Phenyl phosphotriester derivatives of AZT: Variations upon the SATE moiety

Article abstract of DOI:10.1016/j.bmc.2008.06.024

Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3′-azido-2′,3′-dideoxythymidine) are described herein. These p

Full text of DOI:10.1016/j.bmc.2008.06.024

Bioorganic & Medicinal Chemistry 16 (2008) 7321–7329
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Bioorganic & Medicinal Chemistry
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Phenyl phosphotriester derivatives of AZT: Variations upon the SATE moiety
Anne-Laure Villard a, Gaëlle Coussot a, Isabelle Lefebvre a, Patrick Augustijns b, Anne-Marie Aubertin c,
Gilles Gosselin a,d, Suzanne Peyrottes a, Christian Périgaud a,
*
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 Laboratory for Pharmaceutical Technology and Biopharmacy, 3000 Leuven, Belgium
c Université Louis Pasteur, INSERM 544, 67000 Strasbourg, France
d Laboratories Idenix Sarl, Cap Gamma, 1682 rue de la Valsière, 34819 Montpellier Cedex 04, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel
phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 30-azido-20,30-dideoxy-
thymidine) are described herein. These pronucleotides are characterized by the presence of polar func-
tions on the SATE biolabile phosphate protections. Whereas derivatives incorporating an amino
residue in the vicinity of the thioester functionality display low chemical stability, the introduction of
one or two hydroxyl groups on the SATE moieties confers high resistance of the resulting prodrugs
towards esterase hydrolysis. Thus, one of these pronucleotides, the monohydroxylated SATE derivative
of AZT 2, is able to cross a Caco-2 cell monolayer mainly in intact form, probing that further development
is warranted as a possible HIV-pronucleotide candidate.
Received 6 May 2008
Revised 12 June 2008
Accepted 13 June 2008
Available online 18 June 2008
Keywords:
Antiviral agents
Prodrug
Biotransformation
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
were also interested in studying a SATE moiety built up with an
amino acid such as L-valine (compound 5) in order to favour an
Over the last decade, we have demonstrated the potency of
mononucleotide prodrugs (pronucleotides) bearing the S-acyl-
2-thioethyl (SATE) biolabile phosphate protection. Initially deve-
loped with bis(SATE) phosphotriesters,1–3 this approach was
pursued with mixed phosphoester derivatives,4,5, that is, SATE aryl
phosphotriesters6,7 and SATE phosphoramidate diesters.8 Such
pronucleotides were shown to exhibit enhanced activity in cell
culture experiments compared to the corresponding parent nucle-
oside especially in the case when the first phosphorylation step is
hampered.
active transport of the pronucleotide through cell membranes.
Indeed, it is well documented in the literature10–12 that esterifica-
tion of the hydroxyl group at the 50- or 30-position of nucleoside
analogues with L-valine significantly increases their oral
bioavailability.
2. Results and discussion
2.1. Chemistry
As part of the development of the SATE aryl phosphotriesters’
family, we were interested in studying the influence of polar func-
tions introduced on the acyl portion of the SATE group. Indeed,
such a modification would increase the enzymatic stability of the
corresponding pronucleotide because of a higher resistance of
the polar SATE moiety against esterase hydrolysis, as previously
shown in other series of pronucleotides.9 In this study, we succes-
sively introduced one or two hydroxyl functions, as well as amino
groups on the S-pivaloyl-2-thioethyl (tBuSATE) skeleton and eval-
uated the effect of such variations upon the in vitro anti-HIV activ-
ity and enzymatic stability of the resulting phenyl phosphotriester
derivatives of AZT (compounds 2–4, Fig. 1) in comparison with the
corresponding tBuSATE phenyl phosphotriester 1. Furthermore, we
The preparation of the (tBuSATE) phenyl phosphotriester deriv-
ative of AZT 1 has previously been described,13 and the synthesis of
the modified SATE derivatives 2–5 requires the corresponding thi-
oesters 6–9 (Scheme 1). The procedure initially developed1 for the
preparation of the thioester precursors involves thioacids as start-
ing materials. As only few of them are commercially available or as
they need to use gaseous and bad-smelling reagent (i.e., dihydro-
gen sulfide) for their synthesis, we decided to propose an alterna-
tive pathway using a one-pot reaction from carboxylic acids. Our
new procedure to prepare thioesters 6–9 (Scheme 1) involves first
an activation of the corresponding acids 10–13 using a small ex-
cess of 1,10-carbonyl-diimidazole (CDI) in non polar solvents to
avoid the formation of symmetrical anhydrides.14 Then, the addi-
tion of 2-mercaptoethanol was performed at low temperature after
dilution of the reaction mixture to limit transposition of the
S-acyl-2-thioethanol (kinetic isomer) into the corresponding ester
* Corresponding author. Tel.: +33 467 14 38 55; fax: +33 4 67 54 96 10.
E-mail address: perigaud@univ-montp2.fr (C. Périgaud).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.06.024

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A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
Figure 1. Structures of target compounds.
Scheme 1. Synthesis of thioester precursors. Reagents and condition: (a) TrCl, NEt3, CH2Cl2; (b) NaOH aq 30%, dioxan; (c) MeC(OMe)2, APTScat, CH2Cl2, 2 h; (d) CDI, DMF/
toluene or CH2Cl2, 30 min; then HO(CH2)2SH, ꢀ10 °C, 2 h.
(O-acyl-2-thioethanol, thermodynamic isomer).15 The required
acid partners were obtained as follows:
with an isopropylidene group following a previously published
procedure in order to obtain compound 11.16 Compounds 12 and
13 were commercially available. Finally, commercial phenyl
dichlorophosphate was coupled with the various thioesters 6–9
to give the corresponding SATE phenyl phosphorochloridates
(compounds 15–18, Scheme 2), which were pure enough (on the
basis of their 1H and 31P NMR spectra) and directly engaged in
the next step without further purification. Coupling with AZT
was performed in presence of N-methylimidazole and led to
protected SATE phenyl phosphotriesters 19–22 in 63 to 76% yields
over the two steps. Removal of the various protecting groups was
carried out using diluted trifluoroacetic acid (TFA) or aqueous
acetic acid solutions and the target compounds 2–5 were isolated,
The choice of the protecting groups born by the modified SATE
moieties should be done thoughtfully and we decided to use acid-
labile protecting groups (trityl, isopropylidene acetal and tert-
butyloxycarbonyl, tBoc) in order to keep the integrity of the target
compounds. Indeed, they possess base and nucleophile sensitive
functions (thioester, phosphotriester). Consequently, the hydroxyl
function of the 2,2-dimethyl-3-hydroxypropanoic methyl ester
was protected with a trityl group and led to derivative 14, which
was saponified to obtain the desired acid 10 in 81% overall yield
after crystallization. The two hydroxyl functions of the 2,2-
bis(hydroxymethyl) propanoic acid were simultaneously protected
Scheme 2. Synthetic pathway to the SATE aryl phosphotriester derivatives of AZT. Reagents and conditions: (a) PhOP(O)Cl2, NEt3, THF, ꢀ78 °C to rt, 4 h; (b) AZT,
N-methylimidazole, THF, 4 h; (c) 20% TFA in CH2Cl2, ꢀ10 °C to rt, 15 min to 2 h; d) 50% aq AcOH, 4 h.

A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
7323
as mixtures of two diastereoisomers, in high yields after silica gel
column chromatography.
out in CEM-SS cell extracts and culture medium using our previ-
ously described ‘HPLC-on line cleaning’ method.1 Metabolites were
identified using both HPLC/MS coupling and/or standard co-
injections.
2.2. Antiviral activity
In the cell culture medium, the derivatives 1–3 are slowly
hydrolyzed into the corresponding phenyl phosphodiester deriva-
tive of AZT through an esterase mediated activation (Scheme
3).13 A similar decomposition process was observed in cell extracts,
but the rate of hydrolysis of the SATE group was quicker in the lat-
ter medium. Furthermore, the successive introduction of hydroxyl
groups on the acyl residue led to a progressive increase of the sta-
bility of the corresponding SATE moiety against esterase hydrolysis
(Table 2). This variation of half-life in cell extracts from a few hours
for compounds 1 and 2 to several days for derivative 3 may be cor-
related with the drop of the antiviral effect observed for 3 in the
CEM/TKꢀ cell line. As we previously observed,6,9 a relative stability
in extra cellular medium associated with a rapid intracellular
decomposition are key factors for compounds designed to release,
within the cells, the parent 50-mononucleotide through enzymatic
activation. It is reasonable to assume that the enzymatic stability
of compound 3 may not be appropriate in the context of a cell cul-
ture evaluation.
