Novel selective human mitochondrial kinase inhibitors: Design, synthesis and enzymatic activity

Article abstract of DOI:10.1016/j.bmc.2007.01.049

Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at the 2′ position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 2′-O-acyl chain with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC₅₀: 6.4 and 3.8 μM, respectively). The substitution of an ester for a carboxamide moiety at the 2′ position of araT afforded a consistent reduction of the inhibitory activity (25, IC₅₀: 480 μM). On the contrary, modifications at 2′-OH position of araC and araG, have provided inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 2′-O-decanoyl-BVaraU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.

Full text of DOI:10.1016/j.bmc.2007.01.049

Bioorganic & Medicinal Chemistry 15 (2007) 3065–3081
Novel selective human mitochondrial kinase
inhibitors: Design, synthesis and enzymatic activity
Nunzia Ciliberti,a Stefano Manfredini,a,* Angela Angusti,g Elisa Durini,a
Nicola Solaroli,a,b Silvia Vertuani,a Lisa Buzzoni,a Maria Cruz Bonache,a
Efrat Ben-Shalom,c Anna Karlsson,b Ann Saadad,e and Jan Balzarinif
aDepartment of Pharmaceutical Sciences, University of Ferrara, Italy
bDivision of Metabolic Diseases, Department of Laboratory Medicine Karolinska Institute, Novum, S-141 86 Stockholm, Sweden
cPediatric Nephrology, Shaare Zedek Medical Center, Jerusalem, Israel
dMetabolic Disease Unit, Hadassah Medical Center, Jerusalem, Israel
eBen Gurion University of the Negev, Jerusalem, Israel
fRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
gBiotechnology Research Institute (BRI) NRC-CNRC 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2
Received 14 June 2006; accepted 31 January 2007
Available online 2 February 2007
Abstract—Selective and effective TK2 inhibitors can be obtained by introduction of bulky lipophilic chains (acyl or alkyl entities) at
the 20 position of araT and BVaraU, nucleoside analogues naturally endowed with a low TK2 affinity. These derivatives showed a
competitive inhibitory activity against TK2 in micromolar range. BVaraU nucleoside analogues, modified on the 20-O-acyl chain
with a terminal N-Boc amino-group, conserved or increased the inhibitory activity against TK2 (7l and 7m IC50: 6.4 and
3.8 lM, respectively). The substitution of an ester for a carboxamide moiety at the 20 position of araT afforded a consistent reduc-
tion of the inhibitory activity (25, IC50: 480 lM). On the contrary, modifications at 20-OH position of araC and araG, have provided
inactive derivatives against TK2 and dGK, respectively. The biological activity of a representative compound, 20-O-decanoyl-BVa-
raU, was also investigated in normal human fibroblasts and was found to impair mitochondrial function due to TK2 inhibition.
ꢀ 2007 Published by Elsevier Ltd.
1. Introduction
pool especially in non-replicating tissues.5 Mutated
TK2 and dGK are associated with decreased copy num-
ber of mitochondrial DNA (mtDNA) and mitochondrial
respiratory chain (MRC) dysfunction.6,7
Deoxyribonucleoside kinases (dNKs) are key enzymes in
the salvage pathway of mammalian cells.1 dNKs catalyze
the conversion of the nucleosides derived from DNA
degradation, or from the extra-cellular environment, into
the corresponding deoxyribonucleoside monophos-
phates. Human dNKs, thymidine kinase 1 (TK1), thymi-
dine kinase 2 (TK2), deoxycytidine kinase (dCK), and
deoxyguanosine kinase (dGK), have different sub-cellu-
lar localization and variable substrate specificities.2–4
TK2 and dGK are key enzymes in the mitochondrial
nucleoside salvage pathway and are crucial in the main-
tenance of balanced mitochondrial deoxyribonucleotide
TK2 is homologous with dCK, dGK, the recently dis-
covered multisubstrate deoxyribonucleoside kinase from
Drosophila melanogaster (Dm-dNK) and herpes simplex
virus type 1 thymidine kinase (HSV-1 TK).8 TK2 is able
to activate several antiviral nucleoside analogues such as
(E)-5-(2-bromovinyl)-20-deoxyuridine (BVDU), 1-b-D-
arabinofuranosyl-5-(2-bromovinyl)uracil
(BVaraU),
30-azido-20-,30-dideoxythymidine (AZT), and 1-(2-deoxy-
2-fluoro-b-D-arabinofuranosyl)-5-iodouracil (FIAU).9–13
Thus, treatment with these analogues may be accompa-
nied with mitochondrial toxicity of terminally differenti-
ated cells (AZT and FIAU).14,15 Because of this it has
been suggested to exploit TK2 in gene therapy against
malignancies.16,17
Keywords: Design; Synthesis; Nucleoside analogs; Human TK2
inhibitors.
*
Corresponding author. Tel.: +39 0532 291292; fax: +39 0532
291296; e-mail: s.manfredini@unife.it
0968-0896/$ - see front matter ꢀ 2007 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2007.01.049

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N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
The development of potent and selective inhibitors
of TK2 may be helpful to unravel a variety of met-
abolic processes, including the role of TK2 in the
metabolic activation of some antiviral and anticancer
nucleoside analogues, the role of TK2 in the mainte-
nance of the mitochondrial dNTP pool, mtDNA
synthesis and repair, and in the homeostasis of mito-
chondria. Also, specific TK2 inhibitors may elucidate
the differential activities of cytosolic versus mito-
chondrial thymidine kinase in different cell lines or
in the different phases of the cellular replication
cycle.
Intrigued by these interesting preliminary results, fur-
ther investigations were performed to complete the ser-
ies of 20-O-acyl/alkyl substituted arabinofuranosyl
nucleosides and to synthesize a new class of compounds.
In this study we report in detail: (a) the synthetic ap-
proaches applied to achieve the desired chemical struc-
tures; (b) the inhibitory effect of synthesized
derivatives on phosphorylation by deoxyribonucleoside
kinases; (c) the biological effect of one of the most effec-
tive TK2 inhibitors (7e) in tissue culture.
Preliminary data aimed to identify potential dGK inhib-
itors, able to provide information about substrate-spec-
ificity and the physiological role of the mitochondrial
enzyme will be also reported.
So far only few literature reports have described
selective and effective TK2 inhibitors. Among these,
the 50-O-substituted derivatives of thymidine, BVDU,
and their acyclic analogues are able to selectively inhibit
the mitochondrial enzyme, therefore their structural fea-
tures can contribute to desiging new TK2 inhibitors.18–20
Also some ribofuranosylnucleoside analogues highly
functionalized at the 30-position are unexpectedly able
to inhibit the mitochondrial enzyme. This is in complete
disagreement with the available data that the enzyme
recognize, as an efficient substrate, only natural deoxyri-
bonucleosides and unnatural deoxyribonucleoside
analogues.21
2. Chemistry
20-O-Acyl derivatives of BVaraU (compounds 5a, b, d,
and e) and araT (compounds 6d–g) were prepared intro-
ducing the suitable acyl chain, in refluxing pyridine on
the 30,50-O-TIPDS protected arabinonucleosides 324 and
4, as previously reported by us (compound 5c.24) Com-
pound 4 was synthesized as described for the derivative 3.
20-O-(N-Boc-aminoalkanoyl) derivatives of BVaraU
(compounds 5h–m) were obtained in quantitative yield
by reaction of the N-Boc amino acid derivatives with
30,50-O-TIPDS protected BVaraU (3), in presence of
DMAP in CH2Cl2 at room temperature. The TIPSD
cleavage, was performed, following the procedure de-
scribed by us for compounds 7c, e,24 by treatment with
NH4F and Dowexꢁ H+ form in CH3OH, to give the
final products 7a, b, d, h–m, and 8d–g in 30–90% yield
(Scheme 1).
Recently, we have described the inhibitory activity
against TK2 of a series of 20-O-alkylester and ether
derivatives of arabinofuranosyl nucleosides. The bulky
lipophylic entities introduced at the 20-OH of 1-b-D-ara-
binofuranosylthymine (araT) and BVaraU, both not
preferential substrates for the TK2 enzyme, converted
the araT and BVaraU nucleoside analogues to selective
and purely competitive TK2 inhibitors in the micromo-
lar concentration range.22,23
B
B
B
ii
i
O
HO
O
O
O
O
OH
OR
OH
TIPDS
TIPDS
O
O
OH
1,2
5a-e, 5h-m, 6d-g
3,4
O
iii
Br
NH
N
O
B
iv
HO
O
HO
O
OCO(CH2)nNH2.HCl
OR
only for 7h
and 7i
OH
OH
7a-e, 7h-m, 8d-g
9: n=3
10: n=5
1,3,5,7: B=Bromovinyluracil
2,4,6,8: B=Thymine
TIPDS: tetraisopropyldisiloxane-1,3-diyl.
a:R= -COBn; b:R= -CO(p-OCH3-Bn); c:R= -COCH2OCH3; d:R= -CO(CH2)6CH3; e:R= -CO(CH2)8CH3;
f:R= -CO(CH2)3CH3; g:R= -CO(CH2)10CH3; h:R= -CO(CH2)2CH2NH-Boc; i:R= -CO(CH2)4CH2NH-Boc;
l:R= -CO(CH2)6CH2NH-Boc; m:R= -CO(CH2)10CH2NH-Boc;
Scheme 1. Reagents and conditions: (i) TIPDSCl2, pyridine, room temperature; (ii) RCOCl, pyridine, reflux or N-Boc-aa, DMAP, DCC, CH2Cl2,
0 ꢂC to room temperature; (iii) NH4F, CH3OH, Dowexꢁ H+ form 50W · 2 50–100 mesh, room temperature; (iv) HCl (g) Et2O, 0 ꢂC.

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3067
The Boc protecting group was removed from com-
pounds 7h and 7i by bubbling HCl in diethyl ether at
0 ꢂC: after the crystallization step, the hydrochloride
salts 9 and 10 were obtained in yield greater than 90%
(Scheme 1).
The compound 15 was obtained by reaction of
compound 13 with benzyl bromide and NaH in THF
followed by the treatment of the crude mixture with
p-TsOH monohydrate in methanol at room temperature
(overall yield 52%) (Scheme 3). In this case, alkylation at
N-3 was not observed (see below) in the above described
reaction conditions.
Due to the low reactivity of the alkylating reagents, the
20-O-alkyl ethers of araT (15-18) were obtained in
applying different synthetic conditions that required
the synthesis of the unnatural nucleoside 1-b-D-ribofu-
ranosylthymine (11).25,26 To this end, the protection
of 30 and 50 hydroxyl groups was carried out using
1,2-dihydropyrane (DHP) on the 2,20-anhydro-1-(b-D-
arabinofuranosyl)thymine intermediate (12).27 The next
treatment with 0.1 M KOH in EtOH (Scheme 2)28
allowed to obtain the starting compound 13 for the
alkylation reactions in good overall yield (75%).
No product formation was observed, even under reflux-
ing conditions, when the less reactive octyl bromide was
used as alkylating agent. In attempt to obtain the ex-
pected product we modified the reaction conditions
using alkyl bromide in a 1:1 mixture of THF–DMSO
to obtain, as the solely product, the 30,50 THP–protected
N3-alkylated derivative. Crude mixture of this latter
compound was next deprotected, as show in Scheme 3,
providing the final compound 16 in 58% overall yield.
O
O
N
O
CH3
CH3
HN
CH3
HN
HN
O
N
O
O
O
N
HO
i
O
HO
ii, iii
THPO
O
OH
OH
OH
OH
12
OTHP
11
13
Scheme 2. Reagents and conditions: (i) (PhO)2CO, NaHCO3, DMF, 150 ꢂC; (ii) DHP, p-TsOHÆH2O, CH3CN, room temperature; (iii) 0.1 M KOH,
EtOH 95%, room temperature.
O
O
CH3
R'
CH3
R'N
N
O
N
O
N
B
THPO
i or ii
or iii
HO
THPO
iv
O
O
O
OH
OR
OR
OH
15,16,17,18
OTHP
OTHP
13,14
iii
N
N
O
N
N
NH2
HN
O
N
N
O
N
O
N
vi, iv, vii
v
THPO
HO
THPO
O
O
O
O(CH2)7CH3
O(CH2)7CH3
21
O(CH2)7CH3
OTHP
20
OH
OTHP
19
13: B= Thymine
14: B= Uracil
15: R=Bn R'=H
16: R=H; R'=CH3(CH2)7-
17: R= CH3(CH2)7-; R'=H;
18: R=CH3(CH2)7-; R'= CH3(CH2)7-
Scheme 3. Reagents and conditions: (i) NaH, alkyl bromide, THF, room temperature or reflux; (ii) K2CO3, octyl bromide, THF–DMSO, room
temperature; (iii) NaH, I(CH2)7CH3, THF reflux; (iv) CH3OH, p-TsOHÆH2O, room temperature; (v) POCl3, 1,2,4-triazole, TEA, 0 ꢂC to room
temperature; (vi) dioxane and 30% NH4OH (1:1 mixture) room temperature; (vii) Dowexꢁ OHꢀ form 1 · 2 200, H2O.

