Synthesis and evaluation of anti-HIV activity of 3-azido-4-(hydroxymethyl)tetrahydrofuran derivatives containing 2-(thymin-1-yl)methyl, 2-(cytosin-1-yl)methyl or 2-(adenin-9-yl)methyl substituents - A new series of AZT analogues

Article abstract of DOI:10.1039/b009109j

The three monocyclic AZT analogues 6, 7 and 8 {3-azido-4-(hydroxymethyl)tetrahydrofuran derivatives containing 2-[(thymin-1-yl)methyl], 2-[(cytosin-1-yl)methyl] and 2-[(adenin-9-yl)methyl] substituents} are synthesized via methyl 3-azido-3-deoxy-2-O,4-C-methylene-D-ribofuranoside 13 as key intermediate. All nucleoside analogues proved to be devoid of anti-HIV activity in MT-4 cells.

Full text of DOI:10.1039/b009109j

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Synthesis and evaluation of anti-HIV activity of 3-azido-
4-(hydroxymethyl)tetrahydrofuran derivatives containing 2-(thymin-
1-yl)methyl, 2-(cytosin-1-yl)methyl or 2-(adenin-9-yl)methyl
substituents – a new series of AZT analogues
1
Anne G. Olsen,a Claus Nielsenb and Jesper Wengel*†a
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) 14th November 2000, Accepted 22nd January 2001
First published as an Advance Article on the web 16th February 2001
The three monocyclic AZT analogues 6, 7 and 8 {3-azido-4-(hydroxymethyl)tetrahydrofuran derivatives containing
2-[(thymin-1-yl)methyl], 2-[(cytosin-1-yl)methyl] and 2-[(adenin-9-yl)methyl] substituents} are synthesized via methyl
3-azido-3-deoxy-2-O,4-C-methylene--ribofuranoside 13 as key intermediate. All nucleoside analogues proved to be
devoid of anti-HIV activity in MT-4 cells.
conclusion that an S-type conformation is necessary for effi-
Introduction
cient triphosphorylation while subsequent inhibition of HIV-1
3Ј-Azido-3Ј-deoxythymidine (AZT, 1, Fig. 1) was the first drug
RT requires an N-type conformation.9,10
to be approved by the FDA (US Food and Drug Adminis-
We and others have further investigated these results by syn-
tration) for treatment of AIDS patients. AZT and the five
thesizing the two locked AZT analogues 411,12 and 512 (Fig. 1)
approved 2Ј,3Ј-dideoxynucleosides1 are prodrugs which need to
adopting a C3Ј-endo conformation and a C3Ј-exo conform-
be triphosphorylated in vivo at the 5Ј-hydroxy group to success-
ation, respectively. In line with the results of Marquez et al.9,10
fully interact with the virus-specific enzyme HIV-1 Reverse
both 4 and 5 were inactive against HIV-1, thus supporting the
Transcriptase (HIV-1 RT).2 The active triphosphate derivatives
assumption that conformational flexibility of the furanose ring
can either act as competitive inhibitors, preventing incorpor-
is essential for anti-HIV-1 activity.12
ation of the natural substrate, or as an alternative substrate thus
Based on the above we decided to synthesize and study a
causing viral DNA chain termination as no hydroxy group is
series of novel monocyclic AZT analogues 6–8 (Fig. 1). These
present in the 3Ј-position.3 AZT and the other nucleoside ana-
are considered as ring-opened derivatives of 4 and were chosen
logues continue to play an important role in the treatment of
as synthetic targets in an attempt to discover new, conform-
HIV infection but, due in part to the development of resistance
ationally more flexible, lead structures in the search for
improved anti-HIV drugs.
and toxic side effects, they are often administered in combin-
ations with other drugs.4 Therefore, further investigations of the
pharmacological effects of novel nucleosides and the develop-
ment of new drug candidates are important.
