Solid-phase synthesis of 5′-triphosphate 2′-5′- oligoadenylates analogs with 3′-O-biolabile groups and their evaluation as RNase L activators and antiviral drugs

Article abstract of DOI:10.1016/j.bmc.2013.06.008

5′-Triphosphate 2′-5′-oligoadenylate (2-5A) is the central player in the 2-5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2-5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2-5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2-5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2-5A drug in an intact form offer an alternative approach to therapeutic use of 2-5A. Here we have synthesized the trimeric and tetrameric 2-5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2-5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity.

Full text of DOI:10.1016/j.bmc.2013.06.008

Bioorganic & Medicinal Chemistry 21 (2013) 5461–5469
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Solid-phase synthesis of 50-triphosphate 20-50-oligoadenylates
analogs with 30-O-biolabile groups and their evaluation as RNase L
activators and antiviral drugs
Yann Thillier a, Sarah K. Stevens b, Christabel Moy b, Joshua Taylor b, Jean-Jacques Vasseur a,
Leonid Beigelman b, Françoise Debart a,
⇑
a IBMM, UMR 5247, CNRS-UM1-UM2, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier, France
b Alios BioPharma, 260 E. Grand Ave., 2nd Floor, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
50-Triphosphate 20-50-oligoadenylate (2–5A) is the central player in the 2–5A system that is an innate
immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase
L activated by 2–5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2–5A
for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2–5A
with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the
parent 2–5A drug in an intact form offer an alternative approach to therapeutic use of 2–5A. Here we
have synthesized the trimeric and tetrameric 2–5A species bearing hydrophobic and enzymolabile piva-
loyloxymethyl groups at 30-positions and a triphosphate at the 50-end. Both analogs were able to activate
RNase L and the production of the trimer 2–5A (the most active) was scaled up to the milligram scale for
antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog
demonstrated some significant antiviral activity.
Article history:
Received 12 March 2013
Revised 30 May 2013
Accepted 4 June 2013
Available online 13 June 2013
Keywords:
20-50-Oligoadenylate
Pivaloyloxymethyl group
2–5A analogs
RNase L
Influenza virus
Respiratory syncytial virus (RSV)
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
With the aim to develop antiviral therapeutic agents based on
the 2–5A/RNase L pathway, numerous 2–5A analogs have been de-
In response to the presence of pathogens such as viruses, bacte-
ria, parasites or tumor cells, mammalian cells trigger an innate im-
mune response mainly mediated by interferons (IFNs).1 One well-
characterized mechanism of interferon activation to suppress viral
infections is the 2–5A system2 in which unusual short 50-triphos-
phate (TP) 20-50-phosphodiester-linked oligoadenylates (2–5A)
play a key role to modulate RNA degradation in cells.3 These olig-
omers of adenosine (mostly trimers and tetramers) are synthesized
from ATP by several cellular 20-50 oligoadenylate synthetases (OAS)
induced by IFN upon activation by double-stranded viral RNA dur-
ing infection. The 2–5A binds to an intracellular endoribonuclease
RNase L converting it from an inactive monomer to its active dimer
to cleave viral and cellular single-stranded RNA.4–6 This RNA deg-
radation inhibits protein synthesis resulting in cell apoptosis and
thereby suppression of viral replication. The effects of 2–5A are
transient since natural 2–5A are unstable in cells due to the activ-
ities of a 50-phosphatase and a specific 20-50-phosphodiesterase
that cleave 2–5A to AMP and ATP.7
signed and prepared to obtain RNase L activators with improved
properties. Natural 2–5A cannot be used directly as a drug due to
its lack of in vivo stability and cell permeability. A number of mod-
ifications of the nucleobase,8,9 ribose,10,11 internucleotide link-
age12–16 and 50-phosphoryl group17,18 have been introduced in
the 2–5A to enhance the enzymatic stability and the cellular up-
take of these analogs while maintaining or improving the ability
of natural 2–5A to activate RNase L. Structure–activity studies on
these diverse 2–5A analogs could provide knowledge on the struc-
tural requirements of RNase L for the binding to the oligoadenylate
molecule. Among these requirements, the presence of a 50-phos-
phoryl group, a minimum of three adenosine residues linked by
20-50-internucleotidic bonds are critical for the binding to RNase
L19 but to date, no accurate rationale could be ascertained to design
efficient 2–5A analogs with permanent modifications.
An alternative approach to design modified 2–5A is founded on
the prodrug strategy which consists of the temporary modification
of the parent 2–5A by biolabile groups. Protection of 2–5A is aimed
at improving its enzymatic stability in biological fluids, its hydro-
phobicity and therefore its internalization in cells while preserving
its activity after the release of the native 2–5A drug in an intact form.
For many years, the prodrug approach with oligonucleotides20 has
⇑
Corresponding author. Tel.: +33 4 6714 3898; fax: +33 4 6704 2029.
E-mail address: debart@univ-montp2.fr (F. Debart).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.06.008

5462
Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
been mainly developed with the protection of the internucleoside
phosphate linkage to mask the negative charge21–24 and cell uptake
has been improved in several examples.22,25,26 Our initial research in
this field was also focused on the phosphate modification by the S-
acylthioethyl group27 but more recently the ribose 20-OH in RNA
has received our attention to be modified by enzymolabile acyl-
oxymethyl groups.28–30 In the same way Damha and co-workers
investigated the 20-O-acetal levulinic ester (ALE) and various 20-O-
(amino acid) acetalesters as prodrug moieties.31 Our preliminary re-
sults demonstrated that such 20-O-acyloxymethyl uridylates ful-
filled the criteria of nuclease resistance and esterase hydrolysis.28
Among the different evaluated protecting groups, the pivaloyloxym-
ethyl (PivOM) was selected for its chemical stability and its lipo-
philic character to improve cellular uptake. Following this, some
20-OH of mixed-nucleobase RNA have been masked by the PivOM
group to obtain RNA prodrugs. One of them was evaluated in a siRNA
assay and the delivery of such modified siRNA was more effective
than the natural one. For the first time, these data provided a
proof-of concept for a prodrug-based approach for the delivery of
RNA to cells.30
These previous data offer an excellent guidance to design and
synthesize stable and lipophilic 2–5A analogs bearing biolabile Pi-
vOM groups at the 30-position as exogenous RNase L activators for
broad spectrum antiviral activity. It is noteworthy that recently
Lönnberg and co-workers reported on the use of the PivOM for
the synthesis of a 20-50-dinucleoside and a fully protected 20-50-
adenylate trimer in a prodrug strategy to enhance 2–5A cellular
uptake.15,16 These molecules were evaluated neither as RNase L
activators nor antiviral inhibitors but only their enzymatic depro-
tection was investigated. Here, we report the original synthesis
of 50-O-triphosphate 2–5A trimer and tetramer analogs with 30-
OH masked by PivOM (Fig. 1) and their biological properties. First
their RNase L activating ability was tested using a Fluorescence
Resonance Energy Transfer (FRET)-based assay and the trimer ana-
log exhibited the best activation when compared to tetramer.
