Synthesis and biological evaluation of S-acyl-3-thiopropyl prodrugs of N-phosphonoacetyl-L-aspartate (PALA)

Article abstract of DOI:10.1016/j.ejmech.2003.07.002

The synthesis of new prodrugs of PALA characterised by the presence of S-acyl-3-thiopropyl, as enzyme-labile groups on the phosphonate moiety of PALA, is reported. The cytotoxic activities of PALA prodrugs were determined against human cell line (SW1573 lung carcinoma cells). A number of prodrugs bearing S-pivaloyl as acyl groups displayed cytotoxic activity in the same order of magnitude of PALA.

Full text of DOI:10.1016/j.ejmech.2003.07.002

European Journal of Medicinal Chemistry 38 (2003) 883ꢀ891
/
www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of S-acyl-3-thiopropyl prodrugs
of N-phosphonoacetyl- -aspartate (PALA)
L
Vale´rie Gagnard, Alain Leydet *, Ve´ronique Le Mellay, Marielle Aubenque,
Alain More`re, Jean-Louis Montero
´
Laboratoire de chimie biomole´culaire, UMR 5032, Universite´ Montpellier II, ENSCM, 8, rue de l’Ecole normale, 34296 Montpellier cedex 5, France
Received 22 April 2003; received in revised form 22 July 2003; accepted 29 July 2003
Abstract
The synthesis of new prodrugs of PALA characterised by the presence of S-acyl-3-thiopropyl, as enzyme-labile groups on the
phosphonate moiety of PALA, is reported. The cytotoxic activities of PALA prodrugs were determined against human cell line
(SW1573 lung carcinoma cells). A number of prodrugs bearing S-pivaloyl as acyl groups displayed cytotoxic activity in the same
order of magnitude of PALA.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: PALA; prodrugs; S-acyl-3-thiopropyl; lung carcinoma; calculated log P
1. Introduction
ATCase does not seem to be increased by modifying its
structure. Thus, other strategies appear to be necessary
in order to improve the activity of PALA.
For many years, PALA (N-phosphonoacetyl-L-aspar-
tate) has been considered to be of important therapeutic
interest. PALA [1] is a rationally designed transition
state analogue of the aspartate transcarbamylase (AT-
Case) subunit of CAD (Carbamylphosphate, Aspartate
transcarbamylase, Dihydro-orotase), a multifunctional
enzyme required for de novo pyrimidine biosynthesis
and cell growth [2].
The spectrum of activity of PALA is unique in that it
exhibits tumour-inhibitory activity against a number of
transplantable solid murine tumours such as Lewis lung
carcinoma (which is often insensitive to other treat-
ments) and B16 melanoma [1], whereas L1210 and P388
leukaemias are relatively insensitive to the drug [3].
Over the past 25 years, PALA underwent a lot of
structural modifications, motivated by the results ob-
tained during in vivo studies. In order to improve the
affinity of PALA towards ATCase, the different func-
tions of the molecule were modified alternatively or
simultaneously, leading to an important variety of
compounds [4,5]. However, the affinity of PALA for
The de novo pyrimidine biosynthesis takes place in
the cytosol of the cell and PALA has to penetrate inside
the cell to act as an inhibitor of the aspartate transcar-
bamylase [6].
The ionic nature of PALA (Fig. 1) is a limiting factor
for its diffusion through the lipidic bilayer of the cellular
membrane [3b]. The use of vectors such as liposomes
was postulated to allow the penetration of PALA into
the cell [7]. The use of liposomes as vectors leads to a
weak encapsulation of drugs (generally around 5% [7a])
and therefore alternative prodrug approaches can be
used to overcome many problems associated with
bioavailability and permeability of ionic molecules
such as PALA.
The prodrug strategy will temporarily mask the
anionic charges by a lipophilic ester protective group
to enhance the hydrophobicity of the molecule and
therefore improve the cellular membrane permeation.
This protective group will then be selectively cleaved
inside the cell in the presence of esterases.
We have demonstrated [8] that the S-acyl-3-thiopro-
pyl groups (SATP) are new enzyme-labile protective
groups of phosphonate esters. Indeed, SATP can be
* Correspondence and reprints:.
E-mail address: leydet@univ-montp2.fr (A. Leydet).
´
0223-5234/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.07.002

884
V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ
/891
Fig. 1. PALA and its targeted prodrugs.
hydrolysed enzymatically by carboxyesterases which are
more prevalent intracellularly.
The first step of the synthesis was the chloroacetyla-
tion of aspartate dimethylester by addition of chloroa-
cetic anhydride [13] (Fig. 2). The phosphonylation of the
In this study, we have linked the SATP group to the
phosphonate moiety of PALA. One difficulty in prodrug
approach is to find the fair balance between the
lipophilicity necessary, for the transport through the
cellular membrane and the hydrophilicity allowing the
solubilisation in physiological media. The choice of the
protective groups on the PALA carboxylate moieties
was guided by the evaluation of the log P values of
targeted molecules [9] (by the software ACD/LogP,
ChemCAD). We chose monomethylether of polyethyle-
resulting chloroacetylꢀaspartate dimethylester 1 was
/
then achieved according to the Arbuzov’s reaction using
tris(trimethylsilyl)phosphite [14] at 160 8C. The hydro-
lysis in a waterꢀmethanol mixture (1:1) afforded the
/
expected phosphonic acid 2.
The phosphonic acid 2 undergoes activation by oxalyl
chloride in the presence of DMF, followed by the
addition of the S-pivaloyl-3-thiopropanol 6. The
acylthiopropanols were obtained by radical addition of
the corresponding thioacid to the allylic alcohol (Fig. 3).
The calculated log P value of compound 4 was 3.6
(Table 1) and the values of log P generally admitted to
neglycol (nꢁ
/
1ꢀ3) because the hydrophilicity of pro-
/
drugs is improved when they are linked to PEG [10]. We
are presenting here the chemical synthesis of seven
prodrugs of PALA as well as their cytotoxic activities
against SW1573 lung carcinoma cells.
give good bioavailability are usually within the 2ꢀ3.5
/
range. Nevertheless, the solubilisation of compound 4 in
aqueous media requires an additional 5% of DMSO. To
improve the water solubility of these PALA prodrugs,
we decided to introduce polyethyleneglycol monomethy-
2. Chemistry
Among the several syntheses of PALA reported
[1,2a,11], we designed a general synthetic procedure
[12] in which the key step is an Arbuzov’s reaction using
the tris(trimethylsilyl)phosphite. At first, the introduc-
tion of enzyme-labile SATP groups was achieved using
PALA dimethylester derivative 2 to obtain 4 bearing
two S-pivaloyl-3-thiopropyl (tBuSATP) groups on the
phosphonate moiety. In this compound, the phosphonic
acids were masked by an enzyme-labile ester and the
carboxylic moieties of aspartic acid by methyl esters
(Fig. 2).
