Synthesis and in vitro evaluation of S-acyl-3-thiopropyl prodrugs of Foscarnet

Article abstract of DOI:10.1016/j.bmc.2004.01.017

A new enzyme-labile group called S-acyl-3-thiopropyl group (SATP) has been synthesized from allylic esters of phosphonate. After demonstration of the enzyme-labile character of the SATP in cellular extracts, it has been introduced onto the phosphonate moiety of PFA (Foscarnet) to obtain potential lipophilic prodrugs. To ponder the lipophilicity of the triesters of PFA, esters of monomethylether of polyethyleneglycols and of thioglycerol were introduced on the PFA carboxylate moiety. The SATP groups were introduced in an attempt to deliver PFA after bioactivation inside the cells. The PFA prodrugs were evaluated in vitro for their activity against human immunodeficiency viruses (HIV-1 and HIV-2).

Full text of DOI:10.1016/j.bmc.2004.01.017

Bioorganic & Medicinal Chemistry 12 (2004) 1393–1402
Synthesis and in vitro evaluation of S-acyl-3-thiopropyl prodrugs
of Foscarnet
a
a,
a
a
Vale
´
Isabelle Lefe
`
rie Gagnard, Alain Leydet, * Alain More
`
b
re, Jean-Louis Montero,
c
b
c
bvre, Gilles Gosselin, Christophe Pannecouque and Erick De Clercq
aLaboratoire de Chimie Biomole´culaire, UMR 5032, Universite´ Montpellier II,ENSCM, 8 rue de l’Ecole Normale,
4296, Montpellier Cedex 5, France
Laboratoire de Chimie Biomole´culaire de Synthe`se, UMR 5625, Universite´ Montpellier II, Place Euge`ne bataillon,
4 095, Montpellier Cedex 5, France
3
b
3
c
Rega Institute for Medical Research, Katholieke Universiteit Leuven, 10, Minderbroedersstraat, B-3000 Leuven, Belgium
Received 7 March 2003; accepted 13 January 2004
Abstract—A new enzyme-labile group called S-acyl-3-thiopropyl group (SATP) has been synthesized from allylic esters of phos-
phonate. After demonstration of the enzyme-labile character of the SATPin cellular extracts, it has been introduced onto the
phosphonate moiety of PFA (Foscarnet) to obtain potential lipophilic prodrugs. To ponder the lipophilicity of the triesters of PFA,
esters of monomethylether of polyethyleneglycols and of thioglycerol were introduced on the PFA carboxylate moiety. The SATP
groups were introduced in an attempt to deliver PFA after bioactivation inside the cells. The PFA prodrugs were evaluated in vitro
for their activity against human immunodeficiency viruses (HIV-1 and HIV-2).
#
2004 Elsevier Ltd. All rights reserved.
1. Introduction
associated with oral bioavailability and cellular perme-
4
ability of PFA.
The AIDS epidemic has resulted in continuous and
intensive efforts to find effective chemotherapeutic
agents against the human immunodeficiency virus
The prodrug approaches consist in masking the anionic
charges temporarily by a lipophilic ester protective group
to enhance cellular permeation. This protective group will
be selectively cleaved inside the cell.
(
HIV), the causative agent of the disease. Reverse tran-
scriptase and protease are the targets of choice in the
1
,2
replicative cycle of HIV.
For instance, S-acylthioalkyl phosphonic esters can be
hydrolyzed enzymatically by carboxyesterases which
may be more prevalent intracellularly. S-acyl-2-thio-
Phosphonoformate (PFA, foscarnet) is an effective
antiviral agent that has been approved for clinical use in
the treatment of cytomegalovirus retinitis in AIDS
5ꢀ8
9
ethyl (SATE)
and S-acyl-4-thiobutyl (SATB) have
3
patients under the name of Foscavir. PFA inhibits the
been utilized in a prodrug approach to deliver intra-
cellularly the monophosphates of azidothymidine
DNA polymerases of herpes viruses, as well as the
reverse transcriptase of HIV by blocking the pyrophos-
phate site in these enzymes. The efficacy of PFA is
hampered by poor membrane permeability due to its
polyanionic structure at physiological pH. Prodrug
approaches can be used to overcome many problems
0
and other nucleotides with antiviral activities.
0
(AZT), 2 ,3 -dideoxyuridine (d4T) and stavudine (ddU),
8
The SATE and SATB behavior was very different; the
enzymatic decomposition in cell extracts of the SATE
5
group was instantaneous, whereas the SATB group
9
decomposition was not observed.
Keywords: Foscarnet; Prodrugs; S-acyl-3-thiopropyl; HIV-1 and HIV-
It appeared interesting to us to synthesize a new poten-
tially cleavable group by esterase with half-live inter-
mediate between SATE and SATB groups. The S-acyl-
2
*
; Calculated logP.
Corresponding author. Tel.: +33-4-6714-4346; fax: +33-4-6714-
4344; e-mail: leydet@univ-montp2.fr
0
968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.017

1394
V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
0
Figure 3. Reaction scheme of Arbusov s reaction between an alkyl
chloroformate and a trialkyl phosphate.
The interpretation of the results permitted to show the
enzyme-labile character of the SATPgroup. As expec-
ted, the SATPgroup had a half-life ( T1/2) of 41 min
which is intermediate between those of the ethyl (SATE)
and the butyl (SATB) derivatives.
Figure 1. Reaction scheme for model compound.
3-thiopropyl protective group (SATP) could be a good
candidate to fulfill the proposed criterion.
Moreover, new prodrugs of PALA bearing S-acyl-3-
thio-propyl groups (SATP) have shown cytotoxic activ-
ities against human cells lines (SW 1573 lung carcinoma
cells). A number of prodrugs displayed a cytotoxic
activity in the same order of magnitude of PALA. This
biological activities of prodrugs of PALA can be explain
Before applying the SATPgroups to PF A, we had to
verify the enzyme-labile character of SATPgroup on
simple structure model.
1
3
In a preliminary work we synthesized a hydroxy-
resorcylmethyl phosphonate 2 as model for the meta-
bolism studies. The presence of three hydroxyl functions
confers the necessary hydrophilicity for water solubili-
sation [The calculated log Pis 2.8 (software ACD logP-
Chem CAD)]. Compound 2 was obtained by a Pudovik
reaction between the 2,4-resorcylaldehyde and the dia-
by the enzyme-labile character of the SATPgroup.
2. Synthesis
The prodrug strategy was applied to PFA after the cor-
roboration of the enzyme-labile character of the SATP
group. Generally the synthesis of the foscarnet deriv-
atives is achieved according to an Arbusov’s reaction
1
0,11
llyl phosphonate with basic alumina
under sonica-
tion conditions, followed by addition of thiopivaloic
acid on the double bond of allyl groups under UV
irradiation (l=254 nm) (Fig. 1).
1
4
between an alkyl chloroformate and a trialkyl phosphite
this necessitates temperature conditions incompatible
with the thermal stability of allylic esters (Fig. 3).
The decomposition of the model compound 3, achieved
in cellular extracts, mimics the bioconversion of the
model in the intracellular compartment. The kinetics
We opted for a milder method15 that takes place at
room temperature, and in which was used the diallyl-
phosphonate 1 (Fig. 4). The diallyl phosphonate 1, not
ꢁ
was followed over 5 days to 37 C, with a substrate
ꢀ
5
16
concentration of 5.10 M in the cellular extracts in the
presence of 2.5% of DMSO. The analysis was per-
formed by ‘on-line cleaning HPLC-UV-MS’.1 The
decomposition process of compound 3 is indicated in
Figure 2.
commercially available, was prepared
from phos-
phorous trichloride and allylic alcohol in the presence of
two equivalents of pyridine. After distillation under
reduced pressure, the diallyl phosphonate, was obtained
in 75% yield, and kept under argon to avoid its
hydrolysis.
2
0
0
The decomposition of the monoester 3 continues with-
0
0
Compound 1 was characterized by 31PNMR (decou-
out metabolites detection. Indeed the metabolites of 3
are very hydrophilic and they are eliminated during the
cleaning on line’ process.
