Synthesis and inhibitory activity on hepatitis C virus RNA replication of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analogs

Article abstract of DOI:10.1016/j.bmcl.2014.07.019

Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replication in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analog 1a as a novel HCV inhibitor. St

Full text of DOI:10.1016/j.bmcl.2014.07.019

Bioorganic & Medicinal Chemistry Letters 24 (2014) 4276–4280
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and inhibitory activity on hepatitis C virus RNA replication
of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analogs
b
a
a
a
b
Kenji Matsuno a, , Youki Ueda , Miwa Fukuda , Kenji Onoda , Minoru Waki , Masanori Ikeda ,
⇑
Nobuyuki Kato b, Hiroyuki Miyachi a,
⇑
a Medicinal and Bioorganic Chemistry, 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, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, 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:
Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replica-
tion in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-
2-propyl)aniline analog 1a as a novel HCV inhibitor. Structural modifications of 1a provided a series of
sulfonamides 7 with much more potent HCV RNA replication-inhibitory activity than ribavirin. Com-
Received 11 June 2014
Revised 3 July 2014
Accepted 8 July 2014
Available online 15 July 2014
pound 7a showed an additive anti-HCV effect in combination with standard anti-HCV therapy (IFN-a plus
ribavirin). Since 7a generated reactive oxygen species (ROS) in the ORL8 system and its anti-HCV activity
was blocked by vitamin E, its anti-HCV activity may be mediated at least in part by ROS.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus
HCV RNA replication
Anti-HCV agent
Full genome length
ORL8
Hepatitis C virus (HCV), an enveloped, single-stranded RNA
virus, is a major cause of human hepatitis. It is estimated that ca.
150 million people worldwide are chronically infected with this
virus, and more than 350,000 people die every year from hepatitis
C-related liver disease.1 Most infections become persistent and
about 60% of cases progress to chronic liver disease, which in turn
can lead to cirrhosis, hepatocellular carcinoma, and liver failure.
Currently, no anti-HCV vaccine is available, and the standard treat-
ment for chronic hepatitis C consists of pegylated interferon (IFN)-
therapy, but also because of the high mutation and replication
rates of HCV.
The HCV genome consists of 9,600 bases encoding three struc-
tural and seven non-structural proteins.2 So far, drug discovery
programs for anti-HCV agents have focused on NS3 protease and
NS5B polymerase as targets to block HCV replication. Convention-
ally, HCV replication assay has generally been conducted with sub-
genomic HCV replicon systems encoding minimum sequences for
autonomous HCV replication, that is, HCV replicase proteins NS3
to NS5B, with firefly luciferase as the reporter gene.3 However,
we recently developed new screening systems to identify HCV
inhibitors, namely a reporter gene assay system with Renilla
luciferase for replication of genome-length HCV RNA in human
hepatoma-derived HuH-7 cells (OR6)4 and in human hepatoma-
derived Li23 cells (ORL8).5 Figure 1 shows a schematic representation
of the gene organization in the genome-length HCV RNA in
ORL8. These full-genomic systems are considered to be superior
to the conventional sub-genomic systems, and are suitable for
use in chemical-biological approaches to detect different types
of HCV inhibitors due to the different genetic backgrounds of
HuH-7 and Li23 cells.6 By use of these assays, we identified
N-{4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)phenyl}-N-methylb-
enzamide (1a) as a novel HCV inhibitor (Fig. 2). Compound 1a is a
a
in combination with the classical anti-HCV agent ribavirin.
Recently, telaprevir, boceprevir and simeprevir, which are mem-
bers of a new class of HCV NS3-4A serine-protease inhibitors, have
been approved for hepatitis C treatment as combination therapy
with pegylated IFN-a and ribavirin for patients with HCV genotype
⁄⁄⁄1a or 1b. Although these new therapies have increased cure
rates for both previously untreated people and prior non-respond-
ers, there are still non-responders to these treatments. Also, seri-
ous adverse reactions such as skin reaction may require
discontinuation of the combination therapy. Thus, new anti-HCV
agents are still required, not only to extend the coverage of current
⇑
much more potent inhibitor of HCV RNA replication (EC50 0.32
lM)
Corresponding authors.
E-mail address: matz@pharm.okayama-u.ac.jp (K. Matsuno).
than ribavirin (EC50 8.7 M). Interestingly, the ORL8 assay system
l
http://dx.doi.org/10.1016/j.bmcl.2014.07.019
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

