Synthesis and evaluation of anti-HIV-1 activity of 3′-azido-3′-deoxy-2′-O,4′-C-methylene-linked bicyclic thymine nucleosides

Article abstract of DOI:10.1039/b005469k

The two conformationally locked AZT analogues 4 and 5, each containing a 2′-O,4′-C-methylene-linked bicyclic furanose moiety, are synthesized via the 3′-azido-3′-deoxy-4′-C-hydroxymethyl nucleoside 16. The β-D-ribo-configured derivative 4 is shown by NOE

Full text of DOI:10.1039/b005469k

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Synthesis and evaluation of anti-HIV-1 activity of 3Ј-azido-3Ј-
deoxy-2Ј-O,4Ј-C-methylene-linked bicyclic thymine nucleosides
Anne G. Olsen,a Vivek K. Rajwanshi,a Claus Nielsenb and Jesper Wengela,*†
1
a Center for Synthetic Bioorganic Chemistry, Department of Chemistry,
University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark
b Retrovirus Laboratory, Department of Virology, Statens Serum Institut,
DK-2300 Copenhagen, Denmark
Received (in Cambridge, UK) 7th July 2000, Accepted 8th September 2000
First published as an Advance Article on the web 16th October 2000
The two conformationally locked AZT analogues 4 and 5, each containing a 2Ј-O,4Ј-C-methylene-linked bicyclic
furanose moiety, are synthesized via the 3Ј-azido-3Ј-deoxy-4Ј-C-hydroxymethyl nucleoside 16. The β--ribo-
configured derivative 4 is shown by NOE experiments to exist in a north-type (3E, C3Ј-endo) conformation and the
α--xylo-configured derivative 5 in a south-type (3E, C3Ј-exo) conformation. Both nucleosides were devoid of
anti-HIV activity in MT-4 cells.
Introduction
The use of 2Ј,3Ј-dideoxynucleosides to combat HIV infection
remains important in the treatment of AIDS patients. These
nucleosides are prodrugs which need to be triphosphorylated at
the 5Ј-hydroxy group in vivo in order be effective inhibitors of
the HIV-encoded enzyme HIV-1 Reverse Transcriptase (HIV-1
RT).1–3 3Ј-Azido-3Ј-deoxythymidine (AZT, 1, Fig. 1) was the
first anti-HIV drug to be approved for treatment of AIDS but
as with other anti-HIV drugs its use is hampered by toxic side-
effects and the emergence of resistance. Therefore, the collection
of additional knowledge on the pharmacological effect of
various nucleosides and the development of improved anti-HIV
drugs continues to be of immense importance.
A study published by Marquez et al. has furnished relevant
insight into the relationship between conformation of the
furanose ring and anti-HIV activity.4 Thus, the two conform-
ationally restricted bicyclic carbocyclic AZT analogues 2 and
3 (Fig. 1) were synthesized and their cyclopentane moieties
shown to be locked into what corresponds to a north-type
(2E, C2Ј-exo) and a south-type (3E, C3Ј-exo) conformation,
respectively. Whereas the 5Ј-triphosphate of 3 was inactive
as an inhibitor of HIV-1 RT, the inhibitory activity of the
5Ј-triphosphate of the north-type analogue 2 corresponded
closely to that of AZT 5Ј-triphosphate.4 In line with the earlier
proposal that a south-type conformation is essential for
efficient conversion of the nucleoside prodrugs to the bioactive
5Ј-triphosphates,5 these results led to the conclusion that
efficient 5Ј-triphosphorylation and subsequent inhibition of
HIV-1-RT requires conformational flexibility of the pento-
furanose ring (allowing the nucleoside prodrug to adopt a
Fig. 1 Structures of AZT (1) and conformationally restricted bicylic
south-type conformation and the 5Ј-triphosphate drug to adopt
AZT analogues 2–5. The displayed conformations of nucleosides 4 and
5 are those found by molecular modelling (HyperChemTM program,
Polak–Ribiere algorithm), and confirmed herein by NMR experiments
(see Fig. 2).
a north-type conformation).4
To further evaluate the relation between furanose conform-
ation and anti-HIV activity we have synthesized the two con-
formationally locked AZT analogues 4 and 5 each containing
a 2Ј-O,4Ј-C-methylene-linked bicyclic furanose moiety (Fig. 1).
