Discovery of the 2-phenyl-4,5,6,7-Tetrahydro-1 H -indole as a novel anti-hepatitis C virus targeting scaffold

Article abstract of DOI:10.1016/j.ejmech.2015.04.022

Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is now a reality, today's HCV drugs are expensive, and more affordable drugs are still urgently needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-Tetrahydro-1H-indole chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected representative set of derivatives as well as their chemical synthesis are described herein. The most potent compound 39 displayed EC₅₀ values of 7.9 and 2.6 μM in genotype 1b and 2a, respectively. Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3 helicase, IRES mediated translation and selected host factors. Thus, future work will involve both the chemical optimization and target identification of 2-phenyl-4,5,6,7-Tetrahydro-1H-indoles as new anti-HCV agents.

Full text of DOI:10.1016/j.ejmech.2015.04.022

Accepted Manuscript
Discovery of the 2-Phenyl-4,5,6,7-Tetrahydro-1H-indole as a Novel Anti-Hepatitis C
Virus Targeting Scaffold
Ivan A. Andreev, Dinesh Manvar, Maria Letizia Barreca, Dmitry S. Belov, Amartya
Basu, Noreena L. Sweeney, Nina K. Ratmanova, Evgeny R. Lukyanenko, Giuseppe
Manfroni, Violetta Cecchetti, David N. Frick, Andrea Altieri, Neerja Kaushik-Basu,
Alexander V. Kurkin
PII:
S0223-5234(15)00271-8
10.1016/j.ejmech.2015.04.022
EJMECH 7837
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 February 2015
Revised Date: 8 April 2015
Accepted Date: 9 April 2015
Please cite this article as: I.A. Andreev, D. Manvar, M.L. Barreca, D.S. Belov, A. Basu, N.L. Sweeney,
N.K. Ratmanova, E.R. Lukyanenko, G. Manfroni, V. Cecchetti, D.N. Frick, A. Altieri, N. Kaushik-
Basu, A.V. Kurkin, Discovery of the 2-Phenyl-4,5,6,7-Tetrahydro-1H-indole as a Novel Anti-
Hepatitis C Virus Targeting Scaffold, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.04.022.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Discovery of the 2-Phenyl-4,5,6,7-Tetrahydro-1H-indole as
a Novel Anti-Hepatitis C Virus Targeting Scaffold.
a,d,§
b,§
c,*
a,d
b
Ivan A. Andreev , Dinesh Manvar , Maria Letizia Barreca , Dmitry S. Belov , Amartya Basu ,
e
d
a
c
Noreena L. Sweeney , Nina K. Ratmanova , Evgeny R. Lukyanenko , Giuseppe Manfroni , Violetta
c
e
a,*
b,*
d
Cecchetti , David N. Frick , Andrea Altieri , Neerja Kaushik-Basu , Alexander V. Kurkin
a
EDASA Scientific srls., Via Stingi, 37, 66050 San Salvo (CH), Italy
b
Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers, The State University
of New Jersey, New Jersey Medical School, New Jersey 07103, USA
c
Department of Pharmaceutical Sciences, University of Perugia, Via A. Fabretti, 48, 06123 Perugia,
Italy
d
Chemistry Department of Lomonosov Moscow State University, Moscow, 119991, GSP-2,
Leninskie gory, 1/3
e
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer
St., Milwaukee, WI 53211, USA
*
Corresponding authors
E-mail addresses: lbarreca@unipg.it (M.L. Barreca), kaushik@njms.rutgers.edu (N. Kaushik-
Basu), aaltieri@edasascientific.com (A. Altieri)
Phones: +39-075-5855157 (M.L. Barreca), +1-973-972-8653 (N. Kaushik-Basu), +39 334 8572 542
(A. Altieri)
§
equal contribution
Abstract
Although all-oral direct-acting antiviral (DAA) therapy for hepatitis C virus (HCV) treatment is
now a reality, today’s HCV drugs are expensive, and more affordable drugs are still urgently
needed. In this work, we report the identification of the 2-phenyl-4,5,6,7-tetrahydro-1H-indole
chemical scaffold that inhibits cellular replication of HCV genotype 1b and 2a subgenomic
replicons. The anti-HCV genotype 1b and 2a profiling and effects on cell viability of a selected
representative set of derivatives as well as their chemical synthesis are described herein. The most
potent compound 39 displayed EC values of 7.9 and 2.6 µM in genotype 1b and 2a, respectively.
5
0
Biochemical assays showed that derivative 39 had no effect on HCV NS5B polymerase, NS3
helicase, IRES mediated translation and selected host factors. Thus, future work will involve both
the chemical optimization and target identification of 2-phenyl-4,5,6,7-tetrahydro-1H-indoles as
new anti-HCV agents.
1

