4′-α-C-Branched N,O-nucleosides: Synthesis and biological properties

Article abstract of DOI:10.1016/j.bmc.2004.04.041

The synthesis of 4′-α-C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity again

Full text of DOI:10.1016/j.bmc.2004.04.041

Bioorganic & Medicinal Chemistry 12 (2004) 3903–3909
40-a-C-Branched N,O-nucleosides: synthesis and biological
properties
Ugo Chiacchio,a Filippo Genovese,b Daniela Iannazzo,b Anna Piperno,b,*
Paolo Quadrelli,c Corsaro Antonino,a Roberto Romeo,b Vincenza Valverid
and Antonio Mastinod
aDipartimento Scienze Chimiche, Universitꢀa di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
bDipartimento Farmaco-Chimico, Universitꢀa di Messina, Viale Annunziata, 98168 Messina, Italy
cDipartimento di Chimica Organica, Universitꢀa degli Studi di Pavia V.le Taramelli 10, 27100 Pavia, Italy
dDipartimento Scienze Microbiologiche, Genetiche e Molecolari, Universitꢀa di Messina, Salita Sperone 31, 98100 Messina, Italy
Received 12 March 2004; revised 22 April 2004; accepted 30 April 2004
Abstract—The synthesis of 40-a–C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones
with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity
against HSV-1, HSV-2, HTLV-1. Cytotoxicity and apoptotic activity have been also investigated: compound 10c shows moderate
apoptotic activity in Molt-3 cells.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
minor toxicity and improved or more specific biological
activity.
Nucleosides play a fundamental role in modern viral
chemotherapy as potent inhibitors of HIV, the causative
agent of AIDS.1 After being converted to the triphos-
phate form in cells by specific kinases,2 they may either
inhibit HIVreverse transcriptase, or be incorporated
into the growing DNA chain, resulting in chain termi-
nation or disruption.3 In this context, modifications of
the sugar fragment has resulted in the synthesis of
interesting nucleoside analogues such as AZT, ddC and
ddI,4 which have shown remarkable activity towards the
human immunodeficiency virus.
A synthetic strategy is represented by the exploitation of
the heteroatom substitution; thus, a series of com-
pounds, where the sugar moiety has been replaced by
alternative heterocyclic rings, as 3TC and L-20,30-dide-
oxy-30-oxacytidine, proved to be effective in the treat-
ment of HIV/HBV infections and different lymphoid
and solid tumours, respectively.5
The introduction of a side-chain on the sugar led to
several branched nucleosides, which have been evalu-
ated as potential antitumoural or antiviral agents;6 some
However, prolonged therapeutic use of this class of
compounds resulted in the development of resistant
strains and cross-resistance to related nucleosides.
Accordingly, considerable efforts have been made to
synthesize new modified nucleosides characterized by
of them, such as 1-(2-deoxy1-2-methylene-b-
D
-erythro-
pentafuranosyl)cytosine (DMDC),7 1-(2-cyano-2-deoxy-
b-
-arabino-pentafuranosyl)cytosine(CNDAC),8 1-(2-
deoxy-2-fluoro-methylene-b- -erythro-pentafuranosyl)-
-ribo-pentafur-
D
D
cytosine (FMDC),9 1-(3-C-ethynyl-b-
D
anosyl)cytosine (ECyd)10 and its uracil congener (EUr-
d),10 have shown potent in vitro and in vivo
antitumoural activity (Fig. 1).
40a-C-Branched nucleosides, namely 40a-C-cyano-deoxy-
thymidine 1, 40a-C-methyl-20-deoxycytidine 2, 40a-C-
ethynyl-20-deoxycytidine 3 and 40a-C-hydroxymethyl-
Keywords: Modified nucleosides; 1,3-Dipolar cycloaddition; Antiviral
activity; Apoptotic activity; C-Branched nucleosides.
* Corresponding author. Tel.: +39-090-356230; fax: +39-090-6766562;
e-mail: apiperno@pharma.unime.it
deoxythymidine
4 have also been prepared and
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reseraved.
doi:10.1016/j.bmc.2004.04.041

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U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
The cycloaddition reaction between 7 and vinyl acetate,
carried out at room temperature for 36 h, leads with a
complete regioselectivity to isoxazolidine 8 (yield 85%),
which has been purified by flash chromatography
(cyclohexane/diethyl acetate 95:5 as eluant).
The structure of isoxazolidine 8 has been confirmed by
1H NMR experiments. Thus, H4 protons give rise to two
doublets of doublets centred at 2.04 and 2.62 ppm; the
two methylene groups of the substituents at C3 resonate
as four doublets in the range 3.50–3.80 ppm, while H5
proton gives rise to a doublet of doublets at 6.23 ppm.
Figure 1. Modified nucleosides.
The cycloaddition product has been, then, coupled with
€
silylated nucleobases, according to the Vorbruggen
demonstrate to possess strong anti-HIVand antil-
eukaemic activities.11–13
glycosylation methodology.16 The condensations of 8
with silylated thymine, N-acetylcytosine, 5-fluorouracil
and adenine have been performed in acetonitrile, at
room temperature, in the presence of 0.15 equiv of
TMSOTf as catalyst to give after TBAF treatment the
corresponding nucleosides 10a,c–e (Scheme 2).
Recently we have reported that N,O-nucleosides,14
where the sugar moiety has been replaced by an isox-
azolidine ring, exhibit very promising biological prop-
erties. In particular, ADFU, while showing low level of
cytotoxicity, is a good inductor of apoptosis in lym-
phoid and monocytoid cells.15
The molecular structure of the reaction products was
confirmed on the basis of analytical and spectroscopic
data. The 1H NMR spectra, recorded in D2O, show
diagnostic signals for H1, which resonate as doublet of
doublets in the range 5.92–6.01 ppm, while H5 protons
appear as two doublet of doublets centred at 2.70–2.75
and 2.17–2.30 ppm.