Fig. 3. Synthesis of S-acylthiopropanols. Reagents and conditions: (i)
thioacetic acid, AIBN, benzene, reflux; (ii) thiopivaloic acid, AIBN,
THF, UV: lꢁ254 nm.
/
Fig. 2. Synthesis of bis-t BuSATPꢀ
H2OꢀMeOH (1:1); (iv) (ClCO)2, CH2Cl2, DMF; (v) 6, Et3N, CH2Cl2.
/
PALA dimethylester. Reagents and conditions: (i) (ClCH2CO)2O, CH2Cl2, pyridine; (ii) P(OSiMe3)3, 160 8C; (iii)
/

V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ
/891
885
Table 1
Estimated log P values, yield and 31P-NMR analysis of bis (R-SATP) polyethyleneglycol monomethylether N-phosphonoacetyl-
L
-aspartates 23ꢀ28
/
Compounds
R (acyl)
n
Yield%
log Pcalc
9
/
0.6
d
31P (ppm)
23
24
25
26
27
28
Me
Me
Me
t Bu
t Bu
t Bu
1
2
3
1
2
3
16
53
32
25
53
27
1.2
0.4
23.4
23.5
23.6
23.4
23.4
23.6
ꢂ0.3
/
3.6
2.9
2.2
lether groups (nꢁ
aspartic acid.
/
1ꢀ
/
3) on the carboxylate moiety of
After distillation of the tris(trimethylsilyl)phosphite
excess under reduced pressure, reaction of the crude
mixture with oxalyl chloride in dichloromethane in the
presence of catalytic amounts of DMF afforded the bis-
Attempts of esterification were conducted on N-
chloroacetylaspartic acid (Fig. 4, path A), N-benzylox-
ycarbonyl aspartic acid (path B) or even on the aspartic
acid bearing no protective groups on its nitrogen.
Surprisingly (path A), the N-chloroacetylaspartic acid
did not lead to the expected diesters 14, 15 and 16, while
the path B via the benzyloxycarbonyl derivative 7 [15]
provided the best results.
chloro intermediates 20ꢀ/22 [18]. The addition of
acylthiopropanols 5 or 6 on the appropriate derivative
20ꢀ/22 afforded the enzyme-labile prodrugs of PALA
23ꢀ/28 (Fig. 5), the relative calculated lipophilicity of
which is presented in Table 1.
The cytotoxic activities of the prodrugs 4, 23ꢀ
/28 were
determined using SW1573 lung carcinoma cell line. This
human cell line of alveolar lung carcinoma was chosen
because PALA had demonstrated a moderate inhibitory
activity against human LX-1 lung carcinoma cell line
[2c]. The tris-sodium salt of PALA (N-phosphonoace-
The polyethyleneglycol monomethylether groups
were introduced by an esterification reaction involving
DCC/DMAP [16] affording the diesters 8, 9 and 10 in
75ꢀ80% yield.
/
The Cbz group was then removed by catalytic transfer
hydrogenation leading in quantitative yields to 11, 12
and 13, respectively [17]. Chloroacetylation was then
performed using chloroacetic anhydride in pyridine and
the chloroacetylated compounds 14, 15 and 16 were
tyl-L-aspartate) was evaluated in parallel for compar-
ison. Only the prodrugs 4, 26, 27 and 28 bearing S-
pivaloyl-3-thiopropyl enzyme-labile groups on the phos-
phonate moiety displayed cytotoxic activity against
SW1573 lung carcinoma cell line (Table 2).
The prodrugs 4, 26, 27, 28, induced the death of the
cell to the same extent when compared to PALA. The
introduction of S-pivaloyl-3-thiopropyl on the phos-
phonate moiety did not modify the cytotoxic activity by
comparison with PALA. Nevertheless, the cytotoxic
isolated in the range of 60ꢀ
The phosphonylation of chloroacetylated compounds
14ꢀ16 was performed as previously to give the bis-
silylated phosphonates 17ꢀ19 which were not isolated
(Fig. 4).
/80% yields (Fig. 4).
/
/
Fig. 4. Synthesis of N-chloroacetylaspartate (polyethyleneglycol monomethylether) diester. Reagents and conditions: (i) ClCO2Bn, NaHCO3; (ii)
HO(CH2CH2ÃO)n Me, DCC, DMAP, CH2Cl2; (iii) Pd/C, 1,4-cyclohexadiene, EtOH; (iv) (ClCH2CO)2O, CH2Cl2, pyridine.
/

886
V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ891
/
Fig. 5. Synthesis of bis (SATP) derivatives of PALA. Reagents and conditions: (i) P(OSiMe3)3, 160 8C; (ii) (ClCO)2, CH2Cl2, DMF; (iii) 5, Et3N,
CH2Cl2; (iv) 6, Et3N, CH2Cl2.
release of the S-acetyl-3-thiopropyl groups into the
culture medium leading to a diester by-product of
PALA. Indeed, kinetic studies on nucleotidic prodrugs
determined half-live in cellular extracts media to be
shorter for S-acetyl than for S-pivaloyl derivatives [21].
Table 2
Cytotoxic activities of PALA prodrugs against SW1573 lung carcino-
ma cells
b
Compounds
IC50
M
log Pcalc
4
23
3.63ꢃ
/
10ꢂ49
/
5.5ꢃ
/
10ꢂ5 a
3.6
1.2
0.4
c
/
ꢀ
/
/ꢀ
/ꢀ
/ꢀ
3.50ꢃ
3.25ꢃ
3.50ꢃ
2.88ꢃ
24
25
ꢀ/
/
3. Conclusion
ꢀ/
/
ꢂ
/
0.3
3.6
2.9
2.2
26
27
/
10ꢂ49
10ꢂ49
2.5ꢃ
10ꢂ49
7.1ꢃ
10ꢂ49
30.0ꢃ
/
7.9ꢃ
/
10ꢂ5
10ꢂ5
10ꢂ5
/
/
/
A number of new prodrugs of PALA were synthesized
and their cytotoxic activities determined against
SW1573 lung carcinoma cells.
28
PALA (Reference)
/
/
/
/
/
/
10ꢂ5
ꢀ
0 d
/
a
The take home message emerging from this study is
the cytotoxic activity of the SATP prodrugs bearing S-
pivaloyl as acyl groups. This cytotoxic activity against
the SW1573 lung carcinoma cells is close to the PALA
activity and can be explained by a passive transport of
the prodrugs which release PALA inside the cells. It is
noteworthy that only the prodrugs of estimated log P
values higher than 2.5 showed a cytotoxic activity.
