preparation of conjugates by anchoring the
compounds onto the antigenic carrier by direct cross-linking
with glutaraldehyde. Compounds 4 and 6 were prepared to
elicit antibodies against ddIMP 3 and ddAMP 5, respec-
tively, in order to take into account the particular metabolic
pathway for ddI24 9Scheme 1). Compounds 10 and 14 were
Figure 1.
tion of haptens in the picomolar±nanomolar concentration
range. The assay is based on the use of speci®c antibodies
raised against the substance to monitor and of a hapten/
acetylcholinesterase conjugate as tracer.31
Herein, we describe the synthesis of a series of haptens
designed for the production of speci®c antibodies
Scheme 1.
Scheme 2.

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8131
synthesized to raise antibodies against d4TMP 9 and
3TCMP 13.
of the O6-alkylated product 21a and 44% of the N1-regio-
isomer 21b. These two compounds were separated by
chromatography and isomer 21a was treated with piperidine
to provide the 50-hydroxy compound 22a. Phosphorylation
of 22a afforded 23a in 86% yield and further hydrogenolysis
led to the target compound 4. The same reaction sequence
involving isomer 21b has been investigated. Removal of
the 50-protecting group and subsequent phosphorylation
were achieved in 82 and 81% yield, respectively. The
resulting compound 23b is less stable than 23a, presumably
due to the lowered aromaticity of the purine base. Conse-
quently ®nal hydrogenolysis and animal immunizations
were not further considered. It is likely that our attempts
to phosphorylate the N1-alkylated compound 18b failed for
similar reasons as only deglycosylation products were
isolated.
2.1. Synthesis of the ddI and ddA derivatives
The ddI derivative 2 bearing a linker in the O6-position was
prepared in 5 steps, starting from inosine 9Scheme 2). The
®rst 2 steps were realized according to Bhat et al..33 Inosine
was reacted with 2-acetoxy-2-methylpropanoyl bromide
9a-AIB, Mattock's bromide).34,35 Reductive elimination of
the bromoacetate mixture obtained was readily accom-
plished with zinc±copper couple in acetic acid. The result-
ing compound 15 was then treated with azido alcohol 1636
under Mitsunobu conditions37 to yield a mixture of the O6-
and N1-alkylated products. The compounds were separated
by chromatography over silica gel and regioisomers 17a and
17b were obtained as mixtures of two diastereomers. A
further treatment of 17a 9resp. 17b) with methanolic
ammonia yielded compound 18a 9resp. 18b) that was then
hydrogenolyzed over Pd/C. The double bond and the azido
group were reduced simultaneously and compound 2 9resp.
19) was obtained quantitatively.
ddIMP 3 was synthesized using a slightly different pathway
and introducing a variation adapted from a previous work by
Townsend38 9Scheme 4). Inosine was treated with an excess
of Mattock's bromide. At the end of the reaction methyl
alcohol was added to decompose the intermediate ortho-
acetate. The crude material was dried and directly acylated
with acetic anhydride. Puri®cation over silica gel afforded a
mixture of regioisomers 24a and 24b. Treatment with
Zn±Cu couple led to the formation of the unsaturated
nucleoside 25 that was hydrogenated over poisoned
Pd/BaSO4. In that case the use of poisoned catalyst revealed
necessary to avoid partial deglycosylation of the substrate as
previously reported by Manchand in similar cases.39 The
resulting compound 26 was sulfonylated with 2,4,6-tri-
isopropylbenzenesulfonyl chloride 9TPSCl) under the
conditions described by Hata,40 Jones,41 and Noyori.42
Under these conditions however the N1-sulfonylated
Initial attempts to prepare the ddIMP derivative 4 by
phosphorylation of 18a with 16 followed by hydrogenolysis
failed. Under the Mitsunobu reaction conditions 18a rapidly
decomposes and deglycosylation products are obtained
quantitatively. The poor stability of 18a is likely due to
the presence of the double bond in the sugar ring and our
further strategy required its saturation prior to phosphoryl-
ation 9Scheme 3). Thus starting from the earlier precursor
15, the 20,30-double bond was ®rst reduced by hydro-
genolysis33 and compound 20 was alkylated with azido
alcohol 16 under the Mitsunobu conditions37 to yield 54%
Scheme 3.

8132
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
Scheme 4.
Scheme 5.

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8133
Scheme 6.
compound 27b was preferentially obtained whereas the
expected O6-derivative 27a was isolated in only 12%
yield. Treatment of the latter compound with methanolic
ammonia at 08C allowed removal of the 50-protecting
group. The sulfonyl moiety in compound 28 was then
displaced by benzyl alcohol using the conditions developed
by Hata and Noyori.40,42 The resulting compound 29 was
phosphorylated with phosphoric acid dibenzyl ester under
the Mitsunobu conditions37 to yield phosphotriester 30
which was ®nally deprotected by hydrogenolysis to afford
ddIMP 3. Very surprisingly there is no other report on the
chemical synthesis of ddIMP in the literature to our
knowledge.43
2.2. Synthesis of d4T derivatives
The d4T derivatives were prepared from thymidine
9Scheme 6). The nucleoside was readily transformed into
cyclonucleoside 36 according to Horwitz.45,46 That
compound was then alkylated under the Mitsunobu
conditions47 with 5-azido-1-pentanol. The reaction was
quantitative and regioisomer 37a was obtained in 94%
yield 937b:5%). The major regioisomer was treated with
potassium tert-butoxide and opening of the oxetane led to
the d4T derivative 38. A ®nal reduction of the azide
group using a Staudinger reaction48 produced the target
compound 8.
Though ddAMP hapten 6 could be elaborated from 28, the
poor yield obtained in the preparation of the latter
compound led us to look for a more ef®cient route. So we
went a few steps back and prepared compound 6 starting
from the mixture of the earlier intermediates 24a and 24b
9Scheme 5). Chlorination under conditions described by
Robins and Basom44 led to the 6-chloropurines 31a and
31b that were further transformed into compound 32
upon treatment with Zn±Cu couple. The acetyl protecting
group was removed in methanolic ammonia at 08C and 33
was phosphorylated in the same way as 29. The resulting
phosphotriester 34 was then reacted with 4-azido-1-butyl-
amine to yield compound 35 that was ®nally deprotected
by hydrogenolysis and transformed into the target
compound 6. Finally it is worth noting that all our
attempts to synthesize 6 starting from 25 or 26 failed as
chlorination led to complete depurination of the
compounds.
The treatment of 36 under basic conditions furnished d4T 7
as described by Horwitz.45,46 The synthesis of d4TMP 9 has
been already described.49 Mansuri et al. obtained the
nucleotide in 33% yield using phosphorus oxychloride as
phosphorylating agent. In order to avoid puri®cation
problems we proceeded in a different manner 9Scheme 7).
The phosphorylation of 7 with phosphoric acid dibenzyl
ester under Mitsunobu conditions yielded compound 39
that was easily puri®ed over silica gel. Subsequent removal
of the phosphate protecting groups using trimethylsilyl
bromide led to pure d4TMP 9 after simple aqueous work
up. The yield over these two steps was 75%.
The functionalized d4TMP derivative 10 was prepared from
39 according to a procedure that was previously established
for the synthesis of AZT derivatives30,50 9Scheme 8).
Alkylation with 5-azido-1-pentanol under the Mitsunobu
conditions47 led exclusively to the N3-alkylated product
Scheme 7.

8134
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
Scheme 8.
40. This result is slightly different from the one obtained
during alkylation of cyclonucleoside 36 and in agreement
with the total regioselectivity generaly observed with other
thymidine derivatives.30,47,51 Reduction of compound 40
with tri-n-butylphosphine in the presence of water led to
41 that was ®nally deprotected with trimethylsilyl bromide
to yield 10.
4-azido-1-butylamine yielded azido ester 46 that was further
quantitatively reduced with LiAlH4 into 47. An exact
control of the operating conditions was necessary to avoid
reduction of the azide group and straightforward trans-
formation of 46 into 12. The ®nal reduction of the azide
moiety was achieved by hydrogenolysis and compound 12
was obtained in 84% yield. During our attempts to directly
reduce 46 into 12 with LiAlH4 the ®nal puri®cation step
revealed tedious and led to a lower yield when compared
to the two-step procedure.
2.3. Synthesis of 3TC derivatives
The 3TC derivatives were synthesized starting from
compound 42 previously described by Tse52 9Scheme 9).
Bis-tert-butyldimethylsilyl-uracil 43 prepared in situ was
reacted with compound 42 in the presence of trimethylsilyl
iodide to form compound 44. Treatment with TPSCl
quantitatively led to the O-sulfonylated regioisomer 45.
Nucleophilic displacement of the sulfonate moiety with
Compound 14 was prepared starting from intermediate 47
9Scheme 10). Phosphorylation of the primary hydroxyl
group happened to be unexpectedly dif®cult. Reactions
proceeding through an activation of the 50-hydroxyl group
invariably failed and led exclusively to the formation of
cyclonucleoside 51 9Scheme 11). Different activated
Scheme 9.

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8135
Detailed procedures and immunological results will be
reported elsewhere.
4. Experimental
4.1. General
Unless otherwise stated, all chemicals used were of
commercial sources. THF, Et2O and dioxane were distilled
over Na/benzophenone and CH2Cl2 over CaH2, just before
Ê
use. Methanol and DMF were dried over 3 A molecular
sieves previously heated for 12 h at 1808C under reduced
pressure 90.1 mmHg). Triethylamine was dried over Na and
pyridine over KOH. Reactions were monitored by TLC
9Merck precoated plates 0.25 mm, silica gel 60 F254
,
0.040±0.060 mm, 230±400 mesh ASTM). Liquid chroma-
tography was performed on silica gel 60 9Merck, 0.040±
0.060 mm, 230±400 mesh ASTM). Melting points were
measured with a Reichert±Jung Thermo-Galen instrument
1
coupled to an optical microscope. H-, 13C-, and 31P NMR
spectra were recorded on Brucker-WP-200-Sy and
Brucker±Avance-DPX-300 spectrometers, and chemical
shifts d are in ppm relative to an internal reference resulting
from incomplete deuteration of the NMRsolvent 9 1H:
CHCl3 at 7.27 ppm or CD2HOD at 3.31 ppm, the latter for
CDCl3/CD3OD solutions, HDO at 4.63 ppm; 13C: CDCl3 at
77.0 ppm or CD3OD at 49.0 ppm, the latter for CDCl3/
CD3OD solutions; 31P: H3PO4 at 0.00 ppm). 13C NMRspec-
tra in D2O were calibrated at 3.31 ppm by addition of a drop
of CD2HOD. 31P NMRspectra were recorded in the proton-
decoupled mode. IRspectra were recorded on Perkin±
Elmer-1600-FTIRor Perkin±Elmer-Spectrum2000-FTIR
spectrometers, and absorption values n are in cm21. Mass
Spectra 9MS) were recorded on a Finnigan-4600 quadrupole
instrument at chemical ionization. Mass data are reported in
mass units 9m/z). Abbreviations: s, singlet; d, doublet; t,
triplet; h, heptuplet; q, quintuplet; m, multiplet; b, broad.
