Research on L-Nucleosides. Synthesis and biological evaluation of a series of L- and D-2′,3′-Dideoxy-3′-[tris(methylthio)methyl]-β- pentofuranosyl nucleosides

Article abstract of DOI:10.1016/S0968-0896(02)00460-1

Novel nucleoside analogues of both D and L enantiomeric series were prepared by coupling reaction between a 2′,3′-dideoxy-3′-modified furanose moiety and four different nucleobases. Though in all cases anomeric mixtures of nucleosides were obtained, the p

Full text of DOI:10.1016/S0968-0896(02)00460-1

Bioorganic & Medicinal Chemistry 11 (2003) 357–366
Research on L-Nucleosides. Synthesis and Biological Evaluation of
a Series of L- and D-20,30-Dideoxy-30-[tris(methylthio)methyl]-
ꢀ-pentofuranosyl Nucleosides
Claudia Mugnaini,a Maurizio Botta,a,* Massimo Coletta,b Federico Corelli,a,*
Federico Focher,c Stefano Marini,b Michela Lucia Renzullia and Annalisa Verric
a
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via A. Moro, snc, I-53100 Siena, Italy
bIstituto di Genetica Molecolare, CNR, via Abbiategrasso 207, I-27100 Pavia, Italy
c
`
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Universita di Roma Tor Vergata,
via Montpellier 1, I-00133 Rome, Italy
Received 16June 2002; accepted 19 September 2002
Abstract—Novel nucleoside analogues of both d and l enantiomeric series were prepared by coupling reaction between a
20,30-dideoxy-30-modified furanose moiety and four different nucleobases. Though in all cases anomeric mixtures of nucleosides
were obtained, the presence of the sterically bulky 30-tris(methylthio)methyl group allowed a good stereoselectivity level. All the
compounds of both enantiomeric series showed high IC50 values as HSV-1 TK inhibitors and scarce ability to be phosphorylated by
HSV-1 TK. In order to overcome possible problems related to the first phosphorylation step and to facilitate the penetration of the
molecule through the cellular membrane, a monophosphate prodrug containing a long lipophilic chain was synthesized. No
appreciable antiviral activity was exhibited by this molecule.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
against human immunodeficiency virus type 1 (HIV-1)
and hepatitis B virus (HBV) compared to their d-coun-
terparts while exhibiting less toxicity.4 Although only
one l-nucleoside (Lamivudine) has so far gained
approval by FDA for the treatment of AIDS, other
Nucleoside analogues have been the cornerstone of
antiviral therapy over the past 30 years1 and a variety of
strategies have been exploited to design new nucleoside
analogues able to block viral replication without affect-
ing host cellular processes.2 However, the toxicities
associated with certain nucleoside analogues and the
emergence of resistant viral strains warrant the search for
further novel and structurally diverse compounds with
minimally overlapping resistance profile and toxicity.3
It was long supposed that only nucleosides with the
natural b-d-configuration at the 40-position would be
substrates for enzymatic phosphorylation and hence
able to act as antiviral agents. As a result, only sporadic
examples of b-l-nucleosides were reported in the litera-
ture until the early 1990s, when certain l-nucleosides
like 3TC (l-20,30-dideoxy-30-thiacytidine, Lamivudine,
1) and FTC (l-20,30-dideoxy-30-thia-5-fluorocytidine, 2)
(Fig. 1) proved to possess more potent antiviral activity
*Corresponding authors. M. Botta; tel.: +39-0577-234306; fax: +39-
0577-234333; F. Corelli; tel.: +39-0577-234308; fax: +39-0577-
234333; e-mail: botta@unisi.it (M. Botta); corelli@unisi.it (F. Corelli).
Figure 1.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00460-1

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C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
l-enantiomers of nucleoside analogues are currently
under development as antiviral or antitumor agents.5
Therefore, it is of interest to investigate new l-nucleo-
sides in search of antiviral agents with favorable toxicity
profile.6
coupling reaction between pyrimidine bases and
the sugar moiety
v. the tris(methylthio)methyl group is chemically
versatile and can be transformed into different
functionalities.
As part of our efforts to develop new antiviral agents,7
we decided to synthesize a series of l-nucleosides of
general structure 3, characterized by the presence of a
tris(methylthio)methyl group at the 30-position of the
sugar moiety, in order to test them as potential antiviral
agents against herpes simplex virus (HSV) in compar-
ison with their d-enantiomers 4. Several considerations
prompted us to select this type of compounds as syn-
thetic target:
Results and Discussion
Chemistry
For the synthesis of the target nucleoside analogues we
adopted a convergent approach9 based on the coupling
reaction between an appropriately activated sugar moiety
and a silylated pyrimidine base, according to the Vor-
bruggen conditions.10 The a,b-unsaturated lactones 5a
(l-enantiomer) and 5b (d-enantiomer) (Scheme 1) were
prepared following known procedures11 and exploiting
an improved one-pot methodology for the direct con-
version of (R)- and (S)-2,3-O-isopropylideneglycerol into
i. the modification of the pentofuranose ring is one
of the most effective among all the strategies
devised to design new nucleoside analogues
ii. compounds 3 and 4 can be considered as
20,30-dideoxynucleosides just like the majority of
the nucleoside antivirals approved so far
iii. introduction of electronwithdrawing sub-
stituents, such as azido and fluorine, at the
30-position has often enhanced the antiviral
potency8
iv. the presence of a bulky group at the 30-position
can improve the b/a anomeric ratio during the
ethyl
(Z)-3-[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]-2-pro-
penoate and the corresponding (4S)-enantiomer, respec-
tively.12 Starting from 5a, compound 6a was prepared in
81% yield using the same conditions reported by
Hanessian et al.13 for the synthesis of 6b from 5b.
Reduction with DIBAL of the lactones 6a,b to lactols
7a,b, followed by acetylation under standard conditions,
led to the 30-substituted sugar derivatives 8a and 8b,
suitably protected and activated for the coupling step, in
62% overall yield.
Scheme 1.

C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
359
The synthesis of 20-deoxynucleosides represents a rather
difficult task in nucleoside chemistry, since derivatives
of 20-deoxysugars are often unstable and their nucleo-
sidation reaction frequently suffers from low total yield
and scarce stereoselectivity, that leads to unfavorable
ratios between the natural b-anomer and the undesired
a-epimer.10 For the preparation of the target nucleoside
analogues 3 and 4, the Vorbruggen approach was fol-
lowed to couple the activated sugar 8a and 8b with per-
silylated uracil, 5-fluorouracil, thymine, and cytosine,
After chromatographic separation of the anomers,
deprotection of l-b-9a–d and d-b-10a–d with TBAF in
THF afforded the final nucleoside analogues 3a–d and
4a–d in 90–95% yield. The corresponding a-anomers
were not further considered and hence not subjected to
the deprotection step.