All data represent the average value of four separate experiments
each one performed in triplicate.
b
50% inhibitory concentration.
No inhibition observed.
c
d
The software do not give fair estimated log P value for ionic
molecule such PALA.
activity of the prodrug was lost when the S-pivaloyl was
substituted by an S-acetyl in the SATP enzyme-labile
group.
Several experiments confirmed the penetration of
PALA into the cell through endocytosis [19]. On the
other hand, prodrugs of PALA do not follow the same
transport mechanism and they will use a passive
transport mechanism to enter the cell [20]. The inhibi-
tory activity of prodrugs bearing the S-pivaloyl group 4,
4. Experimental protocols
4.1. Chemistry
1H- and 13C-NMR were recorded on a Brucker
apparatus DPX200, AC250 and DRX400. 31P-NMR
spectra were recorded on a Brucker DPX200. Mass
spectra were recorded on a JEOL JMS-DX300. Log P
was estimated by means of the software ACD/LogP
calculator developed by ACD (Advanced Chemistry
Development Inc.) and distributed by the company
ChemCAD. Optical rotations were determined on a
26ꢀ28 could be explained by the passive transport
/
through the cell membrane followed by an esterasic
activity to release the active form of these prodrugs. The
lack of activity observed for the prodrugs bearing the S-
acetyl group 23ꢀ
/
25 could be explained by a less effective
passive transport due to low log P values, or by a quick

V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ
/891
887
Perkinꢀ
/
Elmer polarimeter PE 241. Microanalyses were
solution of NaHCO3, then twice with brine. The organic
layer was dried with sodium sulphate and concentrated
under reduced pressure. The crude product was purified
performed in the analytical department of the CNRS
(Vernaison, Rhoˆne, France). Analyses indicated by the
symbols were within 9
/
0.4% of the theoretical values.
by silica gel column chromatography (petroleum etherꢀ
ethyl acetate 70/30). 1.1 g of compound 6 was obtained
as colourless oil in 56% yield. Rfꢁ0.28 (ethyl acetateꢀ
petroleum ether 50/50). H-NMR CDCl3/250 MHz: d
/
4.2. Dimethyl-O,O-bis(S-pivaloyl-3-thiopropyl)-N-
phosphonoacetyl- -aspartate (4)
/
/
1
L
(ppm): 1.75 (qt, JHH
CH3); 2.38 (s, 1H, OH); 2.93 (t,3JHH
CH2S); 3.57 (t, 3JHH 5.7 Hz, 2H, CH2OH). 13C-NMR
ꢁ/6.4 Hz, 2H, CH2); 2.28 (s, 3H,
3
The dimethyl N-phosphonoacetyl-
L-aspartate (2) [12]
ꢁ
/
6.8 Hz, 2H,
(0.38 g, 1.34 mmol, 1 equiv.), was dissolved in 5 mL of
dichloromethane and 10 drops of DMF were added as a
catalyst. To the solution, cooled 10 min at 0 8C, oxalyl
chloride (0.40 mL, 4.69 mmol, 3.5 equiv.) was added
dropwise and the mixture was stirred for 3 h 30 min at
room temperature. The volatile components were re-
moved under reduced pressure and the P,P-dichloro-N-
ꢁ
/
CDCl3/100 MHz: d (ppm): 25.9 (s, CH2S); 30.9 (s, CH3);
32.8 (s, CH2); 60.8 (s, CH2OH); 197.8 (s, CO).
MS
(Fabꢄ
/
/NBA):
m/zꢁ117
/
[CH3COSCH2CH2CH2]ꢄ; 43 [CH3CO]ꢄ.
4.4. S-Pivaloyl-3-thiopropyl alcohol 6
phosphonoacetyl-L-aspartate (3), obtained as a brown
oil, was rapidly used without further purification.
Complex 3 was dissolved in 10 mL of dichloromethane
and cooled to 0 8C. A mixture of S-pivaloyl-3-thiopro-
pyl alcohol (6) (0.48 g, 2.7 mmol, 2 equiv.) and
triethylamine (1.12 mL, 8.04 mmol, 6 equiv.) in 5 mL
of dichloromethane was added dropwise and the reac-
tion was stirred for 2 h at room temperature. The
mixture was diluted with 150 mL of dichloromethane
and washed three times with a saturated solution of
NaHCO3 and twice with brine. The organic layer was
dried over Na2SO4 and concentrated under reduced
pressure. The crude product was purified by silica gel
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
degazed for 15 min by argon bubbling. The solution was
submitted to UV irradiation at lꢁ254 nm during 3 h
/
under inert atmosphere at room temperature. Then the
mixture was diluted with 200 mL of ethyl acetate,
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 residual oil was purified by silica gel column
column chromatography (ethyl acetateꢀ/methanol 90/10,
chromatography (petroleum etherꢀ
/
ethyl acetate 70/30)
to give 2.9 g of compound 6 as colourless oil in 80%
80/20) to give 0.198 g of compound 4 as yellow oil.
1
Yield: 25%. Rfꢁ
200 MHz: d (ppm): 1.23 (s, 18H, CH3 (tBu)); 1.95 (qt,
3JHH
6.0 Hz, 4H, CH2); 2.8ꢀ3.08 (m, 8H, CH2S,
3H, 2CH3);
/0.28 (ethyl acetate). H-NMR CDCl3/
1
0.57. H-NMR CDCl3/200 MHz: d (ppm):
yield. Rfꢁ
/
3
1.22 (s, 9H, CH3); 1.79 (tt, JHH
3
6.8 Hz, JHH
ꢁ
/
ꢁ5.9
ꢁ6.8 Hz,
/
ꢁ
/
/
3
Hz, 2H, CH2); 2.68 (s, 1H, OH); 2.96 (t, JHH
/
PCH2CO, CH2CO2); 3.70 and 3.76 (2s, 2ꢃ
/
ꢁ
5.9 Hz, 2H, CH2OH). 13C-
NMR CDCl3/100 MHz: d (ppm): 25.2 (s, CH2S); 27.8
/
3
2H, CH2S); 3.61 (t, JHH
3
3
4.18 (m, 4H, POCH2); 4.88 (td, JHH
3
8.0 Hz, 1H, CHCO2); 7.44 (d, JHH
ꢁ
/
4.7 Hz, JHH
ꢁ
/
ꢁ
/
8.0 Hz, 1H, NH).
13C-NMR CDCl3/100 MHz: d (ppm): 24.9 (s, CH2S);
(s, CH3); 32.9 (s, CH2); 46.9 (s, C(tBu)); 60.8 (s, CH2OH);
Na]ꢄ;
208.9 (s, CO). MS (Fabꢄ
/
/NBA): m/zꢁ
/
199 [Mꢄ
/
3
27.8 (s, CH3(tBu)); 30.9 (d, JPC
ꢁ6.3 Hz, CH2); 35.5 (d,
/
177 [Mꢄ
/
H]ꢄ; 85 [tBuCO]ꢄ; 57 [tBu]ꢄ.