On the basis of these findings, we became interested in the
synthesis of 40a-branched-chain N,O-nucleosides in order
to evaluate their biological characteristics; therefore, we
designed a series of new 40-C-substituted N,O-nucleosides
5. In this communication, we describe the synthesis of
these derivatives and their antiviral activities against
herpes simplex virus type 1 (HSV-1) and 2 (HSV-2) as well
as their apototic activity versus Molt-3 differentiated cells.
The reaction route has been also addressed towards the
formation of 40-methyl N,O-nucleosides; thus, treatment
of 1-(tert-butyl-diphenyl-silanyloxy)propan-2-one18 11
with N-methyl hydroxylamine afforded nitrone 12 as a
Z/E mixture (2.5:1 ratio). The subsequent cycloaddition
reaction with vinyl acetate proceeded without any dia-
stereoselectivity, affording isoxazolidines 13a and 13b in
the 1:1 relative ratio (Scheme 3).
2. Chemistry
The synthetic scheme is based on the 1,3-dipolar cyclo-
addition of C-a-disilyloxymethyl-N-methyl nitrone 7
with vinyl acetate, followed by nucleosidation per-
formed with silylated purine and pyrimidine bases.16
The assignment of cis/trans configurations has been
performed on the basis of NOEDS experiments. For
13a, irradiation of H-5 proton induced a positive NOE
effect on the downfield resonance (H-4a) of methylene
protons at C-4 and on the methyl group at C-3; con-
versely, when H-4b was irradiated, a NOE enhancement
Nitrone 7 has been prepared in good yields starting from
1,3-bis-(-t-butyl-dimethyl-silanyloxy)-propan-2-one 6.17
The subsequent treatment with N-methyl hydroxylamine
in toluene, in the presence of triethylamine, gave, after
flash chromatography (chloroform/methanol 98:2 as
eluant), the expected nitrone 7 (90% yield) (Scheme 1).
Scheme 1. Reagents and conditions: (a) NEt3, CH3NHOHÁHCl, Tol-
Scheme 2. Reagents and conditions: (a) silylated base/TMSOTf, 12 h,
rt; (b) TBAF/THF, 1 h, rt; (c) for 9b: MeOH, K2CO3, 1 h, rt.
uene, 2 h, rt. (b) Vinyl acetate, 36 h, rt.

U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
3905
tions tested (e.g. 320 lM), indicating a lack of significant
antiviral activity. Since the biological activity of this
type of inhibitors is often linked to their susceptibility to
phosphorylation by kinases, it would be interesting to
evaluate the phosphate or phosphonate forms of the
synthesized compounds in order to determine whether
they could improve the antiviral activity towards retro-
viruses.
The apoptotic activity was also tested in Molt-3 cells.
Only 10c has shown a moderate activity (32%) at the
concentration 500 lM after 48 h. No significant toxicity,
when evaluated using the classical trypan blue test, was
detected until 3 days of treatment in lymphoid Molt-3
cell line assayed.
Scheme 3. Reagents and conditions: (a) NEt3, CH3NHOHÆHCl, Tol-
uene, 2 h, rt. (b) Vinyl acetate, 36 h, rt.
4. Experimental
4.1. General
Commercially available chemicals and solvents were
reagent grade and used as received. All solvents were
dried according to literature methods. Melting points
were determined with a Kofler apparatus and are
uncorrected. Elemental analyses were performed on a
Perkin–Elmer 240B microanalyzer. NMR spectra were
recorded on a Varian instrument at 300 or 500 MHz
(1H) and at 75 MHz (13C) using deuterochloroform or
deuterated water as solvents; chemical shifts are given in
ppm from TMS as internal standard. NOE difference
spectra were obtained by subtracting alternative right
off-resonance free induction decays (FIDS) from right-
on-resonance-induced FIDS. Thin-layer chromato-
graphic separations were achieved through Merck silica
gel 60-F254 precoated aluminium plates. Preparative
separations were carried out by flash chromatography
using Merck silica gel 0.035–0.070 mm. The identifica-
tion of samples from different experiments was secured
by mixed mps and superimposable NMR spectra.
Scheme 4. Reagents and conditions: (a) silylated base/TMSOTf, 12 h,
rt; (b) TBAF/THF, 1 h, rt; for 14b and 15b: MeOH, K2CO3, 1 h, rt.
was observed for the methylene group linked at C-3.
These data unambiguously indicate a cis relationship
between H-5, H-4a and the methyl substituent at C-3. In
13b, irradiation of H-5 induced a positive NOE effect for
the methylene group at C-3.
Coupling of the diastereoisomeric mixture of 13 with
silylated bases led to a mixture of a- and b-nucleosides
14 and 15 (40:60 ratio), which have been separated by
flash chromatography (Scheme 4).
4.2. General procedure for the synthesis of the nitrone 7
and 12
The stereochemical assignments to the obtained nucleo-
sides have been performed by NOEDS spectroscopy: in
a-derivatives 14a–d, irradiation of H-5a, the downfield
resonance of methylene protons at C-5, induces a posi-
tive NOE effect on H-1 and the CH2OH group at C-4, so
suggesting that these protons are topologically close
together. In contrast, in b-derivatives 15a–d, the NOE
effect was observed between H-1 and the methyl group
at C-4.
A solution of 6 or 11 (19 mmol) N-methyl hydroxyl-
amine hydrochloride (19 mmol) and triethylamine
(19 mmol) in toluene (70 mL) was left to stir 3 h; after
filtration the solvent was evaporated under reduced
pressure, and the residue was subjected to flash-
chromatography (CHCl3/CH3OH 99:1).
4.2.1. [2-{[tert-Butyl(dimethyl)silyl]oxy}-1-({[tert-butyl
(dimethyl)silyl]oxy}methyl)ethylidene](methyl)azane oxide
(7). Starting from 1,3-bis-(t-butyl-dimethyl-silanyloxy)-
propan-2-one, compound 7 was obtained as yellow oil
(90%). 1H NMR (CDCl3): d 0.15 (s, 12H), 0.95 (s, 18H),
3.81 (s, 3H, N–CH3), 4.45 (s, 2H), 4.65 (s, 2H); 13C
NMR (CDCl3): d 5.62, 18.04, 18.10, 22.41, 22.50, 40.01,
66.47, 67.64, 170.48. HRMS (FAB) calcd for [Mþ]
C16H37NO3Si2 347.6489, found 347.6485. Anal.