Based on molecular modelling, NMR and X-ray crystal-
lography of the corresponding nucleoside derivatives6–9 having
a 3Ј-hydroxy group instead of the 3Ј-azido group, the β--ribo-
configured derivative 4 was predicted to exist in a north-type
(3E, C3Ј-endo) conformation and the α--xylo-configured-
derivative 5 in a south-type (3E, C3Ј-exo)‡ conformation (Fig. 1).
These predictions were confirmed by NOE experiments on 4
and 5 (vide supra) and by results reported in a recent com-
‡ The conformation of the furanose ring of nucleoside 5 could also be
given as C3Ј-endo, reflecting the positioning of the 4Ј-C-hydroxymethyl
group and the C3Ј atom at the same face of the plane created by the
atoms C1Ј, C2Ј, C4Ј and O4Ј. However, to allow direct comparison with
nucleosides 1–4 the conformation of 5 is given as C3Ј-exo.
† Present address: Department of Chemistry, University of Southern
Denmark, Campusvej 55, DK-5230 Odense M, Denmark.
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J. Chem. Soc., Perkin Trans. 1, 2000, 3610–3614
This journal is © The Royal Society of Chemistry 2000
DOI: 10.1039/b005469k

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munication10 by Obika et al. who independently synthesized
derivative 4. The 2,5-dioxabicyclo[2.2.1]heptane derivatives 4
and 5 therefore mimic the north- and south-type pentofuranose
conformations of AZT 1, respectively, although the configur-
ation at C4Ј of the α--xylo-configured derivative 5 is opposite
to that of AZT.
was prepared in 78% yield by reaction of 11 with mesyl chloride
and 4-(N,N-dimethyl)aminopyridine (DMAP) in pyridine.
However, attempted conversion of 12 into the 3Ј-azido nucleo-
side 13 (NaN3, DMF), a possible intermediate towards syn-
thesis of the target molecule 4, was unsuccessful in our hands.
Likewise, reaction of the 3Ј-O-trifluoromethanesulfonate
derivative of 11 (data not shown) with sodium azide in
anhydrous DMF failed to give nucleoside 13.
Results and discussion
With two unsuccessful attempts at introducing the azido
substituent at the nucleoside level we decided to use the known
3-azido-3-deoxyfuranose 15,14 easily obtainable from -glucose
via 1,2-O-isopropylidene derivative 14,14 as starting material
(Scheme 3). Reaction of 15 with thymine, N,O-bis(trimethyl-
silyl)acetamide and trimethylsilyl triflate in acetonitrile15,16
afforded nucleoside 13 in 68% yield. As expected from the
presence of a 2-O-acetyl group in 15, the Vorbrüggen-type
coupling reaction15,16 proceeded stereoselectively with no
indication of the formation of the anomeric nucleoside. Com-
plete deacylation of nucleoside 13 by reaction with sodium
methoxide in methanol afforded triol 16 in 85% yield. Mesyl-
ation of 16 using 2.1 equivalents of mesyl chloride in pyridine
afforded an intermediate, tentatively assigned as derivative
17 with the two primary hydroxy groups mesylated, which
subsequently in a 1:1 mixture of 1,4-dioxane and 1 M aqueous
NaOH (room temperature, 15 min; 90 ЊC, 12 h) was directly
converted into the target compound 3Ј-azido-3Ј-deoxy-2Ј-O,4Ј-
C-methylene-β--ribofuranosyl)thymine 4 in 36% yield (calcu-
lated from 16). We have recently reported a similar, successful,
one-pot ring-closure and 5Ј-O-demesylation reaction during the
synthesis of 2Ј-O,4Ј-C-methylene-α--ribofuranosyl)thymine.9
The overall yield for the five-step synthesis of nucleoside 4 from
furanose 14 was 19% as compared with 38% for the reported10
alternative seven-step synthesis likewise starting from furanose
14.
Towards the synthesis of 3Ј-azido-3Ј-deoxy-2Ј-O,4Ј-C-
methylene-α--xylofuranosyl)thymine 5, the tri-O-mesyl inter-
mediate 18 was prepared in 79% yield by trimesylation of
derivative 16 using 3.5 equivalents of mesyl chloride in pyridine.