Analytical HPLC studies were carried out in the gradient
mode 9A: 0.1% TFA in H2O; B: 0.04% TFA in CH3CN;
t0 min A/B 10:0, t60 min A/B 8:2) using a reversed phase
column 9Nucleosil 5C18, 250£4.6 mm, 0.1 mm; ¯ow rate
1 ml/min at 258C) and a photodiode array detector 9LKB
2410, detection at 267 nm).
Scheme 10.
Scheme 11.
phosphorus species were used in standard conditions but
with little success. The best conditions we could ®nd out
involve a double deprotonation of 47 with n-butyllithium
and subsequent reaction with dibenzyl chlorophosphate.
Following that procedure, compound 48 was prepared in
80% yield. The removal of the two benzyl phosphate
protecting groups was carried out using trimethylsilyl
bromide and water. The reaction was quantitative but
proceeded with partial inversion of con®guration at the
anomeric position. That phenomenon has been previously
reported with other nucleotide derivatives.30 The two
isomers 49a and 49b were not separated and ®nal reduction
of the azide groups by hydrogenolysis led to a mixture of
compounds 14 and 50. These two compounds were
separated by HPLC and 14 was obtained in 76% yield.
4.1.1. 20,30-Dideoxy-O6-[1-08-amino-3,6-dioxaoctyl)]ino-
sine 02). Compound 18a 992 mg, 0.23 mmol) and Pd/C
10% 910 mg) in methanol 95 ml) are vigorously stirred
under hydrogen atmosphere for 4 h. The catalyst is ®ltered
off and washed with methanol 95 ml). The ®ltrate is reduced
under vacuum to yield 2 as a white hygroscopic powder
986 mg, 99%). Anal. HPLC tR 52 min. H NMR9CDCl /
1
3
CD3OD 1/1, 300 MHz) d 8.57 9s, 1H); 8.47 9s, 1H); 6.33
9t, Jˆ5.3 Hz, 1H); 4.73 9m, 2H); 4.47 9m, 1H); 3.95±3.89
9m, 3H); 3.77±3.65 9m, 7H); 3.16 9m, 2H); 2.56±2.49 9m,
2H); 2.23±2.08 9m, 2H). 13C NMR9CDCl /CD3OD 1/1,
3. Conclusion
3
75 MHz) d 160.6; 152.4; 151.4; 142.6; 121.9; 87.1; 82.9;
70.8; 70.4; 69.4; 67.2; 66.4; 63.7; 40.1; 33.4; 25.7. MS 9CI/
NH3) m/z 368 [M1H]1. IR9®lm) n 3369; 2930; 1666; 1605;
1472; 1340; 1110.
The different haptens described herein have been coupled to
the carrier protein KLH 9keyhole lympet hemocyanin) using
glutaraldehyde and the conjugates have been injected into
rabbits. The production and selection of monoclonal anti-
bodies were realized using an enzymatic tracer prepared by
anchoring acetylcholinesterase to the different haptens.
4.1.2. 20,30-Dideoxy-50-O-dihydroxyphosphoryl inosine
03). To compound 30 99 mg, 0.017 mmol) in methanol

8136
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
94 ml) are added 4 drops of water and Pd/C 10% 94 mg). The
mixture is vigorously stirred under hydrogen atmosphere for
2 h. The catalyst is ®ltered off, washed with methanol 95 ml)
and water 95 ml). The ®ltrate is reduced in vacuo to yield 3
mmol) in anhydrous dichloromethane 91.5 ml) is treated
dropwise with trimethylsilyl bromide 917 ml, 0.130 mmol)
at room temperature. The mixture is stirred for 10 h before
5% ammonia 9250 ml) is added. The solvent is removed in
vacuo and the residue is solubilized in water 91 ml), washed
twice with ethyl acetate and the aqueous phase is
lyophilized to yield the ammonium salt of 9 98 mg, 99%)
as a white hygroscopic powder. RP-18 TLC 9H2O/CH3CN
as a white hygroscopic powder 94 mg, 82%). Anal. HPLC tR
6 min. H NMR9D O/CD3OD 1/1, 300 MHz) d 8.28 9s,
1
2
1H); 8.01 9s, 1H); 6.19 9dd, Jˆ3.4, 6.8 Hz, 1H); 4.32±
4.25 9m, 1H); 3.93±3.72 9m, 2H); 2.52±2.28 9m, 2H);
2.12±1.74 9m, 2H). 13C NMR 9D2O/CD3OD 1/1, 50 MHz)
d 159.6; 150.3; 146.8; 141.3; 124.7; 86.3; 82.5 9d,
Jˆ8.7 Hz); 66.7 9d, Jˆ4.3 Hz); 33.0; 26.2. 31P NMR
9D2O/CD3OD 1/1, 121.5 MHz) d 3.07. MS 9CI/NH3) m/z
220 [M2H2PO41H]1. IR9®lm) n 3396; 2949; 1603;
1470; 1058.
8/2) Rf 0.8. 1H NMR9D O, 200 MHz) d 7.52 9s, 1H); 6.86
2
9m, 1H); 6.39 9ddd, Jˆ6.2, 1.5, 1.5 Hz, 1H); 5.85 9ddd,
Jˆ6.2, 2.2, 2.2 Hz, 1H); 5.00 9m, 1H); 3.92 9m, 2H); 1.82
9s, 3H). 13C NMR 9D2O, 75 MHz) d 166.1; 152.6; 136.1;
134.0; 125.2; 110.4; 90.8; 85.9 9d, Jˆ8.7 Hz); 65.4; 12.1.
31P NMR9D 2O, 121.5 MHz) d 1.60. MS 9CI/NH3) m/z 208
[M2H2PO41H]1. IR9KBr) n 3425; 3201; 3072; 1696;
1678; 1090.
4.1.3. 20,30-Dideoxy-50-O-dihydroxyphosphoryl-O6-[1-08-
azido-3,6-dioxaoctyl)]inosine 04). Compound 4 924 mg,
51%) is obtained as a white hygroscopic powder from
4.1.7. 30-Deoxy-20,30-didehydro-50-O-dihydroxyphosphoryl-
N3-[1-05-aminopentyl)]thymidine 010). Compound 10 is
obtained as its ammonium salt starting from 41 915 mg,
0.027 mmol) and following the procedure described for
the preparation of compound 9. The crude material is puri-
®ed by preparative RP-18 TLC 9H2O/CH3CN 70/30) to
yield 10 98 mg, 67%) as a white powder. RP-18 TLC
9H2O/CH3CN 7/3) Rf 0.7. Mp .2308C. Anal. HPLC tR
compound 23a following the same procedure as described
for 3. Anal. HPLC tR 43 min. H NMR9D O, 300 MHz) d
1
2
8.53 9s, 1H); 8.26 9s, 1H); 6.23 9d, Jˆ4.1 Hz, 1H); 4.53 9m,
2H); 4.30 9m, 1H); 3.86±3.52 9m, 10H); 2.96 9t, Jˆ5.3 Hz,
2H); 2.54±2.31 9m, 2H); 2.08±1.92 9m, 2H). 13C NMR
9D2O/CD3OD 1/1, 50 MHz) d 161.0; 152.6; 151.7; 143.3;
121.7; 86.2; 82.8; 70.9; 70.4; 67.5; 66.1; 40.0; 33.0; 26.1.
31P NMR9D O/CD3OD 1/1, 121.5 MHz) d 4.79. MS 9CI/
35 min. H NMR 9D2O, 200 MHz) d 7.51 9s, 1H); 6.90 9d,
1
2
NH3) m/z 351 [M2H2PO41H]1. IR9Kbr) n 3397; 3119;
2931; 1601; 1096.
Jˆ2.9 Hz, 1H); 6.38 9dd, Jˆ6.0, 2.9 Hz, 1H); 5.84 9dd,
Jˆ3.7, 2.1 Hz, 1H); 4.99 9m, 1H); 3.94 9m, 2H); 3.82 9t,
Jˆ6.6 Hz, 2H); 3.00 9t, Jˆ6.6 Hz, 2H); 1.80 9s, 3H); 1.54±
1.49 9m, 2H); 1.44±1.39 9m, 2H); 1.23±1.19 9m, 2H). 13C
4.1.4. 20,30-Dideoxy-50-O-dihydroxyphosphoryl-N6-[1-04-
aminobutyl)]adenosine 06). Compound 6 96 mg, 51%) is
obtained as a white hygroscopic powder from compound 35
following the same procedure as described for 3. RP-18
TLC 9H2O/CH3CN 1/1) Rf O.55. Anal. HPLC tR 20 min.
1H NMR 9D2O, 300 MHz) d 8.29 9s, 1H); 8.07 9s, 1H);
6.20 9dd, Jˆ7.2, 3.6 Hz, 1H); 4.32 9m, 1H); 4.02±2.93 9m,
2H); 3.49 9m, 2H); 2.90 9m, 2H); 2.54±2.30 9m, 2H); 2.14±
1.72 9m, 2H); 1.68±1.53 9m, 4H). 13C NMR 9D2O, 75 MHz)
d 149.6; 149.5; 147.5; 134.5; 116.0; 79.4; 76.5; 60.7; 34.2;
27.0; 22.0; 20.7; 20.2; 19.2. 31P NMR 9D2O, 121.5 MHz) d
3.42. MS 9CI/NH3) m/z 290 [M2H2PO41H]1. IR9®lm) n
3401; 2938; 1623; 1083.
NMR9D O, 75 MHz) d 165.7; 152.3; 136.1; 133.9; 125.2;
2
110.5; 90.8; 85.8 9d, Jˆ8.7 Hz); 65.4 9d, Jˆ4.4 Hz); 41.4;
39.1; 28.3; 26.1; 23.0; 12.1. 31P NMR 9D2O, 121.5 MHz) d
1.51. MS 9CI/NH3) m/z 293 [M2H2PO41H]1. IR9KBr) n
3131; 2284; 1696; 1396; 1243.
4.1.8. 50-0S)-{N4-[1-04-Aminobutyl)]cytosin-1-yl}-200R)-
hydroxymethyl-10,30-oxathiolane 012). Compound 12
919 mg, 84%) is prepared from 47 following the procedure
previously described for 2. Anal. HPLC tR 22 min. 1H NMR
9CD3OD, 200 MHz) d 7.94 9d, Jˆ7.6 Hz, 1H); 6.29 9t,
Jˆ5.0 Hz, 1H); 5.86 9d, Jˆ7.6 Hz, 1H); 5.27 9t, Jˆ4.0 Hz,
1H); 3.93±3.86 9m, 2H); 3.42±3.34 9m, 2H); 3.30 9AB part
of ABX syst., JABˆ12.0, JAXˆ5.5, JBXˆ4.7 Hz, nAˆ3.30,
nBˆ3.30, 2H); 2.76 9t, Jˆ6.8 Hz, 2H); 1.67±1.54 9m, 4H).
4.1.5. 30-Deoxy-20,30-didehydro-N3-[1-05-aminopentyl)]-
thymidine 08). Compound 38 916 mg, 0.048 mmol) is
stirred in THF 92 ml) with tri-n-butylphosphine 926 ml,
0.106 mmol) and water 93 ml, 0.144 mmol) at room
temperature. The reaction is monitored by TLC. When at
completion, the solvents are removed under vacuo and the
crude residue is puri®ed by chromatography 9AcOEt/EtOH:
13C NMR9CD OD, 50 MHz) d 165.5; 158.3; 141.0; 96.9;
3
88.8; 87.6; 64.2; 41.4; 41.0; 38.1; 29.4; 27.2. MS 9CI/NH3)
m/z 301 [M1H]1. IR9®lm) n 3279; 2922; 1643; 1574;
1510; 1050.