As a consequence of the biological testing of these
compounds (see below), the question arose whether
their modest antiviral activity might be due to a low rate
and/or extent of conversion to their active (triphos-
phate) forms by cellular enzymes rather than to the
scarce affinity of the triphosphates for the target
enzyme. Since one way to improve the efficiency of
nucleosides should be to bypass at least in part these
phosphorylation steps, we decided to prepare the
monophosphate prodrug 11 (Scheme 3) in which the
phosphate is modified by a hydrophobic moiety in order
to enhance its cell penetration capability.17 As the
nucleoside substrate to be subjected to this strategy we
chose compound 3d because it is easily available as the
b-anomer and shows an interesting antiviral profile. To
this end 9d was N-benzoylated using 4-nitrophenyl
benzoate to give 12 which was selectively deprotected to
give 13. According to the method of nucleoside phos-
phorylation based on phosphoramidite reagents,18
2-cyanoethyl N,N,N0,N0-tetraisopropylphosphoramidite
1417,19 was prepared and used without purification in
the next step. Reaction of 14 with the N-protected
nucleoside 13 in the presence of tetrazole gave the
phosphite triester 15, which was not isolated but directly
oxidized to the corresponding phosphate 16. Although
different reagents have been reported in the literature to
accomplish this oxidation step,17,20 the remarkable
using
trimethylsilyl
trifluoromethanesulfonate
(TMSOTf) as a catalyst in acetonitrile (Scheme 2). All
of the nucleobases employed underwent N-glycosylation
reaction to give protected nucleosides 9a–d and 10a–d as
anomeric mixtures, which could be separated into indi-
vidual anomers by chromatography. In all cases the
overall yield of the coupling reaction was good, ranging
from 55 to 77%, and comparable to those reported in
the literature for other 20-deoxynucleoside analogues,
while the b/a ratios (4/1 to 11/1) were far higher than
those usually obtained for 20-unsubstituted nucleosides
(b/a ratioꢀ1/1),8,14 thus definitely demonstrating the
positive influence of the bulky erythro-30-substituent on
the stereochemical outcome of the reaction.
The structure of a- and b-anomers was assigned on the
basis of their proton NMR spectra, according to the
method previously used by Okabe et al.14 for d-nucleo-
sides and by Lin et al.15 for l-nucleosides. The H-40 pro-
ton of the a-anomer appears at a lower field than that of
the b-anomer, while the H-50 protons of the a-anomers
appear at a higher field than those of the b-anomers.
Contrary to previous findings,16 no relation was found
between anomers stereochemistry and polarity.
Scheme 2. a: R1=OH, R2=H; b: R1=OH, R2=F; c: R1=OH, R2=Me; d: R1=NH2, R2=H.

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C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
Scheme 3.
reactivity of the tris(methylthio)methyl group toward
oxidizing agents and thiophilic promoters suggested the
use of a reagent able to selectively oxidize the phosphite
moiety. According to a very recent literature report,21
ethyl(methyl)dioxirane perfectly met this requirement
and allowed to obtain phosphate 16 in 23% overall
yield. Treatment of 16 with concentrated aqueous
ammonia overnight, followed by TLC purification,
afforded the hexadecyl phosphate 11 (ammonium salt)
in 20% yield.
Based on these findings, the synthesized compounds
l-b-9a–d and d-b-10a–d were evaluated in vitro for their
cytotoxicity against normal lymphocytes and different
tumor lines [NSO cells (plasmocytoma murine cell line),
3T3 cells (murine fibroblast line), and Daudi cells
(human lymphoblastoid cell line)] as well as against
HSV-1 TK and the results are shown in Table 1. All of
the tested compounds proved to be not cytotoxic
against normal and tumor cell lines, with ED50 values
approximately in the range 100–400 mM. On the other
hand, none of the synthesized nucleoside analogues
exhibited any ability to act as substrates or inhibitors of
human thymidine phosphorylase (data not shown), a
potential target for tumor-dependent angiogenesis.25 As
a result, these compounds are not promising as poten-
tial antitumor agents.
Biological studies
The remarkable anti-HSV activity of several nucleoside
analogues, such as Acyclovir and Gancyclovir, is ulti-
mately due to a specific interaction of their 50-triphos-
phates with the viral DNA polymerase, but relies on
their prior phosphorylation to nucleoside mono- or
diphosphates by the virally encoded thymidine kinase
(TK).5c The HSV TKs differ from those of animal cells
both in their greater affinity for the substrate thymidine
(and deoxycytidine) and their wide acceptance of
nucleosides modified either in the pyrimidine ring or in
the sugar moiety as alternative substrates.22 The ability
of purified HSV TKs to recognize and phosphorylate in
vitro the l-enantiomer of the naturally occurring sub-
strate d-b-deoxythymidine has been investigated by
some of us.23 On the other hand, Balzarini et al. studied
the interaction of the same enzyme with the d- and
l-enantiomers of the carbocyclic analogues of (E)-5-(2-
As far as the antiviral activity is concerned, in most
cases l-enantiomers were found to be more potent
Table 1. Biological evaluation of compounds 3–4
Compd
Cytotoxicity assay
Anti-HSV-1 TK
assay
NLa
NSOb
IC50 (mM)
ED50 (mM)
ED50 (mM)
3a
3b
3c
3d
4a
4b
4c
4d
332
207
76105
314
240
205
207
234
398
230
No inhibition
35
227
383
330
77
275
320
45
No inhibition
167
No inhibition
No inhibition
bromovinyl)-20-deoxyuridine
and
5-iodo-20-deoxy-
uridine.24 Both groups demonstrated that HSV TK,
contrary to human TK, is not enantioselective, being
able to phosphorylate with similar efficiency both d-
and l-enantiomers of the tested nucleosides.
aNL, normal lymphocytes.
bNSO, plasmocytoma murine cell line.

C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
361
HSV-1 TK inhibitors than the corresponding d-counter-
parts: in particular l-b-3b and d-b-4b, having 5-fluoro-
uracil as the nucleobase, emerged as the most interesting
compound within both enantiomeric series. However,
when tested as possible substrates of viral TK, following
the in vitro assay and HPLC analysis described in
Experimental, these nucleoside analogues were found
resistant to the phosphorylation activity of the enzyme.
In fact, in assay conditions which allow the phos-
phorylation of 1.2 nmol of the natural substrate thymi-
dine, we did not observe any significant phosphorylation
of the tested nucleoside analogues. Therefore, because of
their high IC50 values (35 and 167 mM, respectively) and
their inability to be phosphorylated by HSV-1 TK, we
did not perform further biological evaluation.26 In con-
clusion, the compounds which show IC50 values against
HSV-1 TK can be only considered non-substrate inhi-
bitors of the enzyme. To our disappointment, neither
phosphate prodrug 11 nor the fully protected triester 16
exhibited any appreciable antiviral activity: they did not
reduce either the incorporation of exogenous [3H]thy-
midine into cellular DNA in HeLa TKꢁ/HSV-1 TK+ or
HSV-1 multiplication in HeLa cells following the meth-
ods described by Salvetti et al.22
Elemental analyses (C, H, N) were performed in house
using a Perkin–Elmer Elemental Analyzer 240C.
(4R,5R)-5-({[1-(tert-Butyl)-1,1-diphenylsilyl]oxy}methyl)-
4-[tri(methylthio)methyl]tetrahydro-furan-2-one (6a). To
a solution of tris(methylthio)methane (1.13 mL, 8.5
mmol) in anhydrous THF (100 mL) kept at ꢁ78 ꢂC
nBuLi (6.4 mL of a 1.6 M solution in hexane, 10.2
mmol) was added over 3–4 min. After 30 min a solution
of 5a (3.0 g, 8.5 mmol) in THF (20 mL) was added
dropwise and the reaction was stirred at ꢁ78 ꢂC for 1 h.
