Synthesis and anti-HIV-1 activity of novel bicyclic nucleoside analogues restricted to an S-type conformation

Article abstract of DOI:10.1039/b004774k

(1S,3R,4S)-3-Hydroxyrnethyl-1-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]hepta ne 9 and the corresponding cytosine derivative 10, nucleoside analogues with a novel bicyclic nucleoside structure 3, were synthesized in a few steps from the known 1-(3′-deoxy-β-D-p

Full text of DOI:10.1039/b004774k

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Synthesis and anti-HIV-1 activity of novel bicyclic nucleoside
analogues restricted to an S-type conformation
Lisbet Kværnø,†a Claus Nielsenb and Richard H. Wightman*a
1
a Department of Chemistry, Heriot-Watt University, Riccarton, Edinburgh, UK EH14 4AS
b Retrovirus Laboratory, Department of Virology, Statens Seruminstitut, DK-2300 Copenhagen,
Denmark
Received (in Cambridge, UK) 14th June 2000, Accepted 6th July 2000
Published on the Web 10th August 2000
(1S,3R,4S)-3-Hydroxymethyl-1-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane 9 and the corresponding cytosine
derivative 10, nucleoside analogues with a novel bicyclic nucleoside structure 3, were synthesized in a few steps
from the known 1-(3Ј-deoxy-β-D-psicofuranosyl)uracil 4. NOE experiments verified the bicyclic nucleosides to
be restricted to the expected S-type furanose conformation while the nucleobase is in an anti-conformation. Both
nucleosides proved to be devoid of anti-HIV activity in MT-4 cells, which further supports the hypothesis that
conformational flexibility of the furanose ring in a nucleoside analogue is necessary to obtain both intracellular
5Ј-triphosphorylation and inhibition of HIV-1 reverse transcriptase.
Introduction
The synthesis and evaluation of novel nucleoside analogues,
which, as their triphosphates, can act as inhibitors of HIV 1
reverse transcriptase (HIV-1 RT), continue to be of immense
importance in AIDS therapy.1–3 3Ј-Azido-3Ј-deoxythymidine
(AZT) was the first nucleoside analogue to show effective inhib-
ition of HIV-1 RT4 and, together with another five nucleoside
analogues, AZT is at present approved by the United States
Food and Drug Administration (FDA) for use in the treatment
of AIDS.5,6 To interact successfully with HIV-1 RT, the nucleo-
side analogue has to be triphosphorylated in the 5Ј-position,
which also appears to be the rate-limiting intracellular step
since different known HIV-1 RT inhibitors showed a similar
inhibitory effect when triphosphorylated in vitro.7 When tri-
Fig. 1 Structures of conformationally restricted bicyclic nucleosides.
1 is restricted to an N-type whilst 2 and 3 are restricted to an S-type
phosphorylated, the nucleoside analogue acts either as a com-
petitive inhibitor (i.e., prevents chain incorporation of natural
substrates) or as a substrate, thus causing viral DNA chain
termination since no 3Ј-hydroxy group is present for chain
elongation.8
conformation.
refuted, since conformational analysis of 2 by X-ray and NMR
studies proved the nucleobase to exist in an unusual syn-
conformation13 which might be unfavourable for both intra-
cellular triphosphorylation and/or HIV-1 RT inhibition. We
therefore decided to further investigate the above-mentioned
hypothesis proposed by Marquez et al. by synthesis of bicyclic
pyrimidine furanosides of the type 3 (Fig. 1) expected to be
restricted entirely to an S-type conformation by the additional
3Ј,1Ј-oxymethylene bridge. An additional desirable property of
3 is its presumed stability towards hydrolysis of the C–N glyco-
sylic bond, since formation of a planar oxonium ion is
unfavourable at the bridgehead in such a small bicyclic system.