100/0±0/100) to yield 8 as a white powder 99 mg, 61%).
Anal. HPLC tR 45 min. H NMR9CDCl , 200 MHz) d
4.1.9. 02)-20-Deoxy-50-O-dihydroxyphosphoryl-N3-[1-05-
aminopentyl)]-30-thiacytidine 014). A mixture of com-
pounds 14 and 50 is obtained from 49a and 49b following
the procedure previously described for 3. Stereoisomer 14
913 mg, 76%) is puri®ed by preparative HPLC 9Zorbax SB
C18, 250£20 mm, 5 mm; ¯ow rate 1 ml/min at 258C;
isocratic mode 0.25 M 9NH4)2CO3/CH3CN 98:2 at pH 7;
tR 7 min). Anal. HPLC tR 7 min. 1H NMR9D 2O,
300 MHz) d 8.23 9d, Jˆ8.3 Hz, 1H); 6.20 9dd, Jˆ5.3,
3.1 Hz, 1H); 6.03 9d, Jˆ8.3 Hz, 1H); 5.36 9m, 1H);
4.27±4.17 9m, 1H); 4.08±3.98 9m, 1H); 3.51±3.44 9m,
1H); 3.42±3.30 9m, 2H); 3.26±3.16 9m, 1H); 2.97±2.85
1
3
7.46 9s, 1H); 7.03 9s, 1H); 6.35 9ddd, Jˆ5.9, 1.9, 1.5 Hz,
1H); 5.87 9ddd, Jˆ5.9, 1.9, 1.5 Hz, 1H); 4.93 9m, 1H); 3.95
9t, Jˆ6.6 Hz, 2H); 3.98±3.75 9m, 2H); 3.16 9dt, Jˆ6.2,
6.1 Hz, 2H); 1.89 9s, 3H); 1.79±1.37 9m, 6H). 13C NMR
9CDCl3/CD3OD 1/1, 50 MHz) d 163.8; 151.6; 134.9;
134.5; 126.3; 109.9; 90.7; 87.2; 63.2; 41.0; 39.9; 26.7;
24.2; 23.6; 13.1. MS 9CI/NH3) m/z 310 [M1H]1. IR9®lm)
n 3322; 2944; 2855; 1700; 1666; 1639; 1467; 1239; 1089.
4.1.6.
phoryl thymidine 09). Compound 39 912 mg, 0.026
30-Deoxy-20,30-didehydro-50-O-dihydroxyphos-
9m, 2H); 1.70±1.55 9m, 4H). 13C NMR9D O/CD3OD 1/1,
2

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8137
50 MHz) d 158.2; 149.6; 143.4; 96.2; 88.1; 87.3; 65.9; 43.5;
42.9; 38.2; 25.0; 24.8. 31P NMR9D 2O, 121.5 MHz) d 1.13.
MS 9CI/NH3) m/z 284 [M2H2PO41H]1. IR9KBr) n 3413;
3107; 2943; 1725; 1660; 1190; 1064.
4.1.13. 20,30-Didehydro-20,30-dideoxy-N1-[1-08-azido-3,6-
dioxaoctyl)]inosine 018b). Compound 17b 956 mg,
0.10 mmol) is stirred with methanolic ammonia 910 ml)
at 608C in a sealed vessel for 2 h. The solvent is removed
under reduced pressure and the crude residue is puri®ed by
chromatography over silica gel 9CHCl3/EtOH 95/5±85/15)
to yield compound 18b as a colorless and viscous oil
943 mg, 99%). 1H NMR9CDCl 3, 200 MHz) d 8.17 9s,
1H), 8.06 9s, 1H); 6.93 9m, 1H); 6.44 9ddd, Jˆ5.8, 1.7,
1.6 Hz, 1H); 5.99 9ddd, Jˆ5.8, 1.8, 1.6 Hz, 1H); 5.05 9m,
1H); 4.22 9t, Jˆ4.8 Hz, 2H); 4.10±4.03 9m, 1H); 3.87±
3.759m, 1H); 3.74 9t, Jˆ4.8 Hz, 2H); 3.60±3.57 9m, 6H),
2.32 9t, Jˆ5.0 Hz, 2H). 13C NMR9CDCl 3, 50 MHz) d
154.8; 148.3; 147.1; 139.3; 134.7; 125.4; 121.0; 88.5;
87.9; 70.4; 70.3; 69.9; 68.5; 63.1; 50.4; 46.3. MS 9CI/
NH3) m/z 392 [M1H]1. IR9®lm) n 3337; 2107; 1693;
1350; 1080.
4.1.10.
20,30-Didehydro-20,30-dideoxy-50-O-02,5,5-tri-
methyl-1,3-dioxolan-4-on-2-yl)-O6-[1-08-azido-3,6-dioxa-
octyl)]inosine 017a). Triphenylphosphine 91.50 g, 5.72
mmol) in anhydrous THF 95 ml) is added dropwise to a
mixture of nucleoside 15 91.95 g, 3.17 mmol), azido alcohol
1636 90.78 g, 4.46 mmol), and DEAD 90.90 ml, 5.71 mmol).
The solution is stirred for 45 min and the solvent is removed
under reduced pressure. The crude residue is chromato-
graphed over silica gel 9AcOEt/EtOH 95/5±0/100) to
yield the O6-alkylated product 17a 91/1 mixture of the two
diastereomers) 90.87 g, 52%) as a glassy solid. TLC
9AcOEt/EtOH 85/15) Rf 0.8. H NMR9CDCl , 200 MHz)
1
3
d 8.46 9s, 1H); 8.09 9s, 1H); 8.06 9s, 1H); 7.08 9dd, Jˆ3.0,
1.5 Hz, 1H); 6.38±6.31 9m, 1H); 6.04 9d, Jˆ3.8 Hz, 1H);
5.06 9m, 1H); 4.70 9t, Jˆ4.9 Hz, 2H); 3.89 9t, Jˆ4.9 Hz,
2H); 3.81±3.75 9m, 8H); 3.30 9t, Jˆ5.0 Hz, 2H); 1.65 and
4.1.14.
20,30-Dideoxy-N1-[1-08-amino-3,6-dioxaoctyl)]-
inosine 019). Compound 19 9186 mg, 98%) is obtained
from 18b following the same procedure as described for
3. For analytical purposes, the compound is puri®ed by
1.64 92s, 3H); 1.47±1.35 9m, 6H). 13C NMR9CDCl
,
3
50 MHz) d 174.2 and 172.2; 160.4; 152.0; 151.7; 140.6 and
140.2; 133.8 and 133.6; 125.9 and 125.7; 125.6 and 125.5;
121.3 and 120.1; 88.2 and 88.1; 84.9 and 84.6; 78.7 and 77.9;
70.6; 70.4; 69.8; 69.0; 66.0 and 65.3; 63.1 and 62.6; 50.4; 25.4
and 25.1; 24.8 and 24.6; 24.4 and 24.1. MS 9CI/NH3) m/z 537
[M1NH4]1. IR 9®lm) n 2936; 2876; 2103; 1801; 1599.
preparative TLC 9EtOH/MeOH 95/5). Anal. HPLC tR
39 min. H NMR9CDCl /CD3OD 1/1, 300 MHz) d 8.06
1
3
9s, 1H); 8.03 9s, 1H); 6.06 9dd, Jˆ5.6, 4.6 Hz, 1H); 4.06±
4.00 9m, 3H); 3.73±3.61 9m, 3H); 3.47±3.41 9m, 7H); 2.92±
2.88 9m, 2H); 2.35±2.48 9m, 2H); 2.02±1.95 9m, 2H). 13C
NMR9CDCl 3/CD3OD 1/1, 75 MHz) d 156.6; 148.2; 146.7;
138.8; 123.9; 85.6; 82.0; 70.0; 69.6; 68.1; 67.0; 63.2; 45.7;
39.4; 32.4; 25.1. MS 9CI/NH3) m/z 368 [M1H]1. IR9®lm) n
3368; 2919; 2496; 1684; 1349; 1108.
4.1.11.
20,30-Didehydro-20,30-dideoxy-50-O-02,5,5-tri-
methyl-1,3-dioxolan-4-on-2-yl)-N1-[1-08-azido-3,6-dioxa-
octyl)]inosine 017b). Compound 17b 90.77 g, 47%) is
obtained as a mixture of two diastereomers during the
4.1.15. 20,30-Dideoxy-50-O-02,5,5-trimethyl-1,3-dioxolan-
4-on-2-yl)inosine 020). Compound 15 93.58 g, 9.88 mmol)
and Pd 95%) on BaSO4 92.70 g) are stirred in a mixture of
methanol 930 ml) and methoxyethanol 940 ml) under
hydrogen atmosphere for 2 h. The catalyst is ®ltered off
and the solvent is removed under reduced pressure. The
crude residue is puri®ed by chromatography over silica
gel 9AcOEt/EtOH 9/1±5/5) to yield 20 91/1 mixture of
two diastereomers) 91.50 g, 42%) as a colorless and glassy
solid. 1H NMR9CDCl 3, 300 MHz) d 8.29 and 8.28 92s, 1H);
8.13 and 8.09 92s, 1H); 6.26 9dd, Jˆ4.2, 4.1 Hz, 1H); 4.35
9m, 1H); 3.78 9AB part of an ABXX0 syst., JABˆ29.6,
1
preparation of 17a. TLC 9AcOEt/EtOH: 85/15) Rf 0.5. H
NMR9CDCl 3, 200 MHz) d 8.08 9s, 1H); 7.91 and 7.89 92s,
1H); 6.97 9m, 1H); 6.39 9d, Jˆ5.9 Hz, 1H); 6.06 9d,
Jˆ5.9 Hz, 1H); 5.08 9m, 1H); 4.24 9t, Jˆ4.4 Hz, 2H);
3.83±3.75 9m, 1H); 3.75 9t, Jˆ4.4 Hz, 2H); 3.69±3.55 9m,
7H); 3.33 9t, Jˆ4.8 Hz, 2H); 1.69 and 1.68 92s, 3H); 1.51±
1.43 9m, 6H). 13C NMR9CDCl , 50 MHz) d 174.4 and
3
172.4; 156.5; 148.5; 147.4; 138.3 and 137.8; 134.1 and
133.9; 125.6 and 125.4; 124.2; 120.3; 88.1; 85.1 and 84.7;
78.8 and 78.0; 70.4; 70.3; 69.4; 68.6; 63.1 and 62.6; 50.5;
46.3; 25.6 and 25.2; 25.1 and 24.7; 24.6 and 24.5. MS 9CI/
NH3) m/z 537 [M1NH4]1. IR9®lm) n 3448; 2872; 2108;
1801; 1696.
