Design of inhibitors of influenza virus membrane fusion: Synthesis, structure-activity relationship and in vitro antiviral activity of a novel indole series

Article abstract of DOI:10.1016/j.antiviral.2013.05.005

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure-activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1. H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.

Full text of DOI:10.1016/j.antiviral.2013.05.005

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Contents lists available at SciVerse ScienceDirect
Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral
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Design of inhibitors of influenza virus membrane fusion: Synthesis,
structure–activity relationship and in vitro antiviral activity of a novel
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indole series
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Q1 Virginia Brancato
, Antonella Peduto , Stephen Wharton , Stephen Martin , Vijaykumar More ,
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Antonia Di Mola , Antonio Massa , Brunella Perfetto , Giovanna Donnarumma , Chiara Schiraldi ,
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a,
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Maria Antonietta Tufano , Mario de Rosa , Rosanna Filosa , Alan Hay
MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
Department of Pharmaceutical and Biomedical Science, University of Salerno, Fisciano, Italy
Department of Experimental Medicine, Microbiology and Clinical Microbiology Section, Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy
Department of Chemistry, University of Salerno, Fisciano, Italy
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Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy
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a r t i c l e i n f o
a b s t r a c t
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Article history:
Available online xxxx
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The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The
low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin
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(HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative
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Keywords:
Arbidol derivatives
Antiviral action
Influenza haemagglutinin
pH stabilization
of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of
the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and
synthesized to further probe the foundation of its antiviral activity and develop the basis for a struc-
ture–activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-
indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arb-
idol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and
preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to
Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however,
the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15,
apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.
Ó 2013 The Authors. Published by Elsevier B.V. All rights reserved.
Fluorescence binding assay
Differential binding to haemagglutinin
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1. Introduction
in the lipid envelope of the virion, the haemagglutinin (HA) and
the neuraminidase (NA). Sixteen subtypes of HA and nine of NA re-
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Influenza is one of the most infectious diseases and is
responsible for very significant morbidity and mortality annually
around the world. The viruses belong to the Orthomyxoviridae
family; they have a segmented, single-stranded, negative-sense
RNA genome and are divided into three genera A, B and C, accord-
ing to their internal components (Palese and Shaw, 2007). The
influenza A viruses are classified by subtype, based on the anti-
genic characteristics of the two surface glycoproteins embedded
sult in the existence of a large number of virus subtypes with dif-
ferent combinations of HA and NA, only a few of which, currently
H1N1 and H3N2, have circulated in the human population (Wright
et al., 2007). Most influenza A viruses are maintained in a vast nat-
ural reservoir in wild waterfowl and shorebirds, from which they
may emerge to cause disease in domestic animals and humans;
influenza B and C viruses principally infect humans.
Vaccination is the mainstay of control and prevention strategies
against influenza and is based on the generation of neutralizing
antibodies against the HA. However, antigenic drift in the HA
necessitates frequent change in vaccine composition, and the time
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
(
6–9 months) involved in producing and delivering vaccines
against newly emergent variants can impact the effectiveness of
this approach (Ampofo et al., 2012). Anti-influenza drugs
complement vaccines and their use is particularly important in
combating the initial stages of an emergent pandemic, when
⇑
Corresponding authors. Tel.: +44 0208 816 2141; fax: +44 0208 906 4477.
E-mail addresses: rfilosa@unisa.it (R. Filosa), ahay@nimr.mrc.ac.uk (A. Hay).
These authors contributed equally to this work.
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Present address: Interdisciplinary Research Centre on Biomaterials (CRIB),
University of Naples Federico II, P. Le Tecchio 80, 80125 Naples, Italy.
0
166-3542/$ - see front matter Ó 2013 The Authors. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.antiviral.2013.05.005
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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vaccines against the novel virus are not available (Hayden and
Pavia, 2006).
1999). More recently, a new class of N-(1-thia-4-azaspiro[4.5]dec-
an-4-yl)carboxamide inhibitors that also show specific activity
against H3 subtype viruses was reported (Vanderlinden et al.,
2010; Zhan et al., 2012). Arbidol, on the other hand, has been
shown to be more broadly effective in inhibiting different influenza
A subtypes (with both group 1 and 2 HAs) and influenza B viruses
(Boriskin et al., 2008).
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The widely available influenza antivirals are currently limited to
two classes of agents: the neuraminidase inhibitors (oseltamivir
and zanamivir) and the M2 inhibitors (amantadine and rimanta-
dine) (Moscona, 2005). However, their effectiveness has been
limited by the emergence of drug-resistant viruses. This is
especially so for the M2 inhibitors, in that the seasonal A(H3N2)
viruses have recently acquired resistance and the seasonal
A(H1N1) viruses were replaced by the amantadine-resistant pan-
demic H1N1 2009 viruses (Deyde et al., 2007; Hay et al., 2008;
Gubareva et al., 2009). A significant increase in amantadine resis-
tance among some highly pathogenic H5N1 viruses have further
limited the potential usefulness of these antivirals (Hurt et al.,
2007).
On the other hand, neuraminidase inhibitor drugs have been
favoured clinically, since they are effective against influenza B as
well as all influenza A NA subtypes, are well tolerated, and appear
to have a higher barrier to resistance emergence (Moscona, 2005).
However, the previous seasonal A(H1N1) viruses, circulating in
2007–2009 acquired oseltamivir-resistance (Collins et al., 2009)
and oseltamivir-resistant variants have occurred sporadically
among the novel A(H1N1)pdm09 viruses that emerged in April
2009 (Hurt et al., 2011); and oseltamivir is the therapeutic agent
favoured by clinicians (Bautista et al., 2010; Ling et al., 2010). There
is therefore an urgent need for more antivirals against influenza;
the only other licensed drugs with anti-influenza activity are
ribavirin, which has been used to only a very limited extent to treat
severe infections, and Arbidol which is marketed in Russia and
China and a few other countries (Boriskin et al., 2008).
Potential novel targets being explored for the development of
new anti-influenza agents include the viral polymerase (and
endonuclease), the non-structural protein NS1 and the HA (Das
et al., 2010). Blocking virus entry into the host cell (Luo, 2012) is
already an effective strategy, as it is the target of the M2 inhibitors
and is the basis of vaccination. The trimeric envelope glycoprotein
HA plays a key role in promoting fusion between the virus and
endosome membranes during virion internalization by endocyto-
sis, as well as being responsible for attachment to sialylglycan
receptors on the cell surface (Skehel and Wiley, 2002). The protein
contains two disulfide-linked polypeptide chains, HA1 and HA2;
the HA1 subunit contains the receptor binding site and HA2 the
N-terminal fusion peptide. The acidic pH in the endosome triggers
a conformational change in the HA whereby the fusion peptide is
inserted into the target membrane promoting fusion between the
virus and endosome membranes. Prior to this event passage of pro-
tons through the M2 channel causes acid-induced dissociation of
the internal ribonucleoprotein (RNP) – matrix (M1) structure to ef-
fect release of the virus RNP into the cytosol for transport into the
nucleus to initiate replication (Hay et al., 2008) Cloroquine and
high concentrations of amantadine, like other acidotropic agents,
can inhibit non-specifically virus infection in vitro by increasing
endosome pH and preventing the low pH-dependent structural
transition (Di Trani et al., 2007; Daniels et al., 1985).