SATE phenyl phosphotriesters 4 and 5 appeared highly unstable
in both media and led to the formation of the same metabolite, that
is, the phenyl phosphodiester derivative of AZT. As the estimated
half-life was less than few minutes, a chemical mechanism of
hydrolysis rather than an enzymatic one was suspected. Further-
more, such chemical instability has been previously reported for
aminoacyloxymethyl ester prodrugs.18,19 Thus, stabilities of both
derivatives 4 and 5 were evaluated in water and in RPMI 1640. Re-
sults are given in Table 3 and compared to the N-Boc protected
All the four SATE phenyl phosphotriesters 2–5 were evaluated
for their anti-HIV activity in three cell lines, in comparison to the
parent nucleoside (AZT) and to the tBuSATE phenyl phosphotries-
ter derivative 1 as reference pronucleotide (Table 1). Whereas all
compounds showed similar submicromolar activity against HIV-1
replication in CEM-SS and MT-4 cell lines, striking differences were
observed in CEM/TKꢀ, thus reflecting their ability to bypass the TK
mediated phosphorylation step and/or to deliver AZTMP within the
cells.17,2 Indeed, derivative 2 bearing only one hydroxyl function on
the SATE residue exhibited EC50 in the micromolar range, demon-
strating its potency to act as a pronucleotide of 50-monophosphate,
whereas the introduction of a second hydroxyl group (compound
3) led to a drastic loss of antiviral activity in the CEM/TKꢀ cell line.
A similar observation could be made for derivatives 4 and 5 incor-
porating a free amino residue. Determination of the log P values of
compounds 1–5, ranging from 2 to 3, showed that any of these
derivatives may be able to enter cells through a passive diffusion
process. Consequently, a lack of permeation of the cell membrane
should not be responsible for the loss of antiviral activity observed
for derivatives 3–5. This prompted us to study the behaviour of
such new series of SATE phenyl phosphotriesters in various biolog-
ical media in order to understand the different parameters in-
volved in the variations of antiviral activities observed for the
three cell lines used.
2.3. Enzymatic and chemical stability
Stability studies of SATE phenyl phosphotriesters 1–5, as well as
N-Boc protected derivatives 21 and 22 (vide infra), were carried
Table 2
Calculated half-lives of the phosphotriester derivatives 1–5 in CEM-SS cell extracts
and culture mediuma
Compound
Table 1
1
2
3
4
21
5
22
Anti-HIV-1 activitya
(lM) in three cell culture systems of target compounds 2–5 in
t1/2 in culture
medium
25.3 d 13.8 d 8.8 d <1 min 14.1 d <1 min 8.2 d
comparison to the parent nucleoside AZT and the tBuSATE phenyl phosphotriester
derivative 1, as a reference pronucleotide
t1/2 in cell extracts 1.5 h
14.8 h 7.4 d 8 min
1.7 d
<1 min 4.4 d
Compound
CEM-SS
MT-4
CEM/TKꢀ
a
All data represent average values for three separate experiments and variability
was lower than 10%.
b
c
b
c
b
c
EC50
CC50
EC50
CC50
EC50
CC50
AZT
1
2
3
4
0.003
0.015
0.006
0.012
0.009
0.001
>100
>10
>130
>150
>120
>120
0.015
0.81
0.03
0.074
0.054
0.004
>100
>10
>130
>150
>120
>120
> 100
0.45
3.9
>100
90
>100
>10
>130
>150
>120
>120
Table 3
Chemical stability of the phosphotriester derivatives 4, 5, 21 and 22 in various mediaa
5
81
Compound
a
4
21
5
22
All data represent average values for at least three separate experiments. The
variation of these results under standard operating procedures is below 10%.
t1/2 in water (pH 6.8)
t1/2 in RPMI 1640 (pH 7.5)
>6 d
6 min
> 20 d
>12 d
>6 d
<1 min
> 40 d
> 9 d
b
EC50: effective concentration or concentration required to inhibit the replication
of HIV-1 by 50%.
c
a
CC50: cytotoxic concentration or concentration required to reduce the viability
All data represent average values for two separate experiments and variability
was lower than 10%.
of uninfected cells by 50%.
Scheme 3. Proposed decomposition pathway for the phosphotriester derivatives of AZT 1–3.13,6

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A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
derivatives 21 and 22. All four compounds were stable in water,
while half-lives observed in RPMI 1640 were similar to the ones
obtained in culture medium.
With regard to the nature of the metabolite observed in all
experiments, that is, the phenyl phosphodiester derivative of
AZT, we dismissed the hypothesis of a nucleophilic attack on the
phosphorus atom. Two mechanisms of decomposition could then
be proposed, involving a nucleophilic attack either (i) on the car-
3 were partially metabolized into the corresponding phenyl phos-
phodiester derivative (Scheme 3), AZTMP, and AZT. These metabo-
lites were identified by co-injection with authentic samples.
Absorptive and exsorptive transport of the pronucleotides 2 and
3 in which one or two hydroxyl functions are present on the acyl
residue are outlined in Figure 2.
Pronucleotide 2 was able to cross the Caco-2 monolayer mainly
in intact form in the absorptive as well as in the exsorptive direc-
tions. When assessing its absorptive transport, the analogue 3
could not be detected in the basolateral compartment and the par-
ent nucleoside was essentially observed. In contrast, the pronucle-
otide 3 appeared in the apical compartment when exsorptive
transport was studied and transport in this direction was threefold
higher than the absorptive one (on the basis of AZT-equivalents).
These data suggest that the dihydroxylated prodrug 3 may be a
substrate for an apically localized efflux carrier modulating its
absorptive transport. Compared to pronucleotide 2, the substrate
properties of 3 for a P-gp-like efflux mechanism, associated with
a lower diffusion across the cell membrane due to a decreased lipo-
philicity (Table 4) may be responsible for the observed result and
might prevent its intestinal transport. Consequently, lipophilic
metabolites (i.e., AZT), resulting from its partial hydrolysis inside
mucosal cells, were only observed in the basolateral compartment.
bon atom in the
a position to the phosphorus atom or (ii) on the
thiocarbonyl function of the SATE chain. This last process may be
favoured by the electron-withdrawing effect of the ammonium
group of the valine and of the a-amino butyric acid in the vicinity
of the thiocarbonyl.18,19 In this respect, the stability of derivatives
21 and 22 may be due to the presence of a bulky and electron do-
nor protecting group, such as the tBoc group. This induced a less
susceptibility of the corresponding SATE moieties towards a nucle-
ophilic attack on the vicinal thiocarbonyl.
The implication of the free amino function (within the SATE
moiety) which could be involved in the decomposition mechanism
through a SNi or an intermolecular nucleophilic attack was also de-
nied considering the pKa of valine being 9.7. Indeed the corre-
sponding amino function mainly exists in its protonated form at
the pH of the RPMI 1640 (7.5).
Finally, we highly suspected the participation of nucleophiles
present in this medium in the hydrolysis process leading to the loss
of the SATE moiety in compounds 4 and 5.
3. Conclusion
Novel SATE phenyl phosphotriesters bearing polar functions
(hydroxyl or amine) on the acyl moiety have been synthesized
and studied. These mononucleotide prodrugs were shown to exhi-
bit greatly to moderately enhanced activity against HIV compared
to the parent nucleoside in vitro. Furthermore, antiviral activity
2.4. Uptake and metabolism in Caco-2 cells
The potential for intestinal absorption was evaluated by assess-
ing the transport properties of the studied pronucleotides in the
Caco-2 system. Considering the behaviour of derivatives incorpo-
rating the amino acid residues, compounds 4 and 5, we did not
examine their transport properties. In addition, due to the rela-
tively low aqueous solubility (57 lg/mL) of tBuSATE phenyl phos-
photriester 1, a reliable quantitative description of its transport
was not possible. Thus, transport of phosphotriesters 2 and 3 was
Table 4
Physicochemical properties of pronucleotides 1, 2 and 3 in comparison with their
parent nucleoside
a
Compound
MW
clogPo/w
Aqueous solubility (g/l)
evaluated at a target concentration of 100 lM and was initiated
1
2
3
AZT
567.56
583.55
599.55
267.24
3.34
2.30
1.76
0.06
0.057
6.64
60.6
by adding the test solution to the apical side (absorptive transport)
or basolateral side (exsorptive transport) of the monolayer. Trans-
port was assessed by measuring concentrations of the pronucleo-
tide and its metabolites in the receiver compartment after an
incubation period of 2 h. During transport, pronucleotides 2 and
29.3
a
Calculated values; logP determination was performed using Advanced Chem-
istry Development (Toronto, Canada) logP dB 4.5 calculations.
25.00
2
3
Pronucleotide
Phenylphos phodiester
metabolite
20.00
15.00
10.00
5.00
AZT
AZTMP
0.00
A/B
B/A
A/B
B/A
Figure 2. Total cumulative absorptive (A/B) and exsorptive (B/A) transport ( SD) of 100
lM of derivatives 2 and 3 across Caco-2 monolayer (2 h incubation). All data are
expressed as percentage of the amount initially added to the apical side (donor compartment).

A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
7325
was mainly retained in kinase-deficient cell lines, which was con-
sistent with the efficient bypass of the first phosphorylation step.