Subsequent treatment of 18 with first a mixture of 1,4-dioxane
and 1 M aq. NaOH (5:2; 90 ЊC, 24 h), to give a putative 5Ј-O-
mesyl intermediate, and then with a mixture of 1 M aq. NaOH,
EtOH and water (1:5:5; 80 ЊC, 24 h; then 90 ЊC, 17 h) effected
formation of the target nucleoside 5 in 40% yield. We have
recently reported a similar conversion for the 3Ј-hydroxy deriv-
ative9 of 18 likewise involving 2Ј-epimerization, cyclization and
demesylation. Mechanistically en route from 18 to 5, a reaction
cascade including 2,2Ј-anhydro-nucleoside formation, hydr-
olysis of this intermediate to give the 2Ј-epimer of 17, and
ring-closure by intramolecular nucleophilic attack followed by
demesylation seems likely. This efficient synthesis of nucleoside
5 underlines the importance of anhydro nucleoside inter-
mediates in nucleoside chemistry. However, the necessity of
2Ј-epimerization and the fact that anhydro-intermediates are
generally unattainable for purine derivatives suggests the
strategy used for synthesis of 5 to be viable for pyrimidine
derivatives only.
Our first approach (Scheme 1) towards the synthesis of 1-(3-
Scheme 1 Reagents and conditions (and yields): i) TrCl, pyridine
(91%); ii) MsCl, pyridine (87%); iii) NaOH, aq. ethanol.
azido-3-deoxy-2-O,4-C-methylene-β--ribofuranosyl)thymine
4 was planned to involve nucleophilic opening of the 2,3Ј-
anhydro nucleoside 9 using azide ion, followed by detritylation.
As the first step, the known bicyclic nucleoside diol 68 was
monotritylated at the 5Ј-position by reaction with less than two
equivalents of trityl chloride in pyridine to give the novel
nucleoside 7 in 91% yield. However, activation of the 3Ј-
hydroxy group by formation of the 3Ј-O-mesyl derivative 8
(mesyl chloride, pyridine; 87% yield) and subsequent treatment
with one equivalent of base following standard conditions
for anhydro-nucleoside formation (aq. NaOH in ethanol)11,12
failed to yield the desired anhydro nucleoside 9. Analogously,
attempted conversion of the 5Ј-O-(tert-butyldimethylsilyl)
derivative of 8 into the corresponding 2,3Ј-anhydro nucleoside,
as well as direct conversion of the 5Ј-O-(tert-butyldimethylsilyl)
derivative of 7 using Mitsunobu conditions, failed (data not
shown). These results are in contrast to those of Marquez
et al. as they successfully used a 2,3Ј-anhydro nucleoside as an
intermediate during synthesis of the north-type conformer 2.4
However, probably because of complete lack of conformational
mobility in the furanose ring of compounds 7 and 8, the
necessary geometry for intramolecular nucleophilic attack at
the 3Ј-position is unattainable.
In a second approach (Scheme 2), the known 1-(2-O-acetyl-
The configuration and conformation of the two bicyclic
1
nucleosides 4 and 5 were evaluated by H NMR and NOE
experiments (Fig. 2). Thus, for the β--ribo-configured deriv-
ative 4, mutual strong NOEs between H6 and H3Ј (8%/8%)
indicate anti conformation of the thymine moiety and C3Ј-endo
conformation of the furanose ring. In addition, comparable
NOEs between the pairs H1Ј and H2Ј (5%/5%) and H2Ј and
H3Ј (5%/4%), together with the fact that these three protons
Scheme 2 Reagents and conditions (and yields): i) H2, 10% Pd/C,
CH3OH (70%); ii) MsCl, DMAP, pyridine (78%); iii) NaN3, DMF.
1
appear as singlets in the H NMR spectrum, indicate a C3Ј-
5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-α--threo-
pentofuranosyl)thymine13 10 was used as starting material.
Debenzylation by catalytic hydrogenation afforded nucleoside
11 in 70% yield. To prepare for introduction of a 3Ј-azido group
with inversion of configuration, the 3Ј-O-mesyl derivative 12
endo conformation of nucleoside 4 in analogy to what was
found earlier for 4 by a similar analysis and by molecular
modelling.10 For the α--xylo-configured derivative 5, mutual
strong NOEs between H6 and one of the two 2Ј-O,4Ј-C-
methylene protons (11%/7%) suggest an anti conformation of
J. Chem. Soc., Perkin Trans. 1, 2000, 3610–3614
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Scheme 3 Reagents and conditions (and yields): i) a, 80% AcOH; b, Ac2O, pyridine (89%); ii) N,O-bis(trimethylsilyl)acetamide, thymine, TMS
triflate, acetonitrile (68%); iii) NaOCH3, CH3OH, (85%); iv) MsCl, pyridine; v) NaOH, aq. 1,4-dioxane (36%, two steps); vi) MsCl, pyridine (79%);
vii) a, NaOH, aq. 1,4-dioxane; b, NaOH, aq. EtOH (40%).