1JPC
C(tBu)); 49.3 (s, CHCO2); 52.5 and 53.2 (2s, 2CH3); 65.6
ꢁ
/
131.0 Hz, PCH2CO); 36.4 (s, CH2CO2); 46.9 (s,
2
and 65.7 (2d, JPC
2
5.6 Hz, POCH2); 164.2 (d, JPC
ꢁ
/
ꢁ
/
4.5. Bis (polyethyleneglycol monomethylether)-N-
benzyloxycarbonyl- -aspartate 8ꢀ10
4.5 Hz, CONH); 171.5 and 171.6 (2s, CO2); 206.9 (2s,
COS). 31P-NMR CDCl3/200 MHz: d (ppm): 24.3.
L
/
MS (Fabꢄ
/
/NBA): m/zꢁ
/
600 [Mꢄ
/
H]1; 622 [Mꢄ
/
A solution of N-benzyloxycarbonyl-
L-aspartic acid 7
Na]ꢄ; 159 [tBuCOSCH2CH2CH2]ꢄ; 57 [tBu]ꢄ. Anal.
C24H42NO10PS2 (C, H, N).
(5 mmol, 1 equiv.) and polyethyleneglycol monomethy-
lether (10 mmol, 2 equiv.) in 8 mL of dichloromethane
was cooled for 10 min at 0 8C. Then DCC (16 mmol, 3.2
equiv.) dissolved in 25 mL of dichloromethane was
added dropwise, followed by catalytic amount of
DMAP. The mixture was stirred overnight at room
temperature and the DCU precipitate was removed by
filtration. The filtrate was washed twice with a 5%
solution of NaHCO3 then twice with 1 M solution of
KHSO4. The organic layer was dried over Na2SO4 and
concentrated under reduced pressure. The crude product
4.3. S-Acetyl-3-thiopropyl alcohol 5
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 degazed 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

888
V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ891
/
was purified by silica gel column chromatography to
give compounds 8ꢀ10 as colourless oil.
C81 ); 156.4 (s, CONH); 170.2 and 171.3 (2s, CO2). MS
(Fabꢄ/NBA): m/zꢁ560 [Mꢄ
H]ꢄ.
/
/
/
/
4.5.1. Bis-(2-methoxy-ethyl)-N-benzyloxycarbonyl-
aspartate (8)
L
-
4.6. Bis(polyethyleneglycol monomethylether)-N-
chloroacetyl- -aspartate 14ꢀ16
L
/
Yield: 75%. Rfꢁ
CDCl3/200 MHz: d (ppm): 3.01 (AB part of an ABX
2.91, 1JAB
system, dAꢁ3.10, dBꢁ 17.3 Hz,
3JAXꢁ3
JBX 4.4 Hz, 2H, CH2CO2); 3.34 and 3.35
(2s, 2ꢃ3H, CH3O); 3.56 (m, 4H, CH2O(2,2?)); 4.22 and
4.30 (2m, 2ꢃ ꢁ4.4
/
0.40 (ethyl acetate). 1H-NMR
To a solution of compounds 8ꢀ10 (3 mmol, 1 equiv.)
/
/
/
ꢁ
/
in 7 mL of ethyl alcohol, were successively added Pd/C
10% (w:w; 1:1) and cyclohexa-1,4-diene (30 mmol, 10
equiv.), stirring was continued for 1 h at room tempera-
ture. After filtration, the filtrate was concentrated under
/
ꢁ
/
/
3
/
2H, CO2CH2(1,1?)); 4.67 (td, JHH
8.8 Hz, 1H, CHCO2); 5.11 (s, 2H, CH2Ph);
5.89 (d, 3JHH 8.6 Hz, 1H, NH); 7.34 (m, 5H, Ph). 13C-
/
3
Hz, JHH
ꢁ
/
reduced pressure. Compounds 11ꢀ13, obtained as pale
/
ꢁ
/
yellow oil in quantitative yield, were used without
further purification. Pyridine (18 mmol, 6 equiv.) was
NMR CDCl3/100 MHz: d (ppm): 36.8 (s, CH2CO2);
49.6 (s, CHCO2); 59.3 (s, CH3O); 64.3 and 65.1 (2s,
CO2CH2(1,1?)); 67.5 (s, CH2Ph); 70.6 (2s, CH2O(2,2?));
added to a solution of 11ꢀ13 (3 mmol, 1 equiv.) in 20
/
mL of dichloromethane. Then chloroacetic anhydride (6
mmol, 2 equiv.) was added to the mixture cooled at 0 8C.
The mixture was stirred for 1 h at room temperature and
then washed three times with cold water. The organic
layer was dried over Na2SO4 and concentrated under
reduced pressure. The crude oil was purified by silica gel
128.5ꢀ
CONH); 170.5 and 170.9 (2s, CO2).
MS (Fabꢄ/NBA): m/zꢁ384 [Mꢄ
H]ꢄ.
/
128.9 (4s, C28, C38, C48); 136.6 (s, C81 ); 156.3 (s,
/
/
/
4.5.2. Bis-12-(2-methoxy-ethoxy)-ethyl]-N-
benzyloxycarbonyl- -aspartate (9)
Yield: 75%. Rfꢁ
0.40 (ethyl acetate). 1H-NMR
CDCl3/400 MHz: d (ppm): 3.00 (AB part of an ABX
system, dAꢁ3.08, dBꢁ 4.7
2.92, 1JAB 17.1 Hz, 3JAX
Hz, 3JBX
4.6 Hz, 2H, CH2CO2); 3.36 and 3.38 (2s, 2ꢃ
column chromatography to give compounds 14ꢀ
pale yellow oil.