K. Matsuno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4276–4280
4277
Figure 1. Schematic illustration of the gene organization in HCV genome-length in ORL8. Abbreviations: RLuc, Renilla luciferase gene; NeoR, neomycin phosphotransferase;
EMCV-IRES, encephalomyocarditis virus internal ribosomal entry site.
synthesized from 3b by benzoylation. Incorporation of substituents
on phenyl rings and phenyl ring replacement were achieved by
using the corresponding commercially available aniline 2, acyl
chloride or sulfonyl chloride. 1,1,1,3,3,3-Hexafluoro-2-(2-hydroxy-
ethoxy)propyl analog 6 was synthesized from THP-protected 5,
which was obtained by alkylation of 1a with commercially avail-
able 2-(2-bromoethoxy)tetrahydro-2H-pyran. The aminomethyl-
Me
N
F3C
HO
O
CF3
1a
EC50 = 0.32µM (ORL8)
EC50 > 20µM (OR6)
ene analog
9 was synthesized by direct incorporation of a
hexafluoroisopropanol moiety into N-benzyl-N-methylaniline.
Figure 2. Structure of 1a and HCV RNA replication-inhibitory activity in the ORL8
All synthetic analogs were assayed for anti-HCV RNA replication
activity in the ORL8 system and EC50 values were determined
(Tables 1 and 2). The values of 50% cytotoxic concentration
(CC50) for Li23 cells were determined by conventional WST-1
and OR6 assay systems.
was more sensitive to 1a than the OR6 system. Herein, we describe
the synthesis of 1a and its derivatives modified at both phenyl rings,
as well as at the hexafluoroisopropanol group and the amide moiety.
The structure–activity relationship of these compounds for HCV RNA
replication was examined, and the mechanism of action is discussed.
The synthetic procedures are outlined in Scheme 1. Briefly, con-
densation of (1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)-aniline
3a with acyl chloride, sulfonyl chloride or isocyanate provided
amides (1a–1u), sulfonamides (7a–7i) or urea (8), respectively.
Compound 3a was obtained as the major product by alkylation
assay, and the values of selectivity index (SI), that is CC50/EC50
were calculated. All assays were conducted in triplicate.
,
The results of modifications at both phenyl rings and at the
hexafluoroisopropanol group are summarized in Table 1. The
incorporation of Me on the left phenyl ring (1b, 1c) reduced anti-
HCV RNA replication activity, and the 3-Me analog was completely
inactive. Methylation of the right phenyl ring (1d–1f) also
decreased anti-HCV activity. Among compounds with replacement
of the right phenyl ring (1g–1q), alkyl analogs showed moderate
activity. Extension of the alkyl chain increased the activity (1g–
1i) and the cyclohexyl analog 1j was as potent as the parent 1a,
with an adequate SI value. Bulky 1-adamantyl analog 1k was a
of aniline
2 with hexafluoroacetone trihydrate under acidic
conditions.7 Compound 3b was also formed as a minor product,
so the regioisomer of the hexafluoroisopropanol group (4) was
F3C
F3C
F3C
F3C
R3
NH
OH
OH
Me
N
R3
N
H
N
a
b
R4
Me
+
F3C
HO
O
(f rom R4=H )
1
R1
3a
3b
(f rom ; R =H)
R1
3b
R1
CF3
2
4
3a
)
(from
b
c
(from R1=H, R3=Me)
d
(from R1=H, R3=Me)
a
(from R1=H, R3=Me, R4=Bn)
R3
N
Me
Me
N
H
N
R2
N
O
R2
O
S
F3C
HO
O
F3C
HO
F3C
HO
O
R1
CF3
1a 1u
CF3
7a-7i
CF3
8
-
(from R1=H, R2=Ph, R3=Me)
e
Me
N
Me
N
Me
N
f
F3C
HO
F3C
O
O
F3C
O
O
THPO
HO
CF3
CF3
CF3
9
5
6
Scheme 1. Synthesis of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analogs. Reagents and conditions: (a) hexafluoro-acetone trihydrate, pTsOH hydrate, toluene,
reflux, 71–93%; (b) R2-COCl, Et3N, toluene or CHCl3, 22–93%; (c) R2-SO2Cl, Et3N, CH2Cl2, quant.; (d) PhNCO, toluene, quant.; (e) THPOCH2CH2Br, Cs2CO3, DMF, 79%; (f) pTsOH
hydrate, iPrOH, H2O, quant.

