Semisynthetic teicoplanin derivatives as new influenza virus binding inhibitors: Synthesis and antiviral studies

Article abstract of DOI:10.1016/j.bmcl.2014.06.018

In order to obtain new, cluster-forming antibiotic compounds, teicoplanin pseudoaglycone derivatives containing two lipophilic n-octyl chains have been synthesized. The compounds proved to be poor antibacterials, but, surprisingly, they exhibited potent anti-influenza virus activity against influenza A strains. This antiviral action was related to inhibition of the binding interaction between the virus and the host cell. Related analogs bearing methyl substituents in lieu of the octyl chains, displayed no anti-influenza virus activity. Hence, an interaction between the active, dually n-octylated compounds and the lipid bilayer of the host cell can be postulated, to explain the observed inhibition of influenza virus attachment.

Full text of DOI:10.1016/j.bmcl.2014.06.018

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3251–3254
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Semisynthetic teicoplanin derivatives as new influenza virus binding
inhibitors: Synthesis and antiviral studies
Ilona Bereczki a, Máté Kicsák a, Laura Dobray a, Anikó Borbás a, Gyula Batta b, Sándor Kéki c,
Éva Nemes Nikodém d, Eszter Ostorházi d, Ferenc Rozgonyi d, Evelien Vanderlinden e, Lieve Naesens e,
,
⇑
Pál Herczegh a,
⇑
a Department of Pharmaceutical Chemistry, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungary
b Department of Organic Chemistry, University of Debrecen, H-4032 Debrecen, Hungary
c Department of Applied Chemistry, University of Debrecen, H-4032 Debrecen, Hungary
d Microbiology Laboratory, Department of Dermatology, Venerology and Dermatooncology, Semmelweis University, Mária u. 41, H-1085 Budapest, Hungary
e Rega Institute for Medical Research, KU 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:
In order to obtain new, cluster-forming antibiotic compounds, teicoplanin pseudoaglycone derivatives
containing two lipophilic n-octyl chains have been synthesized. The compounds proved to be poor anti-
bacterials, but, surprisingly, they exhibited potent anti-influenza virus activity against influenza A strains.
This antiviral action was related to inhibition of the binding interaction between the virus and the host
cell. Related analogs bearing methyl substituents in lieu of the octyl chains, displayed no anti-influenza
virus activity. Hence, an interaction between the active, dually n-octylated compounds and the lipid
bilayer of the host cell can be postulated, to explain the observed inhibition of influenza virus attachment.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 29 April 2014
Revised 6 June 2014
Accepted 8 June 2014
Available online 16 June 2014
Keywords:
Teicoplanin pseudoaglycone
Lipophilic substituents
Aggregation
Influenza virus binding inhibitor
Human influenza A and B viruses cause the annual influenza
epidemics and sporadic pandemics, giving a considerable number
of fatalities.1 The influenza virus has two important antigenic
membrane glycoproteins: hemagglutinin (HA) and neuraminidase
(NA). Both are involved in virus replication. HA is responsible for
initial attachment of the virus to sialic acid-containing receptors
on the host cell, and membrane fusion after virus uptake by endo-
cytosis. NA performs the release of newly formed virions at the end
of the viral life cycle.
Vaccination is an important preventive strategy but, because of
the antigenic drift of influenza viruses, vaccines must be reformu-
lated and readministered every year.2
An alternative and potentially life-saving intervention is the use
of anti-influenza virus drugs. The M2 proton channel inhibitors
amantadine and rimantadine have become of less importance
since the widespread appearance of resistant viral strains.3
The neuraminidase inhibitors oseltamivir and zanamivir are the
standard drugs for treatment and prevention of influenza, but
increasing viral resistance (against oseltamivir in particular4,5
urges development of new anti-influenza medications.