The mixture was then poured into Et2O (250 mL) and
washed with a saturated solution of NH4Cl (300 mL).
The aqueous phase was extracted with Et2O (3ꢃ200
mL) and the combined organic extracts were washed
with brine and dried over anhydrous Na2SO4. Filtration
and removal of the solvent gave 4.5 g of a white solid.
Purification of the crude mixture by column chroma-
tography (petroleum ether/Et2O, 3/1) afforded 6a (3.5 g,
81%) as a white solid: mp 140–142 ꢂC; [a]D20=ꢁ18.2 (c
1
1.10, CHCl3). IR (CHCl3): 1785, 1185, 1120 cmꢁ1. H
NMR (CDCl3): d 7.67–7.63 (m, 4H), 7.47–7.37 (m, 6H),
4.84 (ddd, J=2.7, 2.3, 2.3 Hz, 1H), 3.95 (dd, J=11.4,
2.7 Hz, 1H), 3.62 (dd, J=11.4, 2.3 Hz, 1H), 2.99 (ddd,
J=10.1, 2.8, 2.3 Hz, 1H), 2.95 (dd, J=18.3, 2.8 Hz,
1H), 2.83 (dd, J=18.3, 10.1 Hz, 1H), 2.11 (s, 9H), 1.04
(s, 9H). Anal. calcd for C25H34O3S3Si: C, 59.25; H, 6.76.
Found: C, 59.41; H, 6.74.
Conclusion
In summary, a novel class of l- and d-configuration
nucleoside analogues has been synthesized, which pos-
sess a modified sugar moiety characterized by a bulky
tris(methylthio)methyl group at the 30-position. The
steric hindrance exerted by this group caused the
nucleosidation reaction to occur with a much higher
stereoselectivity than that usually observed for other
20-deoxynucleosides. Although the biological activity of
these compounds is not exciting, it might be possible to
obtain new derivatives endowed with better antiviral
and/or antitumor properties by modifying the chemi-
cally reactive tris(methylthio)methyl group. Studies in
this direction are in progress.
(4R,5R)-5-({[1-(tert-Butyl)-1,1-diphenylsilyl]oxy}methyl)-
4-[tri(methylthio)methyl]tetrahydro-furan-2-ol (7a). To a
solution of 6a (1.35 g, 3.0 mmol) in anhydrous toluene
(37 mL), maintained at ꢁ70 ꢂC, a 1.0 M solution of
DIBAL in hexane (7.1 mL, 7.12 mmol) was added
dropwise during 5 min. The mixture was stirred for 1 h
at ꢁ70 ꢂC, then MeOH (50 mL) was added, and the
mixture allowed to warm to room temperature. The
resulting white precipitate was filtered off and washed
with MeOH. The filtrates were combined and evapo-
rated to give a pale yellow oil (1.3 g, 96%) which was
used without any further purification: [a]2D0=ꢁ12.5
(c 4.40, CHCl3). IR (CHCl3): 3409, 3007, 2951, 2862,
1
1731, 1424 cmꢁ1. H NMR (CDCl3): d 7.72–7.64 (m,
4H), 7.44–7.36(m, 6H), 5.58–5.46(m, 1H), 4.45–4.40
(m, 1H), 4.08 (dd, J=9.2, 3.0 Hz, 1H), 3.90 (dd, J=2.5,
11.3 Hz, 1H), 3.66 (dd, J=2.9, 10.9 Hz, 1H), 3.12–3.01
(m, 1H), 2.59–2.43 (m, 1H), 2.08 (s, 9H), 1.10 (s, 9H).
Experimental
Chemistry
Unless otherwise stated, all reactions were carried out
under an argon atmosphere. Reagents were obtained
from commercial suppliers and used without further
purifications. Merck silica gel 60 was used for both col-
umn chromatogaphy (70–230 mesh) and flash chroma-
tography (230–400 mesh). Melting points were
determined in open capillary tubes on a Gallenkamp
(4S,5S)-5-({[1-(tert-Butyl)-1,1-diphenylsilyl]oxy}methyl)-
4-[tri(methylthio)methyl]tetrahydrofuran-2-ol (7b). Pre-
pared in 95% yield starting from 6b using the same
procedure described for the synthesis of 7a:
[a]2D0=+12.1 (c 4.40, CHCl3).
1
apparatus and are uncorrected. H NMR spectra were
(4R,5R)-5-({[1-(tert-Butyl)-1,1-diphenylsilyl]oxy}methyl)-
4-[tri(methylthio)methyl]tetrahydrofuran-2-yl
measured at 200 MHz with a Bruker AC200F spectro-
meter. Chemical shifts are reported relative to CDCl3 at
d 7.24 ppm and tetramethylsilane at d 0.00 ppm. Infra-
red spectra were recorded on a Perkin–Elmer 1600
FT-IR spectrophotometer. EI and FAB low-resolution
mass spectra were recorded with an electron beam of 70 eV
using a VG 70–250S instrument. Specific rotations were
measured on a Perkin–Elmer Model 343 polarimeter.
acetate
(8a). Lactol 7a (3 g, 5.9 mmol) was dissolved in anhy-
drous CH2Cl2 (14 mL) and then pyridine (4 mL) was
added. Freshly distilled acetyl chloride (1.3 mL, 18.3
mmol) was slowly added to this solution at 0 ꢂC. The
resulting white suspension was stirred at room tem-
perature for 1.5 h. The reaction was then quenched with
saturated aqueous NH4Cl solution (5 mL). The mixture

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C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
was extracted with CH2Cl2 (3ꢃ6mL) and the combined
extract was washed with brine and dried over anhy-
drous Na2SO4. Concentration in vacuo gave 4 g of a
yellow oil. The crude material was subjected to column
chromatography (petroleum ether/AcOEt, 8/1) to pro-
vide 8a (2.1 g, 65%) as a colourless syrup: [a]2D0=ꢁ25.4
(c 3.74, CHCl3). IR (CHCl3): 3682, 3494, 3018, 2860,
J=2.5, 11.7 Hz, 1H), 3.04–2.87 (m, 3H), 2.10 (s, 9H),
1.08 (s, 9H). MS (FAB) m/z 603 [M+H+]. Anal. calcd
for C29H38N2O4S3Si: C, 57.77; H, 6.35; N, 4.65. Found:
C, 57.62; H, 6.49; N, 4.32.
1
L-a-9a. Colourless oil. Rf 0.2. H NMR (CDCl3) d
1
1711, 1427 cmꢁ1. H NMR (CDCl3): d 7.71–7.68 (m,
7.75 (d, J=8.2 Hz, 1H), 7.67–7.59 (m, 4H), 7.49–7.33
(m, 6H), 6.35–6.32 (m, 1H), 5.75 (d, J=7.9 Hz, 1H),
4.46–4.45 (m, 1H), 3.92 (dd, J=11.6, 1.7 Hz, 1H), 3.69
(dd, J=11.5, 3.1 Hz, 1H), 3.18–3.11 (m, 1H), 2.64–2.56
(m, 1H), 2.30–2.21(m, 1H), 2.12 (s, 9H), 1.07 (s, 9H).