HIV activity of 5. For example, efficient 5Ј-O-phosphorylation
in vivo may a priori not be anticipated for nucleoside derivative 5
despite its furanose ring being in a south-type conformation.
Conclusions
Synthesis of the two conformationally locked AZT analogues 4
and 5 has been accomplished. The β--ribo configured deriv-
ative 4 adopts a north-type (3E, C3Ј-endo) conformation, and
the α--xylo-configured derivative 5 a south-type (3E, C3Ј-exo)
conformation, while the thymine moieties adopt anti conform-
ations. Both nucleosides were inactive against HIV-1 in MT-4
cells. These results are in line with the results of Marquez et al.4
indicating that conformational flexibility of the furanose ring
of 5Ј-hydroxy nucleoside prodrugs is essential for anti-HIV-1
activity.
Fig. 2 Key NOE effects (indicated by the curved arrows) observed for
nucleosides 4 and 5. For 4: 8% and 8% (between H6 and H3Ј), 5% and
5% (between H1Ј and H2Ј), 5% and 4% (between H2Ј and H3Ј). For 5:
11% and 7% (between H6 and one of the two H1Љ protons), 7% and 2%
(between H3Ј and the other H1Љ proton), 9% and 8% (between H1Ј and
H2Ј).
the thymine moiety in addition to supporting the assigned
structure and C3Ј-exo conformation of this nucleoside. In
addition, mutual NOEs between the other 2Ј-O,4Ј-C-methylene
proton and H3Ј (7%/2%) and between H1Ј and H2Ј
(9%/8%) add support for the assigned structure and con-
formation of 5. The locked conformations for nucleosides
4 and 5 were further verified by molecular modelling (see
Fig. 1).
The bicyclic nucleosides 4 and 5 were evaluated for antiviral
activity against HIV-1 in MT-4 cells as described earlier.17 Both
compounds were inactive against HIV-1 at 300 µM. Thus,
although derivative 4 is structurally closely related to AZT and
efficiently locked in a north-type conformation corresponding
to the conformation of AZT triphosphate when interacting
with HIV-1-RT, no anti-HIV activity of 4 was found. This,
in line with the earlier reported results, suggests that a south-
type furanose conformation is essential for efficient 5Ј-O-
phosphorylation in vivo.5 As the configuration at C4Ј of the
south-type conformer 5 is of the unnatural -type it is more
difficult to draw conclusions on the basis of the lack of anti-
Experimental
General
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Reactions
were conducted under an atmosphere of nitrogen when
anhydrous solvents were used. During work-up, organic phases
were combined, dried (Na2SO4) and filtered before evaporation.
After column chromatographic purification (glass columns;
silica gel 60, Merck, 0.040–0.063 mm), fractions containing
product were pooled, evaporated to dryness under reduced
pressure, and dried under vacuum to give the product. Petrol-
eum ether of distillation range 60–80 ЊC was used. NMR spec-
tra were recorded on a Varian 400 MHz NMR instrument and
chemical shifts are reported in ppm relative to tetramethylsilane
1
as internal standard for H (400 MHz) and 13C (100.6 MHz)
except for the 13C spectrum (62.9 MHz) of nucleoside 4
which was recorded on a Bruker 250 MHz NMR instrument.
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J. Chem. Soc., Perkin Trans. 1, 2000, 3610–3614

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Coupling constants J are given in Hz. Assignments of
NMR spectra when given are based on 2D spectra. Nuclear
Overhauser enhancement (NOE) experiments were recorded
on a Bruker 250 MHz NMR instrument (nucleoside 4 in
non-degassed CD3OD) and nucleoside 5 [in non-degassed
(CD3)2SO]. Fast-atom bombardment mass spectra (FAB-MS)
were recorded in positive ion mode. Microanalyses were
performed at The Microanalytical Laboratory, Department of
Chemistry, University of Copenhagen.
CH ); δ (CDCl ) 170.5, 166.0, 165.8 (C᎐O), 163.3 (C-4), 150.0
᎐
3 C 3
(C-2), 137.3, 133.3, 129.6, 129.5, 129.0, 128.9, 128.4, 128.1 (C-6,
Bz), 111.8 (C-5), 100.0, 90.5, 86.0, 83.0 (C-1Ј, -2Ј, -3Ј, -4Ј), 63.0,
62.7 (C-5Ј, -1Љ), 20.7 (COCH3), 12.3 (CH3); FAB-MS m/z 539
[M ϩ H]ϩ [Found: (%) C, 60.8; H, 4.9; N, 5.0. C27H26N2O10
requires C, 60.2; H, 4.9; N, 5.2].
