Enantioselective syntheses and cytotoxicity of N,O-nucleosides

Article abstract of DOI:10.1021/jm0308186

Enantiomers of 4′-aza-2′,3′-dideoxynucleosides have been prepared by two different synthetic approaches, on the basis of 1,3-dipolar cycloaddition of a chiral nitrone. Cytotoxicity and apoptotic activity have been investigated. (5′S)-5-Fluoro-1-isoxazolid

Full text of DOI:10.1021/jm0308186

3696
J . Med. Chem. 2003, 46, 3696-3702
En a n tioselective Syn th eses a n d Cytotoxicity of N,O-Nu cleosid es
Ugo Chiacchio,*,† Antonino Corsaro,† Daniela Iannazzo,‡ Anna Piperno,‡ Venerando Pistara`,† Antonio Rescifina,†
Roberto Romeo,‡ Vincenza Valveri,§ Antonio Mastino,*,§ and Giovanni Romeo*,‡
Dipartimento di Scienze Chimiche, Universita` di Catania, Viale Andrea Doria 6, Catania 95125, Italy,
Dipartimento Farmaco-Chimico, Universita` di Messina, Via SS. Annunziata, Messina 98168, Italy, and Dipartimento di
Scienze Microbiologiche, Genetiche e Molecolari, Universita` di Messina, Salita Sperone 31, Messina 98168, Italy
Received February 18, 2003
Enantiomers of 4′-aza-2′,3′-dideoxynucleosides have been prepared by two different synthetic
approaches, on the basis of 1,3-dipolar cycloaddition of a chiral nitrone. Cytotoxicity and
apoptotic activity have been investigated. (5′S)-5-Fluoro-1-isoxazolidin-5-yl-1H-pyrimidine-2,4-
dione [(-)-AdFU], while showing low level of cytotoxicity, is a good inductor of apoptosis on
lymphoid and monocytoid cells, acting as a strong potentiator of Fas-induced cell death.
In tr od u ction
The development of novel drugs in the therapy of
infections caused by viruses and also in the treatment
of certain neoplastic diseases has been remarkably
exploited in these last 10 years.1 In this context,
unnatural nucleoside analogues have emerged as major
therapeutic agents: since the discovery that nucleoside
analogues can effectively protect cells from the lethal
action of some viruses, including the human immuno-
deficiency virus (HIV), herpes simplex virus, hepatitis
C virus, and Cytomegalovirus, several reports have
appeared concerning their synthesis, therapeutic ap-
plications, and mechanism of action.2
In the search for effective, selective, and nontoxic
agents, a variety of strategies has been devised to design
nucleoside analogues. These strategies have involved
several structural modifications of the naturally occur-
ring nucleosides at the level of the sugar moiety and/or
the heterocyclic base. A series of new compounds,
endowed with a relevant biological activity, can be
originated from alteration of the carbohydrate frag-
ment: in this context, the design of novel “ribose” rings
has resulted in the discovery of effective biological
agents with lower toxicity and higher biological effects.
Promising results have been in fact obtained from a new
F igu r e 1. Isoxazolidinyl nucleobases.
generation of nucleoside analogues in which the fura-
nose ring has been replaced by an alternative carbo- or
heterocyclic rings.3,4 Uracil, thymine, cytosine, adenine,
and guanine nucleosides 1 possessing an isoxazolidine
moiety have been recently synthesized, as racemic
mixtures, to investigate their pharmacological activi-
ties;5 in particular (()-AdT has been reported to inhibit
HIV replication in C 8166, with an activity inversely
related to the multeplicity of infection used5c (Figure 1).
To our knowledge, however, no data about the toxicity
of this compound have so far appeared in the literature.
determining the physiological activity of these molecules
suggests the synthesis of enantiomerically pure N,O-
nucleosides in order to investigate their biological
features.
As part of our continuing efforts to develop novel
strategies for preparing heterocyclic nucleosides, we
have focused our attention on the applicability of 1,3-
dipolar cycloadditions of nitrones to the synthesis of
isoxazolidinyl nucleosides 1, unsubstituted at the ni-
trogen atom, in an enantiomeric pure form. Nitrones
containing a chiral auxiliary on the nitrogen atom have
been selected as the most convenient precursors: our
preliminary results have described the synthesis of
enantiomerically pure AdT and AdFU.6 Accordingly, we
report here the extension of the synthetic approach7 to
the asymmetric synthesis of purine and pyrimidine N,O-
nucleosides AdU, AdC, AdFC, and AdA (Figure 1).
However, the consideration that both enantiomeric
purity and absolute configuration are key factors in
* To whom correspondence should be addressed. U. Chiacchio:
E-mail uchiacchio@dipchi.unict.it. Phone +39 095 738 50 14.
† Universita` di Catania.
‡ Dipartimento Farmaco-Chimico, Universita` di Messina.
§ Dipartimento di Scienze Microbiologiche, Genetiche e Molecolari,
Universita` di Messina.
10.1021/jm0308186 CCC: $25.00 © 2003 American Chemical Society
Published on Web 07/04/2003

Synthesis and Cytotoxicity of N,O-Nucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3697
Sch em e 1
Sch em e 2
In particular, we exploit in this paper the use of a
series of vinyl nucleobases8 as dipolarophiles for a one-
pot reaction pathway toward enantiomerically pure 4′-
aza-2′,3′-dideoxyfuranosyl nucleosides. This synthetic
approach has been compared with the two-step meth-
odology based on the Vorbru¨ggen nucleosidation reac-
tion.