MS (FAB) m/z 603 [M+H+]. Anal. calcd for
C29H38N2O4S3Si: C, 57.77; H, 6.35; N, 4.65. Found: C,
57.58; H, 6.21; N, 4.46.
4H), 7.39–7.32 (m, 6H), 6.40–6.31 (m, 1H), 4.50–4.47
(m, 1H), 4.08 (dd, J=2.7, 10.6Hz, 1H), 3.84 (dd,
J=2.7, 11.3 Hz, 1H), 3.27–3.15 (m, 1H), 3.05–2.94 (m,
1H), 2.79–2.65 (m, 1H), 2.16 (s, 9H), 2.14 (s, 9H), 2.06
(s, 3H), 1.82 (s, 3H), 1.07 (s, 9H), 1.05 (s, 9H). Anal.
calcd for C27H38O4S3Si: C, 58.87; H, 6.95. Found: C,
58.69; H, 7.10.
(4S,5S)-5-({[1-(tert-Butyl)-1,1-diphenylsilyl]oxy}methyl)-
4-[tri(methylthio)methyl]tetrahydrofuran-2-yl acetate (8b).
Prepared in 70% yield from 7b as described for 8a: [a]
2D0=+23.9 (c 3.74, CHCl3). Anal. calcd for
C27H38O4S3Si: C, 58.87; H, 6.95. Found: C, 59.12; H,
6.74.
1-[50 -O-tert-Butyldiphenylsilyl-20,30 -dideoxy-30 -C-tris
(methylthio)methyl-ꢀ-D -erythro-pentofuranosyl]-uracil
(D-ꢀ-10a) and 1-[50-O-tert-butyldiphenylsilyl-20,30-di-
deoxy-30-C-tris(methylthio)methyl-ꢁ-D-erythro-pentofur-
anosyl]-uracil (D-ꢁ-10a). These compounds were
prepared in 66 and 10% yield, respectively, as described
for l-b-9a starting from 8b and uracil.
General procedure for the synthesis of nucleosides L–ꢀ-
9a–d and D-ꢀ-10a–d
D-ꢀ-10a. White solid, mp 64–66 ꢂC; [a]D20=+44.5 (c
1.41, CHCl3). MS (FAB) m/z 603 [M+H+]. Anal. calcd
for C29H38N2O4S3Si: C, 57.77; H, 6.35; N, 4.65. Found:
C, 57.88; H, 6.31; N, 4.48.
The appropriate nucleobase (2.2 mmol) was treated
with a large excess of HMDS (6mL) and a catalytic
amount of anhydrous (NH4)2SO4. The white suspension
was refluxed for 2–4 h giving a colourless solution.
Removal of HMDS by codistillation under reduced
pressure with dry toluene (3ꢃ1 mL) gave a white solid
residue. The solid was dissolved in dry CH3CN (6mL),
the solution was cooled to 0 ꢂC and TMSOTf (330 mL,
1.6mmol) was added dropwise to the mixture. Finally,
a solution of 8 (1.1 mmol) in dry CH3CN (3 mL) was
added and the reaction was stirred at room temperature
for 45 min. The mixture was then quenched with a
saturated aqueous solution of NaHCO3 (10 mL) and
extracted with CHCl3 (3ꢃ15 mL). The combined
organic phase was then washed with brine, dried on
anhydrous Na2SO4 and filtered. Evaporation of the
solvent followed by column chromatography gave the
crude nucleoside.
D-ꢁ-10a. Colourless oil. MS (FAB) m/z 603 [M+H+].
Anal. calcd for C29H38N2O4S3Si: C, 57.77; H, 6.35; N,
4.65. Found: C, 57.99; H, 6.24; N, 4.79.
1-[50-O-tert-Butyldiphenylsilyl-20,30-dideoxy-30-C-tris(me-
thylthio)methyl-ꢀ-L -erythro-pentofuranosyl]-5-fluoro-
uracil (L-ꢀ-9b) and 1-[50-O-tert-butyldiphenylsilyl-20,30-
dideoxy-30 -C-tris(methylthio)methyl-ꢁ-L-erythro-pento-
furanosyl]-5-fluorouracil (L-ꢁ-9b). These compounds
were obtained in 54 and 10% yield, respectively, starting
from 8a and 5-fluorouracil after silica gel column
chromatography (CH2Cl2/MeOH, 94/6) followed by
preparative TLC (CH2Cl2/MeOH, 92/8).
L-ꢀ-9b. White solid, mp 73–76 ꢂC (dec). Rf 0.5.
[a]2D0=ꢁ33.6( c 0.69, CHCl3/MeOH, 3/2). IR (CHCl3):
1-[50 -O-tert-Butyldiphenylsilyl-20,30 -dideoxy-30 -C-tris
(methylthio)methyl-ꢀ-L -erythro-pentofuranosyl]-uracil
(L-ꢀ-9a) and 1-[50-O-tert-butyldiphenylsilyl-20,30-dideoxy
- 30 - C - tris(methylthio)methyl - ꢁ - L - erythro - pento-
furanosyl]-uracil (L-ꢁ-9a). These compounds were
obtained starting from 8a and uracil in 63 and 11%
yield, respectively, after silica gel chromatography
(CHCl3/MeOH, 98/2) followed by further purifica-
tion by preparative TLC (three runs, CHCl3/MeOH,
95/5).
1
3012, 2926, 1703, 1465, 1427 cmꢁ1. H NMR (CDCl3):
d 8.01 (d, J=6.0 Hz, 1H), 7.67–7.62 (m, 4H), 7.47–
7.33 (m, 6H), 6.42–6.35 (m, 1H), 4.47–4.45 (m, 1H),
4.06(dd, J=11.8, 1.6Hz, 1H), 3.69 (dd, J=11.7, 2.8
Hz, 1H), 3.08–2.90 (m, 1H), 2.25–2.20 (m, 1H), 2.08–
2.01 (m+s, 1H+9H), 1.08 (s, 9H). MS (FAB) m/z
621 [M+H+]. Anal. calcd for C29H37FN2O4S3Si: C,
56.10; H, 6.01; N, 4.51. Found: C, 56.27; H, 6.12; N,
4.69.
L-ꢀ-9a. White solid, mp 65–67 ꢂC. Rf 0.3.
[a]2D0=ꢁ43.8 (c 1.14, CHCl3). IR (CHCl3): 4201, 3025,
1689 cmꢁ1. 1H NMR (CDCl3): d 9.18 (br s, 1H), 7.88 (d,
J=8.4 Hz, 1H), 7.65–7.59 (m, 4H), 7.41–7.37 (m, 6H),
6.40 (t, J=6.6 Hz, 1H), 5.39 (d, J=8.1 Hz, 1H), 4.45–
4.44 (m, 1H), 4.08 (dd, J=1.5, 11.8 Hz 1H), 3.76(dd,
L-a-9b. Colourless oil. Rf 0.5. H NMR (CDCl3): d
1
7.87 (d, J=6.6 Hz, 1H), 7.67–7.62 (m, 4H), 7.47–7.33
(m, 6H), 6.42–6.35 (m, 1H), 4.47–4.45 (m, 1H), 3.92 (dd,
J=1.8, 11.1 Hz 1H), 3.65 (dd, J=2.8, 11.5 Hz, 1H),
3.20–3.11 (m, 1H), 3.08–2.90 (m, 1H), 2.67–2.60 (m,
1H), 2.12 (s, 9H), 1.07 (s, 9H). MS (FAB) m/z 621

C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
363
[M+H+]. Anal. calcd for C29H37FN2O4S3Si: C, 56.10;
H, 6.01; N, 4.51. Found: C, 56.31; H, 6.13; N, 4.37.
