Special feature of mixed phosphotriester derivatives of cytarabine

Article abstract of DOI:10.1016/j.bmc.2009.07.038

Despite the unquestionable therapeutic interest of bis(SATE) pronucleotides, a presystemic metabolism preventing the delivery of the prodrugs in target cancer cells or tumours may constitute a limitation to the in vivo development of such derivatives. In

Full text of DOI:10.1016/j.bmc.2009.07.038

Bioorganic & Medicinal Chemistry 17 (2009) 6340–6347
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Special feature of mixed phosphotriester derivatives of cytarabine
Marie-Hélène Gouy a, , Lars P. Jordheim b, , Isabelle Lefebvre a, Emeline Cros b, Charles Dumontet b,
a
Suzanne Peyrottes a, , Christian Périgaud
*
a Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS—UM 1 & 2, Université Montpellier 2, Case Courrier 1705, Place E. Bataillon, 34095 Montpellier Cedex 5, France
b Inserm, U590, Lyon, F-69008, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Despite the unquestionable therapeutic interest of bis(SATE) pronucleotides, a presystemic metabolism
preventing the delivery of the prodrugs in target cancer cells or tumours may constitute a limitation
to the in vivo development of such derivatives. In order to overcome these drawbacks several strate-
gies have been envisaged and we report herein the application of the S-acyl-2-thioethyl (SATE) phenyl
Received 9 March 2009
Revised 10 July 2009
Accepted 16 July 2009
Available online 23 July 2009
pronucleotide approach to the well-known cytotoxic nucleoside cytosine-1-b-D-arabinofuranoside (cyt-
arabine, araC). We describe modifications of the SATE moieties with the introduction of polar groups
on the acyl residue, in order to study how these changes affect antitumoral activity and metabolic sta-
bility. Two different synthetic pathways were explored and lead to obtain the corresponding mixed
derivatives in satisfactory yields. Cytotoxicity was studied in murine leukaemia cells L1210 as well
as in cells derived from solid human tumours (Messa and MCF7). Biological evaluation of these com-
pounds in cell culture experiments with nucleoside analogue-sensitive and resistant cell lines showed
that the modified compounds were active at higher concentrations than unmodified cytarabine, yet
were much able to partially reverse resistance due to deficient nucleoside transport or activation.
These results can be correlated with an incomplete decomposition mechanism into the corresponding
50-mononucleotide.
Keywords:
Cytotoxic nucleoside
araC
Prodrug
Biotransformation
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
decreased deoxyribonucleoside kinase activity. Recently, the
bis(SATE) approach was applied to araC and the resulting prodrug
Cytarabine (araC) is a cytotoxic nucleoside used clinically for
the treatment of acute myelocytic leukaemia (AML). Like most
nucleoside analogues, araC is inactive by itself and requires phos-
phorylation to the corresponding triphosphate (araCTP) to exert
its antineoplastic activity by inhibition of nucleic acid biosynthesis.
Variations in metabolic enzymes and membrane transporters have
been shown to alter the cytotoxicity of araC, and different strate-
gies allowing regaining activity have been developed.1 During the
last decade, we and other groups have investigated, especially in
the field of antiviral nucleoside analogues,2–5 the use of mononu-
cleotide prodrugs (namely pronucleotides), bearing the S-acyl-2-
thioethyl (SATE) biolabile phosphate protection.6,7 Such entities
were designed to give rise to the intracellular delivery of the 50-
mononucleotide (i.e., nucleoside 50-monophosphate), which is fur-
ther metabolized to the corresponding triphosphate analogue to
exert activity. Such molecules should therefore be toxic on nucle-
oside analogue-resistant cancer cells with clinically relevant resis-
tance mechanisms such as altered membrane transport or
(Fig. 1, compound 1) was able to partially reverse in vitro resis-
tance.8–10 Despite the unquestionable therapeutic interest of
bis(SATE) pronucleotides,11–13 a presystemic metabolism,14,15 pre-
venting the delivery of the prodrugs in target cancer cells or tu-
mours, may constitute a limitation to the in vivo development of
such derivatives. In order to overcome these drawbacks several
strategies have been envisaged.6 The first one consists in increasing
the enzymatic stability of the esterase-labile phosphate protec-
tions by the introduction of various modifications on the tBuSATE
group, especially polar functions.16 Another possibility is to use a
specific enzymatic activation process in order to target cells or tis-
sues according to preferential transport and distribution or to ob-
tain selective bio-activation.17,18 Attempts illustrating these latter
strategies have been reported in the literature and led to the design
of mixed SATE phosphoesters.6,19,20
On the basis of these results, we investigated other series of
prodrugs combining both approaches (i.e., introduction of polar
function on the tBuSATE pro-moiety and involvement of two dif-
ferent enzymatic systems in the decomposition process, i.e., ester-
ase activity and phosphodiesterase activity, Scheme 2), which have
been firstly experimented on anti-retroviral nucleoside analogues
such as 30-azido-20,30-dideoxythymidine (AZT, zidovudine).19,21
* Corresponding author. Tel.: 33 4 67 14 49 64; fax: +33 4 67 04 20 29.
E-mail address: peyrottes@univ-montp2.fr (S. Peyrottes).
M.-H. Gouy and L.P. Jordheim contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.038

M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
6341
Mixed phosphotriesters
bis(tBuSATE) phosphotriester, compound 1
NH2
N
O
O
O
O
N
O
O P O
O P O araC
O
O
OH
S
R
S
2
OH
araC (Cytarabine)
compound 2
compound 3
compound 4
HO
HO
R =
HO
Figure 1. Chemical structures of araC prodrugs.
Thus, we have recently reported the enhancement of the biological
profile of the corresponding phenyl SATE phosphotriester
derivatives of AZT by modifications of the SATE moiety.22 Here,
we continue our search of biologically active pronucleotides by
applying this approach to the cytotoxic nucleoside analogue araC
(Fig. 1, compounds 2–4).
and both synthetic pathways were explored and compared. While
prodrug formation with the two kinds of phosphorylating reagents
(phosphochloridate22 or phosphoramidate21) was observed, yields
significantly decreased as the scale increased when using phospho-
chloridate intermediates. Thus, the use of phosphoramidite as
phosphorylating reagents led to the desired derivatives in 19–
29% overall yields, in four steps. Furthermore, the PIII approach al-
lows introducing variable aryl substituents whereas the one using
PV is restricted to the use of the commercially available phenyl
phosphorodichloridate.
