Rethinking the old antiviral drug moroxydine: Discovery of novel analogues as anti-hepatitis C virus (HCV) agents

Article abstract of DOI:10.1016/j.bmcl.2015.09.029

The discovery of a novel class of HCV inhibitors is described. The new amidinourea compounds were designed as isosteric analogues of the antiviral drug moroxydine. The two derivatives 11g and 11h showed excellent HCV inhibition activity and viability and proved to inhibit a step(s) of the RNA replication. The new compounds have been synthesized in only three synthetic steps from cheap building blocks and in high yields, thus turning to be promising drug candidates in the development of cheaper HCV treatments.

Full text of DOI:10.1016/j.bmcl.2015.09.029

Accepted Manuscript
Rethinking the old antiviral drug moroxydine: discovery of novel analogues as
anti-hepatitis C virus (HCV) agents
Andrea Magri, Roisin Reilly, Nicolò Scalacci, Marco Radi, Michael Hunter,
Manon Ripoll, Arvind H. Patel, Daniele Castagnolo
PII:
S0960-894X(15)30063-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.09.029
BMCL 23110
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
21 August 2015
9 September 2015
11 September 2015
Please cite this article as: Magri, A., Reilly, R., Scalacci, N., Radi, M., Hunter, M., Ripoll, M., Patel, A.H.,
Castagnolo, D., Rethinking the old antiviral drug moroxydine: discovery of novel analogues as anti-hepatitis C virus
(HCV) agents, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.
2015.09.029
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Andrea Magri,a Roisin Reilly,b Nicolò Scalacci,b Marco Radi,c
Michael Hunter, b Manon Ripoll,b Arvind H. Patela,* and Daniele Castagnolo.b,d,*

Bioorganic & Medicinal Chemistry Letters
jo u r n a l h o m e p a g e : w w w . e ls e v i e r . c o m
Rethinking the old antiviral drug moroxydine: discovery of novel analogues as
anti-hepatitis C virus (HCV) agents
Andrea Magri,a Roisin Reilly,b Nicolò Scalacci,b Marco Radi,c Michael Hunter,b Manon Ripoll,b Arvind H.
Patela,* and Daniele Castagnolo b,d,*
a
MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, 464 Bearsden Road, Glasgow G61 1QH, United
Kingdom.
b
Northumbria University Newcastle, Department of Applied Sciences, Ellison Building, Ellison Place, NE1 8ST Newcastle upon Tyne, United Kingdom.
c P4T Group, Dipartimento di Farmacia, Università degli Studi di Parma, Viale delle Scienze, 27/A, 43124 Parma, Italy
d Current address: Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street SE1 9NH London, United Kingdom
*Corresponding authors: *daniele.castagnolo@kcl.ac.uk, *arvind.patel@glasgow.ac.uk,
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The discovery of a novel class of HCV inhibitors is described. The new amidinourea compounds
were designed as isosteric analogues of the antiviral drug moroxydine. The two derivatives 11g
and 11h showed excellent HCV inhibition activity and viability and proved to inhibit a step(s) of
the RNA replication. The new compounds have been synthesized in only three synthetic steps
from cheap building blocks and in high yields, thus turning to be promising drug candidates in
the development of cheaper HCV treatments.
Keywords:
HCV
Amidinourea
Guanidine
2009 Elsevier Ltd. All rights reserved.
Hepatitis C life cycle
RNA replication
Hepatitis C virus (HCV) is a positive-stranded RNA virus of
the Flaviviridae family. It is a hepatotropic virus causing serious
liver disease, beginning with fibrosis and subsequently slowly
developing to cirrhosis, hepatocellular carcinoma, and liver
failure.1 HCV-associated advanced liver disease is currently the
most common underlying cause of liver transplantation.2 The
world health organization (WHO) estimates that globally around
200 million people are chronically infected with HCV, with 3-4
million new infections occurring every year.3 HCV-related
diseases cause 350,000 people deaths each year and they will
continue to be a serious global health threat for many years to
come because of the chronic nature of the infection, its high
prevalence, and the significant morbidity of the resulting
disease.4 Since 1989, academic and pharmaceutical researchers
have made tremendous progress in understanding the HCV virus
and the complex nature of the diseases caused by HCV infection,
establishing valuable research tools (e.g., the HCV replicon
system) and developing new treatment options for infected
patients. Protective vaccination is not yet available and the
current standard therapy consists of a combined therapy of
injectable pegylated interferon alfa (pegIFNa) and ribavirin
administered for 24-48 weeks depending on the HCV genotype.5
However, this therapy has many limitations, being long,
expensive, toxic, and only effective in around 50% of the patients
for the most common genotype. Recently, several direct acting
antivirals (DAAs) such as telaprevir,6 boceprevir,6 simeprevir7 or
sofosbuvir,8 acting as inhibitors of the viral NS3/4A protease or
NS5B polymerase, have been approved by FDA for the treatment
of HCV infections.
Figure 1. Moroxydine, ribavirin and design of moroxydine
analogues. Structural similarities between amidinoureas and
nucleobases.