Therefore, its synthesis was further improved and scaled up in or-
der to evaluate its antiviral effects on two viral strains, influenza
virus and respiratory syncytial virus (RSV) in cells.
preserve 30-O-PivOM modification. Thus two strategies were possi-
ble: either these analogs would have to be synthesized with differ-
ent protecting groups avoiding RNA deprotection in basic
conditions or a new method of deprotection would have to be
developed while keeping CNE and acyl as standard groups. The first
strategy previously described which consisted in replacing acyl by
silyl groups in nucleobases required a fluoride ions treatment to re-
lease deprotected RNA.30 The major drawback of this strategy was
the tedious multi-step preparation of the corresponding phospho-
ramidites. Consequently in this work CNE, acyl groups and succinyl
linker to the solid support were selected and we have examined
the appropriate conditions for their removal while retaining the Pi-
vOM intact.
2. Chemistry
2.1. Synthesis of 20-O-phosphoramidite 30-O-PivOM adenosine 4
First we prepared the appropriate phosphoramidite 4 from the
30-O-PivOM monomer 2 obtained as a side product during the
non-regiospecific synthesis of 20-O-PivOM adenosine 3 previously
described (Scheme 1).32 Starting from 50-O-dimethoxytrityl (DMTr)
N6-phenoxyacetyl (Pac) adenosine 1, the 20 and 30-hydroxyls were
protected with PivOM via a 20,30-O-dibutylstannylidene intermedi-
ate which was treated with PivOM-chloride to give a mixture of the
30-O-PivOM 2 and 20-O-PivOM 3 derivatives (1/1). The desired slow
eluting 30-isomer 2 was isolated with 30% yield after silica gel chro-
matography. The tritylated 3-PivOM isomer 2 was converted to the
corresponding 20-amidite 4 with 68% yield by using 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite.
2.2. Evaluation of 20-O-phosphoramidite 30-O-PivOM adenosine
4 in the synthesis of a natural 50-triphosphate 2–5
tetraadenylate
With the phosphoramidite 4 in hand, the natural 50-TP 2–5A 5
was prepared on an automated DNA synthesizer to test the effi-
ciency of the assembly. The synthesis was performed on a 1 lmol
The synthesis of 30-O-PivOM 2–5A analogs was performed on
solid support by the phosphoramidite chemistry with a DNA syn-
thesizer. For this purpose a new strategy was developed because
the PivOM as well as the phosphate and nucleobases protecting
groups (cyanoethyl (CNE) and acyl, respectively) used in standard
RNA synthesis are base-labile. Therefore, the final ammonia treat-
ment could not be applied for deprotection of 2–5A analogs to
scale by using the commercially available controlled pore glass
(LCAA-CPG) linked to 50-O-DMTr 20-O-acetyl N6-Pac adenosine
through a 30-O-succinyl linker (Table 1, entry 1). The same elonga-
tion conditions (coupling times 180 s and 50benzylmercaptotetraz-
ole 0.3 M) as used in the case of 20-PivOM amidites were applied
and the average stepwise yield (99%) was comparable. Upon com-
pletion of the assembly, the tetramer still anchored to the support
was 50-functionnalized by a triphosphate following a procedure
published earlier.33 The 50-OH was first converted in 50-H-phos-
phonate which was simultaneously oxidized and activated as a
phosphoroimidazolide derivative. Subsequent substitution of the
imidazole by pyrophosphate gave rise to the fully protected and
supported 50-TP 2–5A. Then a DBU treatment was first applied to
eliminate CNE from phosphates followed by an ammonia treat-
ment to remove Pac and PivOM groups and to release the desired
50-TP 2–5A 5.32 Ion-exchange HPLC and MALDI-TOF-MS analysis
of the crude 5 revealed efficient elongation and 50-functionnaliza-
tion (major peak 49%). To the best of our knowledge this is the first
chemical synthesis of the 50 triphosphorylated 2–5A trimer on the
solid support (Table 1, entry 1).
O
P
O-
O
P
O-
O
P
O-
HO
O
O
O
A
O
O
O
O
P
O
O-
O
O
A
O
O
O
O
P
O
O-
O
O
2.3. Study of suitable deprotection conditions to preserve
PivOM groups using a 50-TP 20-PivOM tetrauridylate model 6
A
O
n
n = 1 or 2
O
O
OH
As mentioned above, one of the aims of this work was to find
RNA deprotection conditions compatible with the preservation of
PivOM groups. In the literature anhydrous organic amines were de-
scribed to cleave and deprotect synthetic oligonucleotides, such
O
Figure 1. Structure of 50-TP 30-O-PivOM 2–5A analogs trimer and tetramer.

Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
5463
O
O
O
O
O
O
NH
NH
NH
N
N
N
N
N
N
N
N
N
Cl
P
O
N
N
N
N
DMTrO
DMTrO
DMTrO
O
O
DBTO / TBAB / PivOM-I
DCE / 70°C / 1h
CN
O
DIEA / CH2Cl2 / 1h
O
O
O
O
OH OH
O
O
O
OH
P
N
O
1
2
CN
4
+
2'-O-PivOM isomer 3
Scheme 1. Synthesis of 50-O-DMTr 30-O-PivOM N6-phenoxyacetyl adenosine 20-O-phosphoramidite 4.
