Peroxisome proliferator-activated receptor delta antagonists inhibit hepatitis C virus RNA replication

Article abstract of DOI:10.1016/j.bmcl.2013.07.005

It has been reported that ligand-mediated transcription factor peroxisome proliferator-activated receptor alpha (hPPARα) is involved in hepatitis C virus (HCV) RNA replication, whereas hPPARγ is not, and the effect of hPPARδ is unknown. Here, we show that hPPARδ-selective antagonists effectively inhibit HCV RNA replication. We describe the design, synthesis and pharmacological evaluation of a series of biphenyl-4-carboxylic acid-type hPPARδ antagonists, including previously reported compounds, as candidate anti-HCV agents. A representative compound (4c) dose-dependently inhibited HCV RNA replication (EC₅₀ 0.22 μM), while exhibiting relatively weak cytotoxicity to the host cells (CC₅₀ 2.5 μM). It also showed an additive and dose-dependent effect on the inhibition of HCV RNA replication by pegylated interferon alpha (Peg-IFNα) alone and by both Peg-IFNα and ribavirin (currently the clinical treatment of choice for HCV infection). Thus, combination of a hPPARδ antagonist with current therapy may improve the efficacy of treatment for HCV infection.

Full text of DOI:10.1016/j.bmcl.2013.07.005

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4774–4778
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peroxisome proliferator-activated receptor delta antagonists inhibit
hepatitis C virus RNA replication
Shintaro Ban a, Youki Ueda b, Masao Ohashi a, Kenji Matsuno a, Masanori Ikeda b, Nobuyuki Kato b,
Hiroyuki Miyachi a,
⇑
a Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan
b Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
It has been reported that ligand-mediated transcription factor peroxisome proliferator-activated receptor
Received 18 June 2013
Revised 3 July 2013
Accepted 5 July 2013
Available online 13 July 2013
alpha (hPPARa) is involved in hepatitis C virus (HCV) RNA replication, whereas hPPARc is not, and the
effect of hPPARd is unknown. Here, we show that hPPARd-selective antagonists effectively inhibit HCV
RNA replication. We describe the design, synthesis and pharmacological evaluation of a series of biphe-
nyl-4-carboxylic acid-type hPPARd antagonists, including previously reported compounds, as candidate
anti-HCV agents. A representative compound (4c) dose-dependently inhibited HCV RNA replication
Keywords:
HCV RNA replication
PPAR delta
Biphenyl-4-carboxylic acid
PPAR delta antagonist
(EC50 0.22
showed an additive and dose-dependent effect on the inhibition of HCV RNA replication by pegylated
interferon alpha (Peg-IFN ) alone and by both Peg-IFN and ribavirin (currently the clinical treatment
lM), while exhibiting relatively weak cytotoxicity to the host cells (CC50 2.5 lM). It also
a
a
of choice for HCV infection). Thus, combination of a hPPARd antagonist with current therapy may
improve the efficacy of treatment for HCV infection.
Ó 2013 Elsevier Ltd. All rights reserved.
Hepatitis C virus (HCV) is a member of the genus Hepacivirus of
the family Flaviviridae, and approximately 180 million people
worldwide were reported to be infected with it in 2007 (World
Health Organization (WHO), 2007). It induces serious chronic hep-
atitis, leading to steatosis, cirrhosis, and ultimately hepatocellular
carcinoma.1 HCV infection is involved in about 50–70% of liver can-
cers, and is the underlying reason behind two–thirds of all liver
transplants in the developed world.1 The standard treatment for
HCV infection is combination therapy with pegylated interferon-
but owing to the rapidly mutating nature of the HCV genome,
drug-resistant mutants readily appear.