The conformation of the furanose ring as described by the
pseudorotation cycle9 seems to be of major importance for
anti-HIV activity. It has been proposed that the preference of
AZT for an extreme S-type (3E) conformation is responsible
for both its efficient triphosphorylation and potent anti-HIV
activity,10,11 while later NMR studies of AZT and thymidine
triphosphates when actually bound to HIV-1 RT revealed both
nucleosides to adopt an N-type (4E) conformation.12 Marquez
and co-workers have recently reported the synthesis of two con-
formationally restricted carbocyclic nucleoside analogues 1 and
2 (Fig. 1) locked into an N-type (2E) and an S-type (3E) con-
formation, respectively, and demonstrated that neither of them
showed anti-HIV activity. However, when converted in vitro to
their 5Ј-triphosphates, the triphosphate of the N-type analogue
1 showed an inhibitory effect similar to that of AZT 5Ј-
triphosphate. These results were generalized to the hypothesis
that an S-type conformation is necessary for triphosphoryl-
ation while an N-type conformation is required for successful
interaction with HIV-1 RT.13
Results and discussion
Starting from the known 1-(3Ј-deoxy-β-D-psicofuranosyl)-
uracil 414,15 (Scheme 1), selective protection of the 6Ј-hydroxy
group was a significant obstacle in the synthesis. Even though
4Ј,6Ј-O-1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS) pro-
tection of 4 has been reported in 53% yield,15 later authors were
only able to reproduce this reaction in 24–26% yield.16 Alter-
natively, 4,4Ј-dimethoxytrityl (DMT) protection of the thymine
analogue of 4 was reported to occur selectively at the
6Ј-position in 56% yield,17 in an approach which also left the
secondary hydroxy group at C-4Ј unprotected. Triol 4 showed
only a moderate selectivity when treated with various amounts
of DMTCl in a pyridine–dichloromethane (DCM) mixture at
However, the potential activity of a nucleoside analogue
restricted to an S-type conformation can still not be completely
† Permanent address: Centre for Synthetic Bioorganic Chemistry,
Department of Chemistry, University of Copenhagen, Universitets-
parken 5, DK-2100 Copenhagen, Denmark.
DOI: 10.1039/b004774k
J. Chem. Soc., Perkin Trans. 1, 2000, 2903–2906
This journal is © The Royal Society of Chemistry 2000
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0 ЊC, and the highest yield obtained of the desired 6Ј-O-DMT-
protected isomer 5 was 37%, using 1.1 mole equivalent of
DMTCl. Surprisingly, when treating 4 with more than two mole
equivalents of DMTCl, even the secondary hydroxy group
reacted to afford the tris-DMT-protected isomer in consider-
able yields. This result was the more surprising since the sub-
sequent selective 1Ј-tosylation of 5 using 3 mole equivalents of
TsCl in a pyridine–dichloromethane mixture at room temper-
ature furnished the monotosyl derivative 6 in 77% yield.
Cyclization to the bicyclic structure 8 was accomplished in 93%
yield by treatment with excess of sodium hydride in anhydrous
DMF for 3 days. The reaction was shown to proceed via the
2,1Ј-anhydronucleoside 7, since the latter was the main product,
isolable in 41% yield, after reaction overnight.18 The structure
of 7 was supported by NMR data; in particular, the signals for
C-2 and C-4 (δC 173.1 and 160.4) were similar in chemical shift
to the values we observed (δ 170.6 and 159.1) for 2,3Ј-anhydro-
1-[1,4,6-tri-O-benzoyl-β-D-fructofuranosyl]uracil,14 an inter-
mediate in the synthesis of 4, but more different from the values
typically observed for normal uracil nucleosides (δ ≈ 165 and
151). Additionally, the signals for the diastereotopic protons at
C-1Ј resonated at δ 4.74 and 5.20, deshielded relative to the
equivalent signals (δ 4.15 and 4.20) in the spectrum of 8.
Removal of the DMT protecting group from 8 proceeded in
91% yield using 80% AcOH to give the final uracil analogue 9 as
the first nucleoside of the bicyclic structure 3. Derivatization to
afford the cytosine analogue 10 via a 4-O-p-nitrophenyl inter-
mediate was accomplished by a one-pot method reported by
Reese and co-workers.19 Thus, nucleoside 9 was successively
treated with trimethylsilyl chloride, triflic anhydride and
p-nitrophenol in a mixture of N-methylpyrrolidine and aceto-
nitrile, followed by conc. ammonia in 1,4-dioxane at 50 ЊC, to
give 10 in 80% yield (Scheme 1).
structure in which the furanose ring was locked in an S-type
conformation. NOE difference spectroscopy experiments on 8
revealed one of the hydrogen atoms at C-3Ј to be close to 6-H
(an NOE of 3% after irradiation of the 3Ј-H) and to one of the
hydrogen atoms at C-6Ј (NOEs of 2% for both irradiations).
