Novel anilinocoumarin derivatives as agents against hepatitis C virus by the induction of IFN-mediated antiviral responses

Article abstract of DOI:10.1039/c2ob26860d

The hepatitis C virus (HCV) is the main cause of progressive liver disease, leading to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Novel anilinocoumarins were synthesized, and their efficacy against HCV replication was evaluated

Full text of DOI:10.1039/c2ob26860d

Organic &
Biomolecular Chemistry
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Novel anilinocoumarin derivatives as agents against
hepatitis C virus by the induction of IFN-mediated
antiviral responses†
Cite this: Org. Biomol. Chem., 2013, 11,
1858
Huang-Kai Peng,a Wei-Chun Chen,b Jin-Ching Lee,b Shiang-Yu Yang,c
Cherng-Chyi Tzeng,d Ying-Ting Lin*b and Shyh-Chyun Yang*a,e
The hepatitis C virus (HCV) is the main cause of progressive liver disease, leading to the development of
liver cirrhosis and hepatocellular carcinoma (HCC). Novel anilinocoumarins were synthesized, and their
efficacy against HCV replication was evaluated. We demonstrated that 3-(3’,4’,5’-trimethoxyanilin-1’-yl)-
methylaminocoumarin (6) exhibited strong anti-HCV activity at protein and RNA levels at non-toxic con-
centrations, with an EC50 value of 12 0.3 μM and a selective index (SI) value of 10. Combined treatment
of compound 6 and interferon-α (IFN) or telaprevir induced a significant decrease in HCV RNA levels,
respectively. We also found that the anti-HCV replication effect of compound 6 was due to the induction
of IFN-mediated antiviral responses. This is the first report demonstrating that coumarins inhibit viral
replication through an IFN-mediated anti-viral response. Collectively, compound 6 possessed potent
activities against HCV replication and could be a new lead compound with higher selectivity and less
toxicity.
Received 21st September 2012,
Accepted 4th January 2013
DOI: 10.1039/c2ob26860d
www.rsc.org/obc
side effects and a limited sustained virological response (SVR)
of 50–70% in patients infected with HCV genotype 1.5,6 An
Introduction
The hepatitis C virus (HCV) is a major cause of liver disease effective vaccine against HCV infection has not yet been devel-
worldwide, including hepatic fibrosis, liver cirrhosis, and oped and there is an increasing need for investigating novel
hepatocellular carcinoma (HCC).1 It is an enveloped virus agents specially targeted toward anti-HCV replication. More-
belonging to the hepacivirus genus of the Flaviviridae family.2 over, drug resistance is a serious problem in the development
The positive single-strand RNA genome flanked by unique of anti-HCV drugs because of the high rate of viral mutation
untranslated regions (UTRs) at 5′ and 3′ ends comprises an and replication.7,8 Because of the side effects and the low cure
open reading frame (ORF).3 The viral structural and nonstruc- rate of the current therapy, safer and more effective anti-HCV
tural proteins generated by post-translational modifications agents need to be discovered or developed. In the present
from the viral genome are processed through host and viral study, we analyzed the anti-HCV activities of synthesized anili-
proteases.4 At present, a combination of pegylated interferon-α nocoumarin derivatives using the HCV replicon system.
(PEG-IFN-α) and ribavirin is the standard clinical therapy for
Most compounds and their pharmacophores have been
HCV-infected patients. However, it is associated with severe designed from natural products during drug discovery.9 In par-
ticular, the broad biological activities of coumarin derivatives,
such as anti-tumor and anti-platelet activities and as oral anti-
thrombotic agents, have been studied extensively.10,11 Recently,
researchers have focused on the anti-viral activities of cou-
marin derivatives. For example, synthetic analogs from natural
products, such as derivatives of novobiocin, clorobiocin,12 and
khellactone, were shown to have potent activities against
human immunodeficiency virus (HIV).13 Similarly, heterobi-
cycle-coumarin conjugated compound I with the –SCH2–
linker was synthesized and found to possess significant anti-
viral activities.14 Studies of heterobicycle-coumarins have
continued, and reports of new anti-HCV agents based on cou-
marin–purine ribofuranoside conjugate II have been published
aSchool of Pharmacy, College of Pharmacy, Kaohsiung Medical University,
Kaohsiung 807, Taiwan. E-mail: scyang@kmu.edu.tw; Fax: +886 7 3210683;
Tel: +886 7 3121101 ext 2662
bDepartment of Biotechnology, College of Life Science, Kaohsiung Medical University,
Kaohsiung 807, Taiwan. E-mail: ytlin@kmu.edu.tw
cSchool of Medicine, College of Medicine, Kaohsiung Medical University,
Kaohsiung 807, Taiwan
dDepartment of Medicinal and Applied Chemistry, College of Life Science,
Kaohsiung Medical University, Kaohsiung 807, Taiwan
eDepartment of Fragrance and Cosmetic Science, College of Pharmacy,
Kaohsiung Medical University, Kaohsiung 807, Taiwan
†Electronic supplementary information (ESI) available: 1H NMR and 13C NMR
spectra for compounds 5–15. See DOI: 10.1039/c2ob26860d
1858 | Org. Biomol. Chem., 2013, 11, 1858–1866
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(Fig. 1).15 Studies of anti-HCV inhibitors containing a hetero- magnetic resonance and confirmed by high-resolution mass
cyclic moiety showed that they exhibited good anti-viral spectrometry and elemental analyses.
activity. However, reports on the inhibitory effects of anilino-
Anilinocoumarins suppressed HCV RNA replication
coumarin derivatives on HCV RNA replication are lacking. This
dearth of information prompted us to design and synthesize
novel lead structures with an aniline moiety conjugated with a
natural coumarin pharmacophore to obtain anilinocoumarin
derivatives with anti-HCV activity.