Derivatives 2 and 3 proved to be more resistant to esterase medi-
ated hydrolysis than the parent tBuSATE phenyl phosphotriester 1
and appeared to deserve further investigations from their biologi-
cal properties. Thus, intestinal absorption study was carried out
and revealed that the monohydroxylated prodrug 2, being able to
cross a Caco-2 cell monolayer in intact form, as the most promising
compound prepared in this series. So, combinations of adequate
aqueous solubility and enzymatic stability of SATE phenyl phos-
photriester derivatives may result in prodrugs with improved
pharmacokinetic properties and contribute to the development of
orally administered pronucleotides.
Inc.; phenyl phosphorodichloridate was purchased from Aldrich;
N -tert-butoxycarbonyl-L-valine (tBoc-Val) and N -tert-butoxy-
carbonyl-amino isobutyric acid (tBoc-Aib) were from Novabio-
chem. tBuSATE phenyl phosphotriester derivative of AZT 113 and
50-monophosphate of AZT (AZTMP)20 were synthesized following
previously published procedures.
a
a
4.1. Biological methods
The origin of the viruses and the techniques used for measuring
inhibition of virus multiplication have been previously described.1
4.2. Transport studies
Material and methods used were as previously described.9
4.3. Stability studies
4. Experimental
Unless otherwise stated, 1H NMR spectra were recorded at
300 MHz and 13C NMR spectra at 75 MHz with proton decoupling
at 25 °C on a Bruker 300 Avance or DRX 400. Chemical shifts are gi-
ven in d values referenced to the residual solvent peak (CDCl3 at
7.26 ppm and 77 ppm, DMSO-d6 at 2.49 ppm and 39.5 ppm) rela-
tive to TMS. Deuterium exchange, decoupling and COSY experi-
ments were performed in order to confirm proton assignments.
Coupling constants, J, are reported in Hertz. Two-dimensional
1H–13C heteronuclear COSY were recorded for the attribution of
13C signals. Unless otherwise stated, 31P NMR spectra were re-
corded 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, GT) as matrix. Melting points were determined in open capil-
lary tubes on a Büchi-545 and are uncorrected. UV spectra were re-
corded 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), visualization of prod-
ucts being accomplished by UV absorbance followed by charring
with 5% ethanolic sulfuric acid with heating for nucleotides. Flash
HPLC analyses were carried out using an improved ‘On-line ISRP
cleaning method’.1 Briefly, the crude sample (80
lL, initial concen-
tration 50
lM) was injected onto the precolumn and eluted firstly
with 20 mM triethylammonium acetate buffer (pH 6.9) for 5 min at
a flow rate of 2 mL/min. Then by activating a six port Rheodine
valve and back-flushing the analytes from the precolumn to the
column, the analytes were eluted with a linear gradient of 0–80%
acetonitrile over 30 min at 1 mL/min flow rate. The decomposition
products were identified by HPLC/MS coupling after calibration
and/or by co-injection with authentic samples (AZTMP, AZT, etc.).
For each incubation time, the calculation of the relative concentra-
tion of each species was related to the peak areas, these data were
considered as the experimental data. The rate constants of disap-
pearance and the half-lives (kinetic data) of the phosphotriester
and phosphodiester derivatives were calculated according to pseu-
do-first order kinetic models and optimized using mono- or poly-
exponential regressions.
For chemical stability studies; buffers and various media were
20 mM TPK (at pH 5.2, 7.2, and 8.2), RPMI 1640 (Gibco BRL) and
culture medium (90% RPMI 1640 and 10% Foetal calf serum from
Gibco BRL inactivated 30 min at 56 °C).
chromatography was carried out using 63–100
lm silica gel
(Merck Art. No. 115101) otherwise 40–63 m silica gel (Merck
l
For biological stability studies; CEM-SS cell extracts were pre-
pared and studies were performed according to published
procedure.1
Art. No. 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 pres-
sure at rt. Moisture sensitive reactions were performed under ar-
gon atmosphere using oven-dried glassware. All aqueous (aq)
solutions were saturated with the specified salt unless otherwise
indicated. Organic solutions were dried over Na2SO4 after workup
and solvents were removed by evaporation at reduced pressure.
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-
4.4. Chemistry
4.4.1. Standard procedure 1: preparation of thioester
precursors 6–9
To a stirred solution of the required acid (10–13, 30 mmol) in a
mixture of toluene and anhydrous N,N-dimethylformamide
(20 mL, 2:1, v/v) for 10 and 11, or anhydrous dichloromethane
(for 12 and 13) was added 1,10-carbonyldiimidazole (1.3 equiv,
39 mmol) under argon. After 30 min at room temperature, the mix-
ture was cooled to ꢀ10 °C, and then diluted with dry toluene
(120 mL) and dry N,N-dimethylformamide (8 mL). 2-Mercap-
toethanol (1.3 equiv, 39 mmol) was added under argon and the
mixture was allowed to stir at ꢀ10 °C. After 2 h, all the volatiles
were removed under reduced pressure; the obtained residue was
dissolved in chloroform (200 mL), and then extracted twice with
water (150 mL). The organic phase was separated, dried over
Na2SO4, filtered and evaporated under reduced pressure to give
the expected S-acyl-2-thioethanol derivatives 6–9.
verse-phase analytical column (Nucleosil, C18
,
150 ꢁ 4.6 mm,
m) equipped with a prefilter, and a precolumn (Nucleosil, C18
m) were used. Detection was monitored at 267 nm. Nucleotidic
5
5
l
l
,
derivatives were eluted using a linear gradient of 0–80% acetoni-
trile in 20 mm triethylammonium acetate buffer (pH 6.9) over
30 min at 1 mL/min flow rate. Electrospray ionization–mass spec-
trometry (ESI-MS) was performed using an SSQ 7000 single quad-
rupole mass spectrometer (Finnigan, San Jose, CA, 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 80 psi) and as auxiliary gas with
a flow rate of 15 U. Under these conditions, full scan data acquisi-
tion was performed from m/z 200 to 800 in centro¨ıd mode and
using a cycle time of 1.0 s. AZT was from Brantford Chemicals
4.4.2. 2,2-Dimethyl-O-3-(triphenylmethyl)-propionyl-
thioethan-2-ol, 6
Standard procedure 1 from 10.8 g of 10 yielded 12.3 g (98%) of 6
obtained as a colourless oil.

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A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
Rf 0.50 (AcOEt/toluene, 2:8, v/v). 1H NMR (CDCl3) d 7.48 (m, 6H,
6H, C(CH3)2). 31P NMR (CDCl3) d 0.51. MS (FAB > 0ꢀ, GT) m/z 559
[MꢀCl]+, (FAB < 0, GT) mþ/z 731 ½M þ ðHNEtþ3 Cl Þ ꢀ Hꢂꢀ, 575
[MꢀCl+O]ꢀ, 489 ½M þ ðHNEt3 ClꢀÞ ꢀ Trꢂꢀ.
Ph), 7.33 (m, 9H, Ph), 3.79 (pq, 2H, CH2CH2O), 3.25 (s, 2H, TrOCH2),
3.14 (t, 2H, J = 6.0 Hz, SCH2CH2), 2.08 (t, 1H, J = 5.7 Hz, OH), 1.30 (s,
6H, C(CH3)2). 13C NMR (CDCl3) d 205.4 (CO), 143.7 (Ph ipso), 128.7,
127.7, 127.0 (3s, Ph), 86.3 ((Ph)3C), 70.1 (TrOCH2), 61.8 (CH2CH2O),
50.9 (C(CH3)2), 31.6 (SCH2CH2), 22.8 (C(CH3)2). IR (CCl4) mmax
1682 cmꢀ1(C@O). MS (FAB > 0, GT) m/z 663 [M+Tr]+, 421 [M+H]+,
(FAB < 0, GT) m/z 375 [MꢀCH2CH2OH]ꢀ. Anal. for (C26H28O3S):
calcd: C, 74.25; H, 6.71; S, 7.62; found: C, 74.05; H, 6.44; S, 7.53.
4.4.8. S-[2-(2,2-Dimethyl-1,3-dioxan-5-yl)propionyl]-2-
thioethyl phenyl phosphorochloridate, 16
Standard procedure 2 from 2.34 g of 7.
1H NMR (CDCl3) d 7.19 (m, 5H, Ph), 4.41 (m, 2H, CH2CH2O), 4.24,
3.72 (2d, 4H, J = 12.2 Hz, CH2), 3.33 (m, 2H, SCH2CH2), 1.45, 1.44
(2s, 6H, C(CH3)2), 1.23 (s, 3H, CH3). 31P NMR (CDCl3) d 0.65. MS
(FAB > 0, GT)ꢀ m/z ꢀ 409 [M+H]+, (FAB < 0, GT) m/zꢀ 545
½M þ ðHNEtþCl Þ ꢀ Hꢂ , 505 ½M þ ðHNEtþClꢀÞ ꢀ CðCH3Þ þ Hꢂ , 389
4.4.3. 2-[(2,2-Dimethyl-1,3-dioxolan)-2-hydroxyethyl]-2-
methylpropionyl-thioethan-2-ol, 7
Standard procedure 1 from 5.22 g of 11 yielded 6.89 g (98%) of 7
obtained as a colourless oil.