NOESY spectra of 9 and 10 also confirmed the proximity of
3Јβ-H and the protons at C-6Ј, whilst 3Јα-H interacted with
1Ј-exo-H. HMBC (Heteronuclear Multiple-Bond Connectivity)
spectra of 9 and 10 showing two- and three-bond H–C corre-
lations were another verification of the expected cyclization
since the pairs of atoms 1Ј-H and C-4Ј, and 4Ј-H and C-1Ј,
appeared in each case to be maximally three bonds apart. The
absence of any coupling constant between 4Ј-H and 5Ј-H in
compounds 8–10 also indicated the conformations of the
furanose rings to be exclusively S-type, even though caution
must be taken since this description does not allow for devi-
ations from the N/S two-state model.20
In contrast to the syn conformation of the carbocyclic
nucleoside 2 (Fig. 1), the nucleobases of 9 and 10 were shown to
adopt anti conformations, since significant NOE interactions
were observed in each case between 3Јβ-H and 6-H. Structural
deviations between 3 and naturally occurring nucleosides
should thus be minimized. Both the uracil and the cytosine
nucleoside analogues, 9 and 10 respectively, were tested for
antiviral activity against HIV-1 in MT-4 cells (refer to Experi-
mental section for further details) but the results showed both
compounds to be completely devoid of anti-HIV activity. This
result further supports the hypothesis of Marquez and co-
workers that a flexible furanose conformation is required to
obtain both efficient triphosphorylation (S-type) and inhibition
of HIV-1 Reverse Transcriptase (N-type).13
Conclusions
Conformational analysis of the nucleoside analogues 8–10 by
NOE spectroscopy supported the assumption of a rigid bicyclic
In summary, a successful synthesis of the novel conformation-
ally restricted bicyclic nucleosides 9 and 10 has been accom-
plished. Both were shown by NOE experiments to be confined
to the expected S-type furanose conformation while the base
adopted an anti conformation as also seen for naturally occur-
ring nucleosides. Their complete lack of anti-HIV activity
provides further support to the theory proposed by Marquez
et al.13 about conformational flexibility of the furanose ring
being essential to obtain both efficient triphosphorylation and
HIV-1 RT inhibition.
Experimental
All reagents were obtained from commercial suppliers and were
used without further purification. Light petroleum of distil-
lation range 40–60 ЊC was used and the silica gel (0.035–0.070
mm) used for column chromatography was purchased from
Fischer Chemicals. After column chromatography, fractions
containing product were pooled, evaporated under reduced
pressure, and the residue was maintained overnight under high
vacuum to give the product. NMR spectra were recorded on
Bruker WH 400 or 200 SY spectrometers. 1H Spectra were
obtained at 400 MHz and 13C spectra at 100 MHz, with
assignments based on 2D experiments, unless otherwise stated.
Coupling constants (J) are measured in Hz. Assignments are
in accordance with standard carbohydrate/nucleoside nomen-
clature (i.e. the furanose skeleton numbered 1Ј to 6Ј) even
though the systematic compound names are given according to
IUPAC nomenclature. Fast-atom bombardment and electro-
spray mass spectra (FAB-MS and ES-MS, respectively) were
recorded in positive-ion mode.
1-[3Ј-Deoxy-6Ј-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-psicofuranosyl]-
uracil 5
Scheme 1 Reagents and conditions (and yields): (a) DMTCl (1.1
equiv.), pyridine, DCM (37%); (b) TsCl, pyridine, DCM (77%);
(c) NaH, DMF, 17 h (41% 7) or 3 days (93% 8); (d) 80% AcOH (91%);
(e) (i) TMSCl, N-methylpyrrolidine, acetonitrile; (ii) Tf2O, 0 ЊC;
(iii) p-nitrophenol; (iv) NH3, 1,4-dioxane, 50 ЊC (80%).
A solution of triol 4 (839 mg, 3.25 mmol) in anhydrous pyridine
(50 ml) was concentrated to ca. half volume under reduced
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pressure, anhydrous DCM (30 ml) was added and the mixture
was cooled to 0 ЊC. 4,4Ј-Dimethoxytrityl chloride (DMTCl)
(1.215 g, 3.58 mmol) was added under an atmosphere of nitro-
gen and, after 4 h at 0 ЊC, the mixture was allowed slowly to
warm to room temperature. After 21 h, MeOH (4 ml) and
DCM (100 ml) were added and the mixture was washed succes-
sively with saturated aq. NaHCO3 (40 ml) and water (40 ml).