2. Results and discussion
2.1. Chemistry
Briefly, preparation of the required thioesters (compounds 5–7,
Scheme 1) was accomplished as previously described,22 using
Synthesis of derivatives 2–4 (Fig. 1) may be performed using
either PV (phosphochloridate) or PIII (phosphoramidite) chemistry
5
O
6
7
TrO
O
R=
OH
R
S
O
2
a
O
O
O P OAraC
O
8
9
3
4
R
S
HO
HO
R=
O
N(iPr)2
TrO
O
O P
R
S
R=
N(iPr)2
HO
10
b
O
d
NBoc2
N
O
O
O P O
O
O
c
N(iPr)2
O
N
OBoc
O
O P
O
R
S
R
S
OBoc
11
12
13
14
15
16
Tr O
O
R=
R=
TrO
O
O
O
Scheme 1. Reagents and conditions: (a) Cl-P[N(iPr)2]2, NEt3, Et2O, 0 °C, 2 h, 60–90%; (b) phenol, 1-H-tetrazole, HN(iPr)2, CH3CN, 85%; (c) tetra(Boc)araC derivative, 1-H-
tetrazole, CH3CN, 2 h, then tBuOOH (1 M) in decan, 0 °C, 2 h, 42–56%; (d) TFA 25% in CH2Cl2, 0 °C then rt, 2 h, 55–90%.

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M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
Esterase mediated
hydrolysis
araC
Phosphodiesterase
mediated hydrolysis
-O
O
P
O
O
P
O
P
O-
-O
O araC
O
O
O araC
R
S
O
O
araCMP
2-4
18
Scheme 2. Expected decomposition pathways for SATE phenyl phosphotriester derivatives of araC 2–4 in biological media.
either pivaloyl chloride, 2,2-bis(hydroxymethyl)propionic acid or
methyl-2, 2-dimethyl-3-hydroxypropionate as starting material.
Thus, phenyl SATE phosphotriester derivatives 2–4 could be
accessible in four steps from the corresponding thioester precur-
sors (Scheme 1). The phosphoramidite strategy has been often
used for the synthesis of SATE pronucleotides.19,21 It involves the
preparation of the mixed phosphorylating agent bearing the two
moieties, that is, the aryl and the SATE counter-parts. In this re-
spect the bis(N,N-diisopropyl) phosphorochloridate was reacted
with thioesters 5–7, leading to the corresponding bis(N,N-diisopro-
pyl) SATE phosphoramidite 8–10, which are then coupled to
phenol in presence of 1-H-tetrazole. Treatment of mixed phospho-
ramidites 11–13 with the protected cytarabine derivative (1-
dCK-deficient Messa 10K cells, resulting in a statistically significant
345-fold decrease in the resistance ratio as compared to araC. On
MCF7 1K cells, in which the resistance to nucleoside analogues is
due to increased ribonucleotide reductase (RNR) activity, com-
pound 2 had approximately the same IC50 as araC. Finally, the
mono- and dihydroxylated derivatives 3 and 4 were less active
than araC and compound 2, and did not show significant reversion
of araC-resistance in any cell model studied.
These results showed some discrepancy with the previous data
obtained with antiviral agents such as AZT containing SATE phenyl
pronucleotides.21 This lower anti-tumour activity observed in the
cytarabine series may be due to the poor cell membrane penetra-
tion of the parent compounds 2–4, a slow kinetic of decomposition,
or an unexpected decomposition mechanism that hampered the
intracellular formation of the 50-mononucleotide (araCMP, Scheme
2). In order to explain our observations, Log P values were deter-
mined and stability studies in biological media were performed
(Table 3). Concerning the Log P value which is commonly consid-
ered as a predictive factor for the passive diffusion of a compound
through cell membranes, it should be in the range of 2–4. Thus,
while the values obtained for compounds 1 and 2 (2.6 and 1.9,
respectively) are in the required range, it is not the case of deriva-
tives 3 and 4 (0.58 and ꢀ0.35, respectively) which appeared as
hydrophilic compounds. This may reflect reduced cell penetration
through a passive diffusion process of these two polar derivatives,
resulting in a low cytotoxic activity as compared to the trans-
porter-dependent penetration of cytarabine.
[4-N,N-20,30-O-tetra-(tert-butyloxycarbonyl)-b-
D-arabinofuranosyl]
cytosine (abbreviated as tetra(Boc)-araC)10 in presence of 1-H-tet-
razole, followed by in situ oxidation of the resulting phosphite
triester with tert-butyl hydroperoxide afforded after purification
by column chromatography the desired protected phosphotriester
derivatives 14–16 in moderate yields. Finally, removal of the pro-
tecting groups using a 25% solution of trifluoroacetic acid in dichlo-
romethane furnished the targeted SATE phenyl phosphotriester
derivatives of araC, 2–4. Due to the chirality of the phosphorus cen-
tre, derivatives 14–16 and 2–4 were isolated as a mixture of two
diastereoisomers (ratio: 1/1) as evidenced from their 31P NMR
spectra.
2.2. Cell-culture evaluation data
The cytotoxicity of mixed derivatives 2–4 was determined and
compared to the activity of araC and compound 1 on nucleoside
analogue-sensitive and resistant cells, using the MTT assay as de-
scribed in Section 4 (Tables 1 and 2). Except for compound 2 on
MCF7 cells, the pronucleotides were less active than araC in all cell
lines. We confirmed the reversion of araC-resistance in dCK-defi-
cient L1210 10K cells with compound 1, and observed a slightly
better activity of compound 2 than araC on these cells. On the hu-
man leukaemia cells CCRF-CEM, compound 2 presented a slightly
better activity than araC and compound 1 on the nucleoside trans-
porter-deficient cell line CEM/ARAC08. Compound 2 also showed
interesting activity on Messa cells, as it was 7.6-fold less active
than araC on parental Messa cells and 66-fold more active on
2.3. Stability studies and decomposition pathways
In view of the biological evaluation results, kinetics and decom-
position pathways of compounds 1–4 were studied in total cell ex-
tracts (TCE) from L1210 cells. This would give indications on the
intracellular behaviour of our compounds, and experiments were
performed by LC/MS coupling.
Stability studies shown that all compounds lost the SATE group
as a first step of the decomposition pathways leading to the ex-
pected phenylphosphodiester for derivatives 2–4 (Scheme 2), and
that the presence of a polar group led to an increased stability of
the parent phosphotriester towards esterase hydrolysis (Table 3).
Thus, the mono- and dihydroxylated derivatives 3 and 4 are
Table 1
Sensitivity (IC50 in lM) for araC and compounds 1–4 in nucleoside analogue-sensitive and resistant cancer cell lines as determined by the cytotoxicity assay
Described mechanism CCRF-CEM
of resistance
CEM/ARAC8C
L1210 wt
L1210 10K Messa wt Messa 10K
MCF7 wt
MCF7 1K
Deficiency in
Deficiency
Deficiency
Increased expression
nucleoside transport26
in dCK9
in dCK27
and activity of RNR28
AraC
0.001 0.000 100c
0
0.00040 0.00015 103 50
1.7 0.3
nd
6333a 2028 0.0040 0.0000
0.040b 0.006
nd
1
2
3
4
0.097 0.055 77b 12
0.020 0.006
0.010 0.000
0.19 0.07
4.7b 0.7
nd
nd
0.017 0.003 55a 16
0.056 0.012 77a 23
43b
>30
>30
7
13
19
6
8
7
3
93b
>30
>30
7
0.00032 0.00024 0.083 0.058
0.024 0.014
0.37a 0.12
0.23 0.09
0.12 0.04
77a 23
0.13 0.03
0.0049 0.0027
Data are mean values of three single experiments standard error. Nd stands for not determined.
a
p <0.05.
p <0.01.
b
c
p <0.001 when compared to IC50 of the corresponding parental cell line with Student’s t-test.