ACCEPTED MANUSCRIPT
Keywords: Hepatitis C Virus, 4,5,6,7-tetrahydro-1H-indole, anti-HCV agents
2

ACCEPTED MANUSCRIPT
1. Introduction
Hepatitis C virus (HCV) infection represents a global health problem that has an associated
high risk for serious liver diseases. On the basis of annual World Health Organization (WHO)
reports, more than 130-150 million people are infected and more than 350,000-500,000 individuals
die from HCV-related liver pathologies each year [1]. To date, at least eleven HCV genotypes (gt)
have been identified. These genotypes can be divided into multiple subtypes. The global
distribution of HCV genotypes varies depending on the particular geographical area. HCV gt 1 is
the most common in North and South America, Europe and Australia [2]. HCV gt 2 is widespread
in America and Europe, while gt 3 is common in Central Asia and Middle East. Finally, HCV gt 4
and gt 5 are found almost exclusively in Africa, and HCV gt 6 is endemic in East and Southeast
Asia [2]. Gt 1 and gt 4 are the hardest to treat and are associated with a particularly aggressive form
of the disease.
HCV was discovered in 1989, and until recently all treatments included some combination of
pegylated interferon-α (pegIFN-α) and ribavirin (RBV), both of which cause debilitating side
effects often worse than HCV symptoms. PEG-IFN/RBV treatment alone has been moderately
successful and is genotype-dependent as only 40-50% of gt 1 and gt 4 patients have achieved a
sustained virological response (SVR) indicative of a cure [3].This treatment regimen remained the
standard-of-care (SOC) until 2011 for gt 1, and until 2014 for the other genotypes. Over the past 20
years, a combination of developments of new models and tools have been able to reveal the
different steps of the HCV life cycle and tremendous drug discovery efforts have allowed the
development of direct-acting antivirals (DAAs) that specifically target HCV proteins. Since 2011,
the new SOC for patients infected with gt 1 is based on a combination of pegIFN-α and RBV with
the first-generation HCV protease inhibitors telaprevir or boceprevir (Figure 1). Although the cure
rates have improved (SVR = 60-80%), the new SOC provides only limited clinical benefit against
HCV gt 2-6 and has resulted in some serious side effects in clinical trials [4, 5]. Consequently, two
new HCV DAAs, simeprevir and sofosbuvir (Figure 1), have been approved in December 2013 in
the United States and in the first half of 2014 in Europe [6-8]. Simeprevir is a second-generation
protease inhibitor that is endowed with a broader genotypic coverage (gt 1, 2 and 4). Its
combination with pegIFN-α and RBV has shown improved SVR and a better tolerance profile [6].
Sofosbuvir, the first nucleotide inhibitor of NS5B polymerase approved by FDA, has paved the way
for all-oral IFN-free therapies, two of which were approved in 2014: Viekira Pak (ombitasvir,
paritaprevir, ritonavir and dasabuvir), and Harvoni (ledipasvir and sofosbuvir) (Figure 1) [9-11].
Viekira Pak and Harvoni are both approved only for adult HCV patients with gt1 infection; they
have displayed >90% SVR and are also effective against other genotype in clinical trials.
3