3. Biological assays
The synthesized compounds were tested for their
antiviral activity against HSV-1, HSV-2 in Vero (Afri-
can Green Monkey) cells, HTLV-1 in human cells
(lymphomonocytes of peripheral blood) and for their
toxicity in vitro. None of these derivatives reached the
inhibitory concentration 50 at the highest concentra-

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U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
Calcd for C16H37NO3Si2: C, 55.28; H, 10.73%; N, 4.03.
Found: C, 55.10; H, 11.02; N, 3.98%.
C24H33NO4Si: C, 67.41%; H, 7.78%; N, 3.27%. Found:
C, 67.15%; H, 7.64%; N, 3.22%. The second eluted
product was (3SR,5SR)-3-[(tert-butyl-diphenyl-silyl)-
oxy-methyl]-2,3-dimethyl-isoxazolidin-5-yl acetate 13b
1
4.2.2. (Z)-(E)(2-{[tert-Butyl(diphenyl)silyl]oxy}-1-methyl-
ethylidene)(methyl)azane oxide (12). Starting from 1-t-
butyl-diphenyl-silanyloxy)propan-2-one, compound 12
was obtained as inseparable mixture of E/Z isomers
(1:2.5). Yellow oil (82% global yield). Z-Isomer 1H
NMR (CDCl3): d 1.05 (s, 9H), 2.18 (s, 3H), 3.65 (s, 3H,
N–CH3), 4.70 (s, 2H), 7.20–7.40 (m, 10H); 13C NMR
(CDCl3): d 19.19, 26.63, 36.70 47.58, 62.65, 127.76,
130.56, 132.69, 135.46, 170.48. E-Isomer: 1H NMR
(CDCl3): d 1.05 (s, 9H), 1.95 (s, 3H), 3.65 (s, 3H, N–
CH3), 4.30 (s, 2H), 7.20–7.40 (m, 10H); 13C NMR
(CDCl3): d 19.19, 26.63, 36.70, 47.77, 62.59, 127.70,
130.72, 132.70, 136.46, 170.40. HRMS (FAB) calcd for
[Mþ] C20H27NO2Si, 369.6135, found 369.6132.
as a sticky oil. H NMR (300 MHz, CDCl3): d 1.08 (s,
9H), 1.25 (s, 3H), 2.07 (s, 3H), 2.73 (s, 3H, N–CH3), 2.74
(m, 2H, H4), 3.62 (d, 1H, J ¼ 10:2 Hz), 3.73 (d, 1H,
J ¼ 10:2 Hz), 6.24 (dd, 1H, J ¼ 3:0, 5.0 Hz, H5), 7.45–
7.65 (m 10H); 13C NMR (75 MHz, CDCl3): d 15.25,
18.30, 21.80, 26.90, 39.20, 46.80, 66.41, 67.90, 94.80,
126.63, 129.70, 132.88, 135.20, 170.42. HRMS (FAB)
calcd for [Mþ] C24H33NO4Si 427.6183, found 427.6180.
Anal. Calcd for C24H33NO4Si: C, 67.41%; H, 7.78%; N,
3.27%. Found: C, 67.10%; H, 7.68%; N, 3.20%.
4.4. General procedure for the synthesis of protected
nucleosides 9a,d
A suspension of the nucleobase (0.62 mmol) in dry
acetonitrile (3 mL) was treated with bis(trimethyl-
silyl)acetamide (2.54 mmol), and refluxed for 15min un-
der stirring. The obtained clear solution was added to a
solution of the isoxazolidine 8 (0.52 mmol) in dry ace-
tonitrile (3 mL); trimethylsilyltriflate (0.2 mmol) was
added dropwise, and the reaction mixture was left under
stirring overnight at rt. After cooling at 0 ꢁC, the solu-
tion was neutralized by careful addition of aqueous 5%
sodium bicarbonate, and then concentrated in vacuo.
After the addition of dichloromethane (8 mL), the
organic phase was separated, washed with water
(2 · 10 mL), dried over sodium sulfate, filtered and
evaporated to dryness. The residue was purified by flash
chromatography (CH2Cl2/MeOH 99:1) to furnish the
nucleosides 9a,d.
4.3. General procedure for the synthesis of isoxazolidines
8 and 13a–b
A solution of nitrones 7 or 12 (5.7 mmol) in vinyl acetate
(30 mL) was left under stirring for 36 h at room tem-
perature; after this period, the reaction mixture was
evaporated under reduced pressure and the residue
purified by flash chromatography (hexane/ethyl acetate
98:2).
4.3.1. 3,3-Bis({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-
methyl-isoxazolidin-5-yl acetate (8). Starting from nit-
rone 7, compound 8 was obtained (85% yield) as a sticky
oil. 1H NMR (300 MHz, CDCl3): d 0.10 (s, 12H), 0.90 (s,
18H), 2.04 (dd, 1H, J ¼ 1:5, 14.1 Hz, H4a), 2.05 (s, 3H),
2.62 (dd, 1H, J ¼ 6:6, 14.1 Hz, H4b), 2.82 (s, 3H, N–
CH3), 3.53 (d, 1H, J ¼ 9:6 Hz), 3.68 (d, 1H, J ¼ 9:9 Hz),
3.76 (d, 1H, J ¼ 9:9 Hz), 3.83 (d, 1H, J ¼ 9:6 Hz), 6.23
(dd, 1H, J ¼ 1:5, 6.6 Hz, H5); 13C NMR (75 MHz,
CDCl3): d 5.96, 18.14, 18.21, 21.50, 23.11, 40.35, 40.91,
62.97, 64.50, 70.05, 95.86, 170.24. HRMS (FAB) calcd
for [Mþ] C20H43NO5Si2 433.7397, found 433.7392. Anal.
Calcd for C20H27NO3Si2: C, 55.38%; H, 9.99%; N,
3.23%. Found: C, 55.10%; H, 9.70%; N, 3.10%.
