Acyclic nucleoside thiophosphonates as potent inhibitors of HIV and HBV replication

Article abstract of DOI:10.1016/j.ejmech.2011.06.034

9-[2-(Thiophosphonomethoxy)ethyl]adenine 3 and (R)-9-[2- (Thiophosphonomethoxy)propyl]adenine 4 were synthesized as the first thiophosphonate nucleosides bearing a sulfur atom at the α-position of the acyclic nucleoside phosphonates PMEA and PMPA. Thiophosphonates S-PMEA 3 and S-PMPA 4 were evaluated for in vitro activity against HIV-1 (subtypes A to G), HIV-2 and HBV-infected cells, and found to exhibit potent antiretroviral activity. We showed that their diphosphate forms S-PMEApp 5 and S-PMPApp 6 are readily incorporated by wild-type (WT) HIV-1 RT into DNA and act as DNA chain terminators. Compounds 3 and 4 were evaluated for in vitro activity against a broad panel of DNA and RNA viruses and displayed beside HIV a moderate activity against herpes simplex virus and vaccinia viruses. In order to measure enzymatic stabilities of the target derivatives 3 and 4, kinetic data and decomposition pathways were studied at 37 °C in several media.

Full text of DOI:10.1016/j.ejmech.2011.06.034

European Journal of Medicinal Chemistry 46 (2011) 4281e4288
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Acyclic nucleoside thiophosphonates as potent inhibitors of HIV and HBV
replication
Karine Barral a, Clément Weck a, Nadine Payrot a, Loic Roux a, Céline Durafour b, Fabien Zoulim b,
,
a,
*
Johan Neyts c, Jan Balzarini c, Bruno Canard a, Stéphane Priet a , Karine Alvarez
*
a Laboratoire d’Architecture et Fonction des Macromolécules Biologiques, UMR CNRS 6098, Equipe “Réplicases Virales: Structure, Mécanisme, et Drug-design”,
Universités Aix-Marseille I et II, Parc scientifique de Luminy, 163 av. de Luminy, 13288 Marseille Cedex 9, France
b INSERM, U871, Université de Lyon, Hospices Civils de Lyon, 69003 Lyon, France
c Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, K.U. Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
9-[2-(Thiophosphonomethoxy)ethyl]adenine 3 and (R)-9-[2-(Thiophosphonomethoxy)propyl]adenine 4
were synthesized as the first thiophosphonate nucleosides bearing a sulfur atom at the a-position of the
Received 16 March 2011
Received in revised form
21 June 2011
Accepted 22 June 2011
Available online 1 July 2011
acyclic nucleoside phosphonates PMEA and PMPA. Thiophosphonates S-PMEA 3 and S-PMPA 4 were
evaluated for in vitro activity against HIV-1 (subtypes A to G), HIV-2 and HBV-infected cells, and found to
exhibit potent antiretroviral activity. We showed that their diphosphate forms S-PMEApp 5 and S-
PMPApp 6 are readily incorporated by wild-type (WT) HIV-1 RT into DNA and act as DNA chain
terminators. Compounds 3 and 4 were evaluated for in vitro activity against a broad panel of DNA and
RNA viruses and displayed beside HIV a moderate activity against herpes simplex virus and vaccinia
viruses. In order to measure enzymatic stabilities of the target derivatives 3 and 4, kinetic data and
decomposition pathways were studied at 37 ꢀC in several media.
Keywords:
Modified nucleotide
Thiophosphonate
Antiviral activity
HIV
Ó 2011 Elsevier Masson SAS. All rights reserved.
HBV
1. Introduction
The acyclic nucleoside phosphonates (ANPs) [2,3] belong to
NtRTIs and contain a stable phosphonate group. Therefore, NtRTIs
The reverse transcriptase (RT) of Human Immunodeficiency
Virus (HIV) converts the single-stranded viral genomic RNA into
double-stranded DNA, which is then integrated into cellular DNA.
Thus, RT plays a key role in the viral life cycle and is therefore an
important target for antiretroviral drugs such as nucleoside (NRTI)
and nucleotide (NtRTI) RT inhibitors [1]. During RT-mediated
conversion of viral genomic RNA into proviral DNA, nucleoside
and nucleotide analogs can fool RT which can use their 50-triphos-
phate form as a substrate. Because these analogs lack a 30-OH group,
DNA chain termination occurs and accounts for an antiretroviral
effect.
require only two phosphorylation steps to be converted into their
diphosphate active form [4]. Hence, NtRTIs bypass the often rate-
limiting nucleoside-kinase step that is known to moderate the
activity of NRTIs [5]. To date, only two ANPs have been approved as
antiretroviral drugs. Adefovir [9-(2-Phosphonomethoxyethyl)
adenine, PMEA] was originally developed for its potential in the
treatment of HIV, but its prodrug adefovir dipivoxil proved to be
nephrotoxic [6] at the dosage required to efficiently inhibit HIV
replication. However, adefovir was subsequently approved for the
treatment of chronic hepatitis B virus infections at a lower dosage of
administration [3]. Tenofovir [(R)-9-(2-Phosphonomethoxypropyl)-
adenine, (R)-PMPA [7], referred hereinafter as PMPA], administered
as the prodrug tenofovir disoproxil fumarate, is nowadays one of the
most widely prescribed anti-HIV drugs in the US and Europe.
Recently, tenofovir was also approved for the treatment of chronic
hepatitis B [8,9].
Abbreviations: HIV, human immunodeficiency virus; HBV, hepatitis B virus; RT,
reverse transcriptase; NRTIs, nucleoside RT inhibitors; NtRTIs, nucleotide RT
inhibitors; dNTP, deoxynucleotide; ANPs, acyclic nucleoside phosphonates; PMEA,
9-[2-phosphonomethoxyethyl]adenine; (R)-PMPA, (R)-9-[2-phosphonomethoxypr-
To widen the antiviral spectrum of ANPs [3], various structural
modifications of PMEA and PMPA have been performed, including
structural diversity at both the side chain [10,11] and the hetero-
cyclic moiety of these drugs [2,12e16]. Little attention has been
given to explore the phosphonate moiety modifications, and efforts
opyl]adenine; PBMC, peripheral blood mononuclear cells; TCID50
culture infectious dose; MOI, multiplicity of infection.
, 50% tissue
* Corresponding authors. Tel.: þ33 491 825 571; fax: þ33 491 266 720.