However, host lipid contents and host lipid homeostasis are also
important factors for the assembly, budding and replication of
many viruses, including HCV.11–13 For example, exogenous admin-
istration of saturated and/or monounsaturated fatty acids en-
hanced HCV RNA replication, while polyunsaturated fatty acids
suppressed it.14 Therefore, we considered that receptor(s) and/or
enzyme(s) associated with lipid biosynthesis might also be impor-
tant target(s) for the development of anti-HCV agents. Sterol regu-
latory element-binding protein-1c (SREBP-1C) is a key protein for
production of saturated and monounsaturated fatty acids, via up-
regulation of the transcription of acyl-CoA carboxylase, fatty acid
synthase and stearoyl-CoA desaturase.15 Consequently, com-
pounds that disrupt the production and/or function of SREBP-1c
might be candidate anti-HCV agents. Here, we focused on meta-
bolic nuclear receptors, especially peroxisome-proliferator acti-
vated receptors (PPARs).
a
(Peg-IFNa) and ribavirin, but this results in a sustained virologi-
cal response (SVR) in only 40–50% of the patients infected with
genotype 1 virus.2 Therefore, there is an urgent need for new
anti-HCV drugs.
HCV has a single-stranded RNA of positive polarity that encodes
a polyprotein with ca. 3000 amino acid residues.3 After maturation,
this is cleaved into at least 10 proteins: structural proteins termed
Core, E1, E2, and p7, and nonstructural proteins termed NS2, NS3,
NS4A, NS4B, NS5A and NS5B (Fig. 2).4 The viral RNA-dependent
RNA polymerase is encoded by NS5b.5 Posttranslational processing
of the nonstructural proteins is catalyzed by the serine protease
NS3,6,7 together with the co-factor NS4A, which promotes anchor-
age on the endoplasmic reticulum membrane.8 These molecules
have been selected as targets of direct-acting antivirals (DAA),9,10
PPARs are ligand-dependent transcription factors belonging to
the nuclear receptor (NR) superfamily. The three subtypes (PPAR
a,
PPARd, and PPAR ) identified to date are differentially expressed in
c
a tissue-specific manner, and play pivotal roles in lipid, lipoprotein,
and glucose homeostasis.16 However, the range of therapeutic po-
tential for hPPAR ligands is currently considered to extend well be-
yond lipid, lipoprotein and glucose homeostasis, and so the biology
and pharmacology of hPPARs are attracting great interest.17–19
⇑
Corresponding author. Tel.: +81 086 251 7930.
E-mail address: miyachi@pharm.okayama-u.ac.jp (H. Miyachi).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.07.005

S. Ban et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4774–4778
4775
RNA interference experiments targeting hPPAR
have indicated that knockdown of hPPAR mRNA inhibits HCV rep-
lication, whereas knockdown of hPPAR
mRNA had no effect.20
perturbation study with small-molecular-weight compounds indi-
cated that high concentration of hPPAR co-antagonist,
a
and hPPAR
c
matic representation of the replicon used in this Letter is depicted
in Figure 1.
a
c
A
Using OR6 cells, we first performed WST-1 cell proliferation as-
say to determine the appropriate concentrations of compounds for
the assay of anti-HCV activity (data not shown).23 The cells were
plated onto 96-well plates (1 Â 103 cells per well) in triplicate
and then treated with each reagent at three or four concentrations
for 72 h. After treatment, the cells were subjected to WST-1 cell
proliferation assay (Takara Bio, Otsu, Japan) according to the man-
ufacturer’s protocol. Based on the assay results, we chose the con-
centration showing a relative activity of approximately 80% as the
maximum concentration for anti-HCV activity assay. For the Renilla
luciferase (RL) assay, 2 Â 104 OR6 cells were plated in 24-well
plates at least in triplicate for each assay and cultured for 24 h.
The cells were treated with the test compounds for 72 h, then har-
vested with Renilla lysis reagent (Promega) and subjected to the RL
assay according to the manufacturer’s protocol. In several cases,
a
c/a
T0070907 (1) (Fig. 2) reduced HCV RNA replication to a comparable
extent to interferon treatment, using a subgenomic HCV replicon,
via the hPPAR
tence of a subtype-specific link between hPPAR
a
pathway.20 These data clearly indicated the exis-
a
and HCV replica-
tion. However, the effect of hPPARd on HCV replication has not
been established. Therefore, here we investigated the effect of
our hPPARd-selective biphenylcarboxylic acid-type ligands on
HCV RNA replication.