4.4.1. 1-[3,3-Bis({[tert-butyl(dimethyl)silyl]oxy}methyl)-
2-methyl-isoxazolidin-5-yl]-5-methylpyrimidine-2,4(1H,
3H)-dione (9a). Starting from 8, compound 9a was
obtained (60% yield) as a white oil. 1H NMR (300 MHz,
CDCl3): d 0.12 (s, 12H), 0.93 (s, 18H), 1.96 (d, 3H,
J ¼ 1:2 Hz), 2.09 (dd, 1H, J ¼ 4:2 and 13.0 Hz, H4), 2.82
(s, 3H, N–CH3), 2.87 (dd, 1H, J ¼ 7:8 and 13.0 Hz, H4),
3.60 (d, 1H, J ¼ 9:6 Hz), 3.63 (d, 1H, J ¼ 9:9 Hz), 3.74
(d, 1H, J ¼ 9:9 Hz), 3.89 (d, 1H, J ¼ 9:6 Hz), 6.10 (dd,
1H, J ¼ 4:2 and 7.8 Hz, H50), 7.71 (q, 1H, J ¼ 1:2 Hz,
H6), 8.60 (br s, 1H, NH); 13C NMR (75 MHz, CDCl3): d
5.59, 12.66, 18.32, 18.64, 25.80, 25.84, 38.59, 43.47,
59.36, 63.63, 69.76, 82.14, 110.36, 136.47, 150.36,
163.72. HRMS (FAB) calcd for [Mþ] C23H45N3O5Si2
483.8027, found 483.8025. Anal. Calcd for
C23H45N3O5Si2: C, 57.10%; H 9.38%; N, 13.22%.
Found: C, 56.95%; H, 9.08%; N, 12.98%.
4.3.2.
3-[(tert-Butyl-diphenyl-silyl)oxy-methyl]-2,3-di-
methyl-isoxazolidin-5-yl acetate (13a–b). Starting from
nitrone 12, isoxazolidines 13a and 13b in 1:1 ratio (88%
global yield) were obtained and separated by flash
chromatography (hexane/ethyl acetate 98:2). The first
eluted product was (3RS,5SR),3-[(tert-butyl-diphenyl-
silyl)oxy-methyl]-2,3-dimethyl-isoxazolidin-5-yl acetate
1
13a as a sticky oil. H NMR (300 MHz, CDCl3): d 1.08
(s, 9H), 1.21 (s, 3H), 2.05 (s, 3H), 2.26 (dd, 1H, J ¼ 3:0,
13.5 Hz, H4a), 2.36 (dd, 1H, J ¼ 6:5, 13.5 H4b), 2.73 (s,
3H, N–CH3), 3.67 (q, 2H, J ¼ 10 Hz), 6.23 (dd, 1H,
J ¼ 3:0, 6.5 Hz, H5), 7.45–7.65 (m 10H); 13C NMR
(75 MHz, CDCl3): d 15.20, 18.36, 21.90, 26.90, 39.18,
46.88, 66.41, 68.80, 94.92, 126.93, 129.71, 132.88,
135.23, 170.45. HRMS (FAB) calcd for [Mþ]
C24H33NO4Si 427.6183, found 427.6177. Anal. Calcd for
4.4.2. N-{1-[3,3-Bis({[tert-butyl(dimethyl)silyl]oxy}meth-
yl)-2-methyl-isoxazolidin-5-yl]-2-oxo-1,2-dihydropyrim-
idin-4-yl}acetamide (9b). Starting from 8, compound 9b
(64% yield) was obtained as a white oil. 1H NMR
(300 MHz, CDCl3): d 0.35 (s, 12H), 0.95 (s, 18H), 2.00
(dd, 1H, J ¼ 3:0 and 14.4 Hz, H4), 2.08 (s, 3H), 3.02 (s,
3H, N–CH3), 3.05 (dd, 1H, J ¼ 7:8 and 14.4 Hz, H4),

U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
3907
3.51 (d, 1H, J ¼ 9:6 Hz), 3.57 (d, 1H, J ¼ 10:2 Hz), 3.69
(d, 1H, J ¼ 10:2 Hz), 3.94 (d, 1H, J ¼ 9:6 Hz), 6.02 (dd,
1H, J ¼ 3:0 and 7.8 Hz, H50), 7.46 (d, 1H, J ¼ 7:5 Hz,
H5), 8.24 (d, 1H, J ¼ 7:5 Hz, H6), 10.21 (br s, 1H, NH);
13C NMR (75 MHz, CDCl3): d )5.67, 18.04, 18.15,
24.13, 25.78, 25.80, 38.71, 44.28, 59.24, 64.11, 68.29,
84.27, 96.16, 145.06, 155.28, 162.85, 171.50. HRMS
(FAB) calcd for [Mþ] C24H46N4O5Si2 526.8278, found
526.8275. Anal. Calcd for C24H46N4O5Si2: C, 54.71%;
H, 8.79%; N, 10.63%. Found: C, 54.62%; H, 8.68%; N,
10.42%.
left under stirring for 2 h. The solvent was then evapo-
rated under reduced pressure, and the residue was flash-
chromatographed (CHCl3/CH3OH 95:5) to give 10e.
4.5.1. 1-[3,3-Bis(hydroxymethyl)-2-methyl-isoxazolidin-5-
yl]-5-methylpyrimidine-2,4(1H,3H)-dione (10a). Starting
from 9a, compound 10a was obtained and crystallized
from acetone/ether. Yield 85%; white solid, mp 108–
110 ꢁC. 1H NMR (300 MHz, D2O): d 1.75 (d, 3H,
J ¼ 1:1 Hz), 2.32 (dd, 1H, J ¼ 4:2 and 14.1 Hz, H4), 2.67
(s, 3H, N–CH3), 2.71 (dd, 1H, J ¼ 8:1 and 14.1 Hz, H4),
3.53 (s, 2H), 3.64 (s, 2H), 5.99 (dd, 1H, J ¼ 4:2 and
8.1 Hz, H50), 7.72 (q, 1H, J ¼ 1:1 Hz, H6); 13C NMR (75
MHz, D2O): d 14.21, 40.06, 43.02, 62.34, 63.47, 72.60,
85.40, 113.48, 140.29, 154.40, 156.35. HRMS (FAB)
calcd for [Mþ] (C11H17N3O5) 271.2741, found 271.2739.