Results and discussion
The relation between the conformation of the furanose ring
as described by the pseudorotational wheel5 and the anti-HIV
activity is a field of current interest. Investigations on solid-
state conformations of nucleosides suggested that an S-type
(south-type; e.g., C2Ј-endo/C3Ј-exo) conformer of AZT is
responsible for both its successful triphosphorylation and
potent anti-HIV activity, thus assuming that a high level of
AZT 5Ј-triphosphate would efficiently inhibit HIV-1 RT.6,7
However, subsequent NMR studies revealed that AZT tri-
phosphate and thymidine triphosphate, when bound to HIV-1
RT, both adopt an N-type (north-type; C4Ј-exo) conformation.8
Marquez et al. have reported the synthesis of the two bicyclic
AZT analogues 2 and 3 (Fig. 1) predominantly adopting an
N-type and S-type conformation, respectively.9 Neither 2 nor
3 showed anti-HIV activity, but when converted to their
5Ј-triphosphates in vitro the N-type analogue 2 showed an anti-
HIV effect similar to that of AZT 5Ј-triphosphate while the
S-type analogue 3 was still inactive. These results led to the
The anomeric methyl furanoside 13 (Scheme 1) was considered
as a potential key intermediate in the synthesis of 6–8 as it has
been reported that a Vorbrüggen-type coupling reaction13,14 on
a related methyl furanoside afforded ring-opened derivatives.15
The favouring of the Lewis acid-mediated ring-opening reac-
tion over the expected cleavage of the anomeric bond was
explained by ring strain due to the bicyclic constitution.15
To prepare 13, the known 3-azido-5-O-benzoyl-4-C-
benzoyloxymethyl-3-deoxy-1,2-O-isopropylidene-α--ribo-
furanose 9 was prepared from -glucose in several steps, as
described.16 The corresponding diol 10 was obtained as previ-
ously reported11 by reaction with sodium methoxide in meth-
anol (94% yield; reported11 86%). Mesylation of 10 using 2.1
equiv. of mesyl chloride in pyridine afforded an intermediate,
tentatively assigned as the di-O-mesyl derivative 11, which was
subsequently treated with HCl in aq. methanol to give the
anomeric mixture of methyl furanosides 12 in 79% yield (calcu-
lated from 10). Treatment of 12 with a mixture of 1 M aq.
NaOH and 1,4-dioxane (room temperature, 1 h) afforded an
intermediate expected to be the bicyclic 5-O-mesylated deriva-
tive, which upon raising the temperature to 85 ЊC was converted
into the anomeric key intermediate 13 in 87% yield (Scheme 1).
† Present address: Department of Chemistry, University of Southern
Denmark, Odense, DK-5230 Odense M, Denmark. Fax: ϩ45 66 15 87
80; E-mail: jwe@chem.sdu.dk
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This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b009109j

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Scheme 1 Reagents, conditions and yields: i) NaOCH3, CH3OH (94%);
ii) MsCl, pyridine; iii) 20% HCl in aq. methanol (79%, two steps);
iv) aq. NaOH, 1,4-dioxane (87%).
sodium methoxide in methanol afforded the target nucleoside
analogue 7 as a diastereoisomeric mixture in 57% yield. The
mixture 7 was not separated into the two diastereoisomers but
was submitted for biological testing as the isolated 1.0 : 1.3
mixture. The coupling of 14a with 6-N-benzoyladenine was
performed under thermodynamic control and furnished the
desired N9 regioisomeric intermediate 16 as a diastereoiso-
meric mixture. The target diastereoisomeric compound 8 was
subsequently obtained by reaction of nucleoside analogue 16
with sodium methoxide in methanol to give the monocyclic
nucleoside derivative 8 (19% yield calculated from 14a) which
was assigned as the N9 regioisomers by comparing the
obtained 13C chemical shift values with published values for
N7 and N9 regioisomeric β--ribofuranosyladenines 17.17 As
with 7, the diastereoisomeric mixture 8 (1.0 : 1.4) was tested as
such.
Fig. 1 Structures of AZT 1, the conformationally restricted bicyclic
AZT analogues 2–5, and the monocyclic nucleoside analogues 6–8.
The bicyclic conformationally locked structures of the anomers
of 13 were verified by the appearance of the H1 and H2
signals as singlets in the H NMR spectra. As expected from
The monocyclic nucleoside analogues 6a, 6b, 7 and 8 were
evaluated for antiviral activity against HIV-1 in MT-4 cells as
described earlier.18 All compounds were inactive against HIV-1
at 300 µM and also proved to be non-toxic to the MT-4 cells at
this concentration. Thus, although the structures of the three
analogues allow considerable conformational flexibility of the
tetrahydrofuran ring, they are devoid of activity. Possible
explanations for the lack of activity, despite the configurations
at all carbon atoms being identical with those of AZT, include
a) the presence of the 1Ј-C-methoxy substituent, the 4Ј-C-
hydroxy group and the 2Ј-O,4Ј-C-methylene bridge, and b) the
less constrained relative positioning of the C5Ј atom and the
thymine moiety compared with AZT. Alone or in combination,
these points may explain unfavorable binding interactions with
relevant receptors.