In order to investigate the putative role of hPPARd in HCV RNA
replication, we used the OR6 assay system, which is the first repli-
cation system for genome-length HCV RNA encoding a reporter
gene for simple monitoring of HCV replication levels.21,22 A sche-
HCV genome length: 9.6 kb RNA
C
E1
E2 p7 NS2
E2 p7 NS2
NS3
NS3
NS4A NS4B NS5A
NS4A NS4B NS5A
NS5B
NS5B
5’
3’
3’
OR6 length: 12 kb RNA
EMCV-
IRES
5’ RLuc NeoR
C
E1
Figure 1. Schematic representation of the replicon (OR6) used in this Letter. Abbreviations: RLuc, Renilla luciferase gene; NeoR, neomycin phosphotransferase; EMCV-IRE,
Sencephalomyocarditis virus internal ribosomal entry site
1
2
5
3
6
4c
Figure 2. Structures of hPPARc/a-co-antagonist, T0070907 (1), hPPARd-selective agonist TIPP-204 (2), hPPARd-partial agonist (3), hPPARd-selective antagonist (4c), hPPARd-
selective antagonist GSK-0660 (5) and hPPAR-pan agonist TIPP-703 (6).
Figure 3. Effects of various hPPARs ligands in the genome-length HCV RNA replication system bearing the Renilla luciferase reporter. (A) Inhibition of replication of genome-
length HCV RNA encoding the Renilla luciferase gene was monitored by luciferase reporter assay. (B) Dose–response relationship of the representative compound 4c for
inhibition of HCV RNA replication (closed circle) and for cell toxicity (closed triangle).

4776
S. Ban et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4774–4778
Figure 4. Structure–activity relationships of biphenyl carboxylic acid derivatives for HCV RNA replication inhibition and for hPPARd/
a-antagonistic activity. Antiviral
ribavirin (RBV) and T0070907 (1) were evaluated as positive controls (HCV RNA replication inhibitor and hPPAR -co antagonist, respectively).
c/a
Scheme 1. Synthetic routes to the present series of compounds 4d–f. Reagents and conditions: (a) benzamide, triethylsilane, TFA, toluene, reflux, 48 h, 30%; (b) 4-
methoxycarbonyl-2-methylbenzeneboronic acid pinacol ester, (Ph3P)2PdCl2, K2CO3, THF, H2O, 60 °C, 5 h, 20%; (c) R1I, K2CO3, DMF, rt, overnight, 62–89%; (d) LiOH, MeOH, H2O,
THF, 100 °C, 3 h, 90–96%.
the 50% cytotoxic concentration (CC50) was determined. The value
of selectivity index (SI) was determined by dividing the CC50 value
by the EC50 value.
The chemical structures of the hPPARs ligands used in our initial
study are depicted in Figure 2, and their effect on HCV replication is
shown in Figure 3.
be involved in HCV RNA replication, in addition to the previously
reported contribution of the hPPAR pathway. Although another
structural class of hPPARd-selective antagonist 5 had no apparent
inhibitory activity on HCV RNA replication, the reason for this
might be that it lacks in vivo bioavailability.
We then examined the effects of our previously reported
hPPARd-selective biphenyl-4-carboxylic acid-type antagonists on
HCV RNA replication (Fig. 4). The alkoxy chain variants, 4d–4f,
were newly prepared according to the method illustrated in
Scheme 1,28 and the hPPARd–antagonistic activity (IC50 value)
a
The hPPARd-selective agonist 2,24 hPPARd-selective partial ago-
nist 3,25 hPPARd-selective antagonist 5 (GSK-0660),26 and hPPAR-
pan agonist 627 all had no effect on HCV RNA replication (their
chemical structures are shown in Fig. 1). On the other hand, the
hPPARd-selective antagonist 4c25 dose-dependently inhibited
HCV RNA replication, while its cytotoxicity to the host cells was
was determined in the presence of 1
lM hPPAR-pan agonist 6.