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N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
Aiming to obtain a more reactive alkylating agent, the
octyl bromide was next converted in the corresponding
iodide derivative under standard conditions (NaI,
CH3CN) and then used in high excess with NaH in
refluxing THF. The crude mixture obtained, subjected
to the THP deprotection step, gave the expected 20-O-
alkylated compound 17 and the N3- and 20-O-bis-alkyl-
ated 18 in 55% and 19% overall yield the respectively
(molar ratio 17/18 = 3:1). The 20-O-octyl derivative of
1-b-D-arabinofuranosylcytosine (AraC) (21) was pre-
pared from the 30,50-O-tetrahydropyranyl-araU (14)28
following and adapting the procedure described in the
literature29 (Scheme 3).
in DMF. In satisfactory yield (61–77%) were obtained
the 20-O-octanoyl and 20-O-pentanoyl araG derivatives
(compounds 28a and 28b). The bis-acylated derivative
of araG (31) was obtained from the intermediate 26,
under the same reaction conditions used for com-
pounds 5 and 6, avoiding protection of –NH2 at the
purinic base.
Compounds 28a, 28b, and 31 were next deprotected at
the sugar ring as previously described, obtaining
compounds 29a, 29b, and 32, respectively. Finally
compounds 29a and 29b were further deprotected at
the 2-amino group following and modifying the method
described by Davisson et al.32 providing the derivatives
30a and 30b in good yield (66–97%) (Scheme 5).
The synthesis of the 20-carbamoyl derivative of araT
(25) was performed starting from compound 22 ob-
tained as reported.30 The next reduction, carried out in
standard conditions (Pd/C under H2 atmosphere), gave
the formation of the amino group on the sugar ring
and, completely unexpected, concomitant cleavage of
the benzoyl group at N-3. The derivative 23 thus ob-
tained was next reacted with activated decanoic acid
that was obtained using N-(3-dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride (EDC) and 1-
hydroxybenzotriazole hydrate (HOBT). The resulting
compound 24 was then deprotected to give the expected
25 (Scheme 4).
3. Biological results and discussion
We started the present study with several aims: (a) to de-
sign selective TK2 inhibitors; (b) to gain insights into
structure–activity relationships (SAR) of the inhibitors
for TK2 versus other nucleoside kinases; (c) to reveal
whether the newly designed TK2 inhibitors can be
uptaken by intact mitochondria; (d) to investigate the
impact of TK2 inhibitors on mitochondrial (dys)
function.
The preparation of 20-O-acyl derivatives of 9-b-D-ara-
binofuranosyl guanosine (araG) was carried out start-
ing from the 30,50-O-TIPDS derivative 26 in turn
obtained modifying a previously reported synthesis.31
The next protection of the 2-amino group (27), per-
formed applying the methodology reported for guano-
sine derivatives,32 allowed the selective introduction of
the acyl chain on the 20 hydroxyl group by using octa-
noyl chloride in refluxing pyridine, or valeric anhydride
New synthetic work has been directed to modify the
more interesting BVaraU and araT acyl derivatives by
introducing an additional hydrophilic (NH2) function
on the acyl moiety (compounds 9 and 10), or by the
isosteric substitution of the pre-existing 20-ester function
of 8e with a carboxamido moiety (compound 25). The
selected modifications introduced on the active mole-
cules new features useful to investigate the TK2 pocket
O
O
Bz
O
CH3
CH3
N
HN
N
O
N
i
O
O
O
O
N3
NH2
TIPDS
TIPDS
O
O
22
23
ii
O
O
N
CH3
CH3
HN
HN
O
N
O
O
TIPDS
HO
iii
O
O
CH2(CH2)7CH3
CH2(CH2)7CH3
NH
NH
O
HO
O
O
24
25
TIPDS: tetraisopropyldisiloxane-1,3-diyl.
Scheme 4. Reagents and conditions: (i) H2, Pd/C, dry CH3OH; (ii) EDC, HOBT, CH3(CH2)8COOH, dry CH2Cl2; (iii) NH4F, dry CH3OH, Dowexꢁ
H+ form 50Wx2 (50–100), room temperature.

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3069
O
N
O
O
N
N
N
N
N
N
NH
NH
NH
NH
NHCO(CH ) CH
3
N
NHCO(CH ) CH
N
2
2 6
3
2 6
i
ii
O
TIPDS
HO
O
TIPDS
O
O
O
OCO(CH ) CH
OH
OCO(CH ) CH
2 6
2 6
3
3
O
O
O
OH
26
31
32
iii
O
O
N
O
N
N
N
N
N
NH
N=CHN(CH )
NH
NH
NH
N
N=CHN(CH )
N
3 2
3 2
N
2
HO
v
iv
ii
O
TIPDS
O
O
O
29a,b
OR
OH
OR
28a,b
TIPDS
O
O
OH
30a,b
27
TIPDS: tetraisopropyldisiloxane-1,3-diyl
a: R= -CO(CH ) CH ; b: R= -CO(CH ) CH ;
2 6
3
2 3
3
Scheme 5. Reagents and conditions: (i) octanoyl chloride, pyridine, reflux; (ii) NH4F, CH3OH, Dowexꢁ H+ form 50Wx2 (50–100 mesh), room
temperature; (iii) CH3CN, DIPEA, N,N-dimethylformamide dimethyl acetal; (iv) octanoyl chloride, pyridine, TEA reflux or DMF, valeric
anhydride, 4-DMAP, room temperature; (v) CH3OH/H2O 2:1, TFA.
with a greater detail. To further investigate the TK2
mitochondrial enzyme, the general approach, based on
the introduction of a lipophilic chain at 20-position of
arabinofuranosyl derivatives, was applied also to araC
(21).33 Similarly, the approach was also extended to
araG in order to explore dGK inhibitors. Furthermore,
inhibitory effects of dThd phosphorylation by deoxyri-
bonucleoside kinases from different origin were studied
in order to characterize the specificity of the different
compounds.
of araT markedly decreases the affinity for all nucleo-
side kinases except for TK2 where a substantially
increased activity was noticed. In fact, the 20-O-deca-
noyl and 20-O-dodecanoyl derivatives (compounds 8e
and 8g) showed pronounced affinity for TK2 (IC50:
27–28 lM) (Table 1). Different results were obtained
when the acyl chain was introduced on the 20-amino-
20-deoxy derivative of araT (compound 25, 480 lM).
The isosteric substitution of the 20-ester function, pres-
ent in compound 8e, by a 20-carboxamido moiety (25)
produced in fact a consistent reduction of the inhibi-
tory activity. The results indicate possible steric
requirements for the activity, indeed the partial double
bond character in the amide causes the linkage to be
more stable than esters and makes that portion of
the molecule more rigid. Thus the molecule may not
fit equally well into the TK2 binding pocket. Alterna-
tively, a hydrogen accepting atom (O) instead of a
hydrogen donating (N) atom may be required at the
20 position of the pentose ring.
AraT shows (Table 1) preferential inhibition of HSV-1
TK and varicella-zoster virus thymidine kinase (VZV-
TK), but also Dm-dNK efficiently recognizes araT as
an alternative substrate. In contrast, mitochondrial
TK2 has at least a 10-fold lower affinity for araT
(IC50: 285 lM) and cytosolic TK1-catalyzed dThd
phosphorylation was not measurably inhibited in the
presence of 1000 lM araT (Table 1). Interestingly,
introduction of acyl or alkyl entities at the 20-position
Table 1. Inhibitory effects of araT derivatives on 2 lM [methyl-3H]dThd phosphorylation by deoxyribonucleoside kinases from different origin
a
Compound
IC50 (lM)
TK1
TK2
285
HSV-1TK
Dm-dNK
VZV-TK
AraT
8d23
8e23
8f23
8g23
1523
16
>1000
>1000
>1000
>1000
P1000
>1000
>500
1
24 3.1
>1000
P1000
>1000
P1000
>1000
>500
65 28
>1000
872
17 8.7
>1000
>1000
>1000
>1000
>1000
>500
120 14
27 2.3
>1000
>1000
>1000
>1000
>500
28
2
801 71
>500
1723
18
>1000
Nd
Nd
120 14
>1000
480
>1000
>1000
>500
>1000
>1000
>500
>1000
>1000
>500
25
a 50% inhibitory concentration or compound concentration required to inhibit the enzyme-catalysed phosphorylation of 2 lM [methyl-3H] dThd by
50%.

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N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
When similar substituents were introduced in BVaraU, a
comparable phenomenon was observed as with the araT
derivatives (Table 2). Indeed, the pronounced affinity of
BVaraU for HSV-1 TK, VZV-TK (IC50: 3.6–3.2 lM),
and Dm-dNK (IC50: 28 lM) is markedly decreased
(10- to >100-fold), whereas TK2 affinity was substan-
tially increased (IC50: 6.3–6.8 lM) for the 20-O-octanoyl
and 20-O-decanoyl derivatives (compounds 7d and 7e).
enzymes. None of these compounds were inhibitory to
any of the nucleoside kinases evaluated (Table 4).
In order to assess the biological activity of the TK2
inhibitors on human tissue we tested 20-O-decanoyl-
BVaraU (7e) on isolated human fibroblasts mitochon-
dria rather than recombinant enzymes. The IC50 against
TK2 was 40 and 97 lM with dThd and dCyd, as sub-
strates, respectively, indicating that higher concentra-
tions are needed for ‘in organello’ inhibition than
in vitro experiments using recombinant TK2. This
occurrence might be explained in view of a possible par-
tial hydrolysis mediated by esterases. Indeed, although
this class of compounds has been tested against pig liver
esterase25 and became stable, we cannot exclude sensi-
tivity towards mitochondrial esterases. Still the inhibi-
tion was specific since control experiments showed that
dGK activity was not inhibited by this compound
(IC50 > 1000 lM). Moreover, this compound did not af-
fect cytochrome c oxidase or succinate dehydrogenase
activities.
Again, there was no appreciable activity against TK1.
Long-chain substituents seemed to be better than
short-chain substituents. This was also the case for the
20-O-octanoyl (7l) and 20-O-dodecanoyl (7m) derivatives
of BVaraU with a terminal NH-Boc moiety that showed
an IC50 of 6.4 and 3.8 lM, respectively, for TK2,
whereas the 20-O-hexanoyl (7i) and 20-O-butanoyl (7h)
NH-Boc derivatives were ꢁ10-fold less effective. Also,
the 20-O-butanoyl (9) and 20-O-hexanoyl (10) derivatives
with a terminal amino moiety, markedly lost affinity for
TK2 with respect to the corresponding NH-Boc pro-
tected derivatives (7h and 7i). These data indicate that
masking of the hydrophilic amino group is very impor-
tant to achieve activity.
We also tested the effect (Fig. 1) of 20-O-decanoyl-BVa-
raU (7e) on mitochondrial function by employing a
selective medium lacking glucose, with a minimal
amount of galactose sufficient to sustain the pentose
phosphate pathway but not ATP production by glycol-
ysis: the ATP production and cell viability are therefore
solely dependent on the MRC in this medium. In order
to sustain MRC deficient cells, a permissive medium,
containing glucose pyruvate and uridine is employed.34
We determined the viability of normal fibroblasts in
Since TK2 is not only endowed with dThd kinase activ-
ity but also with dCyd kinase activity, the 20-octyl deriv-
ative of araC was also synthesized (21), but no
measurable affinity for TK2 was found (Table 3).
Finally, 20-O-acyl-araG derivatives (30a, b and 32) were
synthesized to explore their activity against the araG
mitochondrial enzyme besides the previously mentioned
Table 2. Inhibitory effects of BVaraU derivatives on 2 lM [methyl-3H]dThd phosphorylation by deoxyribonucleoside kinases from different origin
Compound
IC50a(lM)
TK1
TK2
HSV-1TK
Dm-dNK
VZV-TK
BVaraU
7a
>500
Nd
Nd
43 5.8
3.6 1.5
247 54
28 10
419 68
182 98
215 73
178
3.2 1.1
268 2.0
39 12
35 1.5
718 59
845 30
130
74
44 14
3
7b
68
3
7c24
7d
>1000
>1000
>1000
Nd
402 234
6.3 0.5
6.8 0.7
84 1.8
P1000
>1000
7e24
7h
163
55
61
6
8
485 20
P500
133 80
77 24
74 31
P500
160 60
>500
7i
Nd
Nd
277
349
1
1
7l
6.4 2.6
3.8 0.2
187 14
> 500
447
>500
8
7m
9
Nd
>500
>500
>500
3.6 0.4
94 22
17 1.0
322 83
10
a 50% inhibitory concentration or compound concentration required to inhibit the enzyme-catalyzed phosphorylation of 2 lM [methyl-3H] dThd by
50%.
Table 3. Inhibitory effects of araC derivatives on 2 lM [methyl-3H]dThd phosphorylation by deoxyribonucleoside kinases from different origin
Compound
IC50a(lM)
TK1
TK2
HSV-1TK
Dm-dNK
VZV-TK
AraC23
2123
>1000
>1000
>1000
>1000
>1000
>1000
5319
>1000
>1000
>1000
a 50% inhibitory concentration or compound concentration required to inhibit the enzyme-catalyzed phosphorylation of 2 lM [methyl-3H] dThd by
50%.