/
16 as
L
/
/
/
ꢁ
/
ꢁ
/
4.6.1. Bis-(2-methoxy-ethyl)-N-chloroacetyl-
(14)
L
-aspartate
ꢁ
/
/
3H, CH3O); 3.53 (m, 4H, CH2O(5,5?)); 3.62 (m, 4H,
CH2O(4,4?)); 3.69 (m, 4H, CH2O(2,2?)); 4.25 and 4.33 (2m,
Yield: 64%. Rfꢁ
CDCl3/200 MHz: d (ppm): 3.00 (AB part of an ABX
system, dAꢁ3.09, dBꢁ 17.2 Hz,
2.91, 1JAB
3JAXꢁ3
JBX 4.6 Hz, 2H, CH2CO2); 3.29 and 3.31
/
0.55 (ethyl acetate). 1H-NMR
3
3
2ꢃ
8.5 Hz, 1H, CHCO2); 5.12 (s, 2H, CH2Ph); 5.94 (d,
3JHH
8.5 Hz, 1H, NH); 7.34 (m, 5H, Ph).
/
2H, CO2CH2(1,1?)); 4.68 (td, JHH
ꢁ
/
4.6 Hz, JHH
ꢁ
/
/
/
ꢁ
/
/
ꢁ
/
ꢁ
/
(2s, 6H, CH3O); 3.52 (m, 4H, CH2O(2,2?)); 4.06 (s, 2H,
13C-NMR CDCl3/100 MHz: d (ppm): 37.1 (s,
CH2CO2); 50.9 (s, CHCO2); 59.4 (s, CH3O); 64.4 and
65.2 (2s, CO2CH2(1,1?)); 67.5 (s, CH2Ph); 69.2 (2s,
CH2O(2,2?)); 70.8 and 70.9 (2s, CH2O(4,4?)); 72.2 (2s,
CH2Cl); 4.18 and 4.23 (2m, 2H, CO2CH2(1,1?)); 4.84 (td,
3
4.6 Hz, JHH
3JHH
3JHH
ꢁ
/
ꢁ7.8 Hz, 1H, CHCO2); 7.52 (d,
/
ꢁ
7.9 Hz, NH). 13C-NMR CDCl3/100 MHz: d
/
(ppm): 36.4 (s, CH2CO2); 42.6 (s, CH2Cl); 49.4 (s,
CHCO2); 59.3 (s, CH3O); 64.4 and 65.1 (2s,
CO2CH2(1,1?)); 70.4 (2s, CH2O(2,2?)); 164.7 (s, CONH);
CH2O(5,5?)); 128.5ꢀ
156.4 (s, CONH); 170.9 and 171.0 (2s, CO2).
MS (Fabꢄ/NBA): m/zꢁ472 [Mꢄ
H]ꢄ.
/
128.9 (4s, C82 , C38, C48); 136.6 (s, C81 );
/
/
/
170.5 and 170.9 (2s, CO2). MS (Fabꢄ
/
/NBA): m/zꢁ326
/
[Mꢄ
/
H]ꢄ.
4.5.3. Bis 2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl-N-
benzyloxycarbonyl- -aspartate (10)
Yield: 80%. Rfꢁ
0.34 (ethyl acetate). 1H-NMR
CDCl3/400 MHz: d (ppm): 2.97 (AB part of an ABX
system, dAꢁ3.14, dBꢁ 17.1 Hz,
2.80, 1JAB
3JAXꢁ3
JBX 4.9 Hz, 2H, CH2CO2); 3.35 (s, 6H,
CH3O); 3.52 (m, 4H, CH2O(8,8?)); 3.63 (m, 16H,
CH2O(2ꢀ7,2?ꢀ7’)); 4.21 and 4.29 (2m, 2ꢃ2H, CO2CH2);
8.6 Hz, 1H, CHCO2);
L
4.6.2. Bis-[2-(2-methoxy-ethoxy)-ethyl]-N-
chloroacetyl- -aspartate (15)
Yield: 70%. Rfꢁ
0.27 (ethyl acetate). 1H-NMR
CDCl3/200 MHz: d (ppm): 3.00 (AB part of an ABX
system, dAꢁ3.09, dBꢁ 17.3 Hz,
2.91, 1JAB
3JAXꢁ3
JBX 4.6 Hz, 2H, CH2CO2); 3.37 (s, 6H,
/
L
/
/
/
ꢁ
/
/
ꢁ
/
/
/
ꢁ
/
/
ꢁ
/
/
CH3O); 3.53 (m, 4H, CH2O(5,5?)); 3.63 (m, 4H,
CH2O(4,4?)); 3.68 (m, 4H, CH2O(2,2?)); 4.06 (s, 2H,
3
3
4.65 (td, JHH
ꢁ
/
4.6 Hz, JHH
ꢁ
/
3
5.10 (s, 2H, CH2Ph); 5.91 (d, JHH
ꢁ/8.8 Hz, 1H, NH);
ClCH2); 4.25 and 4.37 (2m, 2ꢃ
/
2H, CO2CH2(1,1?));
8.1 Hz, 1H, CHCO2);
8.0 Hz, NH). 13C-NMR CDCl3/100
3
4.85 (td, JHH
3
7.55 (d, JHH
3
7.33 (m, 5H, Ph).
ꢁ
/
4.6 Hz, JHH
ꢁ
/
13C-NMR CDCl3/100 MHz: d (ppm): 36.5 (s,
CH2CO2); 49.3 (s, CHCO2); 59.4 (s, CH3O); 64.5 and
65.1 (2 s, CO2CH2(1,1?)); 67.5 (s, CH2Ph); 69.1 (2s,
CH2O(2,2?)); 71.0 (4s, CH2O(4ꢀ7,4?7?)); 72.3 (s,
ꢁ
/
MHz: d (ppm): 36.6 (s, CH2CO2); 42.7 (s, ClCH2); 49.4
(s, CHCO2); 59.4 (s, CH3O); 64.4 and 65.1 (2s,
CO2CH2(1,1?)); 69.2 (2s, CH2O(2,2?)); 70.8 (2s,
CH2O(4,4?)); 72.2 (2s, CH2O(5,5?)); 164.2 (s, CONH);
CH2O(8,8?)); 128.5ꢀ
128.9 (4s, C82 , C38, C84 ); 136.6 (s,
/

V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ
/891
889
170.3 and 171.5 (2s, CO2). MS (Fabꢄ
/
/NBA): m/zꢁ/414
4.7.1. Bis-(2-methoxy-ethyl)-O,O-bis-(S-acetyl-3-
thiopropyl)-N-phosphonoacetyl- -aspartate (23)
[Mꢄ
/
H]ꢄ.
L
Yield: 16%. Rfꢁ
/
0.50 (ethyl acetateꢀmethanol 90/10).
/
1H-NMR CDCl3/200 MHz: d (ppm): 1.90 (qtd, JHH
ꢁ
/
3
4.6.3. Bis 2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl-N-
chloroacetyl- -aspartate (16)
4
6.9 Hz, JHP
ꢁ2.7 Hz, 4H, CH2); 2.27 (s, 6H, CH3CO);
/
L
2.82ꢀ
/
3.03 (m, 8H, CH2CO2, PCH2CO, CH2S); 3.29 and
Yield: 80%. Rfꢁ
/
0.70 (ethyl acetateꢀmethanol 90/10).