The new anilinocoumarin derivatives, compounds 5–15, were
evaluated for anti-HCV replication activity against Ava5 cells at
0.5–200 μM. Total cellular RNA of compound-treated cells was
subjected to quantitative RT-PCR (qRT-PCR) with specific
primers corresponding to HCV RNA after 3 days. All the
reported compounds were evaluated for cytotoxicity by the
MTS assay. The results for anilinocoumarin derivatives are
summarized in Table 1. In the present study of the variety of
functional groups of anilinocoumarins, compounds 6, 7, 9, 10,
and 11 exhibited anti-HCV activity at <50 μM. Compounds 5, 8,
9, 10, 11, 13, 14, and 15 were found to be less cytotoxic
(>150 μM). The concentration of the compound at which cell
viability was reduced by 50% (CC50), and the concentration of
the compound at which HCV RNA replication was reduced by
50% (EC50) were determined. Values of the selective index (SI;
CC50/EC50) for compounds 6, 7, 9, and 10 were greater than
those for the other compounds. In view of the above-men-
tioned findings, 3-(3′,4′,5′-trimethoxyanilin-1′-yl)methylamino-
coumarin (6) showed the highest SI value of 10 in this series of
Results and discussion
Synthesis of anilinocoumarins
Various syntheses of coumarin derivatives have been
developed.16–20 After careful consideration of the previous
work on the syntheses of coumarin derivatives, we used the
efficient and chemoselective methodology for the preparation
of 3-substituted coumarins.21,22 The reaction mixture was
stirred at room temperature for 14 days with a mixture of
2-hydroxybenzaldehydes (1), t-butyl acrylate (2), and DABCO.
The reaction mixture yielded the corresponding Baylis–
Hillman adduct 3 (40% yields). The reaction of the adduct
with hydrochloric acid and acetic acid at 110 °C then afforded
the 3-(chloromethyl)coumarin 4 in an excellent yield (95%).
Compound 4 was then reacted with various anilines to gene-
rate compounds 5–15 by simple amination. The preparation of
anilinocoumarin derivatives is described in Scheme 1. The
chemical structure was identified by 1H and 13C nuclear
Table 1 Anti-HCV activity of anilinocoumarin derivatives
HCV
Compound
CC50a (μM)
EC50b (μM)
SIc
5
6
7
8
R = H
R = 3′,4′,5′-triOMe
R = 4′-OMe
R = 2′,4′-diMe
R = 2′,6′-diMe
R = 3′-OH
R = 4′-Br, 2′-Cl
R = 4′-COOC2H5
R = 3′-NO2
>200
120
110
>200
152
200
152
128
>200
173
188
90
12
20
>100
24
50
<3
10
5.5
—
6.5
4
3.3
<3
9
10
11
12
13
14
15
45
100
>100
100
85
—
<3
<3
R = 3′,5′-diNO2
R = 4′-Cl, 2′-Me
a Concentration of the compound at which cell viability was reduced
by 50% when compared to the 0.1% DMSO-treated cells, which served
a
mock control. b Inhibitory concentration that reduced viral
Fig. 1 Structures of newly reported anti-HCV compounds and the design
concept of anlinocoumarin derivatives. EWG, electron-withdrawing groups.
EDG, electron-donating groups.
as
replication by 50%. c The selective index was calculated as the ratio of
CC50 versus EC50
.
Scheme 1 The synthesis of anilinocoumarin derivatives. DABCO, 1,4-diazabicyclo[2.2.2]octane.
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anilinocoumarin derivatives examined for their anti-HCV blot analyses with anti-HCV NS5B and anti-GAPDH antibodies,
activity.
which represented the viral protein synthesis and loading
control, respectively. Reduced viral protein expression was cor-
related with increasing concentrations of compound
6
Structure–activity relationship (SAR) of anilinocoumarins
(Fig. 2A). The decrease in HCV RNA replication by compound
6 was dose dependent in both replicon and infectious systems
(Fig. 2B and C). The compound exhibited an EC50 value of 12
0.3 μM and was not cytotoxic. We concluded that compound 6
was a potent lead compound against HCV. In addition, the
inhibition of HCV replication by compound 6 was consistent
with that of the reference compounds, heterobicycle-cou-
marins, which prove the essential role of the pharmacophoric
element of the coumarin ring.
Based on EC50 and CC50 activities, it was intriguing to observe
different substituents (OMe, Me, OH, Cl, Br, NO2, and
COOC2H5) placed at various positions on the phenyl ring.
These substituents and their positions affect anti-HCV activity,
especially the SI value. Overall, substitution of electron-donat-
ing groups such as OMe, Me, and OH on the phenyl ring
resulted in a range of SI values of 4–10. Moreover, addition of
electron density to the phenyl ring by introducing a strong
electron-withdrawing group, such as Cl or NO2, produces a
lower SI value (<3.3). We concluded the brief SAR study by
scrutinizing the EC50, CC50, and SI values of anilinocoumarin
derivatives. Substituents bearing a strong electron-donating
motif on the phenyl ring were crucial for anti-HCV activity. In
addition, more substituents on the phenyl ring are important
because they resulted in a decrease in potency decreased in
the order of compound 10 > compound 7 > compound 9 >
compound 6 (Table 1).