3
3
2
[MꢀCl+O]ꢀ, 331 [MꢀPh]ꢀ, 191 [MꢀSATE]ꢀ.
Rf 0.41 (CH2Cl2/CH3OH, 95:5, v/v). 1H NMR (DMSO-d6) d 4.96 (t,
1H, J = 5.5, OH), 4.06 (d, 2H, J = 12.1 Hz, CH2), 3.68 (d, 2H,
J = 12.1 Hz, CH2), 3.45 (pq, 2H, CH2CH2O), 2.94 (t, 2H, J = 6.6 Hz,
SCH2CH2), 1.35, 1.24 (2s, 6H, C(CH3)2), 1.06 (s, 3H, CH3). 13C NMR
(DMSO-d6) d 202.2 (CO), 97.5 (C(CH3)2), 65.2 (CH2), 59.7 (CH2CH2O),
48.4 (C(CH3)), 30.7 (SCH2CH2), 25.3, 21.9 (2s, C(CH3)2), 18.9 (CH3). IR
4.4.9. S-[(2,2-Dimethyl-N -tert-butyloxycarbonyl) glycinyl]-2-
thioethylphenyl phosphoro chloridate, 17
a
Standard procedure 2 from 2.03 g of 8.
1H NMR (CDCl3) d 7.28 (m, 5H, Ph), 5.0 (sl, 1H, NH), 4.35 (m, 2H,
CH2CH2O), 3.23, 3.22 (2t, 2H, J = 6.6 Hz, SCH2CH2), 1.47 (s, 9H, tBu),
1.43, 1.42 (2s, 6H, C(CH3)2). 31P NMR (CDCl3) d 0.57. MS (FAB > 0,
GT) m/z 839 [2MꢀCl]+, 438 [M+H]+, 382 [MꢀtBu+2H]+, 420
[MꢀCl+H2O]+, 364 [MꢀtBuO]+, 338 [MꢀtBoc+2H]+, (FAB < 0, GT)
m/z 837 [2Mꢀ2Cl+2O+H]ꢀ, 574 ½M þ ðHNEt3þClꢀÞ ꢀ Hꢂꢀ, 498
½M þ ðHNEtþ3 ClꢀÞ ꢀ Phꢂꢀ, 418 [MꢀCl+O]ꢀ, 360 [MꢀPh]ꢀ.
(CCl4) m
max 1676 cmꢀ1 (C@O). Anal. for (C10H18O4S): calcd: C, 51.26;
H, 7.74; S, 13.69; found: C, 51.04; H, 7.88; S, 13.33.
4.4.4. 2-[(tert-Butoxycarbonyl)amino]-2-methylpropionyl-
thioethan-2-ol, 8
a
Standard procedure 1 from 6.10 g of 12 yielded 7.68 g (97%) of 8
obtained as a colourless oil.
4.4.10. S-(N -tert-butyloxycarbonyl-(
L
)-valinyl)-2-thioethyl
phenyl phosphorochloridate, 18
Rf 0.24 (AcOEt/hexane, 4:6, v/v). 1H NMR (DMSO-d6) d 7.55 (bs,
1H, NH), 4.87 (pt, 1H, J = 4.9 Hz, OH), 3.39–3.37 (m, 2H, CH2CH2O),
2.82 (t, 2H, J = 6.7 Hz, SCH2CH2), 1.37 (s, 9H, tBu), 1.30 (1s, 6H,
C(CH3)2). 13C NMR (DMSO-d6) d 204.3 (COS), 154.3 (COO), 78.3
(C(CH3)3), 61.4 (C(CH3)2), 60.1 (CH2CH2O), 30.6 (SCH2CH2), 28.3
Standard procedure 2 from 2.17 g of 9.
1H NMR (CDCl3) d 7.29 (m, 5H, Ph), 4.96 (d, 1H, J = 9.3 Hz,
CHNH), 4.34 (m, 3H, CHNH, CH2CH2O), 3.26 (t, 2H, J = 6.5 Hz,
SCH2CH2), 2.27 (m, 1H, CH(CH3)2), 1.46 (s, 9H, tBu), 1.00, 0.97,
0.91, 0.86 (4s, 6H, CH(CH3)2). 31P NMR (CDCl3)
d 0.60. MS
(C(CH3)3), 25.1 (C(CH3)2). IR (CCl4)
m
max 1700 cmꢀ1(C@O thioester),
(FAB > 0, GT) m/z 452 [M+H]+, 396 [MꢀtBu+2H]+, 352
1727 cmꢀ1(C@O carbamate). Anal. for (C11H21NO4S): calcd: C,
[MꢀtBoc+2H]+, 434ꢀ [MꢀCl+H2O]+, (FAB < 0, ꢀ GT) m/z 588
50.17; H, 8.04; S, 12.18; found: C, 50.54; H, 7.94; S, 12.43.
½M þ ðHNEtþ3 ClꢀÞ ꢀ Hꢂ ,
512
½M þ ðHNEtþ3 Cl Þ ꢀ Phꢂꢀ,
432
[MꢀCl+O]ꢀ, 374 [MꢀPh]ꢀ.
4.4.5. 2-[(tert-Butoxycarbonyl)amino]-3-methylbutionyl-
thioethan- 2-ol, 9
Standard procedure 1 from 6.52 g of 13 yielded 8.16 g (98%) of 9
obtained as a colourless oil.
4.4.11. Standard pocedure 3: preparation of SATE phenyl
phosphotriester derivatives 19–22 of AZT
To a stirred solution of the required SATE phenyl phosphorochl-
oridates (15–18, 6 mmol, 3 equiv) in anhydrous THF (20 mL) were
successively added AZT (0.53 mg, 2.0 mmol) and N-methyl imidaz-
ole (0.95 mL, 12 mmol, 6 eq.). After four hours stirring at room
temperature, the reaction mixture was diluted with dichlorometh-
ane (80 mL) and quenched with diluted 0.1 N HCl solution. The or-
ganic layer was decanted and washed with an aqueous saturated
solution of NaHCO3 and then water. The organic layer was dried
over Na2SO4, filtered and concentrated under vacuum; finally the
residue was purified on silica gel column chromatography.
Rf 0.36 (AcOEt/hexane, 4:6, v/v). 1H NMR (DMSO-d6) d 7.49 (d, 1H,
J = 8.3 Hz, CHNH), 4.91 (t, 1H, J = 5.4 Hz, OH), 3.90 (m, 1H, CHNH),
3.42 (m, 2H, CH2CH2O), 2.87 (m, 2H, SCH2CH2), 2.41–1.98 (m, 1H,
CH(CH3)2), 1.40 (s, 9H, tBu), 0.86, 0.84 (2s, 6H, CH(CH3)2). 13C NMR
(DMSO-d6) d 201.6 (COS), 155.8 (COO), 78.5 (C(CH3)3), 66.1 (CHNH),
59.9 (CH2CH2O), 30.7 (SCH2CH2), 29.7 (CH(CH3)2), 28.2 (C(CH3)3),
19.1, 17.8 (2s, CH(CH3)2). IR (CCl4) m
max 1691 cmꢀ1(C@O thioester),
1726 cmꢀ1(C@O carbamate). Anal. for (C12H22NO4S): calcd: C,
52.15; H, 8.02; S, 11.60; found: C, 52.45; H, 8.34; S, 11.53.
4.4.6. Standard procedure 2: preparation of SATE phenyl
phosphorochloridates 15–18
4.4.12. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(2,2-
dimethyl-3-triphenylmethoxy propionyl)-2-thioethyl]-O00-
phenyl phosphate, 19
Standard procedure 3 from 3.58 g of 15 yielded 1.19 g (72%) of
19 obtained as white foam after silica gel column chromatography
(dichloromethane/methanol, gradient 0 to 1.5%).
To a stirred solution of the required thioesters (6–9, 10 mmol)
and freshly distilled triethylamine (2 equiv, 20 mmol, 2.79 mL) in
anhydrous THF (100 mL) at ꢀ78 °C was added phenyl phosphoro
dichloridate (1.02 equiv, 10.2 mmol, 1.49 mL). The mixture was
stirred at room temperature for 4 h, filtered and the filtrate con-
centrated on vacuum. The residue was diluted in anhydrous carbon
tetrachloride and filtered again, to yield after concentration to the
desired SATE phenyl phosphorochloridate as oils.