/
3.31 (2s, 6H, CH3O); 3.52 (m, 4H, CH2O(2,2?)); 4.09 (m,
1H-NMR CDCl3/400 MHz: d (ppm): 3.01 (AB part of
4H, POCH2); 4.17 and 4.23 (2m, 2ꢃ
/2H, CO2CH2(1,1?));
1
2.93, JAB
an ABX system, dAꢁ
/
3.09, dBꢁ
/
ꢁ17.2 Hz,
/
3
4.82 (td, JHH
3
4.6 Hz, JHH
ꢁ
ꢁ
/
ꢁ8.0 Hz, 1H, CHCO2);
/
3
3JAX
CH3O); 3.55 (m, 4H, CH2O(8,8?)); 3.65 (m, 12H,
CH2O(4ꢀ7,4?ꢀ7?)); 3.70 (m, 4H, CH2O(2,2?)); 4.08 (s, 2H,
ꢁ
/
4.7, JBX
ꢁ4.6 Hz, 2H, CH2CO2); 3.37 (s, 6H,
/
3
7.37 (d, JHH
/
7.9 Hz, NH). 13C-NMR CDCl3/100
3
MHz: d (ppm): 25.6 (s, CH2S); 30.8 (d, JPC
ꢁ/6.3 Hz,
1
CH2); 31.0 (s, CH3CO); 35.4 (d, JPC
ꢁ/131.5 Hz,
ClCH2); 4.22ꢀ
/
4.38 (m, 4H, CO2CH2(1,1?)); 4.88 (td,
PCH2CO); 36.6 (s, CH2CO2); 49.4 (s, CHCO2); 59.3 (s,
CH3O); 64.3 and 65.1 (2s, CO2CH2(1,1?)); 65.4 and 65.5
3
4.6 Hz, JHH
3JHH
3JHH
ꢁ
/
ꢁ8.1 Hz, 1H, CHCO2); 7.62 (d,
/
ꢁ
/
8.0 Hz, NH).13C-NMR CDCl3/100 MHz: d
2
(2d, JPC
2
(d, JPC
ꢁ/6.1 Hz, POCH2); 70.5 (2s, CH2O(2,2?)); 164.2
(ppm): 36.4 (s, CH2CO2); 42.7 (s, ClCH2); 49.3 (s,
CHCO2); 59.4 (s, CH3O); 64.5 and 65.2 (2s,
CO2CH2(1,1?)); 69.1 and 69.2 (2s, CH2O(2,2?)); 70.9 (4s,
CH2O(4ꢀ7,4?ꢀ7?)); 72.3 (s, CH2O(8,8?)); 166.4 (s, CONH);
ꢁ/4.6 Hz, CONH); 170.5 and 170.9 (2s, CO2);
196.0 (2s, COS). 31P-NMR CDCl3/250 MHz: d (ppm):
23.4. MS (Fabꢄ
[MeCOSCH2CH2CH2]ꢄ.
[a]Dꢁ 20.48 (cꢁ
C22H38NO12PS2 (C, H, N).
/
/NBA): m/zꢁ
/
604 [Mꢄ
/
H]ꢄ, 117
170.3 and 171.5 (2s, CO2). MS (Fabꢄ
/
/NBA): m/zꢁ502
/
/
ꢄ
/
/0.2;
CHCl3).
Anal.
[Mꢄ
/
H]ꢄ.
4.7. Bis(polyethyleneglycol monomethylether)-O,O-bis-
(S-acyl-3-thiopropyl)-N-phosphonoacetyl- -aspartate
23ꢀ28
4.7.2. Bis-[2-(2-methoxy-ethoxy)-ethyl]-O,O-bis(S-
-aspartate
(24)
L
acetyl-3-thiopropyl)-N-phosphonoacetyl-
L
/
Yield: 53%. Rfꢁ
/
0.34 (ethyl acetateꢀ
/
methanol 95/5).
3
In a round bottom flask fitted with a distillation
system, a mixture of chloroacetyl- -aspartate 14ꢀ16 (2.0
1H-NMR CDCl3/200 MHz: d (ppm): 1.94 (qtd, JHH
ꢁ
/
4
6.6 Hz, JHP
L
/
ꢁ2.6 Hz, 4H, CH2); 2.31 (s, 6H, CH3CO);
/
mmol, 1 equiv.) and tris(trimethylsilyl)phosphite (3.6
mmol, 1.8 equiv.) was heated at 160 8C. 30 min later the
formed trimethylsilylchloride began to distil. After 2 h,
the mixture was allowed to cool at 60 8C and the excess
of tris(trimethylsilyl)phosphite was removed under re-
2.86ꢀ3.05 (m, 8H, CH2CO2, PCH2CO, CH2S); 3.36 (2s,
/
6H, CH3O); 3.56 (m, 4H, CH2O(5,5?)); 3.61 (m, 4H,
CH2O(4,4?)); 3.67 (m, 4H, CH2O(2,2?)); 4.13 (m, 4H,
POCH2); 4.23 and 4.30 (2m, 22H, CO2CH2(1,1?)); 4.85
3
3
(td, JHH
3
(d, JHH
ꢁ
/
4.8 Hz, JHH
ꢁ
7.4 Hz, NH). 13C-NMR CDCl3/100 MHz: d
ꢁ7.9 Hz, 1H, CHCO2); 7.43
/
duced pressure. Compounds 17ꢀ
pale yellow oil and were used without further purifica-
tion. Derivatives 17ꢀ19 (ꢁ2 mmol, 1 equiv.) were
/19 were obtained as
/
(ppm): 25.6 (s, CH2S); 30.8 (d, 3JPC
1
(s, CH3CO); 35.4 (d, JPC
ꢁ6.4 Hz, CH2); 31.0
/
/
/
ꢁ131.7 Hz, PCH2CO); 36.6
/
dissolved in 7 mL of dichloromethane and 10 drops of
DMF were added as a catalyst. To the solution, cooled
for 10 min at 0 8C, 0.590 mL of oxalyl chloride (7.0
mmol, 3.5 equiv.) was added dropwise and the mixture
was stirred for 3 h 30 min at room temperature. The
volatile components were removed under reduced pres-
(s, CH2CO2); 49.4 (s, CHCO2); 59.4 (s, CH3O); 64.4 and
65.1 (2s, CO2CH2(1,1?)); 65.4 and 65.5 (2d, 2JPC
5.9 Hz,
POCH2); 69.2 (2s, CH2O(2,2?)); 70.8 (2s, CH2O(4,4?)); 72.2
ꢁ
/
2
(2s, CH2O(5,5?)); 164.3 (d, JPC
and 170.9 (2s, CO2); 196.0 (2s, COS). 31P-NMR CDCl3/
250 MHz: d (ppm): 23.5. MS (Fabꢄ/NBA): m/zꢁ692
[Mꢄ 15.28 (cꢁ0.2; CHCl3). Anal.