)
Viral HA is one of the potential targets for drug development.6,7
To obtain potent HA inhibitors, sialoclusters, multivalent analogs of
HA ligands, have been synthesized by covalent attachment of sialic
acid or its oligosaccharide derivatives to polyacrylamids,8–10
liposomes,11,12 chitosan13 or polymersomes.14 Another strategy is
to prepare sialoconjugates capable of forming self-assembled
multivalent ligands.15,16 Recently, it has been discovered that some
synthetic peptides can be used as influenza virus HA traps.17–20
In the course of a detailed study on chemical transformations of
the aglycones of glycopeptide antibiotics, we have identified sev-
eral lipophilic aglycoristocetin derivatives possessing high anti-
influenza virus activity.21–25 Examination of the antiviral mode of
action of one of the most potent derivatives revealed that it traps
the virus in a distinct vesicular compartment, thereby preventing
entry of the virus into the nucleus of the host cell.
Parallel with the aglycoristocetin transformations we did
similar reactions with teicoplanin pseudoaglycone,23,26 but despite
the close structural similarities between the two series of semisyn-
thetic antibiotics, the teicoplanin derivatives did not exhibit
anti-influenza virus activity. However, most of them proved to be
strong antibacterials with outstanding activities against multi-
drug-resistant bacteria.23,26
⇑
Corresponding authors. Tel.: +32 16 337345; fax: +32 16 337340 (L.N.); tel.: +36
52 512 900 22895; fax: +36 52 512 914 (P.H.).
E-mail addresses: lieve.naesens@rega.kuleuven.be (L. Naesens), herczeghp@
gmail.com (P. Herczegh).
http://dx.doi.org/10.1016/j.bmcl.2014.06.018
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3252
I. Bereczki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3251–3254
All of our lipophilic antibiotic derivatives have amphiphilic
similar reaction sequence which was applied for 9 and 10.
First, instead of octylation, methylation was carried out to furnish
3b,30 which was transformed to 4b by reductive opening of the
4,6-O-acetal ring. Introduction of the propargyl-containing linker
to the primary position afforded 6b, which was transformed to 7b
by removal of the p-methoxybenzyl protecting group (Scheme 1).
Finally, the glucose units 6b and 7b were conjugated to teicoplanin
properties and, therefore, the tendency to form large, nano-sized
clusters in water. We postulated that such aggregates can act as
multivalent ligands of surface receptors of bacteria and viruses,
explaining their enhanced biological activities.
In this work, we report on a novel, saccharide-based, versatile,
lipophilic derivatisation of teicoplanin pseudo-aglycone with the
aim of obtaining new antibacterial agents. The new compounds
were found to display unexpected inhibitory activity towards
influenza virus by interfering with virus binding to the host cell.
The synthesis of the carbohydrate auxiliary started from methyl
w
-aglycone via azide–alkyne click reaction to provide 11 and 12,
respectively (Scheme 2).
The aggregation of 9–12 in water was studied by dynamic light
scattering. Formations of clusters with bimodal size-distribution
were observed for 9 and 10. Effective diameter of these clusters
was 62 nm for 9 and 21 nm for 10. Contrary to our expectations,
11 and 12 turned out to be even more prone to form clusters. Effec-
tive diameters of aggregates were 160 nm and 203 nm for 11 and
12, respectively.
When evaluated for antibacterial activity against a panel of bac-
terial strains, compound 10 exhibited modest antibacterial activi-
ties, 9 was totally inactive, and 11 and 12 turned out to be very
modest antibacterials (see Supporting information). These data
were surprising when we consider the favorable antibacterial
a-D-glucopyranoside 1 (Scheme 1). p-Methoxybenzylidenation of 1
by transacetalisation afforded diol 2,27 the two hydroxyls of
which were n-octylated to result in 3a. Regioselective reductive
cleavage of the 4,6-O-acetal ring using the LiAlH4–AlCl3 reagent
combination in a 3:1 ratio (forming AlH3)28 resulted in exclusively
the 4-O-p-metoxybenzyl (PMB) ether 4a. The liberated 6-OH of 4a
was etherified with propargylated bromo-tetraethyleneglycol 529
to give 6a from which the p-methoxybenzyl group was removed
by oxidative cleavage using 2,3-dichloro-5,6-dicyano-1,4-benzo-
quinone (DDQ) as the reagent28a to afford 7a. Both latter compounds
were coupled with an azido derivative of teicoplanin pseudoagly-
cone 823 using a Cu(I) catalyzed 1,3-dipolar cycloaddition click reac-
tion providing amphiphiles 9 and 10 (Scheme 2). To obtain reference
compounds for the biological and aggregation studies, synthesis of
teicoplanin derivatives 11 and 12 containing O-methyl substituents
activity of 13 (Fig. 1), a structurally similar teicoplanin
w-aglycone
derivative bearing one lipophilic n-decyl side chain.23
Even more surprising, we observed that 9 exhibited strong anti-
viral activity against three strains of influenza A virus, including
the 2009 pandemic virus A/H1N1 Virginia/ATCC3/2009. However,
it was inactive against influenza B virus (data not shown). Com-
pound 10 proved to be highly active against two out of the three
investigated influenza A strains. In the course of our extensive
studies on teicoplanin aglycone derivatives,23,26 the herein
reported 9 and 10 were the first compounds possessing anti-
influenza virus activity. Their antiviral EC50 values were in the
on the D-glucose unit was also carried out. We supposed that due to
the lower lipophilicity of methyl groups than that of n-octyls the
tendency to form clusters will be less pronounced for 11 and 12 than
for 9 and 10, and we also hypothesized that the lower cluster-form-
ing ability will be accompanied by lower antibacterial activity.
