Discovery of Imidazo[1,2-α][1,8]naphthyridine Derivatives as Potential HCV Entry Inhibitor

Article abstract of DOI:10.1021/acsmedchemlett.5b00159

RO8191 represents a newly discovered small-molecule IFN-like agent that displays potent anti-HCV activity. With it as lead, a series of compounds bearing an imidazo[1,2-α][1,8]naphthyridine core and an amide bond-linked side chain were designed and synthesized. These compounds were evaluated on HCV cell culture system (HCVcc-hRluc-JFH1), and some of them exhibited remarkable anti-HCV activity (EC₅₀ = 0.017-0.159 μM) and low toxicity (CC₅₀ > 25 μM). Moreover, it was revealed that these newly identified anti-HCV agents exert their antiviral effect through a distinct mechanism of action from that of RO8191 by targeting the viral entry process. Thus, our study provides a starting point for the development of potential HCV entry inhibitor.

Full text of DOI:10.1021/acsmedchemlett.5b00159

Letter
pubs.acs.org/acsmedchemlett
Discovery of Imidazo[1,2-α][1,8]naphthyridine Derivatives as
Potential HCV Entry Inhibitor
Huan Wang,†,∥ Shuo Wang,†,∥ Lili Cheng,† Ligong Chen,†,§ Yongguang Wang,† Jie Qing,‡
Shengdian Huang,† Yuanhao Wang,† Xiaoqiang Lei,† Yunfei Wu,†,§ Zhilong Ma,† Linqi Zhang,*,‡
and Yefeng Tang*,†,§
†Comprehensive AIDS Research Center, and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine,
Tsinghua University, Beijing 100084, China
‡Comprehensive AIDS Research Center, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School
of Medicine, Tsinghua University, Beijing 100084, China
§Collaborative Innovation Center for Biotherapy, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West
China Medical School, Sichuan University, Chengdu 610041, China
S
* Supporting Information
ABSTRACT: RO8191 represents a newly discovered small-
molecule IFN-like agent that displays potent anti-HCV
activity. With it as lead, a series of compounds bearing an
imidazo[1,2-α][1,8]naphthyridine core and an amide bond-
linked side chain were designed and synthesized. These
compounds were evaluated on HCV cell culture system
(HCVcc-hRluc-JFH1), and some of them exhibited remark-
able anti-HCV activity (EC50 = 0.017−0.159 μM) and low toxicity (CC50 > 25 μM). Moreover, it was revealed that these newly
identified anti-HCV agents exert their antiviral effect through a distinct mechanism of action from that of RO8191 by targeting
the viral entry process. Thus, our study provides a starting point for the development of potential HCV entry inhibitor.
KEYWORDS: Hepatitis C virus, anti-HCV agent, RO8191, IFN-like compound, entry inhibitor
epatitis C virus (HCV) is a positive single-stranded RNA
by directly interacting with the type I IFN receptor to drive
IFN-stimulated genes (ISG) expression, which then induces the
antiviral response of innate immune system. In this regard,
RO8191 could be potentially utilized as a small-molecule IFN-
substitute in the traditional IFN-α-based antiviral regimens.14
The appealing nature of RO8191 attracts considerable interest
from pharmaceutical industry. In 2013, GlaxoSmithKline
(GSK) disclosed a systematic structure−activity relationship
(SAR) study on RO8191.15 Although over 100 analogues were
synthesized by varying the structural variants on the A, B, C and
D rings (I, Figure 1), none of the analogues displayed improved
anti-HCV activity, indicating that RO8191 had a relative
narrow window of SAR. In parallel with this seminal work, we
also synthesized a number of RO8191 analogues that bear
modified A, B, C, or D ring.16,17 Unfortunately, such efforts also
met with limited success.
1
Hvirus belonging to the Flaviviridae family.
Over 170
million people have been infected with HCV and thus are at
risk of developing serious liver diseases, such as chronic
hepatitis, liver cirrhosis, and hepatocellular carcinoma.2,3
Historically, the standard care of HCV infection was the
combinations of pegylated interferon-α (Peg-IFN) and ribavirin
(RBV).4 However, its efficiency is largely compromised by
several issues, including the moderate sustained virological
response (SVR), lack of compliance, and severe side effects.5
Over the past decades, great efforts have been devoted to the
development of more effective treatment and prevention of
HCV infection,6−8 which culminated in the recent approval of
several direct acting antivirals (DAA),9,10 the protease
inhibitors11 boceprevir, telaprevir, and simeprevir and the
polymerase inhibitor12 sofosbuvir. These new DAAs, in
combination of Peg-IFN-α and ribavirin, could dramatically
improve the SVR up to 90% across several different HCV
genotypes. Albeit such breakthrough, however, there remains
an unmet need for the development of new anti-HCV
treatments that could have improved resistance profiles and
low side effects.
The above outcomes impelled us to develop the second
generation of RO8191 analogues, as represented by structure II
(Figure 1). The primary idea is to install an amide bond linker
between the imidazo[1,2-α][1,8]naphthyridine scaffold and the
1,3,4-oxadiazole motif. We envisioned that such structural
modification could afford several advantages over the lead
Recently, a small molecule named RO8191 (1) was disclosed
by Konishi and co-workers from Chugai Pharmaceutical Co.