M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
6343
Table 2
(Boc)araC) in presence of triethylamine and subsequent hydrolysis
of the mixed phosphorochloridate.25 Removal of protecting groups
from 17 led to isolated 18 in high yields.
Resistance ratios (RR) for araC and compounds 1–4 in nucleoside analogue-sensitive
and resistant cancer cell lines
RR
CCRF-CEM
L1210
Messa
MCF7
All three derivatives were incubated in presence of purified SVP
at 37 °C and gave rise to the formation of the corresponding nucle-
oside 50-monophosphate with half-lives of 4.6 h, 53 min and
43 min, respectively, for 18, 19 and p-nitrophenyl thymidinyl
phosphate. Thus, in agreement with literature data showing that
20-deoxynucleotides were hydrolyzed faster than the correspond-
ing ribonucleotides, the araC metabolite appeared as a poor sub-
strate of SVP compared to AZT or thymidine.23 Nevertheless, we
observed the enzymatic conversion of phenyl phosphodiester of
araC 18 into araCMP as we early hypothesized.
AraC
100,000
794
3235
1375
641
257,500
235
4300
>158
>231
3725
nd
7.2
>1.6
>5
10
1
2
3
4
nd
259
15
47
RR corresponds to IC50 of resistant cells divided by IC50 of parental cells.
Table 3
Log P valuesa and calculated half-lives (t1/2) of derivatives 1–4 in total cell extracts
3. Conclusion
(TCE) from parental (WT) and resistant (10 K) L1210 cell linesb
A new series of SATE phenyl phosphotriester derivatives of araC
was synthesized. Their cytotoxicity as well as the kinetic and
decomposition mechanism in biological media was determined.
Conversely to what was observed using AZT as nucleosidic model,
araC derivatives were active at significantly higher concentrations
than the parental compound, but were much more efficient that
the latter in lines deficient for nucleoside transport or activation.
These results may be supported by an incomplete decomposition
mechanism of the prodrugs, low diffusion through cell membrane
and inadequate kinetics for cell-culture evaluation. However, the
discrepancies of biological behaviour between araC and AZT deriv-
atives suggest that in order to evaluate the potentiality of this pro-
drug-approach, it should be applied to another and less hydrophilic
cytotoxic nucleoside analogue such as gemcitabine.
Media/compound
1
2
3
4
Log P
2.6 0.05
3.9 h
5.3 h
1.9 0.01
8.3 h
8.3 h
0.58 0.006
3.2 d
3.5 d
ꢀ0.35 0.004
10.7 d
12.0 d
TCE from L1210 wt
TCE from L1210 10K
a
Data are mean values of six single experiments standard error.
All data represent an average values for three separate experiments and vari-
b
ability was lower than 10%.
exhibiting half-lives of 3–12 days in comparison to 4–8 h for the
hydrophobic derivatives 1 and 2.
The decomposition pathway of compound 1 differs from deriv-
atives 2–4 after removal of the SATE moiety (Scheme 2). Indeed,
prodrug 1 was metabolized into araCMP after esterase-mediated
hydrolysis steps in TCE, whereas SATE phenyl phosphotriesters of
araC evolved to a stable metabolite, identified as the corresponding
phenyl phosphodiester derivative (compound 18, Scheme 3).
These unexpected results prompted us to synthesize and to
study the affinity of metabolite 18 towards purified snake venom
phosphodiesterase (SVP) which is commonly used as representa-
tive for the type I phosphodiesterase family, and to compare its
behaviour to the corresponding AZT analogue 19 as well as to
the commercially available well-known SVP-substrate p-nitro-
phenyl thymidinyl phosphate.23,24 Both phenyl phosphodiester
derivatives of araC and AZT (respectively compounds 17 and 19,
Scheme 4) were obtained by coupling the commercial phenyl
phosphorodichloridate with either AZT or protected araC (tetra-
4. Experimental
4.1. Chemistry
Unless otherwise stated, 1H NMR spectra were recorded at
300 MHz and 13C NMR spectra at 75 or 100 MHz with proton
decoupling at 25 °C on a Bruker 300 Avance or DRX 400, respec-
tively. Chemical shifts are given in d values referenced to the resid-
ual solvent peak (CDCl3 at 7.26 ppm and 77 ppm, DMSO-d6 at
2.49 ppm and 39.5 ppm) relative to TMS. Deuterium exchange,
decoupling and COSY experiments were performed in order to con-
firm proton assignments. Coupling constants, J, are reported in
Scheme 3. Typical extracted-ion chromatogram obtained after incubation of compound 3 in TCE for 30 h and corresponding positive-ion ESI spectra of the observed
metabolite (MW = 399).

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M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
NBoc
2
NH
2
N
N
O
P
O
P
N
OBoc
O
N
O
NaO
O
NaO
O
OH
O
O
b
O
O
OBoc
OH
17
18
a
a
O
P
Cl
Cl
O
N
O
NH
O
P
O
NaO
O
O
O
N
19
3
Scheme 4. Reagents and conditions: (a) AZT or tetra(Boc)araC, NEt3, THF, ꢀ78 °C to rt, 4 h, then acetone, Dowex Na+, 41–47%; (b) TFA 25% in CH2Cl2, 0 °C to rt, 1 h, then
Dowex Na+, 85%.
hertz. 2D 1H–13C heteronuclear COSY were recorded for the attri-
bution of 13C signals. Unless otherwise stated, 31P NMR spectra were
recorded at ambient temperature at 121 MHz with proton decou-
pling. Chemical shifts are reported relative to external H3PO4. FAB
mass spectra were recorded in the positive-ion or negative-ion
mode on a JEOL JMS DX 300 using thioglycerol/glycerol (1:1, v/v,
G–T) as matrix. Melting points were determined in open capillary
tubes on a Büchi-545 and are uncorrected. UV spectra were recorded
in ethanol 95 on an Uvikon 931 (Kontron). Elemental analyses were
carried out by the Service de Microanalyses du CNRS, Division de
Vernaison (France). TLC was performed on precoated aluminium
sheets of Silica Gel 60 F254 (Merck, Art. 9385), visualisation of prod-
ucts being accomplished by UV absorbance followed by charring
with 5% ethanolic sulfuric acid with heating for nucleotides. Flash
derivative of araC 1 and tetra (Boc) araC were synthesized follow-
ing previously published procedures.