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3071
Table 4. Inhibitory effects of araC derivatives on 2 lM [methyl-3H]dThd phosphorylation by deoxyribonucleoside kinases from different origin
a
Compound
IC50 (lM)
TK1
TK2
HSV-1 TK
Dm-dNK
VZV-TK
dGK
AraG
30a
30b
32
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
133
>500
>500
>500
a 50% inhibitory concentration or compound concentration required to inhibit the enzyme-catalyzed phosphorylation of 2 lM [methyl-3H]dThd by
50%.
GAL
DMEM UP
BVaraU
90
80
70
60
50
40
30
20
10
0
2'-O-decanoyl-BVaraU
% cell
survival
100
80
60
40
20
0
0
50
μM
100
DMEM UP
GAL
Figure 1. The effect of 20-O-decanoyl-BVaraU (7e) on mitochondrial
function. Stationary, normal fibroblasts were treated with various
concentrations of 20-O-decanoyl-BVaraU (7e) in selective medium
(GAL) and permissive medium (DMEM UP). Viability count was
performed after 1 week of treatment.
Figure 2. The effect of 20-O-decanoyl-BVaraU (7e) and BVaraU on
mitochondrial function. Stationary, normal fibroblasts were treated
with 100 lM of 20-O-decanoyl-BVaraU (7e) and BVaraU in selective
medium (GAL) and permissive medium (DMEM UP). Viability count
was performed after 1 week treatment.
both media in order to differentiate between the specific
effect on MRC and a potential general toxic effect. After
1 week treatment, for concentrations up to 60 lM, the
compound 7e did not alter the cellular viability, regard-
less of the medium, indicating that this compound at
low concentrations is non-toxic and does not alter
MRC function in whole cells. Between 60 and 100 lM
the viability was impaired in selective medium (GAL),
while it was near normal in permissive medium (DMEM
UP). This demonstrates that, at these concentrations, 7e
specifically inhibits MRC function. At concentrations
higher than 100 lM a generally toxic effect was ob-
served, as the survival was impaired also in the permis-
sive medium (Fig. 1). This toxic effect was not due to
solvent as control experiments showed cells treated with
solvent only were not affected (data not shown).
resulted in decreased COX activity and COX/SDH
ratios (Fig. 3). Notably the effect of this compound
was much less on cells with mutated TK2.15
Finally we determined the mtDNA content by real time
PCR in fibroblasts treated, for 1 week, with 100 lM of
20-O-decanoyl-BVaraU (7e). The mtDNA relatively to
nuclear DNA was decreased to 50% of that of untreated
cells, confirming that the mitochondrial dysfunction is a
consequence of mtDNA depletion induced by TK2
inhibition.
4. Conclusion
We have demonstrated that nucleoside analogues such as
araT and BVaraU bearing bulky lipophylic substituents
at the 20 position of the sugar can be efficiently recognised
by TK2 in a highly selective manner. This finding suggests
that TK2, in contrast with the HSV-1 TK, VZV-TK, and
Dm-dNK, must contain a lipophilic pocket or cleft in
which the 20-substituents may fit. Since there is no crystal
structure available for TK2, it is currently unclear how
this lipophylic pocket is created in the presence of the test
compounds. However, the presence of such a putative
pocket may be a useful tool to rationally design new selec-
tive substrate/inhibitors of the mitochondrial TK2, not
affecting TK1 or any of the other nucleoside kinases.
We have also demonstrated that 20-O-decanoyl-BVaraU
(7e) is able to inhibit the TK2 enzyme ‘in organello.’
Differently, as show in Figure 2, at 100 lM the mother
compound BVaraU did not impair the mitochondrial
function.
20-O-Decanoyl-BVaraU-treated cells, grown in permis-
sive medium, were also assayed for the MRC enzymes
cytochrome c oxidase (COX, complex IV) and succinate
dehydrogenase (SDH, Complex II). Since COX contain
subunits encoded by the mtDNA while SDH encoded
solely in the nucleus, COX activity represents an
mtDNA-dependent MRC activity, while SDH activity
serves as an mtDNA-independent control. Treatment
with increasing amount of 20-O-decanoyl-BVaraU (7e)

3072
N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
(10 mL) under argon atmosphere, was dropwise added
% enzyme
activity
COX
SDH
COX/SDH
1,3-dichloro-1,1,3,3 tetraisopropyldisiloxane (TPDSCl2,
320 ll, 1 mmol) and the mixture was stirred at room
temperature for 20 h. The pyridine was evaporated
and coevaporated three times with EtOH and the resi-
due was portioned between EtOAc and H2O. The or-
ganic phase was washed with cold HCl 1N (2 ·
20 mL), H2O (20 mL), saturated NaHCO3 (20 mL),
and brine (20 mL), dried over Na2SO4, filtered, and
evaporated to dryness. The resulting residue was puri-
fied by silica gel column chromatography (eluent:
CH2Cl2/CH3OH, 90/10, v/v) to give 4 (465 mg, 93%)
as a colorless solid (mp >250 ꢂC, dec).
110
100
90
80
70
60
50
40
1H NMR (CDCl3) d (ppm): 0.98–1.18 (m, 28H, TIPDS);
1.90 (s, 3H, CH3); 3.40–3.58 (m, 1H, H40); 3.70–3.81 (m,
1H, H30); 3.92–4.32 (m, 3H, H50, H500, H20); 4.50–4.61
(m, 1H, OH20); 6.05 (d, 1H, J = 6.3 Hz, H10); 7.49 (d,
1H, J = 1.1 Hz, H6); 9.22 (br s, 1H, NH).
0
20
40
60
80
100
μM
Figure 3. The effect of 20-O-decanoyl-BVaraU (7e) on MRC activity.
Stationary, normal fibroblasts were treated with various concentra-
tions of 20-O-decanoyl-BVaraU (7e) in permissive medium for one
week. Cytochrome c oxidase (COX) and succinate dehydrogenase
(SDH) activites were assayed and COX/SDH ratios were calculated.
MALDI-TOFMS: m/z 501.7 Da (M+H)+; 523.7 Da
(M+Na)+; 539.0 Da (M+K)+. C22H40N2O7Si2 requires
500.24.
5.2. General procedure for the acylation of compounds 324
and 4. Synthesis of the compounds 5a, b, d, e and 6d–g
The compound was able to penetrate whole cells, enter
the mitochondria, and subsequently inhibit MRC
(mitochondrial respiratory chain) activity at micromo-
lar concentrations. Therefore TK2 inhibitors must be
carefully used, as an excess may cause mtDNA deple-
tion with subsequent mitochondrial dysfunction.
The acylation of compounds 3 and 4 (0.5 mmol) was per-
formed with the appropriate acyl chloride (1 mmol) in
pyridine, as previously reported by us for compound 5c.24
5.2.1. 1-[20-O-Phenylacetyl-30,50-O-(1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-5(E)-(2-bromo-
vinyl)uracil (5a). Yield 47%; white foam.
5. Experimental
5.1. Chemistry
1H NMR (CDCl3) d (ppm): 0.94–1.28 (m, 28H, TIPDS);
3.51 (s, 2H, CH2CO); 3.76–4.00 (m, 3H, H40, H50 and
H500); 4.39–4.41 (m, 1H, H30); 5.57–5.60 (m, 1H, H20);
6.19 (d, 1H, J = 6.5 Hz, H10); 6.60 (d, 1H, J = 13.5 Hz,
vinyl); 6.75–6.98 (m, 5H, Ph); 7.38 (d, 1H, vinyl); 7.26
(s, 1H, H6); 8.12 (br s, 1H, NH).
Reaction courses were routinely monitored by thin-layer
chromatography (TLC) on silica gel precoated Mache-
rey-Nagel durasil-25, with detection under a 254-nm
UV lamp and/or by spraying the plates with 10%
H2SO4/CH3OH and heating. Column chromatography
was performed with Macherey-Nagel 0.063–0.2 mm/
70–230 mesh silica gel. MALDI-TOFMS (Matrix-as-
sisted laser desorption ionization time-of-flight) spectra
were obtained on a Hewlett–Packard HPG2025A mass
spectrometer operative on a positive linear mode. Nucle-
ar magnetic resonance spectra were determined in
DMSO-d6, D2O and CDCl3 solution with a Brucker
AC-200 spectrometer or Varian MERCURYplus
400 MHz and chemical shifts are presented in ppm from
internal tetramethylsilane as a standard. Melting points
were determined by Kofler melting point apparatus
(Thermovar, C. Reichert AG, Vienna) and are uncor-
rected. Microanalysis, unless indicated, was in agree-
ment with calculated values within 0.4%. All drying
operations were performed over anhydrous sodium sul-
fate or magnesium sulfate; room temperature varied be-
tween 22 and 25 ꢂC.
MALDI-TOFMS: m/z 731.2 Da (M+Na)+; 747.4 Da
(M+K)+. C31H45BrN2O8Si2 requires 708.19.
5.2.2. 1-[20-O-(4-Methoxy)-phenylacetyl-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil (5b). Yield 22%; white foam.
1H NMR (CDCl3) d (ppm): 0.92–1.20 (m, 28H, TIPDS);
3.49 (s, 2H, CH2CO); 3.79–4.07 (m, 6H, CH3, H40, H50
and H500); 4.41–4.43 (m, 1H, H30); 5.57–5.59 (m, 1H,
H20); 6.17 (d, 1H, J = 6.6 Hz, H10); 6.59 (d, 1H,
J = 13.5 Hz, vinyl); 6.77, 7.02 (AB system, 4H,
JAB = 8.6 Hz, Ph); 7.37 (d, 1H, vinyl); 7.27 (s, 1H,
H6); 8.10 (br s, 1H, NH).
MALDI-TOFMS: m/z 762.0 Da (M+Na)+; 777.3 Da
(M+K)+. C32H47BrN2O9Si2 requires 738.20.
5.2.3. 1-[20-O-Octanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabino-furanosyl]-5(E)-(2-bromo-
vinyl)uracil (5d). Yield 90%; yellow oil.
5.1.1.
1-[30,50-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-
diyl)-b-D-arabinofuranosyl]thymine (4). To a suspension
of dried araT (258 mg, 1 mmol) in dry pyridine