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Figure 1.
There are currently many similar HCV DAAs in development, and most target the NS3
protease, NS5B polymerase and NS5A protein. They are undergoing late stages of clinical
development and are close to approval. An up-to-date status of the clinical trials along with
comprehensive overviews of the continued and discontinued HCV-specific DAAs have been
recently described [12].
The main drawback is that the newly approved drugs and/or regimens are very expensive, thus
restricting access for most HCV-infected patients to the new anti-HCV therapies. Another serious
medical issue is the high mutation rate of HCV coupled with the rapid emergence of drug resistance
to the DAAs [13-15]. These observations serve to encourage continuing research in the field of
HCV drug discovery that will lead to the identification of new antiviral agents effective against
HCV.
Thus, it is within this context that we herein report the discovery of a new chemical class of
anti-HCV compounds that have a 2-phenyl-4,5,6,7-tetrahydro-1H-indole core.
2
2
. Results and discussion
.1. Cell-based screening of EDASA compounds: Hit identification
Compounds 1-33 (Figure 2), representative chemotypes of the EDASA Scientific public compound
library (http://www.edasascientific.com/page/catalogue), have been screened for their possible anti-
HCV activity using HCV replicons based on the two most widely studied HCV genotypes (gt 1b
and gt 2a) (Table 1). All compounds, except 24, are racemates.
Gt 1b was studied for many years because it is one of the most resistant to pegIFN-α/RBV
therapy, and the gt 1b (con1) strain was used in the first subgenomic HCV replicons [16]. Gt 2a
exhibits a greater sensitivity than gt 1 to pegIFN-α/RBV treatment, but it was the first to be
replicated in a robust cell culture model [17]. Taking this into consideration for our discovery
campaign, we decided to screen the compounds against both the HCV genotypes.
The compounds were evaluated against Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A cells,
which carry the autonomously replicating HCV RNA of gt 1b and 2a in the firefly
and Renilla luciferase reporters, respectively [18]. During initial screening, the 33 EDASA
Scientific compounds were assayed at 50 µM against both the HCV replicons in reporter assays.
4

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The compounds that inhibited HCV replication by > 50% in the primary assays were then further
evaluated in concentration-response assays. The ability of each compound to inhibit activity in gt 1b
and 2a replicons, and their effect on cell viability are shown in Table 1. The selectivity index (SI)
was calculated as well to estimate the therapeutic potential of the compounds in this system. Only
two compounds (28 and 31) were found to be active against gt 1b (displaying EC values of 24.3
5
0
and 12.4 µM, respectively), although they showed poor SI (< 10). In contrast, a total of 16
compounds were active against gt 2a, with associated EC values in the range from 4.9 to 28.1 µM
5
0
and moderate to good SI. Compound 25 was the most potent among all the tested compounds and
showed EC value of 4.9 µM with a SI > 41. Interestingly, the only two compounds found to be
5
0
active on gt 1b replicons (28 and 31) were also active against gt 2a replicons and exhibited EC50
values of 6.0 and 8.7 µM, respectively, with SI values > 10.
Figure 2.
Table 1: Anti-HCV activities and cytotoxicity of the first 33 EDASA Scientific compounds evaluated on gt
1
b and 2a.
Huh7/Rep-Feo1b
Huh7.5-FGR-JC1-Rluc2A
a
CC50
µM)
Cpd
b
c
b
c
(
Inhibition,
%
EC50
d
Inhibition,
%
EC50
d
SI
SI
(µM)
(µM)
1.1 ±
.9
8.5 ±
.9
1
1
2
>200
>200
19 ± 6
22 ± 9
ND
ND
ND
80 ± 5
51 ± 8
>18
>4
0
4
2
ND
3
3
4
>200
>200
NI
NI
ND
ND
ND
ND
49 ± 6
ND
ND
ND
36 ± 10
ND
3.6 ±
5
6
7
>200
ND
49 ± 12
26 ± 1
NI
ND
ND
ND
ND
ND
ND
66 ± 7
54 ± 4
78 ± 3
>8
ND
>17
4.0
ND
1
1
1
1
2
1.7 ±
>200
0
.9
4.1 ±
.9
5.6 ±
.7
7.3 ±
.2
1.1 ±
.4
8
9
>200
>200
>200
>200
21 ± 2
19 ± 10
21 ± 2
NI
ND
ND
ND
ND
ND
ND
ND
ND
77 ± 7
72 ± 3
77 ± 7
67 ± 5
>14
>13
>12
>9
1
3
1
1
0
1
3
4
1
1
2
3
>200
>200
19 ± 8
NI
ND
ND
ND
ND
NI
ND
ND
ND
ND
14 ± 8
5