Rf 0.45 (CH2Cl2/CH3OH, 95:5, v/v). 1H NMR (DMSO-d6) d 11.4
(bs, 1H, Thy-NH), 7.46, 7.44 (2d, 1H, J = 0.9 Hz, H-6), 7.37–7.16
(m, 20H, Ph, (Ph)3C), 6.13, 6.12 (2t, 1H, J = 6.6 Hz, H-10), 4.44 (m,
1H, H-30), 4.33 (m, 2H, H-50, H-500), 4.16 (m, 2H, CH2CH2O) 4.00
(m, 1H, H-40), 3.17, 3.16 (2t, 3H, J = 6.5 Hz, SCH2CH2), 3.04 (s, 2H,
TrOCH2), 2.35 (m, 2H, H-20, H-200), 1.71 (s, 3H, Thy-CH3), 1.12 (s,
6H, C(CH3)2). 13C NMR (DMSO-d6) d 203.4 (CO), 163.6 (C-4),
150.3 (C-2), 149.9 (d, JPꢀC = 6.7 Hz, Ph ipso), 143.4 (C(Ph)3 ipso),
135.8 (C-6), 129.9, 129.8 (2s, Ph), 128.2, 127.8, 127.0 (3s, C(Ph)3),
125.4 (Ph), 119.9 (d, J = 4.2 Hz, Ph ortho), 109.9 (C-5), 85.8
4.4.7. S-(2,2-Dimethyl-3-triphenylmethoxypropionyl)-2-
thioethyl phenyl phosphorochloridate, 15
Standard procedure 2 from 4.21 g of 6.
1H NMR (CDCl3) d 7.33 (m, 20H, Ph, C(Ph)3), 4.37 (m, 2H,
CH2CH2O), 3.30 (m, 2H, SCH2CH2), 3.20 (s, 2H, TrOCH2), 1.25 (s,

A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
7327
a
4.4.15. O-(30-Azido-20,30- dideoxythymidin-50-yl)-O0-[S-(N -tert-
butyloxycarbonyl-(
phosphate, 22
Standard procedure 3 from 2.71 g of 18 yielded 1.04 g (76%) of
22 obtained as white foam after silica gel column chromatography
(dichloromethane/methanol, gradient 0 to 1.5%).
((Ph)3C), 83.7 (C-10), 81.1 (d, JPꢀC = 7.9 Hz, C-40), 69.5 (TrOCH2), 67.3
(d, JPꢀC = 5.6 Hz, C-50), 66.4 (d, JPꢀC = 5.7 Hz, CH2CH2O,), 59.8, 59.7
(2s, C-30), 50.4 (C(CH3)2), 35.5 (C-20), 28.1 (d, JPꢀC = 7.1 Hz,
SCH2CH2), 22.3 (C(CH3)2), 12.0 (Thy-CH3). 31P NMR (DMSO-d6) d
ꢀ5.24, ꢀ5.46. MS (FAB > 0, GT) m/z 826 [M+H]+, 800 [MꢀN2+3H]+,
127 [BH2]+, (FAB < 0, GT) m/z 1400 [2MꢀAZT]ꢀ, 824 [MꢀH]ꢀ, 748
[MꢀPh]ꢀ, 575 [MꢀAZT]ꢀ, 422 [MꢀSATE]ꢀ, 125 [B]ꢀ. UV (Ethanol
L
)-valinyl)-2-thioethyl]-O00-phenyl
Rf 0.41 (CH2Cl2/CH3OH, 95:5, v/v). 1H NMR (DMSO-d6) d 11.4 (br s,
1H, Thy-NH), 7.57 (d, 1H, J = 8.1 Hz, CHNH), 7.46, 7.44 (2s, 1H, H-6),
7.38 (m, 2H, Ph), 7.22 (m, 3H, Ph), 6.13 (t, 1H, J = 6.6 Hz, H-10), 4.45
(m, 1H, H-30), 4.33 (m, 2H, H-50, H-500), 4.15 (m, 2H, CH2CH2O), 4.01
(m, 1H, H-40), 3.93 (m, 1H, CHNH), 3.11 (pt, 2H, J = 6.3 Hz, SCH2CH2),
2.36 (m, 2H, H-20, H-200), 2.06 (m, 1H, CH(CH3)2), 1.71 (s, 3H, Thy-
CH3), 1.32 (s, 9H, tBu), 0.85, 0.83 (2s, 6H, CH(CH3)2). 13C NMR
(DMSO-d6) d 201.3 (COS), 163.6 (C-4), 155.8 (COO), 150.3 (C-2),
150.0 (d, JPꢀC = 6.7 Hz, Ph ipso), 135.8 (C-6), 129.7, 129.6, 125.4 (3s,
Ph), 119.9 (d, JPꢀC = 3.9 Hz, Ph ortho), 109.9 (C-5), 83.7 (C-10), 81.1
(d, JPꢀC = 7.5 Hz, C-40), 78.7 (C(CH3)3), 67.3 (d, JPꢀC = 5.6 Hz, C-50),
66.4 (d, JPꢀC = 5.4 Hz, CH2CH2O,), 66.2 (CHNH), 59.8, 59.7 (2s, C-30),
35.5 (C-20), 29.6 (CH(CH3)2), 28.0 (d, JPꢀ C = 7.6 Hz, SCH2CH2), 19.0,
17.8 (2s, CH(CH3)2), 12.0 (Thy-CH3). 31P NMR (DMSO-d6) d ꢀ5.45,
ꢀ5.53. MS (FAB > 0, GT) m/z 683 [M+H]+, 657 [MꢀN2+3H]+, 583
[MꢀtBoc+2H]+, 557 [MꢀtBoc-N2+4H]+, 424 [MꢀSATE+2H]+, 398
[MꢀSATE-N2+4H]+, 127 [BH2]+, (FAB < 0, GT) m/z 1363 [2MꢀH]ꢀ,
1287 [2MꢀPh]ꢀ, 1114 [2MꢀAZT]ꢀ, 681 [MꢀH]ꢀ, 605 [MꢀPh]ꢀ,
432 [MꢀAZT]ꢀ, 422 [MꢀSATE]ꢀ, 125 [B]ꢀ. HR-MS (FAB > 0, GT) m/z
calcd. 683.2264 [M+H]+, found 683.2254 [M+H]+. UV (Ethanol 95):
95) kmax (e) = 263 nm (10800), kmin (e) = 240 nm (7400). Anal. for
(C42H44N5O9PS): calcd: C, 61.08; H, 5.37; N, 8.48; found: C, 61.19;
H, 5.46; N, 8.32.
4.4.13. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(2-(2,2-
dimethyl-1,3-dioxan-5-yl)propionyl)-2-thioethyl]-O00-phenyl
phosphate, 20
Standard procedure 3 from 2.45 g of 16 yielded 0.81 g (63%) of
20 obtained as colourless oil after silica gel column chromatogra-
phy (dichloromethane/methanol, gradient 0–2%).
Rf 0.34 (CH2Cl2/CH3OH, 95:5, v/v). 1H NMR (DMSO-d6) d 11.4 (bs,
1H, Thy-NH), 7.46, 7.44 (2d, 1H, J = 0.7 Hz, H-6), 7.38 (m, 2H, Ph),
7.22 (m, 3H, Ph), 6.13 (m, 1H, H-10), 4.46 (m, 1H, H-30), 4.34 (m, 2H,
H-50, H-500), 4.19 (m, 2H, CH2CH2O), 4.04 (m, 3H, CH2, H-40), 3.69 (d,
2H, J = 12.1 Hz, CH2), 3.19, 3.18 (2pt, 2H, J = 6.2 Hz, SCH2CH2), 2.37
(m, 2H, H-20, H-200), 1.72 (s, 3H, Thy-CH3), 1.35, 1.23 (2s, 6H,
C(CH3)2), 1.03 (s, 3H, CH3). 13C NMR (DMSO-d6) d 201.7 (CO), 163.6
(C-4), 150.3 (C-2), 150.0 (d, JPꢀC = 6.8 Hz, Ph ipso), 135.8 (C-6),
129.9, 129.8, 125.4 (3s, Ph), 119.9 (d, JPꢀ C = 4.7 Hz, Ph ortho), 109.9
(C-5), 97.6 (C(CH3)2), 83.7 (C-10), 81.1 (d, JPꢀC = 7.8 Hz, C-40), 67.3 (d,
JPꢀ C = 5.6 Hz, C-50), 66.4 (d, JPꢀC = 5.5 Hz, CH2CH2O,), 59.8, 59.7 (2s,
C-30), 48.5 (C(CH3)), 35.5 (C-20), 28.1 (d, JPꢀC = 7.2 Hz, SCH2CH2),
25.6, 21.5 (2s, C(CH3)2), 18.6 (CH3), 12.1, 12.0 (2s, Thy-CH3). 31P
NMR (DMSO-d6) d ꢀ5.41, ꢀ5.53. MS (FAB > 0, GT) m/z 640 [M+H]+,
614 [MꢀN2+3H]+, 424 [MꢀSATE+2H]+, 127 [BH2]+, (FAB < 0, GT) m/z
1277 [2MꢀH]ꢀ, 1028 [2MꢀAZT]ꢀ, 638 [MꢀH]ꢀ, 562 [MꢀPh]ꢀ, 422
kmax
(e)=266 nm (10900), kmin (e)=235 nm (6900). Anal for
(C28H39N6O10PS): calcd: C, 49.26; H, 5.76; N, 12.31; found: C,
49.55; H, 5.76; N, 12.13.