1-(2-O-Acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-3-O-methyl-
sulfonyl-ꢁ-L-threo-pentofuranosyl)thymine 12
To an ice-cold solution of nucleoside 11 (0.10 g, 0.18 mmol) in
anhydrous pyridine (0.5 cm3) was added mesyl chloride (0.03
cm3, 0.37 mmol). The solution was stirred at room temperature
for 26 h whereupon DMAP (0.01 g, 0.09 mmol) and additional
mesyl chloride (0.01 cm3, 0.13 mmol) were added. After being
stirred at room temperature for 22 h, the reaction mixture was
diluted with dichloromethane (25 cm3), and ice-cold water
(5.0 cm3) was added. The phases were separated and the aqueous
phase was extracted with dichloromethane (3 × 15 cm3). The
combined organic phase was washed successively with satur-
ated aq. sodium hydrogen carbonate (3 × 10 cm3) and brine
(3 × 10 cm3) and evaporated to dryness under reduced pressure.
The residue was purified by silica gel column chromatography
using dichloromethane–methanol (99.5:0.5 to 99:1, v/v) as
eluent to give nucleoside 12 (0.09 g, 78%) as a white solid
material, δH (CDCl3) 8.73 (1H, br s, NH), 8.10–7.30 (11H, m,
6-H, Bz), 6.33 (1H, d, J 6.0, 1Ј-H), 5.70 (1H, dd, J 6.0 and 5.2,
2Ј-H), 5.47 (1H, d, J 5.2, 3Ј-H), 4.90–4.56 (4H, m, 5Ј-, 1Љ-H2),
3.14 (3H, s, Ms), 2.15 (3H, s, COCH3), 1.7 (3H, s, CH3);
FAB-MS m/z 617 [M ϩ H]ϩ [Found: (%) C, 55.0; H, 4.7; N, 4.3.
C28H28N2O12S requires C, 54.6; H, 4.6; N, 4.5].
1-(2-O,4-C-Methylene-5-O-trityl-ꢀ-D-ribofuranosyl)thymine 7
To a solution of 1-(2-O,4-C-methylene-β--ribofuranosyl)-
thymine8 6 (0.39 g, 1.45 mmol) in anhydrous pyridine (8 cm3)
at room temperature was added trityl chloride (0.68 g, 2.32
mmol). The reaction mixture was heated at 110 ЊC for 20 h, and
then evaporated to dryness under reduced pressure. The residue
was dissolved in dichloromethane (35 cm3; containing 0.5%
pyridine, v/v), washing was performed successively with satur-
ated aq. of sodium hydrogen carbonate (3 × 8 cm3) and brine
(3 × 10 cm3), and the organic phase was evaporated to dryness
under reduced pressure. The residue was purified by silica gel
column chromatography using dichloromethane–methanol–
pyridine (98.0:1.5:0.5 to 97.0:2.5:0.5, v/v/v) as eluent to give
an intermediate tentatively assigned as nucleoside 7 (white solid
material, 0.67 g, 91%). FAB-MS m/z 513 [M ϩ H]ϩ. This inter-
mediate was used in the next step without further purification.
1-(2-O,4-C-Methylene-3-O-methylsulfonyl-5-O-trityl-ꢀ-D-ribo-
furanosyl)thymine 8
To an ice-cold solution of nucleoside 7 (0.25 g, 0.49 mmol) in
anhydrous pyridine (3 cm3) was added mesyl chloride (0.06 cm3,
0.73 mmol). The solution was stirred at 10 ЊC for 24 h where-
upon additional mesyl chloride (0.007 cm3, 0.09 mmol) was
added. After being stirred at 10 ЊC for an additional 20 h, the
reaction mixture was concentrated to half-volume, and the
resulting mixture was poured into ice-cold water (125 cm3)
under vigorous stirring to give nucleoside 8 (0.25 g, 87%) as a
white solid material after filtration, washing with water (250
cm3) and coevaporation with anhydrous toluene (3 × 5 cm3);
δH (CDCl3) 8.83 (1H, br s, NH), 7.62–7.25 (16H, m, 6-H, Tr),
5.70 (1H, s, 1Ј-H), 5.08 (1H, s, 3Ј-H), 4.80 (1H, s, 2Ј-H), 3.89–
3.83 (2H, m), 3.62 (1H, d, J 11.1), 3.43 (1H, d, J 11.1), 3.00 (3H,
s, Ms), 1.66 (3H, s, CH3). δC (CDCl3) 163.5 (C-4), 149.7 (C-2),
142.8, 133.6, 128.5, 128.3, 128.1, 127.9, 127.6, 127.2 (C-6, Tr),
111.3 (C-5), 87.6, 87.2, 77.7, 75.2, 72.0, 57.6 (C-1Ј, -2Ј, -3Ј, -4Ј,
-5Ј, -1Љ), 39.0 (Ms), 12.6 (CH3); FAB-MS m/z 591 [M ϩ H]ϩ
[Found: (%) C, 63.4; H, 5.1; N, 4.5. C31H30N2O8S requires C,
63.0; H, 5.1; N, 4.7].