E-mail addresses: Stephane.priet@afmb.univ-mrs.fr (S. Priet), Karine.Alvarez@
afmb.univ-mrs.fr (K. Alvarez).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.06.034

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K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
were mainly focused on prodrug synthesis [17,18]. Therefore, we
decided to explore the different modifications of the phosphonate
group and we designed the substitution of one of the oxygen atoms
bearing by the phosphorus by several groups and atoms. In 2006,
we developed an original method to synthesize an H-phosphinate
intermediate [19] that can easily serve as a precursor to generate
phosphorus-modified nucleotides. This key step was first applied to
For the diphosphate forms synthesis, thiophosphonates 3 and 4
were activated by carbonyldiimidazole in DMF at ambient
temperature. Intermediates obtained reacted with a solution of
0.5 M tributylammonium pyrophosphate in DMF in the presence of
tributylamine, to lead to the thiophosphonate diphosphate deriv-
ative 5 named S-PMEApp (17%) and 6 named S-PMPApp (19%) after
purification and passage through Dowex ion exchange (Naþ).
Compound S-PMEApp 5 is obtained as an enantiomeric mixture,
compound S-PMPApp 6 is obtained as a diastereoisomeric mixture.
Non-thio phosphonate diphosphates PMEApp 5a and PMPApp
6a, derived from PMEA and PMPA, that served as referenced
compounds, were synthesized as described in the literature [15].
synthesize nucleoside
a-boranophosphonate [19e21] and nucleo-
side -selenophosphonate (unstable compounds, data not pub-
a
lished) derived from PMEA and PMPA.
Here, we report the synthesis of novel PMEA and PMPA
derivatives bearing a sulfur atom at the
a-position of acyclic nuc-
leoside phosphonates. -Thiophosphonates S-PMEA 3 and S-PMPA
a
4 (Fig. 1) exhibit robust antiretroviral activity in cells infected by
HIV-1 (LAI strain, and viral isolates from subtypes A to G), HIV-2 as
well as HBV. To confirm the mechanism of action of these novel
compounds on the viral polymerase, we explored in vitro drug
susceptibility of recombinant wild-type (WT) HIV-1 RT from
subtype B to the diphosphate forms S-PMEApp 5 and S-PMPApp 6
(Fig. 1) using steady-state kinetic analyses. In addition, the anti-
viral potential of the compounds 3 and 4 was evaluated by
monitoring the reduction of the cytopathic effect of a broad panel
of DNA and RNA viruses. Then, we evaluated the stability of our
compounds 3 and 4 under conditions mimicking biological fluids.
2.2. Anti-HIV activity of S-PMEA 3 and S-PMPA 4 against wild-type
(WT) HIV-1 (subtype B) and cytotoxicity in cells
To evaluate the activity of the target compounds, a cell-based
antiviral drug susceptibility assay was performed using subtype B
WT HIV-1 (LAI, IIIB) and HIV-2 (ROD) (Table 1). CEM are treated
with increasing concentrations of S-PMEA 3, S-PMPA 4, and refer-
ence compounds PMEA or PMPA, and in vitro infected with 100
TCID50 (50% tissue culture infectious doses) of HIV-1 (IIIB) and HIV-
2 (ROD). T-lymphocyte H9 cells and activated peripheral blood
mononuclear cells (PBMC) are treated with increasing concentra-
tions of S-PMEA 3, S-PMPA 4, and reference compounds PMEA or
PMPA, and in vitro infected, respectively with 125 and 100 TCID50
(50% tissue culture infectious doses) of HIV-1 (LAI). Similar exper-
iments were performed in H9 cells infected with 6250 TCID50 of
HIV-1 (LAI) in order to evaluate the impact of viral load on the anti-
HIV activity of the thiophosphonates. On day 7 post infection,
reverse transcriptase (RT) activity in cell culture medium was
measured to determine the antiviral properties of the compounds
expressed as 50% reduction of viral replication (half maximal
effective concentration, EC50) (H9, PBMC) or on day 3 post infection
(CEM), virus-induced cytopathicity was microscopically recorded
(Table 1). Concomitantly, the antimetabolic effect (50% cytotoxic
concentration, CC50) of each compound was evaluated in CEM, H9
as well as in uninfected PBMC cultures.
These novel antiviral nucleoside
by us [22].
a-thiophosphonates are patented
2. Results and discussion
2.1. Chemistry
Thiophosphonates S-PMEA 3 and S-PMPA 4 are prepared in five
steps. The four first steps were previously published [19] for the
synthesis of
a-boranophosphonate nucleosides and are briefly
described here. The first step consists in specifically substituting
adenine in order to obtain 9-(2-hydroxyethyl)adenine for the
S-PMEA series and (R)-9-(2-hydroxypropyl)adenine for the S-PMPA
series. Intermediates are then coupled by nucleophilic substitution
to diethyl[[(p-toluenesulfonyl)oxy]methyl]phosphonate, prepared
beforehand, to lead to the corresponding diethylphosphonates.
Diethylphosphonates are reduced to phosphines by means of
lithium aluminum hydride and trimethylsilyl chloride in anhydrous
THF at ꢁ78 ꢀC. Then oxidation of the corresponding phosphines, in
the presence of hydrogen peroxide in a water-THF mixture, lead,
with a quantitative yield, to the key intermediates H-phosphinates
1 and 2 (Scheme 1).
Finally, the thiophosphonate derivatives S-PMEA 3 and S-PMPA
4 are obtained with yields of 52% and 47% respectively, in the
presence of BSA and S8/CS2/pyridine in anhydrous pyridine. After
reverse-phase purification, they are dissolved in water and passed
over a Dowex 50WX2 ion exchange column (Naþ form) with
a quantitative yield.
The thiophosphonate compounds S-PMEA 3 and S-PMPA 4
display a good activity (EC50 1.9 mM and 0.38 mM, respectively) in H9
cells infected with 125 TCID50. These results show that thio-
phosphonates are able to penetrate cells, to be phosphorylated and
to efficiently inhibit HIV-1 replication (subtype B). Antiviral activity
is also observed in CEM and in PBMC cell cultures infected with
a similar viral load (100 TCID50), indicating that thiophosphonates
are able to inhibit HIV-1 replication in other T-lymphocyte cell
cultures as well, including primary cells. However, higher EC50
values are obtained with S-PMEA 3 and S-PMPA 4 as well as
reference compounds PMEA and PMPA in CEM- and PBMC-infected
cells, suggesting that the difference between the two cell types
could be attributed to different origin and/or activation states for
each cell-type and therefore different phosphorylation properties
Fig. 1. Structure of thiophosphonates S-PMEA 3 and S-PMPA 4 and their diphosphate forms S-PMEApp 5 and S-PMPApp 6, and the structure of the diphosphate forms PMEApp 5a
and PMPApp 5b derived from PMEA and PMPA.