H]ꢄ. [a]Dꢁ
C26H46NO14PS2 (C, H, N).
ꢁ
/
4.3 Hz, CONH); 170.5
/
/
sure and the P,P-dichloro-N-phosphonoacetyl-
tate 20ꢀ22, obtained as a brown oil, was rapidly used
without further purification. Compounds 20ꢀ22 (ꢁ2
L-aspar-
/
/
ꢄ
/
/
/
/
/
mmol, 1 equiv.) was dissolved in 10 mL of dichlor-
omethane and cooled in an ice bath. A mixture of S-
acyl-3-thiopropyl alcohol 5 or 6 (6.0 mmol, 3 equiv.) and
triethylamine (12.0 mmol, 6 equiv.) in 10 mL of
dichloromethane was added dropwise and the reaction
was stirred for 2 h at room temperature. The mixture
was diluted in 150 mL of dichloromethane and washed
three times with brine. The organic layer was dried over
Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel column chro-
4.7.3. Bis 2-[2-(2-methoxy-ethoxy)-ethoxy]-ethyl-O,O-
bis(S-acetyl-3-thiopropyl)-N-phosphonoacetyl-L-
aspartate (25)
Yield: 32%. Rfꢁ
1H-NMR CDCl3/400 MHz: d (ppm): 1.90 (qt, JHH
6.6 Hz, 4H, CH2); 2.27 (s, 6H, CH3CO); 2.80ꢀ3.00 (m,
/
0.17 (ethyl acetateꢀmethanol 90/10).
/
3
ꢁ
/
/
8H, CH2CO2, PCH2CO, CH2S); 3.31 (s, 6H, CH3O);
3.48 (m, 4H, CH2O(8,8?)); 3.57 (m, 12H, CH2O(4ꢀ7,4?ꢀ7?));
3.62 (m, 4H, CH2O(2,2?)); 4.09 (m, 4H, POCH2); 4.17 and
3
2H, CO2CH2(1,1?)); 4.81 (td, JHH
4.24 (2m, 2ꢃ
/
ꢁ
/
4.8
3
Hz, JHH
3
7.9 Hz, 1H, CHCO2); 7.47 (d, JHH
matography to give compounds 23ꢀ
/
28 as brown oil.
ꢁ
/
ꢁ
/
7.9

890
V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ891
/
Hz, NH). 13C-NMR CDCl3/100 MHz: d (ppm): 25.6 (s,
zꢁ
/
776 [Mꢄ
/
H]ꢄ. [a]Dꢁ
Anal. C32H58NO14PS2 (C, H, N).
/
ꢄ
/
17.68 (cꢁ0.2; CHCl3).
/
3
CH2S); 30.8 (d, JPC
ꢁ6.4 Hz, CH2); 31.0 (s, CH3CO);
/
1
35.7 (d, JPC
49.4 (s, CHCO2); 59.4 (s, CH3O); 64.5 and 65.2 (2s,
ꢁ132.7 Hz, PCH2CO); 36.7 (s, CH2CO2);
/
2
4.7.6. Bis-[2-(2-methoxy-ethoxy)-ethyl]-O,O-bis(S-
pivaloyl-3-thiopropyl)-N-phosphonoacetyl-
(28)
CO2CH2(1,1?)); 65.4 (d, JPC
2JPC
4.4 Hz, POCH2); 69.2 (2s, CH2O(2,2?)); 70.9 (3s,
CH2O(4ꢀ7,4?ꢀ7?)); 72.3 (s, CH2O(8,8?)); 164.4 (d, 2JPC
4.8
Hz, CONH); 170.5 and 170.9 (2s, CO2); 196.0 (2s, COS).
31P-NMR CDCl3/250 MHz: d (ppm): 23.6. MS (Fabꢄ
NBA): m/zꢁ780 [Mꢄ 117
H]ꢄ,
[MeCOSCH2CH2CH2]ꢄ, 43 [MeCO]ꢄ. [a]Dꢁ
17.58
(cꢁ0.2; CHCl3). Anal. C30H54NO16PS2 (C, H, N).
ꢁ
/
4.1 Hz, POCH2); 65.5 (d,
L-aspartate
ꢁ
/
ꢁ
/
Yield: 27%. Rfꢁ
/
0.45 (ethyl acetateꢀmethanol 90/10).
/
1H-NMR CDCl3/400 MHz: d (ppm): 1.16 (s, 18H,
//
CH3(tBu)); 1.86 (qtd, JHH
ꢁ
/
6.6 Hz, 4H, CH2); 2.82ꢀ
/
3
/
/
2.95 (m, 8H, CH2CO2, PCH2CO, CH2S); 3.31 (s, 6H,
CH3O); 3.49 (m, 4H, CH2O(8,8?)); 3.57 (m, 12H,
CH2O(4ꢀ7,4?ꢀ7?)); 3.62 (m, 4H, CH2O(2,2?)); 4.09 (q,
/
ꢄ
/
/
3JHHꢁ3
2ꢃ2H, CO2CH2(1,1?)); 4.80 (td, JHH
8.2 Hz, 1H, CHCO2); 7.45 (d, 3JHH
NMR CDCl3/100 MHz: d (ppm): 25.0 (s, CH2S); 27.8
/
JHP
ꢁ
/
7.1 Hz, POCH2); 4.17 and 4.24 (2m,
4.7.4. Bis-(2-methoxy-ethyl)-O,O-bis(S-pivaloyl-3-
thiopropyl)-N-phosphonoacetyl- -aspartate (26)
Yield: 25%. Rfꢁ0.33 (ethyl acetateꢀmethanol 95/5).