Anal. Calcd for (C11H17N3O5): C, 48.70%; H, 6.32%; N,
15.48%. Found: C, 48.51%; H, 6.25%; N, 15.322%.
4.4.3. 1-[3,3-Bis({[tert-butyl(dimethyl)silyl]oxy}methyl)-2-
methyl-isoxazolidin-5-yl]-5-fluoropyrimidine-2,4(1H,3H)-
dione (9c). Starting from 8, compound 9c (55% yield)
was obtained as a white oil. 1H NMR (300 MHz,
CDCl3): d 0.21 (s, 12H), 0.95 (s, 18H), 2.17 (dd, 1H,
J ¼ 3:3 and 13.2 Hz, H4), 2.78 (s, 3H, N–CH3), 2.84 (dd,
1H, J ¼ 8:1 and 13.2 Hz, H4), 3.58 (d, 1H, J ¼ 10:2 Hz),
3.61 (d, 1H, J ¼ 9:9 Hz), 3.72 (d, 1H, J ¼ 10:2 Hz), 3.85
(d, 1H, J ¼ 9:9 Hz), 6.08 (dd, 1H, J ¼ 3:3 and 8.1 Hz,
H50), 8.13 (d, 1H, J ¼ 6:3 Hz, H6), 9.00 (br s, 1H, NH);
13C NMR (75 MHz, CDCl3): d )5.72, 18.36, 18.45,
27.38, 37.48, 41.22, 59.98, 61.04, 69.95, 82.80, 126.50,
142.27, 151.13, 160.37. HRMS (FAB) calcd for [Mþ]
C22H42FN3O5Si2 503.7655, found 503.7652. Anal. Calcd
for C22H42FN3O5Si2: C, 48.48%; H, 8.40%; N, 8.34%.
Found: C, 48.37%; H, 8.29%; N, 8.27%.
4.5.2. 4-Amino-1-[3,3-bis(hydroxymethyl)-2-methyl isox-
azolidin-5-yl]pyrimidin-2(1H)-one (10b). Starting from
9b, compound 10e was obtained and crystallized from
acetone/ether. Yield 80%; white solid, mp 115–119 ꢁC.
1H NMR (300 MHz, D2O): d 2.20 (dd, 1H, J ¼ 3:9 and
14.1 Hz, H4), 2.67 (s, 3H, N–CH3), 2.75 (dd, 1H, J ¼ 7:8
and 14.1 Hz, H4), 3.48 (s, 2H), 3.65 (s, 2H), 5.86 (d, 1H,
J ¼ 7:5 Hz, H5), 5.89 (dd, 1H, J ¼ 3:9 and 7.8 Hz, H50),
7.80 (d, 1H, J ¼ 7:5 Hz, H6); 13C NMR (75 MHz, D2O):
dC 36.46, 38.54, 49.53, 64.38, 72.9, 100.89, 108.2, 110,9,
118.4, 198.8. HRMS (FAB) calcd for [Mþ] C10H16N4O4
256.4144, found 256.4141. Anal. Calcd for C10H16N4O4:
C, 46.84%; H, 6.28%; N, 21.84%. Found: C, 46.64%; H,
6.02%; N, 21.74%.
4.4.4. 9-[3,3-Bis({[tert-butyl(dimethyl)silyl]oxy}methyl)-
2-methyl-isoxazolidin-5-yl]-9H-purin-6-amine
(9d).
Starting from 8, compound 9d (33% yield) was ob-
tained as a white sticky oil. 1H NMR (300 MHz,
CDCl3): 0.40 (s, 12H), 0.95 (s, 18H), 2.48 (dd, 1H,
J ¼ 7:2 and 13.5 Hz, H4), 2.91 (s, 3H, N–CH3), 2.94 (dd,
1H, J ¼ 7:4 and 13.5 Hz, H4), 3.74 (d, 1H, J ¼ 10:2 Hz),
3.80 (d, 1H, J ¼ 10:5 Hz), 3.90 (d, 1H, J ¼ 10:5 Hz),
4.01 (d, 1H, J ¼ 10:2 Hz), 6.25 (dd, 1H, J ¼ 7:2 and
7.4 Hz, H50), 7.21 (br s, 2H, NH), 7.98 (s, 1H, H8), 8.49
(s, 1H, H2); 13C NMR (75 MHz, CDCl3): d –5.63, 18.25,
18.36, 26.94, 26.98, 39.18, 41.43, 59.67, 64.13, 71.29,
84.31, 121.43, 144.08, 151.99, 153.68, 162.39. HRMS
(FAB) calcd for [Mþ] C23H44N6O3Si2 508.8124, found
508.8120. Anal. Calcd for C23H44N6O3Si2: C, 54.29%;
H, 8.71%; N, 16.52%. Found: C, 54.13%; H, 8.62%; N,
16.44%.
4.5.3. 1-[3,3-Bis(hydroxymethyl)-2-methyl-isoxazolidin-5-
yl]-5-fluoropyrimidine-2,4(1H,3H)-dione (10c). Starting
from 9c, compound 10c was obtained and crystallized
from acetone/ether. Yield 89%; white solid, mp 96–
98 ꢁC. 1H NMR (300 MHz, D2O): 2.28 (dd, 1H, J ¼ 3:6
and 13.4 Hz, H4), 2.64 (s, 3H, N–CH3), 2.69 (dd, 1H,
J ¼ 8:4 and 13.6 Hz, H4), 3.45 (m, 2H), 3.62 (m, 2H),
5.96 (dd, 1H, J ¼ 3:6 and 8.4 Hz, H50), 8.06 (d, 1H,
J ¼ 6:5 Hz, H6); 13C NMR (75 MHz, D2O): d 37.47,
42.10, 59.99, 61.03, 70.00, 83.71, 126.55, 142.30, 151.15,
160.41. HRMS (FAB) calcd for [Mþ] C10H14FN3O5
275.2375, found 275.2372. Anal. Calcd for
C10H14FN3O5: C, 43.64%; H, 5.12%; N, 15.26%. Found:
C, 43.49%; H, 5.02%; N, 15.06%.