K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
4283
Scheme 1. Synthetic Pathway for the Synthesis of compounds 3, 4, 5 and 6. Reagents and conditions: (a) BSA, S8/CS2/pyridine, pyridine, rt, 1 h 30 min; then H2O, then dowex Naþ
exchange after purification. (b) Carbonyldiimidazole, DMF, rt, 4 h; then MeOH; (c) HBu3NþH2PO4ꢁ, Bu3N, DMF, rt, 72 h; then dowex Naþ exchange after purification.
of NtRTI or cellular uptake. The thiophosphonates are also effective
against HIV-2 (Table 1). S-PMPA 4 tends to be somewhat more
active than S-PMEA 3. With a 50-fold higher multiplicity of infec-
tion (MOI) in H9 cells, activity of thiophosphonates as well as the
non-thiophosphonates is affected, as expected, with increased EC50
values from 7- to 25-fold. Thus, these results show that the differ-
ence between thio and non-thiophosphonates remained constant
independently from the MOI virus input. However, it should be
noticed that the standard deviation for a number of EC50 values
were rather high, being possibly due to inter-individual variability
between PBMC donors, and masking potential subtle differences in
antiviral potency between the compounds.
Interestingly, the thiophosphonate modification does not
significantly modify the anti-HIV activity of the acyclic nucleoside
phosphonate compounds, although S-PMEA 3 tends to display
a slightly increased EC50 (1.7- to 4-fold) as compared to PMEA and
S-PMPA 4 a 1.2- to 3.5-fold increased EC50 as compared to PMPA.
Compounds S-PMEA 3 and S-PMPA 4 display cytostatic activity
values (CC50) in PBMC, CEM and H9 cells to the same order of
magnitude as the reference compounds PMEA and PMPA, indi-
cating that the thiophosphonate modification does not measurably
affect the cytotoxicity of target compounds.
Table 2). Thus, the novel acyclic nucleoside thiophosphonates not
only markedly suppress laboratory HIV-1 strains in cell culture but
also a broad variety of clinical isolates in primary PBMC repre-
senting the different viral clades.
2.4. Enzyme susceptibility assays using steady-state kinetics
To confirm that compounds bearing a thiophosphonate modi-
fication target the reverse transcriptase of HIV-1, the relative effi-
ciencies of incorporation of the thiophosphonate diphosphates
S-PMEApp 5 and S-PMPApp 6 and reference compounds PMEApp
5a and PMPApp 6a were addressed using subtype B HIV-1 WT RT in
in vitro enzyme susceptibility assay (Table 3).
The 50% inhibitory concentration (IC50) values obtained in this
assay show that both thiophosphonate diphosphates S-PMEApp 5
(6.8
as their parent compounds PMEApp 5a (7.0
(11.3 M). The S-PMEApp 5 and S-PMPApp 6 compounds are thus
mM) and S-PMPApp 6 (13.9
mM) are equally efficient inhibitors
mM) and PMPApp 6a
m
readily incorporated by HIV-1 RT from subtype B and display
pronounced inhibitory properties against HIV-1 WT RT. Moreover,
the effect of the sulfur atom is seemingly neutral, as also observed
with the compounds 3 and 4 in the virus-infected cells.
2.3. Anti-HIV-1 activity of S-PMEA 3 and S-PMPA 4 in cells infected
by HIV-1 isolates from subtypes A to G
2.5. Anti-HBV activity of S-PMEA 3 and S-PMPA 4 in cell cultures
To evaluate the capacity of the compounds to inhibit the repli-
cation of clinically relevant HIV-1 isolates from different clades,
a cell-based antiviral drug susceptibility assay was performed with
HIV-1 isolates from clades A to G obtained from the NIH AIDS
reagent program (Table 2).
Since PMEA and PMPA are clinically approved to inhibit HBV
replication in infected patients, the compounds were tested in two
cell-based antiviral drug susceptibility assays. The inhibition of WT
HBV replication was assessed in HepAD38 and Huh7 cells treated
with increasing concentrations of S-PMEA 3, S-PMPA 4 and refer-
ence compounds PMEA and PMPA. The amount of viral DNA
produced in the cells was quantified to evaluate the antiviral
properties of the compounds as the 50% reduction of viral DNA
(EC50) (Table 4). Concomitantly, the cytotoxic effect of each
compound was evaluated as well.
Both S-PMEA and S-PMPA suppressed HIV replication in the
submicromolar or low micromolar range (4.49
mM-0.12 mM).
Depending the nature of the virus isolate the EC50 values of the thio
derivatives were somewhat higher or lower than their corre-
sponding PMEA and PMPA derivatives (compare the EC50 ratios in
Table 1
Antiviral effects and cytotoxicity of S-PMEA 3 and S-PMPA 4 and reference compounds PMEA and PMPA, in CEM cells infected by 100 TCID50 of HIV-1(IIIa) or HIV-2(ROD), in H9
cells infected by 125 and 6250 TCID50 of HIV-1-LAI and in activated PBMC infected by 100 TCID50 of HIV-1-LAI.
Compounds
CEM
EC50
H9
PBMC
EC50
PBMC and H9
CC50
M)b
(
m
M)a
EC50
(m
M)a
CC50
(
m
M)b
EC50
(m
M)a
EC50
(
m
M)a
(m
M)a
(m
100 TCID50
HIV-1(IIIB)
100 TCID50
HIV-2(ROD)
125 TCID50
HIV-1-LAI
6250 TCID50
HIV-1-LAI
100 TCID50
HIV-1-LAI
S-PMEA 3
PMEA
S-PMPA 4
PMPA
16 ꢂ 3.4
5.5 ꢂ 1.7
14 ꢂ 1.6
4.1 ꢂ 1.0
23 ꢂ 19
7.0 ꢂ 4.3
7.9 ꢂ 3.0
7.1 ꢂ 5.7
233 ꢂ 5.3
85 ꢂ 19
>250
1.9 ꢂ 1.8
1.1 ꢂ 0.7
17 ꢂ 0.8
7.9 ꢂ 4.2
9.7 ꢂ 0.1
8.3 ꢂ 1.7
14 ꢂ 10
3.2 ꢂ 2.1
4.2 ꢂ 3.9
3.4 ꢂ 3.2
>30
>30
>100
>100
0.38 ꢂ 0.10
0.23 ꢂ 0.05
>250
CEM: HumanT-Lymphoblastoid cells; H9: human lymphocyte cells; PMBC: Peripheral blood mononuclear cells.
a
EC50 or concentration of drug that decreases the HIV replication by 50% (means of EC50 from two blood donors).
CC50 or concentration of drug that reduces the viable cell number by 50% (means of CC50 from two blood donors).
b

4284
K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
Table 2
Table 4
Antiviral effects of S-PMEA 3 and S-PMPA 4 and reference compounds PMEA and
PMPA in the H9 cells infected by HIV-1 isolates from subtypes A, B, C, D, A/E, F and G.
Anti-HBV activities and cytotoxicity in HepAD38 and Huh7 cell cultures of S-PMEA 3
and S-PMPA 4 and reference compounds PMEA and PMPA.