1
pled proton), by a signal at 8.9 ppm. In H NMR the
proton directly bound to the phosphorus appeared at
‘
Figure 2. Decomposition process of model compound 3.

V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
1395
Figure 4. Synthesis of tris (S-pivaloyl-3-thiopropyl)phosphonoformate 5.
6
.88 ppm, as a doublet with a coupling constant of
JHP=703.1 Hz.
achieve a reaction with diallyl phosphonate as it is
described in Figure 4. The chloroformates were pre-
pared in high yields by reaction of polyethyleneglycol
monomethylether on phosgene in toluene (Fig. 5).
1
The synthesis of the triester 4 was then achieved in a
‘
one pot’ procedure (Fig. 4).
It is of note that the use of triphosgene, a safe derivative
of phosgene, did not lead to the chloroformate deriv-
ative but to the chlorinated product via a decarboxyl-
ation step. This has been previously observed in the
The diallyl phosphonate 1 was treated by BSA (N,O-
bis(trimethylsilyl)acetamide), giving the diallyltrimethyl-
silylphosphite intermediate containing a nucleophilic
phosphorus atom. This phosphite was allowed to react
with allyl chloroformate leading to triallyl phosphono-
formate 4 obtained in 98% yield after neutral alumina
column chromatography to avoid the partial cleavage of
the allylic esters.
1
7
Literature with activated alcohol.
Then several attempts to obtain phosphonoformates of
polyethyleneglycol were made. The first strategy con-
cerned the coupling reaction between the diallyl phos-
phonate 1 and the chloroformiate 7 in the presence of
BSA (Fig. 6) following the same procedure described in
Figure 3. By this way the phosphonoformate 9 was
obtained in 67% yield. The next step was the addition of
the thioacid onto the allylic functions. However, this
The triallyl phosphonoformate 4 was allowed to react
with thiopivaloic acid in THF, the radical addition
being initiated with catalytic amounts of AIBN, under
UV irradiation (l=254 nm). This step led to the tris-
(
SATP) phosphonoformate in 64% yield. The structure
addition of the thiopivaloıc acid on the diallylphos-
¨
phonoformate of polyethyleneglycol, did not lead to the
expected product, but to the degradation of the reagents
1
of the phosphonoformates 4 and 5 was proved by H,
1
3
31
C and PNMR spectroscopy and mass spectrometry.
(
Fig. 6).
Unfortunately the enzyme-labile triester 5 was char-
acterized by a weak aqueous solubility, a consequence
of the high log Pvalue (log Pcalc>5). It is worth point-
ing out that the phosphonoformates must be sufficiently
lipophilic to cross the biological membranes but also to
dissolve in physiological liquids. The values of log P
generally admitted to give good bioavailibility should be
within the 2–3.5 range.
In fact, the bis enzyme-labile phosphonates 12, 13 were
prepared directly from the phosphorous trichloride by
addition of the corresponding alcohol 10, 11 in presence
of pyridine in an analogous manner to the diallyl phos-
Table 1. Bis (RSATP) polyethyleneglycol monomethylether phos-
phonoformates
Thus, we prepared PFA derivatives (suiting with calcu-
lated logPincluded within the limit values given above)
by the introduction of hydrophilic groups derived from
polyethylene-glycol monomethylether and glycerol. The
S-acyl groups of SATPwere pivaloyl or acetyl groups
Compd
R acyl
n
Yield%
logPcalcÆ0.6
d
31P
14
15
16
tBu
Me
1
1
2
2
3
3
54
75
66
76
62
75
3.7
1.3
3.4
0.9
3.0
0.6
ꢀ3.5
ꢀ3.5
ꢀ3.5
ꢀ3.5
ꢀ3.5
ꢀ3.5
t
Bu
Me
17
t
1
1
8
9
Bu
Me
(Table 1).
In these compounds the SATPenzyme-labile groups
were introduced on the phosphonic acid moiety and the
hydrophilic groups on the carboxylic acid of PFA.
The introduction of polyethyleneglycol monomethyl-
ether as carboxylic esters requires the synthesis of the
chloroformates of the monomethylether in order to
Figure 5. Synthesis of polyethyleneglycol monomethylether chloro-
formates.

1
396
V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
t
phonate 1, with 91% yield for R= Bu and 81% yield
for R=Me (Fig. 7).
polyethyleneglycol 6, 7 and 8 (Fig. 8) with yields ran-
ging from 54% to 76% after chromatography, depend-
ing on the nature of the acyl group R and the length of
the polyethyleneglycol unit (Table 1).
The structures of the phosphonates 12 and 13 were
3
1
proved by PNMR spectrometry with the presence of
the signal at 8.4 ppm consistent with a H-phosphonate
We also considered the use of the thioglycerol and its
incorporation on the carboxylate moiety of the PFA.
The starting materials, for the preparation of the thio-
glycerol esters, were the bis SATPphosphonates 12 and
13. In the first step of the synthesis were prepared the
allylic esters 20 and 21 by addition of the allyloxy-
carbonyl chloride onto the bis SATPphosphonate 12
1
and by H NMR spectrometry by a doublet at 6.8 ppm
1
corresponding in the H-Pproton ( JHP=700 Hz), as
well as by the presence of the characteristic signals of
the RSATPgroups. hP osphonoformates were then
obtained by condensation of the bis SATPphosphonate
1
2, 13 on the chloroformates of monomethylether of
Figure 6. The direct syntheses of Bis SATPphosphonate fail.
Figure 7. Reaction scheme for the bis enzyme-labile phosphonates.
Figure 8. Reaction scheme for Bis (RSATP) polyethyleneglycol monomethylether phosphonoformates.
Figure 9. Synthesis of thioglycerol derivatives.

V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
1397
and 13, respectively. The addition of the thioglycerol
4. Experimental
onto the allylic esters 19 and 20 was then accomplished
under radical conditions, using AIBN as radical initia-
tor and UV irradiation (l=254 nm), leading to 22 in
4
.1. Chemistry
All chemicals were purchased from Aldrich and used as
received. Tetrahydrofuran (THF) was distilled over
lithium aluminum hydride under nitrogen immediately
prior to use. Diallyphosphite was not commercial and it
was prepared in large quantities following the Kennedy
3
7% yield and 23 in 40% yield, respectively (Fig. 9).
The structures of the phosphonoformates 14–23 were
1
13
31
proved by H, C and PNMR spectroscopy and
mass spectrometry.
1
6
procedure.
Merck silica gel 60 F254 (0.25 mm) plates were employed
for analytical TLC. Compounds were revealed by UV
3
. Biological results
(254 nm), 1% potassium permanganate in water (allylic
The phosphonoformate derivatives 5, 14–19, 22 and 23
bearing the enzyme-labile S-acyl-3-thiopropyl group
were evaluated for their inhibitory effects on the repli-
cation of human immunodeficiency virus (HIV) type 1
compounds), 5% phosphomolybdic acid in ethanol or
molybdene blue for phosphorous compounds. Merck
silica gel 60H, Aldrich alumina (activated neutral,
brockmann I, 150 mesh) and Fluka florisil were used for
column chromatography. Reactions under ultrasound
were performed on a Bransonic 220 ultrasonic cleaning
(
(
strain III ) and type 2 (strain ROD) in MT-4 cells
B
Table 2).
1
13
bath. H and C NMR were recorded on a Brucker
1
3
From the present work, it is obvious that none of the
synthesized phosphonate derivatives 5–23 surpassed
PFA in an in vitro antiviral activity. On the contrary,
they proved inactive against HIV at the highest con-
centration tested (125 mg/mL).
apparatus DPX200, AC250 and DRX400. PNMR
1
spectra were recorded on a Brucker DPX200. For H
1
3
and C we used a numbering system as presented in
Chart 1. Chemical shifts are expressed in ppm (d). Mass
spectra were recorded on a JEOL JMS-DX300. The
matrixes used were m-nitrobenzylic alcohol (NBA) or
glycerol-thioglycerol 1/1. Log Pwas estimated by means
of the software ACD/LogPcalculator developed by
ACD (Advanced Chemistry Development Inc.) and
distributed by the company ChemCAD.