The obtained compounds have been tested for an
evaluation of their toxicity and as inductors of apoptosis.
barrier for the nitrogen inversion is 13.9 kcal/mol: a
value of 16.2 kcal/mol is reported for similar systems.11
Resu lts a n d Discu ssion
Ch em istr y. The chiral nitrone 3, as a not isolated
intermediate, was prepared from D-ribose in four steps.7a
Thus, D-ribose was converted into the 5-O-tert-butyl-
diphenylsilyl-2,3-O-isopropylidene-D-ribofuranose, pro-
tected at the CH2OH group, and reacted with hydrox-
ylamine to give the ribosyl hydroxylamine 2; further
reaction of 2 with formaldehyde and vinyl bases 4,
performed in CHCl3 at 60 °C for 12 h, afforded regiose-
lectively, through unisolated intermediate nitrone 3, a
mixture of two homochiral isoxazolidines 5 and 6,
epimeric at C5′, in a relative ratio 1.5:1 (40% yield)
(Scheme 1).
Purification by HPLC chromatography allowed the
isolation of pure adducts 5a -f and 6a -f. All structures
described were determined by 1H and 13C NMR analysis.
In particular, the stereochemistry of the obtained ad-
ducts was assessed by NOE measurements: in fact, the
positive NOE effect observed for protons 4′′ when
irradiating 1′′, indicates a cis relationship between these
protons. These data confirm that the sugar moiety has
a â configuration in all the nucleosides.
On the basis of the marked preference for the trans
form, the obtained anomers 5 and 6 can be considered
as analogues of R-nucleosides. Thus, the difference of
configuration of their C5′ atoms is compensated by the
nitrogen inversion, and both anomers possess the same
trans disposition of their N2′ and C5′ substituents.
The relative configuration at N2′ and C5′ for nucleo-
sides 5 and 6 has been tentatively assigned as reported
in Scheme 1, according to quantomechanical calcula-
tions. The major stereoisomers 5a -f possess the con-
figuration (5′R) which is more stable than the configu-
ration (5′S) of about 0.8 kcal/mol: this value is in
agreement with the observed R/â ratio.
The synthetic approach toward homochiral N,O-
nucleosides 7 and 8 has been completed by selective
cleavage of the sugar moiety, performed by treatment
with 1.5% aqueous HCl (Scheme 2). Thus, both anomers
7a -f and 8a -f have been obtained with a global yield
of 30% and 10%, respectively, starting from the nitrone
3.
We have recently reported a different synthetic ap-
proach to enantiomerically pure isoxazolidinyl nucleo-
sides 7b,c and 8b,c, based on the 1,3-dipolar cycload-
dition of the transient nitrone 3 with vinyl acetate,
followed by the Vorbru¨ggen nucleosidation with si-
lylated nucleobases.6
Mixtures of diastereomeric invertomers could be
observed for compounds 5 and 6. Variable temperature
NMR measurements, performed until -80 °C, show the
presence of only one set of resonances, suggesting the
existence of only one isomer or a nitrogen inversion
sufficiently fast to impart time-averaged properties to
the observed compounds.
PM39 and AM110 quantomechanical calculations in-
dicate that the N2′-C5′ trans isomers are more stable
than the cis derivatives. Thus, for compound 5c, chosen
as a model compound, an energy difference of 4.3 kcal/
mol was calculated in favor of the trans isomer: this
barrier sufficiently explains the fact that experimentally
only one invertomer was formed. The calculated energy
We have here compared two procedures. Ribosyl
hydroxylamine 2 has been reacted with formaldehyde
and vinyl acetate to afford a mixture of two homochiral
isoxazolidines 9 and 10, epimeric at C5, in a relative
ratio 1.5:1 (90% yield).6 The subsequent coupling with
silylated nucleobases 11a -f, in the presence of TMSOTf
at 0 °C, occurred with a 80% yield and led to nucleosides
5 and 6 (≈1.4:1 ratio), which have been separated by

3698 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Chiacchio et al.
Sch em e 3
Ta ble 1. Evaluation of Cytotoxicity of (-)-AdFU in Lymphoid
(U937) cell lines assayed. However, (-)-AdFU was
shown to specifically induce remarkable levels of apop-
tosis, in Molt-3 and U937 cells. In particular, the
lymphoid cell line was more sensitive to AdFU-induced
apoptosis than the monocytoid cells. In some experi-
ments, induction of apoptosis in Molt-3 and U937 cells
was confirmed by flow cytometry analysis following
propidium iodide staining. Two other monocytoid cell
lines tested showed no sensitivity to AdFU-induced
cytotoxicity.
and Monocytoid Cell Linesa
24 h
48 h
72 h
CC50
(µM)
cell
line
CC50
AC50
CC50
(µM)
AC50
(µM)
AC50
(µM)
(µM)b
(µM)b
Molt-3c
U937
HL-60
THP-1
>1000
>1000
>1000
>1000
329
>1000
>1000
>1000
806
>1000
>1000
N.D.
96
366
687
13
3
N.D.
N.D.
123
>1000
N.D.
N.D.d
N.D.
a
Cells were exposed in optimal culture conditions to concentra-
tions of AdFU ranging from 8 µM to 512 µM or control medium
The kinetic of the cytotoxic activity of (-)-AdFU
clearly suggests that apoptosis is the exclusive form of
cell death induced by this drug in lymphoid and mono-
cytoid cells. Actually, even cytotoxity detected by the
trypan blue test at 72 h can be reasonably attributable
to late apoptosis, rather than to other forms of cell
death.
b
for the times indicated. Cytotoxic concentration 50 (CC50) and
apoptotic concentration 50 (AC50) are the concentrations of the
drug required to cause 50% toxicity, detected by trypan blue
exclusion test, and 50% apoptosis, detected by microscopy analysis,
respectively. Each value is determined from two or more experi-
ments. c Values determined using 5-fluorouracil, in Molt-3 cells,
were as follows: 24 h, CC50 > 1000, AC50 ) 28; 48 h, CC50 ) 718,
d
AC50 ) 16; 72 h, CC50 ) 14, AC50 ) 15. Not done.