Several inhibitors of influenza replication in vitro, including
Arbidol, have been shown to specifically target the fusion-mediat-
ing conformational change in HA by increasing the acid stability of
the protein (Skehel and Wiley, 2002). However, many of them are
subtype specific. For example, a series of benzo- and hydro-qui-
nones, including tert-butyl hydroquinone (TBHQ), were shown to
bind in a pocket of the H3 (group 2) HA and prevent the native
HA from undergoing the low pH-induced conformational change,
but not to bind H1 or H2 (group 1) HAs (Bodian et al., 1993). In con-
trast, a number of other inhibitors, including a quinolizidine-linked
benzamide, were shown to block the HA conformational change of
H1 and H2, but not of H3, subtypes (Luo et al., 1997; Plotch et al.,
The basis of the antiviral efficacy of Arbidol is, however, unclear
since it has proven to be effective in the treatment of several other
respiratory viral infections in addition to influenza and it inhibits
the replication in vitro of a variety of enveloped and non-enveloped
viruses, (Boriskin et al., 2008) including, for example, respiratory
syncytial virus, parainfluenza virus, rhinovirus, and hepatitis B
and C viruses (Brooks et al., 2004; Chai et al., 2006; Pécheur
et al., 2007). This broad spectrum of activity has complicated
interpretation of the molecular basis of Arbidol action, for example,
the relative importance of interaction with the lipid membrane or
with protein components involved in the membrane fusion
process. (Teissier et al., 2011). However, Leneva et al. (2009) have
shown that in the case of influenza, properties of the HA, in partic-
ular the pH of fusion, are major determinants of the sensitivity of
virus replication in vitro, indicating that Arbidol inhibits virus entry
by interacting directly with HA to stabilize it against the low
pH-induced conformational change mediating membrane fusion.
With the aim of identifying novel lead compounds active
against emergent human infectious diseases (Perfetto et al.,
2013; Peduto et al., 2011) and to gain a better understanding of
the structural features of Arbidol important for its (broader) antivi-
ral activity and HA binding properties, and the relationship
between HA binding and antiviral activity, in this paper we report
the design, synthesis and structure–activity (SAR) studies of sev-
eral new ethyl 1H-indole-3-carboxylate derivatives structurally re-
lated to Arbidol. Retaining the indole skeleton, modifications were
introduced in positions 2, 4, 5 and 6 of the heterocyclic ring. The
compounds synthesized can be classified into two different series
(Chart 1). The first series (Series I) includes alcohol derivatives
(6, 7, 15, 16, 18 and 21) which lack an amino substituent in
position 4 (Table 1A). The second series (Series II) comprises com-
pounds 24, 28a–h, 29a–m, 30, 31 and 32 in which the hydroxy
group in position 5 was replaced by different amino substituents
(Chart 1, Table 1B).
Compounds were tested against a range of influenza viruses. Of
all the compounds, ethyl 5-(hydroxymethyl)-1-methyl-2-(pheny-
sulphanylmethyl)-1H-indole-3-carboxylate (15) was identified to
be one of the most potent inhibitors, with a therapeutic index
greater than Arbidol for most viruses tested. To investigate the ba-
sis of its improved activity, we compared the effects of 15 with
those of Arbidol on HA-mediated membrane fusion, assayed by
haemolysis and heterokaryon formation, and their interaction with
HA, in fluorescence quenching and thermal denaturation assays.
While the effects of 15 were somewhat greater than those of Arb-
idol against membrane fusion (and virus replication), the affinity of
binding of 15 to HA was substantially higher and in particular
exhibited a much greater preferential binding to group 2 than to
group 1 HAs.
1
1
1
1
1
1
1
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1
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1
1
1
1
1
1
1
1
1
1
1
1
1
1
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00
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12
13
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15
16
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31
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2. Materials and methods
195
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2.1. Chemical synthesis of the compounds
All reagents were analytical grade and purchased from Sigma–
Aldrich (Milano, Italy). Flash chromatography was performed on
Carlo Erba silica gel 60 (230–400 mesh; CarloErba, Milan, Italy).
TLC was carried out using plates coated with silica gel 60 F
254 nm purchased from Merck (Darmstadt, Germany). 1H NMR
197
198
199
200
201
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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N
O
O
HO
Br
N
S
ARBIDOL
O
O
O
O
n
R
HO
R1
R1
N
N
X
X
SERIES I
SERIES II
Chart 1. Chemical structures of Series I and II compounds.
Table 1A
Inhibition of the replication of different influenza viruses by Arbidol and Series I compounds.
Cpds
Virus tested IC50 (lM)
O
O
n
HO
R
1
N
X
X
n
R
1
H1N1a
H1N1b
H1N1pdmc H3N2d
H3N2e
H4N6f
H7N3g
H8N4h
H11N9i
Bj
6
7
1
1
1
2
H
Br
H
H
Br
Br
0
0
1
1
1
1
CH
CH
CH
CH
CH
CH
2
2
2
3
3
2
SPh
SPh
SPh
31.3 (5.6)
31.3 (2.2)
15.6 (5.2)
–
7.9 (3.8)
–
62.5 (4.5) 31.3 (2.3)
31.3 (1.3) 31.3 (2.3)
62.5 (3.5) 125 (2.3)
15.6 (3.1) 3.9 (2.3)
62.5 (2.8)
31.3 (0.5)
7.8 (2.8)
125 (2.9)
7.9 (2.1)
125 (2.5)
15.6 (1.7)
–
–
–
–
62.3 (2.6)
125 (3.0)
–
–
125 (4.9)
31.3 (3.0)
–
15.6 (1.7)
15.6 (1.9)
–
5
6
8
1
125 (2.3)
–
31.3 (1.9) 7.9 (2.3)
–
–
31.3 (1.6) 31.3(2.5)
–
62.5 (2.5)
–
–
–
–
–
125 (3.5)
7.9 (1.9)
125 (2.5)
–
–
–
125 (2.0)
–
7.9 (1.8)
–
SPh
62.5 (2.4)
125 (2.5)
31.3 (1.9)
ARB
11.8 (4.4)
15.6 (3.1) 23.4 (1.3)
15.6 (1.7) 7.8 (2.5)
31.3 (2.3) 15.6 (1.3)
A dash indicates no effect at 125
lM compound. Standard deviations are in parenthesis.
a
A/Brisbane/59/07.