Hence, synthesis of 11 and 12 was performed starting from 2 in a
range of 1–2 lM (Table 1) and 4- to 20-fold lower than the
compound concentrations causing cytotoxic effects, as estimated
by microscopic examination or cell viability testing (see MCC and
CC50 values, respectively, in Table 1). The methyl-functionalized
derivatives 11 and 12 showed no inhibitory activity against any
of the influenza strains used in this study (data not shown).
In the presence of 9 and 10, influenza virus-induced hemagglu-
tination was inhibited. The concentration to achieve this inhibition
increased with the amount of virus (expressed in hemagglutinating
units) (Table 2). This result suggests that compounds 9 and 10
interfere with virus binding to the host cell. In the absence of virus,
H3CO
HO
HO
O
OCH3
OCH3
H3CO
OCH3
O
O
O
HO
TsOH
DMF
HO
OCH3
OH
OH
2 (93%)
1
C8H17Br
or CH3I
DMF
NaH
9 and 10 caused hemagglutination at 50 lM. Based on the results
H3CO
OH
O
of the hemagglutination assay, we carried out a binding assay on
MDCK cells.22 As shown in Figure 2, a dose-dependent inhibition
of influenza virus binding to the host cells was observed; the con-
LiAlH4-AlCl3, 3:1
CH2Cl2 : Et2O
O
PMBO
RO
O
O
0 °C
centration giving 50% inhibition of virus binding was 4.7
and 7.0 M for 10.
In conclusion, two teicoplanin
lM for 9
OCH3
RO
OCH3
l
OR
OR
R = C8H17 (83%)
3b R = CH3 (83%)
w
-aglycone derivatives with
4a
R = C8H17 (77%)
3a
excellent anti-influenza virus activity have been prepared. The
antiviral mode of action appears to be based on inhibition of the
binding interaction between the virus and the host cell. It is note-
worthy that 9 and 10 as well their O-methyl analogs 11 and 12 can
form nanoaggregates in water. However, the striking difference in
anti-influenza virus activity of these pairs of compounds leads to
the conclusion that the antiviral action of 9 and 10 cannot be
explained by their self-assembled multivalency. Instead, the
observed activity may be explained by the potential interaction
of their double, long alkyl chains with the lipid bilayer of the host
cell membrane. We hypothesize that this anchoring of 9 and 10 on
the surface of the host cell creates a strong shielding effect and/or a
multivalent interaction with the viral HA. As a consequence, virus
attachment is prevented. The short methyl groups of 11 and 12 are
not capable of interaction with the host cell membrane, explaining
their lack of antiviral activity.