The hPPAR –antagonistic activity (% inhibition) of representative
a
relatively weak (EC50, CC50 and the SI were 0.22, 2.5, and 11
lM,
compounds was also determined in the presence of 1 lM hPPAR-
respectively). These data indicated that the hPPARd pathway might
pan agonist 6.

S. Ban et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4774–4778
4777
100
100
10
1
10
1
0.1
0.1
0
0.4
0.8
1.2
1.6
2
0
0.4
0.8
1.2
1.6
2
Figure 5. Additive effect on HCV RNA replication of the representative hPPARd-antagonist 4c (
(Left) closed circle: IFN- 0 IU/mL (IU: international unit), closed triangle: IFN- 1 IU/mL, closed box: IFN-
IFN- 0 IU/mL + RBV 0 M, closed triangle: IFN- 1 IU/mL + RBV 6.25 M, closed box: IFN-
l
M) in combination with IFN
a
alone (left) and with IFN
a
plus ribavirin (right).
a
l
a
a
4 IU/mL), closed diamond: IFN-
a
16 IU/mL. (Right) closed circle:
16 IU/mL + RBV 25 M.
a
a
l
a
4 IU/mL + RBV 12.5
lM, closed diamond: IFN-
a
l
As previously reported, biphenyl-3-carboxylic acids (4a and 4b)
did not exhibit hPPARd–antagonistic activity, but showed hPPARd-
partial agonistic activity, while biphenyl-4-carboxylic acids
(4c–4h) exhibited hPPARd–antagonistic activity.25 Therefore, the
position of the carboxylic acid functionality is critically important
for the antagonistic nature of the present series of compounds,
although the chain length of the central alkoxy group might not crit-
ical for activity, because all the alkoxy variants exhibited compara-
bly potent hPPARd–antagonistic activity (IC50 values reached 10 nM
order). It is interesting that heteroatom-containing carboxylic acids
(4i–4k) did not exhibit apparent hPPARd–antagonistic activity,
although the reason for this is not known.
results indicate that combination of 4c with the current therapy
of choice for the treatment for HCV infection has the potential to
improve the outcome, at least in terms of inhibition of HCV RNA
replication.
In summary, we have established that the hPPARd pathway is
involved in HCV RNA replication, in addition to the hPPARa path-
way. We also found that biphenyl-4-carboxylic acid-type hPPARd-
selective antagonists, such as 4c, effectively inhibited HCV RNA
replication, with low levels of host cell toxicity. Compound 4c
showed a dose-dependent, additive effect on the inhibition of
HCV RNA replication when used in combination with current HCV
therapy. Further structural development studies are under way,
and we are also attempting to isolate mutants resistant to OR6 in
order to clarify the nature of the linkage between the hPPARd path-
way and HCV RNA replication.
In our assay system, 1 exhibited very weak hPPARd–antagonis-
tic activity (IC50 > 10
exhibit apparent hPPARd–antagonistic activity even at the highest
concentration examined (10 M).
We selected compounds 1, 4c–4e as representative compounds
and evaluated their hPPAR –antagonistic activity. However, their
lM), and the antiviral ribavirin (RBV) did not
l
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activity was too weak to permit calculation of IC50 values. Among
these four compounds, 4c was the least effective, and it did not ex-
hibit apparent hPPARa–antagonistic activity even at the highest
examined concentration of 10
lM. Therefore, 4c is the most
hPPARd-selective antagonist tested, and its selectivity ratio ex-
ceeded 1000 in our assay system.
As for HCV RNA replication, 1 exhibited a micromolar order
ED50 value, but host cell toxicity appeared prior to the effect on
HCV RNA replication. Therefore, the nature of the effect of 1 on
HCV RNA replication is uncertain. The antiviral ribavirin (RBV) also
exhibited a micromolar order ED50 value without concomitant host
cell toxicity, and its SI value exceeded 10. hPPARd-inactive com-
pounds showed no response in our assay system, as expected,
while all the biphenyl-4-carboxylic acid-type hPPARd-antagonists
inhibited HCV RNA replication. Indeed, the methyl derivatives at
the R2 position (4c–4f) exhibited submicromolar order EC50 values,
being about 10 times more potent than the currently used anti-
HCV agent RBV or the reported hPPARca-co-antagonist 1. Their
SIs were more than 10 (except for compound 4f), and the host cell
toxicities were relatively weak. These data support the idea that
the hPPARd pathway might be involved in HCV RNA replication.