0
0
JAXˆ10.2, JAX ˆ3.4, JBXˆ10.4, JBX ˆ4.2 Hz, nAˆ3.86,
nBˆ3.70, 2H); 2.56±2.48 9m, 2H); 2.06±2.17 9m, 2H);
1.73 9s, 3H); 1.53 and 1.51 92s, 3H); 1.50 and 1.47 92s,
3H). 13C NMR9CDCl 3, 50 MHz) d 174.6 and 174.5;
159.1; 148.2; 145.1; 138.5 and 138.2; 125.0; 120.4; 85.8
and 85.7; 79.7; 79.2 and 79.0; 65.8 and 65.6; 33.0 and
32.9; 25.9 and 25.8; 25.7 and 25.6; 25.5 and 25.3; 24.7
4.1.12. 20,30-Didehydro-20,30-dideoxy-O6-[1-08-azido-3,6-
dioxaoctyl)]inosine 018a). Compound 17a 9115 mg,
0.221 mmol) is stirred with methanolic ammonia 910 ml)
at room temperature for 8 h. The solvent is removed under
reduced pressure and the crude residue is puri®ed by chro-
matography over silica gel 9AcOEt/EtOH 1/0±9/1) to yield
compound 18a as a colorless and viscous oil 986 mg, 99%).
and 24.6. MS 9CI/NH3) m/z 365 [M1H]1. IR9®lm)
n
2984; 1800; 1700; 1207; 1174.
1H NMR9CDCl , 300 MHz) d 8.49 9s, 1H); 8.12 9s, 1H);
4.1.16. 20,30-Dideoxy-50-O-02,5,5-trimethyl-1,3-dioxolan-
4-on-2-yl)-O6-[1-08-azido-3,6-dioxaoctyl)]inosine 021a).
Compound 21a 90.94 g, 55%) is prepared 91/1 mixture of
two diastereomers) from 20 following the same procedure
as for 17a. Puri®cation is achieved over silica gel 9Et2O/
3
6.98 9m, 1H); 6.47 9m, 1H); 6.02 9m, 1H); 5.11 9m, 1H); 4.77
9dd, Jˆ5.3, 3.1 Hz, 2H); 4.01 9td, Jˆ10.6, 1.9 Hz, 1H); 3.95
9t, Jˆ5.0 Hz, 2H); 3.90 9td, Jˆ12.8, 2.8 Hz, 1H); 3.75±3.64
9m, 6H); 3.36 9t, Jˆ5.3 Hz, 2H). 13C NMR9CDCl
,
3
75 MHz) d 160.8; 151.9; 151.4; 141.4; 134.7; 125.8;
122.4; 90.6; 88.7; 70.8; 70.6; 70.0; 69.2; 66.3; 63.8; 50.7.
MS 9CI/NH3) m/z 392 [M1H]1; 409 [M1NH4]1. IR9®lm)
n 3347; 2873; 2110; 1669; 1601.
CH3CN/AcOEt/EtOH
9AcOEt/EtOH 8/2) Rf 0.8. H NMR9CDCl , 200 MHz) d
60/20/20/0±0/0/25/75).
TLC
1
3
8.49 and 8.48 92s, 1H); 8.27 and 8.26 92s, 1H); 6.34 9dd,
Jˆ5.8, 1.7 Hz, 1H); 4.75 9t, Jˆ4.9 Hz, 2H); 4.39 9m, 1H);

8138
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
3.96 9t, Jˆ4.9 Hz, 2H); 3.84±3.59 9m, 8H); 3.39 and 3.36
92t, Jˆ5.1 Hz, 2H); 2.60±2.50 9m, 2H); 2.26±2.08 9m, 2H);
1.76 9s, 3H); 1.53 and 1.52 92s, 3H); 1.49 9s, 3H). 13C NMR
9CDCl3, 50 MHz) d 174.5; 160.5; 151.8 and 151.7; 151.2;
140.6 and 140.2; 121.9; 120.3; 85.8 and 85.7; 79.6 and 79.0;
78.0; 70.8; 70.6; 70.0; 69.2; 66.1; 63.2 and 62.9; 50.6; 32.9
and 32.8; 30.2 and 29.6; 26.0 and 25.6; 25.5 and 25.3; 24.6
and 24.4. MS 9CI/NH3) m/z 522 [M1H]1. IR9®lm) n 3408;
2939; 2105; 1799; 1741; 1604; 1303.
121.8; 85.5; 79.4 9d, Jˆ8.7 Hz); 70.6; 69.9; 69.4; 69.1;
68.0 9d, Jˆ5.8 Hz); 66.1; 50.5; 32.1; 25.8. 31P NMR
9CDCl3, 121.5 MHz) d 20.04. MS 9CI/NH3) m/z 654
[M1H]1. IR9®lm) n 2950; 2105; 1736; 1597; 1469;
1275; 1036.
4.1.21. 20,30-Dideoxy-50-O-dibenzyloxyphosphoryl-N1-
[1-08-azido-3,6-dioxaoctyl)]inosine 023b). Compound
23b 90.34 g, 81%) is prepared from 22b following the
1
same procedure as for 23a. H NMR9CDCl , 200 MHz) d
3
4.1.17. 20,30-Dideoxy-50-O-02,5,5-trimethyl-1,3-dioxolan-
4-on-2-yl)-N1-[1-08-azido-3,6-dioxaoctyl)]inosine 021b).
That compound 90.77 g, 44%) is obtained as a side product
8.46 9s, 1H); 8.13 9s, 1H); 7.35±7.30 9m, 10H); 6.28 9t,
Jˆ5.1 Hz, 1H); 5.00 9d, Jˆ8.8 Hz, 4H); 4.74 9t, Jˆ4.9 Hz,
2H); 4.30±4.03 9m, 3H); 3.95 9t, Jˆ4.9 Hz, 2H); 3.75±3.62
9m, 6H); 3.35 9t, Jˆ5.1 Hz, 2H); 2.54±2.44 9m, 2H); 2.11±
during the preparation of 21a. TLC 9AcOEt/EtOH 8/2) Rf
0.5. H NMR9CDCl , 300 MHz) d 8.04 9s, 1H); 7.98 9s,
1
2.03 9m, 2H). 13C NMR9CDCl , 50 MHz) d 160.5; 151.8;
3
3
1H); 6.20 9m, 1H); 4.33 9m, 1H); 4.22 9m, 2H); 3.74 9t,
Jˆ4.7 Hz, 2H); 3.67±3.50 9m, 8H); 3.31 9t, Jˆ4.9 Hz,
2H); 2.54±2.45 9m, 2H); 2.17±2.04 9m, 2H); 1.71 9s, 3H);
1.56 and 1.54 92s, 3H); 1.53 and 1.50 92s, 3H). MS 9CI/NH3)
m/z 522 [M1H]1.
151.3; 140.3; 135.4 9d, Jˆ7.1 Hz); 128.6; 128.5; 127.8;
121.8; 85.5; 79.4 9d, Jˆ8.7 Hz); 70.6; 69.9; 69.4; 69.1;
68.0 9d, Jˆ5.8 Hz); 66.1; 50.5; 32.1; 25.8. 31P NMR
9CDCl3, 121.5 MHz) d 20.04. MS 9CI/NH3) m/z 654
[M1H]1. IR9®lm) n 2950; 2105; 1736; 1597; 1469;
1275; 1036.
4.1.18. 20,30-Didehydro-20,30-dideoxy-O6-[1-08-azido-3,6-
dioxaoctyl)]inosine 022a). Piperidine 9890 ml, 9.00 mmol)
is added to compound 21a 9938 mg, 1.80 mmol) in
anhydrous methanol 925 ml). The reaction mixture is
re¯uxed for 5 h and a second portion of piperidine
9890 ml, 9.00 mmol) is added. After an additional 5 h re¯ux-
ing period the reaction is at completion. The solvent is
removed in vacuo and the residue is puri®ed over silica
4.1.22. 9-020,50-Di-O-acetyl-30-bromo-30-deoxy-b-d-xylo-
furanosyl)hypoxanthine and 9-030,50-di-O-acetyl-20-
bromo-30-deoxy-b-d-xylofuranosyl)hypoxanthine 024a
and 24b). A suspension of inosine 95.0 g, 18.6 mmol) in
dry acetonitrile is treated dropwise at 08C with 2-acetoxy-
isobutyryl bromide 98.2 ml, 56.1 mmol). Material dissolves
after 2 h and the mixture is stirred at room temperature for
10 h. Anhydrous methanol 95 ml) is added and after 15 min
the solvents are removed in vacuo. The crude residue is
dissolved in anhydrous pyridine 9120 ml) and acetic
anhydride 911.4 ml, 120.8 mmol) is added dropwise at
room temperature. The reaction mixture is stirred for
30 min before evaporation and the residue is puri®ed by
chromatography 9AcOEt/EtOH 100/0±95/5) to yield a
gel 9AcOEt/EtOH 9/1) to yield 22a 9591 mg, 84%) as a
glassy solid. H NMR9CDCl , 300 MHz) d 8.49 9s, 1H);
1
3
8.09 9s, 1H); 6.17 9t, Jˆ6.3 Hz, 1H); 4.76 9t, Jˆ5.0 Hz, 2H);
4.37±4.33 9m, 1H); 4.04±3.93 9m, 3H); 3.74±3.59 9m, 7H);
3.36 9t, Jˆ5.1 Hz, 2H); 2.75±2.66 9m, 1H); 2.46±2.35 9m,
2H); 2.24±2.14 9m, 1H). 13C NMR9CDCl , 75 MHz) d
3
160.9; 151.6; 150.9; 141.5; 122.8; 87.8; 81.7; 70.8; 70.6;
70.3; 70.0; 66.3; 64.7; 50.6; 32.4; 25.9. MS 9CI/NH3) m/z
394 [M1H]1. IR9®lm) n 3360; 2921; 2101; 1596; 1308.
mixture of the two regioisomers 24a and 24b 97.74 g,
99%, 24a/24b 76/24) as a white foam. H NMR9CDCl ,
1
3
300 MHz) d 8.36 9s, 1Ha); 8.35 9s, 1Hb); 8.25 9s, 1Ha); 8.13
9s, 1Hb); 6.35 9d, Jˆ4.2 Hz, 1Hb); 6.18 9d, Jˆ3.0 Hz, 1Ha);
5.68±5.65 9m, 1Ha); 5.50 9t, Jˆ2.3 Hz, 1Hb); 4.68 9dd,
Jˆ4.2, 2.3 Hz, 1Hb); 4.39±4.53 9m, 4Ha and 2Hb); 4.30
9m, 1Hb); 2.18±1.97 9m, 6Ha and 6Hb). 13C NMR
9CDCl3, 50 MHz) d 171.0 9b); 170.2 9a); 169.0 9a,b);
158.7 9a,b); 148.4 9a); 147.7 9b); 146.0 9a,b); 138.2 9a,b);
124.7 9a,b); 87.8 9a); 85.0 9b); 83.0 9a); 80.6 9b); 78.5 9b);
77.9 9a); 64.7 9a); 64.0 9b); 49.2 9a); 48.8 9b); 24.4 9b); 24.2
9a); 20.5 9b); 20.4 9a). MS 9CI/NH3) m/z 415 and 417
[M1H]1. IR9®lm) n 2982; 1744; 1700; 1371; 1221.
4.1.19. 20,30-Didehydro-20,30-dideoxy-N1-[1-08-azido-3,6-
dioxaoctyl)]inosine 022b). Compound 22b 90.78 g, 82%) is
prepared from 21b following the same procedure as for 22a.