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3073
1H NMR (CDCl3) d (ppm): 0.81 (t, 3H, J = 6.7 Hz,
CH3); 0.94–1.15 (m, 8H, 4 · CH2); 1.18–1.30 (m, 28H,
TIPDS) 1.35–1.65 (m, 2H, CH2CH2CO); 2.10–2.21 (m,
2H, CH2CO); 3.70–3.81 (m, 1H, H40); 3.92–4.15 (m,
2H, H50, H500); 4.30–4.41 (m, 1H, H30); 5.51–5.62 (m,
1H, H20); 6.15 (d, 1H, J = 6.4 Hz, H10); 6.59 (d, 1H,
J = 13.6 Hz, vinyl); 7.35 (d, 1H, J = 13.6 Hz, vinyl);
7.46 (s, 1H, H6); 9.25 (br s, 1H, NH).
2 · CH2); 1,85 (s, 3H, CH3); 2.10–2.23 (m, 2H, CH2CO);
3.69–3.78 (m, 1H, H40); 3.90–4.12 (m, 2H, H50, H500);
4.34–4.45 (m, 1H, H30); 5.42–5.53 (m, 1H, H20); 6.16
(d, 1H, J = 6.6 Hz, H10); 7.22 (s, 1H, H6); 8.03 (br s,
1H, NH).
MALDI-TOFMS: m/z 607.5 Da (M+Na)+; 623.8 Da
(M+K)+. C32H48N2O8Si2 requires 584.86.
MALDI-TOFMS: m/z 739.9 Da (M+Na)+; 755.7 Da
5.2.8. 1-[20-O-Dodecanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabinofuranosyl]thymine (6g).
Yield 95%; yellow oil.
(M+K)+.C31H53BrN2O8Si2 requires 716.25.
5.2.4. 1-[20-O-Decanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabinofuranosyl]-5(E)-(2-bromo-
vinyl)uracil (5e). Yield 69%; colourless solid (mp: 235–
238 ꢂC).
1H NMR (CDCl3) d (ppm): 0.87 (t, 3H, J = 6.1 Hz,
CH3); 0.89–1.13 (m, 28H, TIPDS); 1.12–1.38 (m, 16H,
8 · CH2); 1.39–1.71 (m, 2H, CH2CH2CO); 1.85 (s, 3H,
CH3); 2.09–2.37 (m, 2H, CH2CO); 3.69–3.88 (m, 1H,
H40); 3.90–4.15 (m, 2H, H50, H500); 4.41–4.47 (m, 1H,
H30); 5.43–5.58 (m, 1H, H20); 6.16 (d, 1H, J = 6.5 Hz,
H10); 7.24 (s, 1H, H6); 9.15 (br s, 1H, NH).
1H NMR (CDCl3) d (ppm): 0.88 (t, 3H, J = 6.6 Hz,
CH3); 1.02–1.17 (m, 12H, 6 · CH2); 1.23–1.26 (m,
28H, TIPDS); 1.42–1.58 (m, 2H, CH2CH2CO); 2.21 (t,
2H, J = 7.4 Hz, CH2CO); 3.79–3.98 (m, 1H, H40);
4.06–4.12 (m, 2H, H50, H500); 4.39–4.50 (m, 1H, H30);
5.58–5.69 (m, 1H, H20); 6.23 (d, 1H, J = 6.3 Hz, H10);
6.66 (d, 1H, J = 13.6 Hz, vinyl); 7.43 (d, 1H,
J = 13.6 Hz, vinyl); 7.52 (s, 1H, H6); 9.86 (br s, 1H, NH).
MALDI-TOFMS: m/z 683.8 Da (M+H)+; 705.4 Da
(M+Na)+; 721.4 Da (M+K)+. C34H62N2O8Si2 requires
682.40.
5.3. General procedure for the synthesis of the N-Boc-
aminoacyl derivatives 5h–m
MALDI-TOFMS: m/z 768.1 Da (M+Na)+; 783.4 Da
(M+K)+. C33H57BrN2O8Si2 requires 744.28.
To a stirred solution of the selected N-Boc amino
acid derivative (0.25 mmol) in dry CH2Cl2 (3.5 mL),
5.2.5. 1-[20-O-Octanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabinofuranosyl]thymine (6d).
Yield 50%; yellow oil.
compound
3
(150 mg, 0.25 mmol) and DMAP
(3 mg, 0.03 mmol) were added under argon atmo-
sphere. To the solution, cooled at 0 ꢂC, was then
added N,N0-dicyclohexylcarbodiimide (DCC, 68 mg,
0.33 mmol) and the resulting mixture was reacted at
25 ꢂC for 3–5 h. After this period, the N,N0-dic-
yclohexylurea (DCU), deriving by the reaction, was
filtered off and the solvent was evaporated to dryness.
The resulting residue was dissolved in EtOAc
(15 mL), washed with HCl 0.5 N (10 mL), saturated
NaHCO3 (10 mL), and finally with brine (10 mL).
After drying over anhydrous Na2SO4, the solvent
was evaporated to give, in all cases, the pure 20-O-
aminoacyl derivatives as white foams, in almost
quantitative yield (98–100%).
1H NMR (CDCl3) d (ppm): 0.88 (t, 3H, J = 6.5 Hz, CH3);
1.00–1.19 (m, 10H, 5 · CH2); 1.21–1.41 (m, 28H, TIPDS);
1.43–1.78 (m, 2H, CH2CO); 1.92 (s, 3H, CH3); 3.76–3.81
(m, 1H, H40); 3.95–4.28 (m, 2H, H50, H500); 4.40–4.52 (m,
1H, H30); 5.50–5.60 (m, 1H, H20); 6.23 (d, 1H, J = 6.5 Hz,
H10); 7.29 (s, 1H, H6); 8.12 (br s, 1H, NH).
MALDI-TOFMS: m/z 650.0 Da (M+Na)+; 665.3 Da
(M+K)+. C30H54N2O8Si2 requires 626.34.
5.2.6. 1-[20-O-Decanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabinofuranosyl]thymine (6e).
Yield 58%; yellow oil.
5.3.1. 1-[20-O-(N-Boc-4-aminobuanoyl)-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil (5h). 1H NMR (400 MHz,
CDCl3) d (ppm): 0.99–1.12 (m, 28H, TIPDS); 1.49 (s,
9H, t-Bu); 1.75–1.81 (m, 2H, CH2CH2CO); 2.25–2.30
(m, 2H, CH2CO); 3.05–3.11 (m, 1H, CHH–N); 3.22–
3.29 (m, 1H, CHH–N); 3.86–3.88 (m, 1H, part of
ABX system, H40); 4.01 (dd, 1H, part of ABX system,
JAB = 13.6 Hz, JAX = 2.4 Hz, H50); 4.17–4.20 (m, 1H,
part of ABX system, H500); 4.31–4.35 (m, 1H, H30);
4.80 (br s, 1H, NH–Boc); 5.74–5.76 (m, 1H, H20); 6.14
(d, 1H, J = 6.0 Hz, H10); 6.68 (d, 1H, J = 13.6 Hz, vi-
nyl); 7.47 (d, 1H, vinyl); 7.68 (s, 1H, H6); 8.68 (br s,
1H, NH).
1H NMR (CDCl3) d (ppm): 0.78–0.89 (m, 3H, CH3);
1.00–1.18 (m, 28H, TIPDS); 1.14–1.19 (m, 14H,
7 · CH2); 1.85 (s, 3H, CH3); 2.01–2.48 (m, 2H, CH2CO);
3.65–3.74 (m, 1H, H500); 3.83–4.45 (m, 3H, H50, H40,
H30); 5.42–5.51 (m, 1H, H20); 6.17 (d, 1H, J = 6.4 Hz,
H10); 7.20 (s, 1H, H6); 8.50 (br s, 1H, NH).
MALDI-TOFMS: m/z 677.5 Da (M+Na)+; 693.7 Da
(M+K)+. C32H58N2O8Si2 requires 654.37.
5.2.7. 1-[20-O-Pentanoyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-b-D-arabinofuranosyl]thymine (6f).
Yield 68%; yellow oil.
1H NMR (CDCl3) d (ppm): 0.77 (t, 3H, J = 5.5 Hz,
CH3); 0.87–1.12 (m, 28H, TIPDS); 1.15–1.65 (m, 4H,
MALDI-TOFMS: m/z 798.9 Da (M+Na)+; 814.1 Da
(M+K)+. C32H54BrN3O10Si2 requires 775.25.

3074
N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
5.3.2. 1-[20-O-(N-Boc-6-aminohexanoyl)-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil (5i). 1H NMR (CDCl3) d
(ppm): 0.97–1.15 (m, 28H, TIPDS); 1.46 (s, 9H, t-Bu);
1.45–1.89 (m, 6H, 3 · CH2); 2.19–2.24 (m, 2H, CH2CO);
3.01–3.19 (m, 2H, CH2–N); 3.80–3.85 (m, 1H, part of
ABX system, H40); 4.02, 4.15 (2H, part of ABX system,
JAB = 13.4 Hz, JAX = 2.6 Hz, JBX = 1.8 Hz, H50 and
H500); 4.36–4.45 (m, 1H, H30); 4.82 (br s, 1H, NH–
Boc), 5.62–5.69 (m, 1H, H20); 6.21 (d, 1H, J = 6.4 Hz,
H10); 6.66 (d, 1H, J = 13.6 Hz, vinyl); 7.43 (d, 1H,
J = 13.6 Hz, vinyl); 7.53 (s, 1H, H6); 9.39 (br s, 1H,
NH).
4.12–4.14 (m, 1H, H30); 5.12–5.18 (m, 1H, H20);
5.12–5.23 (m, 1H, OH50); 5.76–5.83 (m, 1H, OH30);
6.12 (d, 1H, J = 5.7 Hz, H10); 6.86 (d, 1H,
J = 13.5 Hz, vinyl); 7.07–7.25 (m, 5H, Ph); 7.27 (d,
1H, J = 13.6 Hz, vinyl); 7.87 (s, 1H, H6); 11.55 (br
s, 1H, NH).
MALDI-TOFMS: m/z 489.9 Da (M+Na)+. C19H19BrN2O7
requires 466.04. Anal. (C19H19BrN2O7) C, H, N.
5.4.2. 1-[20-O-(4-Methoxyphenylacetyl)-b-D-arabinofur-
anosyl]-5(E)-(2-bromovinyl)uracil (7b). Yield 67%; white
foam.
MALDI-TOFMS: m/z 826.4 Da (M+Na)+; 842.7 Da
1H NMR (DMSO-d6) d (ppm): 3.27 (s, 3H, CH3);
3.44 (s, 2H, CH2CO); 3.60–3.64 (m, 2H, H50 and
H500); 3.79–3.82 (m, 1H, H40); 4.14–4.16 (m, 1H,
H30); 5.16–5.20 (m, 1H, OH50); 5.17–5.21 (m, 1H,
H20); 5.85 (br s, 1H, OH30); 6.12 (d, 1H,
J = 5.3 Hz, H10); 6.85 (d, 1H, J = 13.5 Hz, vinyl);
7.77, 7.00 (AB system, 4H, JAB = 8.6 Hz, Ph); 7.27
(d, 1H, J = 13.6 Hz, vinyl); 7.86 (s, 1H, H6); 11.55
(br s, 1H, NH).
(M+K)+. C34H58BrN3O10Si2 requires 803.28.
5.3.3. 1-[20-O-(N-Boc-8-aminooctanoyl)-30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil (5l). 1H NMR (CDCl3) d
(ppm): 1.00–1.16 (m, 28H, TIPDS); 1.30–1.97 (m, 10H,
5 · CH2); 1.44 (s, 9H, t-Bu); 2.16–2.25 (m, 2H, CH2CO);
3.05–3.11 (m, 2H, CH2–N); 3.77–3.84 (m, part of ABX
system, 1H, H40); 4.02, 4.13 (part of ABX system, 2H,
JAB = 13.4 Hz, JAX = 3.0 Hz, JBX = 2.2 Hz, H50 and
H500); 4.38–4.47 (m, 1H, H30); 4.76 (br s, 1H, NH–
Boc), 5.55–5.62 (m, 1H, H20); 6.22 (d, 1H, J = 6.4 Hz,
H10); 6.64 (d, 1H, J = 13.6 Hz, vinyl); 7.41 (d, 1H, vi-
nyl); 7.45 (s, 1H, H6); 9.80 (br s, 1H, NH).
MALDI-TOFMS: m/z 497.6 Da (M+H)+; 520.0 Da
(M+Na)+. C20H21BrN2O8 requires 496.05. Anal.
(C20H21BrN2O8) C, H, N.
5.4.3. 1-(20-O-Octanoyl-b-D-arabinofuranosyl)-5(E)-(2-
bromovinyl)uracil (7d). Yield 58%; yellow foam.
MALDI-TOFMS: m/z 855.0 Da (M+Na)+; 871.2 Da
(M+K)+; 894.1 Da (M+Na+K)+. C36H62BrN3O10Si2
requires 831.32.
1H NMR (CDCl3) d (ppm): 0.81 (t, 3H, J = 6.5 Hz,
CH3); 1.10–1.35 (m, 8H, 4 · CH2); 1.50–1.69 (m, 2H,
CH2CH2CO); 2.27 (t, 2H, J = 7.2 Hz, CH2CO); 3.48–
3.50 (m, 1H, H40); 3.78–4.01 (m, 2H, H50, H500); 4.28–
4.38 (m, 1H, H30); 5.13–5.26 (m, 1H, H20); 5.12–5.50
(m, 1H, OH50); 5.70 (br s, 1H, OH30); 6.20 (d, 1H,
J = 5.7 Hz, H10); 6.60 (d, 1H, J = 13.6 Hz, vinyl); 7.31
(d, 1H, J = 13.6 Hz, vinyl); 7.60 (s, 1H, H6); 9.38 (br
s, 1H, NH).
5.3.4. 1-[20-O-(N-Boc-12-aminododecanoyl)-30,50-O-(1,1,
3,3-tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofurano-
1
syl]-5(E)-(2-bromovinyl)uracil (5m). H NMR (CDCl3) d
(ppm): 1.00–1.16 (m, 28H, TIPDS); 1.21–1.52 (m, 16H,
8 · CH2); 1.44 (s, 9H, t-Bu); 1.62–1.78 (m, 2H, CH2);
2.22–2.29 (m, 2H, CH2CO); 3.04–3.14 (m, 2H, CH2–
N); 3.87–4.05 (m, 3H, H50, H500, H40); 4.36–4.41 (m,
1H, H30); 4.61 (br s, 1H, NH-Boc); 5.24–5.30 (m, 1H,
H20); 6.30 (d, 1H, J = 5.8 Hz, H10); 6.66 (d, 1H,
J = 13.6 Hz, vinyl); 7.39 (d, 1H, vinyl); 7.74 (s, 1H,
H6); 9.31 (br s, 1H, NH).
MALDI-TOFMS: m/z 475.3 Da (M+H)+; 497.3 Da
(M+Na)+; 536.4 Da (M+K)+. C19H27BrN2O7 requires
474.10. Anal. (C19H27BrN2O7) C, H, N.
5.4.4. 1-[20-O-(N-Boc-4-aminobuanoyl)-b-D-arabinofur-
anosyl]-5(E)-(2-bromovinyl)uracil (7h). Yield: 75%; white
foam.
MALDI-TOFMS: m/z 810.4 Da (M+Na)+; 926.2 Da
(M+K)+. C40H70BrN3O10Si2 requires 887.38.
5.4. General procedure for the deprotection of TIPDS.
Synthesis of compounds 7a, b, d, h–m and 8d–g
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.36 (s, 9H,
t-Bu); 1.46–1.53 (m, 2H, CH2CH2CO); 2.09–2.15 (m,
1H, CHHCO); 2.20–2.26 (m, 1H, CHHCO); 2.84–
2.86 (m, 2H, CH2–N); 3.63–3.70 (m, 2H, H50 and
H500); 3.79–3.83 (m, 1H, H40); 4.09–4.13 (m, 1H,
H30); 5.16–5.22 (m, 2H, H20 and OH50); 5.81 (br s,
1H, OH30); 6.13 (d, 1H, J = 4.8 Hz, H10); 6.80 (bt,
1H, J = 5.6 Hz, NH–Boc); 6.88 (d, 1H, J = 13.2 Hz,
vinyl); 7.26 (d, 1H, vinyl); 8.00 (s, 1H, H6); 11.64
(br s, 1H, NH).
The TIPDS cleavage was carried out by treatment of the
protected compound (0.3 mmol) in methanol (10 mL)
with Dowexꢁ H+ form resin (0.25 g) and ammonium
fluoride (0.44 g, 1.2 mmol) as previously reported by
us (compounds 7c and e).24
5.4.1. 1-(20-O-Phenylacetyl-b-D-arabinofuranosyl)-5(E)-
(2-bromovinyl)uracil (7a). Yield 69%; white foam.
MALDI-TOFMS: m/z 556.5 Da (M+Na)+; 573.0 Da
(M+K)+. C20H28BrN3O9 requires 533.10. Anal.
(C20H28BrN3O9) C, H, N.
1H NMR (DMSO-d6) d (ppm): 3.51–3.69 (m, 4H,
CH2CO, H50 and H500); 3.79–3.82 (m, 1H, H40);