4.5. General procedure for the synthesis of deprotected
nucleosides 10a,c–e
A 1 M THF solution of tetrabutyl ammonium fluoride
(2.2 mL, 2.2 mmol) was added to a stirred solution of
protected nucleosides 9a,c,d (1 mmol) in dry THF
(10 mL); after 1 h, the solvent was evaporated and the
residue was flash-chromatographed, using a CHCl3/
MeOH gradient from 98:2 up to 96/4, to furnish the
desilylated nucleosides 10a,c,d. In the case of compound
9b, the TBAF treatment afforded a residue which, after
solvent evaporation, was dissolved in a mixture of
aqueous K2CO3 (5%, 5 mL) and methanol (5 mL) and
4.5.4. 9-[3,3-Bis(hydroxymethyl)-2-methyl-isoxazolidin-5-
yl]-9H-purin-6-amine (10d). Starting from 9d, compound
10d was obtained ad crystallized from acetone/ether.
Yield 82%; white solid, mp 112–113 ꢁC. 1H NMR
(300 MHz, D2O): d 2.6 (dd, 1H, J ¼ 6:8 and 13.3 Hz,
H4), 2.71 (s, 3H, N–CH3), 2.82 (dd, 1H, J ¼ 7:2 and
13.3 Hz, H4), 3.61 (m, 2H), 3.80 (m, 2H), 6.25 (dd, 1H,
J ¼ 6:8, 7.2 Hz, H50), 8.05 (s, 1H, H8), 8.24 (s, 1H, H2);
13C NMR (75 MHz, D2O): d 18.22, 26.73, 39.08, 41.22,
59.53, 64.09, 71.12, 84.00, 121.13, 143.92, 151.45,

3908
U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
153.36, 162.02. HRMS (FAB) calcd for [Mþ].
C11H16N6O3 280.2844, found 280.2840. Anal. Calcd for
C11H16N6O3: C, 47.14; H, 5.75; N, 29.98%. Found: C,
47.01; H, 5.62; N, 29.78%.
from acetone/ether. Mp 169–172 ꢁC, white solid. 1H
NMR (500 MHz, CDCl3): d 1.13 (s, 3H), 2.07 (dd, 1H,
J ¼ 4:5 and 14.5 Hz, H4a), 2.80 (s, 3H, N–CH3), 3.26
(dd, 1H, J ¼ 7:5 and 14.5 Hz, H4b), 3.65 (d, 1H,
J ¼ 11:5 Hz), 3.74 (d, 1H, J ¼ 11:5 Hz), 5.70 (d, 1H,
J ¼ 7:5 Hz), 6.04 (dd, 1H, J ¼ 4:5 and 7.5 Hz) 7.65 (d,
1H, J ¼ 7:5 Hz); 13C NMR (75 MHz, CDCl3): d 14.20,
36.50, 37.18, 50.06, 70.20, 99.95, 107.80, 112.10, 157.20,
166.08.; HRMS (FAB) calcd for [Mþ] C10H16N4O3
240.2630, found 240.2626. Anal. Calcd for C10H16N4O3:
C, 49.99; H, 6.71; N, 21.62%. Found: C, 49.85; H, 6.62;
N, 21.43%.
4.6. General procedure for the synthesis of nucleosides
14a–d and 15a–d
A suspension of nucleobase (2 mmol) in dry acetonitrile
(70 mL) was treated with bis(trimethylsilyl) acetamide
(3.0 mmol) and stirred at rt until a clear solution was
obtained (0.5–6 h). A solution of isoxazolidine 13a–b
(1 mmol) in dry acetonitrile (5 mL) and TMSOTf
(0.1 mmol) was added and the resulting mixture was
stirred at rt overnight. After this time, the mixture was
neutralized by addition of a aqueous 5% NaHCO3 and
then concentrated in vacuo. The aqueous layer was ex-
tracted with ethyl acetate and combined organic layers
were dried over sodium sulfate, filtered and then evap-
orated to dryness. The residue was then dissolved in
THF; TBAF (1.1 mmol) was added and the mixture was
stirred at rt for 1.5 h. The solvent was evaporated and
the residue was flash-chromatographed, using a CHCl3/
MeOH gradient from 98:2 up to 96/4, to furnish the
desilylated nucleosides 14a–d and 15a–d. For the cyto-
sine derivatives, the desilylation process followed the
deacetylation reaction, performed by treatment with a
mixture of aqueous K2CO3 and methanol as above re-
ported for compound 10e.
4.6.3. 5-Fluoro-1-(3-hydroxymethyl-2,3-dimethyl-isoxaz-
olidin-5-yl)-1H-pyrimidin-2,4-dione (14c and 15c). Start-
ing from 13a–b, compounds 14c and 15c in 40:60 ratio
(60% global yield) were obtained and separated by flash
chromatography. The first eluted product was
(3RS,5SR)-5-fluoro-1-(3-hydroxymethyl-2,3-dimethyl-
isoxazolidin-5-yl)-1H-pyrimidin-2,4-dione 15c, crystal-
lized from acetone/ether. Mp 103–105 ꢁC, white solid.