4.1.1. Standard procedure 1: preparation of SATE di(N,N-diiso-
propylamino) phosphoramidites (8–10)
To
a solution of bis(N,N-diisopropyl) phosphorochloridate
(1 equiv) in anhydrous diethyl ether (3 mL/mmol) at 0 °C, a solu-
tion of 5 (3.0 g, 18.77 mmol) or 6 (1.0 g, 2.38 mmol), or 7 (0.6 g,
2.38 mmol) and triethylamine (1.1 equiv) in anhydrous diethyl
ether (2 mL/mmol) was added drop wise. The reaction mixture
was stirred 2 h at room temperature, and then filtrated, and the fil-
trate was concentrated under vacuum. The corresponding oily res-
idue was purified by column silica gel chromatography using
cyclohexane/ethyl acetate/triethylamine (90/9/1, v/v/v) as eluent.
chromatography was carried out using 63–100
lm silica gel (Merck
Art. No. 115101) otherwise 40–63 m silica gel (Merck Art. No.
l
4.1.2. S-Pivaloyl-2-thioethyl di(N,N-diisopropylamino) phospho-
ramidite (8)
6.6 g (90%) of the desired compound were obtained as colour-
less oil.
109385) was used. Thin layer chromatography was carried out using
aluminium supported Silica Gel 60 plates (Merck Art. No. 105554).
Solvents were reagent grade or purified by distillation prior to use,
and solids were dried over P2O5 under reduced pressure at rt. Mois-
ture sensitive reactions were performed under argon atmosphere
using oven-dried glassware. All aqueous (aq) solutions were satu-
rated with the specified salt unless otherwise indicated. Organic
solutions were dried over Na2SO4 after workup and solvents were
removed by evaporation at reduced pressure.
Rf 0.7 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v). NMR 1H (DMSO-d6,
400 MHz) d 3.55 (m, 2H, CH2O), 3.52–3.42 (m, 4H, (CH3)2CHN), 3.02
(t, 2H, J = 6.2, SCH2), 1.16 (1s, 9H, C(CH3)3), 1.11, 1.09 (2d, 24H,
J = 6.5, (CH3)2CHN). NMR 13C (DMSO-d6, 100 MHz) d 205.4 (C@O),
62.3 (d, CH2O), 45.9 (C(CH3)3), 43.8 (d, (CH3)2CHN), 29.9 (d,
SCH2), 26.9 (C(CH3)3), 24.3, 23.5 (2d, (CH3)2CHN). NMR 31P
(DMSO-d6, 81 MHz) d 125.5. MS FAB >0 (GT) m/z 393 (M+H)+,
145 (tBuCOSCH2CH2)+, 85 (tBuCO)+, 57 (tBu)+; FAB <0 (GT) m/z
247 (MꢀtBuSATE)ꢀ, 117 (tBuCOS)ꢀ. Anal. Calcd for C19H41N2O2PS:
C, 58.12; H, 10.53; N, 7.14. Found: C, 57.99; H, 10.31; N, 7.32.
Analytical HPLC studies were carried out on an Alliance 2690
system (Waters, Milford, Massachusetts, USA) equipped with 996
photodiode detector and a Millennium data workstation. A re-
verse-phase analytical column (Hypersil BDS, C18, 100 ꢁ 4.6 mm,
3 lm) equipped with a prefilter, and a precolumn (DeltaPak
Waters, C18
,
5
l
m) were used. Detection was monitored at
4.1.3. S-(2,2-Dimethyl-3-triphenylmethoxypropionyl)-2-thioethyl,
di(N,N-diisopropylamino) phosphorobisamidite (9)
1.4 g (90%) of the desired compound was obtained as colourless
oil.
267 nm. Nucleotidic derivatives were eluted using a linear gradient
of 0–80% acetonitrile in 20 mm triethylammonium acetate buffer
(pH 6.9) over 20 min at 1 mL/min flow rate. Electrospray ionisa-
tion-mass spectrometry (ESI-MS) was performed using a SSQ
7000 single quadrupole mass spectrometer (Finnigan, San Jose,
California, USA) in the negative-ion mode with a spray voltage
at—4.5 kV. The capillary temperature was maintained at 250 °C.
Nitrogen served both as sheath gas (operating pressure of
550 KPa) and as auxiliary gas with a flow rate of 103 KPa. Under
these conditions, full scan data acquisition was performed from
m/z 200 to 800 in centroïd mode and using a cycle time of 1.0 s.
AraC was from Intsel Chimos; bis(N,N-diisopropyl) phosphorochlo-
ridate was purchased from Acros. Bis(tBuSATE) phosphotriester
Rf 0.8 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v). NMR 1H (DMSO-d6,
300 MHz) d 7.29 (m, 15H, Ph), 3.59 (m, 2H, CH2O), 3.48 (m, 4H,
(CH3)2CHN), 3.05 (m, 4H, TrOCH2, SCH2), 1.40 (1s, 6H, C(CH3)2),
1.11, 1.09 (2d, 24H, (CH3)2CHN). NMR 13C (DMSO-d6, 75 MHz) d
204.0 (C@O), 128.2, 127.8, 127.0 (3s, Ph), 69.5 (TrOCH2), 62.3 (d,
CH2O), 50.2 (C(CH3)2), 43.8 (d, (CH3)2CHN), 29.8 (SCH2), 26.3
(C(CH3)2), 24.3, 23.5 (2d, (CH3)2CHN). NMR 31P (DMSO-d6,
121 MHz) d 124.8. MS FAB >0 (GT) m/z 651 (M+H)+, 243 (Tr)+. Anal.
Calcd for C38H55N2O3PS: C, 70.12; H, 8.52; P, 4.76. Found: C, 70.35;
H, 8.66; P, 4.27.

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6345
4.1.4. S-[2-(2,2-Dimethyl-1,3-dioxan-5-yl)propionyl]-2-thioethyl,
di(N,N-diisopropylamino) phosphorobisamidite (10)
0.67 g (61%) of the desired compound was obtained as colour-
less oil.
2H, J = 6.3, SCH2), 1.37, 1.25 (2s, 6H, C(CH3)2), 1.19, 1.10 (2d, 12H,
(CH3)2CHN). NMR 13C (DMSO-d6, 75 MHz) d 200.7 (C@O), 152.8
(Ph ipso), 128.2, 121.1, 118.5, 118.4 (4s, Ph), 96.3 (C(CH3)2), 63.9
(OCH2), 60.6 (d, CH2CH2O), 47.2 (C(CH3)), 41.7 (d, (CH3)2CHN),
28.1 (d, SCH2), 24.2, 20.4 (2s, C(CH3)2), 23.0, 22.9 (2d, (CH3)2CHN),
17.5 (CH3). NMR 31P (DMSO-d6, 121 MHz) d 145.9. Anal. Calcd for
C22H36NO5PS: C, 57.75; H, 7.93; N, 3.06; P, 6.77; S, 7.01. Found:
C, 58.14; H, 8.19; N, 3.16; P, 6.66; S, 7.36.
Rf 0.8 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v). NMR 1H (DMSO-d6,
300 MHz) d 4.15 (d, 2H, J = 12.1, OCH2), 3.61 (m, 4H, OCH2, CH2O),
3.42 (m, 4H, (CH3)2CHN), 3.10 (t, 2H, J = 6.2, SCH2), 1.33, 1.31 (2s,
6H, C(CH3)2), 1.20 (s, 3H, CH3), 1.10, 1.08 (2d, 24H, (CH3)2CHN).