A/PR/8/34.
A/California/7/09.
A/Wuhan/359/95.
A/X31.
A/dk/Czechoslovakia/56.
A/ty/Italy/02.
A/ty/Ontario/68.
b
c
d
e
f
g
h
i
A/dk/Memphis/74.
j
B/Brisbane/60/06.
2
2
2
2
2
02
03
04
05
06
spectra were registered on a Brucker AC 300. Chemical shifts are
reported in ppm. The abbreviations used are follows: s, singlet; d,
doublet; dd double doublet; bs, broad signal. Mass spectrometry
analysis ESI-MS was carried out on a Finnigan LCQ Deca ion trap
instrument.
with iodomethane under basic condition (Cao et al., 2005) in
DMF provided compound 3 which was submitted to radical bro-
mination yielding a mixture of mono and dibromo derivatives 4
and 5, easily separated by chromatography on silica gel. Final
nucleophilic displacement by thiophenol (Trofimov et al., 1993)
gave the desired compounds 6 and 7 (Scheme 1).
Compound 8, prepared as described in the literature, (Wein-
stain et al., 2008) was the common intermediate for the synthesis
of 5-hydroxymethyl-indole derivatives 15, 16, 18 and 21. It was
reacted with two different b-ketoesters 9 and 10 in a copper-cata-
lyzed Ullmann-type coupling reaction (Sellitto et al., 2010;
Tanimori et al., 2007) giving the key intermediates 11 and 12.
Subsequent N-alkylation with iodomethane using Kikugawa’s
procedure afforded compounds 13 and 14 in quantitative yields.
215
216
217
218
219
220
221
222
223
224
225
226
227
228
207
208
209
210
211
212
213
214
2.1.1. General synthesis of Series I (6, 7, 15, 16, 18 and 21)
The title compounds, ethyl-5-hydroxy or 5-hydroxymethyl-2-
methyl-1H-indole-3-carboxylate derivatives were obtained as de-
scribed in Schemes 1–3 (Supporting information). The synthesis
of 5-hydroxyindole derivatives 6 and 7 started from commercially
available ethyl 5-hydroxy-2-methylindole-3-carboxylate (1),
which underwent a protection reaction using acetic anhydride
and pyridine, affording intermediate 2 in good yield. Methylation
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

Table 1B
Inhibition of the replication of different influenza viruses by Series II compounds.
Cpds
Virus tested IC50 (lM)
O
O
R
R
1
N
X
X
R
1
R
H1N1a
H1N1b
H1N1pdmc
H3N2d
H3N2e
H4N6f
H7N3g
H8N4h
H11N9i
Bj
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
H
H
CH
3
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
2
NH
2
–
–
–
–
–
–
–
–
–
–
8a
8b
8c
8d
8e
8f
8g
8h
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
SPh
SPh
SPh
SPh
SPh
SPh
SPh
SPh
N(CH
N(CH
N(CH
Pyrrolidine
N-methyl piperazine
NHBoc-piperazine
N-(2,4 difluoro)phenylpiperazine
Morpholine
N(CH
N(CH
N(CH
Pyrrolidine
N-methyl-piperazine
2-piperazin-1-ylethylamino
N(CH
N(CH
3
2
3
)
2
7.9 (0.8)
3.9 (0.7)
3.9 (0.4)
7.9 (0.9)
7.9 (0.3)
31.3 (2.3)
31.3 (3.3)
31.3 (0.7)
125 (2.0)
62.5 (1.4)
7.9 (0.6)
31.3 (0.7)
31.3 (0.6)
31.3 (1.6)
31.3 (0.4)
31.3 (3.0)
31.3 (1.2)
31.3 (0.7)
31.3 (1.8)
31.3 (2.3)
31.3 (0.9)
62.5 (1.0)
–
15.6 (2.8)
15.6 (1.3)
3.5 (0.4)
31.3 (2.6)
31.3 (1.0)
7.9 (0.2)
3.9 (0.5)
3.9 (0.7)
7.9 (1.1)
7.9 (0.8)
31.3 (1.9)
31.3 (0.3)
31.3 (1.8)
–
125 (1.7)
7.9 (0.6)
31.3 (0.7)
31.3 (1.0)
31.3 (0.5)
31.3 (0.5)
31.3 (0.9)
31.3 (1.2)
31.3 (0.6)
31.3 (0.8)
31.3 (0.4)
31.3 (2.2)
125 (1.2)
–
7.9 (0.2)
7.9 (1.2)
7.9 (0.4)
7.9 (1.0)
7.9 (0.7)
3.9 (0.5)
3.9 (0.4)
15.6 (1.1)
15.6 (0.9)
15.6 (0.5)
62.5 (2.0)
62.5 (0.6)
62.5 (1.4)
–
125 (2.9)
7.9 (0.5)
62.5 (0.9)
62.5 (1.3)
62.5 (1.2)
–
7.9 (0.2)
7.9 (0.7)
7.9 (0.6)
7.9 (0.9)
7.9 (0.3)
31.3 (2.9)
31.3 (2.7)
7.9 (0.9)
46.9 (2.5)
31.3 (1.8)
7.9 (1.3)
7.9 (1.0)
7.9 (1.3)
15.6 (1.4)
15.6 (1.3)
7.9 (0.2)
7.9 (0.4)
7.9 (0.7)
7.9 (0.8)
7.9 (1.2)
31.3 (1.4)
31.3 (1.0)
15.6 (0.2)
125 (1.2)
–
CH
)CH
3
)
2
2 2 2 2 3 2
CH CH N(CH CH )
3
2
3
)
2
62.5 (1.7)
125 (1.2)
7.9 (0.8)
–
125 (2.3)
15.6 (1.2)
15.6 (1.4)
31.3 (0.7)
31.3 (1.3)
–
–
–
–
–
–
–
CH
)CH
)
3 2
125 (0.8)
31.3 (0.8)
–
–
125 (1.6)
15.6 (1.7)
–
–
125 (1.8)
31.3 (1.6)
–
–
2
CH
2
CH
2
N(CH
2
CH
3
)
2
15.6 (1.9)
125 (1.7)
125 (1.7)
15.6 (1.8)
125 (1.6)
125 (2.1)
–
3
)CH
CH
2
CH
)CH
2
N(CH
2
CH
3
)
2
2
3
2
CH N(CH
2
2
CH
3
)
2
–
NHCH
NHCH
NHCH
NHCH
NHCH
2
2
2
2
2
CH
CH
CH
CH
CH
2
2
2
2
2
NHCH
NHBoc
N(CH
3
62.5 (1.2)
62.5 (1.7)
62.5 (0.7)
62.5 (0.8)
15.6 (1.3)
–
3 2
)
OH
9m
0
1
2 2
NHCH CH OH
N(CH
3
)
2
125 (1.9)
125 (2.8)
15.6(2.0)
–
–
–
–
125 (1.3)
–
125 (1.3)
–
125 (1.5)
–
Diazepan
N-methyl-pyrrolidinium iodide
–
125 (1.8)
2
SPh
31.3 (0.5)
31.3 (0.4)
62.5 (0.8)
A dash indicates no effect at 125
lM compound.
a
A/Brisbane/59/07.