1
earlier results,15 the Vorbrüggen-type coupling reaction13,14
between thymine and the bicyclic furanoside 13 afforded
the ring-opened target compound 6 as a crude mixture of
diastereoisomers 6a and 6b in a yield of 61%. After column
chromatographic purification followed by preparative reversed-
phase HPLC, the individual diastereoisomers 6a and 6b were
obtained in low yields but their configuration at the exocyclic
asymmetric carbon atom could not be assigned. As expected,
the strain in the bicyclic structure was relieved during the reac-
tion with silylated thymine and the monocyclic constitution of
6a and 6b was verified by NMR studies (e.g., by H1Ј appearing
as a doublet for both isomers and the presence of both 1Ј-C-
methoxy and 4Ј-C-hydroxy groups). Owing to unsuccessful
attempted Vorbrüggen-type coupling reaction between 13 and
silylated cytosine, it was decided to protect the primary hydroxy
group of 13 by reaction with acetic anhydride in pyridine to
give the 5-O-acetylated product 14a and the corresponding
α-anomer 14b as separated anomers in an overall yield of 88%
(α/β = 1 : 3) after column chromatographic purification. Both
anomers 14a and 14b should be applicable for subsequent
introduction of nucleobases, but because of its predominance,
the β-anomer 14a was coupled with 4-N-acetylcytosine to give
the ring-opened nucleoside analogue 15, protected as a tri-
methylsilyl ether at the tertiary hydroxy group, in 60% yield
as an intermediate diastereoisomeric mixture (Scheme 2).
The complete deprotection of compound 15 by reaction with
Conclusions
The synthesis of a series of novel monocyclic AZT analogues 6,
7 and 8 has been accomplished. All nucleoside analogues pre-
pared were devoid of activity against HIV-1 in MT-4 cells. In
theory, they should be conformationally flexible and be able to
adopt what would correspond to the N-type and the S-type
pentofuranose conformations of AZT, thereby possibly being
5Ј-O-triphosphorylated and eventually acting as inhibitors of
HIV-1 RT. However, the structural requirements for anti-HIV-1
activity are apparently not fulfilled with these novel, rather
drastically modified, AZT analogues.
J. Chem. Soc., Perkin Trans. 1, 2001, 900–904
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Scheme 2 Reagents, conditions and yields: i) N,O-bis(trimethylsilyl)acetamide, thymine, TMS triflate, acetonitrile (61%, 6a ϩ 6b); ii) Ac2O, pyridine
(88%, 14a ϩ 14b); iii) N,O-bis(trimethylsilyl)acetamide, 4-N-acetylcytosine, TMS triflate, acetonitrile (60%); iv) NaOCH3, CH3OH (57%); v) N,O-
bis(trimethylsilyl)acetamide, 6-N-benzoyladenine, TMS triflate, acetonitrile; vi) NaOCH3, CH3OH (19%, two steps).
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 furanose 10 (4.34 g, 94%)
Experimental
General
as a white solid material, δH (CD3OD) 5.82 (1H, d, J 3.9, 1-H),
4.88 (1H, dd, J 3.9, 5.63, 2-H), 4.27 (1H, d, J 5.7, 3-H), 3.90
(1H, d, J 11.9), 3.68 (1H, d, J 11.9), 3.66 (1H, d, J 11.8), 3.56
(1H, d, J 11.9) (together 5-, 1Ј-H2), 1.57 (3H, s, CH3), 1.34 (3H,
s, CH3); δC (CD3OD) 114.6 [CH(CH3)2], 106.0 (C-1), 89.3 (C-4),
81.9 (C-2), 64.0, 63.4, 63.0 (C-3, -5, -1Ј), 26.8, 26.3 (2 × CH3);
FAB-MS m/z 246 [M ϩ H]ϩ. Selected IR signal: νmax 2117 cmϪ1
(azido group). Furanose 10 had been synthesized earlier, but
no data were reported.11
All reagents and solvents were obtained from commercial sup-
pliers and were used without further purification. Petroleum
ether of distillation range 60–80 ЊC was used. Reactions were
conducted under an atmosphere of nitrogen when anhydrous
solvents were used. All reactions were monitored by TLC. Dur-
ing work-up, organic phases were combined and either dried
(Na2SO4) and filtered or co-evaporated with anhydrous toluene
before evaporation. After column chromatographic purification
(glass columns; silica gel 60, Merck, 0.040–0.063 mm), frac-
tions containing product were pooled, evaporated to dryness
under reduced pressure, and dried under vacuum to give the
product. NMR spectra were recorded on a 250 MHz spec-
trometer (1H NMR at 250 MHz and 13C NMR at 62.9 MHz)
for compounds 10–13 and on a 400 MHz spectrometer (1H
NMR at 400 MHz and 13C NMR at 100.6 MHz) for all other
compounds. Chemical shifts are reported in ppm relative to
tetramethylsilane as internal standard, and coupling constants
J are given in Hz. Complete assignments of NMR spectra when
given are based on 2D spectra recorded on a 400 MHz spec-
trometer. The atom numbering used throughout the NMR
assignments follows standard carbohydrate/nucleoside style.
