Synthesis, biological evaluation and mode of action studies of novel amidinourea inhibitors of hepatitis C virus (HCV)

Article abstract of DOI:10.1016/j.bmcl.2019.01.008

Novel amidinourea derivatives have been synthesised and evaluated for their antiviral activity against Hepatitis C Virus (HCV). A compound with an amidinourea-spermine chemical structure, different from that of standard anti-HCV drugs, showed micromolar activity against HCV and excellent viability. Studies on the mode of action revealed that the new compound may act against HCV through the inhibition of IRES-mediated translation.

Full text of DOI:10.1016/j.bmcl.2019.01.008

Accepted Manuscript
Synthesis, Biological Evaluation and Mode of Action Studies of Novel Amidi-
nourea Inhibitors of Hepatitis C Virus (HCV)
Andrea Magri, Omar Mokrane, Kate Lauder, Arvind H. Patel, Daniele
Castagnolo
PII:
S0960-894X(19)30008-3
https://doi.org/10.1016/j.bmcl.2019.01.008
BMCL 26245
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
21 November 2018
4 January 2019
9 January 2019
Please cite this article as: Magri, A., Mokrane, O., Lauder, K., Patel, A.H., Castagnolo, D., Synthesis, Biological
Evaluation and Mode of Action Studies of Novel Amidinourea Inhibitors of Hepatitis C Virus (HCV), Bioorganic
& Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.01.008
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Bioorganic & Medicinal Chemistry Letters
Synthesis, Biological Evaluation and Mode of Action Studies of Novel
Amidinourea Inhibitors of Hepatitis C Virus (HCV)
Andrea Magri,a Omar Mokrane,b Kate Lauder,b Arvind H. Patela and Daniele Castagnolob,*
aMRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, 464 Bearsden Road, Glasgow G61 1QH, United
Kingdom.
bSchool of Cancer and Pharmaceutical Sciences, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, United Kingdom.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Novel amidinourea derivatives have been synthesised and evaluated for their antiviral activity
against Hepatitis C Virus (HCV). A compound with an amidinourea-spermine chemical
structure, different from that of standard anti-HCV drugs, showed micromolar activity against
HCV and excellent viability. Studies on the mode of action revealed that the new compound
may act against HCV through the inhibition of IRES-mediated translation.
Keywords:
HCV
2009 Elsevier Ltd. All rights reserved.
Amidinourea
Polyamine
HCV translation
RNA IRES
*Corresponding Author e-mail: daniele.castagnolo@kcl.ac.uk
Hepatitis C virus (HCV) infection represents a disease of
significant global impact that afflicts around 170 million people
worldwide.1-2 A small proportion of infected people clear the
virus naturally,3 whilst the majority develop chronic hepatitis C
(CHC) which can lead to a spectrum of liver diseases from mild
inflammation to extensive liver fibrosis and cirrhosis, conferring
significant morbidity and mortality to affected individuals.4 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.5 The standard
therapy for HCV infection has been based for more than a decade
on the use of pegylated interferon alpha (PEG-IFNα) and the
antiviral nucleoside analogue ribavirin (RBV).6 Since 2011, a
number of directly acting antivirals (DAAs), such as the protease
inhibitors (PIs) telaprevir, boceprevir and simeprevir,7 and the
viral RNA-dependent RNA polymerase inhibitor sofosbuvir,8-9
have been licensed for use as part of combination therapies for
HCV and CHC infections. These innovative treatment regimens
have revolutionized the field of HCV medicine and provided
optimism to find a cure in HCV patients. However, the fight
against HCV infections is not fully over yet, because of the high
costs associated with the therapies as well as the emergence of
mutant strains resistant to DAA drugs.10-12
putrescine were recently reported to possess inhibitory activity
against HCV,16 we became intrigued by the possibility to merge
both the amindinourea and the polyamine moieties into a hybrid
structure. This resulted led to the design of a series of
amidinourea-polyamine hybrids with general structure B (Figure
1).
We recently reported the design and discovery of new
amidinourea HCV inhibitors with structure A (Figure 1), as
analogues of the antiviral drug moroxydine.13 As a continuation
of this work, the synthesis and biological evaluation of a new
series of amidinourea14 derivatives with general structure B was
planned. Since the natural polyamines15 spermine, spermidine and
Figure 1. Structure of polyamines and anti-HCV
amidinoureas