1H NMR9CDCl , 200 MHz) d 8.04 9s, 1H); 7.97 9s, 1H);
3
7.35±7.29 9m, 10H); 6.20 9dd, Jˆ6.2, 4.8 Hz, 1H); 5.02 9d,
Jˆ9.1 Hz, 4H); 4.23±4.09 9m, 5H); 3.76 9t, Jˆ4.6 Hz, 2H);
3.70±3.57 9m, 6H); 3.35 9t, Jˆ5.1 Hz, 2H); 2.54±2.02 9m,
4H). MS 9CI/NH3) m/z 654 [M1H]1.
4.1.20. 20,30-Dideoxy-50-O-dibenzyloxyphosphoryl-O6-
[1-08-azido-3,6-dioxaoctyl)]inosine 023a). PPh3 9240 mg,
0.92 mmol) in anhydrous THF 94 ml) is added dropwise to
nucleoside 22a 950 mg, 0.13 mmol), phosphoric acid
dibenzyl ester 939 mg, 0.14 mmol), and DEAD 9144 ml,
0.92 mmol) in THF 98 ml). After 2 h the solvent is removed
under reduced pressure and the residue is puri®ed by chro-
matography 9Et2O/AcOEt/EtOH 1/0/0±0/6/4) to yield 23a
971 mg, 86%) as a glassy solid. 1H NMR9CDCl 3, 200 MHz)
d 8.46 9s, 1H); 8.13 9s, 1H); 7.35±7.30 9m, 10H); 6.28 9t,
Jˆ5.1 Hz, 1H); 5.00 9d, Jˆ8.8 Hz, 4H); 4.74 9t, Jˆ4.9 Hz,
2H); 4.30±4.03 9m, 3H); 3.95 9t, Jˆ4.9 Hz, 2H); 3.75±3.62
9m, 6H); 3.35 9t, Jˆ5.1 Hz, 2H); 2.54±2.44 9m, 2H); 2.11±
4.1.23. 50-O-Acetyl-20,30-didehydro-20,30-dideoxyinosine
025). The previous white foam 9500 mg, 1.20 mmol) is
dissolved in anhydrous THF 914 ml) and to the clear solu-
tion is added Zn±Cu couple 90.5 g) and 2 drops of glacial
acetic acid. The reaction mixture is vigorously stirred at
room temperature for 12 h. The resulting suspension is
®ltered over a celite pad. The ®ltrate is reduced under
vacuum and the residue is puri®ed by chromatography
9CHCl3/MeOH 10/0±8/2) to yield 25 9146 mg, 44%) as a
white crystalline powder. H NMR9CDCl , 300 MHz) d
1
3
8.22 9s, 1H); 8.09 9s, 1H); 7.06 9m, 1H); 6.41 9d,
Jˆ6.0 Hz, 1H); 6.13 9d, Jˆ6.0 Hz, 1H); 6.13 9d,
Jˆ6.0 Hz, 1H); 5.18 9m, 1H); 4.35 9AB part of ABX syst.,
2.03 9m, 2H). 13C NMR9CDCl , 50 MHz) d 160.5; 151.8;
3
151.3; 140.3; 135.4 9d, Jˆ7.1 Hz); 128.6; 128.5; 127.8;

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8139
JABˆ12.1, JAXˆ4.1, JBXˆ3.4 Hz, nAˆ4.42, nBˆ4.28, 2H);
9m, 4H); 1.32 9d, Jˆ6.6 Hz, 6H); 1.31 9d, Jˆ6.6 Hz, 6H);
2.07 9s, 3H). 13C NMR9CDCl , 75 MHz) d 159.3; 148.8;
1.30 9d, Jˆ6.6 Hz, 6H). 13C NMR9CDCl , 75 MHz) d
3
3
145.4; 138.3; 133.7; 127.3; 125.9; 88.6; 84.9; 65.4; 21.2.
MS 9CI/NH3) m/z 277 [M1H]1. IR9®lm) n 3062; 1743;
1698.
155.0; 154.4; 153.0; 151.1; 151.0; 144.0; 131.1; 123.9;
123.6; 87.3; 82.2; 63.8; 34.3; 32.9; 29.7; 25.4; 24.5; 23.5.
MS 9CI/NH3) m/z 503 [M1H]1. IR9®lm) n 3360; 2962;
1606; 1589; 1386.
4.1.24. 50-O-Acetyl-20,30-dideoxyinosine 026). Compound
26 9104 mg, 72%) is obtained as a white powder starting
from 25 and following the same procedure as described for
19. Puri®cation is achieved by chromatography over silica
gel 9AcOEt/EtOH 9/1±6/4). 1H NMR9CDCl 3, 300 MHz) d
8.18 9s, 1H); 8.16 9s, 1H); 6.27 9dd, Jˆ5.3, 4.5 Hz, 1H);
4.44±4.21 9m, 3H); 2.59±2.55 9m, 2H); 2.24±2.07 9m,
4.1.28. O6-Benzyl-20,30-dideoxyinosine 029). Benzyl
alcohol 947 ml, 0.45 mmol) and quinuclidine 96 mg,
0.05 mmol) are added to nucleoside 28 925 mg,
0.05 mmol) in anhydrous dichloromethane 92.5 ml). The
solution is stirred for 10 min at 08C then DBU 911 ml,
0.08 mmol) is added and the reaction mixture is allowed
to slowly warm to room temperature for 12 h. The solvent
is removed under reduced pressure and the crude residue is
chromatographed over silica gel 9hexane/AcOEt 1/5±0/1) to
2H); 2.09 9s, 3H). 13C NMR9CDCl , 75 MHz) d 170.3;
3
159.2; 148.2; 144.7; 138.3; 119.9; 85.7; 79.2; 65.6; 29.7;
n
24.5; 21.1. MS 9CI/NH3) m/z 279 [M1H]1. IR9®lm)
2919; 1737; 1696.
1
yield 29 910 mg, 62%) as a white crystalline powder. H
NMR9CDCl , 200 MHz) d 8.54 9s, 1H); 8.07 9s, 1H);
3
4.1.25. 50-O-Acetyl-20,30-dideoxy-O6-02,4,6-triisopropyl-
benzenesulfonyl)inosine 027a). 2,4,6-Triisopropylbenzene-
sulfonyl chloride 92.88 g, 9.52 mmol) in dichloromethane
918 ml) is added dropwise to a mixture of nucleoside 26
91.32 g, 4.75 mmol), 4-DMAP 927 mg, 0.24 mmol), and
anhydrous triethylamine 92.65 ml, 19.0 mmol) in dichloro-
methane 930 ml) at room temperature. The reaction mixture
is stirred for 4 h and saturated aqueous NaHCO3 and NaCl
solutions 915 and 5 ml, respectively) are added. The mixture
is extracted with dichloromethane 95£20 ml). The organic
layer is dried over Na2SO4, reduced in vacuo, and puri®ed
over silica gel 9Et2O/AcOEt 7/3). The O6-sulfonylated
product 27a 9307 mg, 12%) is obtained as a white powder.
TLC 9AcOEt/Et2O 7/3) Rf 0.75. Mp 1138C. 1H NMR
9CDCl3, 300 MHz) d 8.55 9s, 1H); 8.35 9s, 1H); 7.22 9s,
2H); 6.36 9dd, Jˆ6.2, 3.0 Hz, 1H); 4.45±4.25 9m, 5H);
2.92 9h, Jˆ6.8 Hz, 1H); 2.65±2.60 9m, 2H); 2.19±2.08
9m, 4H); 1.33 9d, Jˆ6.6 Hz, 6H); 1.32 9d, Jˆ6.6 Hz, 6H);
6.16 9dd, Jˆ6.9, 5.9 Hz, 1H); 5.68 9s, 2H); 4.39±4.33 9m,
1H); 4.03 9dd, Jˆ12.8, 1.8 Hz, 1H); 3.71±3.59 9m, 1H);
2.81±2.67 9m, 1H); 2.51±2.33 9m, 2H); 2.31±2.15 9m,
1H). 13C NMR9CDCl , 50 MHz) d 160.8; 151.6; 150.9;
3
141.5; 135.9; 128.4; 128.3; 128.2; 123.0; 87.9; 81.7; 68.5;
64.7; 32.4; 25.9. MS 9CI/NH3) m/z 327 [M1H]1; 344
[M1NH4]1. IR9®lm) n 3311; 2922; 1600; 1461; 1344;
1222.
4.1.29.
O6-Benzyl-50-O-dibenzyloxyphosphoryl-20,30-
dideoxyinosine 030). Compound 30 994 mg, 56%) is
prepared starting from nucleoside 29 and following the
same procedure as described for 23a. The compound is
puri®ed by chromatography over silica gel 9Et2O/AcOEt/
EtOH 1/1/0±0/9/1). H NMR9CDCl , 300 MHz) d 8.51
1
3
9s, 1H); 8.15 9s, 1H); 7.40±7.28 9m, 15H); 6.30 9dd,
Jˆ4.9, 5.7 Hz, 1H); 5.68 9s, 2H); 5.01 9AB part of ABX
syst., JABˆ8.7, JAXˆ12.4, JBXˆ12.1 Hz, nAˆ5.04,
nBˆ4.99, 4H); 4.31 9m, 1H); 4.22±4.07 9m, 2H); 2.55±
2.47 9m, 2H); 2.14±2.01 9m, 2H). 13C NMR9CDCl
,
3
1.31 9s, Jˆ6.6 Hz, 6H). 13C NMR9CDCl , 75 MHz) d
3
170.5; 154.7; 154.3; 153.2; 151.1; 151.0; 143.0; 131.1;
123.8; 123.2; 86.1; 79.5; 64.6; 34.2; 32.5; 29.7; 25.7;
24.5; 23.4; 20.7. IR9®lm) n 2955; 1739; 1600; 1561;
1233. MS 9CI/NH3) m/z 545 [M1H]1.
75 MHz) d 160.5; 151.9; 151.4; 140.4; 136.1; 128.8;
128.6; 128.4; 128.3; 128.1; 128.0; 122.0; 85.6; 79.5 9d,
Jˆ8.7 Hz); 69.5 9d, Jˆ5.8 Hz); 68.4; 68.1 9d, Jˆ5.8 Hz);
29.7; 25.9. 31P NMR9CDCl , 121.5 MHz) d 0.58. MS 9CI/
3
4.1.26. 50-O-Acetyl-20,30-dideoxy-N1-02,4,6-triisopropyl-
benzenesulfonyl)inosine 027b). The N1-sulfonylated
compound 27b 9620 mg, 36%) is obtained as the major
NH3) m/z 588 [M1H]1. IR9®lm) n 2922; 1739; 1600; 1455;
1005.
reaction product during the preparation of 27a. TLC
9AcOEt/Et2O 7/3) Rf 0.65. H NMR9CDCl , 300 MHz) d
4.1.30. 6-Chloro-9-020,50-di-O-acetyl-30-bromo-30-deoxy-
b-d-xylo-furanosyl)purine 031a) and 6-chloro-9-030,50-
di-O-acetyl-20-bromo-30-deoxy-b-d-xylofuranosyl)purine
031b). A solution of thionyl chloride 91.98 ml, 27.3 mmol)
and DMF 90.96 ml, 12.4 mmol) in dichloromethane 924 ml)
is added dropwise over 3 h to a mixture of the two regio-
isomers 24a and 24b 91.0 g, 2.4 mmol) in re¯uxing
dichloromethane 960 ml). The reaction mixture is re¯uxed
for 12 h more, cooled down to room temperature and stirred
for 30 min with saturated aqueous NaHCO3. The solution is
extracted with dichloromethane and the organic layer is
dried over Na2SO4 and reduced in vacuo. The crude residue
is puri®ed by chromatography over silica gel 9Et2O) to yield
the two regioisomers 31a 90.71 g, 68%) and 31b 90.23 g,
1
3
8.85 9s, 1H); 8.01 9s, 1H); 7.19 9s, 2H); 6.28 9dd, Jˆ6.4,
2.6 Hz, 1H); 4.44±4.22 9m, 3H); 4.41 9h, Jˆ6.8 Hz, 1H);
2.90 9h, Jˆ6.8 Hz, 1H); 2.51 9m, 2H); 2.28±2.09 9m, 1H);
2.06 9s, 3H); 1.23 9d, Jˆ6.3 Hz, 6H); 1.21 9d, Jˆ6.3 Hz,
6H); 1.20 9d, Jˆ6.3 Hz, 6H). MS 9CI/NH3) m/z 545
[M1H]1.