When mixtures of isomers were obtained, signals obtained for
the major isomer are marked with an asterisk (*). NOE spectra
were recorded on a 250 MHz spectrometer. NOE and 2D
spectra were obtained in the same solvent as the corresponding
1H and 13C spectra. Fast-atom bombardment (high-resolution)
mass spectra [FAB-(HR)MS] were recorded in positive ion
mode. Microanalyses were performed at The Microanalytical
Laboratory, Department of Chemistry, University of
Copenhagen.
3-Azido-3-deoxy-1,2-O-isopropylidene-5-O-mesyl-4-C-mesyl-
oxymethyl-ꢀ-D-erythro-pentofuranose 11
To a stirred solution of furanose 10 (4.25 g, 0.0173 mol)
in anhydrous pyridine (125 cm3) was added a solution of
methanesulfonyl chloride (3.25 cm3, 0.0418 mol) in anhydrous
pyridine (5.0 cm3). After stirring of the mixture for 1.5 h at
room temperature, ice-cold water (50 cm3) was added at 0 ЊC
and the mixture was evaporated to dryness under reduced pres-
sure. The residue was dissolved in dichloromethane (125 cm3),
washed with saturated aq. sodium hydrogen carbonate (3 × 50
cm3) and evaporated to dryness under reduced pressure to give
an intermediate tentatively assigned as 11 which was used with-
out further purification in the next step; δC (CDCl3) 113.4
[CH(CH3)2], 103.8 (C-1), 82.6, 78.5, 67.5, 67.3, 62.8 (C-2, -3, -4,
-5, -1Ј), 37.1, 36.8 (2 × Ms), 25.0, 24.6 (2 × CH3). FAB-MS m/z
402 [M ϩ H]ϩ; selected IR signal: νmax 2120 cmϪ1 (azido group).
Methyl 3-azido-3-deoxy-5-O-mesyl-4-C-mesyloxymethyl-D-
erythro-pentofuranoside 12
Intermediate 11 (6.96 g) was stirred in a mixture of HCl in
methanol (225 cm3; 20% w/w) and water (30 cm3) for 72 h at
room temperature, then neutralized with sodium hydrogen car-
bonate (s). Extraction was performed with dichloromethane
(3 × 100 cm3) and the combined organic phase was washed with
saturated aq. sodium hydrogen carbonate (3 × 100 cm3) and
evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography, using dichloro-
methane–methanol (100 : 0 to 97 : 3 v/v) as eluent, to give the
3-Azido-3-deoxy-4-C-hydroxymethyl-1,2-O-isopropylidene-ꢀ-D-
erythro-pentofuranose 10
To a stirred solution of compound 916 (8.52 g, 0.0188 mol) in
anhydrous methanol (200 cm3) was added sodium methoxide
(2.13 g, 0.0394 mol). The reaction mixture was stirred for 1 h at
room temperature whereupon the mixture was neutralized with
a 7% (w/w) solution of HCl in 1,4-dioxane. After evaporation
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J. Chem. Soc., Perkin Trans. 1, 2001, 900–904

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anomeric furanoside 12 (≈1.0 : 2.5; 5.16 g, 79%, two steps) as a
light yellow oil; δH (CD3OD) 4.91 (d, J 4.2, 1-H), 4.84* (J 12.8,
1-H), 4.50–4.12 (m, 2-, 3-, 5-, 1Ј-H), 3.44 (s, CH3O), 3.38* (s,
CH3), 3.18, 3.15, 3.14 (3 × s, 4 × Ms); δC (CD3OD) 107.9, 103.0
(C-1), 81.7, 81.0, 74.4, 72.2, 69.1, 68.7, 68.6, 68.5, 65.3, 62.7
(C-2, -3, -4, -5, -1Ј), 54.4, 53.9 (CH3O), 35.6, 35.4 (Ms);
FAB-MS m/z 376 [M ϩ H]ϩ; selected IR signal: νmax 2121 cmϪ1
(azido group). To verify the purity of this compound, a copy
of the 13C NMR spectrum was enclosed when submitting this
manuscript.