Moreover, values determined using the free base
5-fluorouracil in Molt-3 cells in parallel, for comparative
purposes, revealed a different profile of potency and
kinetic of cell death induction, suggesting that (-)-
ADFU activity is not due simply to release of 5-FU by
the (-)-ADFU molecule.
On the basis of these results, we have further inves-
tigated the capacity of (-)-AdFU to modulate apoptosis
in Molt-3 lymphoid cells. First, interaction with Fas-
induced apoptosis was investigated. As shown in Figure
2, we found that (-)-AdFU had remarkable Fas-
potentiating activity. Infact, (-)-AdFU at concentrations
which induced low levels of apoptosis was able to
enhance apoptotic cell death triggered by anti-Fas added
to the cells at suboptimal concentrations.
flash chromatography and then by HPLC (Scheme 3).
This reaction pathway showed a global yield of 72%.
Cytotoxicity Assa ys. One of the major limitations
in the use of nucleoside analogues as antiviral drugs is
their toxicity. However, compounds exhibiting reason-
ably low levels of cytotoxicity, preferentially directed
toward the cell types targeted by viral agents causing
chronic infection, could exploit this characteristic in
order to improve their specific antiviral activity also by
eliminating infected cells. For this reason, we tested the
cytotoxicity of the new synthesized compounds using a
conventional viability assay, such as the trypan blue
exclusion test, in cell lines of lymphoid and monocytoid
origin.
Moreover, considering the pivotal role of apoptosis as
a mechanism, triggered by cellular signals as well as
by a variety of drugs, for the controlled removal of dead
cells, we assayed the ability of (-)-AdFU 8b, chosen as
a model compound, to specifically induce apoptosis,
detected by microscopy analysis. Results, expressed as
cytotoxic concentration 50 (CC50, concentration that
causes 50% toxicity by trypan blue) and apoptotic
concentration 50 (AC50, concentration required to cause
apoptosis in 50% of treated cells), respectively, are
reported in Table 1.
To obtain further information on the mechanisms
regulating AdFU-induced apoptosis, the possible in-
volvement of the caspase cascade was investigated using
two approaches. The first approach consisted of adding
synthetic, cell-permeable, noncleavable peptide ana-
logues of caspase substrates to AdFU-treated cultures
to irreversibly inhibit protease activity. Z-VAD-FMK,
pan-caspase, Z-DEVD-FMK, caspase-3 specific, and
Z-LEHD-FMK, caspase-9 specific, inhibitors all signifi-
cantly reduced apoptosis of infected cultures in com-
parison with control cultures (data not shown). More-
over, the level of caspase-8 enzymatic activity was
determined, using a colorimetric assay able to specifi-
cally detect substrate cleavage. Comparison at 24 h of
No significant toxicity, when evaluated using the
classical trypan blue test, was detected until 3 days of
treatment in the lymphoid (Molt-3) and monocytoid

Synthesis and Cytotoxicity of N,O-Nucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3699
The two-step procedure, based on the 1,3-dipolar cy-
cloaddition of the chiral nitrone 4 with vinyl acetate and
the subsequent Vorbru¨ggen nucleosidation, leads to
improved yields.
The cytotoxicity and the apoptotic activity of the
obtained compounds have been investigated. The ob-
tained data indicate that AdFU, while presenting low
levels of cytotoxicity assessed by a conventional assay
to detect viability, such as the trypan blue exclusion test,
is, conversely, a good inductor of cell death by apoptosis
on lymphoid and, less efficiently, monocytoid cells. More
important, AdFU seems to act as a strong potentiator
of Fas-induced cell death, opening new perspectives in
future investigations on its possible use as a therapeutic
agent.
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Kofler
apparatus and are uncorrected. Elemental analyses were
performed with a Perkin-Elmer elemental analyzer. NMR
spectra were recorded on a Varian instrument at 200 or 500
MHz (1H) and at 50 or 125 MHz (13C) using deuteriochloroform
or deuterated methanol as solvent; chemical shifts are given
in ppm from TMS as internal standard. Thin-layer chromato-
graphic separations were performed on Merck silica gel 60-
F
254 precoated aluminum plates. Preparative separations were
carried out by column and flash chromatography using Merck
silica gel 0.063-0.200 mm and 0.035-0.070 mm, respectively,
with chloroform-methanol mixtures as eluents. HPLC puri-
fications were made with a preparative column (Microsorb
Dynamax 100 Å, 21.4 × 250 mm). The purity of all homochiral
compounds has been tested with a Nucleosil Chiral-2, 4 × 250
mm column with mixtures of n-hexane-2-propanol as eluents.
The identification of samples from different experiments was
secured by mixed mps and superimposable NMR spectra.
Sta r tin g Ma ter ia ls. Vinyl bases 4a -f were prepared by
literature methods.8 Formaldehyde, vinyl acetate, uracil,
5-fluorouracil, thymine, cytosine, 5-fluorocytosine, adenine,
and D-ribose were purchased from Aldrich Co. All solvents
were dried according to literature methods.
F igu r e 2. Apoptosis modulation by (-)-AdFU in Molt-3
lymphoid cells. Combination treatment of cell with (-)-AdFU
plus anti-Fas (CH11); Molt-3 cells were incubated with 25, 50,
or 100 ng/mL anti-Fas, either alone or immediately after cells
had been exposed to (-)-AdFU at the concentrations 32 or
64 µM.
Meth od A. Gen er a l P r oced u r e for 1,3-Dip ola r Cyclo-
a d d ition Rea ction s betw een Vin yl Ba ses 4a -f a n d Va -
sella -Typ e Nitr on e 3. A suspension containing vinylbase
4a -f (1 equiv), ribosyl hydroxylamine 2 (1 equiv), and form-
aldehyde (1 equiv) in chloroform (10 mL) was heated in a
sealed vessel at 60 °C under stirring, until the hydroxylamine
was consumed (7-8 h); after this time, 0.5 equiv of hydroxyl-
amine and formaldehyde were added, and the mixture was
left to react for additional 4 h. Removal of the solvent in a
vacuum affords a crude material which was purified by flash
chromatography to give a mixture of homochiral isoxazolidines
5a -f and 6a -f, which after separation by HPLC (2-propanol/
n-hexane) show physical and spectral data listed below.