Viral isolate
Clade
EC50
(m
M)a
EC50
(
m
M)a
EC50 ratiob
Compound
EC50
(
m
M)a
EC50
(
m
M)a
CC50 (m
M)b
S-PMEA 3
PMEA
(PMEA/S-PMEA)
HepAD38 cells
Huh7 cells
4113
2101
2056
1722
2914
4110
1649
2165
2338
3187
A
B
B
B
C
1.64 ꢂ 1.92
0.49 ꢂ 0.74
3.41 ꢂ 2.73
2.80 ꢂ 1.69
0.49 ꢂ 0.36
0.45 ꢂ 0.47
2.77 ꢂ 2.01
0.30 ꢂ 0.13
0.22 ꢂ 0.16
0.88 ꢂ 0.21
17.76 ꢂ 4.23
3.51 ꢂ 0.57
1.02 ꢂ 0.02
1.85 ꢂ 0.19
1.45 ꢂ 1.85
2.74 ꢂ 3.58
0.32 ꢂ 0.10
0.32 ꢂ 0.34
0.19 ꢂ 0.04
0.22 ꢂ 0.11
10.8
7.16
0.29
0.66
2.96
6
0.11
1.06
0.86
0.25
S-PMEA 3
PMEA
S-PMPA 4
PMPA
3.0 ꢂ 0.1
7.6 ꢂ 0.0
2.8 ꢂ 0.7
2.3 ꢂ 0.2
4.5 ꢂ 0.5
6.0 ꢂ 1.0
4.0 ꢂ 0.0
4.2 ꢂ 0.3
> 150
> 150
> 150
> 150
a
EC50: effective concentration of compounds inducing a 50% reduction of the
level of HBV viral DNA in the culture.
C
D
A/E
F
b
CC50: cytotoxic concentration of compounds inducing a 50% reduction of the cell
density.
G
Viral isolate
Clade
EC50
(m
M)
EC50
(
m
M)
EC50 ratio
S-PMPA 4
PMPA
(PMPA/S-PMPA)
Table 5
4113
2101
2056
1722
2914
4110
1649
2165
2338
3187
A
B
B
B
C
4.49 ꢂ 0.99
0.47 ꢂ 0.18
0.45 ꢂ 0.58
0.51 ꢂ 0.44
0.38 ꢂ 0.33
0.54 ꢂ 0.03
0.71 ꢂ 0.12
0.34 ꢂ 0.01
0.20 ꢂ 0.35
0.12 ꢂ 0.11
5.61 ꢂ 6.34
0.24 ꢂ 0.13
1.25 ꢂ 1.82
0.69 ꢂ 0.05
0.34 ꢂ 0.29
0.83 ꢂ 0.19
0.10 ꢂ 0.07
0.51 ꢂ 0.31
0.04 ꢂ 0.01
0.15 ꢂ 0.03
1.25
0.51
2.77
1.35
0.89
1.54
0.14
1.5
Antiviral activities and cytotoxicity of S-PMEA 3 and S-PMPA 4 and the reference
compounds PMEA and PMPA against several DNA viruses in cell culture.
Compound EC50
(
m
M)a
Minimum
cytotoxic
Herpes
Herpes simplex Herpes
virus-1 simplex
Vaccinia
virus
C
concentration
simplex
virus-1 (KOS) (TKꢁ KOS ACVr) virus-2 (G)
(m
M)b
D
A/E
F
S-PMEA 3
PMEA
40 ꢂ 0
89 ꢂ 6
24 ꢂ 2
20 ꢂ 4
80 ꢂ 5
>200
24 ꢂ 1
20 ꢂ 3
48 ꢂ 0
>200
84 ꢂ 6 >200
>200 >200
80 ꢂ 7 >200
>200 >200
0.2
1.25
G
S-PMPA 4 169 ꢂ 13
a
Results are expressed as the EC50 or concentration of drug that decreases the
PMPA
>200
HIV replication by 50%.
a
b
EC50: effective concentration of compounds required to reduce virus-induced
cytopathicity by 50%.
EC50 ratio ¼ EC50
(mM) PMEA/EC50 (mM) S-PMEA 3 and EC50 (mM) PMPA/EC50
(
m
M) S-PMPA 4.
b
Minimum concentration required to cause a microscopically detectable alter-
ation of normal cell morphology.
Similar inhibitory properties are observed for S-PMPA 4 as
compared with PMPA in the two assays, with IC50 values of 2.8 and
M, respectively for S-PMPA 4. The S-PMEA 3 derivative tended to
be slightly more potent (1.5e2.5 fold) than PMEA irrespective of the
cell-type used. These results show that thiophosphonate
compounds are effective to inhibit the replication of WT HBV. No
DNA viruses than the reference compounds although the observed
EC50 values are rather high but consistent with previously
described results [23e25]. Indeed, S-PMPA 4 is able to inhibit
4
m
Herpes simplex virus type 1 and 2 as well as Vaccinia virus (EC50
48e169 M) while the reference compound PMPA exhibits no
activity at 200 M. S-PMEA 3 shows some inhibitory properties
:
m
cytotoxicity is observed up to 150 mM for all compounds tested.
m
against Vaccinia virus in contrast to PMEA. In addition, S-PMEA 3 is
as active as PMEA against the thymidine kinase-deficient HSV-1
and against HSV-2 but is slightly more active against wild-type
HSV-1. These results confirm that PMPA and PMEA are highly
specific for DNA viruses (i.e. encoding a viral DNA polymerase) and
in particular for viruses encoding for a viral reverse transcriptase
like HIV and HBV. The thiophosphonates S-PMEA 3 and S-PMPA 4
mainly display the same specificity as PMEA and PMPA but with
somewhat amplified antiviral spectrum for S-PMPA in particular.
2.6. Evaluation of S-PMEA 3 and S-PMPA 4 against other DNA and
RNA viruses in cell culture
To evaluate the antiviral selectivity of the compounds, the
antiviral activity testing against a panel of RNA and DNA viruses
was performed in cell culture. Coxsackie virus B4, Respiratory
Syncytial virus, Influenza A (H1N1 and H3N2 subtype), Influenza B,
Parainfluenza-3 virus, Reovirus-1, Sindbis virus, Feline Corona
virus, Punta Toro virus, Herpes Simplex virus type 1 and 2 (HSV-1
and 2), Vaccinia virus and Vesicular Stomatitis virus were included
in the study (Table 5).
2.7. Stability studies
None of the compounds exhibit cytotoxicity at concentrations as
To be effective in vivo, any antiviral compound has to be stable
enough to reach the bloodstream and then the infected cell where
its target is localized. Therefore, a stability study with S-PMEA 4 and
S-PMPA 5 was carried out under conditions mimicking biological
fluids, i.e. in complete culture medium (extra-cellular medium) and
in cell extracts (intra-cellular medium) in order to evaluate the
stability spectrum. The degradation kinetics were followed by
analytical HPLC. The apparatus used possesses a specific system for
on-line filtration, which makes it possible to inject a protein-rich
mixture (cell extracts, culture medium, serum) into the analytical
column without prior filtration [26]. For each condition studied, the
whole sample was incubated at 37 ꢀC and at each chosen time
high as 200
mM. No antiviral activity up to 200 mM is observed
against any of the RNA viruses tested, i.e. Coxsackie virus B4,
Respiratory Syncytial virus, Influenza A and B, Parainfluenza-3
virus, Reovirus-1, Sindbis virus, Feline Corona virus, Vesicular
stomatitis virus and Punta Toro virus (data not shown). However,
S-PMEA 3 and S-PMPA 4 display a similar or better activity against
Table 3
Drug susceptibilities of WT HIV-1 RT from subtype B for S-PMEApp 5, S-PMPApp 6,
and reference compounds PMEApp 5a and PMPApp 6a.