Table 1. Synthesis of amidinoureas 11a-o
However, recent studies showed the rapid emergence of resistant
mutants after treatment with the latter drugs.9 Numerous other
DAAs are in advanced stages of development, including next-
generation protease inhibitors and nonstructural protein (NS) 5A
inhibitors.10 Nevertheless, the costs associated with new HCV
treatments are extremely high, making their use in low- and
middle-income countries extremely limited.11-12 With an
escalating number of patients infected with HCV for more than
15 years, many of whom are likely to develop advanced liver
diseases, there is an urgent need for cheaper, more-effective,
less-toxic and less-complex treatment regimens with shorter
duration. Due to our previous studies on guanidine compounds
as antimicrobial agents, we became interested in exploring the
potential of moroxydine 1 (Figure 1) as anti-HCV drug.
Moroxydine is an antiviral drug discovered in the 1950’s which
was shown to be active against RNA viruses such as influenza,
measles and mumps.13 Moroxydine has minor side effects, it is
readily absorbed by the body and does not demonstrate any
toxic build up. Although its mechanism of action is not fully
known, the effects seem to be due to an influence on the virus
host-cell system. However, despite its interesting biological
profile, moroxydine became a mislaid antiviral drug over time
and its potential as a lead molecule for the development of novel
and safer antiviral agents has never been fully explored. In
particular, to the best of our knowledge, no studies on the anti-
HCV activity of moroxydine have been reported so far.
Cmp
11a
n
R
R1
Cmp
11i
n
R
R1
1
H
0
Cl
First, we decided to re-consider moroxydine as an anti-hepatitis
11b
1
H
11j
0
Cl
C
agent. Unfortunately, preliminary biological screening
showed that 1 is inactive against HCV.14 Similarly, a few
moroxydine analogues synthesised by us showed no inhibitory
activity on HCV.14 However, during our preliminary studies, we
observed that the simple replacement of the N4 of moroxydine
with the isosteric O led to amidinourea derivatives endowed
with moderate anti-HCV activity. Amidinoureas are a class of
underexplored compounds15 endowed with antimicrobial
properties and structurally related to nucleobases guanine and
cyctosine (Figure 1).16 Thus, due to our previous experience
with amidinoureas15 and using the moroxydine scaffold as a
template, we decided to synthesise a library of isosters of 1 with
general structure A (Figure 1) and to evaluate their HCV
inhibitory activity. In particular, the addition of different
substituents on the N1 and the replacement of the morpholine
group with more lipophilic groups were explored in order to
increase the druggability of the new derivatives and explore the
chemical space around the amidinourea nucleus. Finally, the
mechanism of action of the novel compounds was also
investigated.
11c
1
1
H
H
11k
11l
0
0
Cl
H
11d
11e
11f
1
H
H
Cl
11m
11n
11o
0
0
0
H
H
H
1
A first series of amidinourea compounds 7a-d bearing different
substituents on C2’ was synthesised according to Scheme 1. The
di-Boc-guanidine 5 was refluxed in THF in the presence of an
appropriate amine leading to the formation of amidinoureas 6a-
d17 which were in turn converted into the deprotected
hydrochloric salts 7a-d by HCl/AcOEt Boc-cleavage.
11g
0
Reagents and conditions: i.
PhCH2NH2 or PhNH2 or p-Cl-
PhNH2, Et3N, DCM, r.t, 14h; ii.
Amine, Et3N, THF, reflux, 14h;
iii. HCl/AcOEt, r.t., 12h.
11h
0
Cl
Scheme 1. Synthesis of amidinoureas 7. Reagents and conditions:
i. Amine, Et3N, THF, reflux, 14h; ii. HCl/AcOEt, r.t., 12h.