Meth od B. Gen er a l P r oced u r e for th e Rea ction be-
tw een Silyla ted Ba ses 11a -f a n d Isoxa zolid in es 9, 10. A
suspension of bases 11a -f (0.62 mmol) in dry acetonitrile (3
mL) was treated with bis(trimethylsilyl)acetamide (BSA) (2.48
mmol; 1.24 mmol for 11f) and refluxed for 15 min under
stirring; for 11f, BSA was added at rt, and the suspension was
stirred for 24 h at rt. To the clear solution obtained were added
dropwise a solution of the epimeric isoxazolidines 9, 106 (0.52
mmol; 0.31 mmol for 11f) in dry acetonitrile (3 mL) and
trimethylsilyl triflate (TMSOTf) (0.78 mmol; 0.06 mmol for
11f), and the reaction mixture was refluxed for 1 h; for 11f,
TMSOTf was added at rt, and the solution was stirred for 6 h
at rt. After being cooled at 0 °C, the solution was neutralized
by careful addition of aqueous 5% sodium bicarbonate and then
concentrated in vacuo. After addition of dichloromethane
(8 mL), the organic phase was separated, washed with water
(2 × 10 mL), dried over sodium sulfate, filtered, and evapo-
rated to dryness. The residue was purified by flash chroma-
F igu r e 3. Dose-dependent activity of caspase-8 by (-)-AdFU;
results are expressed as fold induction of caspase-8 activity
from samples of (-)-AdFU-treated cells at the concentrations
64, 128, or 256 µM with respect to control cells exposed to
medium alone and cultured without (-)-AdFU. Mean values
( SD obtained from three replicate determinations are re-
ported
the absorbance from samples of (-)-AdFU-treated cells
with samples from untreated controls showed a signifi-
cant, dose-dependent increase in protease activity fol-
lowing the treatment (Figure 3). Thus, the involvement
of the caspase-cascade, including apical caspases, was
clearly demonstrated in (-)-AdFU-induced apoptosis.
Con clu sion s
Enantiomers of 4′-aza-2′,3′-dideoxynucleosides have
been prepared by two different synthetic approaches.

3700 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
Chiacchio et al.
tography and then by HPLC (2-propanol/n-hexane) to give the
homochiral isoxazolidines 5a -f and 6a -f.
64.29, 81.45, 82.89, 85.60, 86.20, 93.42, 99.69, 113.25, 127.76,
129.86, 133.08, 133.13, 135.27, 141.14, 155.67, 165.60. Anal.
(C31H40N4O6Si) C, H, N.
(5′S)-4-Am in o-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-b u t yl-d i-
p h en yl-sila n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o-
[3,4-d ][1,3]d ioxol-4-yl]-isoxa zolid in -5-yl}-1H-p yr im id in -
(5′R)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-1H -p yr im id in e-2,4-d ion e
(5a ). Yield 24% (method A), 42% (method B); [R]25 ) +11.66
D
2-on e (6d ). Yield 18% (method A), 28% (method B); [R]25
)
(c 0.6, CHCl3); mp 82-84 °C, white solid from ethyl acetate.
1H NMR (200 MHz, CDCl3): δ 1.07 (s, 9H), 1.35 (s, 3H), 1.53
(s, 3H), 2.17-2.22 (m, 1H, H4′b), 2.76-2.80 (m, 1H, H4′a), 3.04
(dddd, 1H, J ) 1.5, 6.5, 8.5, 10.0 Hz, H3′a), 3.16 (dddd, 1H,
J ) 2.0, 5.5, 8.5, 10.5 Hz, H3′b), 3.71 (m, 2H, H5′′a, H 5′′b), 4.31
(dt, J ) 1.5, 6 Hz, H 4′′), 4.62 (d, 1H, J ) 1.5 Hz, H1′′), 4.69-
4.72 (m, 2H, H2′′, H3′′), 5.59 (d, J ) 8.5 Hz, H5), 6.22 (dd, 1H,
J ) 3.7 Hz, H5′), 7.37-7.44 (m, 5H, aromatic protons), 7.45 (d,
J ) 8.5 Hz, H6), 7.47-7.67 (m, 5H, aromatic protons), 8.66
(bs, 1H, NH); 13C NMR (50 MHz, CDCl3): δ 25.13, 26.80, 26.85,
36.34, 48.67, 64.23, 81.67, 83.02, 84.30, 86.37, 100.02, 102.22,
113.30, 127.77, 127.81, 129.93, 132.99, 133.06, 135.50, 135.54,
139.71, 150.00, 162.94. Anal. (C31H39N3O7Si) C, H, N.
D
+4.96 (c 0.3, CHCl3); pale yellow oil. 1H NMR (200 MHz,
CDCl3): δ 1.06 (s, 9H), 1.35 (s, 3H), 1.54 (s, 3H), 2.24 (dddd,
1H, J ) 3.4, 5.7, 8.5, 13.5 Hz, H4′a), 2.82 (m, 1H, H4′b), 3.09
(m, 2H, H3′), 3.70 (dd, 1H, J ) 7.0, 10.5 Hz, H5′′a), 3.74 (dd,
1H, J ) 5.5, 10.5 Hz, H5′′b), 4.27 (ddd, 1H, J ) 2.5, 6.5, 7.0 Hz,
H
4′′), 4.60 (dd, 1H, J ) 2.5, 6.5 Hz, H3′′), 4.67 (dd, 1H, J ) 2.5,
6.0 Hz, H2′′), 4.79 (d, 1H, J ) 2.5 Hz, H1′′), 5.49 (d, 1H, J ) 7.0
Hz, H5), 5.97 (dd, 1H, J ) 3.0, 7.0 Hz, H5′), 7.37-7.44 (m, 5H,
aromatic protons and H6); 13C NMR (50 MHz, CDCl3): δ 19.22,
25.40, 26.81, 27.15, 29.68, 37.56, 47.99, 64.02, 81.51, 82.77,
85.52, 85.82, 93.16, 99.54, 113.28, 127.77, 129.86, 135.61,
141.38, 155.42, 165.43. Anal. (C31H40N4O6Si) C, H, N.