Compound 6 enhanced expression of IFN response genes
Anti-HCV drugs can be developed focusing on three specific
targets: the internal ribosome entry site (IRES), NS3/4A pro-
tease activity, and NS5B polymerase.23 To evaluate the anti-
HCV action of compound 6, we tested the inhibitory effect of
compound 6 on these three specific steps of the HCV replica-
tion cycle using several reporter-based assays. No significant
inhibition of IRES-mediated translation, NS3/4A protease, or
NS5B polymerase activities was observed by compound 6 treat-
Compound 6 reduced HCV protein synthesis and RNA levels
To study the reduction of HCV protein synthesis and RNA ment (data not shown). We further tested whether compound
levels by compound 6, quantification of HCV RNA and western 6 regulated cellular factors to interfere with HCV replication.
blotting were performed. After incubation of compound 6 in The IFN-mediated anti-viral pathway is an effective cellular
Ava5 cells for 4 days, the treated cells were lysed for western defense against HCV replication.24 To investigate whether
Fig. 2 Inhibition of HCV replication and protein expression in Ava5 cells by compound 6. Ava5 cells were incubated with compound 6 at the indicated concen-
tration (from 2.5 to 150 μM). (A) After four days of incubation, cell lysates were extracted and analyzed by western blotting with anti-NS5B and anti-GAPDH anti-
bodies (a loading control). (B) After three days of incubation, total RNA was extracted from the Ava5 cells to quantify HCV RNA levels by RT-qPCR. Cell viability was
performed simultaneously by the MTS assay. (C) After Huh7 cells were infected with the concentrated virus for 8 hours, the infected cells were treated with com-
pound 6 at the indicated concentrations for 3 days. Total RNA was extracted from the cells to quantify HCV RNA levels by RT-qPCR. The non-linear regression graphs
and an effective concentration of 50% inhibition (EC50) were calculated by the GraphPad Prism5.0 program (Version 5.0, GraphPad Software, Inc. La Jolla, CA). Mock
indicates the treatment with 0.1% DMSO. Results are expressed as means SD (error bar) for triplicate experiments. *P < 0.05; **P < 0.01.
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Synergistic inhibitory effects of compound 6 when used in
combination with IFN, telaprevir, BMS790052 or PSI7977
A combination of PEG-IFN-α with ribavirin is the current
standard therapy against HCV. However, this treatment is
associated with a low cure rate and severe side effects.5 In
addition, direct-acting antiviral (DAA) drugs of HCV attenuated
the sustained viral response rates by approximately 79% in
patients with HCV genotype 1.25 Based on these findings, we
foresaw that compound 6 could be a potent anti-HCV agent in
clinical use if it demonstrates enhanced anti-HCV replication
activity when combined with IFN-α or telaprevir, a currently
used FDA approved protease inhibitor. Besides, we investigated
whether the compound 6 enhanced the anti-HCV replication
combined the other DAA drugs, such as phase 2 oral anti-viral
regiments of HCV polymerase inhibitor, PSI7977 and NS5A
replication complex inhibitor, BMS790052.26,27 To evaluate the
effect of combination treatment of compound 6 with IFN-α,
telaprevir, BMS790052, or PSI7977 on anti-HCV replication
activity, we treated Ava5 cells with various concentrations of
compound 6 and IFN-α or telaprevir at fixed ratios as described
in the materials and methods section. Combination treatment
of compound 6 with IFN-α, telaprevir, BMS790052, or PSI7977
(Fig. 4) increased the reduction of HCV RNA levels (lanes 6–9)
compared with the DMSO vehicle control (lane 1) and treat-
ment with each agent alone (lanes 2–5). To determine the
effects of combination treatment, we used the isobologram
method and the CalcuSyn™ software. CI values of compound
6 combined with IFN, telaprevir, BMS790052 or PSI7977 were
0.8, 0.5, 0.4 and 0.6, respectively. These results strongly
supported the notion that compound 6 could be a good
adjuvant to treat HCV infection.
Fig. 3 Induction of IFN response genes expression in Ava5 cells with com-
pound 6. Ava5 cells were incubated with compound 6 at the indicated concen-
trations (5, 10 and 20 μM). (A) Ava5 cells were transiently transfected with
1.0 μg of the reporter plasmid containing an interferon-stimulated response
element, pISRE-Luc. Subsequently, the transfected cells were treated with the
indicated concentrations of compound 6 for 3 days, and total cell lysates were
analyzed for luciferase activity. (B) Ava5 cells were treated with the indicated
concentrations of compound 6 for 3 days, and total RNA extracts were analyzed
by RT-qPCR to quantify the interferon response genes (OAS1, OAS2, OAS3, and
PKR) RNA expression normalized by cellular GAPDH mRNA. Mock indicates the
treatment with 0.1% DMSO. Results are expressed as means SD (error bar) for
triplicate experiments. *P < 0.05; **P < 0.01.
Further study to prevent HCC
A strategy for preventing chronic HCC in HCV infection was
described by Okamoto et al.28 Previous studies have shown
that coumarin compounds, i.e., osthole, imperatorin, and
furanocoumarins, which are isolated from plants or chemically
synthesized, decrease the levels of plasma alanine aminotrans-
ferase (ALT) that correlates with HCC etiology. Oral adminis-
tration of one of these coumarins, osthole, has been observed
to cause decrease in plasma ALT levels in mice with concana-
valin A-induced hepatitis and anti-Fas antibody-induced
hepatitis. Interestingly, compound 6 is similar to osthole
and may prevent HCC by a different mechanism. Osthole
was observed to normalize plasma ALT levels, and compound
6 is the first discovered coumarin-like compound known
to induce the IFN-mediated anti-viral pathway in HCV replica-
tion. The results of the present study will form the basis of
further study for prevention of the development of HCC in the
future.
compound 6 induced activation of the IFN pathway, we per-
formed a reporter assay using transient Ava5 cells with a firefly
luciferase reporter gene containing the interferon-stimulated
response element (ISRE) to verify the level of induction of the
IFN pathway by compound 6. The Ava5 cells transfected with
pISRE-Luc and treated with compound 6 at various concen-
trations were used in the luciferase assay.