It would seem that the prodrugs of the PFA does not
have an antiviral activity in relation with a pondered
lipophilicity and only the more lipophilic derivative 5
(logPcalc=5.6) would be capable to penetrate inside the
cells to exercise its activity.
Microanalyses were performed in the analytical depart-
ment of the CNRS (Vernaison, Rhoˆ ne, France). C, H
and S elemental analyses were done for most of the
compounds.
This inactivity could be explained by a lack of cellular
permeation and/or by a weak esterase activity of the
MT- 4 cells. The esterase activity may be different from
one cell type to another, and may be most pronounced
in cells of the monocyte/macrophage lineage. It may be
worth examining whether the SATP-PFA prodrugs
would show any anti-HIV activity in these cells.
4.1.1. Diallyl(2,4-dihydroxyphenyl)hydroxymethylphos-
phonate (2). 2,4-Dihydroxybenzaldehyde (2.8 g, 20
mmol, 1 equiv) was dissolved in 3 mL of diallylphos-
phite 1 (20 mmol, 1 equiv) under vigorous stirring. Then
the mixture was completely absorbed on basic alumina
(10 g) and put in an ultrasonic cleaning bath for 10
days. The products were extracted from alumina with
dichloromethane (3Â25 mL). The solvent was evapo-
rated under reduced pressure, the crude product was
purified by a florisil column chromatography (ethyl
acetate/petroleum ether 50/50), and 0.63 g of compound
Table 2. Anti-HIV Activity of phosphonoformate derivatives bearing
enzyme labile SATPgroups
Compd
Strain
IC50
CC50
SI
logPcalc
(
mg/mL)
(mg/mL)
5
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
III
ROD
B
>91
>83.9
76.85
76.85
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
—
<1
5.6
3.7
1.3
3.4
0.9
3.0
0.6
3.4
1.0
<0
<1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
> <1
>8
2
was obtained as colourless oil in 10% yield. R =0.25
f
14
15
16
17
18
19
22
23
B
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
>125
(ethyl acetate/petroleum ether 20/80).
B
B
B
B
B
B
B
Reference
Foscarnet
B
14.35
10.1
—
>12
Chart 1.

1
1
398
V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
H NMR CDCl /200 MHz: d (ppm): 4.48 (m, 4H,
Both first ones metabolites, the thiol (tr=16.58 min)
and the monoester (tr=13.51 min) quickly form, they
were later already observed 15 min of incubation, how-
ever the diester (tr=18.94 min) represents more than
80% of the detected species, it disappears totally only at
the end of 4 h. Mainly, the cleavage of the thioester by
enzymatic way was observed followed by the rearran-
gement that leads to the phosphonate monoester. The
hydrolysis by chemical way of the phosphonic ester is also
observed but it concerns only 40% of the compound.
3
POCH ); 5.31 (m, 4H, CH
2
0
); 5.90 (m, 2H, CH );
(3,3 ) (2)
3
2
5
Hz, 1H, H ); 7.28 (s, 1H, H ); 7.42 (d, JHH=8.5 Hz,
.10 (d, J =11.4 Hz, 1H, CHOH); 6.13 (d, JHH=8.5
HP
3
5
3
3
1
1
H, H ). PNMR CDCl /200 MHz: d (ppm): 24.4
6
3
4
.1.2. Bis(S-pivaloyl-3-thiopropyl)-(2,4-dihydroxyphenyl)-
hydroxymethylphosphonate (3). In a quartz reactor, a
solution of diallyl(2,4-dihydroxyphenyl) hydroxymethyl-
phosphonate 2 (0.63 g, 2.1 mmol, 1 equiv) and thio-
pivaloic acid (0.85 mL, 7.5 mmol, 3.6 equiv) in the
presence of a catalytic amount of AIBN (25 mg) in 18
mL of THF was degassed for 15 min by argon bubbling.
The mixture was submitted to UV irradiation (l=254
nm) for 45 min, under argon atmosphere at room
temperature. Then, the solution was diluted with 80 mL
of ethyl acetate and washed twice with a saturated
The experiment did not allow detecting the formation of
last metabolites because of their too high hydrophilicity
(log P<0) (Table 3).
4.2.1. Triallylphosphonoformate (4). To a solution of
diallylphosphonate 1 (2 mL, 12 mmol, 1 equiv) in 20 mL
of dichloromethane was added N,O-bis(trimethylsilyl)-
acetamide BSA (4.1 mL, 14 mmol, 1.2 equiv) under
nitrogen atmosphere at room temperature. After 3 h, a
solution of allylchloroformate (2.55 mL, 24 mmol, 2
equiv) in 20 mL of dichloromethane was added drop-
wise. Then the reaction was stirred overnight at room
temperature. The solvent was removed under reduced
pressure and the crude product was purified by silica gel
column chromatography (dichloromethane) to give 2.9
solution of NaHCO . The organic layer was dried with
3
sodium sulphate and concentrated under reduced
pressure. The residual oil was purified by silica gel
column chromatography (petroleum ether/ethyl acetate
5
colourless oil in 36% yield. R =0.37 (ethyl acetate/pet-
0/50), and 0.41 g of compound 3 was isolated as
f
1
roleum ether 20/80). H NMR CDCl /200 MHz: d
3
(
(
ppm): 1.20 (s, 18H, CH ); 1.89 (m, 4H, CH ); 2.91
m, 4H, CH S); 4.14 (td, J =7.1 Hz, J =7.2 Hz,
2 HH HP
3 2
3
3
2
4
Hz, 1H, CHOH); 6.38 (d, J =7.7 Hz, 1H, H ); 6.48
(s, 1H, H ); 7.03 (d, J =7.7 Hz, 1H, H ); 8.77 (s,
HH
OH).
H, POCH ); 4.44 (s, 1H, CHOH); 5.10 (d, JHP=11.1
3
g of compound 4 as colourless oil in 98% yield.
1
2
R =0.48 (diethyl ether/petroleum ether 2/1). H NMR
f
HH
5
3
CDCl /200 MHz: d (ppm): 4.67 (m, 6H, POCH and
3
6
3
2
3
CO CH ); 5.23 (d, J =9.9 Hz, 2H, CH (3p)); 5.25 (d,
3
2
2
HH
3
JHH=9.3 Hz, 1H, CH(3c)); 5.32 (dd, JHH=17.1 Hz,
3
1
3
2
C NMR CDCl /100 MHz: d (ppm): 25.0 (s, CH S);
JHH=1.1 Hz, 1H, CH
(3 c)
2JHH=1.1 Hz, 2H, CH
0
); 5.34 (dd, J =17.1 Hz,
HH
); 5.89 (m, 3H, CH ).
(2)
3
2
3
13
2
7.8 (s, CH ); 30.8 (2d, J =5.7 Hz, CH ); 46.9 (s,
2
0
C
3
PC
2
(3 p)
3
C
POCH ); 69.9 (d, J =104.0 Hz, CHOH); 105.3 (s,
C ); 108.5 (s, C ); 113.4 (s, C ); 130.2 (d, JCP=7.4 Hz,
3
C ); 156.7 (d, J =4.6 Hz, C ); 158.4 (s, C ); 208.0 and
6
2
t
); 66.2 and 66.7 (2d, JPC=7.3 and 7.1 Hz,
1
NMR CDCl /100 MHz: d (ppm): 66.9 (d, JPC=5.2 Hz,
ð BuÞ
3
2
CO CH ); 68.9 and 69.0 (2d, JPC=6.4 Hz, POCH2);
119.5 (s, CH2 (3p)); 120.4 (s, CH2(3c)); 131.0 (s, CH(2c));
2
PC
2
2
3
5
1
3
3
1
132.3 (d, JPC=6.0 Hz, CH(2p)); 166.6 (d, JPC=269.5
Hz, CO2).
CP
2
4
08.1 (2s, CO).
3
1
31
PNMR CDCl /200 MHz: d (ppm): 23.9
PNMR CDCl /200 MHz: d (ppm): ꢀ3.4. MS (Fab
3
3
+
;
+;
+/NBA): m/z=493 [2 M+H]
[CH =CH-CH ] .
2 2
247 [M+H]
41
+
t
+
+
MS (Fab +/GT): m/z=535 [M+H] ; 57 [ Bu ] .