4278
K. Matsuno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4276–4280
Table 1
Values of HCV RNA replication-inhibitory activity, cytotoxicity and safety index of 1a–1q, 4 and 6 in ORL8 assay
R3
N
F3C
F3C
Me
N
OH
Me
N
R1
3
2
R2
F3C
O
O
F3C
HO
O
HO
O
CF3
CF3
1a-1q
6
4
Compd no
R1
H
2-Me
3-Me
H
H
H
H
H
H
H
H
H
H
H
H
H
R2
R3
EC50
(lM)
CC50
(lM)
SIa
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
6
Ph
Ph
Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
Me
nPr
nBu
cHex
1-Adamantyl
CH2Ph
2-pyridyl
3-pyridyl
4-pyridyl
2-thienyl
3-thienyl
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.32
1.7
>100
17
5.8
10
65
9.0
87
38
20
26
40
>100
26
20
17
27
>100
63
40
33
44
>20
>20
203
5.3
NDb
2.2
3.4
2.6
2.9
NDb
11
14
3.8
2.3
0.23
1.0
>100
>100
45
8.2
2.6
17
87
17
NDb
NDb
1.4
4.9
13
H
2.6
NDb
NDb
11
>20
4
a
Selectivity index (CC50/EC50).
Not determined.
b
Table 2
As for hexafluoroisopropanol modifications, 2-hydroxyethoxy ana-
Values of HCV RNA replication-inhibitory activity, cytotoxicity and safety index of 1r–
1u, 7a–7i, 8 and 9 in ORL8 assay
log 6 was a weak HCV inhibitor. This result suggests that the acid-
9
ity of OH might be important for anti-HCV activity (calculated pKa
R3
N
of OH group: 7.7 for 1a, 14.4 for 6, respectively). The position of the
hexafluoroisopropanol group appears to be critical, since regio-
isomer 4 was inactive.
R2
1r 1u
Z=CO (
Z=SO2 (7a-7i)
-
)
Z
F3C
HO
8
Z=CONH ( )
Z=CH2 (9)
Inhibitory activities of the amide-modified analogs on HCV RNA
replication are listed in Table 2. Among them, sulfonamide analog
7a showed potent anti-HCV activity. In contrast to the amide ser-
ies, substituents on the right phenyl ring (7b–7g) had little detri-
mental effect, except for OMe analogs (7h–7i), which showed
>10-fold weaker activity and low SI values. Both deletion of the
Me group on the amide moiety (1r) and extension of the alkyl
group (1s–1u) decreased the anti-HCV activity. Insertion of NH
(urea analog 8) and carba replacement of the amide carbonyl (ami-
nomethylene analog 9) also reduced the anti-HCV activity by about
10-fold compared with 1a. It is generally accepted that benzanilide
takes transoid conformation, whereas N-methylated benzanilide
uniquely takes cisoid conformation in aqueous solution.10 We spec-
ulate that adequate proximity of the right phenyl ring (or hydro-
phobic group such as cHex) to the left phenyl ring, namely cisoid
conformation of amide, is favorable for potent inhibition of HCV
RNA replication. This would be consistent with the inactivity of
the 2-pyridyl analog (1m), which would adopt transoid conforma-
tion at low pH.8 Also, this is also supported by the following spec-
ulations; the moderate inhibitor urea 8 takes similar conformation
of 1a, namely cisoid (at –NMeCO–)–transoid (at –CONH–) confor-
mation,11 whereas the inactive benzyl analog 1l might take
complete cofacial conformation of the two phenyl ring due to the
CF3
Compd no
R2
R3
EC50
(lM)
CC50
(
lM)
SIa
1r
1s
1t
1u
7a
7b
7c
7d
7e
7f
7g
7h
7i
Ph
Ph
Ph
Ph
Ph
2-Cl-Ph
2-Br-Ph
2-Me-Ph
3-F-Ph
3-Me-Ph
4-Me-Ph
3-MeO-Ph
4-MeO-Ph
Ph
H
Et
nPr
Bn
4.1
0.78
3.5
12
19
15
7.5
16
15
9.1
12
19
11
15
13
7.9
>20
13
2.9
24
4.3
2.6
84
88
18
33
53
31
34
6.2
5.6
NDb
4.3
2.9
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
0.19
0.17
0.50
0.36
0.36
0.35
0.44
2.1
1.4
8
9
5.7
3.0
Ph
a
Selectivity index (CC50/EC50).
Not determined.
b
moderately active HCV inhibitor, while the benzyl analog 1l was
inactive. This result suggests the importance of the orientation of
the hydrophobic moiety (vide infra). Suggestively, 2-pyridyl analog
1m, whose hydrophilic N atom is located at the most proximal
position to the amide moiety, was completely inactive. The
conformation of the two aromatic rings could differ from those
of other pyridyl analogs (1n–1o) at low pH values, such as that
in endosomes of cells (vide infra).8 Bioisosteric replacement of
the phenyl ring with thiophene (1p–1q) decreased the activity.
p–p
stacking interaction.
Next, the effect of adding 7a to current standard HCV therapy
(IFN- plus ribavirin) was evaluated (Fig. 3). In our ORL8 assay sys-
tem, HCV RNA replication was dose-dependently reduced both by
IFN- alone and by IFN- plus ribavirin. Addition of 7a dose-
dependently enhanced the inhibition of HCV RNA replication by
these agents. Also, addition of 7a to IFN- plus ribavirin was more
a
a
a
a