Compound 6 significantly induced IFN responses by 3.6-
fold at 20 μM compared with the mock control (Fig. 3A). Next,
we identified whether the IFN response genes (e.g., 2′-5′-oligo-
adenylate synthetase (OAS) family and protein kinase R (PKR))
were also induced by compound 6. The induction level of
these genes was analyzed by qRT-PCR in the presence of
different concentrations of compound 6. Compound
6
induced an approximate 3-fold induction of expression of
these genes at 20 μM compared with the mock control
(Fig. 3B). These results were comparable with the reporter-
based assay and indicated that the anti-HCV mechanism of
Conclusions
compound 6 may contribute to enhancement of the IFN- In the present study of new potent anti-HCV inhibitors, we syn-
mediated anti-viral pathway.
thesized anilinocoumarin derivatives and evaluated their anti-
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HCV activities. Our results indicated that replacement of a
strong electron-donating substituent such as OMe on the
2-anilinocoumarin pharmacophore is a promising characteristic
for HCV inhibition. The most potent derivative, 3-(3′,4′,5′-tri-
methoxyanilin-1′-yl)methylaminocoumarin (6), exhibited anti-
HCV activity with an EC50 value of 12 μM and an SI value of
10. Compound 6 also showed synergistic effects on HCV repli-
cation when combined with IFN or telaprevir. This is the first
report detailing the mechanism of action of anilinocoumarins,
in anti-HCV replication through IFN responses. Compound 6
targeted host factors of the IFN pathway. Hence, it is a promis-
ing strategy for circumventing drug resistance and reducing
differential responses according to HCV virus mutations and
different genotypes. We found a novel molecular mechanism
regulated by anilinocoumarin derivatives in liver cells. We
suggest that compound 6 could be a lead compound in anti-
HCV therapy.
Experimental section
General
All melting points were uncorrected. IR absorption spectra
were recorded on a Perkin-Elmer System 2000 FT-IR spectro-
photometer. Proton and carbon-13 NMR spectra were
measured with a Unity Plus-400 or a Mercury Plus-400 spec-
trometer. Carbon multiplicities were obtained from DEPT
experiments. Chemical shifts (δ) and coupling constants (Hz)
were measured with respect to TMS or chloroform-d1. MS and
high-resolution mass spectra (HRMS) were taken on a Thermo-
Finnigan TRACE GC, Waters micromassZQ or JEOL JMS-700
instrument, with a direct inlet system. Elemental analyses were
carried out on the elementar vario EL III elemental analyzer.
General procedure for the synthesis of 3-(anilin-1′-yl)-
methylaminocoumarins 5–15
2-Hydroxybenzaldehyde (1) was reacted with t-butyl acrylate (2)
in the presence of DABCO to afford the corresponding Baylis–
Hillman adduct 3 in 40% yield. Reaction of the adduct with
hydrochloric acid in refluxing acetic acid then afforded the
3-(chloromethyl)coumarin 4 in excellent yield (95%). The
compound 4 was then reacted with various anilines by amin-
ation for an appropriate time. After the completion of the reac-
tion (TLC monitoring), the reaction mixture was neutralized
with saturated NaHCO3 until pH 7 and then partitioned
between H2O (50 mL) and CH2Cl2 (50 mL × 3). The organic
layer was washed with brine, dried over MgSO4, and evapor-
ated in vacuo. The resulting residue was purified by column
chromatography (hexane–ethyl acetate) and crystallized from
EtOH to give 5–15.
Fig. 4 Enhancement inhibition of the compound 6 combined treatment with
IFN, telaprevir, BMS790052, or PSI7977 in HCV replicon cells. Ava5 cells were
treated with the indicated concentration of compound 6 and IFN, telaprevir,
BMS790052, or PSI7977 for 3 days. The total RNA was extracted and quantified
by RT-qPCR analysis. The relative percentage of HCV RNA levels was compared
with the cells incubated with 0.1% DMSO (mock control). The errors reflect the
SD from triplicate experiments. Results are expressed as means SD (error bar)
for triplicate experiments. *P < 0.05; **P < 0.01.
tert-Butyl-3-hydroxy-3-(2-hydroxyphenyl)-2
methylenepro-
panoate (3). Yield: 100 mg, 40%. Mp: 108–110 °C (lit.,22
108–110 °C). 1H NMR (400 MHz, CDCl3) δ: 1.51 (s, 9H,
CH3 × 3), 4.38 (d, J = 4.0 Hz, 1H, OH), 5.49 (s, 1H, vinyl H),
5.69 (d, J = 4.0 Hz, 1H, CH), 6.84 (ddd, J = 1.2, 7.2, 7.6 Hz, 1H,
ArH), 6.91 (dd, J = 1.2, 8.0 Hz, 1H, ArH), 6.97 (dd, J = 1.6,
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7.2 Hz, 1H, ArH), 7.21 (ddd, J = 1.6, 7.6, 8.0 Hz, 1H, ArH), 122, 115, 107, 79. EI-HRMS calcd for C17H15NO3: 281.1052,
8.15 (s, 1H, OH). 13C NMR (100 MHz, CDCl3) δ: 28.0 (CH3 × 3), found: 281.1049. Anal. Calcd for C17H15NO3: C, 72.58; H, 5.37;
73.7 (CH), 82.6 (C), 117.5 (CH), 119.8 (CH), 124.1 (C), 127.0 N, 4.98. Found: C, 72.71; H, 5.50; N, 4.68.
(CH2), 127.8 (CH), 129.5 (CH), 140.8 (C), 156.0 (C), 166.8 (C).
3-(2′,4′-Dimethylanilin-1′-yl)methylaminocoumarin (8). Yield:
EIMS: m/z 250 (M+), 194, 176, 148, 131, 121, 105, 91, 77, 65, 55, 206 mg, 74%. Mp: 146–148 °C. IR (KBr) ν: 3422, 2917, 2394,
1
50, 41.