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3075
5.4.5. 1-[20-O-(N-Boc-6-aminohexanoyl)-b-D-arabinofur-
anosyl]-5(E)-(2-bromovinyl)uracil (7i). Yield: 78%; white
foam.
MALDI-TOFMS: m/z 385.4 Da (M+H)+; 407.2 Da
(M+Na)+; 423.2 Da (M+K)+. C18H28N2O7 requires
384.19. Anal. (C18H28N2O7) C, H, N.
1H NMR (DMSO) d (ppm): 1.05–1.13 (m, 2H, CH2);
1.23–1.37 (m, 4H, 2 · CH2); 1.35 (s, 9H, t-Bu); 2.09–
2.15 (m, 1H, CHH–CO); 2.18–2.24 (m, 1H, CHH–CO);
2.80–2.85 (m, 2H, CH2–N); 3.60–3.71 (m, 2H, H50,
H500); 3.77–3.81 (m, 1H, H40); 4.10–4.11 (m, 1H, H30);
5.09–5.18 (m, 1H, OH50); 5.16–5.18 (m, 1H, H20); 5.79
(d, 1H, J = 4.0 Hz, OH30); 6.13 (d, 1H, J = 5.2 Hz, H10);
6.71 (t, 1H, J = 6.0 Hz, NH–Boc); 6.89 (d, 1H,
J = 13.6 Hz, vinyl); 7.26 (d, 1H, J = 13.6 Hz, vinyl); 7.93
(s, 1H, H6); 11.63 (br s, 1H, NH).
5.4.9.
(8e). Yield 42%; white foam.
1-(20-O-Decanoyl-b-D-arabinofuranosyl)thymine
1H NMR (DMSO) d (ppm): 0.85 (t, 3H, J = 6.8 Hz,
CH3); 1.10–1.28 (m, 12H, 6 · CH2); 1.32–1.40 (m, 2H,
CH2CH2CO); 1.77 (s, 3H, CH3); 2.10–2.28 (m, 2H,
CH2CO); 3.58–3.70 (m, 2H, H50, H500); 3.74–3.78 (m,
1H, H40); 4.11–4.15 (m, 1H, H30); 5.06–5.09 (m, 1H,
OH50); 5.12–5.16 (m, 1H, H20); 5.75–5.77 (m, 1H,
OH30); 6.14 (d, 1H, J = 5.2 Hz, H10); 7.52 (s, 1H, H6);
11.37 (br s, 1H, NH).
MALDI-TOFMS: m/z 584.5 Da (M+Na)+; 601.2 Da
(M+K)+. C22H32BrN3O9 requires 561.13. Anal.
(C22H32BrN3O9) C, H, N.
MALDI-TOFMS: m/z 435.2 Da (M+Na)+; 451.3 Da
(M+K)+. C20H32N2O7 requires 412.22. Anal.
(C20H32N2O7) C, H, N.
5.4.6. 1-[20-O-(N-Boc-8-aminooctanoyl)-b-D-arabinofur-
anosyl]-5(E)-(2-bromovinyl)uracil (7l). Yield: 71%; white
foam.
5.4.10. 1-(20-O-Pentanoyl-b-D-arabinofuranosyl)thymine
(8f). Yield 60%; pale yellow foam.
1H NMR (CDCl3) d (ppm): 1.20–1.24 (m, 6H, 3 · CH2);
1.45 (s, 9H, t-Bu); 1.43–1.67 (m, 4H, 2 · CH2); 2.21–2.30
(m, 2H, CH2CO); 3.04–3.14 (m, 2H, CH2–N); 3.88–4.07
(m, 3H, H50, H500, H40); 4.37–4.43 (m, 1H, H30); 4.79 (br
s, 1H, NH–Boc); 5.24–5.27 (m, 1H, H20); 6.30 (d, 1H,
J = 6.0 Hz, H10); 6.67 (d, 1H, J = 13.8 Hz, vinyl); 7.39
(d, 1H, J = 13.8 Hz, vinyl); 7.77 (s, 1H, H6); 9.21 (br
s, 1H, NH).
1H NMR (CDCl3) d (ppm): 0.79 (t, 3H, J = 7.1 Hz,
CH3); 1.09–1.42 (m, 4H, 2 · CH2); 1.77 (s, 3H, CH3 het-
erocycle); 2.05–2.35 (m, 2H, CH2CO); 3.58–3.81 (m, 3H,
H50, H500, H40); 4.09–4.23 (m, 1H, H30); 5.01–5.23 (m,
2H, OH50, H20); 5.70–5.81 (m, 1H, OH30); 6.14 (d,
1H, J = 5.4 Hz, H10); 7.53 (d, 1H, J = 1 Hz, H6); 11.35
(br s, 1H, NH).
MALDI-TOFMS: m/z 365.2 Da (M+Na)+; 381.3 Da
(M+K)+. C15H22N2O7 requires 342.14. Anal.
(C15H22N2O7) C, H, N.
MALDI-TOFMS: m/z 613.0 Da (M+Na)+; 629.2 Da
(M+K)+, 652.2 Da (M+Na+K)+. C24H36BrN3O9
requires 589.16. Anal. (C24H36BrN3O9) C, H, N.
5.4.11.
mine (8g). Yield 50%; yellow foam.
1-(20-O-Dodecanoyl-b-D-arabinofuranosyl)thy-
5.4.7. 1-[20-O-(N-Boc-12-aminododecanoyl)-b-D-arabino-
furanosyl]-5(E)-(2-bromovinyl)uracil (7m). Yield: 100%;
white foam.
1H NMR (DMSO-d6) d (ppm): 0.85 (t, 3H, J = 6.6 Hz,
CH3); 1.09–1.30 (m, 16H, 8 · CH2); 1.16–1.20 (m, 2H,
CH2CH2CO); 1.77 (s, 3H, CH3); 2.10–2.25 (m, 2H,
CH2CO); 3.56–3.71 (m, 2H, H50, H500); 3.46–3.71 (m,
1H, H40); 4.50–4.85 (m, 1H, H30); 5.06–5.09 (m, 1H,
OH50); 5.61–5.90 (m, 1H, H20); 5.55–5.75 (m, 1H,
OH30); 6.14 (d, 1H, J = 5.2 Hz, H10); 7.52 (s, 1H, H6);
13.33 (br s, 1H, NH).
1H NMR (CDCl3) d: 1.21–1.52 (m, 16H, 8 · CH2); 1.44
(s, 9H, t-Bu); 1.62–1.78 (m, 2H, CH2); 2.22–2.29 (m, 2H,
CH2CO); 3.04–3.14 (m, 2H, CH2–N); 3.87–4.05 (m, 3H,
H50, H500, H40); 4.36–4.41 (m, 1H, H30); 4.61 (br s, 1H,
NH–Boc); 5.24–5.30 (m, 1H, H20); 6.30 (d, 1H,
J = 5.8 Hz, H10); 6.66 (d, 1H, J = 13.6 Hz, vinyl); 7.39
(d, 1H, J = 13.6 Hz, vinyl); 7.74 (s, 1H, H6); 9.31 (br
s, 1H, NH).
MALDI-TOFMS: m/z 463.9 Da (M+Na)+; 379.9 Da
(M+K)+. C22H36N2O7 requires 440.25. Anal.
(C22H36N2O7) C, H, N: H calcd 8.24; found 8.18.
MALDI-TOFMS: m/z 669.0 Da (M+Na)+; 685.2 Da
(M+K)+ C28H44BrN3O9 requires 645.23. Anal.
(C28H44BrN3O9) C, H, N: H calcd 6.86; found 6.83.
5.5. General procedure for the synthesis of 20-O-amino-
acyl derivatives 9, 10
5.4.8.
(8d). Yield 50%; white foam.
1-(20-O-Octanoyl-b-D-arabinofuranosyl)thymine
To a stirred suspension of compound 7h or 7i
(0.5 mmol) in dry diethyl ether (20 mL), HCl was
bubbled at 0 ꢂC until disappearance of the starting
material. The obtained white precipitate was col-
lected by filtration and washed with a minimum
amount of cold diethyl ether. The resulting sticky
solid, in both cases obtained, was crystallized from
CH3OH and diethyl ether to give the derivatives 9
and 10.
1H NMR (DMSO) d (ppm): 0.85 (t, 3H, J = 6.8 Hz,
CH3); 1.08–1.28 (m, 8H, 4 · CH2); 1.30–1.10 (m, 2H,
CH2CH2CO); 1.77 (s, 3H, CH3); 2.02–2.34 (m, 2H,
CH2CO); 3.57–3.69 (m, 2H, H50, H500); 3.71–3.82 (m,
1H, H40); 4.08–4.18 (m, 1H, H30); 5.02–5.19 (m, 2H,
OH50, H20); 5.72–5.83 (m, 1H, OH30); 6.14 (d, 1H,
J = 5.4 Hz, H10); 7.52 (s, 1H, H6); 11.32 (br s, 1H, NH).