IC50
(
m
M)a
IC50
(
m
M)a
IC50
(
m
M)a
IC50 (m
M)a
S-PMEApp 5
PMEApp 5a
S-PMPApp 6
PMPApp 6a
interval, 50 mL were sampled and directly injected into an analytical
WT RT
6.8 ꢂ 2.3
7.0 ꢂ 2.7
13.9 ꢂ 5.4
11.3 ꢂ 3.8
HPLC column where the analytical conditions make it possible to
visualize and properly separate the starting compound from the
degradation products.
a
IC50 values were determined with recombinant RT assayed on activated calf
thymus DNA and averaged (ꢂS.D.) for at least three independent experiments.

K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
4285
Table 6
FAB High Resolution Mass Spectra (HRMS) were recorded in
positive-ion mode on a JEOL SX 102 mass spectrometer using
a cesium ion source and a glycerol/thioglycerol matrix; ESI High
Resolution Mass Spectra were recorded in the negative-ion mode
on a Micromass Q-TOF Waters (Laboratoire de Mesures Physiques
RMN, USTL, Montpellier, France). HPLC was performed on a Waters
600E controller system equipped with a 991 photodiode array
detector (detection 260 nm), auto-injector 717 and on-line degazer.
Eluant A: 0.05 M triethylammonium bicarbonate buffer (pH 7.5);
eluant B: solution A containing 50% of acetonitrile. A stock solution
of triethylammonium bicarbonate buffer TEAB (pH 7.5) 1 M was
prepared by addition of dry-ice in a 1 M triethylamine solution in
Stabilities of S-PMEA 3 and S-PMPA 4 in various media.
Compound
t1/2 complete medium
t1/2 cell extracts
S-PMEA 3
S-PMPA 4
>24 ha
Stablec
>24 hb
Stablec
t1/2: half-life of decomposition.
a
Product 80% intact (degradation into PMEA).
Product 77% intact (degradation into PMPA).
Less than 1% degradation after 24 h.
b
c
The tests under culture medium conditions were carried out
with RPMI medium containing 10% fetal calf serum (complete
medium) and on extracts of CEM cells. The half-lives were calcu-
lated and are displayed in Table 6.
water to reach pH 7.5 and filtered with membrane 0.22 mM GV-type
(Millipore). HPLC and FPLC buffers are prepared daily. Analytical
reverse phase (RP) chromatography was carried out on
a 4.6 ꢃ 100 mm SourceÒ15RPC column or a column X-Terra MS C18
S-PMEA 3 and S-PMPA 4 behave similarly under the conditions
tested (complete culture medium and cell extracts). Compound 3 is
slowly degraded into a single product, identified as being PMEA by
HPLC co-injection. Compound 4 is slowly decomposed into a single
product, identified as being PMPA by HPLC co-injection. This
conversion is due to a de-sulfuration of the PeS bond into a PeO
bond. The difference in behavior observed in complete culture
medium (20% degradation after 24 h) and in cell extracts (less than
1% degradation after 24 h) could be explained by the difference in
enzymatic content between the two media. As described previously
[19], the reference compounds PMEA and PMPA are stable in all
tested media.
(5
m
M, 4.6 ꢃ 250 mm) þ precolumn X-Terra MS C18 (3.5
mM,
3.9 ꢃ 20 mm) and samples were eluted at a flow rate of 1 mL/min
using a linear gradient 0e100% B in 60 min. Preparative purifica-
tions of thiophosphonate and thiophosphonate diphosphate
derivatives were achieved on an ÄKTAprime FPLC (Amersham)
using respectively
a
reverse-phase SourceÒ30RPC column
(18 ꢃ 350 mm) and a DEAE-Sephadex A-25 column (2 ꢃ 30 cm)
with a linear gradient 0e100% B programmed over a 6 h period
with a flow rate of 2 mL/min for thiophosphonate derivatives and
over a 10 h period with a flow rate of 1 mL/min for thiophosphonate
diphosphate derivatives (detection at 254 nm). Compounds {[2-(6-
amino-9H-purin-9-yl)ethoxy]methyl}phosphinic acid
1
and
({[(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phos-
phinic acid 2 were synthesized as described previously [19,22].
3. Conclusion
Novel a-thiophosphonate derivatives S-PMEA 3 and S-PMPA 4
4.1.1. General procedure for the synthesis of {[2-(6-amino-9H-
purin-9-yl)ethoxy]methyl}phosphonothioic acid (3) and ({[(2R)-1-
(6-amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)phosphonothioic
acid (4)
and their diphosphate forms S-PMEApp 5 and S-PMPApp 6 have
been synthesized. Target compounds S-PMEA 3 and S-PMPA 4 were
screened for their antiviral activity in vitro against HIV-1, HIV-2,
HBV and other DNA or RNA viruses in comparison with reference
compounds PMEA and PMPA. Compounds 3 and 4 display potent
activity against HIV-1 and HIV-2, clinically relevant HIV-1 isolates
(clades A to G) and HBV generally to a similar extent as the refer-
ence compounds. S-PMEA 3 and S-PMPA 4 display a moderate
activity against herpes simplex virus and vaccinia virus. The
diphosphate forms 5 and 6 are readily incorporated by WT HIV-1
RT providing the molecular basis of their anti-HIV activity.
Stability studies of derivatives 3 and 4 in culture media and cell
extracts have shown an excellent stability of these derivatives up to
24 h and a unique but slow metabolism pathway in enzymatic
nucleophile-enriched media via the conversion of the PeS bond
into a PeO bond due to a de-sulfuration.
H-phosphinate (1 eq, 0.194 mmol) was dried over phosphorus
pentoxide under vacuum for 5 h then dissolved in anhydrous
pyridine (2 mL). N,O-Bis(trimethylsilyl)acetamide (BSA) (5 eq,
0.972 mmol) was added by syringe and the solution was stirred for
about 1 h at room temperature, under argon. A freshly solution of
elemental sulfur (2 eq, 0.388 mmol) in CS2/pyridine (1/1, 1 mL) was
added, the reaction mixture was stirred for 30 min, and quenched
with deionized water (5 mL). After the solvents were evaporated
under reduced pressure, the residue was purified by reversed-
phase column chromatography (linear gradient 0e100% B).
Product fractions were collected, evaporated to dryness and
lyophilized. Excess triethylammonium bicarbonate was removed
by repeated freeze-drying with deionized water. The residue was
dissolved in water and eluted on a Dowex 50WX2 column (Naþ
exchange) to give the pure compound as disodium salt.