NMR 13C (DMSO-d6, 75 MHz) d 202.0 (C@O), 97.5 (C(CH3)2), 65.2
(OCH2), 62.2 (d, CH2O), 48.4 (C(CH3)), 43.8 (d, (CH3)2CHN), 29.8
(d, SCH2), 26.3, 25.3 (2s, C(CH3)2), 24.3, 23.5 (2d, (CH3)2CHN),
18.8 (CH3). NMR 31P (DMSO-d6, 121 MHz) d 124.3. MS FAB >0
(GT) m/z 465 (M+H)+, 364 (MꢀNiPr2+H)+, 100 (NiPr)+, 43 (iPr)+,
FAB <0 (GT) m/z 362 (MꢀNiPr2ꢀH)ꢀ. Anal. Calcd for C22H45N2O4PS:
C, 56.87; H, 9.76; N, 6.03. Found: C, 56.56; H, 9.72; N, 5.77.
4.1.9. Standard procedure 3: preparation of phenyl SATE phos-
photriesters (14–16)
To a solution of the protected nucleoside (1 equiv) in anhydrous
acetonitrile in presence of molecular sieve 3 Å, were successively
added 1H-tetrazole (4 equiv), the required mixed phosphoramidite
(1.4 equiv), that is, compound 11 (0.5 g, 1.22 mmol), or 12 (0.9 g,
1.40 mmol), or 13 (0.6 g, 1.20 mmol). After one hour stirring at
room temperature under an argon atmosphere, the reaction mix-
ture was cooled down to 0 °C and a tert-butyl hydroperoxide solu-
tion (2.4 equiv, 5 M in decan) was added drop wise. The reaction
proceeded for one hour at room temperature and then diluted in
dichloromethane. The resulting organic layer was washed with
an aqueous solution of Na2S2O3 (10%, w/v), then brine and water.
The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated under vacuum. The residue was purified on silica
gel column chromatography.
4.1.5. Standard procedure 2: preparation of phenyl SATE (N,N-
diisopropylamino) phosphoramidites (11–13)
To a solution of phenol (0.2 g, 2.55 mmol) in anhydrous acetoni-
trile (14 mL) at 0 °C, in presence of molecular sieve 3 Å (2.5 g), was
successively added anhydrous diisopropylamine (0.72 mL, 5.10
mmol), 1-H-tetrazole (0.45 M solution in acetonitrile, 11.3 mL,
5.10 mmol) and the required bisphosphoramidite 8, or 9, or 10
(3.82 mmol). After 15 min at 0 °C, the reaction mixture was stirred
for 2 h at room temperature and then 100 mL of ethyl acetate were
added. The resulting solution was washed with brine and then
water. The organic layer was dried over anhydrous Na2SO4, filtered
and concentrated under vacuum. The residue was purified by col-
umn chromatography using cyclohexane/ethyl acetate/triethyl-
amine (97/2/1, v/v/v) as eluent to give the mixed phenyl SATE
(N,N-diisopropylamino) phosphoramidites 11–13 (ꢂ85%) as col-
ourless oil.
4.1.10. O-[4-N,N-20,30-O-Tetra-(tert-butyloxycarbonyl)-b-
D-arabi-
nofuranosylcytosine]-O0-(S-pivaloyl-2-thioethyl)-O00-phenyl phos-
phate (14)
0.46 g (56%) of the fully protected phosphotriester 14 was ob-
tained as yellow oil after purification on silica gel using a gradient
of methanol (0–1%) in dichloromethane.
Rf 0.8 (CH2Cl2/AcOEt, 7/3, v/v). NMR 1H (DMSO-d6, 400 MHz) d 8.03
(d, 1H, J = 7.6, H-6), 7.43 (m, 2H, Ph), 7.39 (m, 3H, Ph), 7.25 (t, 1H,
H-5), 6.27 (m, 1H, H-10), 5.30 (m, 1H, H-20), 5.15 (m, 1H, H-30), 4.45
(m, 2H, H-50, H-500), 4.38 (m, 1H, H-40), 4.18 (m, 2H, CH2O), 3.15 (t,
2H, SCH2), 1.49 (s, 18H, C(CH3)3, Boc), 1.45, 1.29 (2s, 18H, C(CH3)3,
Boc), 1.15 (s, 9H, C(CH3)3, SATE). NMR 13C (DMSO-d6, 100 MHz) d
205.0 (C@O, SATE), 161.8 (C-4), 152.6, 151.6, 150.8, 150.0 (4s, C-
2 et C@O, Boc), 149.0 (C-6), 129.9 (d, Ph ipso), 125.7, 125.4, 121.5
(3s, Ph), 119.9 (d, Ph ortho), 95.2 (C-5), 84.7 (C-10), 83.4, 83.3 (2s,
C(CH3)3, Boc), 78.0 (C-40), 77.5 (C-30), 76.5 (C-20), 67.1 (CH2O),
66.5 (C-50), 50.8 (C(CH3)3, SATE), 28.6 (SCH2), 28.5, 28.4, 27.6,
27.4 (4s, C(CH3)3, Boc), 27.3 (C(CH3)3, SATE). NMR 31P (DMSO-d6,
100 MHz) d ꢀ5.6, ꢀ5.7. MS FAB >0 (GT) m/z 944 (M+H)+, 844
(MꢀBoc+H)+, 744 (Mꢀ2Boc+H)+, 644 (Mꢀ3Boc+H)+. UV (EtOH
4.1.6. O-Phenyl S-pivaloyl-2-thioethyl N,N-diisopropylamino phos-
phoramidite (11)
Rf 0.8 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v/v). NMR 1H (DMSO-
d6, 300 MHz) d 7.29 (m, 2H, Ph), 7.00 (m, 3H, Ph), 3.70 (m, 4H,
CH2O, (CH3)2CHN), 3.09 (t, 2H, J = 6.4, SCH2), 1.40 (s, 9H, tBu),
1.17, 1.11 (2d, 12H, J = 6.8, (CH3)2CHN). NMR 13C (DMSO-d6,
75 MHz) d 205.3 (C@O), 154.1 (Ph ipso), 129.4, 122.3, 119.7,
119.6 (4s, Ph), 62.0 (d, CH2O), 45.9 (C(CH3)3), 42.9 (d, (CH3)2CHN),
29.4 (d, SCH2), 26.9, 26.8, 26.7 (3s, C(CH3)3), 24.3, 24.1 (2d,
(CH3)2CHN). NMR 31P (DMSO-d6, 121 MHz) d 145.9. MS FAB >0
(GT) m/z 243 (MꢀtBuSATE+H)+, 145 (tBuSATE)+, 57 (tBu)+, FAB <0
(GT) m/z 383 (MꢀH)ꢀ, 291 (MꢀPhOꢀH)ꢀ. Anal. Calcd for
C19H32NO3PS: C, 59.20; H, 8.37; N, 3.63. Found: C, 59.30; H, 8.63;
N, 3.51.
