Design, synthesis and anti-influenza virus activities of terminal modified antisense oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2013.10.129

Four novel terminal modified antisense oligonucleotides (ODNs) were designed, synthesized and tested for their anti-influenza virus activity. Initial biological studies indicated that lipophilic and rimantadin emodificated Flutide exhibited more potent anti-H1N1 activity than Flutide. Among them, lipophilic modificated ODN (Flutide-I) showed the most antiviral activity. The EC₅₀ value of Flutide-I for inhibiting H1N1 induced cytopathic effect (CPE) and H1N1 RNA were respectively (0.26 ± 0.16) μM and (0.11 ± 0.03) μM. The cytotoxicity of these compounds has also been assessed. No significant cytotoxicities were found for any of these compounds with the concentrations up to 20 μM.

Full text of DOI:10.1016/j.tetlet.2013.10.129

Tetrahedron Letters 55 (2014) 94–97
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Design, synthesis and anti-influenza virus activities of terminal
modified antisense oligonucleotides
a,b,
Jingyu Zhang a,b, , Dandan Lu a, , Aixing Li c, Jing Yang a, , Shengqi Wang
⇑
⇑
a Beijing Institute of Radiation Medicine, Beijing 100850, China
b Henan University of Traditional Chinese Medicine, Zhengzhou 450008, China
c School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel terminal modified antisense oligonucleotides (ODNs) were designed, synthesized and tested
for their anti-influenza virus activity. Initial biological studies indicated that lipophilic and rimantadin
emodificated Flutide exhibited more potent anti-H1N1 activity than Flutide. Among them, lipophilic
modificated ODN (Flutide-I) showed the most antiviral activity. The EC50 value of Flutide-I for inhibiting
Received 23 June 2013
Revised 21 October 2013
Accepted 27 October 2013
Available online 5 November 2013
H1N1 induced cytopathic effect (CPE) and H1N1 RNA were respectively (0.26 0.16)
(0.11 0.03) M. The cytotoxicity of these compounds has also been assessed. No significant cytotoxici-
ties were found for any of these compounds with the concentrations up to 20 M.
lM and
l
Keywords:
l
Antisense oligonucleotides
Influenza virus
H1N1
Ó 2013 Elsevier Ltd. All rights reserved.
Terminal modification
Introduction
Flutide (Scheme 1) is a 13-mer antisense phosphorothionate
oligonucleotide (PS-ODNs), which is complementary to the 50 ter-
minal conserved regions of viral RNA found in almost all of the
influenza A virus.11 All of the nonbridging oxygen atoms in the
phophodiester bond are replaced by sulfur in its structure. The
introduction of phosphorothioate linkages into ODNs is primarily
intended to enhance their nuclease resistance.12 Previous studies
have showed that Flutide exhibited the most potent antiviral
activity in vitro and in vivo. It inhibited influenza virus A induced
cytopathic effects in MDCK cells with the EC50 ranging from 2.2
In 1978, Paul Zamecnik and Mary Stephenson reported the first
experiments on antisense mechanisms of gene silencing, using
short synthetic antisense oligonucleotides (ODNs) to inhibit repli-
cation of the Rous sarcoma virus by binding and blocking the
action of 35s RNA, then increasing attention turned to the possible
therapeutic applications of antisense technology.1,2 In 1998, the
first antisense drug, Vitravene (ISIS Pharmaceuticals Inc.), was
approved for retinitis induced by cytomegalovirus.
In recent years, antisense ODNs have been applied to the treat-
mentofa varietyof diseases includingviral infection,3 tumor,4 vessel
restenosis,5 fulminant septic shock,6 asthma, and allergies.7 In Jan
29th, 2013, the U.S. FDA approved the second antisense drug,
Kynamro (mipomersen sodium, ISIS Pharmaceuticals Inc. and
Genzyme Corp.) to treat inherited cholesterol disorder, which is an
oligonucleotide inhibitor for homozygous familial hypercholester-
olemia(HoFH).8 Antisense ODN is designedto hybridizetoa comple-
mentary target sequence of corresponding mRNA, which inhibits
protein expression. Therefore antisense ODNs may display increase
in affinity and selectivity for their nucleic acid targets compared
with traditional drugs.9,10
to 4.4 lM. In the infected mouse model, prolonged mean survival
days and declined virus titres in lung in the Flutide treatment
groups compared with the infected control group, with a dose-
dependent manner.11
Some reports have showed that terminal modification of ODNs
facilitates the cellular import and increases the antiviral activ-
ity.13,14 Therefore, as a continuous research program of our labora-
tory to improve the drugability of Flutide, we now report the
design and synthesis of terminal modificated Flutide (Scheme 2)
and their anti-influenza virus activity.