(2R,3S,4S)-3-Azido-4-hydroxy-4-hydroxymethyl-2-[(S)-
(thymin-1-yl)(methoxy)methyl]tetrahydrofuran 6a and (2R,3S,
4S)-3-azido-4-hydroxy-4-hydroxymethyl-2-[(R)-(thymin-1-yl)-
(methoxy)methyl]tetrahydrofuran 6b
To a stirred solution of the anomeric mixture 13 (0.108 g,
0.537 mmol) and thymine (0.135 g, 1.07 mmol) in anhydrous
acetonitrile (10 cm3) was added N,O-bis(trimethylsilyl)-
acetamide (0.79 cm3, 3.22 mmol). The reaction mixture was
stirred and heated with reflux for 1 h. After cooling to 0 ЊC,
TMS triflate (0.26 cm3, 1.34 mmol) was added dropwise. The
mixture was heated to 75 ЊC and stirring was continued for 48 h
whereupon additional TMS triflate (0.10 cm3, 0.537 mmol)
was added and the reaction mixture was stirred for 72 h at
75 ЊC. Half-saturated aq. sodium hydrogen carbonate (10 cm3)
was added and the mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in methanol–
water (2 : 1 v/v), mixed with silica gel and evaporated to dry-
ness under reduced pressure. The silica gel containing
absorbed product was applied to a silica gel column and
purification was performed using dichloromethane–methanol
(98 : 2 to 97 : 3 v/v) as eluent to give nucleoside analogues
6a ϩ 6b as a diastereoisomeric mixture (0.180 g, 61%). This
mixture was purified by repeated silica gel column chrom-
atography as described above, and by preparative reversed-phase
HPLC (C18 column, eluting with ethanol–water (1 : 40 to 1 : 10
v/v)) to yield the two individual diastereoisomers 6a and 6b
(4.0 mg, 2.2%, tR 43 min; 5.0 mg, 2.8%, tR 66 min) as indi-
vidual but not assigned compounds, both as white solid
materials.
Data for the more polar diastereoisomer: δH ((CD3)2SO) 11.4
(1H, br s, NH), 7.45 (1H, s, 6-H), 5.53 (1H, d, J 6.2, 1Ј-H),
5.41 (1H, br s, OH), 4.90 (1H, br s, OH), 3.88–3.46 (6H,
m, 2Ј-, 3Ј-, 5Ј-, 1Љ-H), 3.27 (3H, s, CH3O), 1.78 (3H, s, CH3);
FAB-HRMS m/z 328.1263 [C12H18N5O6, M ϩ H]ϩ. Calc.
328.1257.
Data for the less polar diastereoisomer: δH ((CD3)2SO)
11.3 (1H, br s, NH), 7.53 (1H, s, 6-H), 5.73 (1H, d, J 7.9,
1Ј-H), 5.40 (1H, br s, OH), 4.91 (1H, br s, OH), 4.04–3.42 (6H,
m, 2Ј-, 3Ј-, 5Ј-, 1Љ-H), 3.28 (3H, s, CH3O), 1.79 (3H, s, CH3);
FAB-HRMS m/z 328.1255 [C12H18N5O6, M ϩ H]ϩ. Calc.
328.1257.
Methyl 3-azido-3-deoxy-2-O,4-C-methylene-D-ribofuranoside 13
Furanoside 12 (5.16 g, 0.0137 mol) was stirred in a mixture of
1,4-dioxane (150 cm3) and 1 M aq. NaOH (137 cm3). After 1 h
at room temperature, analytical TLC (methanol–dichloro-
methane; 1.0 : 12.5 v/v) showed quantitative conversion of start-
ing material into an intermediate with a slightly lower mobility.
The reaction mixture was subsequently stirred at 85 ЊC for 96 h
and then evaporated to dryness under reduced pressure. The
residue was dissolved in methanol–water (1 : 1 v/v), mixed with
silica gel and evaporated to dryness under reduced pressure.
The silica gel containing absorbed product was applied to
a silica gel column and purification was performed using
dichloromethane–methanol (99 : 1 to 98 : 2 v/v) as eluent to give
anomeric furanoside 13 (≈1.0 : 1.8; 2.40 g, 87%) as a clear oil;
δH (CD3OD) 5.10* (s, 1-H), 4.78 (s, 1-H), 4.32* (s, 2-H), 4.23
(s, 2-H), 4.06–3.57 (m, 3-, 5-, 1Ј-H), 3.46* (s, CH3O), 3.39
(s, CH3O); δC (CD3OD) 106.0, 105.7 (C-1), 91.6, 88.5, 80.2,
80.0, 73.0, 72.4, 65.3, 63.8 (C-2, -3, -4, -5), 59.1, 58.8, 56.5,
55.7 (CH3O, C-1Ј); FAB-MS m/z 202 [M ϩ H]ϩ; selected IR
signal: νmax 2114 cmϪ1 (azido group) [Found: (%) C, 41.4; H,
5.8; N, 20.0. C7H11N3O4ؒ0.25H2O requires C, 40.9; H, 5.6; N,
20.4].