1711, 1610, 1515 cm−1. H NMR (400 MHz, CDCl3) δ: 2.22 (s,
3-(Chloromethyl)coumarin (4). Yield: 184 mg, 95%. Mp: 6H, CH3 × 2), 4.13 (bs, 1H, NH), 4.35 (s, 2H, CH2), 6.43 (d, J =
1
113–115 °C (lit.,22 108–110 °C). H NMR (400 MHz, CDCl3) δ: 8.4 Hz, 1H, ArH), 6.88 (d, J = 8.4 Hz, 1H, ArH), 6.93 (s, 1H,
4.56 (d, J = 0.8 Hz, 2H, CH2), 7.32 (ddd, J = 0.8, 6.4, 8.8 Hz, 1H, ArH), 7.24 (dd, J = 7.2, 7.6 Hz, 1H, ArH), 7.34 (d, J = 8.4 Hz, 1H,
ArH), 7.36 (dd, J = 0.8, 8.8 Hz, 1H, ArH), 7.54 (ddd, J = 1.2, 6.4, ArH), 7.42 (dd, J = 1.6, 7.6 Hz, 1H, ArH), 7.48 (ddd, J = 1.6, 7.2,
7.6 Hz, 1H, ArH), 7.57 (dd, J = 1.2, 7.6 Hz, 1H, ArH), 7.90 (s, 8.4 Hz, 1H, ArH), 7.65 (s, 1H, ArH). 13C NMR (100 MHz,
1H, ArH). 13C NMR (100 MHz, CDCl3) δ: 41.0 (CH2), 116.6 CDCl3) δ: 17.5 (CH3), 20.3 (CH3), 43.9 (CH2), 110.2 (CH), 116.5
(CH), 118.8 (C), 124.7 (CH), 125.0 (C), 128.0 (CH), 132.0 (CH), (CH), 119.2 (C), 122.5 (C), 124.4 (CH), 126.5 (C), 126.9 (C),
141.1 (CH), 153.5 (C), 160.1 (C). EIMS: m/z 196 (M+ + 2), 194 127.4 (CH), 127.7 (CH), 131.0 (CH), 131.2 (CH), 138.5 (CH),
(M+), 159, 131, 115, 103, 89, 77, 63, 51.
142.8 (C), 153.1 (C), 161.3 (C). ESIMS: m/z 280 (M + H)+, 262,
3-(Anilin-1′-yl)methylaminocoumarin (5). Yield: 170 mg, 190, 158, 132, 121, 115, 106, 103, 117, 103. EI-HRMS calcd for
68%. Mp: 173–175 °C. IR (KBr) ν: 3406, 2671, 2359, 1906, 1700, C18H17NO2: 279.1259, found: 279.1258. Anal. Calcd for
1510 cm−1 1H NMR (400 MHz, CDCl3) δ: 4.31 (s, 2H, CH2), C18H17NO2: C, 77.40; H, 6.13; N, 5.01. Found: C, 77.08; H, 6.09;
.
4.36 (bs, 1H, NH), 6.64 (dd, J = 1.2, 8.8 Hz, 2H, ArH), 6.74 (tt, N, 4.85.
J = 1.2, 7.6 Hz, 1H, ArH), 7.18 (dd, J = 7.6, 8.8 Hz, 2H, ArH),
3-(2′,6′-Dimethylanilin-1′-yl)methylaminocoumarin (9). Yield:
7.24 (ddd, J = 1.2, 7.6, 7.6 Hz, 1H, ArH), 7.33 (dd, J = 1.2, 195 mg, 70%. Mp: 106–108 °C. IR (KBr) ν: 3393, 3046, 2930,
8.4 Hz, 1H, ArH), 7.49 (ddd, J = 1.2, 7.2, 8.4 Hz, 1H, ArH), 7.68 1713, 1611 cm−1 1H NMR (400 MHz, CDCl3) δ: 2.31 (s, 6H,
.
(s, 1H, ArH). 13C NMR (100 MHz, CDCl3) δ: 43.7 (CH2), 113.0 CH3 × 2), 3.60 (bs, 1H, NH), 4.07 (s, 2H, CH2), 6.84 (t, J =
(CH), 116.5 (CH), 118.1 (CH), 119.2 (C), 124.4 (CH), 126.2 (C), 7.2 Hz, 1H, ArH), 7.00 (d, J = 7.2 Hz, 2H, ArH), 7.27 (ddd, J =
127.7 (CH), 129.4 (CH), 131.1 (CH), 138.6 (CH), 147.2 (C), 153.1 1.2, 7.2, 7.6 Hz, 1H, ArH), 7.34 (dd, J = 1.2, 8.4 Hz, 1H, ArH),
(C), 161.3 (C). ESIMS: m/z 252 (M + H)+, 191, 159, 131, 114, 7.44 (dd, J = 1.6, 7.6 Hz, 1H, ArH), 7.50 (ddd, J = 1.6, 7.2,
102, 96. EI-HRMS calcd for C16H13NO2: 251.0946, found: 8.4 Hz, 1H, ArH), 7.68 (s, 1H, ArH). 13C NMR (100 MHz,
251.0944. Anal. Calcd for C16H13NO2: C, 76.48; H, 5.21; N, CDCl3) δ: 18.4 (CH3 × 2), 47.8 (CH2), 116.6 (CH), 119.2 (C),
5.57. Found: C, 76.26; H, 5.27; N, 5.38.
122.5 (CH), 124.4 (CH), 127.3 (C), 127.7 (CH), 128.9 (CH), 130.0
3-(3′,4′,5′-Trimethoxyanilin-1′-yl)methylaminocoumarin (2C), 131.1 (CH), 139.1 (CH), 144.9 (C), 153.3 (C), 161.6 (C).
(6). Yield: 279 mg, 82%. Mp: 134–136 °C. IR (KBr) ν: 3392, ESIMS: m/z 280 (M + H)+, 190, 158, 132, 121, 115, 106, 104.
1
3054, 2936, 1713, 1605, 1511 cm−1. H NMR (400 MHz, CDCl3) EI-HRMS calcd for C18H17NO2: 279.1259, found: 279.1258.
δ: 3.76 (s, 3H, OCH3), 3.80 (s, 6H, OCH3 × 2), 4.20 (bs, 1H, Anal. Calcd for C18H17NO2: C, 77.40; H, 6.13; N, 5.01. Found:
NH), 4.29 (s, 2H, CH2), 5.89 (s, 2H, ArH), 7.28 (ddd, J = 1.2, 7.2, C, 77.36; H, 6.24; N, 4.90.