The combined organic phase was evaporated under reduced
pressure, coevaporated with anhydrous acetonitrile (2 × 30 ml)
and the residue was subjected to silica gel column chromato-
graphy (8.8 × 5.5 cm) using a gradient of pyridine–MeOH–
DCM (0.5:0.5–4:99–95.5 v/v/v) as eluent to give dimethoxy-
trityl derivative 5 (673 mg, 37%) as a yellowish foam, Rf 0.45
(MeOH–DCM 1:9), after coevaporation with acetonitrile
(6 × 15 ml) and DCM (2 × 15 ml); ES-MS m/z 561 [M ϩ H]ϩ,
583 [M ϩ Na]ϩ; FAB-MS m/z 560 [M]ϩ; δH (CDCl3) 9.87 (1H,
br s, NH), 7.86 (1H, d, J 8.2, 6-H), 7.32–7.15 (9H, m, DMT),
6.79 (4H, dd, J 1.3, 8.8, DMT), 5.47 (1H, d, J 8.2, 5-H), 4.45
(1H, m, 5Ј-H), 4.35 (2H, br s, OH), 4.22 (1H, br s, 4Ј-H), 4.06
(1H, d, J 11.2, 1Ј-Ha), 3.76 (6H, s, OMe), 3.58 (1H, d, J 11.2,
1Ј-Hb), 3.15 (1H, dd, J 4.2, 10.6, 6Ј-Ha), 3.05 (1H, dd, J 5.6,
10.4, 6Ј-Hb), 2.82 (1H, d, J 15.2, 3Ј-Ha), 2.71 (1H, dd, J 5.6,
15.2, 3Ј-Hb); δC (CDCl3) 163.89 (C-4), 158.47 (DMT), 150.38
(C-2), 144.10 (DMT), 140.94 (C-6), 135.13, 135.11, 129.78,
129.75, 127.78, 127.70, 126.91, 113.08 (DMT), 100.98 (C-5),
100.10 (C-2Ј), 89.40 (C-5Ј), 86.74 (Ar2PhC), 72.93 (C-4Ј), 64.98
(C-1Ј), 63.25 (C-6Ј), 55.11 (OMe), 44.08 (C-3Ј).
successively with saturated aq. NaHCO3 (20 ml) and water
(20 ml), evaporated to dryness under reduced pressure and
coevaporated with anhydrous acetonitrile (2 × 20 ml). The resi-
due was purified by silica gel column chromatography (9 × 3.3
cm) and elution with a gradient of pyridine–MeOH–DCM
(0.5:2–6:97.5–93.5 v/v/v) to give anhydronucleoside 7 (74 mg,
41%) as a light brownish foam, Rf 0.29 (MeOH–DCM 7:93)
after coevaporation with anhydrous acetonitrile (4 × 15 ml);
ES-MS m/z 543 [M ϩ H]ϩ, 565 [M ϩ Na]ϩ, 606 [M ϩ CH3-
CN ϩ Na]ϩ; FAB-MS m/z 543 [M ϩ H]ϩ; δH (CDCl3; 200
MHz) 7.40–7.20 (10H, m, DMT, 6-H), 6.85 (4H, d, DMT), 5.68
(1H, d, J 7.5, 5-H), 5.28 (1H, br s, 4Ј- or 5Ј-H), 5.20 (1H, d,
J 10.5, 1Ј-Ha), 4.85 (1H, m, 5Ј- or 4Ј-H), 4.74 (1H, d, J 10.5,
1Ј-Hb), 4.30 (1H, br s, OH), 3.80 (6H, s, OMe), 3.45 (1H, dd,
J 3.0, 10.5, 6Ј-Ha), 3.31 (1H, dd, J 2.5, 10.5, 6Ј-Hb), 2.79 (1H,
dd, J 4.3, 14.0, 3Ј-Ha), 2.70 (1H, br d, J 14, 3Ј-Hb); δC [CD3OD–
CDCl3 (1:1 v/v); 50 MHz] 173.09 (C-2), 160.37 (C-4), 158.51
(DMT), 143.78 (DMT), 134.96, 134.68, 134.38 (DMT, C-6),
130.00, 128.04, 127.82, 127.07, 113.07 (DMT), 109.40 (C-5),
98.21 (C-2Ј), 87.67 (C-5Ј), 87.00 (Ar2PhC), 78.80 (C-1Ј), 71.68
(C-4Ј), 63.63 (C-6Ј), 55.02 (OMe), 43.01 (C-3Ј).