Table 1
Data for synthesized short 50-TP 2–5A, 50-TP 30-PivOM uridylate and 50-TP 2–5A 30-PivOM analogs
Entry ON 50-Sequence-30
Synthesis
scalea
Linker to
support
Crude purityb
(%)
Crude
Purified
Molecular
formula
Calcdem/ Foundem/
materialc
materiald
z
z
1
2
3
4
5
6
7
8
pppA2–5A2–5A2–5
pppUPUPUPU
ppp(AP)2–5(AP)2–5APiv
ppp(AP)2–5(AP)2–5(AP)2–
5APiv
A
0.5
1.0
1.0
1.0
Succinyl
Succinyl
Succinyl
Succinyl
49
64
78
73
154
292
157
132
n.df
n.df
72
C40H51N20O31P6
C54H74N8O70P6
C47H64N15O30P5
C63H85N20O38P6
1493.79
1743.51
1473.96
1916.33
1491
1744
1473
1917
70
5
6
7
7
ppp(AP)2–5(AP)2–5APiv
ppp(AP)2–5(AP)2–5APiv
10.0
10.0
Succinyl
Q-linker
51
70
740
4590
654
1665
C47H64N15O30P5
C47H64N15O30P5
1473.96
1473.96
1474
1476
ppp = triphosphate; P = PivOM; Piv = pivaloyl; 2-5: 20-50 internucleotidic bond.
a
Synthesis scale (lmol).
b
c
d
e
f
Percentage purity of short 50-TP-RNA in the crude as calculated from the integration of the IEX chromatogram.
nmol of total crude material released from support and eluted in the TEAB fraction.
nmol of isolated purified material (from TEAB and THF solutions).
MALDI-TOF mass spectrometry in negative mode.
n.d = not determined because not purified.
DNA or RNA molecules.34,35 The amine function is preferably either
a primary or secondary amine with the ability to nucleophilically
attack a base labile group on the nucleotide. The amine reagent
is used neat or dissolved in an organic solvent that not substan-
tially dissolve the oligonucleotide from the solid support. These
findings revealed that the reagent could be removed from the syn-
thesis column leaving the oligonucleotide retained upon the sup-
port then it was recovered in a buffer without the need of
evaporation. This attractive deprotection method prompted us to
test several amines and consequently to apply a mixture of butyl-
amine in THF (1/1) to a protected and support bound RNA decamer
with 20-O-PivOM groups as a model. MALDI-TOF-MS analysis did
not reveal the presence of the expected fully deprotected RNA
but a peak at m/z = 4166 matched with the decamer without the
CNE and acyl groups and with PivOM groups still in the 20-position.
This data indicated that these deprotection conditions could be
suitable for the PivOM preservation.
2.4. Solid-phase synthesis of 50-TP 30-PivOM 2–5A analogs trimer
7 and tetramer 8 on a 1 mol scale (Table 1, entries 3 and 4)
l
Early studies have shown the requirement of at least three 20,50-
linked adenine residues and a 50-phosphoryl group for the efficient
activation of RNase L.19 The best activation was obtained with the
trimer or the tetramer forms thus we prepared two 50-TP 2–5A
analogs (3- and 4-mers) bearing PivOM groups as prodrugs mole-
cules to evaluate their biological properties.
The synthesis of these analogs 7 and 8 with all the 30-OH pro-
tected by a PivOM group required first the preparation of a support
LCAA-CPG linked to 50-O-DMTr 30-O-PivOM N6-Pac adenosine
through a 20-O-succinyl linker following a literature procedure.36
An efficient loading of 61
lmol/g was obtained. The assembly of
2–5A analogs was achieved on a 1
lmol scale from this support
and the phosphoramidite 4 as described above (Scheme 2). Then
their 50-triphosphorylation was performed followed by their
deprotection with DBU then butylamine. IEX-HPLC analysis of
the two crude materials 7 and 8 showed a major peak representing
a purity of 78% and 73%, respectively (Fig. 2, Table 1, entries 3 and
4). MALDI-TOF MS analyses revealed in both cases one peak at m/z
1501 for 7 and at m/z 1944 for 8 corresponding to the desired ana-
logs and a second peak with a difference of 30 Da related to a side
product. This difference was assigned to the mass of a formalde-
hyde molecule which would correspond to the loss of the oxym-
ethyl linker (CH2O) of PivOM group in adenosine at the 30-end of
2–5A sequence.
Thus we prepared on a 1 l
mol scale a 30-50 tetrauridylate model
from the commercially available (Chemgenes) 20-O-PivOM uridine
phosphoramidite and LCAA-CPG linked to 50-O-DMTr 20-O-Ac U.
This short RNA was functionalized with a 50-TP to mimic the tar-
geted 2–5A analog in order to set up the correct conditions for
the best deprotection and recovery. After assembly and 50-trip-
hosphorylation, the uridylate model compound was first treated
with 1 M DBU for 3 min then with a butylamine/THF solution (1/
1) at room temperature for 1 h. After washing the support with
THF to remove residual amine reagent, the proper 50-TP 20-PivOM
tetrauridylate 6 was eluted with a 50 mM TEAB solution. The crude
material was efficiently recovered (292 nmol) with an estimated
purity of 64% (IEX-HPLC analysis) and 6 was characterized by MAL-
DI-TOF MS (m/z = 1744) (Table 1, entry 2).