4.2.2. Tris(S-pivaloyl-3-thiopropyl)phosphonoformate (5).
In a quartz reactor, a solution of triallylphosphono-
formate 4 (1.03 g, 4.2 mmol, 1 equiv), and thiopivaloic
4
.2. Decomposition and stability studies of model 3
Far-ultraviolet (UV) HPLC-grade acetonitrile was
purchased from Fisons (Loughborough, UK). Triethyl
ammonium acetate buffer was purchased from Dis-
tribio. The purified water was obtained from a Millipore
purifier (Bedford, Massachusetts, USA). The compound
Table 3. The half-lives of the various metabolites
Metabolites
logP(Æ0.6)
Hla
RTb
ꢀ
5
3
3
was incubated (initial concentration 5.10
ꢁ
M) at
7 C either in a full cell extract (pH=7.4). Incubates
were removed at appropriate times and injected as 80
mL samples in a system comprising the cleaning device,
the chromatograph and UV detector, and the mass
spectrometer.
2.8
1.5
27
18.94
16.58
173
The analysis by mass spectrometry of the chromato-
gram identified without ambiguity the various metabo-
lites which were formed in the cellular extract. Spectra
were recorded after 2 h of incubation, the retention
times observed reporting differences of polarity of the
metabolites.
0.5
13.51
a
Hl: Half-lives in min.
RT: retention times in min.
b

V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
1399
acid (2.5 mL, 22.7 mmol, 5.4 equiv) in the presence of a
catalytic amount of AIBN (50 mg) in 35 mL of THF
was degassed for 15 min by argon bubbling. The solu-
tion was submitted to UV irradiation at l=254 nm for
3JHH=10.4 Hz, 2JHH=1.1 Hz, 2H, CH (3)); 5.34
4
2
3
(qd, J = J =1.4 Hz, JHH=17.1 Hz, 2H, CH
HH
0
);
HH
(3 )
1
3
5.91 (m, 2H, CH (2)). C NMR CDCl /100 MHz: d
3
3
(ppm): 59.5 (s, CH O); 65.1 (d, J =5.0 Hz, CO CH );
2
3
PC
2
2
3
h, under inert atmosphere at room temperature. Then
66.8 (d, J =6.1 Hz, POCH ); 68.9 (s, CH O
2
0
); 71.1
); 119.1 (s, CH2 (3)); 132.5
PC
2
(2 )
the mixture was diluted with 60 mL of ethyl acetate and
washed twice with a saturated solution of NaHCO3.
The organic layer was dried with sodium sulphate and
concentrated under reduced pressure. The residual mix-
ture was purified by silica gel column chromatography
(s, CH O
2
0
); 72.3 (s, CH O
2
0
(4 )
(5 )
3
1
(d, J =6.0 Hz, CH (2)); 166.8 (d, JPC=269.9 Hz,
PC
3
1
CO2). PNMR CDCl /200 MHz: d (ppm): ꢀ4.2. MS
3
+
(Fab +/NBA): m/z=309 [M+H ] , 41 [CH =CH-
2
+
CH ] .
2
(
compound 5 as pale yellow oil in 64% yield. R =0.48
petroleum ether/ethyl acetate 80/20) to give 1.6 g of
4.2.5. S-pivaloyl-3-thiopropyl alcohol (10). In a quartz
reactor, a solution of allyl alcohol (1.4 mL, 37.8 mmol,
1.8 equiv), and thiopivaloic acid (4.2 mL, 37.8 mmol, 1.8
equiv) in the presence of a catalytic amount of AIBN
(50 mg) in 150 mL of THF was degassed for 15 min by
argon bubbling. The solution was submitted to UV
irradiation at l=254 nm for 3 h under inert atmosphere
at room temperature. Then the mixture was diluted with
200 mL of ethyl acetate, washed twice with a saturated
f
1
(
diethyl ether/petroleum ether 2/1). H NMR CDCl /
3
t
2
00 MHz: d (ppm): 1.19 (s, 27H, CH ); 1.96 (q ,
3
3
3
JHH=6.6 Hz, 6H, CH ); 2.88 (t, JHH=7.1 Hz, 2H,
3
2
CH2S (c)); 2.91 (t, J =7.1 Hz, 4H, CH S (p)); 4.27 (m,
HH
6
2
1
3
H, POCH and CO CH ). C NMR CDCl /100 MHz:
2 2 2 3
d (ppm) : 24.8 and 25.1 (2 s, CH S); 27.7 and 27.8 (2 s,
2
3
CH ); 29.0 (s, CH
t
); 30.8 (d, JPC=6.1 Hz, CH2 (p));
3
3
2 (c)
4
6.8 (s, C ); 64.8 (d, J =4.7 Hz, CO CH ); 67.3 (d,
PC 2 2
JPC=6.5 Hz, POCH ); 166.8 (d, JPC=270.2 Hz, CO2);
06.6 (2 s, COS). PNMR CDCl /200 MHz: d (ppm):
3.5. MS (Fab+/GT): m/z=601 [M+H] ; 57 [ Bu] .
Anal. calcd for C2 H O PS (600.79): C, 49.98; H,
ð BuÞ
2
1
solution of NaHCO , then twice with brine. The organic
3
2
3
1
2
layer was dried with sodium sulphate and concentrated
under reduced pressure. The residual oil was purified by
silica gel column chromatography (petroleum ether/
ethyl acetate 70/30) to give 2.9 g of compound 10 as
3
+
t
+
ꢀ
5
45
8
3
7
.55; S, 16.01; found: C, 49.79; H, 7.60; S, 15.88.
1
colourless oil in 80% yield. R =0.57. H NMR CDCl /
f
3
4
.2.3. 5-methoxy-3-oxapentylchloroformate (7). Diethyl-
200 MHz: d (ppm): 1.22 (s, 9H, CH ); 1.79 (tt,
3 3
3
eneglycol monomethylether (0.70 mL, 6.0 mmol, 1
equiv) was added drop wise under inert atmosphere to
4
equiv) cooled 10 min in an ice bath. The mixture was
stirred 4 h at room temperature and then the solvents
were removed under reduced pressure. The compound 7
was obtained as colourless oil in quantitative yield. H
NMR CDCl /400 MHz: d (ppm): 3.32 (s, 3H, CH O);
JHH=6.8 Hz, J =5.9 Hz, 2H, CH ); 2.68 (s, 1H,
HH 2
3
OH); 2.96 (t, JHH=6.8 Hz, 2H, CH S); 3.61 (t,
2
3
13
.0 mL of phosgene at 20% in toluene (8.1 mmol, 1.35
JHH=5.9 Hz, 2H, CH OH).
2
C NMR CDCl3/
100 MHz: d (ppm): 25.2 (s, CH S); 27.8 (s, CH ); 32.9 (s,
2
3
CH ); 46.9 (s, C
2
t
); 60.8 (s, CH OH); 208.9 (s, CO).
2
ð BuÞ
+
;
+;
MS (Fab +/NBA): m/z=199 [M+Na] 177 [M+H]
t
1
t
+;
+
85 [ BuCO] 57 [ Bu] .
3
3
3
.49 (m, 2H, CH O ); 3.59 (m, 2H, CH O ); 3.70 (m,
(4)
4.2.6. S-acyl-3-thiopropyl alcohol (11). A solution of
allyl alcohol (1.0 mL, 14.7 mmol, 1 equiv), thioacetic
acid (2.1 mL, 26.9 mmol, 2 equiv) and AIBN (2.41 g,
14.7 mmol, 1 equiv) in 20 mL of benzene was degassed
for 15 min by argon bubbling and then heated to reflux
for 4 h. The mixture was cooled to room temperature,
washed twice with a saturated solution of NaHCO3,
then twice with brine. The organic layer was dried with
sodium sulphate and concentrated under reduced pres-
sure. The crude product was purified by silica gel col-
umn chromatography (petroleum ether/ethyl acetate 70/
30), 1.1 g of compound 11 was obtained as colourless oil
2
(5)
2
1
3
2
H, CH O
2
); 4.40 (m, 2H, CO CH ). C NMR
2 2
(2)
CDCl /100 MHz: d (ppm): 59.5 (s, CH O); 68.7 (s,
CH O ); 71.1 (s, CH O
CH O ); 151.2 (s, CO ). The chloroformate of mono-
(5)
methylether of monoethyleneglycol (6) and of triethyl-
eneglycol (8) were obtained according to the
aforementioned procedure in quantitative yield and
used in the next step without purification.