A/PR/8/34.
A/California/7/09.
A/Wuhan/359/95.
A/X31.
A/dk/Czechoslovakia/56.
A/ty/Italy/02.
A/ty/Ontario/68.
b
c
d
e
f
g
h
i
A/dk/Memphis/74.
j
B/Brisbane/60/06.

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5
2
2
2
2
2
2
2
2
2
2
29
30
31
32
33
34
35
36
37
38
Final deprotection using TBAF in THF (Corey et al., 1977) furnished
the 5-hydroxymethyl derivatives 15 and 16 in excellent yields
(Scheme 2). Treatment of 14 with 1 equiv. of NBS (Trofimov
et al., 1993) allowed us to obtain monobromo 17 which was fur-
ther deprotected, as previously described, to give compound 18.
Using 2 equiv. of NBS, intermediate 14 underwent dibromination
in positions 2 and 6 and conversion of the hydroxy-methyl group
in position 5 into aldehyde to yield 19. Reduction with NaBH4
and subsequent treatment with thiophenol in EtOH permitted us
to recover compound 21 (Scheme 3).
test agent. MDCK cells were seeded in 96-well plates and incu-
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
bated for 24 h in a humidified atmosphere at 5% CO . The cells
2
were exposed to twofold serial dilutions of compound, dissolved
in DMEM with 5% FCS, ranging from 250 lM to 3.9 lM, and incu-
bated for 24 h and 48 h. Cells were then washed with phosphate
buffered saline (PBS) and incubated for 2 h with neutral red
(3-amino-7-dimethylamino-2-methyl-phenazine hydrochloride-
Sigma, N4638) dissolved in serum-free DMEM at a final concentra-
tion of 40 lg/ml. Cells were washed again with PBS to remove the
dead cells and the dye was extracted from the intact cells in 1% gla-
cial acetic acid in 50% ethanol (Glacial acetic acid, Sigma, 537020;
Ethanol, Riedel-de Haen, 32294). The fluorescence was read in a
spectrofluorimeter with excitation and emission wavelengths of
530 nm and 645 nm, respectively. Three independent experiments
were performed and the CC50 (drug concentration required to
reduce cell viability by 50%) was calculated at 24 h and 48 h.
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
2.1.2. General synthesis of Series II (24, 28a–h, 29a–n, 30, 31, 32)
The title compounds, ethyl-5-aminomethyl-1-methyl-1H-in-
dole-3-carboxylate derivatives were obtained as described in
Schemes 4–6 (Supporting information). The synthesis of primary
amine 24 is described in Scheme 4. Ethyl 1,2-dimethyl-5-(hydroxy-
methyl)-1H-indole-3-carboxylate (16) was treated with PPh3 in a
mixture of CCl and DMF at room temperature, affording the corre-
4
2.4. Plaque reduction assay
306
sponding chloromethyl derivative 22 (Hübner et al., 2000). Gabriel
synthesis and subsequent hydrazinolysis furnished ethyl 5-(ami-
nomethyl)-1,2-dimethyl-1H-indole-3-carboxylate 24 in good yield
(Roubini et al., 1991).
The virus plaque assay, using low-viscosity overlay medium in a
96-well format, (Matrosovich et al., 2006) was used to measure the
inhibition of virus replication by Arbidol and its analogues. Briefly,
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
3
Amines 28a–h, 29 a–n and 30 were synthesized according to
the procedure described in Scheme 5 (Supporting information).
Oxidation of alcohols 15, 16 and 18 with pyridinium dichromate
led to the formation of aldehydes 25–27 in high yields. Subsequent
reductive amination with several aliphatic amines, using
NaBH(OAc)3 as reducing agent, provided the desired compounds
28a–h, 29a–n and 30 in excellent yields. Ethyl 5-(1,4-diazepan-
1-ylmethyl)-1,2-dimethyl-indole-3-carboxylate 31 was synthe-
sized from Boc deprotection of amine 29n using TFA in CH Cl
MDCK cells 3 Â 10 cells per well were seeded in 96-well plates in
DMEM, containing 10% FCS, 100 U/ml penicillin, 100 lg/ml
streptomycin, and were incubated at 37 °C until 90% confluence.
Twofold serial dilutions of each compound from 125 M to
3.9 M were added to duplicate wells. After incubation for
30 min at 37 °C, 100 l of virus-containing allantoic fluid (approx-
l
l
l
imately 0.1 PFU/cell) was added to each well, except uninfected
control cells. Each microtitre plate included uninfected control
wells, virus-infected control wells and virus-infected wells
containing compound. After 3 h of infection, cells were overlaid
with an equal volume of DMEM medium supplemented with
2
2
(Schemes 6 in Supporting information). Conversion of ethyl 1-
methyl-2-(phenylsulfanylmethyl)-5-(pyrrolidin-1-ylmethyl)-1H-
indole-3-carboxylate 29d into a quaternary ammonium salt 32 was
performed using methyl iodide in ethyl acetate.
0.2% Bovine Serum Albumin, 1.2% (w/v) Avicel, and 2.5
TPCK-trypsin (Sigma T-1426).
lg/ml
After 24 h incubation at 37 °C in a humidified atmosphere with
5% CO , cells were washed and fixed by adding 50 l of cold 4%
2
63
2.2. Cells and viruses
l
2
paraformaldehyde in PBS. Cells were incubated for 1 h with a
mouse monoclonal antibody against influenza type A or B nucleo-
protein and then with a peroxidase-labelled anti-mouse antibody,
diluted 1:4000 and 1:1000, respectively, in ELISA buffer, containing
10% horse serum, 0.1% Tween-80 in PBS. Cells were then incubated
with True Blue™ substrate (KPL, Gaithersburg, USA), 0.03% H O ,
2
2
2
2
2
2
64
65
66
67
68
69
Madin-Darby canine kidney (MDCK) cells were cultured in
Dulbecco’s Modified Eagle Medium (Sigma D6429), supplemented
with 10% foetal calf serum (FCS) inactivated at 56 °C for 1 h,
penicillin (100 U/ml) and streptomycin (100
lg/ml) (Sigma
P0781), in a humidified atmosphere with 5% CO2.