Actually upon butylamine treatment once the succinyl linker
was cleaved, free 20-OH in cis-position of 30-PivOM could lead to
a transesterification reaction of pivaloyl (Piv) moiety with loss of
formaldehyde and a mixture of 30-Piv and 20-Piv was probably

5464
Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
O
P
H
-O
O
APac
HO
APac
O
O
TMS
O
P
H
PivOMO
OPh
O
P
O
PivOMO
O
P
O
N
PhO
O
OCNE
O
OCNE
DMTrO
APac
1)
TMS
H3C
O
O
APac
APac
Pyridine / rt / 30 min
O
O
Imidazole / CCl4
PivOM
O
O
P
CYCLE
2) TEAB 100 mM
rt / 45 min
OCNE
CH3CN / NEt3
30 °C / 5 h
PivOMO
O
P
PivOMO
O
P
CNEO
N
O
OCNE
O
O
O
APac
APac
4
O
O
n
n
O
O
O
O
PivOM
PivOMO
APac
O
NH
NH
O
O
O
HO P
O-
O
P
O
P
O
P
O
P
O
O P
O
HO
O
O
O
O
O
O
N
P
O
APac
A
O-
O-
O-
O-
O-
O
O-
N
O
PivOMO
O
P
O
O
O
O
P
O
PivOMO
O
P
O
O
O-
O
OCNE
O
O
OCNE
O
APac
APac
A
TBAPP / DMF
18 h / 30 °C
O
1) DBU / CH3CN
O
2) BuNH2 / THF
(1:1) / 1h
PivOMO
O
P
O
O
O
O
P
PivOMO
O
P
O
O-
O
OCNE
O
O
OCNE
O
O
O
APac
O
APac
A
O
n
n
n
O
O
O
O
OH
PivOMO
O
PivOMO
O
NH
NH
7 n = 1
O
O
O
8
n = 2
Scheme 2. Solid-phase automated synthesis of 50-TP 30-O-PivOM 2–5A analogs trimer 7 and tetramer.
70 nmol were recovered. Their MALDI-TOF MS analysis showed a
unique peak at m/z 1916.75 for the tetramer analog 8 attesting
the exclusive presence of Piv groups on 20 or 30-OH of the 30-termi-
nal adenosine. For the trimer 7 a major peak at m/z 1473 was ob-
served but a second peak at m/z 1502 indicated also the presence
of the PivOM group (Fig. 3, Table 1, entry 3). Nevertheless the alter-
ation of 30-PivOM in 20 or 30-Piv should not affect the prodrug prop-
erties of the 2–5A analogs since the pivaloyl ester should be also
cleaved by cell esterases. Both 2–5 analogs 7 and 8 were tested
in a FRET-based assay for their ability to activate RNase L (see Sec-
tion 3).
2.5. Solid-phase synthesis of 50-TP 30-PivOM 2–5A analogs trimer
7 on a 10 lmol scale (Table 1, entries 5 and 6)
With the aim to obtain sufficient amount of the trimer analog 7
for antiviral evaluation in cells, its automated production from 4
was scaled up to 10 l
mol. After assembly completion, the 50-func-
tionalization with triphosphate moiety was performed in the same
manner as above. Then a DBU solution was applied to remove CNE
of the trimer followed by a dry solution of butylamine in THF to
cleave the succinyl linker and to deprotect nucleobases. The crude
material (740 nmol) was recovered by elution with a TEAB solution
and IEX-HPLC analysis revealed a purity of 51% for the trimer ana-
log 7 in the crude material (Table 1, entry 5). The recovery yield
(7.4%) was very low therefore we checked the content of the wash-
ing THF solution (2143 nmol) to find some desired material (with
24% purity) which was worthwhile to purify. Both THF and TEAB
solutions were separately IEX-HPLC purified to afford larger total
Figure 2. IEX-HPLC and MALDI-TOF MS analyses of the crude material 2–5A trimer
7 and tetramer 8 analogs after deprotection upon butylamine/THF treatment.
obtained at the 30-end (Scheme 3). However these different species
20-Piv, 30-Piv and 30-PivOM have close lipophilicity and could not
be separated by HPLC. The major peak of each crude material
was isolated by IEX-HPLC semi-preparative with 96% purity and

Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
5465
O
O
O
O
A
A
A
A
O
O
O
O
O
O
O
P
O
O
O
P
O
O
O
O
P
O
O
O
O
P
O
O
O-
O-
O
O-
O
O-
O
O
O
O
O
O
A
A
A
A
O
O
BuNH2 / THF
O
O
O
OH
OH
O
O
HO
O
O
O
OH
O
O
O
O
H
H
3'-PivOM
3'-O-Piv
2'-O-Piv
Scheme 3. Transesterification side-reaction on the 30-terminal adenosine of 2–5A analogs upon butylamine treatment.
3. Biological evaluation of 2–5A 30-PivOM analogs
3.1. RNase L activation evaluated by FRET-based assay
The synthesized trimer 7 and tetramer 8 analogs were evalu-
ated in vitro for activation of RNase L using a FRET-based enzy-
matic assay.38 The ability of the natural 2–5A trimer and 2–5A
analogs 7 and 8 to activate RNase L was determined by monitoring
the cleavage of a synthetic 50-[FAM] 36-mer RNA-[BHQ-1]. This
RNA substrate corresponds to a segment of the intergenic region
of RSV genomic RNA containing several sites potentially cleaved
by RNase L (UpUp and UpAp). Upon RNA cleavage the fluorescent
FAM group is released from the BHQ quencher. Recombinant hu-
man RNase L expressed from a baculovirus vector in insect cells
was used in this assay. Fluorescence was measured in a continuous
mode at 535 nm for 50 min under excitation at 485 nm. Concentra-
tion of 2–5A molecules required for cleavage of half of the syn-
thetic RNA substrate (EC50) was determined. The EC50 of the
natural 2–5A trimer was 6.1 nM whereas the EC50 values were
19.1 and 241 nM for the trimer 7 and the tetramer 8 analogs,
respectively. The EC50 of both analogs were in the nmol range.
Whereas the trimer 7 resulted only in a minimal decrease in activ-
ity, the tetramer 8 exhibited a moderate activation of RNase L with
a 40-fold drop. However, it is noteworthy that this experiment did
not take in account the advantage provided by the prodrug ap-
proach since no esterase was present to remove the PivOM groups
and to reconstitute the parent 2–5A.
Figure 3. IEX-HPLC and MALDI-TOF MS analyses of purified 2–5A trimer 7 and
tetramer 8 analogs.
quantities of 7 (654 nmol). When compared to the synthesis of the
uridylate trimer 6, the recovery of the crude material 7 was about
2–4 times lower (29.2%, 15.7% and 7.4%, see Table 1 entries 2, 3 and
5). The difference between these two syntheses was the 20-OH Pi-
vOM (for 7) or acetyl (for 6) protection of the ribonucleoside linked
through succinyl linker to the support. Acetyl group was removed
by butylamine whereas PivOM remained on the 20-OH which could
not assist the cleavage of the succinyl linker. This hypothesis could
explain the difference in the amount of recovered crude material.