95%) kmax1 294 nm (e 4800), kmax2 235 nm (e 11,400).
4.1.7. O-Phenyl S-(2,2-dimethyl-3-triphenylmethoxypropionyl)-
2-thioethyl N,N-diisopropyl- aminophosphoramidite (12)
Rf 0.8 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v/v). NMR 1H (DMSO-
d6, 300 MHz) d 7.29 (m, 17H, Ph, Tr), 7.00 (m, 3H, Ph), 3.71 (m,
4H, CH2O, (CH3)2CHN), 3.12 (t, 2H, SCH2), 3.05 (s, 2H, TrOCH2),
1.17 (s, 6H, C(CH3)2), 1.11, 1.09 (2s, 12H, (CH3)2CHN). NMR 13C
(DMSO-d6, 75 MHz) d 205.3 (C@O), 155.7 (Ph ipso), 145.1, 131.1,
129.8, 129.4, 128.6 124.0, 121.4 (7s, Ph), 87.4 ((Ph)3C), 71.2
(TrOCH2), 63.7 (d, CH2O), 51.9 (C(CH3)2), 44.5 (d, (CH3)2CHN),
30.9 (d, SCH2), 25.9 (d, (CH3)2CHN), 24.0 (C(CH3)2). NMR 31P
(DMSO-d6, 121 MHz) d 145.9. MS FAB >0 (GT) m/z 643 (M+H)+,
243 (Tr)+, FAB <0 (GT) m/z 641 (MꢀH)ꢀ.
4.1.11. O-[4-N,N-20,30-O-Tetra-(tert-butyloxycarbonyl)-b-
D-arabi-
nofuranosylcytosine]-O0-[S-(2,2-dimethyl-3-triphenyl methoxy-
propionyl)-2-thioethyl)-O00-phenyl phosphate (15)
0.55 g (46%) of the fully protected phosphotriester 15 was ob-
tained as yellow oil after purification on silica gel using a gradient
of methanol (0–1%) in dichloromethane.
Rf 0.8 (CH2Cl2/AcOEt, 8/2, v/v). NMR 1H (DMSO-d6, 300 MHz) d
8.02 (d, 1H, J = 7.4, H-6), 7.32–7.21 (m, 20H, Ph, (Ph)3C), 6.83 (t,
1H, H-5), 6.25 (m, 1H, H-10), 5.30 (m, 1H, H-20), 5.15 (m, 1H, H-
30), 4.40 (m, 3H, H-40, H-50, H-500), 4.16 (m, 2H, CH2CH2O), 3.19
(m, 2H, SCH2), 3.05 (m, 2H, TrOCH2), 1.59, 1.49, 1.42, 1.29 (4s,
36H, C(CH3)3, Boc), 1.15 (s, 6H, C(CH3)2, SATE). NMR 13C (DMSO-
d6, 75 MHz) d 203.8 (C@O, SATE), 162.3 (C-4), 153.0, 152.0, 151.2,
150.4 (4s, C-2 et C@O, Boc), 149.4 (C-6), 147.2, 143.8 (2s, Ph),
128.6, 128.2, 127.5, 125.8 (4s, Ph), 120.3 (d, Ph ortho), 95.7 (C-5),
86.2 ((Ph)3C), 85.7 (C-10), 83.8, 83.7 (2s, C(CH3)3, Boc), 79.3 (C-40),
78.0 (C-30), 76.0 (C-20), 69.9 (TrOCH2), 67.0 (CH2CH2O), 66.9 (C-
50), 50.8 (C(CH3)2, SATE), 28.5 (SCH2), 28.5, 27.8, 27.6, 27.3 (4s,
4.1.8. O-Phenyl S-[2-(2,2-dimethyl-1,3-dioxan-5-yl)propionyl]-
2-thioethyl N,N-diisopropyl-aminophosphoramidite (13)
Rf 0.8 (cyclohexane/AcOEt/Et3N, 8/1/1, v/v/v). NMR 1H(DMSO-
d6, 300 MHz) d 7.29 (m, 2H, Ph), 7.00 (m, 3H, Ph), 4.08, 3.70 (2d,
4H, J = 12,1, OCH2), 3.70 (m, 4H, CH2CH2O, (CH3)2CHN), 3.13 (t,

6346
M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
C(CH3)3, Boc), 22.7 (C(CH3)2, SATE). NMR 31P (DMSO-d6, 121 MHz) d
ꢀ6.62, ꢀ6.65. MS FAB >0 (GT) m/z 1202 (M+H)+, 859 (MꢀTrꢀBoc)+,
FAB >0 (NBA) m/z 1202 (M+H)+, 1102 (MꢀBoc+H)+, 1002
(Mꢀ2Boc+H)+, 243 (Tr)+, 77 (C6H5)+, FAB <0 (NBA) m/z 1124
(MꢀC6H5)ꢀ, 1024 (MꢀBocꢀC6H5)ꢀ, 798 (MꢀSATE)ꢀ, 698
4.1.15. O-[b-D
-Arabinofuranosylcytosine]-O0-[S-(2,2-dimethyl-
3-hydroxypropionyl)-2-thioethyl]-O00-phenyl phosphate (3)
0.14 g (55%) of targeted compound was obtained as a white
powder after two columns chromatography; firstly on silica gel
using a gradient of methanol (0–10%) in dichloromethane and then
on reverse phase (RP 18, using a gradient of acetonitrile (0–30%) in
water).
(MꢀBocꢀSATE)ꢀ, 575 (Mꢀnu)ꢀ. UV (EtOH 95%) kmax 297 nm (
e
5200). Anal. Calcd for C6H76N3O18PS, 0.4 C6H14: C, 61.73; H, 6.41;
N, 3.41; P, 2.51; S, 2.60. Found: C, 61.74; H, 6.71; N, 3.48; P, 2.43;
S, 2.77.
Rf 0.4 (CH2Cl2/MeOH, 85/15, v/v). NMR 1H (DMSO-d6, 300 MHz)
d 7.55 (d, 1H, H-6), 7.42 (m, 2H, Ph), 7.23 (m, 3H, Ph), 7.10, 7.05 (2s,
2H, NH2), 6.11 (d, 1H, H-10), 5.61 (m, 3H, H-5, H-30, OH), 4.94 (m,
1H, CH2OH), 4.31 (m, 2H, H-50, H-500), 4.19 (m, 2H, CH2O), 3.99
(m, 2H, H-20, H-40), 3.90 (m, 1H, OH), 3.42 (m, 2H, CH2OH), 3.13
(m, 2H, SCH2), 1.12 (s, 6H, C(CH3)2). NMR 13C (DMSO-d6, 75 MHz)
d 203.9 (C@O, SATE), 165.6 (C-4), 155.0 (C-2), 142.8 (d, C-6),
129.9 (d, Ph ipso), 125.3 (Ph), 119.9 (d, Ph), 92.5 (C-5), 86.5 (C-
10), 82.5 (d, C-40), 76.3 (C-30), 74.0 (C-20), 68.3 (CH2OH), 67.9 (C-
50), 66.3 (d, CH2O), 51.7 (C(CH3)2, SATE), 28.0 (d, SCH2), 21.8
(C(CH3)2, SATE). NMR 31P (DMSO-d6, 121 MHz) d ꢀ6.4, ꢀ6.5. MS
FAB >0 (GT) m/z 1119 (2 M+H)+, 560 (M+H)+, FAB <0 (GT) m/z
558 (MꢀH)ꢀ, 398 (MꢀSATE)ꢀ. HR-MS (C22H31N3O10PS) (M+H)+:
4.1.12. O-[4-N,N-20,30-O-Tetra-(tert-butyloxycarbonyl)-b-
D-arabi-
nofuranosylcytosine]-O0-[S-(2-(2,2-dimethyl-1,3-dioxan-5-yl)pro-
pionyl)-2-thioethyl]-O00-phenyl phosphate (16)
0.37 g (42%) of the fully protected phosphotriester 16 was ob-
tained as yellow oil after purification on silica gel using a gradient
of methanol (0–1%) in dichloromethane.