D-ꢀ-10c. [a]2D0=+25.0 (c 1.90, CHCl3). MS (FAB) m/
z 617 [M+H+]. Anal. calcd for C30H40N2O4S3Si: C,
58.41; H, 6.54; N, 4.54. Found: C, 58.25; H, 6.68; N,
4.72.
1-[50-O-tert-Butyldiphenylsilyl-20,30-dideoxy-30-C-tris(me-
thylthio)methyl-ꢀ-D -erythro-pentofuranosyl]-5-fluoro-
uracil (D-ꢀ-10b) and 1-[50-O-tert-butyldiphenylsilyl-20,30-
dideoxy-30-C-tris(methylthio)methyl-ꢁ-D-erythro-pento-
furanosyl]-5-fluorouracil (D-ꢁ-10b). Prepared in 53 and
10% yield, respectively, as described for l-b-9b starting
from 8b and 5-fluorouracil.
D-a-10c. MS (FAB) m/z 617 [M+H+]. Anal. calcd
for C30H40N2O4S3Si: C, 58.41; H, 6.54; N, 4.54. Found:
C, 58.09; H, 6.33; N, 4.75.
1-[50-O-tert-Butyldiphenylsilyl-20,30-dideoxy-30-C-tris(me-
thylthio)methyl - ꢀ - L - erythro - pentofuranosyl] - cytosine
(L-ꢀ-9d) and 1-[50-O-tert-butyldiphenylsilyl-20,30-dideoxy -
30 -C-tris(methylthio)methyl-ꢁ-L-erythro-pentofuranosyl]-
cytosine (L-ꢁ-9d). Obtained by reacting 8a with cyto-
sine in 51 and 4.5% yield, respectively, after purifica-
tion by silica gel chromatography (CHCl3/MeOH, 9/1)
followed by preparative TLC (six runs, CHCl3/MeOH,
9/1).
D-ꢀ-10b. White solid, mp 75–77 ꢂC (dec);
[a]2D0=+39.2 (c 0.71, CHCl3/MeOH, 3/2). MS (FAB)
m/z 621 [M+H+]. Anal. calcd for C29H37FN2O4S3Si:
C, 56.10; H, 6.01; N, 4.51. Found: C, 56.41; H, 6.13; N,
4.25.
D-a-10b. MS (FAB) m/z 621 [M+H+]. Anal. calcd
for C29H37FN2O4S3Si: C, 56.10; H, 6.01; N, 4.51.
Found: C, 55.94; H, 6.22; N, 4.39.
L-ꢀ-9d. White solid, mp 96 ꢂC (dec); Rf 0.5.
[a]2D0=ꢁ59.5 (c 0.42, CHCl3). IR (CHCl3): 3419, 3022,
1-[50 -O-tert-Butyldiphenylsilyl-20,30 -dideoxy-30 -C-tris
(methylthio)methyl-ꢀ-L-erythro-pentofuranosyl]-thymine
(L-ꢀ-9c) and 1-[50-O-tert-butyldiphenylsilyl-20,30-dideoxy
- 30 - C - tris(methylthio)methyl - ꢁ - L - erythro - pento-
furanosyl]-thymine (L-ꢁ-9c). Prepared in 55 and 14%
yield, respectively, starting from 8a and thymine after
purification by silica gel chromatography (CHCl3/
MeOH, 99/1) followed by preparative TLC (five runs,
CHCl3/MeOH, 98/2).
2399, 2346, 1643, 1478 cmꢁ1. H NMR (CDCl3): d 7.97
1
(d, J=7.2 Hz, 1H), 7.64–7.61 (m, 4H), 7.40–7.32 (m,
6H), 6.37–6.31 (m, 1H), 5.38 (d, J=7.5 Hz, 1H), 4.45–
4.44 (m, 1H), 4.06(dd, J=1.5, 11.6Hz, 1H), 3.78 (dd,
J=2.8, 11.6, 1H), 3.05–2.89 (m, 2H), 2.25–1.73 (m+s,
1H+1H), 1.06(s, 9H). MS (FAB) m/z 602 [M+H+].
Anal. calcd for C29H39N3O3S3Si: C, 57.87; H, 6.53; N,
6.98. Found: C, 57.65; H, 6.70; N, 6.62.
1
L-a-9d. Colourless oil. Rf 0.4. H NMR (CDCl3): d
L-ꢀ-9c. White solid, mp 56 ꢂC (dec); Rf 0.3; [a]D20=
ꢁ52.0 (c 2.50, CHCl3). IR (CHCl3): 4214, 3379, 3009,
7.76(d, J=6.9 Hz, 1H), 7.66–7.60 (m, 4H), 7.46–7.32
(m, 6H), 6.34–6.15 (m, 1H), 5.75 (d, J=6.9 Hz, 1H),
4.48 (m, 1H), 3.89 (dd, J=11.1, 2.5 Hz, 1H), 3.67 (dd,
J=10.9, 2.6Hz, 1H), 3.05–2.92 (m, 2H), 2.09 (s, 10H),
1.04 (s, 9H). MS (FAB) m/z 602 [M+H+]. Anal. calcd
for C29H39N3O3S3Si: C, 57.87; H, 6.53; N, 6.98. Found:
C, 58.25; H, 6.43; N, 7.30.
1
1689 cmꢁ1. H NMR (CDCl3): d 8.55 (br s, 1H), 7.66–
7.63 (m, 4H), 7.57 (s, 1H), 7.45–7.27 (m, 6H), 6.44 (dd,
J=6.5, 8.0 Hz, 1H), 4.47–4.46 (m, 1H), 4.09 (dd, J=1.3,
11.6Hz, 1H), 3.75 (dd, J=2.2, 11.2 Hz 1H), 3.17–3.06
(m, 1H), 2.88 (ddd, J=1.6, 5.9, 13.8 Hz, 1H), 2.12 (s,
9H), 2.05–1.93 (m, 1H), 1.59 (s, 3H), 1.09 (s, 9H). MS
(FAB) m/z 617 [M+H+]. Anal. calcd for
C30H40N2O4S3Si: C, 58.41; H, 6.54; N, 4.54. Found: C,
58.25; H, 6.69; N, 4.39.
1-[50 -O-tert-Butyldiphenylsilyl-20,30 -dideoxy-30 -C-tris
(methylthio)methyl-ꢀ-D-erythro-pentofuranosyl]-cytosine
(D-ꢀ-10d) and 1-[50-O-tert-butyldiphenylsilyl-20,30-di-
deoxy-30-C-tris(methylthio)methyl-ꢁ-D-erythro-pentofur-
anosyl]-cytosine (D-ꢁ-10d). Prepared in 57 and 5%
yield, respectively, as described for l-b-9d.