3076
N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
5.5.1. 1-[20-O-(4-Aminobutanoyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil hydrochloride (9). Yield 60%;
white hygroscopic solid (mp: 200–203 ꢂC).
MALDI-TOFMS: m/z 449.5 Da (M+Na)+; 465.4 Da
(M+K)+. C20H30N2O8 requires 426.20.
5.5.4. 1-(20-O-Benzyl-b-D-arabinofuranosyl)thymine (15).
To a solution of compound 13 (100 mg, 0.23 mmol) in
anhydrous THF (3 mL) 60% NaH (27 mg, 0.58 mmol)
and, after 10 min, benzyl bromide (70 lL, 0.58 mmol)
were added and the mixture was stirred at room temper-
ature for 16 h under argon atmosphere. Then the solvent
was evaporated and the residue dissolved in CH2Cl2
(20 mL) was washed with aqueous saturated NH4Cl
solution (10 mL) and H2O (10 mL). The organic layer,
dried (Na2SO4) and evaporated to dryness, gave a crude
residue that was used for the next step, without any fur-
ther purification. The above mentioned residue was dis-
solved in CH3OH (4 mL) and p-TsOH monohydrate
(115 mg, 0.514 mmol) was added and the mixture was
vigorously stirred at room temperature for 4 h. After
the evaporation the crude residue was purified by silica
gel column chromatography (eluent: CH2Cl2/CH3OH,
98/2 ! 90/10, v/v) to give 15 (42 mg, overall yield
52%) as a yellow oil.
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.64–1.72 (m,
2H, CH2CH2CO); 2.16–2.24 (m, 1H, CHH–N); 2.37–
2.45 (m, 1H, CHH–N); 2.70–2.75 (m, 2H, CH2CO);
3.61–3.72 (m, 2H, H50 and H500); 3.79–3.83 (m, 1H,
H40); 4.11–4.15 (m, 1H, H30); 5.21–5.26 (m, 2H, H20
and OH50); 5.81 (d, 1H, J = 5.2 Hz, OH30); 6.11 (d,
1H, J = 5.6 Hz, H10); 6.88 (d, 1H, J = 13.6 Hz, vinyl);
7.25 (d, 1H, J = 13.6 Hz, vinyl); 7.76 (br s, 3H, NH3þ);
8.05 (s, 1H, H6); 11.62 (s, 1H, NH).
MALDI-TOFMS: m/z: 435.5 Da (M+H)+; 457.7 Da
(M+Na)+ C15H20BrN3O7 Æ HCl requires 433.05. Anal.
(C15H20BrN3O7 Æ HCl) C, H, N.
5.5.2. 1-[20-O-(6-Aminohexanoyl)-b-D-arabinofuranosyl]-
5(E)-(2-bromovinyl)uracil hydrochloride (10). Yield 90%;
white hygroscopic solid (mp: 198–200 ꢂC).
1H NMR (400 MHz, DMSO-d6) d (ppm): 1.17–1.48 (m,
6H, 3 · CH2); 2.09–2.16 (m, 1H, CHH–CO); 2.23–2.30
(m, 1H, CHH–CO); 2.68–2.70 (m, 2H, CH2–N); 3.57–
3.67 (m, 2H, H50 and H500); 3.80–3.81 (m, 1H, H40);
4.11–4.13 (m, 1H, H30); 5.20–5.26 (m, 2H, H20 and
OH50); 5.80 (br s, 1H, OH30); 6.13 (d, 1H, J = 4.8 Hz,
H10); 6.91 (d, 1H, J = 13.2 Hz, vinyl); 7.26 (d, 1H,
J = 13.2 Hz, vinyl); 7.81 (br s, 3H, NH3þ); 7.98 (s, 1H,
H6); 11.65 (s, 1H, NH).
1H NMR (DMSO-d6) d (ppm): 1.74 (s, 3H, CH3); 3.55–
3.73 (m, 3H, H500, H50, H40); 4.04–4.10 (m, 2H, H30,
H20); 4.41, 4.58 (d, AB system, 2H, J = 11.9 Hz, CH2–
Ph); 5.02–5.11 (m, 1H, OH50); 5.56–5.59 (m, 1H,
OH30); 6.17 (d, 1H, J = 5.3 Hz, H10); 7.10–7.45 (m,
5H, Ph); 7.62 (s, 1H, H6): 11.33 (br s, 1H, NH).
MALDI-TOFMS: m/z 371.2 Da (M+Na)+; 387.4 Da
(M+K)+. C17H20N2O6 requires 348.13. Anal.
(C17H20N2O6) C, H, N.
MALDI-TOFMS: m/z 463.4 Da (M+H)+; 485.5 Da
(M+Na)+. C17H24BrN3O7ÆHCl requires 461.08. Anal.
(C17H24BrN3O7ÆHCl) C, H, N.
5.5.5. N3-Octyl-1-(b-D-arabinofuranosyl)thymine (16).
Compound 13 (120 mg, 0.28 mmol) was dissolved in
a 1:1 mixture of anhydrous THF–DMSO (3 mL) and
to the stirred solution octyl bromide (67 ll, 0.33 mmol)
and K2CO3 (46 mg, 0.33 mmol) were added. The reac-
tion mixture was kept at room temperature under
argon atmosphere for 6 h, then the mixture was diluted
with H2O, neutralized with 2 N HCl (30 mL), and
extracted with EtOAc. The organic phase, dried
(Na2SO4) and evaporated to dryness, gave a crude
residue that was used, without any further purification,
for the next step.
5.5.3. 1-(30,50-O-Tetrahydropyran-2-yl-b-D-arabinofur-
anosyl)thymine (13). Compound 1226 (316 mg,
1.32 mmol) was dissolved in dry CH3CN (12 mL)
and dihydropyrane (DHP, 1.14 mL, 13.2 mmol) and
p-toluenesulfonic acid monohydrate (p-TsOH, 24 mg,
0.13 mmol) were added. After 3 h at room tempera-
ture the solvent was evaporated and the resulting res-
idue, dissolved in CH2Cl2 (10 mL), was washed with
saturated NaHCO3 (10 mL). The organic layer was
dried (Na2SO4) and evaporated to give a crude residue
(700 mg) which was dissolved in a solution of 0.1 M
KOH in 95% EtOH (12 mL); then the obtained mix-
ture was heated at reflux conditions for 4 h. The sol-
vent was then evaporated and the residue, dissolved
in EtOAc (20 mL), was washed with brine (20 mL),
dried over Na2SO4, filtered, and evaporated. The
resulting crude yellow oil was purified by silica gel col-
umn chromathography (eluent: petroleum ether/
EtOAc, 70/30 ! 0/100, v/v) to give 13 (420 mg, overall
yield 75%) as yellow oil.
The above mentioned residue (100 mg) was dissolved
in CH3OH (4 mL) and p-TsOH monohydrate
(115 mg, 0.514 mmol) was added. The mixture was
vigorously stirred at room temperature for 4 h. After
evaporation the crude residue was purified by silica
gel column chromatography (eluent: CH2Cl2/CH3OH,
98/2 ! 94/6, v/v) to give 16 (60 mg, overall yield
58%) as a yellow oil.
1H NMR (DMSO-d6)
d
(ppm): 0.85 (t, 3H,
1H NMR (CDCl3) d (ppm): 1.42–1.75 (m, 12H, 6 · CH2
THP); 1.87 (s, 3H, CH3); 3.40–3.55 (m, 2H, H50, H500);
3.60–3.90 (m, 4H, 2 · CH2O THP); 3.92–4.29 (m, 3H,
H40, H30, H20); 4.62–4.90 (m, 2H, 2 · CHO THP);
5.65–5.77 (m, 1H, OH20); 5.97 (d, 1H, J = 6.0 Hz,
H10); 7.35 (s, 1H, H6); 11.31 (br s, 1H, NH).
J = 6.0 Hz, CH3); 1.12–1.34 (m, 10H, 5 · CH2); 1.40–
1.59 (m, 2H, CH2CH2N); 1.80 (s, 3H, CH3); 3.58–
3.82 (m, 4H, H50, H500, CH2N); 3.86–4.19 (m, 3H,
H40, H30, H20); 5.05–5.13 (m, 1H, OH50); 5.41–5.58
(m, 2H, OH30, OH20); 6.01 (d, 1H, J = 4.6 Hz, H10);
7.57 (s, 1H, H6).

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3077
MALDI-TOFMS: m/z 471.9 Da (M+H)+; 393.2 Da
(M+Na)+; 409.4 Da (M+K)+. C18H30N2O6 requires
370.21. Anal. (C18H30N2O6) C, H, N.
at room temperature under argon atmosphere. After
10 min a solution of freshly prepared octyl iodide
(324 mg 1.35 mmol) in anhydrous THF (2.5 mL) was
added and the mixture was heated at refluxing condi-
tions for 24 h. The solvent was evaporated and the res-
idue dissolved in CH2Cl2 (20 mL), washed with
aqueous saturated NH4Cl solution (10 mL) and H2O
(10 mL). The organic phase, dried over MgSO4 and
evaporated to dryness, gave a crude residue that was
purified by silica gel column chromatography (eluent:
petroleum ether/EtOAc, 90/10 ! 0/100, v/v) to give 19
(295 mg, 50%) as a thick white oil.
5.5.6. 1-(20-O-Octyl-b-D-arabinofuranosyl)thymine (17)
and N3-octyl-1-(20-O-octyl-b-D-arabinofuranosyl)thymine
(18). To a stirred solution of compound 13 (188 mg,
0.44 mmol) in anhydrous THF (2.5 mL) 60% NaH
(70 mg, 1.75 mmol) was added at room temperature un-
der argon atmosphere. After 10 min a solution of freshly
prepared octyl iodide (425 mg, 1.75 mmol) in anhydrous
THF (2.5 mL) was added, and the mixture was heated at
70 ꢂC for 24 h. The solvent was evaporated and the res-
idue, dissolved in CH2Cl2 (20 mL), was washed with
aqueous saturated NH4Cl solution (10 mL), and H2O
(10 mL). The organic phase was then dried (Na2SO4)
and evaporated to dryness to give crude residue that
was used, for the next step, without any further purifica-
tion. The above mentioned residue (110 mg) was dis-
solved in CH3OH (8 mL) and treated with p-TsOH
monohydrate (196 mg, 0.88 mmol), and the mixture
was vigorously stirred at room temperature for 4 h.
After evaporation of the solvent the resulting crude res-
idue was purified by silica gel column chromatography
(eluent: CH2Cl2/CH3OH, 98/2 ! 90/10, v/v) to give a
mixture of the expected hydroxyl derivative (17), to-
gether with the N3, 20-O-bis-alkylated product (18) (mo-
lar ratio 17/18 = 3:1).
1H NMR (DMSO-d6) d: 0.83 (t, 3H, J = 6.0 Hz, CH3);
1.15–1.30 (m, 12H, 6 · CH2); 1.32–1.79 (m, 12H,
6 · CH2 THP); 3.22–3.64 (m, 4H, H500, H50, CH2O);
3.66–4.32 (m, 7H, 2 · CH2O THP, H40, H30, H20);
4.59–4.88 (m, 2H, 2 · CHO THP); 5.56–5.64 (m, 1H,
H5); 6.18–6.20 (m, 1H, H10); 7.52–7.58 (m, 1H, H6);
11.40 (br s, 1H, NH).
MALDI-TOFMS: m/z 561.9 Da (M+Na)+; 577.8 Da
(M+K)+. C27H44N2O8 requires 524.31.
5.5.10. 4-(1,2,4-Triazol-1-y1)-1-(20-O-octyl-30,50-O-tetra-
hydropyran-2-yl-b-D-arabinofuranosyl)uracil (20). 1,2,4-
Triazole (355 mg, 5 mmol) and anhydrous POCl3
(79 ll, 0.86 mmol) were dissolved, under argon
atmosphere, in dry CH3CN (2 mL) and to the mixture,
cooled at 0 ꢂC, was dropwise added dry triethylamine
(TEA, 0.6 mL, 4.29 mmol). Compound 19 (150 mg,
0.286 mmol) dissolved in dry CH3CN (1.5 mL) was
added to the above prepared mixture and the obtained
suspension was stirred at room temperature for 15 h.
The solvent was then evaporated to dryness and the res-
idue dissolved in CH2Cl2 (20 mL) washed with NaHCO3
(20 mL), and brine (20 mL). After drying (Na2SO4), the
organic layer was evaporated, and the obtained crude
residue was purified by silica gel column chromatogra-
phy (eluent: petroleum ether/Et2O, 50/50 ! 20/80, v/v)
to give 20 (81 mg, 50%) as a thick white oil. The com-
pound has been used immediately for the next prepara-
tion due to its relative instability.
5.5.7. 1-(20-O-Octyl-b-D-arabinofuranosyl)thymine (17).
Yield 55% with respect to 13; yellow oil.
1H NMR (DMSO-d6)
d
(ppm): 0.84 (t, 3H,
J = 6.4 Hz, CH3); 1.05–1.42 (m, 12H, 6 · CH2); 1.75
(s, 3H, CH3); 3.11–3.70 (m, 5H, H500, H50, CH2O,
H40); 3.82–3.91 (m, 1H, H30); 3.93–4.07 (m, 1H,
H20); 4.09–5.08 (m, 1H, OH50); 5.51–5.59 (m, 1H,
OH30); 6.12 (d, 1H, J = 5.5 Hz, H10); 7.48 (s, 1H,
H6); 11.31 (br s, 1H, NH).
MALDI-TOFMS: m/z 393.3 Da (M+Na)+; 409.2 Da
(M+K)+. C18H30N2O6 requires 370.21. Anal.
(C18H30N2O6) C, H, N.
5.5.8. N3-Octyl-1-(20-O-octyl-b-D-arabino-furanosyl)thy-
mine (18). Yield 17% with respect to 13; yellow oil.
1H NMR (CDCl3) d: 0.78–0.88 (m, 3H, CH3); 1.11–1.30
(m, 12H, 6 · CH2); 1.61–1.82 (m, 12H, 6 · CH2 THP);
3.25–3.63 (m, 4H, H500, H50, CH2O); 3.60–4.39 (m, 7H,
2 · CH2O THP, H40, H30, H20); 4.62–4.88 (m, 2H,
2 · CHO THP); 6.35 (m, 1H, H10); 6.98–6.03 (m, 1H,
H5); 8.13 (s, 1H, H3 triazole); 8.33–8.48 (m, 1H, H6);
8.50 (s, 1H, H5 triazole).
1H NMR (DMSO d6) d (ppm): 0.79–0.90 (m, 6H,
2 · CH3); 1.05–1.34 (m, 22H, 11 · CH2); 1.40–1.59 (m,
2H, CH2CH2O); 1.80 (s, 3H, CH3); 3.11–3.35 (m, 2H,
CH2N); 3.52–3.83 (m, 5H, H40, H500, H50, CH2O);
3.90–4.07 (m, 2H, H30, H20); 5.02–5.11 (m, 1H, OH50);
5.53–5.60 (m, 1H, OH30); 6.15 (d, 1H, J = 5.5 Hz,
H10); 7.58 (s, 1H, H6).
MALDI-TOFMS: m/z 598.9 Da (M+Na)+; 414.8
(M+K)+. C29H45N5O7 requires 575.33.
MALDI-TOFMS: m/z 505.3 Da (M+Na)+; 521.8 Da
(M+K)+. C26H46N2O6 requires 482.34. Anal.
(C26H46N2O6) C, H, N.
5.5.11. 1-(20-O-Octyl-b-D-arabinofuranosyl)cytosine (21).
To a stirred solution of compound 20 (120 mg,
0.2 mmol) in dioxane (4 mL), 4 mL of 30% NH4OH
was added at room temperature. After 20 h the solvent
was evaporated and the crude residue (about 110 mg)
without any further purification, was dissolved in
4 mL CH3OH and to the resulting solution, p-toluen-
sulfonic acid monohydrate (70 mg, 0.368 mmol) was
5.5.9. 1-(20-O-Octyl-30,50-O-tetrahydropyran-2-yl-b-D-
arabinofuranosyl)uracil (19). To a stirred solution of
compound 1428 (465 mg, 1.13 mmol) in anhydrous
THF (9.5 mL), 60% NaH (125 mg, 5.2 mmol) was added