To conclude, these novel acyclic nucleoside thiophosphonates
display promising antiviral activities. Our future work will consist
in the improvement of their pharmacokinetic properties by pro-
drug forms synthesis to make them potential candidates for HIV
and HBV therapy.
4.1.1.1. {[2-(6-Amino-9H-purin-9-yl)ethoxy]methyl}phosphonothioic
acid (3). 1H NMR (D2O)
d: 8.08 (s, 1H, H-8), 7.93 (s, 1H, H-2), 4.23 (t,
J ¼ 4.8 Hz, 2H, CH2N), 3.87 (t, J ¼ 4.8 Hz, 2H, CH2O), 3.60 (d,
4. Experimental
J ¼ 5.2 Hz, 2H, CH2P). 13C NMR (D2O)
d: 152.23, 149.12, 146.98,
141.98, 116.38, 73.01 (d, JCP ¼ 120.0 Hz), 68.57 (d, JCP ¼ 10.6 Hz),
4.1. Chemistry
42.27. 31P NMR (D2O)
d
: 60.67 (t, J ¼ 5.2 Hz). HRMS (TOF, ESI-) calcd
for C8H11N5O3PS (M)ꢁ 288.0320, found 288.0347. HRMS (FABþ)
calcd for C8H13N5O3PS (M þ H)þ 290.0477, found 290.0463. HPLC
purity >98%; white powder after freeze-drying. Yield ¼ 52%.
The 1H NMR, 13C NMR and 31P NMR (Nuclear Magnetic Reso-
nance) spectra were determined with a BRUKER AMX 250 MHz,
a BRUKER Avance DPX 400 MHz and a BRUKER Avance III 600 MHz.
Chemical shifts are expressed in ppm and coupling constants (J) are
in hertz (s ¼ singlet, bs ¼ broad singlet, d ¼ doublet, dd ¼ double
doublet, ddd ¼ doubleedouble doublet, t ¼ triplet, dt ¼ double
triplet, tt ¼ triple triplet, m ¼ multiplet, dm ¼ double multiplet).
4.1.1.2. ({[(2R)-1-(6-Amino-9H-purin-9-yl)propan-2-yl]oxy}methyl)
phosphonothioic acid (4). 1H NMR (D2O)
d: 8.16 (s, 1H, H-8), 8.07 (s,
1H, H-2), 4.29 (dd, J ¼ 14.6 Hz and J ¼ 3.1 Hz, 1H, CHaN), 4.10 dd,
J ¼ 14.6 Hz and J ¼ 6.6 Hz, 1H, CHbN), 3.95 (m, 1H, CHO), 3.63 (dd,

4286
K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
J ¼ 12.6 Hz and J ¼ 5.8 Hz, 1H, CHaP), 3.49 (dd, J ¼ 12.6 Hz and
4.2.2. Drug susceptibilityassays with recombinant subtype B HIV-1 RT
Standard RT activity was assayed using 250 g/mL of activated
calf thymus DNA. To determine IC50 values for S-PMEApp 5,
S-PMPApp 6, PMEApp 5a or PMPApp 6a, reactions were performed
J ¼ 6.0 Hz, 1H, CHbP), 1.05 (d, J ¼ 6.1 Hz, 3H, CH3). 13C NMR (D2O)
d:
m
152.65, 149.12, 147.25, 142.25, 116.26, 74.46 (d, JCP ¼ 10.7 Hz), 70.89
(d, JCP ¼ 121.1 Hz), 46.39, 14.73. 31P NMR (D2O) d: 61.60 (t, J
4.8 Hz). HRMS (TOF, ESI-) calcd for C9H13N5O3PS (M)ꢁ 302.0477,
found 302.0465. HRMS (FABþ) calcd for C9H15N5O3PS (M þ H)þ
304.0633, found 304.0623. HPLC purity >98%; white powder after
freeze-drying. Yield ¼ 47%.
with 10 nM (active sites) of HIV-1 RT and 5
mM of each dNTP con-
taining 100
m
Ci/mmol of 3H-dTTP for 15 min with increasing
amounts of inhibitors. Each aliquot was spotted in duplicate on
DE81 ion-exchange paper discs, and the discs were washed three
times for 10 min in 0.3 M ammonium formate, pH 8.0, two times in
ethanol, and dried. The radioactivity bound to the filters was
determined by liquid scintillation counting. Values of IC50 are the
average from at least three independent experiments.
4.1.2. General procedure for the synthesis of ({[({[(Rp/Sp)-2-(6-
amino-9H-purin-9-yl)ethoxy]methyl}(sulfanyl)phosphoryl)
oxy](hydroxy)phosphoryl}oxy)phosphonic acid (5) and
[({[({[(2R,Rp/2R,Sp)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy}
methyl)(sulfanyl)phosphoryl]oxy} (hydroxy)phosphoryl)oxy]
phosphonic acid (6)
4.3. Antiviral activity in cells
Thiophosphonate (1 eq, 0.069 mmol) was dissolved in DMF
(2 mL) and treated with 1,10-carbonyldiimidazole (4 eq,
0.278 mmol). The resulting mixture was stirred at room tempera-
ture for 24 h. Excess of CDI was decomposed by addition of anhy-
4.3.1. Cells and viruses
Peripheral blood mononuclear cells (PBMC) from healthy
donors were isolated and stimulated with
1
mg/mL of
phytohemagglutinin-P (PHA-P, Sigma) and 5 IU/mL of human
interleukin-2 (IL-2, Invitrogen) for 3 days. PBMCs, CEM and H9 cells
were grown in RPMI-1640 medium supplemented with antibiotics,
and 10% fetal calf serum (FCS). The human hepatoma cell line
HepAD38 [28], stably transfected by an HBV-expressing construct
under the control of tetracyclin, was grown in DMEM/HAM’s F12
(50/50) culture medium supplemented with 10% (FCS), 100 IU/mL
penicillin, 50 mg/mL streptomycin, 400 mg/mL G418 and 0.3 mg/mL
tetracycline. The human hepatoma cell line Huh7 was growth in
DMEM culture medium supplemented with 10% (FCS), 100 IU/mL
drous methanol (8
Anhydrous tri-n-butylamine (50
phosphate (700 L of a 0.5 M solution in DMF) were added and the
mL) and stirring was continued for 30 min.
mL) and tributylammonium pyro-
m
mixture was stirred at room temperature for 3 days. The reaction
was stopped by the addition of 5 mL of cold water. The solvent was
removed under vacuum, the residue dissolved in water, and the
solution applied to a DEAE-Sephadex column (linear gradient
0e100% B). The appropriate fractions were collected, evaporated to
dryness and lyophilized. The residue was dissolved in water and
passed through a Dowex 50WX2 (Naþ form) column to give the
pure compound as tetrasodium salt.
penicillin, 50
mg/mL streptomycin. Human embryonic lung HEL,
simian kidney Vero, feline kidney CrFK and human cervix carci-
noma HeLa cells were propagated in minimal essential medium
4.1.2.1. ({[({[(Rp/Sp)-2-(6-Amino-9H-purin-9-yl)ethoxy]methyl}
(sulfanyl)phosphoryl)oxy](hydroxy) phosphoryl}oxy)phosphonic acid
(MEM) supplemented with 10% FCS, 2 mM L-glutamine, and 0.075%
bicarbonate. The HIV-1-LAI strain was described previously [29].