Rf 0.8 (CH2Cl2/AcOEt, 7/3, v/v). NMR 1H (DMSO-d6, 300 MHz) d
8.02 (d, 1H, H-6), 7.40 (m, 2H, Ph), 7.20 (m, 3H, Ph), 6.83 (t, 1H,
H-5), 6.27 (m, 1H, H-10), 5.30 (m, 1H, H-20), 5.15 (m, 1H, H-30),
4.40 (m, 3H, H-40, H-50, H-500), 4.20 (m, 2H, CH2O), 4.08 (m, 2H,
OCH2), 3.71 (m, 2H, OCH2), 3.20 (m, 2H, SCH2), 1.49, 1.45 (2s,
27H, C(CH3)3, Boc), 1.39, 1.29 (2s, 6H, C(CH3)2), 1.28 (s, 9H,
C(CH3)3, Boc), 1.05 (s, 3H, CH3). NMR 13C (DMSO-d6, 75 MHz) d
201.7 (C@O, SATE), 161.8 (C-4), 152.6, 151.5, 150.8, 149.9 (4s, C-
2 and C@O, Boc), 149.0 (C-6), 129.9 (Ph ipso), 125.4 (Ph), 119.9
(d, Ph ortho), 97.6 (C(CH3)2), 95.3 (C-5), 84.7 (C-10), 83.4, 83.3 (2s,
C(CH3)3, Boc), 79.0 (C-40), 77.5 (C-30), 76.5 (C-20), 67.0 (CH2CH2O),
65.1 (C-50), 48.5 (C(CH3)), 27.9 (SCH2CH2), 27.1, 26.9 (2s, C(CH3)3,
Boc), 25.6, 21.5 (2s, C(CH3)2), 18.6 (CH3). NMR 31P (DMSO-d6,
121 MHz) d ꢀ6.59, ꢀ6.64. MS FAB >0 (NBA) m/z 1015 (M)+, 915
(MꢀBoc)+, 815 (Mꢀ2Boc)+, FAB <0 (NBA) m/z 798 (MꢀSATE)ꢀ,
560.1468; found: 560.1479. UV (EtOH 95%) kmax1 272 nm (
e
9800), kmax2 229 ( 12,400). Anal. Calcd for C22H30N3O10PS (0.7
e
mol of H2O): C, 46.18; H, 5.53; N, 7.34; P, 5.41. Found: C, 46.00;
H, 5.47; N, 7.32; P, 5.19.
4.1.16. O-[b-D
-Arabinofuranosylcytosine]-O0-[S-(2,2-hydroxy-
methyl)propionyl)-2-thioethyl]-O00-phenyl phosphate (4)
0.18 g (89%) of targeted compound was obtained as a white
powder after chromatography on a reverse-phase column (RP
18), using a gradient of acetonitrile (0–20%) in water.
Rf 0.4 (CH2Cl2/MeOH, 85/15, v/v). NMR 1H (DMSO-d6, 300 MHz)
d 7.54 (t, 1H, H-6), 7.41 (m, 2H, Ph), 7.25 (m, 3H, Ph), 7.09, 7.04 (2s,
2H, NH2), 6.11 (d, 1H, H-10), 5.61 (m, 3H, H-5, OH-30, OH-20), 4.80
(m, 2H, CH2OH), 4.40 (m, 2H, H-50, H-500), 4.15 (m, 2H, CH2O),
3.99 (m, 2H, H-20, H-30), 3.90 (m, 1H, H-40), 3.57, 3.47 (2 m, 4H,
CH2OH), 3.13 (m, 2H, SCH2), 1.12 (s, 3H, CH3). NMR 13C (DMSO-
d6, 75 MHz) d 202.5 (C@O, SATE), 165.6 (C-4), 155.0 (C-2), 142.8
(C-6), 129.9 (d, Ph ipso), 125.3 (Ph), 119.9 (d, Ph), 92.5 (C-5), 86.5
(C-10), 82.5 (C-30), 76.3 (C-40), 74.0 (C-20), 67.8 (C-50), 66.3 (d,
CH2O), 64.3 (d, CH2OH), 57.5 (C(CH3)), 27.8 (SCH2), 16.8 (CH3).
NMR 31P (DMSO-d6, 121 MHz) d ꢀ6.38, ꢀ6.42. MS FAB >0 (GT)
m/z 1151 (2 M+H)+, 576 (M+H)+, FAB <0 (GT) m/z 575 (MꢀH)ꢀ.
HR-MS (C22H31N3O11PS) (M+H)+: 576.1417; found: 576.1414. UV
698 (MꢀBocꢀSATE)ꢀ. UV (EtOH 95%) kmax1 294 nm (
e 6300), kmax2
238 nm (e 13,600).
4.1.13. Standard procedure 4: Preparation of phenyl SATE
phosphotriester derivatives of araC (2–4)
To a solution of the phosphotriester derivative 15 (0.5 g,
0.53 mmol), or 16 (0.5 g, 0.46 mmol), or 17 (0.37 g, 0.36 mmol) in
dichloromethane (10 mL/mmol), at 0 °C, was added drop wise a tri-
fluoroacetic acid solution (50%, v/v) in dichloromethane (10 mL/
mmol). The reaction mixture was stirred 15 min at 0 °C and then
one hour at room temperature. Solvents were evaporated under
vacuum and the crude was purified.
(EtOH 95%) kmax 274 nm (e 10,700). Anal. Calcd for C22H30N3O11PS
(1 mol of H2O): C, 44.52; H, 5.43; N, 7.08; P, 5.22; S, 5.40. Found: C,
44.21; H, 5.34; N, 6.95; P, 5.18; S, 5.41.
4.1.14. O-[b-
D
-Arabinofuranosylcytosine]-O0-(S-pivaloyl-2-
thioethyl)-O00-phenyl phosphate (2)
0.19 g (67%) of targeted compound was obtained as a white
powder after two columns chromatography; firstly on silica gel
using a gradient of methanol (0–10%) in dichloromethane and then
on reverse phase (RP 18, using a gradient of acetonitrile (0–40%) in
water).