1-(2-O-Acetyl-3-azido-5-O-benzoyl-4-C-benzoyloxymethyl-3-
deoxy-ꢀ-D-erythro-pentofuranosyl)thymine 13
To a stirred solution of the anomeric mixture 1,2-O-acetyl-3-
azido-5-O-benzoyl-4-C-benzoyloxymethyl-3-deoxy--erythro-
pentofuranose14 15 (0.64 g, 1.28 mmol) and thymine (0.32 g,
2.57 mmol) in anhydrous acetonitrile (15 cm3) was added N,O-
bis(trimethylsilyl)acetamide (2.19 cm3, 8.98 mmol). The reac-
tion mixture was stirred and heated under reflux for 1 h. After
cooling of the mixture to 0 ЊC, TMS triflate (0.67 cm3, 3.47
mmol) was added dropwise. After being stirred for 10 min at
0 ЊC, the mixture was heated to 60 ЊC and stirring was con-
tinued for 16 h. The reaction mixture was evaporated to dryness
under reduced pressure, and the residue was dissolved in ethyl
acetate (50 cm3), washed with saturated aq. sodium hydrogen
carbonate (3 × 20 cm3) and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using ethyl acetate–petroleum ether (2:3 to
1:1, v/v) as eluent to give nucleoside 13 (0.49 g, 68%) as a white
foam, δH (CDCl3) 8.83 (1H, s, NH), 8.08–7.44 (10H, m, Bz),
7.02 (1H, s, 6-H), 5.90 (1H, d, J 4.8, 1Ј-H), 5.75 (1H, dd, J 4.8
and 6.6, 2Ј-H), 4.92 (1H, d J 6.6, 3Ј-H), 4.88–4.39 (4H, m, 5Ј-,
1Љ-H2), 2.20 (3H, s, COCH3), 1.72 (3H, s, CH3); δC (CDCl3)
1-(2-O-Acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-ꢁ-L-threo-
pentofuranosyl)thymine 11
1-(2-O-Acetyl-5-O-benzoyl-4-C-benzoyloxymethyl-3-O-benzyl-
α--threo-pentofuranosyl)thymine13 10 (1.00 g, 1.60 mmol) was
dissolved in methanol (4.0 cm3) and 10% Pd/C (0.25 g) sus-
pended in methanol (3.0 cm3) was added. The mixture was
degassed and then stirred for 21 h under an atmosphere of
hydrogen. Additional Pd/C (0.75 g) suspended in methanol (4
cm3) was added followed by degassing and subsequent stirring
for 46 h under an atmosphere of hydrogen. The mixture was
filtered [silica gel; washing was performed using dichloro-
methane–methanol (250 cm3; 1:1, v/v], and the combined
filtrate was evaporated to dryness under reduced pressure.
The residue was subjected to column chromatography on silica
gel using dichloromethane–methanol (99:1, v/v) as eluent to
give nucleoside 11 (0.60 g, 70%) as a white solid material,
δH (CDCl3) 9.03 (1H, br s, NH), 8.06–7.27 (11H, m, 6-H, Bz),
5.92 (1H, d, J 3.8, 1Ј-H), 5.35–5.37 (1H, m, 2Ј-H), 4.80–4.43
(5H, m, 3Ј-H, 5Ј-, 1Љ-H2), 2.13 (3H, s, COCH3), 2.13 (3H, s,
170.0, 165.9, 165.8 (C᎐O), 163.3 (C-4), 149.8 (C-2), 133.6,
᎐
133.3, 129.7, 129.6, 129.2, 128.9, 128.6, 128.4 (Bz), 136.8 (C-6),
111.7 (C-5), 90.0 (C-1Ј), 84.1 (C-4Ј), 74.5 (C-2Ј), 65.0, 62.8, 62.7
(C-3Ј, -5Ј, -1Љ), 20.4 (COCH3), 12.0 (CH3); FAB-MS m/z 564
[M ϩ H]ϩ. Selected IR signal: νmax 2118 cmϪ1 (azido group)
[Found: (%) C, 57.3; H, 4.8; N, 11.6. C27H25N5O9ؒ0.5EtOAc
requires C, 57.3; H, 4.8; N, 11.5].