1H NMR (500 MHz, CDCl3): d 1.25 (s, 3H), 2.64 (m,
2H, H4), 2.65 (s, 3H, N–CH3), 3.65 (m, 2H), 6.04 (dd,
1H, J ¼ 5:9 and 6 Hz, H5), 7.94 (d, 1H J ¼ 6:5 Hz) 8.26
(br s, 1H) 13C NMR (75 MHz, CDCl3): d 15.0, 37.16,
45.77, 64.44, 67.35, 83.50, 124.77, 125.50, 156.70,
157.60. HRMS (FAB) calcd for [Mþ] C10H14FN3O4
259.2381, found 259.2376. Anal. Calcd for
C10H14FN3O4: C, 46.33%; H, 5.44%; N, 16.20%. Found:
C, 46.28%; H, 5.33%; N, 16.10%. The second eluted
product was (3SR,5SR)-5-fluoro-1-(3-hydroxymethyl-
2,3-dimethyl-isoxazolidin-5-yl)-1H-pyrimidin-2,4-dione
14c, crystallized from acetone/ether. Mp 96–98 ꢁC, white
4.6.1. 1-(3-Hydroxymethyl-2,3-dimethyl-isoxazolidin-5-
ylmethyl)-1H-pyrimidine-2,4-dione (14a and 15a). Start-
ing from 13a–b, compounds 14a and 15a in 40:60 ratio
(56% global yield) were obtained and separated by flash
chromatography. The first eluted product was
(3SR,5SR)-1-(3-hydroxymethyl-2,3-dimethyl-isoxazoli-
din-5-ylmethyl)-1H-pyrimidine-2,4-dione 15a. The sec-
ond eluted product was (3RS,5SR)-1-(3-hydroxymethyl-
2,3-dimethyl-isoxazolidin-5-ylmethyl)-1H-pyrimidine-2,4-
dione19 14a.
1
solid. H NMR (500 MHz, CDCl3): d 1.21 (s, 3H), 1.59
(sb 1H, OH), 2.11 (dd, 1H, J ¼ 4:5 and 14 Hz, H4a), 2.77
(s, 3H, N–CH3), 3.10 (dd, 1H, J ¼ 7:5 and 14 Hz, H4b),
3.63 (d, 1H, J ¼ 11 Hz), 3.77 (d, 1H, J ¼ 11 Hz), 6.06
( dd, 1H, J ¼ 4:5 and 7.5 Hz, H5), 7.94 (d, 1H,
J ¼ 6 Hz), 8.62 (sb, 1H); 13C NMR (75 MHz, CDCl3): d
15.10, 37.18, 45.70, 64.60, 68.35, 83.70, 125.10, 125.60,
156.76, 157.58; HRMS (FAB) calcd for [Mþ]
C10H14FN3O4 259.2381, found 259.2378. Anal. Calcd
for C10H14FN3O4: C, 46.33%; H, 5.44%; N, 16.20%.
Found: C, 46.13%; H, 5.29%; N, 16.02%.
4.6.2. 4-Amino-1-(3-hydroxymethyl-2,3-dimethyl-isoxaz-
olidin-5-yl)1H-pyrimidin-2-one (14b and 15b). Starting
from 13a–b,compounds 14b and 15b in 40:60 ratio (50%
global yield) were obtained and separated by flash
chromatography. The first eluted product was
(3RS,5SR)-4-amino-1-(3-hydroxymethyl-2,3-dimethyl-
isoxazolidin-5-yl)1H-pyrimidin-2-one 15b crystallized
from acetone/ether. Mp 163–166 ꢁC, white solid. 1H
NMR (500 MHz, CDCl3): d 1.22 (s, 3H), 2.51 (m, 2H,
H4), 2.52 (s, 3H, N–CH3), 3.72 (s, 2H), 5.70 (d, 1H,
J ¼ 7:5 Hz), 5.96 (dd, 1H, J ¼ 5:1 and 7.3 Hz) 7.65 (d,
1H, J ¼ 7:5 Hz); 13C NMR (75 MHz, CDCl3): d 14.10,
36.60, 37.20, 51.01, 72.09, 99.90, 108.80, 111.30, 158.20,
164.06. HRMS (FAB) calcd for [Mþ] C10H16N4O3
240.2630, found 240.2627. Anal. Calcd for C10H16N4O3:
C, 49.99%; H, 6.71%; N, 21.62%. Found: C, 49.89%; H,
6.58%; N, 21.43%. The second eluted product was
(3SR,5SR)-4-amino-1-(3-hydroxymethyl-2,3-dimethyl-
isoxazolidin-5-yl)1H-pyrimidin-2-one 14b, crystallized
4.6.4. 9-[3-(Hydroxymethyl)-2,3-dimethylisoxazolidin-5-
yl]-9H-purin-6-amine (14d and 15d). Starting from 13a–
b, compounds 14d and 15d in 40:60 ratio (40% global
yield) were obtained and separated by flash chroma-
tography. The first eluted product was (3RS,5SR)9-[3-
(hydroxymethyl)-2,3-dimethyl-isoxazolidin-5-yl]-9H-pu-
1
rin-6-amine 15d, as a sticky oil. H NMR (300 MHz,
D2O): d 1.09 (s, 3H), 2.62 (s, 3H, N–CH3), 2.98 (m, 2H),
3.59 (s, 2H), 6.21 (dd, 1H, J ¼ 4:2 and 7.9 Hz), 8.26 (s,
1H), 8.33 (s, 1H); 13C NMR (75 MHz, D2O): d
15.30,38.01, 41.30, 62.50, 64.70, 84.30, 120.90, 144.73,
148.90, 153.35, 155.40. HRMS (FAB) calcd for [Mþ]
C11H16N6O2 264.2860, found 264.2855. Anal. Calcd for
C11H16N6O2: C, 49.99%; H, 6.10%; N, 37.80%. Found:
C, 49.82%; H, 6.01%; N, 37.72%. The second eluted
product
was
(3SR,5SR)9-[3-(hydroxymethyl)-2,3-

U. Chiacchio et al. / Bioorg. Med. Chem. 12 (2004) 3903–3909
3909
Chem. 1992, 267, 13938; (b) Grove, K. L.; Guo, X.; Lui, S.
H.; Gao, Z.; Chu, C. K.; Cheng, Y. C. Cancer Res. 1995,
55, 3008.
dimethyl-isoxazolidin-5-yl]-9H-purin-6-amine 14d as a
sticky oil. 1H NMR (300 MHz, D2O): d 1H NMR
(300 MHz, D2O): d 1.15 (s, 3H), 2.61 (s, 3H, N–CH3),
2.73 (dd, 1H, J ¼ 7:9 and 13.7 Hz H4a), 2.83 (dd, 1H
6. Nomura, M.; Shuto, S.; Tanaka, M.; Saaki, T.; Shuichi,
M.; Shigeta, S.; Matsuda, A. J. Med. Chem. 1999, 42,
2901.