K. Matsuno et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4276–4280
4279
Figure 3. Additive effect of 7a on HCV RNA replication in combination with IFN-
(IU: international unit), box: IFN- 4 IU/mL), diamond: IFN- 16 IU/mL. (Right) circle: vehicle, triangle: IFN-
13 M. Open symbols in the broken lines show the values expected as an additive anti-HCV effect, and closed symbols in the solid lines show the values obtained by the ORL8
assay. Assays were conducted in triplicate.
a
alone (left) and with IFN-
a
+ ribavirin (right). (Left) circle: vehicle, triangle: IFN-
a
1 IU/mL
a
a
a
1 IU/mL + ribavirin 6.3 M, box: IFN-a 4 IU/mL + ribavirin
l
l
In conclusion, N-{4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-
propyl)phenyl}-N-methylbenzamide (1a) was identified as a novel
HCV inhibitor by use of our recently developed assay system for
full-genome-length HCV RNA replication in Li23 cells (ORL8).
Structural modifications of 1a provided a series of sulfonamides
7 that showed potent inhibition of HCV RNA replication. Com-
pound 7a showed an additive anti-HCV effect in combination with
standard HCV therapy (IFN-a plus ribavirin). The anti-HCV action
of 7a may be mediated by ROS production, since it was abrogated
by vitamin E.
Acknowledgments
Figure 4. Effect of vitamin E on anti-HCV activity of 7a. 1–4: in the absence of 7a,
5–8: in the presence of 5 M 7a (60% inhibitory concentration for HCV RNA
replication). Assays were conducted in triplicate.
l
This work was supported by the Drug Discovery for Intractable
Infectious Diseases Project, Graduate School of Medicine, Dentistry
and Pharmaceutical Sciences, Okayama University from the Minis-
try of Education, Culture, Sports, Science and Technology (MEXT).
The authors appreciate Mr. Yoshiaki Masuda of PharmaDesign
Inc., Tokyo for the pKa calculations. The authors thank the Division
of Instrumental Analysis, Okayama University for the NMR
measurements.
effective than addition to IFN-
a alone at the same concentration of
IFN- . These observations indicate that combination of 7a with the
a
current standard therapy might improve the cure rate of HCV
infection.
Finally, the effect of vitamin E on the anti-HCV activity of 7a
was evaluated. As we previously reported, vitamin E strongly
enhances HCV RNA replication.12 Here, we examined the effect of
vitamin E at various concentrations on HCV RNA replication in
the presence of 7a at the 60% inhibitory concentration level. As
shown in Figure 4, vitamin E enhanced HCV RNA replication and
dose-dependently blocked the anti-HCV activity of 7a. We specu-
late that the 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline
analogs synthesized in this study have nuclear receptor-modulat-
ing activity, since structurally similar T0901317 is known to be a
modulator of multiple nuclear receptors (NRs), including liver X
References and notes
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receptor (LXR),13 retinoic acid receptor-related orphan receptor
14
gamma (ROR
c)
and farnesoid X receptor (FXR).15 Modulation of
NRs produces reactive oxygen species (ROS) in various cells.16,17
Indeed, 7a dose-dependently produced ROS in the ORL8 assay sys-
tem as determined by FACS analysis (data not shown). Therefore,
we speculated that the mechanism of the anti-HCV activity of 7a
involves inhibition of HCV RNA replication by ROS produced via
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antioxidant vitamin E blocked the anti-HCV activity of 7a. Detailed
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