Results and discussion
Flutide and modified ODNs are solid phase synthesized with the
phosphoramidite approach. The whole synthetic process has been
completely automated with DNA synthesizer (ABI3900).
As shown in Scheme 3, 2-(4-aminobutyl)propane-1,3-diol 3
was prepared from 4-bromobutyronitrile 1 and diethyl malonate
⇑
Corresponding authors. Tel.: +86 1066931423 (S. Wang).
E-mail addresses: jingyang0511@sina.com (J. Yang), sqwang@bmi.ac.cn
(S. Wang).
Co-first author: Jingyu Zhang and Dandan Lu contributed equally adequate to this
work.
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.10.129

J. Zhang et al. / Tetrahedron Letters 55 (2014) 94–97
95
NH2
N
O
HN
N
O
O
N
H
HO
O
O
NH2
N
H
H
H
H
H
H
O
H
H
H
O
P
O
P
HN
Na+-
S
O
Na+-
S
O
H
N
O
O
O
N
H
O
O
O
O
H
H
H
H
H
NH2
N
H
H
HN
O
P
H
O
P
Na+-
S
Na+-
S
O
H
O
H
O
O
N
H
N
O
O
O
O
O
O
H
H
H
H
H
HN
O
P
H
H
HN
O
P
Na+-
S
O
H
Na+-
S
O
H
O
N
H
O
O
N
H
O
O
O
H
H
H
O
NH2
N
H
H
N
N
O
P
NH
NH2
N
N
O
P
H
+-S
O
Na
N
O
Na+-S
O
H
N
O
O
O
O
H
H
H
NH2
N
H
H
H
H
H
HN
O
P
H
O
P
Na+-S
O
Na+-
S
O
H
O
N
H
N
O
O
O
O
O
O
H
H
H
H
H
H
H
O
P
H
HN
O
P
Na+-
S
O
Na+-
S
O
H
O
O
N
H
O
O
H
H
H
OH
Scheme 1. Structure of Flutide.
OH
OH
S
P
O
S
O
O
O
O
Flutide
O
P
O
Flutide
N
H
N
H
3'
5'
5'
3'
R
R
O
H
N
O
Flutide = 5'-CCTTGTTTCTACT-3'
or
R=
Scheme 2.
2
via condensation reaction and reduction reaction succes-
pylphos-phoramidite 7 in 78% isolated yield. Then, the terminal
modified Flutide-II was synthesized on a 1.0 mol scale by using
sively.15 Treatment of behenic acid 4 and 3 with 1-ethyl-3-(3-di-
methyl-aminopropyl) carbodiimide hydrochloride in acetonitrile
gave N-(6-hydroxy-5-(hydroxymethyl)hexyl)docosanamide 5 in
88% isolated yield. Subsequent selective protection of one
primary hydroxy group of 5 with 4,40-dimethoxytrityl group
(DMT) provided 6 in 37% yield. Next, 6 was treated with 2-cyano-
ethyl N,N,N0,N0-tetraisopropylphosphordiamidite ((iPr2N)2POCH2
CH2 CN) together with diisopropylammonium and tetrazolide in
dry acetonitrile to afford 2-((bis(4-methoxyphenyl)(phenyl)
methoxy)methyl)-6-docosanamidohexyl(2-cyanoethyl) diisopro-
l
7, as a kind of modified phosphoramidite, which coupled to the 50
termini of Flutide following the standard PS-ODN synthesis pro-
cess with the commercially DNA phosphoramidites (Sigma–Al-
drich) and Unylinker™ 200 solid support (Nitto Denko Cor.). On
the other hand, 6 was treated with butanedioic anhydride in
tetrahydrofuran at ambient temperature for 12 h. Then NH2
group of the Controlled Pore Glass (CPG) was added to the reac-
tion system to afforded 8, which was used as the solid support
for the synthesis of Flutide-I. Rimantadine terminal modified