3
3
and CO CH ); 72.3 (s,
2
(2)
2
(4)
2
2
2
2
4
.2.4. Diallyl-(5-methoxy-3-oxapentyloxycarbonyl)-phos-
phonate (9). N,O-bis(trimethylsilyl) acetamide BSA
0.88 mL, 3.6 mmol, 1.2 equiv) was added to a solution
(
in 56% yield. R =0.28 (ethyl acetate/petroleum ether
f
1
t
of diallylphosphonate 1 (3.0 mmol, 1 equiv) in 5 mL of
anhydrous dichloromethane, under nitrogen atmos-
phere at room temperature. After 3 h the chloroformate
of monomethylether of diethyleneglycol 7 (9.0 mmol, 3
equiv) in 5 mL of dichloromethane was added dropwise.
The reaction was stirred overnight at room temperature.
The solvent was removed under reduced pressure and
the crude product was purified by silica gel column
chromatography (ethyl acetate/petroleum ether 50/50).
50/50). H NMR CDCl /250 MHz: d (ppm): 1.75 (q ,
3
3
JHH=6.4 Hz, 2H, CH ); 2.28 (s, 3H, CH ); 2.38 (s, 1H,
3
2
3
OH); 2.93 (t, JHH=6.8 Hz, 2H, CH S); 3.57 (t,
2
3
13
JHH=5.7 Hz, 2H, CH OH). C NMR CDCl3/
2
100 MHz: d (ppm): 25.9 (s, CH S); 30.9 (s, CH ); 32.8 (s,
2
3
CH ); 60.8 (s, CH OH); 197.8 (s, CO).
2
2
+
MS (Fab +/NBA): m/z=117 [CH COSCH CH CH ] ;
2
43 [CH CO] .
3
3
2
2
+
A total amount of 0.615 g of compound 9 was isolated
1
as colourless oil. Yield 67%.R =0.51(ethyl acetate). H
f
4.2.7. Bis(S-pivaloyl-3-thiopropyl)phosphonate (12). A
mixture of S-pivaloyl-3-thiopropyl alcohol 10 (0.83 g,
4.7 mmol, 2 equiv) and pyridine (0.375 mL) was added
dropwise to a solution of phosphorus trichloride (0.2
NMR CDCl /400 MHz: (ppm): 3.30 (s, 3H, CH O);
3
3
3
.47 (m, 2H, CH O
2
0
); 3.57 (m, 2H, CH O ); 3.69
0
(5 )
2
(4 )
(
m, 2H, CH O
2
0
); 4.35 (m, 2H, CO CH ); 4.66 (q,
(2 ) 2 2
3
3
ꢁ
JHH= JHP=6.4 Hz, 4H, POCH2); 5.22 (dd,
mL, 2.33 mmol, 1 equiv) in 2 mL of cyclohexane at 0 C.

1400
V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
3
After 15 min at room temperature, S-pivaloyl-3-thio-
propyl alcohol (0.4g, 2.3 mmol, 1 equiv) in a minimum
of cyclohexane was added. The mixture was stirred
during 4 h 30 min, the formed precipitate was removed
by filtration and washed with cyclohexane. The filtrate
was concentrated, the residue was diluted in dichloro-
methane and washed twice with brine. The organic layer
was dried with sodium sulphate and the solvent was
removed under reduced pressure. The purification of the
crude product by silica gel column chromatography
CH ); 2.80 (t, J =7.1 Hz, 4H, CH S); 3.23 (s, 3H,
HH
2
2
CH O); 3.51 (m, 2H, CH O (2)); 4.15 (q,
3
JHH= J =6.5 Hz, 4H, POCH ); 4.26 (m, 2H,
HP
CO CH ). C NMR CDCl /100 MHz: d (ppm): 24.9 (s,
2
3
3
2
1
3
2
2
3
3
CH S); 27.8 (s, CH
t
t
); 30.8 (d, J =6.1 Hz, CH );
PC 2
3
2
3 ð BuÞ
46.9 (s, C
CO CH ); 67.4 (d,
); 59.3 (s, CH O); 65.0 (d, JPC=5.1 Hz,
3
ð BuÞ
2
J =6.3 Hz, POCH ); 70.1 (s,
PC 2
2
2
1
CH O ); 166.9 (d, J =268.0 Hz, CO ); 206.9 (s,
2
(2)
1
PC
2
3
COS). PNMR CDCl /250 MHz: d (ppm): ꢀ3.5. MS
3
+
(Fab +/NBA): m/z=501 [M+H] . Anal. calcd for C
H O PS (500.61): C, 47.98; H, 7.45; S, 12.81; found:
37 8 2
2
0
(
compound 12 as colourless oil in 91% yield. R =0.43.
ethyl acetate/petroleum ether 50/50) gave 850 mg of
C, 47.81; H, 7.50; S, 12.68.
f
1
H NMR CDCl /250 MHz: (ppm): 1.16 (s, 18H, CH );
3
.89 (q , J =6.6 Hz, 4H, CH ); 2.87 (t, JHH=7.1 Hz,
HH
H, CH S); 4.07 (td, J =6.2 Hz, J =8.1 Hz, 4H,
2
POCH ); 6.77 (d, JHP=699.6 Hz, 1H, PH). C NMR
2
3
t 3
3
1
4
4.3.3. [bis(S-acyl-3-thiopropyl)](2-methoxyethyloxycarb-
onyl) phosphonate (15). The yield was 75%. Chromato-
graphic conditions: ethyl acetate/petroleum ether 50/
2
3
3
HH
HP
1
13
1
CDCl /100 MHz: (ppm): 24.9 (s, CH S); 27.7 (s, CH );
3
50.R =0.40. H NMR CDCl /100 MHz: d (ppm): 1.78
3
2
f
3
3
t 3
3
0.8 (d, J =6.6 Hz, CH ); 46.8 (s, C ); 64.4 (d,
31
JPC=5.6 Hz, POCH ); 206.9 (s, COS). PNMR CDCl /
t
(q , J =6.5 Hz, 4H, CH ); 2.10 (s, 6H, CH CO); 2.75
HH 2 3
3
PC
2
ð BuÞ
2
(t, J =7.1 Hz, 4H, CH S); 3.14 (s, 3H, CH O); 3.41
HH 2 3
2
3
3
3
2
50 MHz: d (ppm): 8.4. MS (Fab +/NBA): m/z=399
(m, 2H, CH O ); 4.06 (q, J = JHP=6.4 Hz, 4H,
2 (2) HH
POCH ); 4.18 (m, 2H, CO CH ). C NMR CDCl /
2 2 2 3
00 MHz: d (ppm): 36.2 (s, CH S); 40.3 (d, JPC=4.9
+
t
+
t
+
13
[
M+H ] , 159 [ BuCOSCH CH CH ] , 57 [ Bu] .
2
2
2
3
1
2
4
.2.8. Bis(S-acyl-3-thiopropyl)phosphonate (13). Com-
Hz, CH ); 40.6 (s, CH CO); 62.9 (s, CH O); 67.4 (d,
3
2 3 3
2
pound 13 was prepared according to the above proce-
dure (compound 12) from 0.48 mL of phosphorus
trichloride (5.47 mmol, 1 equiv) in 10 mL of cyclohex-
ane, 2.20 g of S-acyl-3-thiopropyl alcohol 11 (16.4
mmol, 3 equiv) and 0.88 mL of pyridine (10.9 mmol, 2
equiv). After purification by silica gel column chroma-
tography (eluent: ethyl acetate/petroleum ether 50/50).
JPC=4.0 Hz, CO CH ); 69.1 (d, JPC=4.8 Hz,
2 2
1
POCH ); 71.4 (s, CH O ); 147.7 (d, JPC=213.7 Hz,
(2)
2
2
3
1
CO ); 170.6 (s, COS). PNMR CDCl /250 MHz: d
2
3
+
(ppm): ꢀ3.5. MS (Fab +/NBA): m/z=417 [M+H ] .