Table 2. Inhibition of HCV life cycle (µM) using the Standard Assay and the Fresh Assay
Standard Assay HCVcc (µM)
Fresh Assay HCVcc (µM)
Cmp
a
b
a
b
EC50
CC50
92.9
324
SIc
EC50
CC50
49.3
325
SIc
21.9
52.0
45.7
4.25
6.23
1.40
6.17
35.3
2.41
7.99
9.19
14.4
6c
7c
64.0
34.7
11f
11g
11h
11i
>30
63.0
73.0
>2.68
6.06
1.30
1.69
13.9
39.7
34.2
77.0
30.4
20.2
5.54
11.2
10.4
9.8
7.45
59.5
9.35
30.4
0.45
>30
26.4
>30
>20
0.50
2.82
>1
8.70
4.70
33.5
n.d.
>30
21.0
30.5
n.d.
>3.4
4.40
0.91
n.d.
11l
11n
Ribavirin
Telaprevir22
44
a
Comparison of Standard-Assay/ Fresh-Assay inhibition data (EC50 and CC50), Antiviral activity on HCV life cycle. EC50: effective
b
c
concentration to achieve 50% of HCV inhibition in 72 h. Cellular cytotoxicity on naïve cells. CC50: 50% cytotoxic concentration. Selectivity
Index.
A second series of compounds 11a-o bearing substituents on C2’
and N1 of the amidinourea backbone was synthesized as reported
on Table 1. Benzyl amine was reacted with the thiopseudourea 8a
affording the benzylguanidine 9a in 92% yield. The less
nucleophilic anilines were reacted with the more reactive triflic-
guanidine 8b in DCM at room temperature affording
arylguanidines 9b-c in excellent yield. The substituted guanidines
9a-c were finally reacted with an appropriate amine in refluxing
THF affording amidinoureas 10a-o which by treatment with
HCl/AcOEt led to the desired 11a-o as hydrochloric salts. All the
compounds were evaluated for their ability to inhibit HCV.
Compounds 6b-c, 7a-d, 10c-d and 11a-o were initially screened
at the concentration of 10 µM in order to test their effect on cell
viability, and their activity against the genotype 2a cell culture
infectious HCV (HCVcc) strain JFH-1.18 The strain JFH-1
represents the best model to screen drugs since it is able to
efficiently complete a full life cycle in cultured human hepatoma
cells Huh-7: beginning from initiating infection by entry into
cells, followed by replication of the viral RNA genome and
assembly and secretion of new infectious virus particles.18 This
assay allowed us to evaluate the antiviral effects of the new
compounds in all the viral steps during natural infection and to
select a focused number of hit compounds for further studies.
Cells were first pre-incubated with our compounds for 1h,
infected with HCVcc in the presence of the drugs for 3h, then
washed and incubated with fresh drugs for further 3 days. All the
compounds showed an excellent cell viability profile, comparable
with the drug-carrier control DMSO, and were therefore
considered not cytotoxic. Activity data proved that only eight
compounds (6c, 7c, 11f, 11g, 11h, 11i, 11l, 11n) showed a good
inhibitory effect against HCV life cycle (~50%). Based on these
results, all further experiments were carried out on this focused
set of compounds. The selected compounds were then tested on
HCVcc system to determine the EC50 values. Surprisingly, poor
EC50 values were observed for most of the compounds.
Derivative 6c showed inhibitory activity at 22 µM and a
Selectivity Index (SI) of 4.25 (Table 2, Standard Assay column).
Similarly, compounds 7c and 11f-n proved to be poorly active,
with SI ranging from 1.40 to 7.45. Based on these results we
hypothesized that the low antiviral effect observed on the HCVcc
system could be related to a reduced uptake of the drug or to the
rapid metabolization and deactivation of the compounds by the
host cell machinery. Thus, keeping in mind the excellent viability
of our compounds, we decided to set up a novel “fresh-addition”
(FA) assay in which drugs were added every 24h post-infection
to the cell medium at 1/10th of the initial concentration used (See
supplementary info Table 1S). The inhibitory effects were
evaluated at 3 days post-infection.