Even if the use of succinyl linkage allowed synthesis of the desired
trimer analog 7, we investigated the use of the hydroquinone-O,O0-
diacetic acid (Q-linker) known to be more labile in basic condi-
tions.37 First we prepared CPG solid support derivatized with the
3.2. Antiviral activity of the 2–5A 30-PivOM trimer
The antiviral activity of the trimer 2–5A analog was first deter-
mined by evaluating the inhibition of influenza A/WSN/33 (H1N1)
virus in human lung epithelial cells. After 72 h incubation, the anti-
viral inhibitory effect was measured by the reduction of the lumi-
nescence signal from the determination of the viral RNA level in
using a Real-Time PCR a one-step quantitative RT-PCR assay.39,40
An EC50 value was obtained from the concentration of active com-
pound causing a reduction of half of the viral RNA production and
the cytotoxic effect (CC50) was evaluated by spectrophotometry
determination of ATP levels in a cell viability assay (Fig. 4). The effi-
30-PivOM adenosine 2 through the Q-linker with a 41
ing. As previously with the succinyl linker, the trimer analog
assembly was performed at 10 mol scale then the functionaliza-
lmol/g load-
l
tion at the 50-end with the triphosphate moiety and finally the
deprotection were carried out. The synthesis was more efficient
since the purity of the desired trimer 7 was estimated by HPLC
at 70% in the 2000 nmol of crude material released in TEAB solu-
tion (Table 1, entry 6). THF solution was analyzed and trimer 7
was present at 28% in the crude material representing 2590 nmol.
The analog 7 was further isolated after HPLC purification with high
purity and in satisfactory quantities (1665 nmol, 16.7%). As ex-
pected, recovery of material was more efficient with Q-linker than
with succinyl linker upon butylamine treatment. Both batches of
the trimer analog 7 were pooled to give rise 3.73 mg of total mate-
rial for antiviral studies.
cacy of the trimer analog (EC50 = 14.6
lM) was quite similar to the
efficacy of a control compound ALS-480 (EC50 = 3.2
l
M) which
serves as reference in Alios BioPharma assays. However the trimer
analog exhibited marginal specific activity in cell culture because
the window between EC50 and CC50 values was narrow. Indeed,
the EC50 = 14.6 lM, CC50 = 47 lM window is only approximately
3-fold making it likely that cell killing is contributing significantly
to the observed antiviral effect in the assay. In comparison the
activity of ALS-480 is clearly not mediated via killing cells as the
EC50 = 3.2
lM and CC50 = 194 lM window is large (selectivity in-
dex 60). Nevertheless, it is noteworthy that no transfection agent

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Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
Figure 4. Determination of EC50 and CC50 of 50-TP 30-PivOM 2–5A trimer in human lung epithelial cells infected by influenza virus.
was used in this assay and the 2–5A 30-PivOM analog could be ac-
tive proving the benefit of the biolabile groups on the increase of
the time life in cytoplasm and cell penetration.
4. Conclusion
In conclusion, we succeeded in the synthesis of a prodrug of 2–
5A trimer bearing biolabile groups with significant ability to acti-
vate RNase L and a noteworthy antiviral activity vs. RSV. This
promising data is encouraging to pursue the prodrug approach to
increase cellular delivery of 2–5A molecules.
Secondly, the antiviral inhibitory effect of the 30-PivOM 2–5A tri-
mer was evaluated in a Vero cell line (CCL-81) infected by the RSV
strain A2.41,42 In this cytopathic effect (CPE)-based assay the trimer
2–5A analog demonstrated similar activity to the positive control
ALS-480. In both cases an antiviral activity was noticed from drug
concentrations of 15 nM and above (Fig. 5). The control compound
ALS-480 exhibited a higher cytotoxicity since tested alone the blue
5. Material and methods
color disappeared at 33.3
lM whereas for the trimer analog as up to
5.1. General experimental procedures
100 M compound concentrations, no reduction in cell viability
l
was observed inthe absenceof RSV (bluecolor). However, inthe pres-
ence of RSV the cellular cytotoxicity was synergistically enhanced for
both ALS-480 (1.23 lM) and the trimer analog (33.3 lM) (Fig. 5, dot-
ted red line). Nevertheless its activity window was larger than the one
of the ALS-480 reference compound. This data suggests that the 50-TP
30-PivOM2–5Atrimerwasable toactivate RNase L intheVero cellline
and exhibited a noteworthy antiviral activity.
CH3CN, pyridine and triethylamine were distilled over calcium
hydride. Dichloromethane and carbon tetrachloride were distilled
over phosphorus pentoxide. All reactions were performed in anhy-
drous conditions under argon. The NMR experiments were accom-
plished on a Bruker DRX 400 spectrometer at 20 °C. HRMS analyses
were obtained with electrospray ionization (ESI) in positive mode
on a Q-TOF Micromass spectrometer.
Analytical and semi-preparative high performance liquid chroma-
tographies were performed on a Dionex DX600 HPLC systemor a Dio-
nex U 3000 HPLC system equipped with anion-exchange DNAPac PA
100 columns (4 ꢀ 250 mm for analysis or 9 ꢀ 250 mm, Dionex). The
following HPLC solvent systems were used: 5% CH3CN in 25 mM
Tris–HClbuffer,pH8(bufferA)and5%CH3CNcontaining400 mMNa-
ClO4 in 25 mM Tris–HCl buffer, pH 8 (buffer B). Flow rates were 1.5
and 5 mL minꢁ1 for analysis and semi-preparative purposes, respec-
tively.MALDI-TOFmassspectrawere recorded on a Voyager-DEspec-
trometer (Perseptive Biosystems, USA) using a 10:1 (m/m) mixture of
2,4,6-trihydroxyacetophenone/ammonium citrate as a saturated
solution inacetonitrile/water(1:1,v/v)forthe matrix. Analyticalsam-
ples were mixed with the matrix in a 1:5 (v/v) ratio, crystallized on a
100-well stainless steel plate and analyzed. UV quantitation of RNAs
was performed on a Varian Cary 300 Bio UV/Visible spectrometer by
measuring absorbance at 260 nm.