(1S,3R,4S)-3-(4,4Ј-Dimethoxytrityloxymethyl)-1-(uracil-1-yl)-
2,5-dioxabicyclo[2.2.1]heptane 8
To a solution of sulfonate 6 (277 mg, 0.388 mmol) in anhydrous
DMF (10 ml) at 0 ЊC under a nitrogen atmosphere was added
NaH [60% (w/w); 62 mg, 1.55 mmol] and the mixture was
stirred at room temperature for 3 days. EtOAc (100 ml) was
added, the mixture was washed with water (2 × 30 ml) and the
organic phase was evaporated to dryness under reduced pres-
sure and coevaporated successively with acetonitrile (50 ml) and
n-hexane (2 × 50 ml). The residue was purified by silica gel
column chromatography (11 × 3.3 cm) with a gradient of
pyridine–MeOH–DCM (0.5:0.5–2:99–97.5 v/v/v) as eluent to
give the bicyclonucleoside 8 (196 mg, 93.3%) as a light yellowish
foam, Rf 0.49 (MeOH–DCM 7:93), after coevaporation with
anhydrous acetonitrile (4 × 15 ml), n-hexane–DCM (1:4;
2 × 15 ml) and DCM (2 × 15 ml); m/z (FAB) 542 [M]ϩ, 543
[M ϩ H]ϩ, 565 [M ϩ Na]ϩ; Found: m/z (FAB) 542.20762 (Mϩ),
543.21302 (MHϩ). Calc. for C31H30N2O7: M, 542.20530. Calc.
for C31H31N2O7: m/z, 543.21313; δH (CDCl3) 9.14 (1H, br s,
NH), 7.55 (1H, d, J 8.2 , 6-H), 7.40–7.20 (9H, m, DMT), 6.84–
6.81 (4H, m, DMT), 5.73 (1H, d, J 8.2, 5-H), 4.48 (1H, d, J 2.2,
4Ј-H), 4.35 (1H, dd, J 4.6, 6.0, 5Ј-H), 4.20 (1H, d, J 7.9, 1Ј-Ha),
4.15 (1H, d, J 7.7, 1Ј-Hb), 3.78 (6H, s, OMe), 3.21 (1H, dd, J 4.5,
10.4, 6Ј-Ha), 3.03 (1H, dd, J 6.4, 10.3, 6Ј-Hb), 2.57 (1H, dd,
J 2.3, 10.0, 3Ј-Ha), 2.22 (1H, d, J 10.1, 3Ј-Hb); δC (CDCl3)
162.85 (C-4), 158.45 (DMT), 149.38 (C-2), 144.23 (DMT),
141.46 (C-6), 135.41, 135.36, 129.84, 127.92, 127.75, 126.83,
113.05 (DMT), 102.49 (C-5), 95.76 (C-2Ј), 86.40 (Ar2PhC),
83.92 (C-5Ј), 77.67 (C-4Ј), 72.97 (C-1Ј), 63.40 (C-6Ј), 55.10
(OMe), 37.19 (C-3Ј).