1
L-a-9c. Colourless oil. Rf 0.4. H NMR (CDCl3): d
9.05 (bs, 1H), 7.66–7.59 (m, 4H), 7.63 (s, 1H), 7.46–7.32
(m, 6H), 6.42–6.39 (m, 1H), 4.48–4.46 (m, 1H), 3.91 (dd,
J=10.9, 1.9 Hz, 1H), 3.67 (dd, J=12.6, 2.2 Hz, 1H),
3.13–3.11 (m, 1H), 2.59–2.52 (m, 1H), 2.33–2.17 (m, 1H),
2.12 (s, 9H), 1.93 (s, 3H), 1.07 (s, 9H). MS (FAB) m/z 617
[M+H+]. Anal. calcd for C30H40N2O4S3Si: C, 58.41; H,
6.54; N, 4.54. Found: C, 58.64; H, 6.43; N, 4.73.
D-ꢀ-10d. White solid, mp 94 ꢂC (dec); [a]D20=+58.4 (c
1.12, CHCl3). MS (FAB) m/z 602 [M+H+]. Anal. calcd
for C29H39N3O3S3Si: C, 57.87; H, 6.53; N, 6.98. Found:
C, 58.15; H, 6.33; N, 7.22.
1-[50-O-tert-Butyldiphenylsilyl-20,30-dideoxy-30-C-tris(me-
thylthio)methyl-ꢀ-D-erythro-pentofuranosyl]-thymine (D-
ꢀ-10c) and 1-[50-O-tert-butyldiphenylsilyl-20,30-dideoxy-
30-C-tris(methylthio)methyl-ꢁ-D-erythro-pentofuranosyl]-
thymine (D-ꢁ-10c). Prepared in 60 and 14% yield,
respectively, starting from 8b and thymine as described
for l-b-9c.
D-a-10d. Colourless oil. MS (FAB) m/z 602 [M+H+].
Anal. calcd for C29H39N3O3S3Si: C, 57.87; H, 6.53; N,
6.98. Found: C, 58.22; H, 6.32; N, 7.28.
General procedure for the synthesis of nucleosides L-ꢀ-
3a–d and D-ꢀ-4a–d

364
C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
A solution of 9 or 10 (0.12 mmol) in dry THF (1 mL)
was treated with a 1 M solution of TBAF in THF (0.28
mmol) and the mixture was stirred at room temperature
for 1 h. Evaporation of the solvent under reduced pres-
sure gave the crude nucleoside.
[M+H+]. Anal. calcd for C14H22N2O4S3: C, 44.42; H,
5.86; N, 7.40. Found: C, 44.64; H, 5.89; N, 7.37.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-D-erythro-
pentofuranosyl]-thymine (D-ꢀ-4c). Prepared in 98% yield
as described for l-b-3c: mp 180–182 ꢂC; [a]D20=+6.0 (c
0.38, CHCl3). MS (FAB) m/z 379 [M+H+]. Anal. calcd
for C14H22N2O4S3: C, 44.42; H, 5.86; N, 7.40. Found:
C, 44.57; H, 5.76; N, 7.22.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-L-erythro-
pentofuranosyl]-uracil (L-ꢀ-3a). This compound was
obtained in 95% yield starting from l-b-9a as a white
solid after purification by silica gel chromatography
(CHCl3/MeOH, 9/1): mp 212–214 ꢂC; [a]D20=ꢁ19.1 (c
0.31, CHCl3/MeOH, 3/2). IR (CHCl3): 3013, 2392,
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-L-erythro-
pentofuranosyl]-cytosine (L-ꢀ-3d). Prepared in 90% yield
starting from l-b-9d. White solid after purification by
silica gel chromatography (CHCl3/MeOH, 9/1): mp
69 ꢂC (dec); [a]D20=ꢁ25.0 (c 0.60, CHCl3). IR (CHCl3):
4214, 3406, 3327, 3009, 1650 cmꢁ1. 1H NMR (CDCl3): d
7.77 (d, J=7.2 Hz, 1H), 6.95 (br s, 1H), 6.15 (t, J=6.4
Hz, 1H), 5.82 (d, J=7.2 Hz, 1H), 4.48–4.43 (m, 1H),
3.95 (dd, J=2.1, 10.9 Hz, 1H), 3.74 (dd, J=1.8, 11.1
Hz, 1H), 3.31–3.12 (m, 1H), 3.11–2.92 (m, 1H), 2.87–
2.68 (m, 1H), 2.15 (s, 9H). MS (FAB) m/z 364
[M+H+]. Anal. calcd for C13H21N3O3S3: C, 42.95; H,
5.82; N, 11.56. Found: C, 42.74; H, 5.90; N, 11.39.
1
1687, 1518, 1462 cmꢁ1. H NMR (CDCl3+few drops
of CD3OD): d 7.69 (d, J=8.0 Hz, 1H), 6.25–6.15 (dd,
J=6.6, 13.3 Hz, 1H), 5.67 (d, J=8.1, 1H), 4.45–4.43
(m, 1H), 4.01 (dd, J=2.1, 11.3 Hz, 1H), 3.83 (dd,
J=2.3, 11.0 Hz, 1H), 3.09–2.99 (m, 1H), 2.95–2.78 (m,
1H), 2.75–2.53 (m, 1H), 2.19 (s, 9H). MS (FAB) m/z
365 [M+H+]. Anal. calcd for C13H20N2O4S3: C,
42.84; H, 5.53; N, 7.69. Found: C, 43.01; H, 5.62; N,
7.44.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-D-erythro-
pentofuranosyl]-uracil (D-ꢀ-4a). Prepared in 97% yield
as
described
for
l-b-3a:
mp
213–214 ꢂC;
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-D-erythro-
pentofuranosyl]-cytosine(D-ꢀ-4d). Prepared in 97% yield
as described for l-b-3d: mp 72 ꢂC (dec); [a]D20=+27.5 (c
0.69, CHCl3). MS (FAB) m/z 364 [M+H+]. Anal. calcd
for C13H21N3O3S3: C, 42.95; H, 5.82; N, 11.56. Found:
C, 42.72; H, 5.98; N, 11.69
[a]2D0=+21.3 (c 0.81, CHCl3/MeOH, 3/2). MS (FAB)
m/z 365 [M+H+]. Anal. calcd for C13H20N2O4S3:
C, 42.84; H, 5.53; N, 7.69. Found: C, 43.12; H,
5.43; N, 7.41.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-L-erythro-
pentofuranosyl]-5-fluorouracil (L-ꢀ-3b). Starting from
l-b-9b this compound was obtained in 72% yield as a
white solid after purification by silica gel chroma-
tography (CH2Cl2/MeOH, 92/8): mp 155–158 ꢂC;
[a]2D0=ꢁ39.5 (c 0.38, CHCl3). IR (CHCl3): 3378, 3020,
N-Benzoyl-1-[50-O-tert-butyldiphenylsilyl-20,30-dideoxy-
30-C-tris(methylthio)methyl-ꢀ-L-erythro-pentofuranosyl]-
cytosine (12). To a solution of 9d (201 mg, 0.33 mmol)
in anydrous DMF (5 mL) 4-nitrophenyl benzoate (121
mg, 0.49 mmol) and HOBt (23 mg, 0.16mmol) was
added and the reaction was stirred overnight at room
temperature. The reaction was quenched with aqueous
Na2CO3 (3 mL) and the mixture was extracted with
CH2Cl2 (3ꢃ3 mL). The combined organic layers were
then washed with small amounts of water (5ꢃ2 mL),
brine (6mL) and then dried over anydrous Na 2SO4.