5.2. Chemistry
5.2.1. 50-O-(4,40-Dimethoxytrityl)-30-O-pivaloyloxymethyl-N6-
phenoxyacetyl-adenosine 2
Compound 2 was prepared according to a reported procedure.32
The crude mixture of both isomers 2 and 3 was subjected to silica
Figure 5. Inhibition of RSV A2 replication in Vero cell line in a CPE-based assay.

Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
5467
gel column chromatography with a step gradient of acetone (0–
40%) in dichloromethane with 1% pyridine. The second-eluted iso-
mer was the desired compound 2 and was obtained as yellow foam
after evaporation of the solvent. 1H NMR (400 MHz, HH-COSY,
dissolved in anhydrous pyridine (14 mL) and stirred at room tem-
perature for 6 h. Then pyridine solution was evaporated to oil,
redissolved in CH2Cl2 and H2O extractions were carried out. The
mixture obtained after drying of the extract over Na2SO4 and re-
moval of the solvent to yield crude yellow foam. HRMS (ESI)+ m/z
calcd for C55H56N5O15 (M+H)+ 1026.3759, found 1026.3773.
CDCl3): d 9.38 (s, 1H, NH), 8.70 (s,1H, H-2), 8.20 (s, 1H, H-8),
3
0
0
7.34-7.03 (m, 18 H, H ar, DMTr, Pac), 6.01 (d, JH1 /H2 = 5.6 Hz),
1H, H-10), 5.38, 5.35 (2dAB, JAB = 6.4 Hz, 1H+1H, OCH2O), 4.94 (t,
3
JH2 /H3 ,H1 = 5.2 Hz, 1H, H-20), 4.83 (s, 2H, NHCOCH2Ph), 4.56 (dd,
5.2.5. 50-O-(4,40-Dimethoxytrityl)-30-O-pivaloyloxymethyl-20-O-
Q-CPG-N6-phenoxyacetyl-adenosine
0
0
0
3
3
JH3 /H2 ,H4 = 4 Hz, H-30), 4.32 (q, JH4 /H5 ,H5 ,H3 = 3.7 Hz, H-40), 3.46
0
0
0
0
0
00
0
2
3
2
(dd, JH5 /H5 = 10.4 Hz; JH5 /H4 = 4 Hz, 1H, H-50), 3.26 (dd, JH5 /
To a dried 50 mL tube flask was added LCAA-CPG (1 g), unpuri-
0
00
0
00
3
= 10.8 Hz; JH5 /H4 = 4 Hz, 1H, H-500), 1.17 (s, 9H, OCOC(CH3)3).
fied
20-O-hydroquinone-O,O0-diacetylhemiester
derivative
00
H50
13C NMR (100 MHz, CDCl3): d 177.8 (OC@O), 166.6 (NHCO),
(551 mg, 0.5 mmol, 1 equiv), 4-dimethylaminopyridine (12.4 mg,
0.1 mmol, 0.2 equiv), 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide hydrochloride (192 mg, 1 mmol, 2 equiv) in anhydrous pyr-
idine (10 mL). The flask was shaken at room temperature for 6 h.
The CPG was filtered off, washed with CH2Cl2 and dried.
158.6, 157.0, (Cq, Car), 157.0 (Cq, Pac), 152.5 (C2), 151.5 (C6),
148.4, 144.4 (Cq, Car), 142.2 (C8), 130.0, 129.9, 128.2, 128.0,
127.9, 126.9, 125.3, 122.5, 114.9, 113.2 (CH, Car), 123.1 (C5), 89.4
0
0
0
(OCH2O), 88.8 (C1 ), 86.6 (Cq, DMTr), 83.0 (C4 ), 79.0 (C2 ), 74.2
0
0
(C3 ), 68.2 (NHCOCH2OPh), 62.9 (C5 ), 55.2 (OCH3, DMTr), 38.8
(Cq, OCOC(CH3)3), 27.0 (OCOC(CH3)3). HRMS (ESI+) m/z calcd for
C45H48N5O10 (M+H)+ 818.3400, found 818.3401.
For the capping step: in a 50 mL tube flask, CPG was mixed with
5 mL of a solution of 5% phenoxyacetic anhydride in tetrahydrofu-
ran and pyridine and 5 mL of a solution 10% methylimidazole in
THF. The flask was shaken at room temperature for 1 h. The CPG
was filtered off, washed with CH2Cl2 and dried over P2O5. Nucleo-
side loading was determined by DMTr analysis at 498 nm and was
5.2.2. 50-O-(4,40-Dimethoxytrityl)-30-O-pivaloyloxymethyl-20-O-
(2-cyanoethyl-N,N-diisopropylphosphoramidite) N6-
phenoxyacetyl-adenosine 4
40 lmol/g.
Compound 2 (2 g, 2.45 mmol, 1 equiv) was dried by 3 coevapo-
rations with anhydrous CH3CN. Then the residue was dissolved in
anhydrous CH2Cl2 (25 mL) and a mixture of N,N-diisopropylethyl-
5.2.6. Synthesis of 2–5A sequences on solid-support
RNA synthesis was performed on an ABI 394 synthesizer (Ap-
amine (770
lL, 4.4 mmol, 1.8 equiv), 2-cyanoethyl N,N-dii-
plied Biosystems) at 1 or 10 lmol scale using the prepared solid-
soproplylchlorophosphoramidite (814
l
L, 3.67 mmol, 1.5 equiv)
supports with a succinyl or a Q-Linker. 2–5A sequences were
assembled in Twist oligonucleotide synthesis columns (Glen re-
search) with the phosphoramidite building block 4. Phosphorami-
dite 4 was vacuum dried prior to its dissolution in extra dry
acetonitrile (Biosolve) at 0.1 M. For the coupling reaction, the acti-
vator was 5-benzylmercaptotetrazole (BMT, Chemgenes) used at
0.3 M concentration. Dichloroacetic acid (3% in CH2Cl2) (Glen re-
search) was the detritylation reagent. The capping step was per-
formed with a mixture of 5% phenoxyacetic anhydride (Pac2O) in
THF and 10% N-methylimidazole in THF (Link Technologies). The
oxidizing solution was 0.1 M iodine in THF/pyridine/H2O
(78:20:2; v/v/v) (Link Technologies). After 2–5A assembly comple-
tion, the column was removed from the synthesizer and dried un-
der a stream of argon.
in CH2Cl2 (2 mL) was added dropwise. The mixture was stirred un-
der argon at room temperature for 2 h. After completion, ethyl ace-
tate was added, the reaction mixture was poured into saturated
NaHCO3 solution and AcOEt extractions were carried out. The mix-
ture obtained after drying of the extract over Na2SO4 and removal
of the solvent was purified by silica gel column chromatography
with an isocratic gradient of CH2Cl2 and AcOEt (1:1) with 1% pyri-
dine. The desired phosphoramidite 4 was obtained as white foam
after evaporation of the solvent. (1.7 g, 1.67 mmol, 68%). 31P NMR
(121 MHz, CDCl3): d 151.39, 151.09. HRMS (ESI)+ m/z calcd for
C
54H65N7O11P (M+H)+ 1018.4467, found 1018.4480.