96
J. Zhang et al. / Tetrahedron Letters 55 (2014) 94–97
OH
ODMT
O
ODMT
OH
O
O
3'
5'
Flutide
S
P
O
f
H3C
O
H3C
O
e
H3C
O
( )
( )
( )
( )
N
H
N
H
( )
( )
20
20
3
O
N
H
3
P
N
CN
20
3
7
6
Flutide-II
O
d
d
O
OEt
OEt
H3C
OH
OH
OH
OH
( )
OH
20
O
NC
b
a
4
NC
Br
H3C
( )
( )
H2N
N
H
5
O
20
3
c
2
1
3
OH
ODMT
O
ODMT
OH
O
O
O
O
3'
Flutide
5'
S
P
O
f
H3C
O
g
h
H3C
H3C
( )
( )
N
( )
( )
O
N
N
H
CPG
( )
( )
20
3
N
H
3
H
20
H
20
3
O
8
6
Flutide-I
Flutide = 5' CCTTGTTTCTACT 3'
DMT=
OCH3
H3CO
Scheme 3. Reagents and conditions: (a) Na, diethyl malonate, CH3CH2OH, 85%; (b) LiAlH4, THF, 93%; (c) CH3CN, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, rt to 50 °C, 88%; (d) 4,40-dimethoxytrityl chloride, dimethylaminopyridine, pyridine, 0 °C, 37%; (e) (iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide,
CH3CN, THF, 78%; (f) DNA synthesizer for Flutide-I and Flutide-II; (g) butanedioic anhydride, THF, 0 °C to rt; (h) 2,3,4,5,6-pentafluorophenol, NH2-CPG, THF, rt.
O
OH
OH
OH
OH
O
O
O
H
N
H
N
H
N
NH2
N
H2N
( )
OH
O
N
H
3
3
b
O
a
O
O
O
c
12
d
10
9
11
ODMT
OH
ODMT
OH
S
O
O
O
H
N
H
N
H
N
O
O
O
P
O
Flutide
( )
( )
3
N
N
P
CN
( )
3
N
H
3'
f
3
5'
H
H
O
e
O
N
O
O
13
Flutide-III
14
g
h
OH
S
O
ODMT
O
H
N
O
O
O
P
O
Flutide
5'
H
N
( )
N
H
3
CPG
3'
( )
O
N
H
N
H
f
3
O
O
O
Flutide-IV
15
Flutide
= 5' CCTTGTTTCTACT 3'
DMT=
OCH3
H3CO
Scheme 4. Reagents and conditions: (a) CH3CN, butanedioic anhydride, reflux, 99%; (b) THF, HoSu, DCC, rt, 97%; (c) CH3CN, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride, rt to 50 °C, 83%; (d) 4,40-dimethoxytrityl chloride, dimethylaminopyridine, pyridine, 0 °C, 63%; (e) (iPr2N)2POCH2CH2CN, diisopropylammonium tetrazolide,
CH3CN, THF, 78%; (f) DNA synthesizer for Flutide-III and Flutide-IV; (g) butanedioic anhydride, THF, 0 °C to rt; (h) 2,3,4,5,6-pentafluorophenol, NH2-CPG, THF, rt.
Flutide (Flutide-III and Flutide-IV) was prepared employed the
similar strategy as shown in Scheme 4.
The cleavage of the synthesized terminal modified ODNs
(Flutide-I–Flutide-IV) from the solid support, and the deprotection
of the nucleotides were carried out with 28% aqueous ammonia at
60 °C for 8 h. The solution was evaporated under reduced pressure
at room temperature to remove ammonia, and the residue was di-
luted with 0.10 M ammonium acetate (50 mL). The solution was

J. Zhang et al. / Tetrahedron Letters 55 (2014) 94–97
97
In addition, both 30 and 50 terminal modified Flutides showed
significant increase of antiviral activity. And we conjecture that
the modification on both ends is more conducive to improve the
antiviral activity. The above speculation content needs to be fur-
ther studied.