Scheme 1. Synthesis of amidinoureas derivatives 3a-c, 5, 6, 8a-b and 10
In this work, the synthesis, biological evaluation and mode of
action studies of new polyamine amidinourea B as inhibitors of
HCV is described.
correlation between the presence of an amidinourea moiety on a
polyamine backbone with the antiviral activity. The alkylated S-
methylisothiourea derivatives 12a-c were synthesised via
Mitsunobu reaction according to previously reported
procedures.24 Reaction of 12a-c with 1,8-diaminooctane 1b and
the triamine 14, followed by Boc cleavage with HCl/AcOEt led
to guanidine derivatives 13a-b and 15a-b.
The synthesis of a set of amidinourea compounds derived
from the diamines putrescine 1a and 1,8-diaminooctane 1b was
first planned. The diamines 1a-b were reacted with N,N′-di-Boc-
S-methylisothiourea in DCM at room temperature affording the
Boc-guanidines 2a-b.17 The latters were reacted with an
appropriate amine (allylamine, benzylamine and p-Cl-aniline)
and then treated with HCl/AcOEt solution to afford the
corresponding amindinoureas 3a-c. Scheme 1. Putrescine was
also converted into the monoguanidine derivative 4 which in turn
led the amidinoureas 5 and 6 through reaction with allylamine,
benzoyl chloride and Boc deprotection as shown in Scheme 1.
The choice of an allyl substituent on the amidinourea moiety
arose from preliminary data and previous work on similar
amidinourea derivatives endowed with antimicrobial activity.18-19
Amidinoureas 8a-b bearing a spermine and spermidine
backbone respectively were synthesised in a similar way as
reported in Scheme 1. The guanylated intermediates 7a-b,
obtained via guanylation of the appropriate polyamine, were
reacted with allyamine in refluxing THF affording, after Boc
deprotection, the derivatives 8a-b. The secondary amine groups
in 7a were also converted into tertiary amines via reductive
amination reaction leading to 9, in order to explore the
importance of an NH moiety for the antiviral activity. The
reaction of 9 with allyl amine and Boc-derotection led to
amidinourea 10.
Scheme 2. Synthesis of guanylated polyamines 13a-b and
15a-b
All the compounds 3a-c, 5, 6, 8a-b, 10, 13a-b and 15a-b were
assayed for their potential cytotoxicity at a single dose of 10 µM
on Huh7 cells. Interestingly, all compounds proved to be not
toxic, with the exception of 3a and 3b which exhibited high
cellular toxicity and therefore were excluded from further
Finally, a set of guanidine derivatives 13a-b and 15a-b were
synthesised as described in Scheme 2 with the aim to evaluate a

experiments (Figure 2a). The non-cytotoxic compounds were
then assayed as potential HCV antiviral agents, specifically as
potential HCV entry or replication inhibitors.
Based on the results of the initial screening which showed a
good inhibitory activity and low toxicity for 8a, and intrigued by
its innovative and uncommon chemical structure when compared
to standard anti-HCV agents, we decided to further investigate
this compound try to elucidate its mode of action.
Initially, the antiviral activity of amidinourea and guanidine
derivatives on HCV entry using HCV pseudo-particles (HCVpp)
at a single dose of 10 µM was evaluated. All the compounds
showed no entry effect, in contrast with the great inhibition
detected from the positive control bafilomycin (Figure 2b). The
same compounds were then tested on HCV transient replication
using a sub-genomic HCV replicon. Accordingly, Huh7 cells
were electroporated with HCV RNA and seeded in the presence
of the potential inhibitors at the concentration of 10 µM for 24h.
As shown in Figure 2c, the spermine-amidinourea derivative 8a
was the sole active molecule, exhibiting a strong antiviral activity
(90%). Since only one compound showed antiviral activity, a
Structure Activity Relationship (SAR) study was not possible.
However, it looks clear that the anti-HCV activity must be
associated to the presence of a spermine backbone mainly.
Moreover, the amine groups in the spermine chain must be
secondary, as no activity was observed for the tertiary amine
derivative 10.
We initially validated our findings by evaluation of compound
8a on the fully infectious HCV cell-cultured, using the JFH-1
clone.20 Following viral inoculation for 3h, Huh7-J20 infected
cells were exposed to 8a treatment for a period of 72h with the
same dose-response concentrations tested for transient replicons.
A good cellular viability was observed, with a moderate
cytotoxicity detected only at the concentration of 30 µM and a
predicted CC50 value of 81.1 µM. The compound also showed a
good antiviral activity on fully infectious HCV cell-cultured,
with an IC50 value of 12.3 µM (Figure 3b), in line with the
antiviral effect observed on the HCV replicon. In order to
understand whether the slightly reduced antiviral activity of 8a
was due to a structure-related issue affecting the drug entry or
drug stability or to a different mechanism of action, the
amidinourea 8a was re-tested against the transient replicon at the
concentration of 10 µM, allowing the replication to establish for
24 h before treating with 8a for an additional 24h. Interestingly, a
reduced antiviral activity (~45%, Figure 4a) compared to
immediate exposure to the compounds (~90%) was observed,
suggesting a potential role of 8a in the early stage of HCV
replication. With the aim to explore this hypothesis, Huh7 cells
transfected with wild-type replicon RNA (WT) were seeded in
the presence of the compound 8a and monitored at 2, 4, 8 and
24h. Interestingly, a strong antiviral phenotype was observed
after only 2 h and maintained throughout the experiment (Figure
4b). Thus, we decided to evaluate the efficacy of 8a against a
replication-defective replicon (GND), which, once transfected, is
only able to be translated to generate mature proteins, resulting in
a loss of signal in 24h. As it is evident from Figure 4c, 8a
exhibited a consistent antiviral activity against GND, showing a
constant reduction from
2 to 8 hours post-transfection.
Comparing inhibition levels between WT and GND showed same
antiviral efficacy at every time point considered (Figure 4d),
suggesting that the antiviral effect of 8a is exerted through
inhibition of viral translation. We finally evaluated the effect of
8a on a stable replicon cell line, persistently expressing HCV
subgenomic genome (Huh7-J17).21 A moderate effect was
detected on viral replication, with the maximal antiviral effect
exerted in the first 24h (45%) (Figure 4e).
Figure 2. Antiviral activity and cytotoxicity of amidinourea
and guanidine compounds. a) Huh7 cells were treated with 10
M of each compound and incubated for 24h before evaluating
cell viability. b) Huh7 cells were treated with each compound and
the concentration of 10 M for 1h and then infected with HCVpp
for 3h in the presence of the compounds. Viral entry was detected
72h post infection. c) Huh7 cells, electroporated with HCV
subgenomic RNA, were seeded and treated with 10 M of each
compound for 24h before measuring luciferase.
However, despite the disappointing screening results, the
amidinourea 8a showed an excellent antiviral profile and thus it
was further characterised performing a full dose response scale
on the transient replication using a starting concentration of 30
µM, following 1:3 dilutions (Figure 3a). A significant dose
dependency was observed, with an IC50 of 2.65 µM. Again, no
effects on the cellular viability were detected, except a modest
toxic effect at 30 µM.
Figure 3. Dose response scale curves for amidinourea 8a on
HCV replicon or HCVcc. a) Huh7 cells were electroporated with
HCV replicon RNA and exposed to 8a for 24h. b) Huh7-J20 cells