4.4.16. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(2,2-
dimethyl-3- hydroxypropionyl)-2-thioethyl]-O00-phenyl
phosphate, 2
[MꢀSATE]ꢀ, 389 [MꢀAZT]ꢀ, 125 [B]ꢀ. UV (Ethanol 95) kmax
(
e
) =
To a stirred solution of protected derivative 19 (0.61 g,
263 nm (9700), kmin
(e
) = 240 nm (6300). Anal. for (C26H34N5O10PS):
0.74 mmol) in dry dichloromethane (4 mL) was added a solution
of TFA (4 mL, 50% in dichloromethane) drop wise at 0 °C. The reac-
tion reached completion in 15 min and the reaction mixture was
quenched by adding a saturated aqueous solution of NaHCO3
(ꢃ5 mL), then diluted with dichloromethane (50 mL). After separa-
tion of the phases, the organic layer was washed with a saturated
aqueous solution of NaHCO3 and then brine. The organic layer was
dried over Na2SO4, filtered and concentrated under vacuum. Purifi-
cation of the residue by silica gel chromatography (dichlorometh-
ane/methanol, gradient 2 to 5%) afforded the desired compound
2(0.367 g, 85%) as a colourless oil.
calcd: C, 48.32; H, 5.36; N, 10.95; found: C, 48.77; H, 5.47; N, 10.78.
4.4.14. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-((2,2-
a
dimethyl-N -tert-butyloxycarbonyl) glycinyl)-2-thioethyl]-O00-
phenyl phosphate, 21
Standard procedure 3 from 2.62 g of 17 yielded 0.923 g (69%) of
21 obtained as white foam after silica gel column chromatography
(dichloromethane/methanol, gradient 0–1.5%).
Rf 0.39 (CH2Cl2/CH3OH, 95:5, v/v). 1H NMR (DMSO-d6) d 11.4
(bs, 1H, Thy-NH), 7.6 (bs, 1H, NH), 7.46, 7.44 (s and d, 1H,
J = 0.8 Hz, H-6), 7.38 (m, 2H, Ph), 7.22 (m, 3H, Ph), 6.12 (t, 1H,
J = 6.6 Hz, H-10), 4.45 (m, 1H, H-30), 4.34 (m, 2H, H-50, H-50), 4.11
(m, 2H, CH2CH2O), 4.00 (m, 1H, H-40), 3.08 (m, 2H, SCH2CH2),
2.36 (m, 2H, H-20, H-200), 1.71 (s, 3H, Thy-CH3), 1.36 (s, 9H, tBu),
1.29 (2s, 6H, C(CH3)2). 13C NMR (DMSO-d6) d 203.7 (COS), 163.6
(C-4), 154.3 (COO), 150.3 (C-2), 150.0 (d, JPꢀC = 6.7 Hz, Ph ipso),
135.8 (C-6), 130.0, 129.9, 125.4 (3s, Ph), 119.9 (d, JPꢀC = 4.6 Hz,
Ph ortho), 109.9 (C-5), 83.7 (C-10), 81.1 (d, JPꢀC = 7.8 Hz, C-40),
78.5 (C(CH3)3), 67.3 (d, JPꢀC = 5.5 Hz, C-50), 66.5 (d, JPꢀC = 5.8 Hz,
CH2CH2O,), 61.4 (C(CH3)2), 59.9, 59.8 (2s, C-30), 35.6 (C-20), 28.2
(C(CH3)3), 27.9 (d, JPꢀC = 6.5 Hz, SCH2CH2), 24.9 (C(CH3)2), 12.1,
12.0 (2s, Thy-CH3). 31P NMR (DMSO-d6) d ꢀ5.38, ꢀ5.56. MS
(FAB > 0, GT) m/z 669 [M+H]+, 643 [MꢀN2+3H]+, 569
[MꢀtBoc+2H]+, 543 [MꢀtBoc-N2+4H]+, 424 [MꢀtBocAibSATE+2H]+,
398 [MꢀtBocValSATE-N2+4H]+, 127 [BH2]+, (FAB < 0, GT) m/z 1335
[2MꢀH]ꢀ, 1259 [2MꢀPh]ꢀ, 1086 [2MꢀAZT]ꢀ, 667 [MꢀH]ꢀ, 591
[MꢀPh]ꢀ, 422 [MꢀtBocAibSATE]ꢀ, 418 [MꢀAZT]ꢀ, 218 [tBocAib-
SATE-CH2CH2]ꢀ, 125 [B]ꢀ. HR-MS (FAB > 0, GT) m/z calcd.
Rf 0.41 (CH2Cl2/CH3OH, 9:1, v/v). 1H NMR (DMSO-d6) d 11.3 (bs,
1H, Thy-NH), 7.46, 7.44 (2d, 1H, J = 0.9 Hz, H-6), 7.38 (m, 2H, Ph),
7.22 (m, 3H, Ph), 6.13, 6.12 (2t, 1H, J = 6.7 Hz, H-10), 4.91 (t, 1H,
J = 5.2 Hz, OH), 4.46 (m, 1H, H-30), 4.34 (m, 2H, H-50, H-500), 4.16
(m, 2H, CH2CH2O) 4.02 (m, 1H, H-40), 3.42 (m, 2H, CH2OH), 3.12,
3.11 (2t, 2H, J = 6.4 Hz, SCH2CH2), 2.37 (m, 2H, H-20, H-200), 1.72
(1s, 3H, Thy-CH3), 1.10, 1.09 (2s, 6H, C(CH3)2). 13C NMR (DMSO-
d6) d 203.8 (CO), 163.6 (C-4), 150.3 (C-2), 150.0 (d, JPꢀC = 6.7 Hz,
Ph ipso), 135.8 (C-6), 129.9, 129.8, 125.4 (3s, Ph), 119.9 (d,
J = 4.6 Hz, Ph ortho), 109.9 (C-5), 83.7 (C-10), 81.1 (d, JPꢀC=7.7 Hz,
C-40), 68.3 (CH2OH), 67.3 (d, JPꢀC = 5.7 Hz, C-50), 66.5 (d,
JPꢀC = 5.7 Hz, CH2CH2O,), 59.8, 59.7 (2s, C-30), 51.7 (C(CH3)2), 35.5
(C-20), 28.0 (d, JPꢀC = 7.6 Hz, SCH2CH2), 21.8 (C(CH3)2), 12.1, 12.0
(2s, Thy-CH3). 31P NMR (DMSO-d6) d ꢀ5.41, ꢀ5.53. MS (FAB > 0,
GT) m/z 584 [M+H]+, 558 [MꢀN2+3H]+, 424 [MꢀSATE+2H]+, 127
[BH2]+, (FAB < 0, GT) m/z 1748 [3MꢀH]ꢀ, 1165 [2MꢀH]ꢀ, 1005
[2MꢀSATE]ꢀ, 916 [2MꢀAZT]ꢀ, 582 [MꢀH]ꢀ, 506 [MꢀPh]ꢀ, 422
[MꢀSATE]ꢀ, 333 [MꢀAZT]ꢀ, 125 [B]ꢀ. UV (Ethanol 95) kmax
669.2108 [M+H]+, found 669.2130 [M+H]+. UV (Ethanol 95) kmax
263 nm (9900), kmin )=233 nm (5300). Anal. for (C27H37N6O10PS):
calcd: C, 48.50; H, 5.58; N, 12.57; found: C, 48.20; H, 5.25; N, 12.36.
(e)=
(e
) = 263 nm (10200), kmin
(e) = 240 nm (6500). Anal for
(e
(C23H30N5O9PS): calcd: C, 47.34; H, 5.18; N, 12.00; found: C,
47.63; H, 5.34; N, 11.81.

7328
A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
4.4.17. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(2,2-
hydroxymethyl)propionyl)-2-thioethyl]-O00-phenyl phosphate, 3
A stirred solution of derivative 20 (0.911 g, 1.42 mmol) in 50%
aqueous acetic acid (16 mL) was maintained at room temperature
for 4 h, then the reaction mixture was concentrated under vacuum
and co-evaporated with toluene. Purification of the residue by silica
gel chromatography (dichloromethane/methanol, gradient 2–4%)
afforded the desired compound 3 (0.652 g, 76%) as a colourless oil.
