Potent hepatitis C virus NS5A inhibitors containing a benzidine core

Article abstract of DOI:10.1021/ml4003293

Here we report the discovery of a series of potent hepatitis C virus (HCV) NS5A inhibitors based on the benzidine prolinamide backbone. Taking a simple synthetic route, we developed a novel inhibitor structure, which allows easy modification, and through optimization of the capping group, we identified compound 6 with highly potent anti-HCV activity. Compound 6 is nontoxic and is anticipated to be an effective HCV drug candidate.

Full text of DOI:10.1021/ml4003293

Letter
pubs.acs.org/acsmedchemlett
Potent Hepatitis C Virus NS5A Inhibitors Containing a Benzidine Core
Il Hak Bae,† Jin Kyu Choi,† Chieyeon Chough,† Sun Ju Keum,‡ Heesun Kim,‡ Sung Key Jang,*,‡
and B. Moon Kim*,†
†Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, South Korea
‡Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, South Korea
S
* Supporting Information
ABSTRACT: Here we report the discovery of a series of potent
hepatitis C virus (HCV) NS5A inhibitors based on the benzidine
prolinamide backbone. Taking a simple synthetic route, we developed
a novel inhibitor structure, which allows easy modification, and
through optimization of the capping group, we identified compound
6 with highly potent anti-HCV activity. Compound 6 is nontoxic and
is anticipated to be an effective HCV drug candidate.
KEYWORDS: HCV, NS5A inhibitor, benzidine, antiviral agent, structure−activity relationship
pproximately 170 million people, including almost 4
Amillion in the United States, are estimated to be infected
with hepatitis C virus (HCV).1,2 It has been estimated that 75−
85% of those infected with HCV will develop chronic hepatitis
and more serious diseases such as liver cirrhosis and
hepatocellular carcinoma.3 Until recently, therapies for HCV
patients have consisted of subcutaneous injections of pegylated
interferon-α (PEG-IFN-α) in combination with oral doses of
ribavirin (RBV).4 This interferon based therapy has a limited
sustained virologic response (SVR), especially in patients
infected with genotype 1 HCV. In 2011, the US Food and
Drug Administration (FDA) approved boceprevir (Merck) and
telaprevir (Vertex Pharmaceuticals and Johnson & Johnson) as
antiviral agents directly targeted against HCV NS3/4A
protease, in combination with PEG-IFN-α and RBV. In
genotype 1 patients, very promising results have been reported
when either telaprevir or boceprevir are added to the standard
of care.5 Although the introduction of these direct-acting
antivirals (DAAs) into the regimen improves therapeutic
outcome, their possible limitations include a low genetic
barrier, which may result in the appearance of drug-resistant
mutants during long-term treatment. The development of
effective and safe small molecule antiviral agents aimed at a
variety of gene targets is therefore warranted.6
HCV, which belongs to the Hepacivirus genus in the
Flaviviridae family, contains a 9.8 kb, single-stranded positive
sense RNA genome encoding a polyprotein of approximately
3000 amino acids. This polyprotein encompasses structural
proteins (Core, E1, and E2), virion particles, and nonstructural
proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B)
participating in RNA replication.7−9 Although the precise
enzymatic role of NS5A has not been clarified, it has been
shown to be essential for HCV replication, virus assembly, and
the host immune response related to viral resistance to IFN-α
therapy. NS5A is zinc-binding phosphoprotein (56−58 kDa),
consisting of 447 amino acids,10,11 and associated with the
membrane through an N-terminal amphipathic α-helix region.12
The three principle domains in NS5A are domain I, containing
a zinc binding motif required for viral RNA replication;13
domain II, which interacts with NS5B and cellular proteins such
as PKR and PI3K; and domain III, which plays a role in
infectious virus assembly, but not in RNA replication.14
A novel NS5A inhibitor, daclatasvir (1, shown above; BMS-
790052), was recently reported to exhibit strong anti-HCV
activity, particularly in the case of the HCV genotype 1. The
effective concentration (EC50) of daclatasvir was shown to be in
the picomolar range in vitro, and in clinical trials, a single dose
treatment of 100 mg reduced the HCV RNA level about 3.3
log10 without apparent toxicity.15,16 Subsequently, pharmaceut-
ical companies and numerous research groups have focused on
the development of a series of NS5A inhibitors.17−21 In
addition to daclatasvir, currently known candidates in this series
include GS-5885, ABT-267, PPI-461, AZD-7295, BMS-824393,
ACH-2928, IDX-719, PPI-1301, and EDP-239.22 Daclatasvir
(1) has a biphenyl core connected to an imidazole moiety, a
proline moiety, and a capping group of amino acid derivatives.