1-[3Ј-Deoxy-6Ј-O-(4,4Ј-dimethoxytrityl)-1Ј-O-(p-tolylsulfonyl)-
ꢀ-D-psicofuranosyl]uracil 6
The diol 5 (443 mg, 0.790 mmol) was dissolved in anhydrous
pyridine (15 ml) and the solution was concentrated to ca. half
volume under reduced pressure. Anhydrous DCM (25 ml) was
added and the mixture was cooled to 0 ЊC. Toluene-p-sulfonyl
chloride (447 mg, 2.34 mmol) was added and the mixture was
stirred at room temperature under an atmosphere of nitrogen
for 2 days. EtOAc (100 ml) was added and the mixture was
washed successively with saturated aq. NaHCO3 (25 ml) and
water (2 × 25 ml). The organic phase was evaporated to dryness
under reduced pressure, coevaporated with acetonitrile (2 × 40
ml) and the residue was subjected to silica gel column chrom-
atography (12.5 × 3.3 cm) using a gradient of pyridine–MeOH–
DCM (0.5:1–2:98.5–97.5 v/v/v) as eluent to give the tosyl ester
6 (432 mg, 76.5%) as a yellowish foam, Rf (EtOAc–DCM, 3:1)
0.50, after coevaporation with acetonitrile (5 × 15 ml) and
DCM (2 × 15 ml); ES-MS m/z 737 [M ϩ Na]ϩ; FAB-MS m/z
714 [M]ϩ; δH (CDCl3) 8.78 (1H, d, J 1.9, NH), 7.73 (1H, d, J 8.3,
6-H), 7.70 (2H, d, J 8.6, Ts), 7.30–7.17 (11H, m, DMT, Ts), 6.79
(4H, d, J 9.0, DMT), 5.48 (1H, dd, J 2.3, 8.3, 5-H), 4.55 (1H, d,
J 10.7, 1Ј-Ha), 4.43 (1H, d, J 10.7, 1Ј-Hb), 4.30 (1H, br s, 4Ј-H),
4.28 (1H, m, 5Ј-H), 3.76 (6H, s, OMe), 3.21 (1H, dd, J 4.1, 10.7,
6Ј-Ha), 3.15 (1H, dd, J 4.4, 10.7, 6Ј-Hb), 2.74 (1H, dd, J 5.8,
15.3, 3Ј-Ha), 2.58 (1H, dd, J 1.8, 15.1, 3Ј-Hb), 2.41 (3H, s, Ts),
2.32 (1H, d, J 3.4, OH); δC (CDCl3) 163.41 (C-4), 158.67
(DMT), 149.77 (C-2), 145.35 (Ts), 144.19 (DMT), 141.29 (C-6),
135.20, 135.14 (DMT), 132.51 (Ts), 129.91, 129.14, 127.94,
127.85, 127.78, 127.09 (DMT, Ts), 113.2 (DMT), 100.86 (C-5),
96.96 (C-2Ј), 88.67 (C-5Ј), 86.87 (Ar2PhC), 72.54 (C-4Ј), 70.70
(C-1Ј), 63.01 (C-6Ј), 55.25 (OMe), 44.56 (C-3Ј), 21.66 (Ts).
(1S,3R,4S)-3-Hydroxymethyl-1-(uracil-1-yl)-2,5-dioxabicyclo-
[2.2.1]heptane 9
A solution of the dimethoxytrityl derivative 8 (101 mg, 0.186
mmol) in AcOH–water (4:1; 5 ml) was stirred for 40 min, after
which the solution was evaporated to dryness under reduced
pressure and the residue was coevaporated with anhydrous
toluene (2 × 10 ml). The residue was purified by silica gel col-
umn chromatography (7.3 × 3.3 cm) with MeOH–DCM (1:19
to 1:9) as eluent to give the diol 9 (41 mg, 91%) as a white solid,
Rf 0.32 (MeOH in DCM, 1:9); m/z (NH3 CI) 241 [M ϩ H]ϩ,
258 [M ϩ NH4]ϩ; m/z (EI, perfluorotributylamine reference)
240.074597 (Mϩ). Calc. for C10H12N2O5: M, 240.074622;
δH (CD3OD–CDCl3 1:1) 7.79 (1H, d, J 8.2, 6-H), 5.72 (1H, d,
J 8.2, 5-H), 4.51 (1H, d, J4Ј,3Јα 1.8, 4Ј-H), 4.33 (1H, d, J 7.9,
1Ј-endo-H), 4.18 (1H, dd, J 4.7, 6.1, 5Ј-H), 4.14 (1H, d, J 7.9,
1Ј-exo-H), 3.56 (1H, dd, J 4.7, 12.1, 6Ј-Ha), 3.49 (1H, dd, J 6.1,
2,1Ј-Anhydro-1-[3Ј-deoxy-6Ј-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-psico-
furanosyl]uracil 7
A solution of sulfonate 6 (240 mg, 0.336 mmol) in anhydrous
DMF (3 ml) was added to a suspension of NaH [60% (w/w);
45 mg, 1.13 mmol] in anhydrous DMF (5 ml) at 0 ЊC under a
nitrogen atmosphere. After stirring of the mixture at room
temperature for 19 h, water (25 ml) and saturated aq. NaHCO3
(10 ml) were added and the mixture was extracted with EtOAc
(40 ϩ 3 × 20 ml). The combined organic phase was washed
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12.1, 6Ј-Hb), 2.47 (1H, d, J 10.0, 3Јβ-H), 2.43 (1H, dd, J 2.3,
10.0, 3Јα-H); δC (CD3OD–CDCl3 1:1) 165.63 (C-4), 151.39
(C-2), 143.91 (C-6), 102.92 (C-5), 97.19 (C-2Ј), 86.48 (C-5Ј),
78.35 (C-4Ј), 74.16 (C-1Ј), 62.66 (C-6Ј), 37.87 (C-3Ј).