4.1.27.
20,30-Dideoxy-O6-02,4,6-triisopropylbenzene-
sulfonyl)inosine 028). Compound 28 is obtained starting
from 27 and following the procedure previously described
for 18a, unless the reaction is carried out at 08C for 5 h.
Puri®cation over silica gel 9hexane/AcOEt 1/1) yields 28
as a white powder 951 mg, 54%). 1H NMR9CDCl
,
3
22%) as white crystalline powders. 31a: TLC 9Et2O) Rf
0.55. H NMR9CDCl , 300 MHz) d 8.78 9s, 1H); 8.62 9s,
1
300 MHz) d 8.55 9s, 1H); 8.47 9s, 1H); 7.22 9s, 2H); 6.27
9dd, Jˆ6.0, 5.7 Hz, 1H); 4.38±4.29 9m, 3H); 4.05±3.99 9m,
1H); 3.70±3.63 9m, 1H); 2.92 9h, Jˆ6.8 Hz, 1H); 2.64±2.13
3
1H); 6.31 9d, Jˆ2.3 Hz, 1H); 5.78 9dd, Jˆ1.9, 1.5 Hz, 1H);
4.62±4.48 9m, 4H); 2.19 9s, 3H); 2.13 9s, 3H). 13C NMR

8140
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
9CDCl3, 50 MHz) d 170.0; 168.7; 152.0; 150.9; 150.8;
143.0; 131.5; 88.1; 82.5; 78.8; 64.5; 48.6; 20.5; 20.3. IR
9®lm) n 2986; 1751; 1561; 1216; 1043. MS 9CI/NH3) m/z
are stirred at room temperature for 5 h in anhydrous metha-
nol 92.5 ml). The solvent is removed under reduced pressure
and the crude residue is chromatographed over silica gel
9AcOEt/EtOH 1/0±95.7/2.5) to yield 35 917 mg, 42%) as
1
435 [M1H]1. 31b: TLC 9Et2O) Rf 0.4. H NMR9CDCl ,
3
300 MHz) d 8.77 9s, 1H); 8.48 9s, 1H); 6.49 9d, Jˆ2.6 Hz,
1H); 5.63 9dd, Jˆ3.1, 2.9 Hz, 1H); 4.78 9dd, Jˆ4.1, 2.6 Hz,
1H); 4.62±4.48 9m, 3H); 2.18 9s, 3H); 2.16 9s, 3H). 13C
NMR9CDCl 3, 50 MHz) d 172.1; 169.2; 151.9; 150.4;
150.3; 143.4; 131.7; 85.2; 81.1; 78.8; 64.1; 50.8; 20.9;
20.4. MS 9CI/NH3) m/z 435 [M1H]1. IR9®lm) n 2986;
1751; 1561; 1216; 1043.
a glassy solid. A fraction of starting material 913 mg,
37%) is recovered. H NMR9CDCl , 300 MHz) d 8.40 9s,
1
3
1H); 7.89 9s, 1H); 7.37±7.28 9m, 10H); 7.08 9m, 1H); 6.28
9ddd, Jˆ5.6, 1.7, 1.7 Hz, 1H); 5.03 9m, 1H); 4.98 9d,
Jˆ8.6 Hz, 4H); 4.11 9AB part of ABXX0 syst., JABˆ11.3,
0
0
JAXˆ6.6, JAX ˆ3.4, JBXˆ6.2, JBX ˆ3.4 Hz, nAˆ4.16,
nBˆ4.07, 2H); 3.67±3.73 9m, 2H); 3.34 9t, Jˆ6.4 Hz,
2H); 1.68±1.82 9m, 4H). 13C NMR9CDCl , 75 MHz) d
3
4.1.31. 6-Chloro-9-050-O-acetyl-20,30-dideoxy-b-d-glycero-
pent-20-enofuranosyl)purine 032). Compound 32 is
prepared starting from the mixture of regioisomers 31a
and 31b and following the same procedure as described
for 25. Chromatography over silica gel 9Et2O/AcOEt 1/0±
155.4; 154.9; 138.1; 135.4; 132.9; 129.5; 128.9; 128.6;
128.0; 126.4; 119.6; 88.0; 85.1 9d, Jˆ8.3 Hz); 69.4 9d,
Jˆ5.5 Hz); 67.5 9d, Jˆ5.5 Hz); 51.1; 27.0; 26.4; 26.3. 31P
NMR9CDCl , 121.5 MHz) d 0.41. MS 9CI/NH3) m/z 592
3
[M1H]1. IR9®lm) n 3308; 2096; 1619; 1278; 1008.
1
1/1) yields 32 9104 mg, 49%) as a glassy solid. H NMR
9CDCl3, 300 MHz) d 8.75 9s, 1H); 8.31 9s, 1H); 7.16 9ddd,
Jˆ3.0, 1.5, 1.5 Hz, 1H); 6.43 9ddd, Jˆ6.0, 1.9, 1.5 Hz, 1H);
6.17 9ddd, Jˆ6.0, 1.9, 1.9 Hz, 1H); 5.19 9m, 1H); 4.29 9AB
part of ABX syst., JABˆ12.4, JAXˆ3.8, JBXˆ3.0 Hz,
4.1.35. N1-03,5-Anhydro-2-deoxy-b-d-pentafuranosyl)-
N3[-1-05-azidopentyl)]thymidine 037a). DEAD 9127 ml,
0.806 mmol) is added to triphenylphosphine 9211 mg,
0.806 mmol) in anhydrous THF 91 ml). The resulting solu-
tion is added dropwise to a mixture of oxetane 3645,46
9100 mg, 0.446 mmol) and 5-azido-1-pentanol 969 mg,
0.535 mmol) in THF 95 ml). The solution is stirred at
room temperature for 90 min before the solvent is removed
under vacuum. The residue is puri®ed by chromatography
over silica gel 9Et2O/AcOEt/EtOH 1/0/0±0/9/1) to yield 37a
nAˆ4.35, nBˆ4.22, 2H); 2.05 9s, 3H). 13C NMR9CDCl ,
3
75 MHz) d 169.8; 151.6; 151.0; 150.2; 143.1; 133.6; 131.3;
125.2; 88.2; 84.8; 63.9; 20.3. MS 9CI/NH3) m/z 295
[M1H]1; 312 [M1NH4]1. IR9®lm) n 3506; 3404; 2943;
2813; 2355; 1572; 1237.
4.1.32. 6-Chloro-9-020,30-dideoxy-b-d-glycero-pent-20-
enofuranosyl)purine 033). The compound is prepared
from 32 and following the same procedure as described
for 28. The crude material is puri®ed by chromatography
9Et2O/AcOEt 9/1±0/1) to yield 33 9221 mg, 96%) as a white
crystalline powder. Mp 1908C. 1H NMR9CDCl 3, 300 MHz)
d 8.73 9s, 1H); 8.54 9s, 1H); 7.12 9broad s, 1H); 6.50 9broad
d, Jˆ6.0 Hz, 1H); 6.07 9broad d, Jˆ6.0 Hz, 1H); 3.95 9AB
part of ABX syst., JABˆ12.4, JAXˆ2.3, JBXˆ2.6 Hz,
9142 mg, 94%) as a white powder. TLC 9AcOEt/EtOH 95/5)
1
Rf 0.6. H NMR9CDCl , 200 MHz) d 7.97 9d, Jˆ1.5 Hz,
3
1H); 6.70 9dd, Jˆ5.3, 5.2 Hz, 1H); 5.51 9dt, Jˆ2.9, 1.8 Hz,
1H); 4.93 9dt, Jˆ1.8, 4.0 Hz, 1H); 4.45 9AB part of ABX
syst., JABˆ8.2, JAXˆ3.8, JBXˆ1.7 Hz, nAˆ4.76, nBˆ4.14,
2H); 3.92 9t, Jˆ7.5 Hz, 2H); 3.23 9t, Jˆ6.8 Hz, 2H); 2.47
9m, 2H); 1.91 9d, Jˆ1.5 Hz, 3H); 1.71±1.54 9m, 4H); 1.46±
1.31 9m, 2H). 13C NMR9CDCl , 50 MHz) d 163.1; 151.7;
3
134.2; 110.6; 89.4; 87.1; 80.4; 75.8; 51.0; 41.0; 37.9; 28.3;
26.8; 23.9; 13.2. MS 9CI/NH3) m/z 336 [M1H]1; 353
[M1NH4]1. IR9®lm) n 2942; 2097; 1666; 1461; 1267.
nAˆ4.01, nBˆ3.88, 2H). 13C NMR9CDCl , 75 MHz) d
3
151.9; 151.1; 151.0; 144.7; 135.2; 131.8; 125.1; 89.6;
88.6; 63.2. MS 9CI/NH3) m/z 253 [M1H]1; 270
[M1NH4]1.
4.1.36. N1-03,5-Anhydro-2-deoxy-b-d-pentafuranosyl)-
O4-[1-05-azidopentyl)]thymidine 037b). Compound 37b
97 mg, 5%) is a side product isolated in the preparation of
4.1.33. 6-Chloro-9-050-O-dibenzyloxyphosphoryl-20,30-
dideoxy-b-d-glycero-pent-20-enofuranosyl)purine 034).