(5′S)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-1H -p yr im id in e-2,4-d ion e
(5′R)-4-Am in o-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-b u t yl-d i-
p h en yl-sila n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o-
[3,4-d ][1,3]d ioxol-4-yl]-isoxa zolid in -5-yl}-5-flu or o-1H-p y-
(6a ). Yield 16% (method A), 30% (method B); [R]25 ) -29.9
D
r im id in -2-on e (5e). Yield 37%; [R]25 ) +117.65 (c 1.4,
D
1
(c 0.6, CHCl3); mp 68-70 °C, white solid from ethyl acetate.
1H NMR (200 MHz, CDCl3): δ 1.07 (s, 9H), 1.34 (s, 3H), 1.54
(s, 3H), 2.19-2.25 (m, 1H, H4′b), 2.73-2.80 (m, 1H, H4′a), 3.14-
3.17 (m, 2H, H3′a, H3′b), 3.70-3.74 (m, 2H, H5′′a, H5′′b), 4.26 (dt,
J ) 3.0, 6.0 Hz, H4′′), 4.58 (dd, 1H, J ) 4, 6.0 Hz, H3′′), 4.68
(dd, 1H, J ) 2.5, 6.5 Hz, H2′′), 4.81 (d, 1H, J ) 2.5 Hz, H1′′),
5.61 (d, 1H, J ) 8.5 Hz, H5), 5.94 (dd, J ) 3.0, 7.5 Hz, H5′),
7.36-7.46 (m, 5H, aromatic protons), 7.59 (d, 1H, J ) 8.5 Hz,
H6), 7.65-7.68 (m, 5H, aromatic protons), 8.54 (bs, 1H, NH);
13C NMR (50 MHz, CDCl3): δ 25.38, 26.78, 27.17, 37.10, 48.15,
64.08, 81.23, 82.77, 84.33, 85.98, 99.02, 102.16, 113.38, 127.75,
127.77, 129.85, 129.85, 129.91, 133.13, 133.20, 135.56, 139.99,
149.91, 163.00. Anal. (C31H39N3O7Si) C, H, N.
CHCl3); amorphous solid. H NMR (200 MHz, CDCl3): δ 1.06
(s, 9H), 1.35 (s, 3H), 1.53 (s, 3H), 2.21-2.26 (m, 1H, H4′b), 2.75-
2.82 (m, 1H, H4′a), 3.01-3.12 (m, 2H, H3′a, H3′b), 3.72 (ddd, 2H,
J ) 6, 11 Hz, H5′′a, H5′′b), 4.28 (t, 1H, J ) 6 Hz, H4′′), 4.61 (s,
1H, H1′′), 4.71 (s, 2H, H2′′, H3′′), 6.14 (dd, 1H, J ) 2.5, 8 Hz,
H5′), 7.37-7.39 (m, 5H, aromatics H),7.41 (d, 1H, J ) 7.5 Hz,
H6), 7.58-7.71 (m, 5H, aromatics H). 13C NMR (50 MHz,
CDCl3): δ 25.13, 25.32, 26.78, 36.54, 47.74, 64.17, 81.41, 82.90,
85.63, 85.21, 113.29, 127.75, 129.87, 135.54, 138.71, 153.74,
157.82, 158.10. Anal. (C31H39FN4O6Si) C, H, N.
(5′S)-4-Am in o-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-b u t yl-d i-
p h en yl-sila n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o-
[3,4-d ][1,3]d ioxol-4-yl]-isoxa zolid in -5-yl}-5-flu or o-1H-p y-
(5′R)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-5-flu or o-1H -p yr im id in e-
2,4-d ion e (5b). Yield 23% (method A), 46% (method B).6
Physical and spectroscopic data are identical to these previ-
ously reported.6
(5′S)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-5-flu or o-1H -p yr im id in e-
2,4-d ion e (6b). Yield 17% (method A), 26% (method B).6
Physical and spectroscopic data are identical to these previ-
ously reported.6
r im id in -2-on e (6e). Yield 18% (method A), 34% (method B);
1
[R]25 ) -47.3 (c 2.5, CHCl3); amorphous solid. H NMR (200
D
MHz, CDCl3): δ 1.06 (s, 9H), 1.35 (s, 3H), 1.54 (s, 3H), 2.23-
2.28 (m, 1H, H4′b), 3.07-3.17 (m, 1H, H4′a), 3.67-3.76 (m, 2H,
H
H
H
5′′a, H5′′b), 4.20-4.27 (m, 1H, H4′′), 4.59 (dd, 1H, J ) 3, 6 Hz,
3′′), 4.69 (dd, 1H, J ) 2.5, 6 Hz, H2′′), 4.81 (d, 1H, J ) 2.5 Hz,
1′′), 5.93 (dd, 1H, J ) 4.5, 8 Hz, H5′), 7.36-7.44 (m, 5H,
aromatics H), 7.61-7.66 (m, 5H, aromatics H), 7.72 (d, 1H,
J ) 6 Hz, H6); 13C NMR (50 MHz, CDCl3): δ 25.42, 26.82,
27.20, 26.68, 37.40, 47.74, 64.07, 81.39, 82.71, 85.54, 85.82,
99.22, 113.36, 127.74, 129.85, 133.19, 135.57, 153.62, 157.48,
157.74. Anal. (C31H39FN4O6Si) C, H, N.
(5′R)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-5-m eth yl-1H-p yr im id in e-
2,4-d ion e (5c). Yield 25% (method A), 42% (method B).6
Physical and spectroscopic data are identical to these previ-
ously reported.6
(5′S)-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-5-m eth yl-1H-p yr im id in e-
2,4-d ion e (6c). Yield 15% (method A), 30% (method B).6
Physical and spectroscopic data are identical to these previ-
ously reported.6
(5′R)-9-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
d ioxol-4-yl]-isoxa zolid in -5-yl}-9H-p u r in -6-yla m in e (5f).