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2
0.6 ±
1
1
1
4
5
6
85.6 ± 5.9
<25
17 ± 3
88 ± 2
39 ± 4
ND
ND
ND
ND
ND
ND
65 ± 5
99 ± 1
60 ± 6
4
2.9
ND
ND
>9
2
2
2.5 ±
>200
3
.8
8.1 ±
.8
1
7
>200
NI
ND
ND
62 ± 1
>7
4
1
1
2
2
2
8
9
0
1
2
>200
>200
>200
<25
NI
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
55 ± 9
46 ± 8
29 ± 8
99 ± 1
44 ± 9
ND
ND
ND
ND
ND
ND
ND
ND
ND
NI
54 ± 8
92 ± 1
f
>200
NI
ND
2
4.8 ±
4
2
2
2
3
4
5
>200
>200
>200
45 ± 6
19 ± 9
NI
ND
ND
ND
ND
ND
ND
ND
61 ± 9
85 ± 9
73 ± 3
>8
.5
.3 ±
.5
.9 ±
.4
7.4 ±
.8
ND
7
>27
>41
0
4
0
1
2
2
2
6
7
8
>200
>200
NI
NI
ND
ND
5
76 ± 7
43 ± 10
98 ± 2
>11
ND
>19
0
ND
2
1
4.3 ±
6.0 ±
1.0
114.7± 14.6
73 ± 9
1.2
2
3
9
0
>200
>200
NI
NI
ND
ND
ND
NI
ND
ND
ND
ND
ND
28 ± 4
2.4 ±
8.7 ±
1.9
3
1
109.9 ± 2.9
66 ± 9
9
88 ± 8
13
1.0
3
3
2
3
>200
>200
50 ± 4
45 ± 2
ND
ND
ND
25 ± 5
ND
ND
ND
ND
48 ± 11
ND
a
CC50 values were determined in Huh7.5 parental cells by the MTS assay. CC50 = is the concentration required to reduce
the bioreduction of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
into formazan by 50%. The reported value represents the means ± SD of data derived from three independent experiments.
b
c
Anti-HCV activity of the compounds were carried out at 50 µM in preliminary screening. The inhibition data from 8-12
quarter log dilutions were used to generate the dose response curves. EC50 = the effective concentration required to inhibit
virus induced cytopathic effect by 50%. The reported values represent the means ± SD of data derived from three
d
independent experiments. SI: selectivity index ratio of CC50 to EC50 . ND: not determined. NI: no inhibition.
Overall, compound 31, having a 2-phenyl-4,5,6,7-tetrahydro-1H-indole scaffold, emerged as a
hit compound, displaying low cytotoxicity (CC = 109.9 µM) and promising anti-HCV activity in
5
0
replicon reporter cells of both the genotype 1b (EC50 = 12.4 µM) and 2a (EC50 = 8.7 µM).
Following on from this, we had eleven more analogues of 31 available at EDASA Scientific that we
decided to further evaluate for their anti-HCV activities (34-44, Figure 3).
Figure 3.
6

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2
.2. Synthesis of derivatives 31, 34-44
Recently, we have developed a two-step one-pot synthetic methodology, which leads to
4,5,6,7-tetrahydro-1H-indoles with a wide range of substituents, including chiral moieties, both at
C-2 and at the N-1 positions [19]. This synthetic sequence was successfully applied to achieve
derivatives 31, 34-44 (Scheme 1).
Scheme 1.
This one-pot Sonogashira cross-coupling/5-endo-dig cyclization procedure was used as a
flexible and versatile synthetic approach. Thus, the trans-stereoselective and highly regioselective
nucleophilic epoxide ring opening of 45 with different amines was followed by a subsequent one-
pot Pd-catalyzed arylation/cyclization. This short sequence allowed the variation of substituents
both at the nitrogen atom and at the C-2 position of the pyrrole ring, along with a judicial design
and a fast preparation of the most promising tetrahydroindole derivatives. Furthermore, it utilized
mild conditions and inexpensive catalysts, being highly tolerant to a range of functional groups and
readily scalable to provide sufficient amounts of tetrahydroindoles on gram scales in a good to
excellent yields to effectively assemble the tetrahydroindole compound array for further screening.
The full report on the synthetic sequence as well as compound characterization is presented in
Supporting Information.
2.3. Cell-based assays of compounds 34-44
The anti-HCV activities of the new analogues of 31 (34-44) are shown in Table 2. The cell-based
assays revealed that, out of the eleven compounds tested, eight derivatives in gt 1b and ten
compounds in gt 2a showed >60% inhibition during preliminary screening.
7