Methyl 5-O-acetyl-3-azido-3-deoxy-2-O,4-C-methylene-ꢀ-D-
ribofuranoside 14a and methyl 5-O-acetyl-3-azido-3-deoxy-2-
O,4-C-methylene-ꢁ-D-ribofuranoside 14b
To a stirred solution of furanoside 13 (2.33 g, 0.0116 mol) in
anhydrous pyridine (75 cm3) was added acetic anhydride (2.74
cm3, 0.0290 mol). After stirring of the mixture for 5 h at room
temperature, ice-cold water (60 cm3) was added at 0 ЊC and the
mixture was evaporated to dryness under reduced pressure. The
residue was dissolved in ethyl acetate (150 cm3), washed with
saturated aq. sodium hydrogen carbonate (3 × 50 cm3) and
evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography, using ethyl
acetate–petroleum ether (0 : 100 to 30 : 70 v/v) as eluent, to give
the β-anomer 14a and the corresponding α-anomer 14b as
individual compounds (14a : 14b = 3 : 1; 2.48 g combined yield,
88%), both as clear oils.
Data for 14a: δH (CD3OD) 4.79 (1H, s, 1-H), 4.45 (1H, d,
J 12.5, 5- or 1Ј-H), 4.32 (1H, d, J 12.5, 1Ј- or 5-H), 4.25 (1H, s,
2-H), 4.09 (1H, s, 3-H), 3.83 (1H, d, J 7.9, 5- or 1Ј-H), 3.62 (1H,
d, J 7.9, 1Ј- or 5-H), 3.37 (3H, s, CH3O), 2.08 (3H, s, CH3CO);
δC (CD3OD) 172.1 (COCH3), 106.2 (C-1), 85.9 (C-4), 80.1
(C-2), 72.4 (C-5 or -1Ј), 64.4 (C-3), 61.0 (C-1Ј or -5), 55.7
(CH3O), 20.6 (CH3CO); FAB-MS m/z 244 [M ϩ H]ϩ [Found:
(%) C, 43.9; H, 5.5; N, 17.1. C9H13N3O6 requires C, 43.6; H, 5.5;
N, 17.0].
Data for 14b: δH (CD3OD) 5.11 (1H, s, 1-H), 4.35 (1H, s,
2-H), 4.39, 4.29 (2H, 2 × d, J 12.7, 5- or 1Ј-H2), 4.08 (1H, s,
3-H), 3.88, 3.83 (2H, 2 × d, J 8.2, 1Ј- or 5-H2), 3.45 (3H, s,
CH3O), 2.08 (3H, s, CH3CO); δC (CD3OD) 172.0 (COCH3),
105.9 (C-1), 88.8 (C-4), 79.9 (C-2), 73.0 (C-5 or -1Ј), 65.9 (C-3),
61.4 (C-1Ј or -5), 56.6 (CH3O), 20.6 (CH3CO); FAB-MS
m/z 244 [M ϩ H]ϩ [Found: (%) C, 43.8; H, 5.7; N, 17.2.
C9H13N3O6ؒ0.25H2O requires C, 43.6; H, 5.5; N, 17.0].
Data for the anomeric mixture: Selected IR signal: νmax 2115
cmϪ1 (azido group).
(2R,3S,4R)-4-Acetoxymethyl-2-[(4-N-acetylcytosin-1-yl)-
(methoxy)methyl]-3-azido-4-trimethylsilyloxytetrahydrofuran
15
To a stirred solution of furanoside 14a (0.161 g, 0.662 mmol)
and 4-N-acetylcytosine (0.203 g, 1.32 mmol) in anhydrous
acetonitrile (15 cm3) was added N,O-bis(trimethylsilyl)-
acetamide (0.97 cm3, 3.97 mmol). The reaction mixture was
stirred and heated with reflux for 1 h. After cooling to 0 ЊC, the
mixture was treated dropwise with TMS triflate (0.38 cm3, 1.99
mmol). After being stirred for 10 min at 0 ЊC, the mixture was
heated to 60 ЊC and stirring was continued for 48 h and sub-
sequently at 70 ЊC for 24 h. Additional TMS triflate (0.12 cm3,
0.662 mmol) was added and the reaction mixture was stirred for
20 h at 70 ЊC. Half-saturated aq. sodium hydrogen carbonate
(10 cm3) was added and the mixture was evaporated to dryness
under reduced pressure. The residue was dissolved in ethyl
acetate, mixed with silica gel and evaporated to dryness under
reduced pressure. The silica gel containing absorbed product
was applied to a silica gel column and purification was per-
formed using dichloromethane–methanol (100 : 0 to 97.5 : 2.5
v/v) as eluent to give the diastereoisomeric intermediate 15 as a
light yellow oil (0.187 g, 60%); δC (CD3OD) 173.0, 172.1, 172.1,
164.5, 164.4 (3 × C᎐O), 159.3, 158.6, 146.4, 146.1, 98.9, 98.6
᎐
(C-4, -5, -6), 88.6, 88.0, 84.6, 84.4, 84.0, 82.8, 75.3, 74.8, 72.6,
71.3, 66.4, 66.3 (C-1Ј, -2Ј, -3Ј, -4Ј, -5Ј, -1Љ), 58.1, 57.8 (CH3O),
24.7, 22.2, 21.0, 20.9 (2 × CH C᎐O), 1.93 (TMS); selected IR
᎐
3
signal: νmax 2113 cmϪ1 (azido group).