Anal. calcd for C H O PS (416.45): C, 40.38; H, 6.05;
2
1
4
25
8
S, 15.40; found: C, 40.28; H, 6.10; S, 15.28.
1
8
.41 g of compound 13 was isolated as colourless oil in
1
4.3.4. [bis(S-pivaloyl-3-thiopropyl)](5-methoxy-3-oxapen-
tyloxycarbonyl)phosphonate (16). The yield was 66%.
Chromatographic conditions: ethyl acetate/petroleum
1% yield. R =0.28. H NMR CDCl /400 MHz: d
f
3
t
3
(
CH ); 2.90 (t, JHH=7.0 Hz, 4H, CH S); 4.07 (td,
ppm): 1.91 (q , J =6.6 Hz, 4H, CH ); 2.28 (s, 6H,
3
HH 2
1
ether 50/50.R =0.43 (ethyl acetate). H NMR CDCl /
f
3
2
3
3
JHH=6.1 Hz, 3J =8.2 Hz, 4H, POCH ); 6.77 (d,
t
400 MHz: d (ppm): 1.16 (s, 18H, CH
t
); 1.92 (q ,
JHH=6.6 Hz, 4H, CH ); 2.88 (t, J =7.1 Hz, 4H,
HP
2
3 ð BuÞ
1JHP=701.4 Hz, 1H, PH). C NMR CDCl /100 MHz:
13
3
3
3
2
HH
3
d (ppm): 25.6 (s, CH S); 30.7 (d, JPC=6.6 Hz, CH2);
CH S); 3.31 (s, 3H, CH O); 3.47 (m, 2H, CH O (5)); 3.58
2 3 2
(m, 2H, CH O ); 3.70 (m, 2H, CH O (2)); 4.22 (q,
2 (4) 2
2
2
3
1.1 (s, CH ); 64.5 (d, J =5.7 Hz, POCH ); 195.9 (s,
3
3
PC
2
1
3
3
COS). PNMR CDCl 3/200 MHz: d (ppm): 9.1.MS
JHH= J =6.5 Hz, 4H, POCH ); 4.35 ( m, 2H,
2
HP
13
+
(
Fab +/NBA): m/z=315 [M+H] .
CO CH ). C NMR CDCl /100 MHz: d (ppm): 24.9 (s,
2
2
3
3
CH S); 27.8 (s, CH
t
t
); 30.8 (d, J =6.2 Hz, CH );
PC 2
3
2
3 ð BuÞ
46.9 (s, C
CO CH ); 67.4 (d, JPC=6.0 Hz, POCH ); 68.9 (s,
); 59.5 (s, CH O); 65.2 (d, JPC=4.9 Hz,
3
ð BuÞ
4.3. Bis(RSATP)polyethyleneglycol monomethylether
phosphonoformates 14–19
2
2
2
2
CH O ); 71.0 (s, CH O ); 72.3 (s, CH O (5)); 166.9
2
(2)
2
(4)
2
1
31
4
.3.1. General procedure. N,O-bis(trimethylsilyl)acet-
(d, J =270.0 Hz, CO ); 206.9 (s, COS). PNMR
PC 2
amide BSA (0.15 mL, 0.6 mmol, 1.2 equiv) was added to
a solution of bis(S-acyl-3-thiopropyl)phosphonate (0.5
mmol, 1 equiv) in 2 mL of dichloromethane, under
nitrogen atmosphere at room temperature. After 3 h the
CDCl /250 MHz: d (ppm): ꢀ3.5. MS (Fab +/NBA):
3
+
m/z=545 [M+H] . Anal. calcd for C H O PS
9
2
2
41
2
(544.66): C, 48.51; H, 7.59; S, 11.77; found: C, 48.39; H,
7.63; S, 11.65.
chloroformate
of
monomethylether
of
poly-
ethyleneglycol (1.5 mmol, 3 equiv) in 2 mL of dichloro-
methane was added dropwise. The reaction was stirred
overnight at room temperature. The solvent was
removed under reduced pressure and the crude product
was purified by silica gel column chromatography with
ethyl acetate—petroleum ether as eluent. The product
was isolated as colourless oil.
4.3.5. [bis(S-acyl-3-thiopropyl)](5-methoxy-3-oxapenty-
loxycarbonyl) phosphonate (17). The yield was 76%.
Chromatographic conditions: ethyl acetate/petroleum
1
ether 50/50 then 80/20. R =0.57 (ethyl acetate).
f
H
t
3
NMR CDCl /400 MHz: d (ppm): 1.78 (q , JHH=6.4
3
3
Hz, 4H, CH ); 2.11 (s, 6H, CH CO); 2.76 (t, JHH=7.1
Hz, 4H, CH S); 3.15 (s, 3H, CH O); 3.31 (m, 2H,
2
3
2
3
CH O
2
); 3.42 (m, 2H, CH O (4)); 3.53 (m, 2H,
2
(5)
3
3
4.3.2. [bis(S-pivaloyl-3-thiopropyl)](2-methoxyethyloxy-
carbonyl)phosphonate (14). The yield was 54%. Chro-
CH O ); 4.23 (q, J = JHP=6.4 Hz, 4H, POCH2);
2 (2) HH
1
3
4.34 (m, 2H, CO CH ). C NMR CDCl /100 MHz: d
3
2
2
3
matographic conditions: ethyl acetate/petroleum ether
1
(ppm): 36.2 (s, CH S); 40.3 (d, J =5.0 Hz, CH ); 40.6
2 PC 2
3
5
1
0/50.R =0.40. H NMR CDCl /400 MHz: d (ppm):
(s, CH CO); 63.0 (s, CH O); 67.5 (d, JPC=3.9 Hz,
3 3
CO CH ); 69.2 (d, JPC=4.9 Hz, POCH ); 70.4 (s,
2 2 2
f
3
t
3
2
.08 (s, 18H, CH3
t
); 1.84 (q , JHH=6.6 Hz, 4H,
ð BuÞ

V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
1401
CH O ); 72.1 (s, CH O ); 73.1 (s, CH O (5)); 147.7
2
4.3.9. [bis(S-acyl-3-thiopropyl)](allyloxycarbonyl)phosph-
onate (21). The same procedure was followed as for
compound 20. The yield was 83%. Chromatographic
conditions: ethyl acetate/petroleum ether 50/50.
(2)
2
(4)
2
1
31
(
d, J =213.0 Hz, CO ); 170.6 (s, COS). PNMR
PC 2
CDCl /250 MHz: d (ppm): ꢀ3.5. MS (Fab +/NBA): m/
3
+
z=461 [M+H] . Anal. calcd for C H O PS (460.50):
9
1
6
29
2
1
t
C,41.73;H,6.35;S,13.93;found:C,41.58;H,6.40;S,13.82.
R =0.60. H NMR CDCl /400 MHz: d (ppm): 2.01 (q ,
f
3
3
JHH=6,6 Hz, 4H, CH ); 2,27 (s, 6H, CH ); 2,92 (t,
2
3
3J =7,1 Hz, 4H, CH S); 4,22 (q, J = JHP=6,5
3
3
4.3.6. [bis(S-pivaloyl-3-thiopropyl)](8-methoxy-3,6-dioxao-
ctyloxycarbonyl)phosphonate (18). The yield was 62%.