7.6 Hz, 1H, ArH), 7.35 (dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.46 (dd,
3-(3′-Hydroxyanilin-1′-yl)methylaminocoumarin (10). Yield:
J = 1.2, 7.6 Hz, 1H, ArH), 7.51 (ddd, J = 1.2, 7.2, 8.4 Hz, 1H, 202 mg, 76%. Mp: 133–135 °C. IR (KBr) ν: 3404, 3061, 2925,
1
ArH), 7.71 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) δ: 44.0 1697, 1604, 1502 cm−1. H NMR (400 MHz, acetone-d6) δ: 4.21
(CH2), 56.0 (OCH3 × 2), 61.6 (OCH3), 90.8 (CH × 2), 116.5 (CH), (d, J = 1.6 Hz, 2H, CH2), 5.50 (bs, 1H, NH), 6.13 (dd, J = 2.0, 8.0
119.1 (C), 124.5 (CH), 126.4 (C), 127.7 (CH), 130.6 (C), 131.2 Hz, 1H, ArH), 6.17 (t, J = 2.0 Hz, 1H, ArH), 6.19 (dd, J = 2.0,
(CH), 138.8 (CH), 144.0 (C), 153.1 (C), 154.1 (C × 2), 161.3 (C). 8.4 Hz, 1H, ArH), 6.89–6.93 (m, 1H, ArH), 7.30 (ddd, J = 1.2,
ESIMS: m/z 342 (M + H)+, 310, 279, 183, 168, 159, 140, 115, 7.2, 8.0 Hz, 1H, ArH), 7.34 (dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.56
109, 97. EI-HRMS calcd for C19H19NO5: 341.1263, found: (ddd, J = 1.2, 7.2, 8.4 Hz, 1H, ArH), 7.63 (dd, J = 1.2, 8.0 Hz,
341.1265. Anal. Calcd for C19H19NO5: C, 66.85; H, 5.61; N, 1H, ArH), 7.82 (s, 1H, ArH), 7.95 (s, 1H, OH). 13C NMR
4.10. Found: C, 66.98; H, 5.37; N, 4.05.
(100 MHz, CDCl3) δ: 44.4 (CH2), 101.2 (CH), 106.2 (CH), 117.6
3-(4′-Methoxyanilin-1′-yl)methylaminocoumarin (7). Yield: (CH), 121.0 (C), 125.9 (CH), 128.6 (C), 129.5 (CH), 131.0 (C),
219 mg, 78%. Mp: 129–131 °C. IR (KBr) ν: 3400, 2925, 2060, 131.3 (CH), 131.4 (CH), 132.5 (CH), 139.4 (CH), 151.2 (C), 154.7
1
1710, 1610, 1513 cm−1. H NMR (400 MHz, CDCl3) δ: 3.73 (s, (C), 161.9 (C). ESIMS: m/z 268 (M + H)+, 249, 228, 190, 158,
3H, OCH3), 4.12 (bs, 1H, NH), 4.26 (s, 2H, CH2), 6.60 (d, J = 121, 116, 84, 73. EI-HRMS calcd for C16H13NO3: 267.0895,
8.8 Hz, 2H, ArH), 6.77 (d, J = 8.8 Hz, 2H, ArH), 7.25 (ddd, J = found: 267.0896. Anal. Calcd for C16H13NO3: C, 71.90; H, 4.90;
1.2, 7.2, 7.6 Hz, 1H, ArH), 7.33 (dd, J = 1.2, 8.4 Hz, 1H, ArH), N, 5.24. Found: C, 71.77; H, 5.00; N, 5.15.
7.43 (dd, J = 1.6, 7.6 Hz, 1H, ArH), 7.49 (ddd, J = 1.6, 7.2,
8.4 Hz, 1H, ArH), 7.68 (s, 1H, ArH). 13C NMR (100 MHz, (11). Yield: 360 mg, 57%. Mp: 137–139 °C. IR (KBr) ν: 3423,
CDCl3) δ: 44.6 (CH2), 55.7 (OCH3), 114.4 (CH), 115.0 (CH), 2069, 1637, 1501 cm−1 1H NMR (400 MHz, CDCl3) δ: 4.37
3-(4′-Bromo-2′-methylanilin-1′-yl)methylaminocoumarin
.
116.5 (CH), 119.2 (C), 124.4 (CH), 126.6 (C), 127.7 (CH), 131.0 (d, J = 6.0 Hz, 2H, CH2), 4.97 (t, J = 6.0 Hz, 1H, NH), 6.43 (d, J =
(CH), 138.7 (CH), 141.3 (C), 152.5 (C), 135.1 (C), 161.3 (C). 8.8 Hz, 1H, ArH), 7.17 (dd, J = 2.4, 8.8 Hz, 1H, ArH), 7.27 (ddd,
ESIMS: m/z 282 (M + H)+, 266, 226, 190, 158, 136, 134, 130, J = 1.2, 7.2, 7.6 Hz, 1H, ArH), 7.35 (dd, J = 1.2, 8.4 Hz, 1H,
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ArH), 7.42 (d, J = 2.4 Hz, 1H, ArH), 7.44 (dd, J = 1.6, 7.6 Hz, 1H,
3-(4′-Chloro-6′-methylanilin-1′-yl)methylaminocoumarin
ArH), 7.51 (ddd, J = 1.6, 7.2, 8.4 Hz, 1H, ArH), 7.58 (s, 1H, (15). Yield: 182 mg, 61%. Mp: 156–158 °C. IR (KBr) ν: 3429,
1
ArH). 13C NMR (100 MHz, CDCl3) δ: 43.0 (CH2), 108.6 (C), 2922, 2097, 1506, 1610, 1505 cm−1. H NMR (400 MHz, CDCl3)
112.5 (CH), 116.5 (CH), 118.9 (C), 120.1 (C), 124.6 (CH), 125.2 δ: 2.21 (s, 3H, CH3), 4.26 (bs, 1H, NH), 4.35 (s, 2H, CH2), 6.43
(C), 127.8 (CH), 130.7 (CH), 131.4 (CH), 131.6 (CH), 138.4 (CH), (d, J = 8.4 Hz, 1H, ArH), 7.02 (dd, J = 2.4, 8.4 Hz, 1H, ArH), 7.07
142.2 (C), 153.1 (C), 161.0 (C). ESIMS: m/z 369 (M + H + 4)+, (d, J = 2.4 Hz, 1H, ArH), 7.26 (ddd, J = 1.2, 7.2, 8.0 Hz, 1H,
367 (M + H + 2)+, 365 (M + H)+, 335, 330, 284, 267, 245, 120, ArH), 7.35 (dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.44 (dd, J = 1.6,
195, 181, 151, 120, 96, 81. EI-HRMS calcd for C16H11BrClNO2: 8.0 Hz, 1H, ArH), 7.51 (ddd, J = 1.6, 7.2, 8.4 Hz, 1H, ArH), 7.61
632.9662, found: 362.9663. Anal. Calcd for C16H11BrClNO2: C, (s, 1H, ArH). 13C NMR (100 MHz, CDCl3) δ: 17.4 (CH3), 43.8
52.70; H, 3.04; N, 3.84. Found: C, 52.67; H, 3.02; N, 3.86.