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Table 2: Anti-HCV activities and cytotoxicity of analogues of 31 evaluated on gt 1b and 2a.
a
Huh7/Rep-Feo1b
Huh7.5-FGR-JC1-Rluc2A
CC50
µM)
Cpd
(
b
c
d
b
c
d
Inhibition, % EC50 (µM) SI
Inhibition, % EC50 (µM) SI
3
3
3
3
3
3
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
>200
45.6 ± 6.1
<25
71 ± 6
81 ± 3
92 ± 1
50 ± 8
37 ± 18
95 ± 4
75 ± 7
35 ± 6
99 ± 1
96 ± 2
74 ± 4
29.2 ± 1.2
35.8 ± 3.4
ND
>7
66 ± 10
96 ± 3
99 ± 1
83 ± 3
48 ± 11
99 ± 1
98 ± 2
69 ± 2
96 ± 2
99 ± 1
95 ± 3
12.3 ± 1.0
9.9 ± 1.6
ND
>16
>5
ND
>10
ND
32
27
4
>1
ND
ND
ND
10
155.6± 11
>200
ND
15.4 ± 3.9
ND
ND
80.8 ± 3.1
>200
7.9 ± 0.5
15.0 ± 1.3
ND
2.6 ± 0.4
7.3 ± 1.4
32.1 ± 4.1
4.9 ± 0.3
6.5 ± 0.6
13.7 ± 2.1
13
118.8 ± 2.8
137.4 ± 1.0
48.9 ± 1.7
84.3 ± 1.3
ND
12
11.8 ± 0.6
9.2 ± 0.6
13.2 ± 1.4
28
8
5
6
6
a
CC50 values were determined in Huh7.5 parental cells by the MTS assay. CC50 = is the concentration required to reduce
the bioreduction of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
into formazan by 50%. The reported value represents the means ± SD of data derived from three independent experiments.
b
c
Anti-HCV activity of the compounds were carried out at 50 µM in preliminary screening. The inhibition data from 8-12
quarter log dilutions were used to generate the dose response curves. EC50 = the effective concentration required to inhibit
virus induced cytopathic effect by 50%. The reported values represent the means ± SD of data derived from three
d
independent experiments. SI: selectivity index ratio of CC50 to EC . ND: not determined.
50
All these compounds except one were then evaluated for their EC and SI values; in fact,
5
0
derivative 36 exerted its HCV replication inhibition at toxic concentration (CC < 25 µM) and thus
50
it was not submitted to EC evaluation.
50
Taking into account the obtained biological data (Table 2), some preliminary SAR can be
proposed for this new class of anti-HCV agents.
Derivatives 39, 40 and 42, all having a N-benzyl substitution at the tetrahydroindole core,
showed the higher anti-HCV activities with SI values ranging from 10 to 13 for gt 1b, and from 27
to 32 for gt 2a. Among them, compound 39 was found to be the most potent in both gts displaying
EC50 values of 7.9 µM (1b) and 2.6 µM (2a). Compared to 39, derivative 34, having a para-
fluorophenyl group at the nitrogen atom, showed a nearly 3.7 and 4.7 fold reduction in anti-HCV
activity for gt 1b and 2a replicon reporter assays, respectively. Furthermore, when the N-benzyl
substituent of the tetrahydroindole nucleus was replaced with non-aromatic groups, the anti-HCV
activity on gt 1b was completely lost (37, 38, and 41) or a non selective antiviral effect (i.e. low SI
value) was obtained (44); the analysis on gt 2a provided similar conclusions with the exception of
derivative 37 which turned out to be active.
8