4.2. Biological methods
4.2.1. Cell culture
Human sarcoma cells Messa, human T-lymphoblastoid leukae-
mia cells CCRF-CEM and murine leukaemia cells L1210 were grown
in RPMI media (Gibco, Cergy Pontoise, France) and human breast
adenocarcinoma cells MCF7 were grown in DMEM media (Gibco,
Cergy Pontoise, France), all containing
(200 UI/ml), streptomycin (200 g/ml) (Gibco, Cergy Pontoise,
France) and 10% foetal bovine serum (Gibco, Cergy Pontoise,
France). All cells were grown at 37 °C in presence of 5% CO2. Devel-
opment and characterisation of deoxynucleoside analogue-resis-
tant sub-cell lines of Messa (Messa 10K), CCRF-CEM (CEM/
ARAC8C), L1210 (L1210 10K) and MCF7 (MCF7 1K) have been re-
ported elsewhere.26–28,29
Rf 0.4 (CH2Cl2/MeOH, 85/15, v/v). NMR 1H (DMSO-d6, 300 MHz)
d 7.54 (t, 1H, J = 7.5, H-6), 7.41 (m, 2H, Ph), 7.24 (m, 3H, Ph), 7.10,
7.05 (2s, 2H, NH2), 6.12 (m, 1H, H-10), 5.63 (m, 3H, H-5, H-30, OH),
4.35 (m, 2H, H-50, H-500), 4.19 (m, 2H, CH2O), 3.93 (m, 3H, H-20, H-40,
OH), 3.15 (m, 2H, SCH2), 1.18 (s, 9H, C(CH3)3). NMR 13C (DMSO-d6,
75 MHz) d 205.0 (C@O, SATE), 165.6 (C-4), 155.0 (C-2), 142.7 (C-6),
129.9 (d, Ph ipso), 125.3 (Ph), 119.9 (d, Ph), 92.4 (C-5), 86.5 (C-10),
82.5 (C-40), 76.3 (C-30), 74.0 (C-20), 67.8 (C-50), 66.3 (d, CH2O), 46.0
(C(CH3)3, SATE), 28.0 (SCH2), 26.8 (C(CH3)3, SATE). NMR 31P (DMSO-
d6, 121 MHz) d ꢀ6.4, ꢀ6.5. MS FAB >0 (GT) m/z 1087 (2 M+H)+, 544
(M+H)+, 112 (BH2)+, 57 (tBu)+. HR-MS (C22H31N3O9PS) calcd
(M+H)+: 544.1519; found: 544.1547. UV (EtOH 95%) kmax 272 nm
L-glutamine, penicillin
l
(e
7400). Anal. Calcd for C22H30N3O9PS (0.7 mol of H2O): C, 47.51;
4.2.2. Cytotoxicity studies
H, 5.69; N, 7.56; P, 5.77; S, 5.57. Found: C, 47.16; H, 5.65; N,
For adherent cells (MCF7 and Messa), 3000 cells were plated per
7.46; P, 5.29; S, 5.55.
well in 96-well plates (Becton Dickinson) in a volume of 100 ll and

M.-H. Gouy et al. / Bioorg. Med. Chem. 17 (2009) 6340–6347
6347
incubated for 24 h at 37 °C before drugs were added at different
concentrations. For suspension cells (CCRF-CEM and L1210),
is also grateful to MENRT (Ministère de l’Education Nationale, de
la Recherche et des Technologies) for a PhD grant.
100
containing 100
After incubation at 37 °C for 3 days, MTT (100
St. Quentin Fallavier, France) was added and, after 2 h of incubation
at 37 °C, the supernatant was replaced with 100 l isopropanol/
HCl/H20 (v/v/v, 90/1/9) and spectrophotometric determination of
optical density was performed using a micro plate reader (Labsys-
tem Multiskanner RC). The inhibitory concentration 50 (IC50) was
defined as the concentration inhibiting proliferation to a level
equal to 50% of that of controls and the resistance ratio (RR) was
the ratio between the IC50 of the deoxynucleoside analogue-resis-
tant cell line and the IC50 of the sensitive parental cell line. The sta-
tistical significance between IC50s and RRs was determined using
Student’s t-test and MicrosoftÒ Office Excel 2003 (Microsoft
Corporation).
l
l with 10,000 cells were added to each well of 96-well plates
l media with different concentrations of drugs.
g, Sigma–Aldrich,
l
References and notes
l
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l
4.3. Stability studies
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HPLC analyses were carried out using an improved ‘On-line ISRP
cleaning method’. Briefly, the crude sample (80
ll, initial concen-
tration 50 m) was injected onto the precolumn and eluted firstly
l
with 20 mM triethylammonium acetate buffer (pH 6.9) for 5 min at
a flow rate of 2 mL/min. Then a six port Rheodine valve was acti-
vated and a linear gradient from 0% to 80% acetonitrile over
20 min was run at 1 mL/min flow rate. In these conditions, analytes
were back-flushed from the precolumn to the analytical column
and eluted. The decomposition products were identified by HPLC/
MS coupling after calibration and/or by co-injection with authentic
samples (AraCMP, AZTMP, 18, etc.). For each incubation time, the
calculation of the relative concentration of each species was re-
lated to the peak areas, these data were considered as the experi-
mental data. The rate constants of disappearance and the half-lives
(kinetic data) of the phosphotriester and phosphodiester deriva-
tives were calculated according to pseudo-first order kinetic mod-
els and optimized using mono- or poly-exponential regressions.
For stability studies in biological media; cell extracts were pre-
pared and studies were performed according to published
procedure.29
For stability studies with purified SVP from Sigma; the borate
buffer (pH 9.0) was prepared following the procedure of Clark &
Lub.30 In a vial containing 950
l
l of the SVP solution in the borate
g/mL) was added 50 l of the studied compound in
solution in water (1 mM initial concentration). The mixture was
incubated at 37 °C. At the required times, aliquots (80 l) were
buffer (2.4
l
l
l
sampled and directly injected onto the previously described HPLC
system.
27. Jordheim, L. P.; Galmarini, C. M.; Dumontet, C. Cancer Chemother. Pharmacol.
2006, 58, 547.
28. Jordheim, L. P.; Guittet, O.; Lepoivre, M.; Galmarini, C. M.; Dumontet, C. Mol.
Cancer Ther. 2005, 4, 1268.
Acknowledgments
29. Lefebvre, I.; Périgaud, C.; Pompon, A.; Aubertin, A.-M.; Girardet, J.-L.; Kirn, A.;
Gosselin, G.; Imbach, J.-L. J. Med. Chem. 1995, 38, 3941.
30. Bates, R. G.; Paabo, M. In Handbook of Biochemistry. Physical and Chemical Data;
p J227.
We thank ‘Association pour la Recherche contre le Cancer’ (ARC,
France), Astri og Birger Torsteds legat and Olav Raagholt og Gerd
Meidel Raagholts stiftelse for forskning for funding. M.-H. Gouy