HH 2 HH
3
4
Hz, 4H, POCH ); 4,70 (dd, J =6,0 Hz, JHP=1,2
2 HH
Hz, 2H, CO CH ); 5,26 (dd, J =10,5 Hz, JHH=1,1
2 2 HH
Hz, 1H, CH (3)); 5,32 (dd, J =17,2 Hz, JHH=1,1 Hz
HH
1H, CH
3
2
Chromatographic conditions: ethyl acetate/petroleum
1
3
2
ether 60/40. R =0.70 (ethyl acetate). H NMR CDCl /
f
3
t
13
4
00 MHz: (ppm): 1.16 (s, 18H, CH3
3
t
); 1.92 (q ,
JHH=6.6 Hz, 4H, CH ); 2.88 (t, J =7.1 Hz, 4H,
0
); 5,88 (m, 1H, CH (2)). C NMR CDCl /
(3 ) 3
ð BuÞ
3
3
100 MHz: d (ppm): 25,5 (s, CH S); 30,7 (d, JPC=6,1
2
HH
2
3
CH S); 3.31 (s, 3H, CH O); 3.48 (m, 2H, CH O (8)); 3.58
2
Hz, CH ); 31,1 (s, CH ); 66,9 (d, JPC=5,2 Hz,
2
3
2
2
3
(
m, 6H, CH O
2
); 3.70 (m, 2H, CH O (2)); 4.23 (q,
2
CO CH ); 67,2 (d, J =6,1 Hz, POCH ); 120,6 (s,
2 2 PC 2
(4,5,7)
3
3
1
JHH= J =6.5 Hz, 4H, POCH ); 4.34 (m, 2H,
HP
CH2 (3)); 131,0 (s, CH ); 166,6 (d, JPC=270,3 Hz,
CO ); 195,9 (s, COS). PNMR CDCl /250 MHz: d
2
(2)
31
1
3
CO CH ). C NMR CDCl /100 MHz: d (ppm): 24.9 (s,
2
2
3
2
3
3
+
CH S); 27.8 (s, CH
t
t
); 30.8 (d, J =6.4 Hz, CH );
3
(ppm): ꢀ3.5. MS (Fab +/NBA): m/z=399 [M+H] ,
2 2 2 2 2
2
3ð BuÞ
PC
2
+
+
4
6.8 (s, C
CO CH ); 67.4 (d, JPC=6.5 Hz, POCH ); 68.8 (s,
); 59.4 (s, CH O); 65.2 (d, JPC=4.9 Hz,
2
117 [MeCOSCH CH CH ] , 41 [CH =CH-CH ] .
ð BuÞ
3
2
2
2
CH O ); 71.0 (m, CH O
2
); 72.3 (s, CH O (8));
2
4.3.10.
thioheptyloxycarbonyl)phosphonate (22). In a quartz
reactor, solution of [bis(S-pivaloyl-3-thiopropy-
[bis(S-pivaloyl-3-thiopropyl)](6,7-dihydroxy-4-
(2)
1
2
(4,5,7)
31
1
66.8 (d, J =270.3 Hz, CO ); 206.8 (s, COS). PNMR
PC 2
CDCl /250 MHz: d (ppm): ꢀ3.5. MS (Fab +/NBA):
a
3
+
t
+
m/z=589 [M+H] , 159 [ BuCOSCH CH CH ] . Anal.
2
l)](allyloxycarbonyl) phosphonate 19 (0.388 g, 0.81
mmol, 1 equiv) and 3-mercapto-1,2-propanediol (0.119
mL, 1.45 mmol, 1.8 equiv) in the presence of a catalytic
amount of AIBN (8 mg, 0.048 mmol; 0.06 equiv) in 20
mL of THF was degassed for 15 min by argon bubbling.
The solution was submitted to UV irradiation at l=254
nm during 2 h, under inert atmosphere at room temp-
erature. The solvent was removed under reduced pres-
sure and the crude product was purified by silica gel
column chromatography (ethyl acetate). 0.178 g of
compound 22 was isolated as colourless oil. Yield 37%.
2
2
calcd for C24 H O PS (588.71): C, 48.96; H, 7.70; S,
10
45
2
1
0.89; found: C, 48.85; H, 7.74; S, 10.78.
4.3.7. [bis(S-acyl-3-thiopropyl)](8-methoxy-3,6-dioxaoctyl-
oxycarbonyl)phosphonate (19). The yield was 75%.
Chromatographic conditions: ethyl acetate/petroleum
1
ether 70/30. R =0.40 (ethyl acetate). H NMR CDCl /
3
f
t
3
4
2
3
00 MHz: d (ppm): 1.94 (q , JHH=6.5 Hz, 4H, CH2);
3
.27 (s, 6H, CH CO); 2.92 (t, J =7.1 Hz, 4H, CH S);
.31 (s, 3H, CH O); 3.48 (m, 2H, CH O (8)); 3.58 (m,
3
HH
2
3
2
1
6
H, CH O
2
); 3.69 (m, 2H, CH O (2)); 4.23 (q,
2
R =0.38 (ethyl acetate). H NMR CDCl /400 MHz: d
(4,5,7)
f
3
3
3
JHH= J =6.4 Hz, 4H, POCH ); 4.34 (m, 2H,
HP
CO CH ). C NMR CDCl /100 MHz: d (ppm): 36.2 (s,
(ppm): 1.16 (s, 18H, CH
2.52–2.66 (m, 6H, CH S
t
); 1.94 (m, 6H, CH
0
);
and OH); 2.88 (t,
2
3 ð BuÞ
2 (2,2 )
1
3
2
2
3
2
(3,5)
3
3
CH S); 40.3 (d, J =5.1 Hz, CH ); 40.6 (s, CH CO);
3
JHH=7.1 Hz, 4H, CH2S
0
); 3.58 (AB part of an ABX
(3 )
2
PC
2
3
1
3
6
2.9 (s, CH O); 67.5 (d, J =3.9 Hz, CO CH ); 69.1
2
system, d =3.66, d =3.51, J =11.3 Hz, JAX=3.7
3
3
PC
2
A
B
AB
2
(d, J =5.1 Hz, POCH ); 70.4 (s, CH O ); 72.1 (m,
2
CH O
Hz, CO ); 170.6 (s, COS). PNMR CDCl /250 MHz:
2
Hz, J =6.1 Hz, 2H, CH OH); 3.74 (m, 1H, CHOH);
BX 2
PC
2
(2)
1
3
3
); 73.1 (s, CH O ); 147.7 (d, JPC=213.0
2
4.22 (q, JHH= J =6.5 Hz, 4H, POCH ); 4.33 (t,
JHH=5.9 Hz, 2H, CO CH ).
2
2
(4,5,7)
(8)
HP
2
3
1
3
1
3
2
+
(
Anal. calcd for C H O PS (504.55): C, 42.85; H,
ppm): ꢀ3.5. MS (Fab +/NBA): m/z=505 [M+H] .
3
C NMR CDCl /100 MHz: d (ppm): 24.9 (s, CH S
0
);
(3 )
1
8
33 10
2
3
2
6
.59; S, 12.71; found: C, 42.76; H, 6.62; S, 12.61.
27.8 (s, CH
3
t
); 28.8 et 28.9 (2s, CH2S (3,5)); 30.8 (d,
0
3
JPC=6.3 Hz, CH
ð BuÞ
); 35.9 (s, CH2 (2)); 46.9 (s, C
t
);
2 (2 )
ð BuÞ
3
4
.3.8. [bis(S - pivaloyl - 3 - thiopropyl)](allyloxycarbonyl)-
64.9 (d, J =4.6 Hz, CO CH ); 65.7 (s, CH OH); 67.5
PC 2 2 2
(d, J =6.5 Hz, POCH ); 70.9 (s, CHOH); 166.7 (d,
PC 2
JPC=269.9 Hz, CO ); 207.0 (s, COS). PNMR
2
2
phosphonate (20). Compound 20 was prepared accord-
ing to the above procedure (compounds 14–19) with
allyl chloroformate. The yield was 80%. Chromato-
graphic conditions: ethyl acetate/petroleum ether 70/30.
1
31
CDCl /250 MHz: d (ppm): ꢀ3.6. MS (Fab+/NBA):
3
+
t
+
m/z=591 [M+H] , 159 [ BuCOSCH CH CH ] .