The compound is prepared from 33 following the same
procedure as described for 30. The crude material is puri®ed
by chromatography 9AcOEt/EtOH 95/5) to yield 34 935 mg,
36%) as a glassy solid. 1H NMR9CDCl 3, 300 MHz) d 8.73
9s, 1H); 8.27 9s, 1H); 7.35±7.26 9m, 10H); 7.14 9ddd, Jˆ3.0,
1.5, 1.5 Hz, 1H); 6.32 9ddd, Jˆ6.0, 1.9, 1.5 Hz, 1H); 6.05
9ddd, Jˆ6.0, 2.3, 1.5 Hz, 1H), 5.08±4.89 9m, 5H); 4.14 9AB
1
37a. TLC 9AcOEt/EtOH 95/5) Rf 0.3. H NMR9CDCl ,
3
200 MHz) d 8.10 9s, 1H); 6.72 9dd, Jˆ6.6, 4.0 Hz, 1H);
5.53 9m, 1H); 5.02 9m, 1H); 4.51 9AB part of ABX syst.,
JABˆ8.2, JAXˆ3.8, JBXˆ1.7 Hz, nAˆ4.30, nBˆ4.22, 2H);
4.42 9t, Jˆ6.6 Hz, 2H); 3.30 9t, Jˆ6.6 Hz, 2H); 2.58±2.53
9m, 2H); 1.98 9s, 3H); 1.38±1.92 9m, 6H). 13C NMR9CDCl 3,
50 MHz) d 170.4; 156.7; 139.9; 105.2; 90.5; 87.2; 81.1;
75.6; 67.0; 51.2; 39.3; 28.5; 28.1; 23.2; 12.5. MS 9CI/
NH3) m/z 336 [M1H]1. IR9®lm) n 2924; 2098; 1674.
part of ABXX0 syst., JABˆ11.5, JAXˆ6.8, JAX ˆ3.4,
0
JBXˆ6.4, JBX ˆ3.4 Hz, nAˆ4.19, nBˆ4.08, 2H). 13C NMR
0
9CDCl3, 75 MHz) d 152.1; 151.4; 150.9; 143.6; 135.3 9d,
Jˆ5.3 Hz); 133.6; 131.7; 128.5; 128.0; 127.9; 125.8; 88.5;
85.6 9d, Jˆ7.3 Hz); 69.5; 67.0 9d, Jˆ5.8 Hz). 31P NMR
9CDCl3, 121.5 MHz) d 0.21. MS 9CI/NH3) m/z 514
[M1H]1. IR9®lm) n 3485; 1590; 1561; 1273; 1191.
4.1.37. N3-[1-05-Azidopentyl)]-30-deoxy-20,30-didehydro-
thymidine 038). Nucleoside 37a 933 mg, 0.1 mmol) and
potassium tert-butoxide 912 mg, 0.1 mmol) are stirred in
anhydrous DMSO 92 ml) at room temperature for 4 h.
DMSO is removed in vacuo. The residue is solubilized in
water 90.5 ml) and saturated aqueous NH4Cl 93 ml) is added.
The mixture is extracted with dichloromethane and ethyl
acetate. The organic layer is dried over Na2SO4, reduced
under vacuum and puri®ed by chromatography 9Et2O/
AcOEt/EtOH 6/4/0±0/9/1) to yield 38 916 mg, 59%) as a
4.1.34.
phoryl-20,30-didehydro-20,30-dideoxyadenosine
N6-[1-04-Azidobutyl)]-50-O-dibenzyloxyphos-
035).
Compound 34 935 mg, 0.07 mmol), 4-azido-1-butylamine
98 mg, 0.07 mmol), and triethylamine 910 ml, 0.07 mmol)

T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
8141
1
white crystalline powder. H NMR9CDCl , 200 MHz) d
0
0
syst., JABˆ11.6, JAXˆ6.0, JAX ˆ3.0, JBXˆ5.9, JBX ˆ2.6 Hz,
nAˆ4.19, nBˆ4.15, 2H); 3.91 9t, Jˆ6.8 Hz, 2H); 3.16 9dt,
Jˆ6.8, 5.6 Hz, 2H); 1.81 9s, 3H); 1.63±1.51 9m, 4H); 1.41±
3
7.41 9d, Jˆ1.5 Hz, 1H); 7.05 9ddd, Jˆ3.3, 1.5, 1.5 Hz,
1H); 6.33 9ddd, Jˆ5.8, 1.8, 1.5 Hz, 1H); 5.87 9ddd, Jˆ5.8,
2.2, 1.5 Hz, 1H); 4.93 9m, 1H); 3.95 9t, Jˆ6.9 Hz, 2H);
3.99±3.83 9m, 1H); 3.95 9t, Jˆ7.3 Hz, 2H); 3.85±3.73 9m,
1H); 3.27 9t, Jˆ6.9 Hz, 2H); 1.89 9d, Jˆ1.5 Hz, 3H); 1.74±
1.29 9m, 2H). 13C NMR9CDCl , 75 MHz) d 163.4; 151.4;
3
135.3; 133.9 9d, Jˆ10.1 Hz); 132.8; 128.6; 128.4; 128.0;
127.6; 110.5; 90.2; 84.4; 69.5 9d, Jˆ5.8 Hz); 67.4; 41.0;
,
3
1.58 9m, 4H); 1.50±1.38 9m, 2H). 13C NMR9CDCl
,
3
40.3; 29.3; 26.9; 24.2; 12.9. 31P NMR9CDCl
50 MHz) d 163.4; 152.3; 134.3; 134.1; 126.7; 110.2; 90.7;
86.9; 63.6; 51.2; 41.1; 28.5; 27.0; 24.0. MS 9CI/NH3) m/z
336 [M1H]1; 353 [M1NH4]1. IR9®lm) n 3467; 2933;
2100; 1694; 1667; 1633; 1467.
121.5 MHz) d 0.71. MS 9CI/NH3) m/z 571 [M1H]1; 588
[M1NH4]1. IR9®lm) n 3366; 2931; 1701; 1666; 1637;
1461; 1249; 1008.
4.1.41.
500S)-0Uracil-1-yl)-10,30-oxathiolane-200R)-car-
4.1.38. 30-Deoxy-20,30-didehydro-50-O-dibenzyloxyphos-
phoryl thymidine 039). The preparation is similar to that
described for 23a, starting from d4T.46 The crude reaction
mixture is puri®ed by chromatography 9Et2O/AcOEt/EtOH
boxylic acid 01R,2S,5R)-menthyl ester 044). 2,4,6-
Collidine 93.47 ml, 26.23 mmol) and tert-butyldimethylsilyl
tri¯ate 95.51 ml, 24 mmol) are successively added dropwise
to uracil 91.28 g, 11.45 mmol) in anhydrous dichloro-
methane 911 ml). The resulting solution is stirred at room
temperature for 5 min before acetate 4252 93.00 g,
9.09 mmol) in dichloromethane 911 ml) is added, followed
by dropwise addition of trimethylsilyl iodide 91.42 ml,
10.00 mmol). The reaction mixture is stirred for 20 h and
then decomposed by addition of saturated aqueous Na2S2O3
920 ml) and water 910 ml). The mixture is extracted with
dichloromethane 9150 ml) and the organic layer is washed
with brine, and reduced in vacuo. The residue is solubilized
in ether 9150 ml) and saturated aqueous NaHCO3 990 ml) is
carefully added under vigorous stirring, followed with
n-hexane 935 ml) and cyclohexane 935 ml). Stirring is main-
tained for 20 min. The precipitate that formed is ®ltered,
washed with n-hexane/cyclohexane and recrystallized in
n-hexane/ethyl acetate/methanol to yield 44 92.63 g, 76%)
as a white powder. Mp 1918C. 1H NMR9CDCl 3, 300 MHz)
d 8.29 9d, Jˆ7.9 Hz, 1H); 6.42 9dd, Jˆ7.9, 4.9 Hz, 1H);
5.78 9d, Jˆ7.9 Hz, 1H); 5.40 9s, 1H); 4.72 9dt, Jˆ4.1,
10.9 Hz, 1H); 3.27 9AB part of ABX syst., JABˆ12.1,
JAXˆ4.9, JBXˆ7.9 Hz, nAˆ3.41, nBˆ3.12, 2H); 2.03±1.83
9m, 2H); 1.70±1.62 9m, 2H); 1.53±1.32 9m, 2H); 1.10±0.80
9m, 3H); 0.89 9d, Jˆ6.4 Hz, 3H); 0.87 9d, Jˆ6.4 Hz, 3H);
1/0/0±0/9/1) to yield compound 39 9313 mg, 75%) as a
white powder. Mp 1038C. H NMR9CDCl , 300 MHz) d
1
3
7.62±7.31 9m, 11H); 6.99 9ddd, Jˆ1.9, 1.9, 1.1 Hz, 1H);
6.17 9ddd, Jˆ5.7, 1.9, 1.5 Hz, 1H); 5.82 9ddd, Jˆ5.7, 1.5,
1.1 Hz, 1H); 5.04 9AB part of ABX syst., JABˆ16.8,
JAXˆ8.7, JBXˆ9.0 Hz, nAˆ5.06, nBˆ5.02, 4H); 4.93±4.87
9m, 1H); 4.16 9AB part of ABXX0 syst., JABˆ11.7, JAXˆ6.2,
0
0
JAX ˆ2.6, JBXˆ6.2, JBX ˆ2.6 Hz, nAˆ4.19, nBˆ4.13, 2H);
1.81 9d, Jˆ0.8 Hz, 3H). 13C NMR9CDCl , 50 MHz) d
3
163.5; 150.6; 135.9; 135.4; 132.9; 128.8; 128.7; 128.1;
127.4; 111.3; 89.4; 84.5 9d, Jˆ8.7 Hz); 69.6; 69.5; 12.1.
31P NMR9CDCl , 121.5 MHz) d 0.75. MS 9CI/NH3) m/z
3
502 [M1NH4]1. IR9®lm) n 3189; 3067; 2922; 1689; 1461;
1250; 1005.
4.1.39. N3-[1-05-Azidopentyl)]-30-deoxy-20,30-didehydro-
50-O-dibenzyloxyphosphoryl thymidine 040). The
preparation is similar to that described for 37a, starting
from 39. The crude reaction mixture is puri®ed by chroma-
tography 9Et2O/AcOEt/EtOH 1/0/0±0/9/1) to yield
1
compound 40 9142 mg, 47%) as a white powder. H NMR
9CDCl3, 300 MHz) d 7.31±7.28 9m, 10H); 7.24 9d,
Jˆ1.2 Hz, 1H); 7.02 9ddd, Jˆ1.9, 1.7, 1.5 Hz, 1H); 6.13
9ddd, Jˆ5.7, 1.7, 1.5 Hz, 1H); 5.79 9d, Jˆ5.7 Hz, 1H);
5.00 9AB part of ABX syst., JABˆ11.7, JAXˆ8.6,
JBXˆ8.8 Hz, nAˆ5.01, nBˆ4.99, 4H); 4.87 9m, 1H); 4.12
0.73 9d, Jˆ6.8 Hz, 3H). 13C NMR9CDCl , 50 MHz) d
3
169.7; 163.4; 150.4; 140.3; 102.9; 89.1; 77.6; 76.8; 47.0;
40.7; 35.1; 34.0; 31.4; 26.0; 23.1; 21.9; 20.7; 16.0. MS 9CI/
NH3) m/z 383 [M1H]1; 400 [M1NH4]1. IR9®lm) n 3201;
2942; 1713; 1678.