J ¼ 4:9 and 13.7 Hz H4b), 3.62 (s, 2H), 6.22 (dd, 1H,
13
J ¼ 4:9 and 7.9 Hz), 8.15 (s, 1H), 8.21 (s, 1H);
C
7. (a) Takenuki, K.; Matsuda, A.; Ueda, T.; Sasaki, T.; Fujii,
A.; Yamagami, K. J. Med. Chem. 1988, 31, 1063; (b)
Takenuki, K.; Matsuda, A.; Ueda, T.; Sasaki, T. J. Med.
Chem. 1991, 34, 812; (c) Fujii, A.; Yamagami, K.; Arita,
M.; Okumoto, T.; Sakata, S.; Matsuda, A.; Ueda, T.;
Sasaki, T. Cancer Res. 1991, 51, 2319; (d) Lin, T.-S.; Luo,
M. Z.; Liu, M. C.; Clarkatzenburg, R. H.; Cheng, Y. C.;
Prusoff, W. H.; Mancini, W. R.; Birnbaum, G. I.; Gabe,
E.; Giziewicz, J. J. Med. Chem. 1991, 34, 2607; (e) Baker,
C. H.; Banzon, J.; Bollinger, J. M.; Stubbe, J.; Samano, V.;
Robins, M. J. J. Med. Chem. 1991, 34, 1879; (f) Cory, A.
H.; Samano, V.; Robins, M. J.; Cory, J. G. Biochem.
Pharmacol. 1994, 47, 365.
8. (a) Matsuda, A.; Nakajima, Y.; Azuma, A.; Tanaka, M.;
Sasaki, T. J. Med. Chem. 1991, 34, 2917; (b) Azuma, A.;
Nakajima, Y.; Nishizono, N.; Minakava, N.; Suzuki, M.;
Hanaoka, K.; Kobayashi, T.; Sasaki, T.; Marsuda, A.
J. Med. Chem. 1993, 36, 4183; (c) Tanaka, M.; Matsuda,
A.; Terao, T.; Sasaki, T. Cancer Lett. 1992, 64, 67; (d)
Azuma, A.; Hanaoka, K.; Kurihara, A.; Kobayashi, T.;
Miyauchi, S.; Kamo, N.; Tanaka, M.; Sasaki, T.; Mat-
suda, A. J. Med. Chem. 1995, 38, 3391; (e) Matsuda, A.;
Azuma, A. Nucleos. Nucleot. 1995, 14, 461.
9. McCarthy, J. R.; Matthews, D. P.; Stemerick, D. M.;
Huber, E. W.; Bey, P.; Lippert, B. J.; Snyder, R. D.;
Sunkara, P. S. J. Am. Chem. Soc. 1991, 113, 7439.
10. Hattori, H.; Tanaka, M.; Fukushima, M.; Sasaki, T.;
Matsuda, A. J. Med. Chem. 1996, 39, 5005.
11. O-Yang, C.; Wu, H. Y.; Fraser-Smith, E. B.; Walker,
K. A. M. Tetrahedron Lett. 1992, 33, 37.
12. O-Yang, C.; Kurz, W.; Eugui, E. M.; McRoberts, M. J.;
Verheyden, J. P. H.; Kurz, L. J.; Walker, K. A. M.
Tetrahedron Lett. 1992, 33, 41.
NMR (75 MHz, D2O: d; 16.20, 37.98, 41.02, 61.01,
64.55, 84.10,120.66, 144.15, 147.20, 153.12, 155.36.
HRMS (FAB) calcd for [Mþ] C11H16N6O2 264.2860,
found 264.2856. Anal. Calcd for C11H16N6O2: C,
49.99%; H, 6.10%; N, 37.80%. Found: C, 49.87%; H,
5.99%; N, 37.67%.
4.7. Antiviral and cytotoxicity assay for HSV
The newly synthesized nucleosides were evaluated for
their activity against HSV-1 and HSV-2 by plaque
reduction assay in VERO cells using a methodology
reported in literature.20 Cytotoxicity assays were con-
ducted in rapidly dividing Vero cells, as reported.20
4.8. Evaluation of toxicity and apoptosis
Toxicity was evaluated by a standard viability assay,
using the trypan blue exclusion test. Normally, apop-
tosis was evaluated by morphological analysis of the
cells, performed following staining with acridine orange
as previously described.21 Briefly, over 600 cells, includ-
ing those showing typical apoptotic characteristics, were
counted using a fluorescence microscope. The identifi-
cation of apoptotic cells was based on the presence of
uniformly stained nuclei showing chromatin condensa-
tion and nuclear fragmentation. In some experiments,
apoptosis was detected by flow cytometric analysis of
isolated nuclei, following staining with propidium
iodide, on a Becton Dickinson FAC Scan Analytic Flow
Cytometer, as previously described.22
13. Kodama, E. I.; Kohgo, S.; Kitano, K.; Machida, H.;
Gatanaga, H.; Shigeta, S.; Matsuoka, M.; Ohrui, H.;
Mitsuya, H. Antimicrob. Agents Chemother. 2001, 45,
1539.
ꢀ
14. (a) Iannazzo, D.; Piperno, A.; Pistara, V.; Rescifina, A.;
Romeo, R. Tetrahedron 2002, 58, 581; (b) Chiacchio, U.;
Borrello, L.; Merino, P.; Iannazzo, D.; Piperno, A.;
Rescifina, A.; Richichi, B.; Romeo, G. Tetrahedron:
Asymmetry 2003, 14, 2419.
Acknowledgements
Financial supports by University of Messina, Catania and
MIUR (FIRB and PRIN) are gratefully acknowledged.
15. Chiacchio, U.; Corsaro, A.; Iannazzo, D.; Piperno, A.;
ꢀ
Pistara, V.; Rescifina, A.; Romeo, R.; Valveri, V.; Mas-
tino, A.; Romeo, G. J. Med. Chem. 2003, 46, 3696.
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