Filtration and evaporation gave 250 mg of the crude
product which was purified by column chromatography
(CHCl3/MeOH, 97/3) to give 12 (170 mg, 73%) as a
white solid: mp 180 ꢂC (dec); [a]2D0=ꢁ29.1 (c 0.24,
1
2362, 1707, 1422 cmꢁ1. H NMR (CDCl3): d 8.39 (br
s, 1H), 8.02 (d, J=6.4 Hz, 1H), 6.29–6.22 (m, 1H),
4.49–4.45 (m, 1H), 4.05 (dd, J=1.8, 11.4 Hz, 1H),
3.86(dd, J=2.3, 11.3 Hz, 1H), 3.09–3.01 (m, 1H),
2.99–2.82 (m, 1H), 2.19–2.11 (m+s, 1H+9H). MS
(FAB) m/z 383 [M+H+]. Anal. calcd for
C13H19FN2O4S3: C, 40.82; H, 5.01; N, 7.32. Found:
C, 41.00; H, 5.12; N, 7.44.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-D-erythro-
pentofuranosyl]-5-fluorouracil (D-ꢀ-4b). Prepared in
81% yield as described for l-b-3b: mp 181–183 ꢂC;
[a]2D0=+38.2 (c 0.32, CHCl3). MS (FAB) m/z 383
[M+H+]. Anal. calcd for C13H19FN2O4S3: C, 40.82; H,
5.01; N, 7.32. Found: C, 40.62; H, 4.90; N, 7.19.
1
CHCl3). IR (CHCl3): 3025, 2402, 1660 cmꢁ1. H NMR
(CDCl3): d 8.36(d, J=7.6Hz, 1H), 7.89 (d, J=7.9 Hz,
1H+1H), 7.65–7.37 (m, 14H), 6.36 (t, J=6.2 Hz, 1H),
4.58–4.52 (m, 1H), 4.11–4.06(m, 1H), 3.80 (dd, J=3.2,
11.6Hz), 3.23–3.11 (m, 1H), 2.97–2.86(m, 1H), 2.17–
2.00 (s+m, 9H+ 1H), 1.09 (s, 9H). MS (FAB) m/z 707
[M+H+]. Anal. calcd for C36H43N3O4S3Si: C, 61.24;
H, 6.14; N, 5.95. Found: C, 60.90; H, 6.20; N, 5.68.
1-[20,30-Dideoxy-30-C-tris(methylthio)methyl-ꢀ-L-erythro-
pentofuranosyl]-thymine (L-ꢀ-3c). This compound was
obtained starting from l-b-9c in 90% yield as a white
solid after purification by silica gel chromatography
(CH2Cl2/MeOH, 94/6): mp 187 ꢂC (dec); [a]D20=ꢁ10.4 (c
0.48, CHCl3). IR (CHCl3): 4214, 3459, 3300, 3022, 1676,
N-Benzoyl-1-[20,30 -dideoxy-30 -C-tris(methylthio)methyl-
ꢀ-L-erythro-pentofuranosyl]-cytosine (13). To a solution
of 12 (163 mg, 0.23 mmol) in anydrous THF (1 mL) a
1 M solution of TBAF in THF (570 mL, 0.57 mmol) was
added and the reaction was stirred for 2 h. The solvent
was evaporated to give 380 mg of an orange oil which
was purified by column chromatography (CHCl3/
MeOH, 95/5) to give 13 (81 mg, 75%) as a white solid:
1
1464 cmꢁ1. H NMR (CDCl3): d 8.93 (br s, 1H), 7.44–
7.38 (m, 1H), 6.21–6.13 (m, 1H), 4.40–4.38 (m, 1H), 3.99
(dd, J=2.5, 11.6Hz, 1H), 3.77 (dd, J=3.2, 11.9 Hz,
1H), 3.12–3.00 (m, 1H), 2.97–2.71 (m, 1H), 2.34–2.18
(m+s, 1H+9H), 1.88 (s, 3H). MS (FAB) m/z 379

C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
365
mp 88 ꢂC (dec); [a]D20=+19.0 (c 0.21, CHCl3). IR
When nucleoside analogues were tested as possible sub-
strates of HSV-1 TK, 100mM of each compound was
incubated at 37 ꢂC for 20 min in in a mixture (25 mL)
containing 30 mM HEPES K+, pH 7.5, 6mM MgCl 2, 6
mM ATP, 0.5 mM dithiothreitol (DTT) and 10 mg of
HSV-1 TK. Samples were then heated at 100 ꢂC for 5
min and centrifuged 15 min at 10,000 rpm in an
Eppendorf bench fuge. Supernatants were transfered in
a new tube for subsequent HPLC analysis.
1
(CHCl3): 3025, 2403, 1696 cmꢁ1. H NMR (CDCl3): d
8.43 (d, J=7.5 Hz, 1H), 7.82 (d, J=7.3 Hz, 1H+1H),
7.57–7.38 (m, 4H), 6.21 (t, J=6.0 Hz, 1H), 4.57–4.55
(m, 1H), 4.10–4.04 (m, 1H), 3.87 (dd, J=3.0, 12.0 Hz,
1H), 3.08–2.94 (m, 2H), 2.33–2.07 (m+s, 1H+9H). MS
(FAB) m/z 468 [M+H+]. Anal. calcd for
C20H25N3O4S3: C, 51.37; H, 5.39; N, 8.99. Found: C,
51.72; H, 5.03; N, 9.32.
N-Benzoyl-1-[20,30-dideoxy-30-C-tris(methylthio)methyl-
ꢀ-L-erythro-pentofuranosyl]-cytosine-50-(2-cyanoethyl hex-
adecyl phosphate) (16). To a solution of 14 (55 mg, 0.13
mmol) in anhydrous THF (1 mL) a solution of 13 (63
mg, 0.13 mmol) in anhydrous THF (1 mL) and a 0.45 M
solution of tetrazole in CH3CN (290 mL, 0.13 mmol)
were added. The reaction mixture was stirred overnight
at room temperature and then 0.1 M solution of ethyl-
(methyl)dioxirane in CH2Cl2 (1.3 mL, 0.13 mmol) was
added. After 2 h the solvent was removed to give 184
mg of yellow oil which was purified by column chro-
matography (CHCl3/MeOH, 7/1) to afford 16 (25 mg,
23%) as a colourless oil: [a]2D0=ꢁ20.0 (c 0.50, CHCl3).
IR (CHCl3): 3024, 2399, 1709 cmꢁ1. 1H NMR (CDCl3):
d 8.22 (d, J=7.4 Hz, 1H), 7.90 (d, J=7.2 Hz, 1H+1H),
7.59–7.45 (m, 4H), 6.26 (t, J=5.4 Hz, 1H), 4.72–4.62
(m, 1H), 4.35–4.20 (m, 2H), 4.08–4.13 (m, 2H), 3.19–
3.02 (m, 1H), 3.82–3.01 (m, 1H), 2.8–2.71 (m, 2H), 2.20
(s, 9H), 2.04–2.12 (m, 1H), 1.58–1.63 (m, 2H), 1.24–1.22
(m, 28H), 0.87–0.78 (m, 3H). MS (FAB) m/z 826
[M+H+]. Anal. calcd for C39H61N4O7PS3: C, 56.77; H,
7.45; N, 6.79. Found: C, 57.05; H, 7.12; N, 7.12.