4b R = CH3 (81%)
NaH
DMF
O
O
Br
O
5
O
O
O
R2O
4
O
R1O
OCH3
OR1
R = C8H17, R2 = PMB (75%)
1
6a
6b
1
2
DDQ
R = CH3, R = PMB (74%)
DDQ
CH2Cl2/H2O
9 : 1
7a R1 = C8H17, R2 = H (97%)
1
2
7b
R = CH3, R = H (96%)
Scheme 1. Functionalization of methyl
chains and a propargyl-containing linker.
a
-
D-glucopyranoside with lipophilic side

I. Bereczki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3251–3254
3253
OH OH
HO
OH
Cl
O
H
O
O
O
AcHN
O
Cl
O
O
H
N
H
N
O
N3
N
H
N
N
H
H
H
O
H
HN
O
O
HOOC
HO
OH
OH
OH
HO
8
CuI
Et3N
+ 6a or 6b or 7a or 7b
OR1
OR1
OR2
H3CO
O
OH OH
O
HO
OH
Cl
O
O
O
O
4
AcHN
H
O
O
Cl
O
O
H
H
N
N
O
N
N
N
H
N
H
N
H
N
H
H
O
HN
O
O
HOOC
HO
OH
OH
OH
HO
9 R1 = C8H17, R2 = PMB (37%)
10
R
1 = C8H17, R2 = H (48%)
11 R1 = CH3, R2 = PMB (53%)
12 R1 = CH3, R2 = H (50%)
Scheme 2. Synthesis of teicoplanin
w-aglycone derivatives 9–12 bearing variously substituted glucose unit at the N-terminal of the glycopeptide core.
HO
OH
Cl
HO
O
O
O
O
H
H
O
O
HO
Cl
O
AcHN
O
O
N
NH
NH
N
N
H
N
N
H
N
H
H
O
O
O
HN
HOOC
HO
OH
OH
HO
OH
13
Figure 1. Previously prepared teicoplanin pseudoaglycone derivative with high antibacterial activity.23
Table 1
Anti-influenza virus activity of 9 and 10 in MDCK cell cultures
Compound
Cytotoxicity (
lM)
Antiviral EC50 (lM)
Influenza A/H1N1
(A/PR/8/34)
Influenza A/H1N1
(A/Virginia/ATCC3/2009)
Influenza A/H3N2
(A/HK/7/87)
CC50
MCC
CPE
MTS
CPE
MTS
CPE
MTS
9
10
11
12
>100
>100
>500
>500
20
0.80
>100
0.47
6.8
212
4.6
1.2
1.4
1.8
45
12
>500
>500
<0.80
1.3
20
11
>500
>500
1.8
2.3
8.9
6.8
0.45
0.051
1.2
2.2
9.0
7.3
0.50
0.046
P4.0
>100
P20
>500
P500
>100
0.50
21
111
5.3
Zanamivir
Ribavirin
Amantadine
Rimantadine
Abbreviations used: MDCK, Madin–Darby canine kidney; EC50, 50% effective concentration, or compound concentration required to give 50% inhibition of virus replication, as
estimated by microscopic scoring of the cytopathic effect (CPE) or the colorimetric TS cell viability assay; TS, tetrazolium salt; CC50, 50% cytotoxic concentration, that is, the
concentration that reduces cell viability by 50% according to the colorimetric MTS assay; MTS, 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophe-
nyl)tetrazolium, inner salt; MCC, minimal cytotoxic concentration, that is, the compound concentration that causes minimal alterations in cell morphology, as assessed by
microscopy.

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I. Bereczki et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3251–3254
Table 2
Inhibitory effect of 9 and 10 on influenza virus-induced hemagglutination
Compound
RBC incubated with virus at
2 HA-U
RBC incubated without virus
4 HA-U
1 HA-U
Minimal concentration (lM),causing hemagglutination inhibition
Minimal concentration (lM), causing hemagglutination
9
10
12
3.1
6.3
0.78
0.78
0.39
50
50
Abbreviations used: RBC, red blood cells (from chicken); HA-U, hemagglutinating units.
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Acknowledgments
}
}
The study was supported by the Hungarian Research Fund
(OTKA K 109208), the University of Debrecen (Bridging Fund to
P.H.) and a grant from the Geconcerteerde Onderzoeksacties
(GOA/10/014) from the KU Leuven. The authors wish to thank
Wim van Dam and Stijn Stevens for their dedicated technical assis-
tance. We are indebted to Ádám Fizil for the NMR measurements.
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