2
2
The influenza viruses were from stocks held by the WHO
Collaborating Centre for Reference and Research on Influenza, at NIMR:
A/Puerto Rico/8/34(H1N1) (PR8), A/Brisbane/59/2007(H1N1), A/Califor-
nia/7/2009(H1N1pdm09), A/Singapore/1/57(H2N2), A/Wuhan/359/
95(H3N2), recombinant virus X31(H3N2) (A/Aichi/2/68 Â PR8), A/
duck/Czechoslovakia/56(H4N6), A/turkey/Italy/214845/2002(H7N3),
A/turkey/Ontario/6118/68(H8N4), A/duck/Memphis/546/74(H11N9),
B/Brisbane/60/2006, and four mutants of X31(H3N2), three with in-
creased pH of fusion, 1a (HA2 D112G), 2a (HA2 R54K) and ab4 (HA1
H17R), resistant to high concentrations of amantadine (Daniels et al.,
1985), and the antibody escape mutant V9A (HA1 G218R) (Daniels
et al., 1987). The attenuated recombinant virus RG14(H5N1) (A/Viet-
nam/1194/2004 Â PR8) was obtained from the National Institute for
Biological Standards and Control. Virus stocks were grown in the allan-
toic cavities of 11-day-old embryonated hen eggs for use in all the exper-
iments. The allantoic fluid was clarified by low-speed centrifugation and
the virus titre determined by haemagglutination (HA) assay, and stored
at À80 °C.
until colour developed. Plaques were counted and the IC50 value
(concentration of compound required to reduce the number/size
of plaques by 50%) was calculated (Sullivan et al., 2012). Three
independent experiments were performed.
2
70
2
2
2
2
2
2
2
2
2
71
72
73
74
75
76
77
78
79
2.5. Haemolysis inhibition assay
335
Virus-induced haemolysis was estimated as described previ-
ously using human erythrocytes (Wharton et al., 1994; Steinhauer
et al., 1995). Wild type X31 and mutants 1a, 2a, ab4 and V9A were
used. Each influenza virus was added to 1 ml 1% human red blood
cells (RBC) in PBS and was incubated at 37 °C in the presence or
336
337
338
339
340
341
342
343
344
345
346
2
80
2
2
2
2
2
2
81
82
83
84
85
86
absence of 40 lM Arbidol or 15. The pH was then varied over the
range 4.6 to 7.0 using citrate buffer (0.15 M sodium citrate pH
3.5). After 30 min incubation, cell debris and un-lysed cells were
removed by centrifugation at 2000g for 10 min and the absorbance
of the released haemoglobin in the supernatant was read at
520 nm.
2
87
2.3. Evaluation of cell viability by neutral red assay
2.6. Heterokaryon assay of fusion between cells expressing X31 HA
347
2
2
88
89
The neutral red assay determines the accumulation of the neu-
tral red dye in the lysosomes of viable cells after incubation with
Chinese Hamster Ovary (CHO) cells transfected with the X31 HA
gene (Godley et al., 1992) were grown in 24-well plates. Confluent
348
349
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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V. Brancato et al. / Antiviral Research xxx (2013) xxx–xxx
3
3
3
3
3
50
51
52
53
54
monolayers were treated with TPCK-treated trypsin for 5 min at
37 °C, in order to the cleave HA0 to the fusion active HA1/HA2,
Table 2
Cytotoxicities of Arbidol and its derivatives in MDCK cells.
and then with 40
lM 15 or Arbidol for 30 min at 37 °C. Cells were
CC50 lM
then incubated with citrate buffer at different pH values and het-
erokaryon formation was monitored microscopically every 10 min.
Compounds
24 h
48 h
Compounds
24 h
48 h
Arbidol
6
7
115.2
LC
125
LC
LC
LC
LC
LC
117
LC
31.3
LC
LC
LC
29a
29b
29c
29d
29e
29f
29g
29h
29i
29j
29k
29l
29m
30
LC
LC
52.0
LC
LC
179.4
239
239
LC
LC
LC
LC
LC
49.7
LC
LC
89.5
183
186.3
LC
LC
LC
LC
LC
LC
193
LC
a
3
55
2.7. Purification of HA
1
1
1
2
5
6
8
1
356
357
358
359
360
361
The soluble ectodomain of HA (BHA) was released from purified
virus, grown in hen eggs, by incubation with the protease brome-
lain (1:10 (w/w) bromelain:virus) for 16 h at 37 °C in 0.1 M Tris/
HCl pH 7.5, 50 mM 2-mercaptoethanol, and was purified by su-
crose gradient sedimentation and ion exchange chromatography
as previously described (Ha et al., 2002).
LC
LC
24
28a
42.6
30.5
50.4
18.9
HC
90.3
71.8
175
225
147
2
2
2
2
2
2
2
8b
8c
8d
8e
8f
b
HC
49.3
24.6
LC
LC
LC
LC
LC
LC
LC
3
62
2.8. Tryptophan fluorescence assay
8g
8h
31
32
LC
3
3
3
3
3
3
3
63
64
65
66
67
68
69
Increasing amounts of 15 or Arbidol (from 5 mM stock solutions
in DMSO) were added to BHA (0.2 lM in PBS pH 6.5) at 20 °C and
tryptophan fluorescence was measured using a Jasco FP-6300 spec-
trofluorimeter with excitation at 285 nm and emission at 340 nm.
Fluorescence intensities were corrected for inner filter effects and
a
LC: low cytotoxicity, CC50 > 250
HC: high cytotoxicity, CC50 < 3.9
l
l
M.
M.
b
thiophenyl by a methyl in position 2 of the indole ring reduced
significantly the toxicity (24, 29a–b, 29d–m, 30 and 31), except
for compound 29c which retained toxicity similar to its analogue
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
equilibrium dissociation constants (K ) were determined by non-
d
linear least-squares fitting to a one-site binding model.
2
8c, demonstrating the high cytotoxic potential of this particular
amine.
Analysis of the structure–activity relationships revealed that
3
70
2.9. Thermal shift assay
371
372
373
374
375
376
377
378
379
380
The assay was performed as previously described (Niesen et al.,
the alcohol derivatives 6 and 7 were less active than the lead com-
pound Arbidol against all virus strains, indicating the importance
of the amine at position 4 for Arbidol activity; while the presence
2007). BHA (0.4
Arbidol (final concentration 50
l
M in PBS pH 6.5) was incubated with 15 or
M, 1% DMSO), or with1% DMSO,
l
for 15 min at 25 °C. SYPRO Orange (Sigma; 5X) was added and
the temperature of the preparation was increased at 5 °C per
min, and the fluorescence measured every 2 °C increase (excitation
470 nm, emission 570 nm) using a Jasco FP-6300 instrument. The
Tm was estimated as the temperature at the midpoint of the change
in SYPRO fluorescence brought about by thermal unfolding of the
BHA.