3078
N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
added at room temperature. After 4 h the solvent was
evaporated and the crude residue, dissolved in H2O,
was treated with Dowexꢁ OHꢀ form 1 · 2–400 resin
up to pH 7. The filtered solution was then evaporated
and the resulting yellow residue was purified by silica
gel column chromatography (eluent: CH2Cl2/CH3OH,
95/5 ! 90/10, v/v) to give compound 21 (32 mg, overall
yield 46%) as a white solid (mp: 89–92 ꢂC).
6 · CH2); 1.35–1.41 (m, 2H, COCH2CH2); 1.77 (s, 3H,
CH3 heterocycle); 1.98 (t, 2H, J = 7.6 Hz, COCH2);
3.78–3.81 (m, 1H, H40); 3.94, 4.05 (part of ABX system,
2H, JAB = 12.8 Hz, JAX = 2.8 Hz, JBX = 3.2 Hz, H50,
H500); 4.18–4.23 (m, 1H, H30); 4.63–4.69 (m, 1H, H20);
5.98 (d, 1H, J = 7.2 Hz, H10); 7.22 (s, 1H, H6); 8.02
(d, 1H, J = 8.8 Hz, NH); 11.25 (br s, 1H, NH).
MALDI-TOFMS: m/z 676.8 Da (M+Na)+; 692.2
1H NMR (DMSO-d6) d: 0.84 (t, 3H, J = 6.0 Hz, CH3);
1.14–1.30 (m, 12H, 6 · CH2); 3.12–3.16 (m, 2H,
CH2O); 3.50–3.56 (m, 2H, H500, H50); 3.65–3.68 (m,
1H, H40); 3.77–3.81 (m, 1H, H30); 3.94–3.96 (m, 1H,
H20); 4.88 (t, 1H, J = 4.0 Hz, OH50); 5.48 (d, 1H,
J = 2.0 Hz, OH30); 5.66 (d, 1H, J = 7.4 Hz, H5); 6.12
(d, 1H, J = 6.0 Hz, H10); 7.05–7.12 (m, 2H, NH2); 7.51
(d, 1H, J = 7.4 Hz, H6).
(M+K)+. C32H59N3O7Si2 requires 653.39.
5.5.14. 1-(20-N-Nonylcarboxamido-20deoxy-b-D-arabino-
furanosyl)thymine (25). The TIPDS cleavage of the com-
pound 24 was carried out as previously described for the
compounds 7 and 8.
Yield 80%; white foam.
MALDI-TOFMS: m/z 356.4 Da (M+H)+; 378.9 Da
(M+Na)+; 394.2 (M+K)+. C17H29N3O5 requires
355.21. Anal. (C17H29N3O5) C, H, N.
1H NMR (DMSO) d (ppm): 0.85 (t, 2H, J = 7.6 Hz,
CH3); 1.08–1.28 (m, 12H, 6 · CH2); 1.74 (s, 3H, CH3
heterocycle); 1.95 (t, 2H, J = 7.6 Hz, CH2); 3.55–3.82
(m, 3H, H40, H50, H500); 4.06 (m, 1H, H30); 4.31 (m,
1H, H20); 5.29 (br s, 1H, OH50); 5.57 (d, 1H,
J = 5.6 Hz, OH30); 6.03 (d, 1H, J = 6.4 Hz, H10); 7.48
(s, 1H, H6); 7.94 (d, 1H, J = 8.0 Hz, NH); 11.16 (s,
1H, NH heterocycle).
5.5.12. 1-[20-Amino-20-deoxy-30,50-O-(1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]thymine (23).
A solution of 2230 (100 mg, 0.16 mmol) in dry methanol
(5 mL) was vigorously stirred in a hydrogen atmosphere
with 10% palladium-on-carbon catalyst for 2 h. The
mixture was then filtered on Celite pad and evaporated
under vacuo. The crude residue was purified by silica
gel column chromatography (eluent CH2Cl2/CH3OH,
99/1, v/v) to give the expected product 23 as a white
foam (32 mg, 40%).
MALDI-TOFMS: m/z 434.3 Da (M+Na)+; 450.8
(M+K)+. C20H33N3O6 requires 411.24. Anal.
(C20H33N3O6) C, H, N.
5.5.15. N2-(Dimethylamino)methylen-9-[30,50- O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
guanine (27). To a stirred suspension of compound 2631
(315 mg, 0.6 mmol), in dry CH3CN (5 mL), N,N-diiso-
propylethylamine (DIPEA, 300 lL, 1.8 mmol) and
N,N-dimethylformamide dimethyl acetal (DMFDA,
240 lL, 1.8 mmol) were added under argon atmosphere.
After 15 h the yellow solution was evaporated to dryness
and the crude residue was purified by silica gel column
chromatography (eluent: CH2Cl2/CH3OH, 98/2 ! 95/
5, v/v) to give the expected product 27 (243 mg, 70%)
as a colorless solid (mp: 221–225 ꢂC).
1H NMR (DMSO) d (ppm): 0.97–1.07 (m, 28H, TIPDS);
1.73 (s, 3H, CH3); 3.56 (t, 1H, J = 7.6 Hz, H30); 3.66–3.69
(m, 1H, H40); 3.90–4.20 (m, 3H, H50, H500, H20); 5.90 (d,
1H, J = 6.8 Hz, H10); 7.27 (d, 1H, J = 1.1 Hz, H6); 9.18
(br s, 2H, NH2); 11.30 (br s, 1H, NH).
MALDI-TOFMS: m/z 501.0 Da (M+H)+; 522.6 Da
(M+Na)+; 538.8 (M+K)+. C22H41N3O6Si2 requires
499.25.
5.5.13.
1-[20-N-Nonylcarboxamido-20-deoxy-30,50-O-
(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-D-arabino-
furanosyl]thymine (24). A mixture of decanoic acid
(96 mg, 0.56 mmol), N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDC) (130 mg,
0.67 mmol), and 1-hydroxybenzotriazole hydrate
(HOBT) (83 mg, 0.62 mmol) in 20 mL of dry CH2Cl2
was stirred at room temperature for 1 h. In a separate
flask, compound 21 (280 mg, 0.56 mmol) was dissolved
in 10 mL of dry CH2Cl2 and the resulting solution was
added to the stirred active ester mixture. After 24 h at
room temperature the organic layer was washed with sat-
urated NH4Cl (4 · 20 mL) and then dried over Na2SO4.
The solvent was evaporated to dryness, and the obtained
residue was purified by silica gel column chromatography
(eluent: CH2Cl2/CH3OH, 99/1, v/v) to give the expected
compound 24 (200 mg, 54%) as a white foam.
1H NMR (DMSO) d (ppm): 0.98–1.18 (m, 28H, TIP-
DS); 3.02 (s, 3H, N–CH3); 3.14 (s, 3H, N–CH3); 3.73–
3.81 (m, 1H, H40); 3.78–4.07 (m, 2H, H50, H500); 4.25–
4.35 (m, 1H, H30); 4.41–4.52 (m, 1H, H20); 5.78–5.81
(m, 1H, OH20); 6.12 (d, 1H, J = 6.6 Hz, H10); 7.71 (s,
1H, CH–N); 8.56 (s, 1H, H8); 11.31 (br s, 1H, NH).
MALDI-TOFMS: m/z 581.4 Da (M+H)+; 603.5
(M+Na)+; 619.3 Da (M+K)+. C25H44N6O6Si2 requires
580.29.
5.5.16. N2-(Dimethylamino)methylen-9-[20-O-octanoyl-30,
50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-D-ara-
binofuranosyl]guanine (28a). Compound 28a was ob-
tained from the compound 27 adapting the procedure
described in the literature.35 Compound 27 (200 mg,
0.35 mmol) was dissolved in dry pyridine (4 mL) and
to the stirred solution TEA (125 lL, 0.09 mmol) and
octanoyl chloride (175 lL, 0.63 mmol) were added.
1H NMR (400 MHz, DMSO-d6) d (ppm): 0.87 (t, 3H,
J = 7.2 Hz, CH3); 0.94–1.24 (m, 40H, TIPDS,