J. Chem. Soc., Perkin Trans. 1, 2001, 900–904
903

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(2R,3S,4S)-3-Azido-2-[(cytosin-1-yl)(methoxy)methyl]-4-
hydroxy-4-hydroxymethyltetrahydrofuran 7
19%, 2 steps) as a white solid material, δH(CD3OD) 8.33 (s),
8.28, 8.26* (2 × s) (together 8-, 2-H), 5.88 (d, J 6.2, 1Ј-H), 5.87*
(d, J 7.9, 1Ј-H), 4.56–3.66 (m, 2Ј-, 3Ј-, 5Ј-H, 1Љ-H2), 3.41 (s,
CH3O), 3.40* (s, CH3O); δC (CD3OD) 157.5, 157.5, 154.3,
154.1, 151.6, 151.1, 141.3, 141.2 (C-6, -2, -4, -8), 120.1, 120.1
(C-5), 87.9, 87.7, 85.4, 85.2, 83.3, 82.7, 75.9, 75,9, 71.9,
71.1, 64.0, 63.9 (C-1Ј, -2Ј, -3Ј, -4Ј, -5Ј, -1Љ), 57.6, 57.4 (CH3O);
FAB-HRMS m/z 337.1360. Calc. 337.1373 [M ϩ H]ϩ; selected
IR signal: νmax 2113 cmϪ1 (azido group).
To a stirred solution of derivative 15 (50.0 mg, 0.117 mmol) in
anhydrous methanol (5 cm3) was added sodium methoxide
(22.0 mg, 0.410 mmol). The reaction mixture was stirred for 3 h
at room temperature whereupon the mixture was neutral-
ized with a 7% (w/w) solution of HCl in 1,4-dioxane. After
evaporation to dryness under reduced pressure, the residue
was purified by silica gel column chromatography, using
dichloromethane–methanol (95 : 5 to 92 : 8 v/v) as eluent, to
give the diastereoisomeric nucleoside analogue 7 (ratio between
diastereoisomers = 1.0 : 1.3; 21.0 mg, 57%) as a white solid
material; δH (CD3OD) 7.64 (d, J 7.5, 6-H), 7.62* (d, J 7.5, 6-H),
5.97 (d, J 7.5, 5-H), 5.96* (d, J 7.5, 5-H), 5.93 (d, J 8.4, 1Ј-H),
5.80* (d, J 5.7, 1Ј-H), 4.08–3.59 (m, 2Ј-, 3Ј-, 5Ј-, 1Љ-H), 3.36* (s,
CH3O), 3.34 (s, CH3O); δC (CD3OD) 167.6, 167.5 (C-4), 159.7,
159.0 (C-2), 142.7, 142.4 (C-6), 97.0, 96.7 (C-5), 88.0, 87.5
(C-1Ј), 85.4, 84.8, 82.9, 82.7, 75.7, 75.6, 71.9, 71.0, 64.2, 64.0
(C-2Ј, -3Ј, -4Ј, -5Ј, -1Љ), 57.4, 57.2 (CH3O); FAB-HRMS m/z
313.1243. Calc. 313.1260 [M ϩ H]ϩ; selected IR signal: νmax
2113 cmϪ1 (azido group).
Acknowledgements
Dedicated to the memory of Professor Göran Magnusson.
The Danish Natural Science Research Council, The Danish
Technical Research Council, and Exiqon A/S are thanked for
financial support.
References
1 Information about FDA-approved anti-HIV drugs can be found at
www.niaid.nih.gov/daids/dtpdb
2 E. De Clercq, Collect. Czech. Chem. Commun., 1998, 63, 449.
3 E. De Clercq, Antiviral Res., 1989, 12, 1.
4 E. De Clercq, J. Med. Chem., 1995, 38, 2491.
5 C. S. Altona and M. Sundaralingam, J. Am. Chem. Soc., 1972, 94,
8205.