2
2
2
1
R =0.75. H NMR CDCl /250 MHz: d (ppm): 1.25 (s,
Anal. calcd for C23 H O PS (590.75): C, 46.76; H,
43 9 3
7.34; S, 16.28; found: C, 46.69; H, 7.39; S, 16.19.
f
3
t 3
1
8H, CH3
JHH=7.1 Hz, 4H, CH S); 4.31 (q, J = J =6.5 Hz,
t
); 2.01 (q , J =6.7 Hz, 4H, CH ); 2.95 (t,
3
ð BuÞ
HH
2
3
3
2
HH
HP
4
4
2 HH HP
3
4
Hz, CO CH ); 5.38 (m, 2H, CH ); 5.93 (m, 1H, CH
H, POCH ); 4.77 (qd, J = J =1.3 Hz, JHH=5.9
0
4.3.11. [bis(S-acyl-3-thiopropyl)](6,7-dihydroxy-4-thio-
heptyloxycarbonyl)phosphonate (23). Compound 23 was
prepared according to the above procedure (compound
22) from 0.309 g of [bis(S-acyl-3-thiopropyl)](allyloxy-
carbonyl)phosphonate 21 (0.78 mmol, 1 equiv) and 0.12
mL of 3-mercapto-1,2-propanediol (1.40 mmol, 1.8 equiv)
in the presence of 7.6 mg of AIBN (0.047 mmol; 0.06
equiv) in 20 mL of THF . After purification by silica gel
column chromatography (eluent: ethyl acetate), 0.156 g
of compound 22 was obtained as colourless oil in 40%
2
2
(3,3 )
1
3
(2))
. C NMR CDCl /100 MHz: d (ppm): 25.1 (s,
CH S); 27.8 (s, CH ); 30.7 (d, J =6.1 Hz, CH ); 66.8
(d, JPC=5.0 Hz, CO CH ); 67.4 (d, JPC=6.4 Hz,
2
POCH ); 120.4 (s, CH
3
3
2
3
3
PC
2
2
2
); 131.0 (s, CH (2)); 166.6 (d,
31
2
2 (3)
1
JPC=269.8 Hz, CO ); 206.9 (s, COS). PNMRCDCl3/
2
2
50 MHz: d (ppm): ꢀ3.5. MS (Fab +/NBA): m/z=483
+
+
t
+
t
+
[M+H] , 159 [ BuCOSCH CH CH ] , 57 [ Bu] , 41[
2 2 2
CH =CH-CH ] .
2
2

1402
V. Gagnard et al. / Bioorg. Med. Chem. 12 (2004) 1393–1402
1
yield. R =0.33 (ethyl acetate/ethanol 98/2). H NMR
f
3. Ferguson, C. G.; Gorin, B. I.; Thatcher, G. R. J. J. Org.
Chem. 2000, 65, 1218.
CDCl /400 MHz: d (ppm): 1.95 (m, 6H, CH
0
); 2.26
and OH);
3
2 (2,2 )
4
. (a) Meier, C.; Aubertin, A. M.; De Monte, M.; Faraj, A.;
Sommadossi, J. P.; Perigaud, C.; Imbach, J. L.; Gosselin,
G. Antiviral Chem. Chemother. 1998, 9, 41. (b) Briggs,
A. D.; Camplo, M.; Freeman, S.; Lundstrom, J.; Pring,
B. G. Tetrahedron 1996, 52, 14937.
. Lefebvre, I.; Perigaud, C.; Pompon, A.; Aubertin, A. M.;
(
2
s, 6H, CH ); 2.53 – 2.66 (m, 6H, CH S
3
3
2
(3,5)
´
.88 (t, JHH=7.1 Hz, 4H, CH2S
0
); 3.58 (AB part of
(3 )
an ABX system, d =3.66, d =3.51, JAB=11.3 Hz,
1
A
B
3
3
¨
JAX=3.7 Hz, J =6.1 Hz, 2H, CH OH); 3.73 (m,
3
BX
2
3
1
POCH ); 4.33 (t, JHH=5.9 Hz, 2H, CO CH ).
H, CHOH); 4.21 (q, JHH= J =6.5 Hz, 4H,
HP
5
`
´
3
13
C
);
2
2
2
Girardet, J. L.; Kirn, A.; Gosselin, G.; Imbach, J. L.
J. Med. Chem. 1995, 38, 3941.
6. Girardet, J. L.; Perigaud, C.; Aubertin, A. M.; Gosselin,
´
G.; Kirn, A.; Imbach, J. L. Bioorg. Med. Chem. Lett.
1995, 5, 2981.
´ `
7. Perigaud, C.; Gosselin, G.; Lefebvre, I.; Girardet, J. L.;
NMR CDCl / 100 MHz: d (ppm): 25.5 (s, CH S
28.8 and 28.9 (2s, CH2S (3,5)); 30.7 (d, JPC=6.2 Hz,
0
CH ); 31.0 (s, CH ); 35.9 (s, CH2 (2)); 64.8 (d,
JPC=4.5 Hz, CO CH ); 65.7 (s, CH OH); 67.3 (d,
2
JPC=6.5 Hz, POCH ); 70.9 (s, CHOH); 166.7 (d,
2
1J =270.0 Hz, CO ); 195.8 (s, COS). 31PNMR
0
3
2
(3 )
3
2
(2 )
3
3
2
2
2
Benzaria, S.; Barber, I.; Imbach, J. L. Bioorg. Med. Chem.
Lett. 1993, 13, 2521.
PC
2
CDCl /250 MHz: d (ppm): -ꢀ3.6. MS (Fab +/NBA):
3
8
. (a) Benzaria, S.; Pelicano, H.; Johnson, R.; Maury, G.;
Imbach, J. L.; Aubertin, A. M.; Obert, G.; Gosselin, G. J.
Med. Chem. 1996, 39, 4958. (b) Benzaria, S.; Girardet,
+
+
m/z=507 [M+H] , 117 [CH COSCH CH CH ] , 43
2
3
2
2
+
[
4
1
CH CO] . Anal. calcd for C H O PS (506.59): C,
3 17 31 9 3
0.30; H, 6.17; S, 18.99; found: C, 40.19; H, 6.20; S,
8.78.
J. L.; Perigaud, C.; Aubertin, A. M.; Pelicano, H.; Maury,
´
G.; Gosselin, G.; Kirn, A.; Imbach, J. L. Nucleic Acids
Symposium Series 1994, 31, 129.
9
. Egron, D.; Pe
Imbach, J. L. Bulletin des Socie´t e´ s Chimiques Belges 1997,
06, 461.
10. Texier-Boullet, F.; Foucaud, A. Synthesis 1982, 916.
´
rigaud, C.; Gosselin, G.; Aubertin, A. M.;
4
.4. Biological studies
1
Anti-HIV assays were conducted following the proce-
1
8
dure described previously.
1
1. Frechette, R. F.; Ackerman, C.; Beers, S.; Look, R.;
Moore, J. Bioorg. Med. Chem. Lett. 1997, 7, 2169.
1
2. Lefe
Gosselin, G.; Imbach, J. L. LC-GC International 1997, 9,
02.
3. Gagnard, V.; Leydet, A.; Le Mellay, V.; Aubenque, M.;
bvre, I.; Pompon, A.; Valette, G.; Perigaud, C.;
` ´
Acknowledgements
The authors gratefully acknowledge the Ministe
6
`
re de
1
l’Education Nationale de la Recherche et de la Techno-
logie for a fellowship to V. G. and the Laboratoires
mayoly-SPINDLER for financial support.
More
883.
14. Nore
`
re, A.; Montero, J. L. Eur. J. Med. Chem. 2003, 38,
´
n, J. O.; Helgstrand, E.; Johansson, N. G.; Misiorny,
A.; Stening, G. J. Med. Chem. 1983, 26, 264.
¨
5. Briggs, A. D.; Camplo, M.; Freeman, S.; Lundstrom, J.;
1
References and notes
Pring, B. G. Tetrahedron 1996, 52, 14937.
6. Kennedy, J. . Patent Brit 778,077 Chem. Abstr 1957, 1957,
1
1
2
. Helgstand, E.; Eriksson, B.; Johansson, N. G.; Lannero
¨
,
B.; Larsson, A.; Misiorny, A.; Noren, J. O.; Sjoberg, B.;
51 17980b.
17. Goren, Z.; Heeg, M. J.; Mobashery, S. J. Org. Chem.
1991, 56, 7186.
18. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols,
D.; Herdewijn, P.; Desmyter, J.; De Clercq, E. J. Virol.
Methods 1988, 20, 309.
¨
Sternberg, K.; Stening, G.; Stidh, S.; Oberg, B. Science
978, 201, 819.
. De Clercq, E. Collect. Czech. Chem. Comm. 1998, 63,
49.
1
4