Ltd., which displays remarkable anti-HCV activity (EC50 = 0.2
μM).13 More importantly, RO8191 exerts its antiviral activity
Received: April 17, 2015
Accepted: July 26, 2015
© XXXX American Chemical Society
A
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The anti-HCV activities of the synthesized compounds were
first evaluated on the HCV cell culture system (HCVcc-hRluc-
JFH1) with HCV genotype 2a JFH-1 virus containing a
humanized Rellina luciferase reporter gene (Table 1). RO8191
(EC50 = 0.203 μM) were employed as positive control. First, an
array of compounds bearing different heterocycles (e.g.,
piperidine, pyrrolidine, morpholine, or piperazin-2-one) (5a−
5h) were examined. To our delight, while most of them failed
to give promising results, 5e displayed remarkable anti-HCV
activity (EC50 = 0.102 μM), which was 2-fold more potent than
RO8191 (EC50 = 0.203 μM). It seemed that the amine moiety
on the C-3 position of the piperidine ring played a crucial role
for its activity since the other analogues attached with a −OH,
−CO2Me, or −CH2OH functionality displayed no or weak
activity. This hypothesis was further validated by another group
of piperazine-derived compounds (5i−5l). As shown, 5i and 5j,
which bear a secondary amine group, displayed even better
anti-HCV activity than 5e. Comparably, for 5k and 5l, which
carry a tertiary amine, inferior activity was obtained. Following
this clue, various other analogues bearing cyclic or acyclic amine
(5m−5w) were synthesized and evaluated, from which we
could briefly summarize the key elements of the SAR. First, the
analogues with a primary or secondary amine functionality
showed better activity than those with a tertiary one (e.g., 5m−
5p vs 5q). Second, the length of the tether between the amine
and amide moieties plays a significant role. As shown, while
most of the compounds with a two-carbon tether (5m−5o)
displayed highly potent activity, those with a longer tether
(three to five carbons, e.g., 5r−5t) afforded decreased potency.
Third, it seems that the flexibility of the tether also has
influence on the activity. Indeed, different from the aliphatic
amine-derived analogues (5m−5t), several aniline-derived ones
(5u−5w) proved to be inactive at the concentration of 20 μM.
Of note, all of the above compounds displayed no obvious
cytotoxicity (CC50 > 25 μM).
Figure 1. Structure of RO8191 and its analogues.
structure of RO8191. First, it could provide an additional
hydrogen bond acceptor, which may facilitate the interaction
between the molecule and its biological target. Second, it will
increase the molecular flexibility and hydrophilicity, thus
improving its pharmacokinetics (e.g., water solubility and oral
bioavailability). Last but not least, the second generation
analogues are more synthetically accessible and prone to
derivatization. Based on this design, we completed the synthesis
of a series of compounds that featured an imidazo[1,2-
α][1,8]naphthyridine scaffold coupled with an amide bond-
derived cyclic or acyclic chain. Fortunately, it turned out that
some of these compounds exhibited remarkable anti-HCV
activity (EC50 = 0.02−0.05 μM), which were 5−10-fold more
potent than RO8191. More interestingly, the preliminary
mechanism of action (MOA) investigations revealed that
these newly identified anti-HCV compounds most likely
functioned as HCV entry inhibitor instead of the expected
IFN-like agent.
The general synthetic route toward the designed compounds
is depicted in Scheme 1. Thus, 2-amino-5,7-dis-
Besides the HCV cell culture system, those active
compounds with EC50 < 0.2 μM (5e, 5i−5k, and 5m−5q)
were further evaluated with HCV replicon cells, which possess a
luciferase reporter gene and express the HCV genotype 1b
subgenomic replicon. To our surprise, all of the examined
compounds display no inhibition on HCV replicons. In sharp
contrast, RO8191 was found to be active in both systems. This
result indicated that the newly synthesized analogues may exert
their anti-HCV activity through a distinct mechanism of action
different from that of RO8191.
a
Scheme 1. Synthesis of Compounds 5a−5w
To get deep insight into the plausible mechanism of action,
further investigations were carried out with the most active
compound 5o. First, we explored whether it could function as
RO8191 to drive the IFN-stimulated genes (ISG) expression.
For this end, we transiently transfected IFN-stimulated
response element (ISRE) reporter gene into different cells
(Vero, Huh7, Huh7.5.1, and HepG2), which serves as an
enhancer to promote transcriptional induction by interferon. It
was shown that while both RO8191 and IFN-α could induced
the ISRE activation, compound 5o showed no effects (Figure
S1, SI).