5.2.3. 50-O-(4,40-Dimethoxytrityl)-30-O-pivaloyloxymethyl-20-O-
succinyl-CPG N6-phenoxyacetyl-adenosine
To a dried microwave vial was added succinylated LCAA-CPG
(650 mg), adenosine derivative 2 (213 mg, 0.26 mmol, 1 equiv),
4-dimethylaminopyridine (32 mg, 0.26 mmol, 1 equiv), 1-(3-
5.2.7. 50-Triphosphorylation of 5–10
The solid-supported 1 or 10 l
mol 50-H-phosphonate, 50-phosp-
horoimidazolide derivatives, 50-triphosphate as well as tris(tri-n-
butylammonium) hydrogen pyrophosphate were synthesized as
described previously.33
dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride
(100 mg, 52 mmol, 2 equiv) in anhydrous CH3CN (5 mL). The reac-
tion mixture was heated over microwave irradiation at 60 °C for
1 h. The CPG was filtered off, washed with CH2Cl2 and dried.
For the capping step: in a 50 mL tube flask, CPG was mixed with
2.5 mL of a solution of 5% phenoxyacetic anhydride in tetrahydro-
furan and pyridine and 2.5 mL of a solution 10% methylimidazole
in THF. The flask was shaken at room temperature for 1 h. The
CPG was filtered off, washed with CH2Cl2 and dried over P2O5.
Nucleoside loading was determined by DMTr analysis at 498 nm
5.2.8. Deprotection and release of 50-TP sequences 6–10
5.2.8.1. 1
lmol Synthesis scale (6–8).
2 mL of a 1 M DBU
solution in anhydrous CH3CN was applied to the column for
3 min. Then the solution was removed and the solid-support was
washed with anhydrous CH3CN followed by a 1 min flush with ar-
gon and dried under vacuum over P2O5 for 2 h. Then 2 mL of a dry
solution of butylamine/THF 1:1 (v/v) was applied to the synthesis
column using two glass syringes filled of 4 Å molecular sieves (5
beads each). The solution was pushed back and forth through the
synthesis column for 2 min and left to react 2 h at 30 °C. The solu-
tion was removed and the column was rinsed with 1 mL of dry THF
followed by a 1 min gently flush with argon. The crude material
was eluted with 2 mL of 50 mM TEAB in a 50 mL round-bottomed
flask. The mixture was evaporated under reduced pressure to dry-
ness and the residue was co-evaporated three times with 1 mL of
water. The residue was dissolved in water (1.5 mL divided in three
portions for flask rinse: 0.8, 0.4, 0.3 mL) and transferred to 2 mL
Eppendorf-vials then lyophilized from water.
and was 62 lmol/g.
5.2.4. 50-O-(4,40-Dimethoxytrityl)-30-O-pivaloyloxymethyl-20-O-
hydroquinone-O,O0-diacetylhemiester-N6-phenoxyacetyl-
adenosine
Adenosine derivative 2 (1.67 g, 2 mmol, 1 equiv) was dried by 3
coevaporations with anhydrous pyridine. Then the residue 2,
hydroquinone-O,O0-diacetic acid (543 mg, 2.4 mmol, 1.2 equiv), 4-
dimethylaminopyridine (24 mg, 0.2 mmol, 0.2 equiv), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide
(382 mg, 2 mmol, 1 equiv) and triethylamine (200
hydrochloride
L) were
l

5468
Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
5.2.8.2. 10
lmol Synthesis scale (7).
The same procedure as
fetal bovine serum (FBS) and penicillin–streptomycin (100 U/mL)
above was applied with 5 mL of DBU solution and 5 mL of butyl-
amine/THF(1:1)solution. Thesolutionwasremovedfromthecolumn
and was collected in a 100 ml round bottom flask filled with 10 mL of
50 mM TEAB. A first elution was performed with 2 mL of dry THF in
this same flask, followed by a 1 min gently flush with argon. Then a
second elution was performed with 5 mL of 50 mM TEAB in a 50 mL
flask. Both mixtures were evaporated under reduced pressure to dry-
ness and the residues were co-evaporated three times with 1 mL of
water. Each residue was dissolved in water (1.5 mL divided in three
portions for flask rinse: 0.8, 0.4, 0.3 mL) and transferred to 2 mL
Eppendorf-vials then lyophilized from water.
and maintained at 37 °C, 5% CO2. The laboratory-adapted influenza
strain A/WSN/33 (H1N1) was obtained from (ATCC). This virus has
been previously propagated by infecting 10-day-old embryonated
chicken eggs. Allantoic fluid containing virus was harvested 48 h
post-inoculation. Titer of virus stock was determined by plaque as-
say on MDCK cell monolayers.
A549 cells were prepared and plated in 24-well culture plates at
a density of approximately 1 ꢀ 105 cells/well and incubated for
24 h at 37 °C. The next day the cells were washed twice with PBS
and then dosed with serially diluted compound (1:3), after incu-
bating at 37 °C for 1 h, the cells were challenged with influenza
A/WSN/33 virus. Cells were then incubated at 37 °C for 72 h. After
72 h incubation the A549 cell supernatant media was collected and
centrifuged to remove cellular debris. The supernatant was then
divided into aliquots and stored at ꢁ80 °C. Influenza viral RNA
was extracted from the A549 cell supernatant using the QIAamp
viral RNA mini kit (Qiagen, GmbH, Hilden, Germany) and viral
RNA quantity was assessed by OD260. Viral RNA was then stored
at ꢁ80 °C. To assess the level of influenza virus RNA in the cellular
supernatant a Real-Time PCR a one-step quantitative RT-PCR assay
was employed, this assay makes use of minor groove binder (MGB)
probe technology. Real-Time PCR using primers and probe target-
ing a highly conserved region of the matrix gene (M2) of influenza
type A viruses were used. The required concentration of compound
to reduce the 50% viral copy number (EC50) was calculated by
regression analysis of the dose–response curves generated from
these data.