HPLC separation of nucleosides and nucleotides
The reversed-phase chromatography method employing
the HPLC system (SHIMADZU) was used in order to
separate nucleosides from nucleotides. A 4.6mm ꢃ25
cm ALLTIMA C18-NUC 100A 5U (Alltech) column
was used at room temperature in the following condi-
tions: injection volume, 20 mL; detection, UV 260 nm;
eluents of the linear gradient (40 min): buffer A (20 mM
KH2PO4, pH 7.5), buffer B (20 mM KH2PO4, pH 5.2,
60% methanol). Flow rate: 0.5 mL/min.
Cytotoxitity assay
Cell lines. All cell lines were obtained from ATCC. The
cells were cultured in RPMI 1640 supplemented with
5% FCS, 0.1 mM glutamine, 1% penicillin and strep-
tomycin. Cells were grown in Nunc clone plastic bottles
(TedNunc, Roskilde, Denmark) and split twice weekly
at different cell densities according to standard proce-
dures. 3T3 cells were grown as monolayer and were split
by using trypsin. Perypheral blood mononuclear cells
(MNC) were separated from heparinized whole blood
obtained from healthy donor on a Ficoll-Hypaque gra-
dient as previously described.27 MNC thus obtained
were washed twice with RPMI 1640 supplemented with
10% FCS, glutamine and antibiotics, suspended at
200,000 viable cells/mL in medium containing, as mito-
gen, 5 mg/mL PHA (Sigma) and used in toxicity tests.
Ammonium salt of 1-[20,30-dideoxy-30-C-tris(methylthio)-
methyl-ꢀ-L-erythro-pentofuranosyl]-cytosine-50 -(hexa-
decyl phosphate) (11). Treatment of 16 (20 mg, 0.024
mmol) with concentrated aqueous ammonia (2.5 mL)
overnight and purification by preparative TLC (CHCl3/
MeOH/NH4OH, 12/4/1) gave 11 (5 mg, 30%) as a col-
1
ourless oil. H NMR (DMSO): d 7.79 (d, J=7.2 Hz,
1H), 6.25–6.17 (m, 1H), 5.97 (d, J=7.1 Hz, 1H), 4.51–
4.43 (m, 1H), 3.96–3.68 (m, 2H+2H), 2.92–2.72 (m,
1H+1H), 2.38–2.24 (m, 1H), 2.16(s, 9H), 1.46–1.23 (m,
28H), 0.93–0.87 (m, 3H). MS (ES) m/z 666 [M+]. Anal.
calcd for C29H53N3O6PS3: C, 51.37; H, 5.39; N, 8.99.
Found: C, 51.70; H, 5.58; N, 8.68.
Chemicals.
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was purchased from
Sigma. It was dissolved at a concentration of 5 mg/mL
in sterile PBS at room temperature and the solution was
further sterilized by filtration and stored at 4 ꢂC in a
dark bottle. SDS was obtained from Sigma and DMF
was purchaised from Fluka. Lysis buffer was prepared
as follows: 20% w/v of SDS was dissolved at 37 ꢂC in a
solution of 50% of each DMF and demineralized water;
pH was adjusted to 4.7 by adding 2.5% of an 80%
solution of acetic acid and 2.5% 1 N HCl.
Biological studies
Thymidine kinase assay. HSV-1 TK was purified and
assayed as previously described:22 briefly, enzyme was
incubated at 37 ꢂC for 30 min in a mixture (25 mL) con-
taining 30 mM HEPES [4-(2-hydroxyethyl)-1-piper-
Toxicity tests. Cell were plated at different concentra-
tions on flat-botton 96-well microplates (0.1 mL/well).
Lymphocytes were plated out at 20,000 cells/well; 3T3
cells (murine fibroblast line) were plated at 10,000 cells/
well; NSO cells (plasmocytoma murine cell line) were pla-
ted out at 3000 cells/well; Daudi cells (human lympho-
blastoid cell line) were plated at 3000 cells/well. 12 h after
plating, different concentrations of each compound were
added to each well. After 48 h, MTT assay was performed
to analyze cytotoxicity of the different compounds. Some
experiments were performed by using confluent cells:
azineethanesulfonic acid] K+, pH 7.5, 6mM MgCl , 6
2
mM ATP, 0.5 mM dithiothreitol (DTT), and 0.8 mM
[3H-methyl]-dThd (2200 cpm/pmol). The reaction was
terminated by spotting 20 mL of the incubation mixture
on a 25-mm DEAE paper disk (DE-81 paper, What-
man). The disk was washed with an excess of 1 mM
ammonium formate (pH 3.6) in order to remove
unconverted nucleoside, then with ethanol. [3H]-TMP
was estimated by scintillation counting in 1 mL of
BetamaxTM (ICN-Biomedicals).

366
C. Mugnaini et al. / Bioorg. Med. Chem. 11 (2003) 357–366
compounds were added on 3T3 monolayer 3 days after
plating. Tests were then run as described above. Any com-
pound was tested in triplicate and any experiment has been
performed at least twice. Calculated ED50 values for NSO
and normal lymphocytes are reported in Table 1. SD never
excedeed 5% of the calculated dose. Results obtained were
similar for all used lines.
M. C.; Manetti, F.; Corelli, F.; Garaci, E.; Botta, M. J. Med.
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Bittman, R. Ciganek, E., Curran, D., Denmark, S. E., Hage-
dus, L., Joyce, R. M., Martinelli, M. J., McCombie, S. W.,
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MTT/formazan extraction procedure. 20 mL of the 5 mg/
mL stock solution of MTT were added to each well;
after 2 h of incubation at 37 ꢂC, 100 mL of the extraction
buffer were added. After an overnight incubation at
37 ꢂC, the optical densities at 570 nm were measured
using a Titer-Tech 96-well multiscanner, employing the
extraction buffer as the blank. S.D. never exceded 5%
of the mean value. The results obtained have been used
to calculate ED50 values.
11. For 5a (a) Ishihara, J.; Miyakawa, J.; Tsujimoto, T,;
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Acknowledgements
12. (a) Marianne, J.; Kvarnstrom, I.; Svensson, S. C. T.;
Classon, B.; Samuelsson, B. Synthesis 1993, 129. (b) Hafele,
B.; Jager, V. Liebigs Ann. Chem. 1987, 85.
These investigations were supported by Grants from
Istituto Superiore di Sanita, Roma, Italy (II Programma
Nazionale di Ricerca sull’AIDS-1998, grant no.40B.69,
and III Programma Nazionale di Ricerca sull’AIDS-
1999, grant no.40C.65), the University of Siena (PAR
1999 and 2001) and the CNR project ‘Biotecnologie’.
We thank Andrea Lossani for his precious technical
support. M.B. wishes to thank the Merck Research
Laboratories for the 2001 Academic Development Pro-
gram (ADP) Chemistry Award.
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