Table 1
Anti-influenza virus (H1N1) activity and cellular toxicity of terminal modificated
ODNs (Flutide-I–Flutide-IV) and Flutide
a
b
Compounds
EC50
(
l
M)
EC50
H1N1, RNA
(
l
M)
CC50 (lM)
H1N1, CPE
Flutide-I
Flutide-II
Flutide-III
Flutide-IV
Flutide
0.26 0.16
0.72 0.17
0.28 0.10
0.54 0.24
1.74 0.91
0.11 0.03
0.17 0.04
0.26 0.02
0.39 0.06
2.15 0.87
>20
>20
>20
>20
>20
In summary, four novel terminal modificated PS-ODNs were de-
signed and synthesized and their anti-influenza virus activities
were evaluated in vitro. Initial biological studies indicated that
lipophilic terminal modification at the 30 end of Flutide (Flutide-
I) showed the most promising activity against H1N1 replication.
Further investigations of the biological activities are currently
underway.
a
EC50
CC50
(
l
M) is the concentration that inhibits IV by 50%.
M) is the concentration of the compound at which 50% of the cells were
b
(
l
destroyed.
Acknowledgments
placed on the C18 cartridge column and the failure sequences were
eluted by use of 10% acetonitrile/0.10 M ammonium acetate. After
being washed with 0.1 M ammonium acetate and water, the col-
umn was treated with aqueous 2% DCA to remove the DMT group,
washed with 0.10 M ammonium acetate and water. The target oli-
gonucleotide was eluted by use of 30% acetonitrile/water and the
fractions containing the target were lyophilized to give the crude
oligonucleotide. Pure material was obtained by being purified on
an anion-exchange HPLC by use of 0–50% gradient of 1.0 M NaCl
in 25 mM sodium phosphate–10% acetonitrile. The salts were
removed by use of the C18 cartridge column to give the pure oligo-
nucleotide after being lyophilized to dryness.
We gratefully acknowledge the Beijing Proteome Research Cen-
ter for their kind support in MALDL-TOF-MS analysis. This work
was supported by two Grants from the National Natural Science
Foundation of China (Nos. 81230089 and 30901825) and two
Grants from the National Science and Technology Major Projects
(Nos. 2011ZX09102-009-09 and 2012ZX09301003-001-004) and
Doctor Foundation (BSJJ2009-07) of Henan University of Tradi-
tional Chinese Medicine.
Supplementary data
The anti-influenza virus activity of terminal modificated Flutide
and the primary ODN Flutide were evaluated by detecting cyto-
pathic effect (CPE) using CCK8 assay and detecting viral replication
using qRT-PCR analysis. Influenza virus-infected MDCK cells were
treated with series of two fold dilution of terminal modificated Flu-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2013.10.
129.
References and notes
tide or the primary ODN Flutide started at 0.10–4.0 lM for 48 h.
CPE was measured by cell viability with CCK8 assay according to
the manufacturer’s instructions. The virus RNAs from cell medium
were isolated and subjected to qRT-PCR described as previous
reports.16 The EC50 values were calculated and listed in Table 1.
Flutide showed significant anti-H1N1 (A/Jingfang/01/1986
(H1N1)) activity, which is the same as the previous reports.11 Lipo-
philic and rimantadine terminal modification could increase the
anti-H1N1 activity. Among them, Flutide-I showed the most potent
anti-H1N1 activity. Compared with Flutide, the inhibiting activity
of H1N1 induced CPE of Flutide-I increased 6.69 folds, the inhibit-
ing activity of H1N1 RNA increased 19.55 folds. In addition, the
cytotoxicity of terminal modificated Flutide had been assessed by
cell proliferation assay. MDCK cells were treated with series dilu-
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tions of the compounds started at 1.0–20
were then used. As showed in Table 1, no significant cytotoxicity
was found for any of them with concentrations up to 20 M.
lM for 48 h. CCK8 assay
l
We speculated that the possible mechanism is that the terminal
lipophilic and rimantadine modification changed the polarity of
Flutide, which is an ASODN. From the structure and many reports,
the polarity of ASODN is large, which may result in low cellular
uptake.13,14 In this study, terminal lipophilic and rimantadine mod-
ification makes the molecule Flutide less polar and thus the cellular
uptake might be improved via better transmembrane ability of
antisense ODN–rimantadine conjugate and antisense ODN–lipoid
conjugate. Therefore, the dosage was decreased and anti-influenza
virus activity was strengthened obviously.