1-(3-Azido-3-deoxy-4-C-hydroxymethyl-ꢀ-D-erythro-pento-
furanosyl)thymine 16
To a stirred solution of nucleoside 13 (1.00 g, 1.77 mmol) in
anhydrous methanol (20 cm3) was added sodium methoxide
(0.58 g, 10.6 mmol). The reaction mixture was stirred for 30 min
at room temperature, and was then neutralized with a 7% (w/w)
solution of HCl in 1,4-dioxane. After evaporation to dryness
J. Chem. Soc., Perkin Trans. 1, 2000, 3610–3614
3613

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1-(3-Azido-3-deoxy-2-O,4-C-methylene-ꢁ-L-xylofuranosyl)-
thymine 5
under reduced pressure, the residue was purified by silica gel
column chromatography using dichloromethane–methanol
(96:4 to 94:6, v/v) as eluent to give nucleoside 16 (0.47 g, 85%)
as a white foam, δH (CD3OD) 7.78 (1H, s, 6-H), 5.93 (1H, d,
J 6.6, 1Ј-H), 4.69 (1H, m, 2Ј-H), 4.31 (1H, d, J 6.1, 3Ј-H), 3.78–
3.60 (4H, m, 5Ј-, 1Љ-H2), 1.88 (3H, s, CH3); δC (CD3OD) 164.3
(C-4), 150.8 (C-2), 136.5 (C-6), 110.0 (C-5), 87.6 (C-1Ј), 86.4
(C-4Ј), 74.0 (C-2Ј), 63.6 (C-3Ј), 63.2, 61.7 (C-5Ј, -1Љ), 10.5
(CH3); FAB-MS m/z 314 [M ϩ H]ϩ. Selected IR signal: νmax
2117 cmϪ1 (azido group) [Found: (%) C, 40.2; H, 4.8; N, 20.4.
C11H15N5O6ؒH2O requires C, 39.9; H, 5.2; N, 21.1].
Nucleoside 18 (20.0 mg, 0.0365 mmol) was stirred in a mixture
of 1,4-dioxane (1.0 cm3) and 1 M NaOH (0.4 cm3). After 15
min at room temperature, analytical TLC (methanol–dichloro-
methane; 2:23, v/v) showed no conversion. The reaction
mixture was subsequently stirred at 90 ЊC for 24 h and then
evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography using dichloro-
methane–methanol (97.5:2.5 to 97:3, v/v) as eluent to give
an intermediate. This intermediate was stirred in a mixture of
water–ethanol (1 cm3; 1:1 v/v) and 2 M NaOH (0.2 cm3) at
80 ЊC for 24 h and subsequently at 90 ЊC for 17 h. After neutral-
ization using a 7% (w/w) solution of HCl in 1,4-dioxane, the
mixture was evaporated to dryness under reduced pressure. The
residue was purified by silica gel column chromatography using
dichloromethane–methanol (97:3, v/v) as eluent to give nucleo-
side 5 (4.3 mg, 40%) as a white solid material, δH [(CD3)2SO]
11.3 (1H, br s, NH), 7.61 (1H, s, 6-H), 5.91 (1H, s, 1Ј-H), 5.26
(1H, dd, J 5.5 and 5.7, OH), 4.60 (1H, d, J 2.7, 3Ј-H), 4.54 (1H,
d, J 2.6, 2Ј-H), 4.09 (1H, d, J 8.8, 5Ј-Ha), 4.03 (1H, d, J 8.8,
5Ј-Hb), 3.78 (2H, m, 1Љ-H2), 1.82 (3H, s, CH3); δC [(CD3)2SO]
163.8 (C-4), 150.3 (C-2), 136.0 (C-6), 108.3 (C-5), 89.3, 88.5
(C-1Ј, C-4Ј), 76.8 (C-2Ј), 73.6 (C-5Ј), 65.7 (C-3Ј), 56.9 (C-1Љ),
12.3 (CH3); FAB-MS m/z 296 [M ϩ H]ϩ. Selected IR signal:
νmax 2123 cmϪ1 (azido group). To verify the purity of this com-
pound, a copy of the 13C NMR spectrum was enclosed when
submitting this manuscript.