were infected with fully infectious HCV (JFH1), treated for 72h
and then harvested to evaluate viral replication. Grey dots and
line: cellular viability; black dots and line: viral replication.
Figure 4. Effect of amidinourea 8a on early stage of viral replication and on initial processing and IRES-mediated translation. a) Huh7
cells were electroporated with HCV subgenomic RNA and immediately treated with 8a (left bar) or incubated for 24h before being
treated with 8a for 24h (centre bar). b-c) Time course of 8a on HCV replicon (b) or replication defective GND (c). Huh7 cells were
transfected with HCV RNA and seeded in the presence of 8a. Viral replication/translation was evaluated at 2, 4, 8 and 24h post
transfection. d) Antiviral effect of 8a at each time point evaluated compared to untreated cells. e) Huh7 cells, stably expressing HCV
replicon RNA, were treated with 8a and the viral replicon was evaluated at 24, 48 and 72h. f) Huh7 cells were transfected with HCV
replicon RNA and immediately treated with 8a for 8 or 24h. g) Huh7 were transfected with a plasmid encoding for firefly luciferase
under the control of HCV IRES and co-transfected with a plasmid encoding for the Renilla luciferase to normalise for transfection
efficiency
progress in our labs to fully confirm the mode of action and to
design analogues of 8a bearing a spermine backbone to be tested
against wild-type and resistant HCV.
It was speculated that 8a could affect the protein translation or
processing in HCV replication cycle. The viral protein processing
was thus investigated, to establish whether the HCV inhibition
was determining an increased amount of unprocessed
polyprotein, due to a block in the processing, or a reduced
Acknowledgments
amount of mature protein, indicating in this case an impaired
translation.
We gratefully acknowledge the EPSRC UK National Mass
For this purpose, Huh7 cells, transfected with HCV replicon
RNA, were treated with 8a for 8 or 24h and then assayed by
western blot to evaluate the viral protein NS5A. As reported in
Figure 4f, we detected a significantly reduced amount of NS5A,
while no accumulation of unprocessed polyprotein was detected,
suggesting a role for 8a in affecting viral translation. To confirm
our hypothesis, the effect of 8a on the HCV IRES-mediated
translation was finally evaluated. To this scope, Huh7 cells were
co-transfected with two plasmids: 1) one expressing firefly
luciferase under the control of HCV IRES and 2) another
expressing renilla firefly under the control of TK promoter to
normalise data for transfection efficiency. Cells were exposed to
8a for 24h before assaying for dual luciferase. Results showed a
moderate, although significant, inhibition (40%, Figure 4g) of
firefly translation, supporting the hypothesis that 8a is controlling
HCV replication through a modulation of its IRES-mediated
translation. It is noteworthy, that some of the known HCV IRES
inhibitors described in the literature to date bear a guanidine/bis-
guanidine moiety,22-24 clearly indicating a key role of this, and
similar groups like amidinoureas, for antiviral activity.
Spectrometry Facility for providing the mass spectrometry data.
Royal Society (RG160870) is acknowledged for financial support
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Synthesis, biological evaluation and mode of action
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