Viral isolates from clades A, B, C, D, A/E, F and G were obtained
through the AIDS Research and Reference Reagent Program, Divi-
sion of AIDS, NIAID, NIH. Herpes simplex virus type 1 (HSV-1) (KOS
and KOS ACV TK-), HSV-2 (G), vaccinia virus, vesicular stomatitis
virus, Coxsackie virus B4, respiratory syncitial virus, parainfluenza-
3 virus, reovirus-1, Sindbis virus and Punta Toro virus were
described elsewhere [19].
(5). 1H NMR (D2O)
d: 8.40 (s, 1H, H-8), 8.32 (s, 1H, H-2), 4.52 (t,
J ¼ 5.0 Hz, 2H, CH2N), 4.10 (t, J ¼ 5.0 Hz, 2H, CH2O), 3.97 (dd,
J ¼ 13.8 Hz and J ¼ 4.2 Hz, 1H, CHaP), 3.93 (dd, J ¼ 13.2 Hz and
J ¼ 4.8 Hz,1H, CHbP). 31P NMR (D2O)
d
: 62.06 (d, 2J ¼ 33.0 Hz), ꢁ10.94
(d, 2J ¼ 19.8 Hz), ꢁ23.53 (d, 2J ¼ 33.0 Hz and 2J ¼ 19.8 Hz). HRMS (TOF,
ESI-) calcd for C8H13N5O9P3S (M)ꢁ 447.9647, found 447.9656. HPLC
purity >99%; white powder after freeze-drying. Yield ¼ 17%.
4.1.2.2. [({[({[(2R,Rp/2R,Sp)-1-(6-Amino-9H-purin-9-yl)propan-2-yl]
4.3.2. Anti-HIV assay in cells
oxy}methyl)(sulfanyl)phosphoryl]oxy}
(hydroxy)phosphoryl)oxy]
PHA-P-activated PBMC (1.5 ꢃ 105 cells) or H9 cells (5 ꢃ 105 cells)
treated for 30 min by increasing concentrations of S-PMEA 3,
S-PMPA 4, and reference compounds PMEA and PMPA (generously
provided by Dr Antonin Holý from Prague, Czech Republic) were
infected, respectively, with 100 or 125 TCID50 (50% tissue culture
infectious doses) of the HIV-1-LAI strain. Supernatants were
collected at day 7 post infection and stored at ꢁ20 ꢀC. Viral repli-
cation was measured by quantifying reverse transcriptase (RT)
activity in cell culture supernatants by use of the RetroSys RT
Activity Kit (Innovagen). Cytotoxicity of the compounds was eval-
uated in uninfected PHA-P-activated PBMC and H9 cells by MTT
assay (Sigma) on day 7. Experiments were performed in triplicate
and repeated with another blood donor for PBMCs. Data analyses
were performed using SoftMaxÒPro 4.6 microcomputer software.
Percent of inhibition of RT activity or cell viability were plotted
versus compound concentration and fitted with quadratic curves to
phosphonic acid (6). 1H NMR (D2O)
d
: 1H NMR (D2O)
d
: 8.42 (s, 1H,
H-8), 8.32 (s, 1H, H-2), 4.52 (dd, J ¼ 14.8 Hz and J ¼ 3.1 Hz, 1H,
CHaN), 4.33 (ddd, J ¼ 14.8 Hz, J ¼ 6.6 Hz and J ¼ 2.8 Hz, 1H, CHbN),
4.14 (m, 1H, CHO), 3.99 (m, 1H, CHaP), 3.84 (m, 1H, CHbP), 1.23 (d,
J ¼ 6.2 Hz, 3H, CH3). 31P NMR (D2O)
d
: 62.18 (d, 2J ¼ 33.5 Hz), 62.02
(d, 2J
¼
33.3 Hz), ꢁ10.60 (d, 2J
¼ 19.9 Hz), ꢁ10.96 (d,
2J ¼ 20.0 Hz), ꢁ23.1-(ꢁ)23.6 (m). HRMS (TOF, ESI-) calcd for
C9H15N5O9P3S (M)ꢁ 461.9803, found 461.9802. HPLC purity >99%;
white powder after freeze-drying. Yield ¼ 19%.
4.2. Reverse transcriptase assays
4.2.1. HIV-RT plasmid constructions, enzyme preparations and
reagents
The recombinant clade B HIV-1 WT RT were co-expressed with
HIV-1 protease in Escherichia coli in order to obtain p66/p51 het-
erodimers, which were later purified using affinity chromatography
as described [27]. Enzymes were quantitated by active-site titration
before biochemical studies. Activated Calf Thymus DNA was
purchased from GE Healthcare. [3H]-labeled deoxythymidine 50-
triphosphate was purchased from Perkin Elmer.
determine 50% effective concentrations (EC50
concentrations (CC50).
) and cytotoxic
The methodology of the anti-HIV assays was as follows: human
CEM (w3 ꢃ 105 cells/cm3) cells were infected with 100 CCID50 of
HIV(IIIB) or HIV-2(ROD)/mL and seeded in 200
mL wells of
a microtiter plate containing appropriate dilutions of the test
compounds. After 4 days of incubation at 37 ꢀC, HIV-induced CEM

K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
4287
giant cell formation was examined microscopically. The 50%
effective concentration (EC50) was defined as the compound
concentration required to inhibit syncytia formation by 50%. The
50% cytostatic concentration (CC50) was defined as the compound
concentration required to inhibit CEM cell proliferation by 50% in
mock-infected cell cultures.
syncytial virus were assayed in HeLa cell cultures. Parainfluenza-3
virus, reovirus-1, Sindbis virus and Punta Toro virus were assayed
in Vero cell cultures. Feline corona viruses were assayed in CrFK cell
cultures. Cells were grown to confluency in microtiter trays and
were inoculated with 100 times the 50% cell culture infective dose.
Compounds S-PMEA 3, S-PMPA 4, and reference compounds PMEA
or PMPA, were added after a 1 to 2-h virus adsorption period. The
virus-induced cytopathic effect (CPE) was recorded microscopically
at 3 days post infection and was expressed as percentage of the
untreated controls. The 50% effective concentrations (EC50) were
derived from graphical plots. The minimal toxic concentration
(MTC) was defined as the minimal concentration that resulted in
a microscopically detectable alteration of cell morphology. The MTC
was determined in uninfected confluent cell cultures that were
incubated, akin to the cultures used for the antiviral assays, with
serial dilutions of the compounds for the same time period. Cultures
were inspected microscopically for alteration of cell morphology.