1
Yield 29%; [R]25 ) +83.7 (c 0.9, CHCl3); amorphous solid. H
D
NMR (200 MHz, CDCl3): δ 0.92 (s, 9H), 1.34 (s, 3H), 1.53 (s,
3H), 2.60-2.75 (m, 1H, H4′b), 2.88-3.03 (m, 1H, H4′a), 3.23-
3.35 (m, 1H, H3′a), 3.37-3.51 (m, 1H, H3′b), 3.65-3.77 (m, 2H,
H
5′′a, H5′′b), 4.23 (dt, J ) 1.5, 4.5 Hz, H 4′′), 4.70 (d, 1H, J ) 2
Hz, H1′′), 4.60-4.73 (m, 2H, H2′′, H3′′), 5.97 (bs, 2H, NH2), 6.47
(dd, J ) 2.4, 7.2 Hz, H5′), 7.39-7.46 (m, 5H, aromatics H),
7.47-7.68 (m, 5H, aromatics H), 8.07 (s, 1H, H2), 8.34 (s, 1H,
H8); 13C NMR (50 MHz, CDCl3): δ 25.08, 25.80, 26.82, 35.68,
48.35, 63.52, 80.82, 82.95, 83.05, 86.51, 99.70, 113.26, 127.77,
129.93, 132.99,145,22, 153.48, 155.47. Anal. (C32H40N6O5Si) C,
H, N.
(5′R)-4-Am in o-1-{2-[(3a R,4R,6R,6a S)-6-(ter t-b u t yl-d i-
p h en yl-sila n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o-
[3,4-d ][1,3]d ioxol-4-yl]-isoxa zolid in -5-yl}-1H-p yr im id in -
2-on e (5d ). Yield 22% (method A), 44% (method B); [R]25
)
(5′S)-9-{2-[(3a R,4R,6R,6a S)-6-(ter t-Bu t yl-d ip h en yl-si-
la n yloxym eth yl)-2,2-d im eth yl-tetr a h yd r o-fu r o[3,4-d ][1,3]-
D
-14.6 (c 1.02, CHCl3); mp 80-82 °C, yellow solid from ethyl
acetate. 1H NMR (200 MHz, CDCl3): δ 1.06 (s, 9H), 1.35 (s,
3H), 1.53 (s, 3H), 2.23 (dddd, 1H, J ) 1.5, 2.5, 7.0, 13.5 Hz,
d ioxol-4-yl]-isoxa zolid in -5-yl}-9H-p u r in -6-yla m in e (6f).
1
Yield 25%; [R]25 ) -49.5 (c 1.1, CHCl3); amorphous solid. H
D
H
H
4′a), 2.79 (dq, 1H, J ) 6.0, 7.0 Hz, H4′b), 3.07 (m, 2H, H3′a
,
NMR (200 MHz, CDCl3): δ 0.90 (s, 9H), 1.32 (s, 3H), 1.54 (s,
3H), 2.63-2.73 (m, 1H, H4′b), 2.93-3.02 (m, 1H, H4′a), 3.24-
3.34 (m, 2H, H3′a), 3.37-3.55 (m, 1H, H3′b), 4.13-4.26 (m, 2H,
3′b), 3.72 (ddd, 2H, J ) 6.0, 7.0, 11.0, Hz, H5′′), 4.28 (dt, 1H,
J ) 1.5, 5.5 Hz, H4′′), 4.65 (d, 1H, J ) 2.0 Hz, H1′′), 4.71 (m,
2H, H2′′ and H3′′), 5.53 (d, 1H, J ) 7.0 Hz, H5), 6.21 (dd, 1H,
J ) 2.5, 7.0 Hz, H5′), 7.36-7.44 (m, 5H, aromatic protons), 7.56
(d, 1H, J ) 7.0 Hz, H6), 7.64-7.66 (m, 5H, aromatic protons);
13C NMR (50 MHz, CDCl3): δ 19.21, 25.17, 26.79, 36.63, 47.81,
H
H
5′′a, H5′′b), 4.21 (dt, J ) 2, 5 Hz, H4′′), 4.43-4.68 (m, 2H, H2′′,
3′′), 4.85 (d, 1H, J ) 2.6 Hz, H1′′), 6.24 (bs, 2H, NH2), 6.43
(dd, J ) 2.6, 7.4 Hz, H5′), 7.36-7.46 (m, 5H, aromatics H),
7.65-7.68 (m, 5H, aromatics H), 8.02 (s, 1H, H2), 8.29 (s, 1H,

Synthesis and Cytotoxicity of N,O-Nucleosides
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17 3701
(5′R)-9-Isoxa zolid in -5-yl-9H-p u r in -6-yla m in e (7f). Yield
H8); 13C NMR (50 MHz, CDCl3): δ 25.34, 26.79, 27.17, 36.60,
48.67, 63.50, 81.52, 82.84, 83.33, 85.99, 99.84, 113.04, 127.75,
129.85, 133.13, 145.43, 153.12, 155.51. Anal. (C32H40N6O5Si)
C, H, N.
71%; [R]25 ) +24.6 (c 1.4, CHCl3); amorphous solid. Anal.
D
(C8H10N6O) C, H, N. Physical and spectroscopic data are
identical to these previously reported.