1H-NMR CDCl3/200 MHz: d (ppm): 1.16 (s, 18H,
3
3
/
ꢁ
/
4.9 Hz, JHH
ꢁ
/
L
ꢁ
/
7.9 Hz, NH). 13C-
/
/
3
CH3(tBu)); 1.87 (qtd, JHH
ꢁ/6.9 Hz, JHP
ꢁ2.5 Hz, 4H,
/
3
4
(s, CH3(tBu)); 30.9 (d, JPC
1JPC
132.7 Hz, PCH2CO); 36.6 (s, CH2CO2); 46.8 (s,
(tBu)); 49.4 (s, CHCO2); 59.4 (2s, CH3O); 64.5 and 65.2
ꢁ/6.1 Hz, CH2); 35.5 (d,
ꢁ
/
CH2); 2.80ꢀ3.02 (m, 8H, CH2CO2, PCH2CO, CH2S);
/
C
3.29 and 3.31 (2s, 6H, CH3O); 3.52 (m, 4H, CH2O(2,2?));
4.08 (m, 4H, POCH2); 4.17 and 4.23 (2m, 22H,
2
(2s, CO2CH2(1,1?)); 65.7 (2d, JPC
69.2 (2s, CH2O(2,2?)); 70.9 and 70.9 (2s, CH2O(4ꢀ7,4?ꢀ7?));
ꢁ4.1 Hz, POCH2);
/
3
CO2CH2(1,1?)); 4.82 (td, JHH
3
4.6 Hz, JHH
ꢁ
/
ꢁ8.0 Hz,
/
2
3
1H, CHCO2); 7.38 (d, JHH
ꢁ
/
8.0 Hz, NH). 13C-NMR
CDCl3/100 MHz: d (ppm): 25.0 (s, CH2S); 27.8 (s,
72.3 (2s, CH2O(8,8?)); 164.4 (d, JPC
170.5 and 170.9 (2s, CO2); 208.0 (s, COS). 31P-NMR
CDCl3/250 MHz: d (ppm): 23.6. MS (Fabꢄ/NBA): m/
zꢁ864 [Mꢄ
H]ꢄ, 159 [tBuCOSCH2CH2CH2]ꢄ. [a]Dꢁ
18.18 (cꢁ0.2; CHCl3). Anal. C36H66NO16PS2 (C, H,
N).
ꢁ/4.7 Hz, CONH);
3
CH3(tBu)); 30.9 (d, JPC
1
6.2 Hz, CH2); 35.5 (d, JPC
/
ꢁ
/
ꢁ
/
/
/
/
131.5 Hz, PCH2CO); 36.6 (s, CH2CO2); 46.8 (s, C(tBu));
49.4 (s, CHCO2); 59.3 (s, CH3O); 64.3 (s, CO2CH2(1?));
ꢄ
/
/
2
65.1 (s, CO2CH2(1)); 65.6 and 65.7 (2d, JPC
2
POCH2); 70.4 (2s, CH2O(2,2?)); 164.2 (d, JPC
ꢁ
/
6.3 Hz,
4.2 Hz,
ꢁ
/
CONH); 170.5 and 170.9 (2s, CO2); 206.9 (2s, COS).
31P-NMR CDCl3/250 MHz: d (ppm): 23.4. MS (Fabꢄ
NBA): m/zꢁ688 [Mꢄ 159
H]ꢄ,
[tBuCOSCH2CH2CH2]ꢄ, 85 [tBuCO]ꢄ. [a]Dꢁ
19.58
(cꢁ0.2; CHCl3). Anal. C28H50NO12PS2 (C, H, N).
//
5. Biological studies
/
/
/
ꢄ
/
/
5.1. Cell culture
4.7.5. Bis-[2-(2-methoxy-ethoxy)-ethyl]-O,O-bis(S-
L-aspartate
SW1573 lung carcinoma cell line [22] (obtained from
ATCC) was maintained in Dubelcco’s modified Eagle
medium supplemented with 2 mM glutamine, 10% fetal
calf serum and gentamycin at 37 8C in 5% CO2
humidified atmosphere for one week. After trypsination
pivaloyl-3-thiopropyl)-N-phosphonoacetyl-
(27)
Yield: 53%. Rfꢁ
/
0.30 (ethyl acetateꢀmethanol 95/5).
/
1H-NMR CDCl3/200 MHz: d (ppm): 1.16 (s, 18H,
CH3(tBu)); 1.87 (qtd, JHH
ꢁ
/
ꢁ
/
2.7 Hz, 4H,
3
4
6.6 Hz, JHP
cells were seeded at 2ꢃ
104 cells/well in 96 wells plate
and maintained in medium for24 h.
/
CH2); 2.80ꢀ3.00 (m, 8H, CH2CO2, PCH2CO, CH2S);
/
3.31 and 3.32 (2s, 6H, CH3O); 3.47 (m, 4H, CH2O(5,5?));
3.56 (m, 4H, CH2O(4,4?)); 3.62 (m, 4H, CH2O(2,2?)); 4.08
(m, 4H, POCH2); 4.18 and 4.27 (2m, 2ꢃ
/
2H,
7.9 Hz,
7.9 Hz, NH). 13C-NMR
5.2. Cytotoxicity assay
3
CO2CH2(1,1?)); 4.80 (td, JHH
3
ꢁ
/
4.7 Hz, JHH
ꢁ
/
3
1H, CHCO2); 7.38 (d, JHH
ꢁ
/
Cells were treated with PALA or derivatives at
various concentrations for 48 h. Medium was replaced
by the one containing neutral red for 4 h. After two
washes in phosphate buffered saline (PBS), cells were
lysed in 50% ethanol, 1% acetic acid solution and
incubated for 15 min at room temperature. Absorbance
at 540 nm was determined and used for the measure-
ment of the proportion of surviving cells. The data
represent the average value of four separate experiments
each one performed in triplicate.
CDCl3/100 MHz: d (ppm): 25.0 (s, CH2S); 28.8 (s,
1
6.3 Hz, CH2); 35.4 (d, JPC
3
CH3(tBu)); 30.9 (d, JPC
ꢁ
/
ꢁ
/
132.3 Hz, PCH2CO); 36.6 (s, CH2CO2); 46.8 (s, C(tBu));
49.4 (s, CHCO2); 59.4 (2s, CH3O); 64.4 and 65.1 (2s,
2
CO2CH2(1,1?)); 65.6 (2d, JPC
and 69.3 (2s, CH2O(2,2?)); 70.8 and 70.9 (2s, CH2O(4,4?));
ꢁ6.1 Hz, POCH2); 69.2
/
2
72.2 (2s, CH2O(5,5?)); 164.3 (d, JPC
170.5 and 170.9 (2s, CO2); 206.9 (s, COS). 31P-NMR
CDCl3/250 MHz: d (ppm): 23.4. MS (Fabꢄ/NBA): m/
ꢁ
/
4.6 Hz, CONH);
/

V. Gagnard et al. / European Journal of Medicinal Chemistry 38 (2003) 883ꢀ
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891
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Acknowledgements
/
The authors gratefully acknowledge the Ministe´re de
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.
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