1-(3-Azido-3-deoxy-5-O-mesyl-4-C-mesyloxymethyl-ꢀ-D-
erythro-pentofuranosyl)thymine 17
To a stirred solution of nucleoside 16 (0.082 g, 0.26 mmol) in
anhydrous pyridine (10 cm3) at Ϫ20 ЊC was added a solution of
mesyl chloride (0.04 cm3, 0.55 mmol) in anhydrous pyridine (1.0
cm3) dropwise during 45 min. After 2 h, ice-cold water (6.0 cm3)
was added and the mixture was evaporated to dryness under
reduced pressure to give an intermediate tentatively assigned as
crude 17, which after co-evaporation with anhydrous toluene
was used without further purification in the next step.
1-(3-Azido-3-deoxy-2-O,4-C-methylene-ꢀ-D-ribofuranosyl)-
thymine 4
Intermediate 17 (0.122 g, 0.26 mmol) was stirred in a mixture
of 1,4-dioxane (4.0 cm3) and 1 M aq. NaOH (2.25 cm3). After
15 min at room temperature, analytical TLC (methanol–
dichloromethane; 1:19, v/v) showed quantitative conversion of
starting material into an intermediate with a slightly lower
mobility. The reaction mixture was subsequently stirred at
90 ЊC for 12 h, then neutralized with a 7% (w/w) solution of
HCl in 1,4-dioxane and finally evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane–methanol (97:3, v/v)
as eluent to give nucleoside 4 (28 mg, 36%, two steps) as a white
solid material, δH (CD3OD) 7.70 (1H, s, 6-H), 5.59 (1H, s, 1Ј-H),
4.57 (1H, s, 2Ј-H), 4.02 (1H, s, 3Ј-H), 3.95–3.76 (4H, m, 5Ј-, 1Љ-
H2), 1.89 (3H, s, CH3). These δH (CD3OD) data are in accord
with previously published δH (CD3OD) data;10 δC (CD3OD)
164.2 (C-4), 150.0 (C-2), 134.6 (C-6), 109.1 (C-5), 89.2 (C-4Ј),
86.5 (C-1Ј), 78.1 (C-2Ј), 59.5 (C-3Ј), 70.6, 55.5 (C-5Ј, -1Љ), 10.8
(CH3); FAB-HRMS m/z 296.0995. Calc. 296.0995 [M ϩ H]ϩ.
Selected IR signal: νmax 2120 cmϪ1 (azido group).
Acknowledgements
The Danish Natural Science Research Council and The
Danish Technical Research Council are thanked for financial
support.
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1-(3-Azido-3-deoxy-2,5-di-O-mesyl-4-C-mesyloxymethyl-ꢀ-D-
erythro-pentofuranosyl)thymine 18
To a stirred solution of nucleoside 16 (206 mg, 0.66 mmol)
in anhydrous pyridine (10 cm3) was added a solution of mesyl
chloride (0.18 ml, 2.30 mmol) in anhydrous pyridine (1 cm3).
After stirring of the mixture for 1.5 h, ice-cold water (8 cm3)
was added and the mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in dichloromethane
(50 cm3), washed with saturated aq. sodium hydrogen carbon-
ate (3 × 20 cm3) and evaporated to dryness under reduced
pressure. The residue was purified by silica gel column chrom-
atography using dichloromethane–methanol (49:1, v/v) as
eluent to give nucleoside 18 (283 mg, 79%) as a white solid
material, δH [(CD3)2SO] 11.54 (1H, s, NH), 7.55 (1H, s, 6-H),
6.06 (1H, d, J 5.0, 1Ј-H), 5.68 (1H, dd, J 5.0 and 6.6, 2Ј-H), 5.03
(1H, d, J 6.6, 3Ј-H), 4.43 (4H, m, 5Ј-, 1Љ-H2), 3.37, 3.31, 3.28
(9H, 3 × s, Ms), 1.79 (3H, s, CH3); δC [(CD3)2SO] 163.7 (C-4),
150.5 (C-2), 136.6 (C-6), 110.5 (C-5), 87.8 (C-1Ј), 82.7 (C-4Ј),
78.1 (C-2Ј), 68.1, 67.4 (C-5Ј, -1Љ), 61.5 (C-3Ј), 37.8, 37.0, 37.0
(3 × Ms), 12.1 (CH3); FAB-MS m/z 548 [M ϩ H]ϩ.
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3614
J. Chem. Soc., Perkin Trans. 1, 2000, 3610–3614