(5′S)-9-Isoxa zolid in -5-yl-9H-p u r in -6-yla m in e (8f). Yield
Gen er a l P r oced u r e for Hyd r olysis of Hom och ir a l
Isoxa zolid in es 5a -f a n d 6a -f. Isoxazolidines 5a -f and
6a -f were dissolved in a 1.5% (weight by weight) HCl solution
in EtOH (2.5 mL), and the reaction mixture was stirred at
room temperature for 3 h. The solution was brought to pH 10
by adding aqueous 10% sodium carbonate and extracted with
dichloromethane (2 × 10 mL). The organic phase, dried over
sodium sulfate, filtered, and evaporated to dryness. The
residue was purified by radial chromatography (chloroform/
methanol 9:1) to furnish homochiral N,O-nucleosides 7a -f and
8a -f.
69%; [R]25 ) -21.4 (c 1.4, CHCl3); amorphous solid. Anal.
D
(C8H10N6O) C, H, N. Physical and spectroscopic data are
identical to these previously reported.
Eva lu a tion of Toxicity a n d Ap op tosis. Toxicity was
evaluated by a standard viability assay, using the trypan blue
exclusion test. Normally, apoptosis was evaluated by morpho-
logical analysis of the cells, performed following staining with
acridine orange as previously described.12 Briefly, over 600
cells, including those showing typical apoptotic characteristics,
were counted using a fluorescence microscope. The identifica-
tion of apoptotic cells was based on the presence of uniformly
stained nuclei showing chromatin condensation and nuclear
fragmentation. In some experiments, apoptosis was detected
by flow cytometry analysis of isolated nuclei, following staining
(5′R)-1-Isoxa zolid in -5-yl-1H-p yr im id in e-2,4-d ion e (7a ).
Yield 91%; [R]25 ) +97.5 (c 0.19; CH3OH); mp 183-185 °C,
D
white solid from methanol. 1H NMR (200 MHz, CDCl3): δ 2.62
(m, 1H, H2′b), 3.12 (m, 1H, H3′a), 3.87 (m, 1H, H3′b), 5.82 (d,
1H, J ) 7.5 Hz, H5), 6.19 (dd, 1H, J ) 3.1,7.9 Hz, H5′), 7.41 (d,
1H, J ) 7.5 Hz, H6), 10.28 (bs, 1H, NH); 13C NMR (50 MHz,
CDCl3): δ 30.98, 47.34, 85.52, 100.80, 102.18, 151.06, 164.22.
Anal. (C7H9N3O3) C, H, N.
with propidium iodide, on
a Becton Dickinson FACScan
Analytic Flow Cytometer, as previously described.13
To evaluate Fas-induced apoptosis, anti-human Fas anti-
body (clone CH11, Upstate Biotechnology Inc., Lake Placid,
NY) was added at various concentrations to the untreated or
AdFU-treated cells in 96-well plates. Cultures were then
incubated for 24 h at 37 °C.
Assa y for Ca sp a se-8 Activity a n d Ca sp a se-In h ibition
Exp er im en ts. Caspase-8 activity was assayed using the
ApoAlert colorimetric assay kit (Clontech, Palo Alto, CA),
according to the manufacturer’s instructions. Enzyme activity
was expressed as fold increase with respect to control cells.
To inhibit protease activity, synthetic, cell-permeable, non-
cleavable peptide analogues of caspase substrates were added
to infected and control cultures. Z-VAD-FMK, Z-DEVD-FMK
and Z-LEHD-FMK (Calbiochem, San Diego, CA) irreversible
inhibitors were utilized.
(5′S)-1-Isoxa zolid in -5-yl-1H-p yr im id in e-2,4-d ion e (8a ).
Yield 92%; [R]25 ) -84.7 (c 0.23; CH3OH); mp 183-185 °C,
D
white solid from methanol. Anal. (C7H9N3O3) C, H, N.
(5′R)-5-F lu or o-1-isoxa zolid in -5-yl-1H -p yr im id in e-2,4-
d ion e (7b). Yield 90%; [R]25 ) +52.4 (c 0.43; CH3OH); mp
D
155-157 °C (lit.6 153-155 °C), white solid from methanol.
Anal. (C7H8FN3O3) C, H, N. Physical and spectroscopic data
are identical to these previously reported.6
(5′S)-5-F lu or o-1-isoxa zolid in -5-yl-1H -p yr im id in e-2,4-
d ion e (8b). Yield 89%; [R]25 ) -63.8 (c 0.17; CH3OH); white
D
solid from methanol, mp 155-157 °C (lit.6 153-155 °C). Anal.
(C7H8FN3O3) C, H, N. Physical and spectroscopic data are
identical to these previously reported.6
Ack n ow led gm en t. We thank the M.I.U.R. and
C.N.R. for their partial financial support.
(5′R)-1-Isoxa zolid in -5-yl-5-m eth yl-1H-p yr im id in e-2,4-
d ion e (7c). Yield 93%; [R]25 ) -82.7 (c 0.21; CH3OH); mp
D
201-203 °C, white solid from methanol (lit.6 202-204 °C).
Anal. (C8H11N3O3) C, H, N. Physical and spectroscopic data
are identical to these previously reported.6
Refer en ces
(1) De Clercq, E. New Developments in Anti-HIV Chemotherapy.
Biochim. Biophys. Acta 2002, 1587, 258-275.
(5′S)-1-Isoxa zolid in -5-yl-5-m et h yl-1H -p yr im id in e-2,4-
d ion e (8c). Yield 91%; [R]25 ) +94.3 (c 0.14; CH3OH); mp
(2) De Clercq, E. Strategies in the Design of Antiviral drugs. Nat.
Rev. Drug Discovery 2002, 1, 13-25.
D
201-203 °C, white solid from methanol (lit.6 202-204 °C).