In 2012, Schinazi and co-workers reported on inhibitors
containing a variety of extended biphenyl linkers with small
modifications at both ends of the inhibitors.23−25 Here, we
report on a new class of potent inhibitors based upon a
benzidine prolinamide skeleton.
Special Issue: HCV Therapies
Received: August 25, 2013
Accepted: December 4, 2013
Published: December 4, 2013
© 2013 American Chemical Society
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Letter
The symmetrical structure of the benzidine scaffold greatly
simplified our synthetic route. Coupling of Boc-L-proline with
benzidine resulted in excellent yield of an almost pure product,
obviating the need for column chromatographic purification.
After removal of the Boc group using trifluoroacetic acid (TFA)
in CH2Cl2, a series of capping groups was added to the free
Table 1. Structure−Activity Relationships of Inhibitors
Containing a Benzidine Prolinamide Skeleton against HCV
Type 2a
amine using either peptide coupling (EDCI and Hunig’s base in
̈
CH2Cl2) or reductive amination, to furnish the target
compounds, 4−11 (Scheme 1).26,27
a
Scheme 1. Synthesis of Benzidine Prolinamide Skeleton
a
Reagents and conditions: (a) N-Boc-L-proline, EDC, DCM, 94%; (b)
TFA, DCM; (c) capping group, EDC, DIPEA, DCM 22−49% (2
steps).
To determine the inhibitory activities (EC50’s) of the
compounds, dose−response experiments were performed
using an infectious HCV cell culture system (HCVcc) (see
also Supporting Information). JFH 5a-Rluc-ad34 is a derivative
of JFH1 containing a Renilla luciferase reporter and cell culture
adaptive mutations.28 Huh 7.5.1 cells were inoculated with the
HCVcc for 3 h and then cultivated for 3 d in the presence of
the indicated compounds at serially diluted concentrations.
After cell harvesting, EC50’s were calculated by sigma plot
analysis of the luciferase activities in the cells.
The assay data indicate that the inhibitory activity of these
compounds is greatly dependent upon the structure of the
flanking end groups (Table 1).29,30 When both enantiomers of
N-methyloxycarbonyl-protected valine were attached, a clear
difference in inhibitory activity between the two epimers was
observed, indicating the importance of the stereochemistry of
the capping group. The inhibitory activity of the D-isomer
(compound 4) was 8-fold higher than that of the L-epimer
(compound 5, Table 1, entries 1 and 2, respectively). Next, we
identified the optimal capping group through a series of
structure−activity relationship (SAR) studies. When phenyl-
glycine derivatives were incorporated on both ends, extremely
high inhibitory activities were observed, regardless of the N-
protecting groups. The highest inhibition was observed for the
compound containing an N-methyloxycarbonylphenylglycine
group (Table 1, entry 3), while N,N-dimethyl- and N,N-diethyl
phenylglycine derivatives also had inhibitory EC50’s in the low
nanomolar range. Compounds 6−8 were potent inhibitors of
HCV proliferation, with EC50’s of 260 pM, 2.3 nM, and 2.2 nM
in infectious HCVcc, respectively, and none had detectable
cytotoxic effects at 25 μM of concentration (Table 1, entries 3−
5, respectively). In contrast, compound 9 which contains a N-
methyloxycarbonyl-L-alanine moiety, had no detectable inhib-
ition at 1 μM. Inhibitors capped with N-methyloxycarbonyl-L-
tert-leucine and N-2(R)-tetrahydrofurylcarbonyl groups in-
hibited HCV proliferation with EC50’s of 24 and 400 nM
inhibition, respectively (entries 7 and 8, Table 1).