(7) or absence (6) of bromine had little effect. The insertion of a
methylene spacer between the indole ring and hydroxyl group
caused a substantial increase in activity of compound 15 (relative
to 6), which proved to be the most potent compound of Series I
(Table 1A). It was very potent against almost all the subtypes of
influenza A tested, with IC50 values ranging from 3.9 to 31.3 M,
l
except for A/ty/Italy/2002(H7N3) and the influenza B virus. In
particular, its inhibitory activity was better than Arbidol against
four virus strains, A/California/7/2009(H1N1pdm), A/X31(H3N2),
A/dk/Czechoslovakia/56(H4N6) and A/dk/Memphis/74(H11N9),
and was comparable against A/Wuhan/359/95(H3N2). The greater
potency of compound 15 is accentuated by its lower cytotoxicity
than Arbidol; consequently the therapeutic indices are generally
higher than those of the lead compound (Table 3A).
The insertion of the methylene spacer in 7, which retains the Br
substituent, resulted in loss of activity of compound 21. Compound
18, in which the thiophenyl substituent at position 2 was replaced
by a methyl group and bromine was retained in position 6, was
even more potent than Arbidol against some of the viruses, while
16, which lacks the Br moiety, was much less active (Tables 1A
and 3A).
3
81
3. Results
3
3
82
83
3.1. Inhibition of influenza virus replication by 1H
-indole-3-carboxylate derivatives
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
Arbidol and its derivatives were tested at different concentra-
tions (from 3.9
lM to 125
l
M) for their ability to inhibit influenza
virus replication in MDCK cells, in a plaque reduction assay. Differ-
ent types and subtypes of influenza viruses were used initially:
three different strains of subtype A(H1N1) and two of subtype
A(H3N2), and single strains of subtypes H4N6, H7N3, H8N4 and
H11N9, and a type B virus. Differences were observed in the sensi-
tivities of the different viruses to particular derivatives as well as in
the effectiveness of different compounds, expressed as IC50 (Tables
1A and 1B). In later tests, only three viruses, two H1N1 and one
H3N2 subtype A viruses, were used, those which had shown higher
susceptibility to inhibition.
Compounds of the ‘amino’ Series II, in which the hydroxyl group
at position 5 is replaced by an amine, that have a methyl group in
position 2 (24, 29a–b, 29d–m, 30, 31) exhibited low activity or
were completely inactive, except for compound 29c that showed
The cytotoxicities of the compounds (from 3.9
lM to 250
l
M)
good potency (IC50 ranged from 7.9 lM to 31.3 lM) against all
in MDCK cells were assessed at 24 and 48 h using a neutral red
viability assay. The results are expressed as the CC50 (the concen-
tration of compound causing 50% reduction in cell viability) (Table
2). No toxicity was observed with the alcohol derivatives (Series I)
virus strains, but was very cytotoxic (Tables 1A and 2). Compounds
28a–e, which retained the thiophenyl substituent, also exhibited
greater potency than Arbidol, but were more cytotoxic.
For compounds with low cytotoxicity and high inhibitory activ-
ity, the therapeutic indices (TI; defined as the ratio between the
CC50 and the IC50) are shown in Tables 3A and 3B. Based on these
data, compound 15 was selected for further experiments because
it showed a better therapeutic index for most viruses tested,
compared to Arbidol and the others analogues, except for 18 which
at concentrations below 250 lM, except for compound 7 that was
slightly cytotoxic. In contrast, as regards the compounds of Series
II, most with a thiophenyl ring in position 2 (28a–e) were highly
cytotoxic. The toxicity disappeared when a positive charge was in-
serted into the amino substituent (32 vs 28d). The replacement of
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

AVR 3203
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V. Brancato et al. / Antiviral Research xxx (2013) xxx–xxx
7
Table 3A
Therapeutic indices (TI) of Arbidol and Series I compounds in inhibition of the replication of influenza viruses.
Cpds
Virus tested TI
O
O
n
HO
R
1
N
X
X
n
R
1
H1N1a
H1N1b
H1N1pdmc
H3N2d
H3N2e
H4N6f
H7N3g
H8N4h
H11N9i
Bj
6
7
H
Br
H
H
Br
Br
0
0
1
1
1
1
10.0
CH
CH
CH
CH
CH
CH
2
2
2
3
3
2
SPh
SPh
SPh
8.0
4.0
16.0
–
4.0
1.0
8.0
–
8.0
8.0
16.0
–
8.0
2.0
16.0
–
8.0
1.0
64.1
–
–
4.0
7.5
4.0
4.0
25.0
2.0
31.6
2.0
7.5
4.0
1.0
8.0
2.0
31.6
2.0
15
16
18
21
2.0
–
8.0
8.0
–
31.6
2.0
3.7
4.0
–
–
31.6
2.0
31.6
–
SPh
ARB
7.5
5.0
7.5
15.0
3.7
–
a
b
c
d
e
f
A/Brisbane/59/07.
A/PR/8/34.
A/California/7/09.
A/Wuhan/359/95.
A/X31.
A/dk/Czechoslovakia/56.
A/ty/Italy/02.
A/ty/Ontario/68.
g
h
i
A/dk/Memphis/74.
B/Brisbane/60/06.
j
Table 3B
Therapeutic indices of Arbidol and Series II compounds in inhibition of the replication of influenza viruses.
Cpds Virus tested TI
O
O
R
R
1
N
X
H1N1a
H1N1b
H1N1pdmc
H3N2d
H3N2e
H4N6f
H7N3g
H8N4h
H11N9i
Bj
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
8a
8b
8d
8e
8f
8g
8h
9c
9d
9e
9f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
SPh
N(CH
N(CH
Pyrrolidine
N-methyl piperazine
NHBoc-piperazine
N-(2,4 difluoro)phenylpiperazine
Morpholine
3
)
2
6.4
7.8
11.4
9.1
8.0
8.0
8.0
6.6
8.0
8.0
5.7
7.6
7.6
8.0
8.0
8.0
8.0
8.0
4.0
–
3.2
2.0
2.9
2.3
6.4
7.8
11.4
9.1
8.0
8.0
8.0
6.6
8.0
8.0
5.7
7.6
7.6
8.0
8.0
8.0
8.0
8.0
2.0
–
6.4
3.9
11.4
9.1
12.9
7.8
5.8
4.6
4.0
4.0
4.0
6.6
4.0
4.0
2.9
–
6.4
3.9
11.4
9.1
1.6
1.0
1.9
2.3
6.4
3.9
5.8
4.6
6.4
3.9
11.4
9.1
1.6
1.0
0.7
–
SPh
SPh
SPh
SPh
SPh
SPh
SPh
2 3 2
CH )
N(CH
Pyrrolidine
N-methyl-piperazine
2-piperazin-1-ylethylamino
3
)CH
2
CH
2
CH
2
N(CH
2
CH
3
)
2
6.6
–
3.3
8.0
8.0
3.3
2.0
2.0
3.3
2.0
2.0
1.7
–
–
3.3
–
–
1.7
–
–
9g
9h
9i
N(CH
N(CH
3
)CH
CH
2
CH
)CH
2
N(CH
CH N(CH
2
CH
3
)
2
2
3
2
2
2
CH
3
)
2
–
NHCH
NHCH
NHCH
NHCH
NHCH
2
2
2
2
2
CH
CH
CH
CH
CH
2
2
2
2
2
NHCH
NHBoc
N(CH
3
4.0
4.0
4.0
4.0
16.0
–
–
4.0
15.0
9j
9 k
9 l
9 m
0
1
2
3 2
)
OH
2 2
NHCH CH OH
N(CH
3
)
2
2.0
2.0
16.0
–
–
–
–
2.0
2.0
–
2.0
–
2.0
–
Diazepan
N-methyl-pyrrolidinium iodide
8.0
10.0
8.0
5.0
ARB
7.5
7.5
3.7
7.5
7.5
–
3.7
a
b
c
d
e
f
A/Brisbane/59/07.