2 h. Cultures were maintained with the test compound for 6
days in parallel with virus-infected control cultures without
compound added. Expression of HIV in the culture medium
was quantitated by HIV-1 antigen detection ELISA.23 Com-
pounds mediating less than 30% reduction of antigen expres-
sion were considered without biological activity.
(1S,3R,4S)-1-(Cytosin-1-yl)-3-hydroxymethyl-2,5-
dioxabicyclo[2.2.1]heptane 10
The uracil derivative 9 (35 mg, 0.146 mmol) was dissolved in
anhydrous acetonitrile (3 ml) containing N-methylpyrrolidine
(0.5 ml, 4.79 mmol) under a nitrogen atmosphere and tri-
methylsilyl chloride (0.10 ml, 0.79 mmol) was added. After 1 h,
the reaction mixture was cooled to 0 ЊC and triflic anhydride
(0.10 ml, 0.59 mmol) was added. After 25 min, additional triflic
anhydride (0.10 ml, 0.59 mmol) was added and, after a total of
70 min, p-nitrophenol (110 mg, 0.79 mmol) was added and the
mixture was stirred at room temperature for 4.5 h. Saturated aq.
NaHCO3 (10 ml) was added and the mixture was extracted with
DCM (3 × 10 ml). The combined organic phase was evaporated
to dryness under reduced pressure and coevaporated with
anhydrous acetonitrile (2 × 10 ml). The residue was dissolved in
1,4-dioxane (5 ml) containing conc. aq. ammonia (1.5 ml) and
the solution was heated in a sealed flask at 50 ЊC for 24 h. The
mixture was evaporated to dryness under reduced pressure,
coevaporated with abs. EtOH (4 × 5 ml) and anhydrous
acetonitrile (10 ml), and the residue was purified by silica
gel column chromatography (11.5 × 2 cm) and elution with
MeOH–DCM (1:9 to 1:4) to give the cytosine analogue 10 (28
mg, 80%) as a light brownish solid, Rf 0.25 (MeOH–DCM 1:4);
m/z (FAB, NBA ϩ PEG300 matrix) 240.0988 (MHϩ). Calc. for
C10H14N3O4: m/z, 240.0984; δH (CD3OD) 7.76 (1H, d, J 7.6,
6-H), 5.84 (1H, d, J 7.6, 5-H), 4.40 (1H, d, J4Ј,3Јα 2.1, 4Ј-H), 4.21
(1H, d, J 7.7, 1Ј-endo-H), 4.06 (1H, t, J ≈ 5.5, 5Ј-H), 4.05 (1H, d,
J 7.8, 1Ј-exo-H), 3.44 (1H, dd, J 4.9, 12.0, 6Ј-Ha), 3.38 (1H, dd,
J 6.2, 12.0, 6Ј-Hb), 2.39 (1H, dd, J 2.4, 10.0, 3Јα-H), 2.29 (1H, d,
J 10.0, 3Јβ-H); δC (CD3OD) 167.51 (C-4), 158.16 (C-2), 144.92
(C-6), 98.15 (C-2Ј), 96.52 (C-5), 86.85 (C-5Ј), 78.66 (C-4Ј),
74.44 (C-1Ј), 63.03 (C-6Ј), 38.04 (C-3Ј).
Acknowledgements
We thank EPSRC for access to facilities at the National Mass
Spectrometry Service Centre, University of Wales, Swansea.
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Virus and cells
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Inhibition of HIV-1 replication
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