Since the current treatment for HCV infection is Peg-IFNa com-
bined with ribavirin, we next evaluated the effect on HCV RNA rep-
lication of the representative hPPARd-antagonist 4c in combination
with Peg-IFN
shown in Figure 5, HCV RNA was reduced by Peg-IFN
by Peg-IFN in combination with ribavirin, in a dose-dependent
a
alone, or with both Peg-IFN
a
and ribavirin. As
a
alone, and
a
manner. Furthermore, addition of 4c dose-dependently increased
the inhibition of HCV RNA replication by these agents. These
22. Ikeda, M.; Abe, K.; Yamada, M.; Dansako, H.; Naka, K.; Kato, N. Hepatology 2006,
44, 117.

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476.1485; found 476.1466 (M+H)+.
24. Kasuga, J.; Nakagome, I.; Aoyama, A.; Sako, K.; Ishizawa, M.; Ogura, M.;
Makishima, M.; Hirono, S.; Hashimoto, Y.; Miyachi, H. Bioorg. Med. Chem. 2007,
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28. The physicochemical properties of the compounds 4d–4f are as follows: 4-[4-
ethoxy-3-({[2-fluoro-4-(trifluoromethyl)benzoyl]amino}methyl)phenyl]-3-
methyl-benzoic acid (4d): mp 260–261 °C. 1H NMR (400 MHz, DMSO) d 12.86 (s,
1H), 8.94 (dd, J = 6.0, 6.0 Hz, 1H), 7.83–7.74 (m, 4H), 7.65 (d, J = 8.0, 1H), 7.27–
7.22 (m, 3H), 7.07 (d, J = 9.2 Hz, 1H), 4.49 (d, J = 5.6 Hz, 2H), 4.12 (q, J = 6.8 Hz,
4-[3-({[2-Fluoro-4-(trifluoromethyl)benzoyl]amino}methyl)-4-propoxyphenyl]-3-
methyl-benzoic acid (4e): mp 224–225 °C. 1H NMR (400 MHz, DMSO) d 12.87 (s,
1H), 8.92 (dd, J = 6.0, 6.0 Hz, 1H), 7.85–7.76 (m, 4H), 7.65 (d, J = 8.0 Hz, 1H),
7.27–7.24 (m, 3H), 7.07 (d, J = 8.8 Hz, 1H), 4.51 (d, J = 5.6 Hz, 2H), 4.03 (t,
J = 6.4 Hz, 2H), 2.27 (s, 3H), 1.96–1.86 (m, 2H), 1.11 (t, J = 7.2 Hz, 3H). HRMS
(FAB) calcd for C26H24F4NO4 490.1642; found 490.1647 (M+H)+.
4-[3-({[2-Fluoro-4-(trifluoromethyl)benzoyl]amino}methyl)-4-pentoxyphenyl]-3-
methyl-benzoic acid (4f): mp 228–230 °C. 1H NMR (400 MHz, DMSO) d 12.87 (s,
1H), 8.93 (dd, J = 5.6, 5.6 Hz, 1H), 7.83–7.76 (m, 4H), 7.65 (d, J = 8.0 Hz, 1H),
7.27–7.24 (m, 3H), 7.06 (d, J = 9.2 Hz, 1H), 4.50 (d, J = 5.6 Hz, 2H), 4.05 (t,
J = 6.4 Hz, 2H), 2.27 (s, 3H), 1.80–1.73 (m, 2H), 1.48–1.31 (m, 4H), 0.88 (t,
J = 7.2 Hz, 3H). HRMS (FAB) calcd for C28H28F4NO4 518.1956; found 518.1965
(M+H)+.