(2R,3S,4S)-2-[(Adenin-9-yl)(methoxy)methyl]-3-azido-4-
hydroxy-4-hydroxymethyltetrahydrofuran 8
To a stirred solution of furanoside 14 (75.0 mg, 0.308 mmol)
and 6-N-benzoyladenine (0.148 g, 0.617 mmol) in anhydrous
acetonitrile (10 cm3) was added N,O-bis(trimethylsilyl)-
acetamide (0.45 cm3, 1.85 mmol). The reaction mixture was
stirred and heated under reflux for 1 h. After the mixture had
cooled to 0 ЊC, TMS triflate (0.18 cm3, 0.925 mmol) was added
dropwise. After being stirred for 10 min at 0 ЊC, the mixture was
heated to 70 ЊC and stirring was continued for 90 h. Half-
saturated aq. sodium hydrogen carbonate (5 cm3) was added
and the mixture was evaporated to dryness under reduced pres-
sure. The residue was dissolved in ethyl acetate, mixed with
silica gel, and evaporated to dryness under reduced pressure.
The silica gel containing absorbed product was applied to a
silica gel column and purification was performed using
dichloromethane–methanol (98.5 : 1.5 v/v) as eluent to give an
intermediate tentatively assigned as 16 (74.0 mg).
To a stirred solution of this intermediate (64.0 mg) in
anhydrous methanol (5 cm3) was added sodium methoxide
(22.0 mg, 0.410 mmol). The reaction mixture was stirred for 3 h
at room temperature, additional sodium methoxide (11.0 mg,
0.202 mmol) was added, and stirring was continued for 19 h
whereupon the mixture was neutralized with a 7% (w/w) solu-
tion of HCl in 1,4-dioxane. After evaporation to dryness under
reduced pressure, the residue was purified by silica gel column
chromatography, using dichloromethane–methanol (97.5 : 2.5
to 95 : 5 v/v) as eluent, to give the diastereoisomeric nucleoside
analogue 8 (ratio between diastereoisomers = 1.0 : 1.4; 17 mg,
6 P. Van Roey, E. W. Taylor, C. K. Chu and R. F. Schinazi, Ann. N.Y.
Acad. Sci., 1990, 616, 29.
7 P. Van Roey, J. M. Salerno, C. K. Chu and R. F. Schinazi, Proc.
Natl. Acad. Sci. USA, 1989, 86, 3929.
8 G. R. Painter, A. E. Aulabaugh and C. W. Andrews, Biochem.
Biophys. Res. Commun., 1993, 191, 1166.
9 V. E. Marquez, A. Ezzitouni, P. Russ, M. A. Siddiqui, H. Ford,
R. J. Feldman, H. Mitsuya, C. George and J. J. Barchi, J. Am. Chem.
Soc., 1998, 120, 2780.
10 V. E. Marquez, A. Ezzitouni, P. Russ, M. A. Siddiqui, H. Ford,
R. J. Feldman, H. Mitsuya, C. George and J. J. Barchi, Nucleosides,
Nucleotides, 1998, 17, 1881.
11 S. Obika, J. Andoh, T. Sugimoto, K. Miyashita and T. Imanishi,
Tetrahedron Lett., 1999, 40, 6465.
12 A. G. Olsen, V. K. Rajwanshi, C. Nielsen and J. Wengel, J. Chem.
Soc., Perkin Trans. 1, 2000, 3610. The furanose conformation of
compound 5 could alternatively be assigned as N-type (C3Ј-endo,
3E) because of its -configuration. However, for a direct comparison
with the conformations of the natural DNA/RNA monomers, the
furanose conformation of 5 is considered herein as equivalent to an
S-type (C3Ј-exo, 3E) conformation.
13 H. Vorbrüggen, K. Krolikiewicz and B. Bennua, Chem. Ber., 1981,
114, 1234.
14 H. Vorbrüggen and G. Höfle, Chem. Ber., 1981, 114, 1256.
15 P. Nielsen and J. Wengel, Chem. Commun., 1998, 2645.
16 S. A. Surzhykov and A. A. Krayevsky, Nucleosides, Nucleotides,
1994, 13, 2283.
17 M. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica and
L. B. Townsend, J. Am. Chem. Soc., 1975, 97, 4627.
18 K. Danel, E. B. Pedersen and C. Nielsen, J. Med. Chem., 1998, 41,
191.
904
J. Chem. Soc., Perkin Trans. 1, 2001, 900–904