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
3079
The solution was heated at 50 ꢂC for 4 h. The solvent
was then evaporated and the crude residue dissolved in
CH2Cl2 and washed with saturated NaHCO3 (2 ·
20 mL) then dried (MgSO4) and evaporated to dryness.
The resulting crude residue was purified by silica gel
column chromatography (eluent: CH2Cl2/CH3OH, 95/
5, v/v) to give the expected product 28a (151 mg, 61%)
as a yellow foam.
OH50); 5.82–5.84 (m, 1H, OH30); 6.30 (d, 1H,
J = 6.1 Hz, H10); 7.93 (1H, CH–N); 8.54 (s, 1H, H8);
11.34 (br s, 1H, NH).
MALDI-TOFMS: m/z 465.4 Da (M+H)+; 487.2
(M+Na)+; 503.4 Da (M+K)+. C21H32N6O6 requires
464.24.
5.5.20. N2-(Dimethylamino)methylen-9-(20-O-pentanoyl-
b-D-arabinofuranosyl)guanine (29b). Yield 56%; yellow
foam.
1H NMR (CDCl3) d (ppm): 0.82–0.92 (m, 3H, CH3);
1.00–1.32 (m, 38H, TIPDS, 5 · CH2); 1.80–2.08 (m,
2H, CH2CO); 3.10 (s, 3H, N–CH3); 3.20 (s, 3H, N–
CH3); 3.81–4.18 (m, 3H, H50, H40, H500); 4.23–4.67 (m,
1H, H30); 5.51–5.58 (m, 1H, H20); 6.40 (d, 1H,
J = 6.34 Hz, H10); 7.90 (1H, CH@N); 8.61 (s, 1H, H8);
9.53 (br s, 1H, NH).
1H NMR (DMSO) d (ppm): 0.69 (t, 3H, J = 7.1 Hz,
CH3); 1.05–1.25 (m, 4H, 2 · CH2); 1.85–2.18 (m, 2H,
CH2CO); 3.03 (s, 3H, N–CH3); 3.16 (s, 3H, N–CH3);
3.59–3.94 (m, 3H, H50, H40, H500); 4.38–4.50 (m, 1H,
H30); 5.04–5.10 (m, 1H, H20); 5.25–5.31 (m, 1H,
OH50); 5.81–5.84 (m, 1H, OH30); 6.30 (d, 1H,
J = 4.4 Hz, H10); 7.94 (1H, CH–N); 8.54 (s, 1H, H8);
11.34 (br s, 1H, NH).
MALDI-TOFMS: m/z 707.4 Da (M+H)+; 729.4
(M+Na)+; 745.3 Da (M + K)+. C33H58N6O7Si2 requires
706.39.
5.5.17. N2-(Dimethylamino)methylen-9-[20-O-pentanoyl-
30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-b-D-
arabinofuranosyl]guanine (28b). Compound 27 (200 mg,
0.35 mmol) was dissolved in dry DMF (5 mL) and to
the stirred solution 4-DMAP (10 mg, 0.02 mmol) and
valeric anhydride (92 lL, 0.49 mmol) were added. The
solution was reacted at room temperature for 5 h. The
solvent was then evaporated and the crude residue dis-
solved in Et2O and washed with saturated H2O (2 ·
20 mL) then dried (MgSO4) and evaporated to dryness.
The resulting crude residue was purified by silica gel
column chromatography (eluent: CH2Cl2/CH3OH, 97/
3, v/v) to give the expected product 28b (173 mg, 77%)
as a yellow foam.
MALDI-TOFMS: m/z 423.3 Da (M+H)+; 445.4
(M+Na)+; 461.5 Da (M+K)+. C18H26N6O6 requires
422.19.
5.5.21. 9-(20-O-Octanoyl-b-D-arabinofuranosyl)guanine
(30a). Compound 29a (80 mg, 0.17 mmol) was dissolved
in 410 lL of a 2:1 CH3OH: H2O mixture and to the stir-
red solution 44 lL of TFA (0.57 mmol) was added at
room temperature. After 14 h half of the volume of
the solution was evaporated in vacuo and to the solution
were added a 2:1 mixture EtOH/H2O and 17 mg
(0.21 mmol) NH4HCO3. The mixture was then evapo-
rated to dryness and the crude residue was purified by
silica gel column chromatography (eluent: CH2Cl2/
CH3OH, 98/2 ! 90/10, v/v) to give the expected product
30a (46 mg, yield 66%) as a yellow foam.
1H NMR (DMSO) d (ppm): 0.64–0.72 (m, 3H, CH3);
0.08–1.20 (m, 30H, TIPDS, CH2CH3); 1.78–2.23 (m,
4H, CH2CH2CO); 3.03 (s, 3H, N–CH3); 3.14 (s, 3H,
N–CH3); 3.93–4.02 (m, 2H, H50, H40); 4.09–4.20 (m,
1H, H500); 4.56–4.64 (m, 1H, H30); 5.56–5.64 (m, 1H,
H20); 6.31 (d, 1H, J = 6.1 Hz, H10); 7.79 (s, 1H, CH@N);
8.49 (s, 1H, H8); 11.41 (br s, 1H, NH).
1H NMR (DMSO) d (ppm): 0.83 (t, 3H, J = 7.2 Hz,
CH3); 1.05–1.29 (m, 10H, 5 · CH2); 1.89–2.18 (m, 2H,
CH2CO); 3.58–3.87 (m, 3H, H500, H40, H50); 4.28–4.40
(m, 1H, H30); 5.00–5.06 (m, 1H, H20); 5.18–5.25 (m,
1H, OH50); 5.80–5.83 (m, 1H, OH30); 6.16 (d, 1H,
J = 5.8 Hz, H10); 6.50 (br s, 2H, NH2); 7.78 (s, 1H,
H8); 10.60 (br s, 1H, NH).
MALDI-TOFMS: m/z 665.8 Da (M+H)+; 687.3
(M+Na)+; 703.5 Da (M + K)+. C30H52N6O7Si2 requires
664.34.
MALDI-TOFMS: m/z 410.3 Da (M+H)+; 432.2 Da
(M+Na)+; 448.2 Da (M+K)+. C18H27N5O6 requires
409.20. Anal. (C18H27N5O6) C, H, N.
5.5.18. N2-(Dimethylamino)methylen-9-(20-O-acyl-b-D-
arabinofuranosyl)guanine. General procedure. (Com-
pounds 29a and b). The TIPDS cleavage of compounds
28a and b was carried out as previously described for
the compounds 7 and 8.
5.5.22. 9-(20-O-Pentanoyl-b-D-arabinofuranosyl)guanine
(30b). Compound 29b was deprotected with TFA in a
2:1 CH3OH/H2O mixture, as described for compound
30a.
5.5.19. N2-(Dimethylamino)methylen-9-(20-O-octanoyl-b-
D-arabinofuranosyl)guanine (29a). Yield 40%; yellow
foam.
Yield 97%; yellow foam.
1H NMR (DMSO) d (ppm): 0.73 (t, 3H, J = 7.2 Hz, CH3);
0.92–1.33 (m, 4H, 2 · CH2); 1.89–2.21 (m, 2H, CH2CO);
3.60–3.92 (m, 3H, H50, H500, H40), 4.30–4.42 (m, 1H,
H30); 5.05 (br s, 1H, OH50); 5.20–5.28 (m, 1H, H20);
5.80 (br s, 1H, OH30); 6.16 (d, 1H, J = 5.8 Hz, H10);
6.53 (br s, 2H, NH2); 7.79 (s, 1H, H8); 8.85 (br s, 1H, NH).
1H NMR (DMSO) d (ppm): 0.83 (t, 3H, J = 6.5 Hz,
CH3); 0.90–1.28 (m, 10H, 5 · CH2); 1.80–2.20 (m, 2H,
CH2CO); 3.03 (s, 3H, N–CH3); 3.16 (s, 3H, N–CH3);
3.55–3.85 (m, 3H, H50, H40, H500); 4.36–4.44 (m, 1H,
H30); 5.03–5.10 (m, 1H, H20); 5.25–5.31 (m, 1H,

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N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
MALDI-TOFMS: m/z 368.4 Da (M+H)+; 390.6 Da
(M+Na)+; 406.7 Da (M+K)+. C15H21N5O6 requires
367.15 Da. Anal. (C15H21N5O6) C, H, N.
for 5 min in 1 mM ammonium formate, once for
1 min in H2O, and once for 5 min in ethanol. The radio-
activity was determined by scintillation counting.
5.5.23. N2-Octanoyl-9-[20-O-octanoyl-(30,50-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)-b-D-arabinofuranosyl]-
guanine (31). Compound 31 was obtained from the
compound 26 using the general acylation method
previously described for compounds 5 and 6.
6.2. Tissue cultures
Primary fibroblasts were derived from forearm skin
biopsies from healthy volunteers (with informed con-
sent). Cells were grown in a permissive medium:
DMEM (Dulbecco’s modified Eagle’s medium) with
4.5 g/l glucose, 10% fetal calf serum in the presence
of 50 lg/ml uridine and 110 lg/mL (DMEM UP) or
in a restrictive medium: glucose free RPMI (Roswell
Park Memorial Institute) supplied with 50 lM galact-
ose and 10% dialyzed fetal calf serum (GAL) at 37 ꢂC
in 5% CO2.33 Viability was assessed by trypan blue
exclusion.
Yield 53%; pale yellow oil.
1H NMR (DMSO) d (ppm): 0.75–0.87 (m, 6H, 2 · CH3);
0.90–1.40 (m, 44H, TIPDS, 8 · CH2); 1.55–1.89 (m, 4H,
2 · CH2CH2CO); 2.35–2.50 (m, 4H, 2 · CH2CO); 3.85–
3.97 (m, 1H, H40); 4.08–4.20 (m, 2H, H50, H500); 4.61–
4.83 (m, 1H, H30); 4.45–4.55 (m, 1H, H20); 6.27 (d,
1H, J = 6.3 Hz, H10); 7.74 (s, 1H, H8); 8.86 (br s, 1H,
NHCO); 11.21 (br s, 1H, NH).
6.3. Mitochondrial isolation
MALDI-TOFMS: m/z 778.5 Da (M+H)+; 800.4
(M+Na)+; 816.5 Da (M+K)+. C38H67N5O8Si2 requires
777.45.
Mitochondria were isolated from fibroblasts homoge-
nized by nitrogen cavitation followed by differential cen-
trifugation in sucrose buffer.36
5.5.24.
N2-Octanoyl-9-(20-O-octanoyl-b-D-arabinofur-
6.4. Enzymatic assays in fibroblast mitochondria
anosyl)guanine (32). The cleavage of TIPDS of the com-
pound 31 (320 mg, 0.4 mmol) was carried out as
previously described for compounds 7 and 8, to give
compound 32 (86 mg, 40%) as a pale yellow oil.
Cytochrome c oxidase was measured by spectrophoto-
meter, monitoring the rate of oxidation of cytochrome
c at 550 nm and succinate dehydrogenase by following
phenazine methosulfate-mediated reduction of dichlo-
rophenolindophenol at 600 nm.37,38
1H NMR (DMSO) d (ppm): 0.77–0.92 (m, 6H, 2 · CH3);
1.01–1.38 (m, 16H, 8 · CH2); 1.49–1.69 (m, 4H,
2 · CH2CH2CO); 1.83–2.25 (m, 4H, 2 · CH2CO); 3.43–
3.90 (m, 3H, H50, H40, H500); 4.32–4.46 (m, 1H, H30);
5.09–5.18 (m, 1H, OH50); 5.31–5.47 (m, 1H, H20); 5.87–
5.92 (m, 1H, OH30); 6.26 (d, 1H, J = 5.96 Hz, H10); 8.14
(s, 1H, H8); 8.75 (br s, 1H, NHCO).
TK2 activity in isolated fibroblast mitochondria was
determined by radiochemical methods measuring the
phosphorylation of [methyl-3H]dThd and [3H]dCyd
while dGK was measured with [3H]dado.38,2
6.5. Real time PCR
MALDI-TOFMS: m/z 558.6 (M+Na)+; 574.3 Da
(M+K)+. C26H41N5O7 requires 535.30. Anal.
(C26H41N5O7) C, H, N.
Mitochondrial DNA was quantified by real-time quanti-
tative PCR using Taqman probes from Applied Biosys-
tems UK. The mtDNA to nuclear DNA ratio was
calculated and compared to control samples.39
6. Biological materials and methods
6.1. Enzyme assays
Acknowledgments
The radiolabeled substrate [methyl-3H]dThd (70 Ci/
mmol) was obtained from Amersham Pharmacia Bio-
tech. The cDNAs of TK1, TK2, Dm-dNK, and HSV-1
TK were inserted in the pGEX-5X-1 vector, expressed
as fusion proteins to glutathione S-transferase, and puri-
fied. The activity of the purified recombinant nucleoside
kinases was assayed in a 50 ll reaction mixture contain-
ing 50 mM Tris–HCl, pH 8.0, 2.5 mM MgCl2, 10 mM
dithiothreitol, 0.5 mM CHAPS (3-[(3-Cholamidopro-
pyl)dimethylammonio]-1-propanesulfonate), 3 mg/mL
bovine serum albumin, 2.5 mM ATP and 0.25 lCi
[methyl-3H]dThd. The samples were incubated at 37 ꢂC
for 30 min in the presence or absence of different con-
centrations of the test compounds. Aliquots of 45 ll of
the reaction mixtures were spotted on Whatman DE-
81 filter paper disks. The filters were washed 3 times
A part of this work was supported by research grants
from the Israeli Academy of Sciences No. 406/02 and
Israeli Ministry of Health No. 5307 (A.S.) and by the
University of Ferrara (FAR 2005-2006, project
CAN2005-2006) Also, the European Commission
(QLRT-2001-01004) (J.B. and A.K.) and the Flemish
‘Fonds voor Wetenschappelijk Onderzoek’ (FWO
G-0267.04) (J.B.) provided financial support to J.B.
and A.K.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.01.049.

N. Ciliberti et al. / Bioorg. Med. Chem. 15 (2007) 3065–3081
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