(CH2), 111.1 (CH), 116.5 (CH), 119.0 (C), 122.2 (C), 124.2 (C),
3-(4′-Ethoxycarbonylanilin-1′-yl)methylaminocoumarin 124.5 (CH), 125.8 (C), 126.8 (CH), 127.8 (CH), 130.1 (CH), 131.3
(12). Yield: 203 mg, 63%. Mp: 179–181 °C. IR (KBr) ν: 3375, (CH), 138.7 (CH), 143.7 (C), 153.1 (C), 161.2 (C). ESIMS: m/z
2364, 1685, 1560, 1536 cm−1 1H NMR (400 MHz, CDCl3) δ: 302 (M + H + 2)+, 300 (M + H)+, 264, 191, 158, 132, 61.
.
1.35 (t, J = 7.2 Hz, 3H, CH3), 4.31 (q, J = 7.2 Hz, 2H, CH2CH3), EI-HRMS calcd for C17H14ClNO2: 299.0713, found: 299.0715.
4.37 (d, J = 4.4 Hz, 2H, CH2), 4.82 (bs, 1H, NH), 6.61 (d, J = Anal. Calcd for C17H14ClNO2: C, 68.12; H, 4.71; N, 4.67. Found:
9.2 Hz, 2H, ArH), 7.26 (ddd, J = 1.2, 7.2, 7.6 Hz, 1H, ArH), 7.34 C, 67.99; H, 4.89; N, 4.43.
(dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.43 (dd, J = 1.6, 7.6 Hz, 1H,
ArH), 7.50 (ddd, J = 1.6, 7.2, 8.4 Hz, 1H, ArH), 7.63 (s, 1H, Cell culture and reagents
ArH), 7.88 (d, J = 9.2 Hz, 2H, ArH). 13C NMR (100 MHz, CDCl3)
Huh7 cells and Ava5 cells were hapatoma cell lines cultured as
δ: 14.4 (CH3), 43.1 (CH2), 60.3 (CH2), 111.9 (CH), 116.6 (CH),
described previously.29 Interferon-α (Roferon©-A) was pur-
118.9 (C), 119.8 (C), 124.6 (CH), 125.4 (C), 127.8 (CH), 131.4
chased from Hoffmann-La Roche Inc. (Nutley, NJ, USA). Tela-
previr was purchased from Legend Star International Co., Ltd.
A series of compounds of anilinocoumarin derivatives were
(CH), 131.6 (CH), 138.8 (CH), 150.9 (C), 153.1 (C), 161.2 (C),
166.2 (C). ESIMS: m/z 324 (M + H)+, 310, 278, 222, 191, 159,
132, 115, 103, 97. EI-HRMS calcd for C19H17NO4: 323.1158,
dissolved in DMSO from Sigma-Aldrich. The final concen-
found: 323.1160. Anal. Calcd for C19H17NO4: C, 70.58; H, 5.30;
tration of DMSO in all reactions was maintained at 0.1% in all
N, 4.33. Found: C, 70.28; H, 5.45; N, 4.23.
experiments.
3-(3′-Nitroanilin-1′-yl)methylaminocoumarin (13). Yield:
177 mg, 60%. Mp: 147–149 °C. IR (KBr) ν: 3380, 2136, 1662,
Quantification of hepatitis C virus RNAs and western blotting
1420 cm−1. 1H NMR (400 MHz, DMSO-d6) δ: 4.23 (d, J = 4.8 Hz,
The standard procedure of western blotting and quantitative
2H, CH2), 4.65 (bs, 1H, NH), 6.95–6.98 (m, 1H, ArH), 7.03 (dd,
J = 2.4, 8.0 Hz, 1H, ArH), 7.32 (dd, J = 7.2, 7.6 Hz, 1H, ArH),
7.35 (d, J = 8.0 Hz, 1H, ArH), 7.38–7.42 (m, 1H, ArH), 7.44 (t,
real-time RT-PCR (qRT-PCR) was performed as described pre-
viously.28 The total RNA of treated Ava5 cells was extracted and
detected using the ABI Step One Real-Time PCR-System (ABI,
Warrington, UK) with HCV-specific primers and glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) primers; GAPDH
was used as an endogenous reference gene. The treated Ava5
cells were lysed in RIPA buffer, and the lysates were analyzed
by western blotting. The membranes were probed with an anti-
NS5B antibody (1 : 5000 dilution; Abcam, Cambridge, MA,
J = 2.4 Hz, 1H, ArH), 7.59 (ddd, J = 1.2, 7.2, 8.0 Hz, 1H, ArH),
7.70 (d, J = 8.0 Hz, 1H, ArH), 7.86 (s, 1H, ArH). 13C NMR
(100 MHz, DMSO-d6) δ: 42.0 (CH2), 106.0 (CH), 110.4 (CH),
116.0 (CH), 118.2 (CH), 118.9 (C), 124.5 (CH), 125.5 (C), 128.2
(CH), 130.1 (CH), 131.1 (CH), 138.1 (CH), 148.8 (C), 149.2 (C),
152.6 (C), 160.2 (C). ESIMS: m/z 297 (M + H)+, 279, 191, 159,
132, 116, 97. EI-HRMS calcd for C16H12N2O4: 296.0797, found:
USA) or an anti-GAPDH antibody (1 : 10 000 dilution; GeneTex,
296.0797. Anal. Calcd for C16H12N2O4: C, 64.86; H, 4.08; N,
Irvine, CA, USA). The signals were detected using an ECL
9.46. Found: C, 64.93; H, 4.26; N, 9.07.
detection kit (PerkinElmer, CT).
3-(3′,5′-Dinitroanilin-1′-yl)methylaminocoumarin (14). Yield:
204 mg, 60%. Mp: 217–219 °C. IR (KBr) ν: 3397, 2919, 2311,
Preparation of HCV derived from cell culture (HCVcc) and
quantification of hepatitis C virus RNAs
1695, 1536 cm−1 1H NMR (400 MHz, CDCl3) δ: 4.25 (d, J =
.
6.0 Hz, 2H, CH2), 6.93 (bs, 1H, NH), 7.31 (ddd, J = 1.2, 7.2,
7.6 Hz, 1H, ArH), 7.36 (dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.58 The pcDNA6/TR-Tight/JFH1-FL/AR DNA (kindly provided by
(dd, J = 1.6, 7.6 Hz, 1H, ArH), 7.61 (ddd, J = 1.6, 7.2, 8.4 Hz, Professor Takaji Wakita)30 was in vitro transcripted by a MEGA-
1H, ArH), 7.92 (d, J = 2.0 Hz, 2H, ArH), 8.00 (s, 1H, ArH), 8.10 script® Kit. The RNA was transfected into Huh7.5 cells by a
(t, J = 3.0 Hz, 1H, ArH). 13C NMR (100 MHz, CDCl3) lipofection method to produce HCVcc. The culture medium
δ: 44.2 (CH2), 106.7 (CH), 113.1 (CH), 117.7 (CH), 120.8 (C), was harvested and the virus particle concentrated using poly-
126.0 (CH), 126.7 (C), 129.7 (CH), 132.9 (CH), 140.2 (CH), ethylene glycol-8000 (Sigma-Aldrich, USA). The HCVcc infectiv-
151.1 (C), 151.9 (C), 154.9 (C), 161.8 (C). ESIMS: m/z 342 ity titer was determined by immunostaining the core protein.
(M + H)+, 296, 250, 238, 148, 121, 105. EI-HRMS calcd for The Huh7 cells were infected with the concentrated virus for
C16H11N3O6: 341.0648, found: 341.0650. Anal. Calcd for 8 h and treated with different concentrations of compounds
C16H11N3O6: C, 56.31; H, 3.25; N, 12.31. Found: C, 56.30; H, for 3 days. The total RNAs were collected and the relative virus
3.36; N, 12.31.
RNA quantified by qRT-PCR.
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Analyses of combination treatment
Notes and references
Ava5 cells were treated with diluted compound 6 (1.25, 2.5, 5,
10, and 20 μM) in combination with/without diluted IFN-α
(7.5, 15, 30 and 60 U mL−1), telaprevir (0.1, 0.2, 0.4 and
0.8 μM), BMS790052 (1, 2, 4 and 8 pM), and PSI7977 (10, 20,
40 and 80 nM) at fixed ratios. After 3 days of incubation, total
cellular RNA was collected and analyzed by qRT-PCR, using
endogenous cellular GAPDH as a loading control. Values of
the combination index (CI) were analyzed using CalcuSyn™
software according to the manufacturer’s instructions.29
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Cytotoxicity assay
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derivatives for 4 days. Cell viability was determined by the
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
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Acknowledgements
Financial support of this work by the National Science Council 22 P. T. Kaye, M. A. Musa and X. W. Nocanda, Synthesis, 2003,
of the Republic of China is gratefully acknowledged. We also 531.
thank the National Center for High-Performance Computing 23 E. A. Schaefer and R. T. Chung, Gastroenterology, 2012, 142,
for providing computer resources and chemical database ser- 1340.
vices. This work was supported by the National Science 24 I. W. Cheney, V. C. H. Lai, W. Zhong, T. Brodhag,
Council of Taiwan (grant numbers NSC101-2113-M-037-008-
MY3 and NSC 100-2311-B-037-001) and Kaohsiung Medical
S. Dempsey, C. Lim, Z. Hong, J. Y. N. Lau and R. C. Tam,
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University Research Foundation, Taiwan (KMUER014). We are 25 P. J. Pockros, Expert Opin. Biol. Ther., 2011, 11, 1611.
grateful to Dr. Charles Rice (Rockefeller University and Aapth, 26 M. J. Sofia, D. Bao, W. Chang, J. Du, D. Nagarathnam,
LCC, USA) for kindly supporting Con1b replicon plasmid,
Human hepatoma cell (Huh-7) and HCV subgenomic replicon-
containing cell line (Ava5) and Dr. T. Wakita (National Institute
of Infectious Diseases, Japan) for providing the JFH1 plasmid.
S. Rachakonda, P. G. Reddy, B. S. Ross, P. Wang,
H. R. Zhang, S. Bansal, C. Espiritu, M. Keilman, A. M. Lam,
H. M. Steuer, C. Niu, M. J. Otto and P. A. Furman, J. Med.
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