To assess the inhibitory activity of compounds on HCV
replication, we measured EC50’s using an HCV replicon
containing a HCV nonstructural protein, NS3-NS5B, and the
Renilla luciferase reporter gene.31,32 Compound 6 showed the
most potent inhibition in the replicon assay (EC50 = 28 pM),
while compounds 7 (EC50 = 0.43 nM) and 8 (EC50 = 0.27 nM)
also exhibited subnanomolar inhibition (Table 2, entries 1−3).
Table 2. Antiviral Activity in an HCV Genotype 1b Replicon
Assay
HCV
replicon
SI
entry compound
(type 1b) cytotoxicity (cytotoxicity/EC50 × 1000)
1
2
3
6
7
8
0.028 nM
0.43 nM
0.27 nM
>25 μM
>25 μM
>25 μM
>893
>58
>93
Since compound 6 exhibited the highest inhibitory activity
both in HCVcc infection and in the replicon system (Table 2,
entry 1), we next focused our attention on further evaluation of
this compound.33 First, to evaluate the potential cardiac toxicity
of compound 6 through inhibition of the inward rectifying
voltage gated potassium channel encoded by the hERG gene,
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ACS Medicinal Chemistry Letters
Letter
we carried out a hERG assay.34,35 Compared to the astemizole
control (EC50 = 1.9 nM), compound 6 (EC50 = 9.8 μM) was
shown to bind poorly to the hERG membrane preparations,
suggesting that compound 6 would have minimal cardiac
toxicity (Table 3).
compound 6 and sofosbuvir have a distinct additive effect on
the proliferation of HCV, presumably due to the independent
modes of action of compound 6 and sofosbuvir.
In summary, we have developed a series of inhibitors based
on a new benzidine prolinamide core structure, several of which
have extremely high anti-HCV activity. SAR studies using a
variety of terminal capping groups showed particularly high
inhibitory activities for inhibitors containing phenyl glycine
capping groups, of which compound 6 was the most potent.
Moreover, subsequent studies demonstrated that compound 6
has a desirable cardiac toxicity, rat plasma stability, and low
inhibitory activity against representative Cyp450 enzymes. Our
data indicate that compound 6 is a potent, safe lead compound
that warrants further study for its potential in anti-HCV
therapy.
Table 3. hERG Ligand Binding Assay
compound
EC50 (μM)
6
9.8
control (astemizole)
0.0019
Next, a rat plasma stability test36,37 of compound 6 (Table 4)
showed that more than 99% of the compound was intact after 4
h, indicating high in vivo stability (Table 4).
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, characterizations, spectra and HPLC
traces of compounds 2−11, and experimental protocol used to
evaluate HCV inhibitors in cell and replicon assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Table 4. Stability of Compound 6 in Rat Plasma
■
S
compound 6 (2 μM)
0.5 h (%)
>99
1 h (%)
>99
4 h (%)
>99
6
Next, to evaluate potential drug−drug interactions of
compound 6, we carried out CYP450 screening (Table 5).38
Table 5. CYP450 Screening Assay
AUTHOR INFORMATION
Corresponding Authors
*E-mail: kimbm@snu.ac.kr.
*E-mail: sungkey@postech.ac.kr.
■
compound
1A2
2C9
2D6
3A4
a
6
18.12
98.60
0
0
23.87
95.34
b
inhibitor
75.79
95.07
Funding
a
b
CYP inhibition (%) of compound 6 at 10 μM. 1A2: α-
This research was supported by the Bio R&D Program (No.
2012-054974), through the National Research Foundation
funded by the MEST.
naphthoflavone, 2C9: sulfaphenazole, 2D6: quinidine, 3A4: ketocona-
zole.
Notes
A compound exhibiting greater than 50% inhibition of CYP450
activity at a concentration of 10 μM is classified as a CYP450
enzyme inhibitor. CYP1A2 (18.12% inhibition) and CYP3A4
(23.87% inhibition) were weakly inhibited in the presence of 10
μM compound 6, while CYP2C9 and CYP2D6 retained
maximal activity under the same conditions.
To determine whether compound 6 would have a synergistic
or additive anti-HCV effect in combination with other HCV
inhibition therapy, we treated replicon with compound 6 in the
presence of a 125 nM of sofosbuvir39,40 and monitored the
effect on HCV proliferation (Figure 1). Our data show that
The authors declare no competing financial interest.
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