Rf 0.29 (CH2Cl2/CH3OH, 93:7, v/v). 1H NMR (DMSO-d6) d 11.3
(bs, 1H, Thy-NH), 7.46, 7.44 (2s, 1H, H-6), 7.38 (m, 2H, Ph), 7.22
(m, 3H, Ph), 6.13 (m, 1H, H-10), 4.76 (t, 2H, JOH, H = 5.4 Hz, OH),
4.46 (m, 1H, H-30), 4.34 (m, 2H, H-50, H-500), 4.16 (m, 2H, CH2CH2O),
4.02 (m, 1H, H-40), 3.54 (m, 2H, CH2OH), 3.43 (pdd, 2H, Jvici-
nal = 10.7 Hz, JH,OH = 5.4 Hz, CH2OH), 3.12 (m, 2H, SCH2CH2), 2.37
(m, 2H, H-20, H-200), 1.72 (1s, 3H, Thy-CH3), 1.12, 1.11 (2s, 3H,
CH3). 13C NMR (DMSO-d6) d 202.5 (CO), 163.6 (C-4), 150.3 (C-2),
150.0 (d, JPꢀC = 6.8 Hz, Ph ipso), 135.8 (C-6), 129.9, 129.8, 125.4
(3s, Ph), 120.0 (d, JPꢀC = 4.6 Hz, Ph ortho), 109.9 (C-5), 83.7 (C-10),
81.1 (d, JPꢀC = 7.8 Hz, C-40), 67.2 (d, JPꢀ C = 4.6 Hz, C-50), 66.5 (d,
JPꢀC = 5.8 Hz, CH2CH2O,), 64.3 (CH2OH), 59.8, 59.7 (2s, C-30), 57.5
(C(CH3)), 35.5 (C-20), 27.8 (d, JPꢀC = 7.4 Hz, SCH2CH2), 16.8 (CH3),
12.0 (Thy-CH3). 31P NMR (DMSO-d6) d ꢀ5.40, ꢀ5.51. MS (FAB > 0,
GT) m/z 600 [M+H]+, 574 [MꢀN2+3H]+, 424 [MꢀSATE+2H]+, 127
[BH2]+, (FAB < 0, GT) m/z 1796 [3MꢀH]ꢀ, 1197 [2MꢀH]ꢀ, 948
[2MꢀAZT]ꢀ, 598 [MꢀH]ꢀ, 522 [MꢀPh]ꢀ, 422 [MꢀSATE]ꢀ, 349
of TFA (2 mL, 50% in dichloromethane) drop wise at 0 °C. The reac-
tion reached completion in 2 h and the reaction mixture was con-
centrated and co-evaporated with toluene. The residue was dried
under high vacuum and afforded the expected compound
5(0.207 g, 99%) as a white foam.
Rf 0.39 (CH2Cl2/CH3OH, 9:1, v/v). 1H NMR (DMSO-d6) d 11.4 (bs,
1H, Thy-NH), 8.3 (bs, 3H, NHþ3 ), 7.48, 7.45 (2s, 1H, H-6), 7.39 (m,
2H, Ph), 7.23 (m, 3H, Ph), 6.13 (1t, 1H, J = 6.6 Hz, H-10), 4.46 (m, 1H,
H-30), 4.36 (m, 2H, H-50, H-500), 4.24 (m, 3H, CH2CH2O, CHNHþ3 ),
4.00 (m, 1H, H-40), 3.34 (m, 2H, SCH2CH2), 2.37 (m, 2H, H-20, H-200),
2.16 (m, 1H, CH(CH3)2), 1.72 (s, 3H, Thy-CH3), 0.97, 0.96, 0.93, 0.91
(4s, 6H, CH(CH3)2). 13C NMR (DMSO-d6) d 195.9 (COS), 163.6 (C-4),
150.3 (C-2), 149.9 (d, JPꢀC = 6.8 Hz, Ph ipso), 135.9 (C-6), 130.0,
129.9, 125.5 (3s, Ph), 120.0 (m, Ph ortho), 109.9 (C-5), 83.8 (C-10),
81.1 (d, JPꢀC = 7.7 Hz, C-40), 67.4 (C-50), 66.0 (d, JPꢀ C = 5.5 Hz,
CH2CH2O), 63.4 ðCHNHþ3 Þ, 59.8 (C-30), 35.5 (C-20), 30.1 (CH(CH3)2),
28.8 (d, JPꢀC = 7.5 Hz, SCH2CH2), 18.0, 17.2 (CH(CH3)2), 12.1 (Thy-
CH3).31P NMR (DMSO-d6) d ꢀ5.41, ꢀ5.46. MS (FAB > 0, GT)m/z
1165 [2Mꢀ2CF3COO-H]+, 583 [MꢀCF3COO]+, 557 [MꢀCF3COO–
N2+2H]+, 424 [MꢀCF3COO–SATE+2H]+, 334 [MꢀCF3COO–AZT+H]+,
(FAB < 0, GT) m/z 1163 [2Mꢀ2CF3COOꢀ3H]ꢀ, 695 [MꢀH]ꢀ, 581
[MꢀCF3COOꢀ2H]ꢀ, 505 [MꢀCF3COO–Ph–H]ꢀ, 422 [MꢀCF3COO–
SATE]ꢀ, 332 [MꢀCF3COO-AZT-H]ꢀ, 125 [B]ꢀ. HR-MS (FAB > 0, GT)
m/z calcd. 583.1740 [MꢀCF3COO]+, found 583.1729 [MꢀCF3COO]+.
UV (Ethanol 95) kmax (e) = 263 nm (8600), kmin (e)=232 nm (4600).
[MꢀAZT]ꢀ, 125 [B]ꢀ. UV (Ethanol 95) kmax
(e) = 263 nm (8600), kmin
(e
) = 240 nm (5600). Anal for (C23H30N5O10PS): calcd: C, 46.08; H,
4.4.20. 2,2-Dimethyl-O-3-(triphenylmethyl)-propanoic acid, 10
A suspension of 2,2-dimethyl-3-O-(triphenylmethyl)-propanoic
acid methyl ester 14 (7.0 g, 16.6 mmol) in an aqueous solution of
NaOH (147 mL, 30%) and dioxan (147 mL) was heated under reflux
for 14 h. The reaction mixture was then cooled to room temperature.
Thedioxanand the aqueouslayerswere separated. The dioxanphase
5.04; N, 11.68; found: C, 46.11; H, 5.20; N, 11.36.
4.4.18. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(2,2-
dimethylglycinyl)-2-thioethyl]-O00-phenyl phosphate
(trifluoroacetate salt), 4
To
a
stirred solution of protected derivative 21 (0.20 g,
was acidified using an aqueous solution of HCl 1N until pH 3. The or-
0.30 mmol) in dry dichloromethane (2 mL) was added a solution
of TFA (2 mL, 50% in dichloromethane) drop wise at 0 °C. The reac-
tion reached completion in 2 h and the reaction mixture was con-
centrated and co-evaporated with toluene. The residue was dried
under high vacuum and afforded the expected compound 4
(0.20 g, 98%) as a white foam.
ganicphasewasextractedwithdichloromethane(20 mL), driedover
sodium sulfate, and filtered. Evaporation under reduced pressure of
all the volatiles gave yellow oil which solidified upon standing. The
crude solid was purified by crystallization (dichloromethane/petro-
leum ether, 95:5) to give 2,2-dimethyl-3-O-(triphenylmethyl)-
propanoic acid 10 (5.20 g, 87%) as colourless crystal.
Rf 0.42 (CH2Cl2/CH3OH, 9:1, v/v). 1H NMR (DMSO-d6) d 11.4 (br s,
1H, Thy-NH), 8.5 (bs, 3H, NHþ3 ), 7.48, 7.45 (2d, 1H, J = 0.7 Hz, H-6),
7.40 (m, 2H, Ph), 7.23 (m, 3H, Ph), 6.14, 6.13 (2t, 1H, J = 6.7 Hz, H-
10), 4.46 (m, 1H, H-30), 4.34 (m, 2H, H-50, H-500), 4.24 (m, 2H, CH2CH2O),
4.00 (m, 1H, H-40), 3.29 (m, 2H, SCH2CH2), 2.38 (m, 2H, H-20, H-200),
1.73 (s, 3H, Thy-CH3), 1.49, 1.48 (2s, 6H, C(CH3)2). 13C NMR (DMSO-
d6) d 199.1 (COS), 163.6 (C-4), 150.4 (C-2), 149.9 (d, JPꢀC = 6.6 Hz, Ph
ipso), 136.0 (C-6), 130.0, 129.9, 125.5 (3s, Ph), 120.0 (m, Ph ortho),
110.0, 109.9 (2s, C-5), 83.7 (C-10), 81.1 (d, JPꢀC = 7.7 Hz, C-40), 67.4
(d, JPꢀ C = 4.8 Hz, C-50), 65.9 (d, JPꢀC = 5.7 Hz, CH2CH2O), 61.7
(C(CH3)2), 59.7 (C-30), 35.5 (C-20), 28.8 (d, JPꢀ C = 7.4 Hz, SCH2CH2),
24.0 (C(CH3)2), 12.1, 12.0 (2s, Thy-CH3). 31P NMR (DMSO-d6) d
ꢀ5.39, ꢀ5.45. MS (FAB > 0, GT) m/z 1137 [2Mꢀ2CF3COO-H]+, 1111
Rf 0.5 (cyclohexane/AcOEt, 8:2, v/v). m.p.: 167 °C. 1H NMR
(CDCl3) d 7.41(m, 6H, Ph), 7.27 (m, 9H, Ph), 3.19 (s, 2H, OCH2),
1.24 (s, 6H, CH3). 13C NMR (CDCl3) d 181.9 (CO), 143.3 (Ph ipso),
128.7, 127.0, 127.8 (Ph), 86.5 ((Ph)3C), 69.4 (OCH2), 43.4
(C(CH3)2), 22.5 (C(CH3)2). IR (CCl4) mmax 1706 cmꢀ1 (C@O). MS
(FAB > 0, GT) m/z361 [M+H]+, (FAB < 0, GT) m/z710 [2MꢀH]ꢀ, 359
[MꢀH]ꢀ, 117 [MꢀTr]ꢀ. Anal. for (C24H24O3): calcd: C, 79.97; H,
6.71; found: C, 79.66; H, 6.80.