9AB part of ABXX0 syst., JABˆ11.5, JAXˆ5.8, JAX ˆ3.0,
0
0
JBXˆ5.8, JBX ˆ3.0 Hz, nAˆ4.15, nBˆ4.09, 2H); 3.91 9t,
Jˆ7.5 Hz, 2H); 3.21 9t, Jˆ6.8 Hz, 2H); 1.81 9d, Jˆ1.2 Hz,
3H); 1.67±1.55 9m, 4H); 1.44±1.34 9m, 2H). 13C NMR
9CDCl3, 75 MHz) d 163.2; 151.2; 133.8; 133.0; 132.6;
128.6; 128.5; 128.1; 127.4; 110.3; 90.0; 84.2 9d,
Jˆ8.7 Hz); 69.4 9d, Jˆ5.8 Hz); 67.2 9d, Jˆ5.8 Hz); 51.0;
40.9; 28.2; 26.8; 23.8; 12.7. 31P NMR9CDCl 3, 121.5 MHz)
d 0.66. MS 9CI/NH3) m/z 613 [M1NH4]1. IR9®lm) n 3444;
2922; 2089; 1700; 1667; 1639; 1439.
4.1.42.
100S)-[O4-02,4,6-Triisopropylbenzenesulfonyl)-
uracil-1-yl]-10,30-oxathiolane-200R)-carboxylic acid 01R,
2S,5R)-menthyl ester 045). A mixture of menthyl ester 44
9704 mg, 1.84 mmol), 4-DMAP 910 mg, 0.09 mmol), and
triethylamine 91.02 ml, 7.37 mmol) in anhydrous dichloro-
methane 910 ml) is treated with 2,4,6-triisopropylbenzene-
sulfonyl chloride 91.12 g, 3.69 mmol). The solution is
stirred for 12 h at room temperature before the solvent is
removed under vacuum. The crude residue is puri®ed by
chromatography 9n-hexane/Et2O 7/3) to yield 45 91.19 g,
4.1.40. N3-[1-05-Aminopentyl)]-30-deoxy-20,30-didehydro-
50-O-dibenzyloxyphosphoryl thymidine 041). The
preparation is similar to that described for 8, starting from
40. The crude reaction mixture is puri®ed by preparative
TLC 9AcOEt/EtOH 85/15) to yield compound 41 935 mg,
56%) as a glassy solid. 1H NMR9CDCl 3, 300 MHz) d 7.37±
7.30 9m, 10H); 7.24 9s, 1H); 7.04 9ddd, Jˆ1.9, 1.7, 1.5 Hz,
1H); 6.16 9d, Jˆ6.0 Hz, 1H); 5.82 9m, 1H); 5.02 9AB part of
ABX syst., JABˆ11.7, JAXˆ8.7, JBXˆ8.7 Hz, nAˆ5.05,
nBˆ4.99, 4H); 4.90 9m, 1H); 4.17 9AB part of ABXX0
1
99%) as a white crystalline powder. Mp 134±1358C. H
NMR9CDCl , 200 MHz) d 8.82 9d, Jˆ7.3 Hz, 1H); 7.20
3
9m, 2H); 6.28 9dd, Jˆ5.1, 5.0 Hz, 1H); 6.11 9d, Jˆ7.3 Hz,
1H); 5.50 9s, 1H); 4.75 9dt, Jˆ4.4, 10.6 Hz, 1H); 4.26 9h,
Jˆ6.9 Hz, 2H); 3.40 9AB part of ABX syst., JABˆ12.4,
JAXˆ4.8, JBXˆ5.5 Hz, nAˆ3.64, nBˆ3.17, 2H); 2.90 9h,
Jˆ6.9 Hz, 1H); 2.08±2.01 9m, 1H); 1.97±1.86 9m, 1H);
1.77±1.68 9m, 2H); 1.58±1.37 9m, 3H); 1.32 9d,
Jˆ6.8 Hz, 3H); 1.29 9d, Jˆ6.0 Hz, 3H); 1.27 9d,

8142
T. Brossette et al. / Tetrahedron 57<2001) 8129±8143
1
a glassy solid. H NMR9CD OD, 300 MHz) d 7.68 9d,
Jˆ7.1 Hz, 3H); 1.13±0.84 9m, 3H); 0.94 9d, Jˆ6.4 Hz, 3H);
3
0.91 9d, Jˆ6.8 Hz, 3H). 13C NMR9CDCl , 50 MHz) d
Jˆ7.5 Hz, 1H); 7.35±7.37 9m, 10H); 6,30 9t, Jˆ5.7,
5.6 Hz, 1H); 5.76 9d, Jˆ7.5 Hz, 1H); 5.34 9m, 1H); 5.08
9dd, Jˆ8.7, 2.3 Hz, 4H); 4.28 9m, 2H); 3.48±3.33 9m,
4H); 3.25 9AB part of ABX syst., JABˆ11.7, JAXˆ5.3,
JBXˆ6.0 Hz, nAˆ3.45, nBˆ3.06, 2H); 1.66±1.61 9m, 4H).
13C NMR9CD 3OD, 50 MHz) d 165.4; 158.1; 140.6; 137.1;
137.0; 129.8; 129.7; 129.2; 97.3; 89.1; 83.3 9d, Jˆ7.2 Hz);
71.0 9d, Jˆ5.8 Hz); 69.6 9d, Jˆ5.8 Hz); 52.1; 41.0; 37.2;
3
169.3; 167.3; 154.4; 153.7; 151.2; 146.8; 130.5; 124.0;
94.9; 90.9; 79.8; 77.0; 46.9; 40.6; 37.0; 34.1; 33.9; 31.3;
29.6; 26.0; 24.5; 24.3; 23.4; 23.1; 21.8; 20.6; 16.0. MS
9CI/NH3) m/z 649 [M1H]1. IR9®lm) n 2954; 2928; 1737;
1682; 1545; 1189.
4.1.43.
500S)-{N4-[1-04-Azidobutyl)cytosin]-1-yl}-10,30-
oxathiolane-200R)-carboxylic acid 01R,2S,5R)-menthyl
ester 046). Compound 45 9300 mg, 0.463 mmol) and a
large excess of 4-azido-1-butylamine 9200 ml) in dichloro-
methane 90.8 ml) are stirred at 08C for 45 min. The solvent
is removed under reduced pressure and the crude residue is
dissolved in dichloromethane 90.4 ml). Ether is added until
precipitation occurs and the solution is kept at 2108C for
12 h. The precipitate is ®ltered, washed with ether and dried
27.3. 31P NMR9CD OD, 121.5 MHz) d 20.30. MS 9CI/
3
NH3) m/z 588 [M1H]1. IR9®lm) n 3272; 2931; 2096;
1725; 1642; 1507; 1278; 1014.
4.1.46. 500S)-{N4-[1-04-Azidobutyl)]cytosin-1-yl}-200R)-
dihydroxyphosphoryloxymethyl-10,30-oxathiolane 049a
and 49b). The compound is prepared starting from 48 and
following the same procedure as described for 9. Compound
49 914 mg, 99%) is obtained as a mixture of epimers 949a/
49b 26/74) as determined by NMR. 1H NMR 9D2O,
300 MHz) d 8.32 9d, Jˆ7.9 Hz, 1Hb); 8.09 9d, Jˆ7.9 Hz,
1Ha); 6.27 9d, Jˆ7.9 Hz, 1Hb); 6.20 9dd, Jˆ5.3, 3.0 Hz,
1Ha and 1Hb); 6.04 9d, Jˆ7.9 Hz, 1Ha); 4.27±4.17 9m,
1Ha and 1Hb); 4.08±3.99 9m, 1Ha and 1Hb); 3.53±3.45
9m, 1Ha and 1Hb); 3.38±3.30 9m, 2Ha and 2Hb); 3.28±
3.17 9m, 3Ha and 3Hb); 1.72±1.48 9m, 4Ha and 4Hb). 13C
under vacuum to yield 46 9169 mg, 76%) as a white crystal-
line solid. Mp 191±1928C. H NMR9CDCl , 300 MHz) d
1
3
8.21 9d, Jˆ7.5 Hz, 1H); 6.50 9dd, Jˆ6.4, 6.3 Hz, 1H); 5.73
9d, Jˆ7.5 Hz, 1H); 5.43 9s, 1H); 4.74 9dt, Jˆ4.1, 10.7 Hz,
1H); 3.55±3.46 9m, 3H); 3.36±3.28 9m, 2H); 3.13±3.05 9m,
1H); 1.89±2.07 9m, 2H); 1.75±1.62 9m, 6H); 1.56±1.39 9m,
2H); 1.10±0.85 9m, 3H); 0.91 9d, Jˆ6.0 Hz, 3H); 0.90 9d,
Jˆ6.0 Hz, 3H); 0.76 9d, Jˆ6.7 Hz, 3H). 13C NMR9CDCl ,
3
NMR9D O/CD3OD 1/1, 50 MHz) d 158.2 9b); 157.9 9a);
2
75 MHz) d 169.9; 163.8; 156.0; 140.0; 95.4; 90.2; 77.9;
76.6; 51.0; 47.0; 40.7; 40.2; 36.0; 34.0; 31.4; 26.4; 26.2;
26.0; 23.2; 21.9; 20.7; 16.0. MS 9CI/NH3) m/z 479
[M1H]1. IR9®lm) n 2943; 2097; 1707; 1654; 1449.
149.6 9a); 149.1 9b); 146.5 9b); 143.2 9a); 96.2 9a); 92.4 9b);
88.1 9a and b); 87.0 9d, Jˆ8.7 Hz, b); 86.6 9d, Jˆ7.2 Hz, a);
66.1 9d, Jˆ4.3 Hz, a and b); 51.5 9a and b); 43.7 9b); 43.2
9a); 38.5 9b); 38.1 9a); 26.4 9b); 26.3 9a); 26.2 9b); 25.5 9a).
31P NMR9D 2O, 50 MHz) d 1.59 9b); 1.11 9a). MS 9CI/NH3)
m/z 310 [M2H2PO41H]1. IR9®lm) n 3411; 2944; 2100;
1722; 1661.
4.1.44. 500S)-{N4-[1-04-Azidobutyl)]cytosin-1-yl}-200R)-
hydroxymethyl-10,30-oxathiolane
047).
Lithium
aluminium hydride 913 mg, 0.349 mmol) is added at 08C
to 46 960 mg, 0.126 mmol) in anhydrous THF 93 ml). The
reaction mixture is stirred for 10 min and then decomposed
by addition of wet ether 92 ml), methanol 92 ml), and silica
gel 91 g). The slurry is stirred for 1 h at room temperature
then ®ltered. The ®ltrate is reduced under vacuum and puri-
®ed by chromatography 9AcOEt/EtOH 1/0±9/1) to yield 47
941 mg, 99%) as a glassy solid. 1H NMR9CD 3OD,
300 MHz) d 7.93 9d, Jˆ7.5 Hz, 1H); 6.29 9dd, Jˆ4.9,
4.8 Hz, 1H); 5.84 9d, Jˆ7.5 Hz, 1H); 5.26 9dd, Jˆ4.1,
3.8 Hz, 1H); 3.89 9AB part of ABX syst., JABˆ12.4,
JAXˆ3.8, JBXˆ4.1 Hz, nAˆ3.92, nBˆ3.85, 2H); 3.40 9t,
Jˆ6.4 Hz, 2H); 3.33 9t, Jˆ6.4 Hz, 2H); 3.30 9AB part of
ABX syst., JABˆ11.9, JAXˆ5.7, JBXˆ4.9 Hz, nAˆ3.48,
Acknowledgements
This work was supported by a grant from ANRS 9Agence
Nationale de Recherche sur le Sida, France). The authors
wish to thank Alain Valleix for collecting MS data.
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