A/PR/8/34.
A/California/7/09.
A/Wuhan/359/95.
A/X31.
A/dk/Czechoslovakia/56.
A/ty/Italy/02.
A/ty/Ontario/68.
g
h
i
A/dk/Memphis/74.
B/Brisbane/60/06.
j
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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Table 4
Influence of single amino acid substitutions in HA of X31 on the effects of 15 and
Arbidol on the pH of fusion, determined by haemolysis assay, and on the IC50 for
inhibition of virus replication.
Virus
pH of fusion
Control
D
pH of fusion
IC50
Arbidol 15
Arbidol
15
X31
5.2
6.0
5.7
6.1
À0.3
À0.3
À0.2
À0.7
À0.4
À0.4 7.8 (2.3)
3.9 (2.3)
1
2
a (HA2 D112G)
a (HA2 R54K)
À0.5 62.5 (6.4) 62.5 (6.8)
À0.2 62.5 (2.2) 62.5 (6.9)
À0.7 62.5 (5.8) 125 (12.3)
À0.5 31.3 (9.1) 31.3 (9.1)
ab4 (HA1 H17R)
V9A (HA1 G218R) 5.5
4
50
51
was very potent against several subtypes, but showed no activity
against influenza A(H3N2) and B viruses.
4
Fig. 2. Quenching of tryptophan fluorescence of X31 BHA by 15.
4
4
52
53
3.2. Activities of 15 and Arbidol against the replication of X31 fusion
mutants
but simply shifted the pH at which fusion occurred. Only in the
case of mutant V9A was the pH of haemolysis reduced below that
of wild type X31, which correlates with the lower IC50 against rep-
lication of this mutant. The marked difference in the magnitude of
the effects on the mutants 2a and ab4, one lower and the other
greater than on wild type virus, respectively, suggests differing ef-
fects of the mutations on inhibitor interaction, as well as HA
stability.
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
4
4
4
4
4
4
4
4
4
54
55
56
57
58
59
60
61
62
To better understand the interaction of compound 15 with the
virus HA, relative to that of Arbidol, we compared their activities
against mutants of X31 with increased fusion pH. The results
(Table 4) show that, as for Arbidol, inhibition of virus replication
by 15 was substantially reduced, with IC50 8 to 32-fold higher than
for wild type X31. Thus the amino acid substitutions which reduce
the acid stability of the HA had a similar effect on inhibition by 15
and Arbidol, consistent with a similar mechanism of action, and
possibly interaction with a similar site on the protein.
Equivalent results were obtained for the effects of the inhibitors
on the pH-dependence of heterokaryon formation of CHO cells
expressing the X31 HA, in the absence or presence of 15 or Arbidol
(
Fig. 1). At pH 5.2 there was little observable effect of either pH or
4
63
3.3. Inhibition of HA-mediated membrane fusion
inhibitor on the appearance or integrity of the cell monolayer. At
pH 5.0 heterokaryon formation was evident only in the minus
inhibitor control, while at pH 4.8 heterokaryon formation was evi-
dent in the presence of Arbidol (40
1
4
4
4
4
4
4
4
4
4
4
4
4
64
65
66
67
68
69
70
71
72
73
74
75
The influence of 15 and Arbidol on HA-mediated membrane fu-
sion was studied using two assays, which monitor the pH-depen-
dence of virus-induced haemolysis and of HA-mediated
heterokaryon formation. The former assay allowed analysis of the
effects of the amino acid substitutions in HA on the pH of fusion
as well as on the influence of the inhibitors (Table 4).
lM), but not in the presence of
5 (40 M), indicating a greater effect of the latter in increasing
l
acid stability. Thus 15, like Arbidol, altered the pH of HA-mediated
membrane fusion by increasing the acid stability of the HA.
For each virus, addition of 40 lM of 15 or Arbidol caused a de-
crease in the pH at which 50% haemolysis occurred, the decrease
due to 15 being somewhat greater than that due to Arbidol for
two of the four viruses, consistent with its greater potency against
wild type virus replication. Thus the amino acid substitutions in
the mutant HAs did not abolish the interaction of the inhibitor,
3.4. Binding of 15 and Arbidol to HA
495
Two assays were used to monitor the interaction of 15 and Arb-
idol with purified HA: quenching of tryptophan fluorescence and
fluorescence-based thermal shift assays. Fig. 2 shows the decrease
496
497
498
Fig. 1. Effects of inhibitors on the pH-dependence of heterokaryon formation of CHO cells expressing X31 HA, in the presence and absence of 15 or Arbidol.
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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9
Table 5
activity against virus replication. For example, the removal of the
amine at position 4 and Br at position 6 was complemented by
insertion of a methylene extension of the hydroxy group at posi-
tion 5 of 15, while retention of the Br substituent was necessary
to complement the removal of the thiophenyl group from position
2 in 18. Although compounds in which the hydroxyl of 15 was re-
placed by an amino substituent retained inhibitory activity, they
were in general more cytotoxic.
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
Binding of Arbidol and 15 to purified BHAs of different subtypes, determined by
quenching of tryptophan fluorescence. Results are expressed as Kd in
standard deviation).
lM (+/À
Virus
HA subtype
Arbidol
15
X31
H3
H4
H1
H1
H2
H5
5.6 (0.7)
0.032 (0.006)
0.078 (0.013)
13.6 (2.1)
7.0 (1.2)
24.8 (5.2)
A/dk/Czechoslovakia/56
PR8
A/Brisbane/59/2007
A/Singapore/1/57
RG14 (A/Vietnam/1194/2004)
7.9 (1.5)
41.9 (5.2)
18.8 (2.9)
44.3 (7.1)
28.3 (6.1)
On the other hand, it is apparent that the degree of inhibition
of the replication of different viruses, in terms of IC50, do not di-
rectly reflect differential interaction with HA or its consequences.