The different behavior of our compounds in the HCV cell
culture and replicon system implied that they likely exerted
their function in the entry stage of HCV infection. To validate
this hypothesis, we conducted the time-of-addition assays to
characterize the kinetics of compound activity. Bafilomycin A1,
an inhibitor of endosomal acidification, which blocks the final
fusion step between the virus envelope and the endosomal
a
Reagents and conditions: (a) 1,1,1,5,5,5-hexafluoropentane-2,4-dione,
acetic acid, and sulfuric acid, reflux, 12 h, 63%; (b) methyl
bromopyruvate, acetone, reflux, 12 h, 38%; (c) LiOH, THF/H2O
(v/v: 1:1), 50 °C, 6 h, 90%; (d) amine component, HATU, DIPEA,
DMF, r.t., 1−6 h, 15−68%.
(trifluoromethyl)-1,8-naphthyridine 2 was prepared from the
commercially available 2,6-diaminopyridine 1, 1,1,1,5,5,5-
hexafluoropentane-2,4-dione via an acid-promoted cycliza-
tion.18 Subsequently, 2 reacted with methyl bromopyruvate in
refluxing acetone to yield the 2,4-bis(trifluoromethyl)imidazo-
[1,2-a][1,8]naphthyridine-8-carboxylate 3.19 Hydrolysis of 3 in
the presence of LiOH/THF/H2O provided the acid 4, which
was then coupled with different amines using HATU in DMF
to provide the corresponding amides 5.20
B
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membrane, was tested in a parallel assay to define the end point
of the entry process. As reported, the inhibition of bafilomycin
A1 lasted about 3 h, indicating that HCVcc entry and fusion
were completed within this time frame (Figure 2a).21,22 In
Table 1. Anti-HCV Activity of Compounds 5a−5w
Figure 2. Kinetics of inhibition by 5o as an HCV entry inhibitor. (a)
Time-course of inhibition. (b) Inhibitory effect on attachment and
postattachment stages.
analogy to bafilomycin, the kinetics of 5o activity revealed that
the compound inhibition was exerted within 3 h of infection,
indicating that it is an HCV entry inhibitor.
Furthermore, we turned to explore whether 5o blocks the
initial attachment step to heparan sulfate proteoglycans
(HSPGs) or a downstream event in the HCV entry process.
The hog intestine heparin and bafilomycin A1 were employed
as control inhibitors. As previously reported, heparin only had
effects on the 4 °C attachment step, while bafilomycin was only
effective during the postattachment stage.23,24 It was shown that
compound 5o showed little effect on HCVcc attached to
HSPGs step, but exerted over 90% inhibition on postattach-
ment infection phase (Figure 2b). Taken together, 5o most
likely blocks an event in HCV entry that lies downstream of the
attachment to HSPGs, either prior to or during virus-cell fusion
stage.
Besides HCV virus, compound 5o was also evaluated in a
panel of other viruses including HBV, EV71, HIV, and MERS
(Table 2). However, no obvious inhibition was observed at the
concentration of 20 μM, showing the antiviral specificity of our
compounds as the HCV inhibitor.
a
Table 2. Activity of 5o in Other Virus Infection Assays
a
a
virus
cell line
EC50 (μM)
CC50 (μM)
HBV
EV71
HIV
HepAD38
RD
>20
>20
>20
>20
>20
>20
>20
>20
Ghost
Huh7
MERS
a
b
a
The inhibition of virus luciferase activity. The reduction of viable
Experiments were done in triplicate with each data point having four
parallel test wells.
c
cell number. Calculated by Graphpad Prism 5 software.
C
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Finally, the pharmacokinetic profile of 5o was evaluated in
rats. The time-course of 5o in plasma following a single oral
dose (100 mg/kg) to rats is shown in Figure S2 (Supporting
Information), and the key pharmacokinetic parameters were
summarized in Table 3. Overall, it was shown that 5o was orally
ABBREVIATIONS
■
HCV, hepatitis C virus; Peg-IFN, pegylated interferon-α; SVR,
sustained virological response; DAA, direct acting antivirals;
ISG, IFN-stimulated genes; SAR, structure−activity relation-
ship; ISRE, IFN-stimulated response element; HSPGs, heparan
sulfate proteoglycans
a
Table 3. Pharmacokinetic Parameters of 5o
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pharmacokinetic parameters
compound 5o
■
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2.93 0.60
18.98 2.49
19.12 2.64
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In conclusion, with the known small molecule IFN-like agent
RO8191 as lead, we have designed and synthesized a series of
imidazo[1,2-α][1,8]naphthyridine derivatives, some of which
exhibited significant anti-HCV activity (EC50 = 0.02−0.1 μM).
Further investigations revealed that these compounds exerted
their anti-HCV effect in the viral entry stage, which is distinct
from that of RO8191. While the majority of the recently
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00159.
Experimental procedures, characterization data, and
NMR-mass spectrum for all new compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: yefengtang@tsinghua.edu.cn.
*E-mail: zhanglinqi@tsinghua.edu.cn.
Author Contributions
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∥These authors (H.W. and S.W.) contributed equally to this
work. All authors have given approval to the final version of the
manuscript.
(17) Unpublished results. For details, see Table S-1 (SI).
Funding
́
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We gratefully acknowledge the financial supports from the
NSFC (21272133, 81470839) and Beijing Natural Science
Foundation (2132037).
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Notes
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