5.2.9. Purification and desalting of 50-TP sequences 5–8
The crude 50-TP sequences (5–8) were analyzed by anion ex-
change HPLC using a 0–30% linear gradient of buffer B in buffer A,
and they were characterizedby MALDI-TOF spectrometry. The crude
mixtures (7–8) were then purified by semi-preparative IEX-HPLC
with a 0–30% linear gradient of buffer B in buffer A. The pure frac-
tions of 50-TP 2–5A were pooled in a 100 mL round-bottomed flask
and were concentrated to dryness under reduced pressure. The res-
idues were dissolved in 100 mM TEAB buffer, pH 8 (8 mL divided in
three portions for flask rinse: 5, 2, 1 mL) and were loaded on a C18
cartridge (Waters, Sep-PakÒ). Elution was performed with 10 mL
of 50 mM TEAB then with 10 mL of 50% CH3CN in 12.5 mM TEAB.
The second fraction containing the desired compound was collected
in a 100 mL round-bottomed flask and was freeze-dried. The residue
was dissolved in 1.5 mL water (divided in 3 portions of 0.8, 0.4,
0.3 mL for flask rinse) and transferred to a 2 mL Eppendorf-vial
and lyophilized from water. Lyophilized 50 TP-RNAs were stored at
ꢁ20 °C for several months without any degradation.
Cellular cytotoxicity was assessed using an ATP-based assay
readout. A549 cells (5000 cells/well) were grown in 96-well plates
for 24 h. The medium was then replaced with that containing seri-
ally diluted compounds and the cells are further incubated for 72 h.
The cytotoxicity of the compounds was measured using a Cell Ti-
ter-GloÒ Luminescent Cell Viability Assay (ATP-based cell viability
assay) (Promega, Madison, WI). Cellular efficacy and toxicity of
oseltamivir was tested in parallel as control for all experiments.
5.3. FRET assay
The activation of RNase L endonuclease activity can be detected
in a real-time Förster resonance energy transfer (FRET) assay. This
assay utilizes a 36 nucleotide synthetic oligoribonucleotide sub-
strate: 6-FAM-UUA UCA AAU UCU UAU UUG CCC CAU UUU UUU
GGU UUA-BHQ-1 with a fluorescence emitter (6-FAM) at the 50-
end and a quencher (BHQ-1) at the 30-end. The RNA sequence cor-
responds to a segment of the intergenic region of respiratory syn-
cytial virus (RSV) genomic RNA, chosen because it contains several
cleavage sites for RNase L (UU or UA). Upon RNA cleavage, the fluo-
rescent group is released from the quencher. The recombinant hu-
man RNase L expressed from a baculovirus vector in insect cells
was used in this assay at an effective concentration of 20 nM, to-
5.4.2. Respiratory syncytial virus CPE assay
The host cell line Vero (CCL-81, ATCC) was propagated in Dul-
becco’s Modified Eagle Medium (DMEM, Invitrogen) with 5% Fetal
Bovine Serum and 200 Units/ml Penicillin/Streptomycin at 37 °C,
5% CO2. The day before the experiment, the cells were trypsinized,
counted and plated at 30,000 cell/well in 96-well plates. On the
day of the experiment, test compounds were serially diluted in
serum free DMEM media and then added to the plated cells.
The wells designated for positive control (cells only without
virus) and negative control (cells with virus but no drug protec-
tion) were not dosed. After 2 h pre-incubation with compounds,
RSV A2 (VR-1540, ATCC) at the MOI of 0.05 was added to the
cells, except for the positive controls. The plate was then incu-
bated at 37 °C, 5% CO2 and observed daily for the cytopathic ef-
fects (CPE) in the cells. When the negative controls achieved
full or near full CPE, the culture media was removed from the
wells and Hucker’s Crystal Violet Solution (Fisher Scientific) was
gether with 200 nM FRET RNA in a final volume of 10
buffer [25 mM Tris–HCl (pH 7.4), 100 mM KCl, 10 mM MgCl2,
50 M ATP, 7 mM 2-mercaptoethanol, and 0.005% tween 20]. 2–
lL cleavage
l
5A compounds were added with a 384-well black polypropylene
plate. Fluorescence was measured in a continuous mode up to
50 min with a Wallac 1420 Victor3V multilabel counter (PerkinEl-
mer Life Sciences, Shelton, CT)(excitation 485 nm; emission
535 nm). False positives were eliminated by screening the com-
pounds in parallel in the absence of RNase L. Measured EC50 in
the RNase L activation assay is defined as the concentration at
which the emitted fluorescence reaches half of its maximum rate
added to the cells at 50 lL per well and further incubated for
5 min at room temperature. The stained plate was then washed
extensively to remove the background stain, air dried and
scanned. The cytotoxicity assay of compounds was performed
simultaneously with a CPE assay and was carried out the same
way as respective CPE assay except that no virus was added to
the cells.
(Vmax). The overall catalytic rate of the enzyme, expressed as kcat
,
is directly calculated by dividing Vmax by the enzyme concentra-
tion. Compounds 7 and 8 were run in duplicate with natural 2–5A.
5.4. Antiviral activity
Acknowledgments
5.4.1. Influenza virus assay
Human lung epithelial cells (A549) are routinely cultured in
Dulbeco’s Modified Eagle Medium (DMEM) containing 10% (v/v)
Y. Thillier thanks the Ministère National de la Recherche et de la
Technologie for the award of a research studentship.

Y. Thillier et al. / Bioorg. Med. Chem. 21 (2013) 5461–5469
5469
24. Ausín, C.; Kauffman, J. S.; Duff, R. J.; Shivaprasad, S.; Beaucage, S. L. Tetrahedron
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