Thus although 15 or Arbidol increase the acid stability of mutant
HAs with elevated pH of fusion, e.g. mutant 1a (fusion pH 6.0) by
a similar degree to that of wild type HA (fusion pH 5.2), they
cause poor inhibition of virus replication. This is readily under-
stood in terms of the pH of fusion of the HA in relation to the
pH of the endosome which triggers the conformational change
in HA to promote fusion. The conformational change in the sta-
bilized, inhibitor bound, mutant HA (for 1a, 2a and ab4) occurs
at a pH higher than that of the wild type HA and is thus still
triggered by the endosomal pH and hence refractory to the sta-
bilizing effects of the ligands. Such an effect may account for
the somewhat greater sensitivity of the replication of X31 (fusion
pH 5.2) than of A/dk/Czechoslovakia/56 (fusion pH 6.1), the HAs
of which bind 15 and Arbidol with similar affinities; however,
the inhibition of A/dk/Czechoslovakia/56 replication is not as se-
verely impaired by the high fusion pH as for mutant ab4. Differ-
ences in the effects of inhibitor binding to mutant HAs 1a and
ab4 (with similar fusion pH to A/dk/Czechoslovakia/56) on the
pH of fusion also indicate that other features of the HA influence
the consequences of ligand binding, both in terms of pH of fu-
sion and inhibition of virus replication. In the absence of a differ-
ence in the pH of fusion of PR8 and X31 HAs, both estimated to
be about 5.5 from a combination of comparative haemolysis and
other assays (S. Wharton, unpublished), the somewhat greater
sensitivity of X31 replication to 15 may reflect to a small extent
the 430-fold greater affinity of 15 for the X31 HA than PR8 HA. It
is evident, however, that differences in affinity of inhibitor bind-
ing are not reflected in the relative degree of inhibition of virus
replication and that inhibition of different A subtype viruses by
15 did not show any clear segregation between those with group
1 or group 2 HAs, although 15 was somewhat more active
against the H3 and H4 (group 2), than against H1, H8 and H11
(group 1) viruses. The much greater binding affinity of 15 than
Arbidol to the group 2 HAs at pH 6.5 was not reflected in a
marked increase in potency of 15 compared to Arbidol against
group 2 virus replication.
24.5 (8.6)
Fig. 3. Thermal shift of X31 BHA in the presence and absence of 15 or Arbidol.
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
in Trp fluorescence (at 340 nm) of X31 HA with increasing concen-
tration of compound 15. The maximum decrease in fluorescence
was similar for the different HAs tested, within the range 32–43%
for 15 and 36–52% for Arbidol. Table 5 compares the affinities of
binding of 15 with those determined for Arbidol (Liu et al., in prep-
aration) to HAs of different subtypes. While the affinities of binding
of 15 to group 1 HAs, H1, H2 and H5, were up to 3-fold greater than
those for Arbidol, the affinities for group 2 HAs were 100- and 175-
fold greater, for H3 and H4, respectively. Thus 15 had a much great-
er preference (up to 750-fold) for binding to group 2 HAs than to
group 1 HAs; differential binding by Arbidol was only up to 8-fold.
The thermal shift assay, which measures the influence of li-
gands on the thermal stability of the protein, also showed that
15 exerted greater stabilization of X31 HA (Fig. 3), and had greater
affinity of binding, than Arbidol; T s were 66 °C and 64.5 °C,
respectively, compared with 63 °C for wild type HA. The shift in
Tm of 3 °C by 15 was similar to that caused by TBHQ (Russell
et al., 2008). Similar Tm values were obtained by monitoring dena-
turation by Trp fluorescence, but interpretation of the data was
complicated by the quenching effects of the ligands.
An explanation for the greater inhibition of the replication of
group 2 than group 1 viruses by TBHQ was provided by crystallo-
graphic data of a X31 HA-TBHQ complex which indicated less
space to accommodate the inhibitor in the H1 HA structure com-
pared with H3 HA (Russell et al., 2008). In the absence of equiva-
lent structural information on the interaction of compound 15
and Arbidol with HA we have no explanation for the different rel-
ative binding of 15 and Arbidol to Group 2 and group 1 HAs, or for
the differences observed in inhibition of virus replication, which
m
occurred at a similar concentration (5–10 lM) (Bodian et al.,
1993) for TBHQ as for 15 and Arbidol.
Since the fluorescence intensity of the conserved Trp92 of
HA2 (solvent inaccessible) was shown to be substantially greater
than that of other Trp residues in HA (Wharton et al., 1988), it is
likely that it is the fluorescence of this residue which is
quenched by Arbidol and 15. Furthermore, the proximity of
Trp92 to the TBHQ binding site (Russell et al., 2008) suggests
that 15 and Arbidol may bind to a similar site. Due to the intrin-
sic fluorescence of TBHQ it was not possible, however, to use
5
19
4. Discussion
520
521
522
On the one hand, while many of the derivatives of Arbidol
exhibited reduced inhibitory activity, significant changes to the
structure of Arbidol could be made while retaining inhibitory
Please cite this article in press as: Brancato, V., et al. Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship
and in vitro antiviral activity of a novel indole series. Antiviral Res. (2013), http://dx.doi.org/10.1016/j.antiviral.2013.05.005

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664
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669
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671
672
673
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715
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Q3 723
724
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731
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733
734
735
736
737
738
739
740
741
742
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
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99
00
01
02
03
04
05
06
07
this assay to compare the relative binding affinities of 15 and
TBHQ to the different HAs.
Arginine 54, substituted in HA2 of mutant 2a, contributes to the
TBHQ binding site. The reduced stabilising effect of 15 on the R54K
HA (0.2 pH units) compared to the other mutants or wild type HAs
(0.4–0.7 pH units) (Table 4) is consistent with a direct effect of the
mutation on the binding of 15 to a site close to that bound by
TBHQ. In contrast substitution in the other residues, in particular
H17R in HA1, located close to the fusion peptide and involved in
stabilising its location, tend to accentuate the effect of 15, rather
than impede its interaction.
In view of its greater sensitivity to differences in structure of
inhibitor and HA, the direct binding assay should prove more illu-
minating in future SAR studies. Further information including
structural data on ligand-HA complexes will be required to under-
stand the bases for the differential specificities of binding of arb-
idol and its derivatives and differences in the spectrum of
inhibition of influenza virus replication by different classes of fu-
sion inhibitors.
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6
08
Acknowledgements
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Work at the National Institute for Medical Research was funded
by the Medical Research Council of the UK. We thank Yi Pu Lin and
Johannes Kloess for assistance.
6
12
Appendix A. Supplementary data
6
6
6
13
14
15
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.antiviral.2013.05.
005.
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