Anal. (C8H11N3O3) C, H, N. Physical and spectroscopic data
are identical to these previously reported.6
(3) De Muys J .-M.; Gordeau, H.; Nguyen-Ba, N.; Taylor, D. L.;
Ahmed, P. S.; Mansour, T.; Locas, C.; Richard, N.; Wainberg,
M. A.; Rando, R. F. Anti-Human Immunodeficiency Virus Type
1
Activity, Intracellular Metabolism, and Pharmacokinetic
(5′S)-4-Am in o-1-isoxa zolid in -5-yl-1H-p yr im id in -2-on e
(7d ). Yield 94%; [R]25 ) -12.7 (c 0.3; CH3OH); mp 190-194
Evaluation of 2′-Deoxy-3′-oxa-4′-thiocytidine. Antimicrob. Agents
Chemother. 1999, 43, 1835-1844.
D
°C, amorphous solid. 1H NMR (200 MHz, CD3OD): δ 2.39-
2.54 (m, 1H, H4′a), 2.57-2.71 (m, 1H, H4′b), 3.11-3.34 (m, 2H,
H3′a, H3′b), 5.89 (d, 1H, J ) 7.4 Hz, H5), 5.98 (dd, J ) 3.6, 7.2
Hz, H5′), 7.63 (d, 1H, J ) 7.4 Hz, H6); 13C NMR (50 MHz, CD3-
OD): δ 37.42, 49.52, 90.96, 95.89, 143.75, 158.34, 167.37. Anal.
(C7H10N4O2) C, H, N.
(4) (a) Pan, S.; Amankulor, N. M.; Zhao, K. Syntheses of Isoxazolinyl
and Isoxazolidinyl Nucleoside Analogues. Tetrahedron 1998, 54,
6587-6604. (b) Merino, P.; Franco, S.; Merchan, F. L.; Tejero,
T. Stereodivergent Approaches to the Synthesis of Isoxazolidine
Analogues of R-Amino Acid Nucleosides. Total Synthesis of
Isoxazolidinyl Deoxypolyoxin C and Uracil Polyoxin C. J . Org.
Chem. 2000, 65, 5575-5589. (c) Adams, D. R.; Boyd, A. S. F.;
Ferguson, R.; Grierson, D. S.; Monneret, C. Heterocyclic Nucleo-
side Analogues by Cycloaddition Reactions of 1-Vinylthymine
with 1,3-Dipoles. Nucleosides Nucleotides 1998, 17, 1053-1075.
(d) Fischer, R.; Druckova´, A.; Fisˇera, L.; Ryba´r, A.; Hametner,
C.; Cyran´ski, M. New Chiral Nitrones in the Synthesis of
Modified Nucleosides. Synlett 2002, 1113-1117.
(5′R)-4-Am in o-1-isoxa zolid in -5-yl-1H-p yr im id in -2-on e
(8d ). Yield 89%; [R]25D ) +15.3 (c 0.55; CH3OH); mp 190-194
°C, amorphous solid. 1H NMR (200 MHz, CD3OD): δ 2.42-
2.52 (m, 1H, H4′a), 2.56-2.73 (m, 1H, H4′b), 3.19-3.30 (d, 1H,
J ) 7.4 Hz, H5), 5.97 (m, 1H, H5′), 7.63 (d, 1H J ) 7.4 Hz, H6);
13C NMR (50 MHz, CD3OD): δ 37.49, 49.28, 90.84, 95.87,
143.91, 158.38, 167.81. Anal. (C7H10N4O2) C, H, N.
(5) (a) Colacino, E.; Converso, A.; Liguori, A.; Napoli, A.; Siciliano,
C.; Sindona, G. Simple and Efficient Routes for the Preparation
of Isoxazolidinyl Nucleosides Containing Cytosine and 5-Meth-
ylcytosine as New Potential Anti-HIV Drugs. Tetrahedron 2001,
57, 8551-8557. (b) Leggio, A.; Liguori, A.; Procopio, A.; Siciliano,
C.; Sindona, G. Synthesis of 4′-Aza Analogues of 2′,3′-Dideoxy-
thymidine by 1,3-Dipolar Cycloadditions of Nitrones to 1-N-
Vinylthymine. Tetrahedron Lett. 1996, 37, 1277-1280. (c)
Leggio, A.; Liguori, A.; Procopio, A.; Siciliano, C.; Sindona, G. A
Novel Class of 4′-Aza Analogues of 2′,3′-Dideoxynucleosides as
Potential Anti-HIV Drugs. Nucleosides Nucleotides 1997, 16,
1515-1518. (d) Dalpozzo, R.; De Nino, A.; Maiuolo, L.; Procopio,
A.; De Munno, G.; Sindona, G. 9-Vinylguanine: an Easy Access
to Aza-Analogues of 2′,3′-Dideoxyguanosine. Tetrahedron 2001,
57, 4035-4038.
(5′R)-4-Am m in o-5-flu or o-1-isossa zolid in -5-yl-1H -p y-
r im id in -2-on e (7e). Yield 94%; [R]25 ) +12.7 (c 0.3; CH3-
D
OH); amorphous solid. 1H NMR (200 MHz, CD3OD): δ 2.42
(m, 1H, H4′a), 2.64(m, 1H, H4′b), 3.20 (m, 2H, H3′a, H3′b), 5.98
(dd, 1H, J ) 3.5, 7 Hz, H5′), 7.62 (d, 1H, J ) 6 Hz, H6). 13C
NMR (50 MHz, CD3OD): δ 37.42, 46.42, 90.96, 128.21, 143.75,
158.34, 167.37. Anal. (C7H9FN4O2) C, H, N.
(5′S)-4-Am m in o-5-flu or o-1-isossa zolid in -5-yl-1H-p yr im -
id in -2-on e (8e). Yield 89%; [R]25 ) -15.3 (c 0.35; CH3OH);
D
amorphous solid. Anal. (C7H9FN4O2) C, H, N.

3702 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 17
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