4.3.3. Anti-HBV assay in cells
The tetracycline-responsive hepatoma cell line HepAD38, stably
transfected with a cDNA copy of the pregenomic RNA of wild-type
HBV virus, was used [28]. Withdrawal of tetracycline from the
culture medium resulted in the initiation of viral replication. The
cells (5 ꢃ105) were seeded in 48-well plates and were induced after
2e3 days for viral production by washing with PBS and were fed
with 200 mL culture medium without tetracycline and G418 with or
without the antiviral compounds. Mediumwas changed after 3 days.
The antiviral effect was quantified by measuring levels of intracel-
lular viral DNA at day 6 post induction, by a real time quantitative
PCR (Q-PCR) method. Total cellular DNAwas extracted from the cells
by means of a commercial kit. The Q-PCR was performed in a reac-
4.4. Stability studies
tion volume of 25
mL using the TaqMan Universal PCR Master Mix
4.4.1. Media and preparation of cell extracts
with forward primer (50-CCG TCT GTG CCT TCT CAT CTG-30; final
concentration: 600 nM), reversed primer (50-AGT CCA AGA GTY CTC
TTA TRY AAG ACC TT-30; final concentration: 600 nM), and Taqman
probe (6-FAM-CCG TGT GCA CTT CGC TTC ACC TCT GC-TAMRA; final
concentration 150 nM). The reaction was analyzed using an ABI
Prism SDS 7000 apparatus. A plasmid containing the full-length
insert of the HBV genome was used to prepare the standard curve.
The amount of viral DNA produced in treated cultures was expressed
as a percentage of the untreated samples. Compounds thatexhibited
selective antiviral activity in a first assay were retested to confirm
the activity. The cytostatic effect of the various compounds was
assessed employing the parent hepatoma cell line HepG2. The effect
of the compounds on exponentially growing HepG2 cells was eval-
uated by means of the MTS method. Briefly, cells were seeded in 96-
well plates at a density of 3000 cells/well and were allowed to
proliferate for 3 days in the absence or presence of compounds
S-PMEA3, S-PMPA 4, and referencecompoundsPMEAorPMPA, after
which the optical density was measured with a 96-well plate reader
and cell density was determined.
CEM-SS cells extract was prepared as previously described
[19,26]. Briefly, exponentially growing CEM-SS cells were recovered
by centrifugation (500g), washed with PBS, and resuspended in
10 mM TriseHCl pH 7.4, 140 mM KCl, at the concentration of
30 ꢃ 106 cells/mL. Cells were lyzed by ultrasonic treatment and
cellular debris were removed by centrifugation (10,000g, 20 min).
The supernatant containing soluble proteins (3 mg/mL) was stored
at ꢁ80 ꢀC. Kinetic data of decomposition for S-PMEA 3 and S-PMPA
4 was studied at 37 ꢀC (a) in culture medium (RPMI-1640 con-
taining 10% heat-inactivated fetal calf serum), and (b) in total cell
extract (CEM-SS cell). For each kinetic study, the compound solu-
tion is diluted with a freshly thawed aliquot of the considered
medium to obtain a final concentration of 0.1 mM and the mixture
is incubated at 37 ꢀC. At the desired time, an aliquot is removed and
immediately frozen at ꢁ80 ꢀC for further HPLC analysis, as previ-
ously described [19].
4.4.2. HPLC analysis
We used a previously described on-line HPLC cleaning method
[26]. Studies were performed on a column Nova-pak C18 (4 mm,
3.9 ꢃ 150 mm) þ precolumn Nova-pack C18 (100 Å) with an on-line
filtration system. Samples were eluted using a linear gradient of
0.05 M triethylammonium bicarbonate buffer in 100% water (pH
7.5) (buffer A) to a 0.05 M triethylammonium bicarbonate buffer in
50% acetonitrile (buffer B), programmed over a 60 min period with
a flow rate of 1 mL/min and detection at 260 nm. The crude sample
(50 mL) is injected into the precolumn and eluted with buffer A
during 5 min. Then, the switching valve for connecting the pre-
column to the column is activated and a linear gradient from 0%
buffer B to 20% over 40 min is applied. The retention times are
25.6 min for S-PMEA 3, 29.5 min for S-PMPA 4, 24.3 min for PMEA,
and 27.7 min for PMPA. The amount of remaining parent compound
at each time point was used to determine the half-life of the
compound. The product of decomposition from parent derivative is
determined by comparison with authentic samples and reference
compounds.
For the Anti-HBV assay in the Huh7 cell line, the plasmid
pTriEXMod-HBV, which contains 1.1 IU of the HBV genome, enabled
after cell transfection the production of pregenomic RNA under the
control of the chicken beta actin promoter and therefore triggers
HBV DNA synthesis [30]. Huh7 cells (3 ꢃ 105) were seeded in 12-
well plates and were transfected 1 day post plating with 0.5 mg/
well of pTriEXMod-HBV plasmid containing wt HBV genome, using
the Fugene 6 transfection reagent (Roche) according to the manu-
facturer’s instructions. Sixty hours post transfection, treatment
with increasing concentrations (0, 5, 10, 25, 50, 100 mM) of S-PMEA
3, S-PMPA 4, and reference compounds PMEA or PMPA, was started.
Medium was changed daily, and drug administration was per-
formed from day 3 to day 8 post transfection. Cells were then lysed
for analysis of intracellular viral DNA. HBV DNA was purified from
intracellular core particles as described earlier [31], separated on
agarose gels, analyzed by Southern Blot hybridization, and quan-
tified after PhosphorImager scanning to determine the inhibitory
concentration-50 (IC50) after comparison with standard that is an
isogenic wild-type strain or wild-type quasispecies [32]. Cultures
were inspected microscopically for alteration of cell morphology.
Acknowledgment
This work was supported by grant from the French “Agence
Nationale de Recherche Contre le Sida et les hépatites virales”
(ANRS), by SIDACTION and the K.U. Leuven (GOA 10/014). The
authors wish to thank Dr A. Holý for providing the reference
compounds. We wish to thank Dr. K. Parra, A. Lebrun and Dr G.
4.3.4. Antiviral assay against a panel of DNA and RNA viruses
Herpes simplex virus type 1 (HSV-1) (KOS and KOS ACV TKꢁ),
HSV-2 (G), vaccinia virus and vesicular stomatitis virus were
assayed in HEL cell cultures. Coxsackie virus B4 and respiratory

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K. Barral et al. / European Journal of Medicinal Chemistry 46 (2011) 4281e4288
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Supplementary data associated with this article can be found, in
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[22] B. Canard, K. Alvarez, K. Barral, J.L. Romette, J. Neyts, J. Balzarini, WO/2008/
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