4.4.21. 5-Carboxy-2,2,5-trimethyl-1,3-dioxane, 11
To a solution of 2,2-bis(hydroxymethyl) propionic acid (20 g,
149.1 mmol) in anhydrous dichloromethane (150 mL) were added
2,2-dimethoxypropane (55.5 mL, 447.3 mmol) and APTS monohy-
drate (p-toluene sulfonic acid, 0.284 g, 1.49 mmol). The reaction
mixture was allowed to stir at room temperature under argon for
4:30 min then neutralized with triethylamine (10 mL). After
10 min, dichloromethane was added (500 mL) and the mixture
was washed once with water (100 mL). The aqueous phase was ex-
tracted three times with dichloromethane (300 mL). The organic
phases were combined, dried over Na2SO4, filtered and concen-
trated under reduced pressure. 5-Carboxy-2,2,5-trimethyl-1,3-
dioxane 11 was isolated as colourless crystals (16.1 g, 62%) after
crystallization in hexane/diethyl ether (9:1, v/v).
[(2M-2CF3COO-N2+H)+,
888
[2Mꢀ2CF3COO-AZT]+,
569
[MꢀCF3COO]+, 543 [MꢀCF3COO-N2+2H]+, 424 [MꢀCF3COO-SA-
TE+2H]+, 320 [MꢀCF3COO-AZT+H]+, 127 [BH2]+, (FAB < 0, GT) m/z
1135 [2Mꢀ2CF3COO-3H]ꢀ, 681 [MꢀH]ꢀ, 567 [MꢀCF3COO–2H]ꢀ,
491
[MꢀCF3COO–Ph–H]ꢀ,
422
[MꢀCF3COO–SATE]ꢀ,
318
[MꢀCF3COO–AZT–H]ꢀ, 125 [B]ꢀ. HR-MS (FAB > 0, GT) m/z calcd.
569.1583 [MꢀCF3COO]+, found 569.1588 [MꢀCF3COO]+. UV (Ethanol
95) kmax (e)=263 nm (9600), kmin (e) = 232 nm (3600).
4.4.19. O -(30-Azido-20,30-dideoxythymidin-50-yl)-O0-[S-(
L)-
valinyl-2- thioethyl]-O00-phenyl phosphate (trifluoroacetate
Rf 0.52 (dichloromethane/methanol, 9:1, v/v). m.p.: 120 °C. 1H
NMR (DMSO-d6) d 3.99 (d, 2H, J = 11.5 Hz, CH2), 3.54 (d, 2H,
J = 11.5 Hz, CH2), 1.33, 1.25 (2s, 6H, C(CH3)2), 1.06 (s, 3H, CH3).
13C NMR (DMSO-d6) d 175.5 (CO), 97.2 (C(CH3)2), 65.2 (CH2), 40.7
salt), 5
To
a stirred solution of protected derivative 22 (0.205 g,
0.30 mmol) in dry dichloromethane (2 mL) was added a solution

A.-L. Villard et al. / Bioorg. Med. Chem. 16 (2008) 7321–7329
7329
(C(CH3)2), 24.6, 22.7 (2s, C(CH3)2), 18.2 (CH3). IR (CCl4) mmax
References and notes
1707 cmꢀ1 (C@O). MS (FAB > 0, GT) m/z 175 [M+H]+, (FAB < 0, GT)
m/z 173 [MꢀH]ꢀ. Anal. for (C8H10O4), calcd: C, 55.16; H, 8.10;
found: C, 54.97; H, 8.13.
1. 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.
2. Périgaud, C.; Gosselin, G.; Imbach, J.-L. In Biomedical Chemistry. Applying
Chemical Principles to the Understanding and Treatment of Disease; Torrence, P. F.,
Ed.; John Wiley & Sons Inc.: New York, 2000; p 115.
3. Bazzanini, R.; Gouy, M. H.; Peyrottes, S.; Gosselin, G.; Périgaud, C.; Manfredini,
S. Nucl. Nucl. Nucleic Acids 2005, 24, 1635.
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A.-M.; Périgaud, C. J. Med. Chem. 2003, 46, 782.
8. Egron, D.; Imbach, J.-L.; Gosselin, G.; Aubertin, A.-M.; Périgaud, C. J. Med. Chem.
2003, 46, 4564.
9. Shafiee, M.; Deferme, S.; Villard, A.-L.; Egron, D.; Gosselin, G.; Imbach, J.-L.;
Lioux, T.; Pompon, A.; Varray, S.; Aubertin, A.-M.; Van Den Mooter, G.; Kinget,
R.; Périgaud, C.; Augustinjs, P. J. Pharm. Sci. 2001, 90, 448.
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Sadée, W.; Amidon, G. L. Pharm. Res. 1998, 15, 1154.
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Mathieu, S.; Moussa, A.; Bridges, E. G.; Strandring, D. N.; Sommadossi, J. P.;
Storer, R.; Gosselin, G. J. Med. Chem. 2006, 2, 6614.
13. Schlienger, N.; Beltran, T.; Périgaud, C.; Lefebvre, I.; Pompon, A.; Aubertin,
A.-M.; Gosselin, G.; Imbach, J. L. Bioorg. Med. Chem. Lett. 1998, 8, 3003.
14. Morton, R. C.; Mangroo, D.; Gerber, G. E. Can. J. Chem. 1987, 66, 1701.
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4.4.22. 2,2-Dimethyl-O-3-(triphenylmethyl)-propionic acid
methyl ester, 14
To
a solution of methyl-2,2-dimethyl-3-hydroxypropionate
(1 mL, 7.84 mmol) in dry dichloromethane (63 mL) were added tri-
ethylamine (1.54 mL, 10.97 mmol), triphenylmethane chloride
(2.62 g, 9.4 mmol) and DMAP (0.096 g, 0.74 mmol). The reaction
mixture was heated under reflux overnight then cooled to room
temperature and diluted with dichloromethane (150 mL). The or-
ganic phase was washed twice with an aqueous solution of NaH-
CO3 (10%, 100 mL) and once with water (100 mL). The organic
phase was dried over sodium sulfate, filtered and evaporated under
reduced pressure. A yellow-orange oil was obtained which was
purified by column chromatography on silica gel (n-hexane/dichlo-
romethane, 6:4, v/v) to give 2,2-dimethyl-3-trityloxy-propionic
acid methyl ester 14 (2.75 g, 94%) as a colourless oil.
Rf 0.25 (n-hexane/dichloromethane, 6:4, v/v). 1H NMR (DMSO-
d6) d 7.3 (m, 15H, Ph), 3.61 (s, 3H, OCH3), 3.02 (s, 2H, OCH2), 1.10
(s, 6H, CH3). 13C NMR (DMSO-d6) d 175.9 (CO), 143.3 (Ph ipso),
127.9, 127.2 (2s, Ph ortho and meta), 126.3 (Ph para), 85.5
((Ph)3C), 69.4 (OCH2), 54.8 (OCH3), 42.8 (C(CH3)2), 18.7 (C(CH3)2).
MS (FAB > 0, GT) m/z 277 [MꢀTr–OMe]+, 243 [Tr]+, (FAB < 0, GT)
m/z 275 [MꢀTr–OMe]ꢀ.
Acknowledgments
19. Bundgaard, H. Drugs Fut. 1991, 16, 443.
20. Gosselin, G.; Périgaud, C.; Lefebvre, I.; Pompon, A.; Aubertin, A.-M.; Kirn, A.;
Szabo, T.; Stawinski, J.; Imbach, J.-L. Antiviral Res. 1993, 22, 143.
We thank ‘‘Agence Nationale de Recherches sur le SIDA” (ANRS,
France) for funding. A.-L.V. is also grateful to ANRS for a PhD grant.