Figure 2. Dose-response curves for compounds 6c (top left), 11g (top right), 11f (bottom left), 11h (bottom right).
introduced into cells, but they lack nucleotide sequences
encoding the viral structural protein core, E1 and E2, and as such
they are virus assembly defective and therefore unable to undergo
its full life cycle. Furthermore, a foreign gene, such as that
expressing the reporter firefly luciferase, can be inserted in-frame
into the coding region of the SGR. The amounts of luciferase
produced by the replicon directly correlate with viral RNA
replication levels. Therefore, this system allows easy
quantification of viral replication.20
We reasoned that if the drugs are rapidly metabolized then
replenishing the cell medium with a smaller dose every 24h
might conceivably lead to an increase in their antiviral activity.
We did not expect the compounds to be cytotoxic, given their
excellent viability profiles. We first tested the compound 6c
under this ‘Fresh assay or FA’ condition. As shown in Figure 2
(top left panel), a good improvement in viral inhibition was
observed with a significant drop in the EC50 value (~ 3-fold
decrease) from 21.9 µM to 6.17 µM. Noteworthy, no significant
changes in the cell viability were observed and the SI raised up to
7.99 (Table 2). These data confirmed the hypothesis that a fast
metabolism of the compound must be responsible for the low
activity data reported in Table 2 (Standard Assay column). The
administration of fresh portion of 6c proved not to be a problem
in term of cytotoxicity thus corroborating the validity of our FA
model. We next applied the FA model to the other compounds.
As shown in Table 2 (Fresh Assay column), all compounds
showed a good viability profile with CC50 values in the range of
30-80 µM. Moreover, the EC50 values of all the compounds
showed a significant reduction as previously observed for 6c,
with the exception of compound 7c (Table 2). In particular,
compound 11g showed an excellent antiviral profile with EC50
value of 1.30 µM and SI = 30.4 (Table 2 and Figure 2) as well as
amidinourea 11h that inhibited HCV at 1.69 µM with a good SI =
20.2 (Table 2 and Figure 2).
Figure 3. Inhibition of HCV at 10 M. Comparison of HCV Life
Cycle inhibition (red column) and SGR-JFH inhibition (blue
columns).
100
50
0
Cmpds
HCV life cycle inhibition
SGR-JFH inhibition
Herein, we used the JFH1-luc replicon expressing luciferase
gene as reporter.21 In our assay, sub-genomic RNA was delivered
into cells by electroporation and drugs were immediately added
to transfected cells at the concentration of 10 µM. Luciferase
readings and viability data were obtained after 24 h of incubation.
Results showed that two compounds (11f and 11g) were able to
inhibit HCV replication in this model by up to 50%, perfectly
correlating with HCVcc inhibition data (Figure 3). Again, as
expected, all the compounds were found to be non-cytotoxic,
although lower viability was detected, probably due to an
increase in drug intake arising from the effect of electroporation.
Further studies are in progress in our laboratories to elucidate
more precisely the mode of action of these novel anti-HCV
molecules and to try to identify the precise step where they
interfere with the HCV life cycle.
Remarkably, both 11g and 11h proved to be more active than the
antiviral drug ribavirin, which showed a poor activity and high
cytotoxicity (SI = 1) both in the standard as well as in the FA
model. In general, all the compounds bearing an aryl-piperazine
group showed an improved anti-HCV activity as well as the
presence of an aryl substituent on N1 in place of a benzyl group
was beneficial for the antiviral activity. Finally, the presence of a
p-Cl-phenyl ring on N1 led to compounds with increased activity.
We then decided to investigate the mechanism of action of
amidinoureas on HCV infection. We first explored the possible
interaction of compounds 11 with the HCV entry using a HCV
pseudotyped particle (HCVpp) assay.19 Unsurprisingly, from
these experiments emerged that none of the amidinourea
compounds affect the entry of the virus (data not shown).
According to the previous results and due to the structural
similarity of amidinoureas with nucleobases (depicted in Figure
1), we hypothesized that the novel compounds are likely to
inhibit HCV by interfering with the viral RNA replication. We
therefore used the HCV sub-genomic replicon (SGR) system to
test the ability of our compounds to block viral RNA. Sub-
genomic replicons are translation and replication competent when
In conclusion, we have identified a series of novel and
promising antiviral compounds active against HCV. The novel
molecules have been designed as analogues of the antiviral agent
moroxydine, through the replacement of the biguanide core with
the isosteric amidinourea backbone. The novel compounds
proved to be non-cytotoxic and highly active after repeated
administration. Due to their rapid metabolization, a novel

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AM gratefully acknowledges Compagnia San Paolo (Italy) for
financial support (PhD fellowship). We gratefully acknowledge
the EPSRC UK National Mass Spectrometry Facility for
providing the mass spectrometry data. Work in AHP’s lab was
funded by the Medical Research Council, UK. DC thanks the
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Supplementary Material
Experimental procedures and full characterization (1H-NMR,
13C-NM, MS, HRMS) of the new compounds. Biological
experimental procedures.
Click here to remove instruction text...
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