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When analyzing the biological data for the whole subset of N-benzyl derivatives (i.e.
compounds 31, 35, 39, 40, 42 and 43), the key role of the aryl substituent at the C-2 position
became evident. An unsubstitued phenyl (39) as well as a para-substituted phenyl (i.e. 31: NH , 40:
2
NO and 42: OCH ) were both well tolerated; conversely, the presence of either meta-disubstituents
2
3
(35) or ortho-OH (43) substituent led to compounds endowed with high toxicity. Moreover, the
replacement of the phenyl (39) with a 3-pyridinyl ring (36) was also responsible for the increased
cytoxicity (CC = 80.8 µM vs CC < 25 µM, respectively).
5
0
50
In order to further validate the anti-HCV activity of these compounds, hit 39 was selected
and tested as a representative candidate against a reporter free cell culture system. To achieve this,
we treated MH-14 cells carrying stably replicating HCV sub genomic replicon gt 1b with compound
39 and the HCV RNA was quantitated using standard quantitative RT-PCR methods. Notably, 39
inhibited the HCV replication in a dose-dependent manner and exhibited EC value of 3.13 µM
50
(
Figure 4), which was quite similar to the value obtained in the replicon reporter cells (i.e. EC =
50
7.9 µM).
Overall, the results clearly indicated that promising anti-HCV activity coupled with no
apparent cytotoxic effects were obtained when the 2-phenyl-4,5,6,7-tetrahydro-1H-indole scaffold
was properly functionalized.
Figure 4.
2
3
.4. Molecular target investigation
Next, we carried out target investigation for the most active tetrahydro-1H-indoles (i.e., 31, 34,
9, 40 and 42). Towards this end, we tested the compounds for their ability to inhibit the activity of
two HCV viral proteins, i.e. NS5B polymerase and NS3 helicase. These two targets were chosen as
first choice because indole derivatives have been reported in literature as both HCV NS5B
polymerase and NS3 helicase inhibitors [20, 21].
We utilized a standard primer-dependent elongation assay to test whether the compounds
possessed anti-NS5B RNA-dependent RNA polymerase (RdRp) activity [22, 23]. The compounds
were investigated at 50 µM concentration in the preliminary assay. The results clearly revealed that
9

ACCEPTED MANUSCRIPT
none of the compounds was inhibitory to NS5B RdRp activity (data not shown), thus ruling out the
possibility of possessing anti-HCV activity by targeting this protein.
The five compounds were also tested in HCV NS3 helicase assays as described previously [24].
None of the compounds inhibited the ability of the NS3 helicase to unwind a DNA substrate even at
concentrations as high as 500 µM (data not shown). However, high concentrations of compound 31
inhibited the ability of NS3 helicase to cleave ATP in the presence of RNA. About 420 µM of 31
inhibited HCV helicase catalyzed ATP hydrolysis by 50% (see FigS1 Supporting Information).
Apart from targeting HCV proteins, small molecules known to interfere with HCV Internal
Ribosome Entry Site (IRES)-mediated translation have been documented [25,26]. We therefore
investigated if the observed anti-HCV activity of the 2-phenyl-tetrahydro-1H-indole scaffold could
be due to the down-regulation of HCV IRES-mediated translation. Using compound 39 as
representative, our results displayed that this compound had no effect on HCV IRES mediated
translation (data not shown).
We also tested the possibility that compound 39 could function as a potential activator or
suppressor of host-factor’s that facilitate HCV replication. Towards this end, we carried-out cell
based assays in which reporter plasmids of cyclooxygenase-2, heme oxygenase-1, interferon-
stimulated response element or anti-oxidant response element were transfected, and the ability of
derivative 39 to modulate the activation or suppression of the corresponding host-factors at three
varying compound concentrations (5, 10 and 25 ꢀM) were investigated. Our results revealed that 39
had no effect in these reporter mediated assays, thus ruling out the specified host factors as targets
of the 2-phenyl-4,5,6,7-tetrahydro-1H-indole core.
3. Conclusion
Overall, these results highlight the identification of 2-phenyl-4,5,6,7-tetrahydro-1H-indole
scaffold as a new anti-HCV chemotype.
1
0

ACCEPTED MANUSCRIPT
Preliminary SAR highlighted the key role of both the substituents on the 2-phenyl ring and the
N-1 benzyl moiety in modulating cytotoxicity and activity, respectively, with derivatives 39, 40 and
42 being the best hits within this first series of 2-phenyl-4,5,6,7-tetrahydro-1H-indoles.
While the present study has revealed a novel chemotype worthy of further investigation, the
exact mechanism by which these derivatives inhibit HCV replication remains to be clarified.
4
4
. Experimental section
.1. Cell culture
Huh7/Rep-Feo1b and Huh7.5-FGR-JC1-Rluc2A replicon reporter cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 5% antibiotic and
0.5 mg/ mL G418. Huh 7.5 cells were grown similarly as above without G418. All cells were
o
cultured at 37 C in 5% humidified CO .
2
4.2. NS5B RdRp assay
Recombinant HCV NS5B bearing hexa-histidine tag at N-terminal was expressed in E. coli and
purified as previously described [23, 27]. The anti-NS5B RdRp activity of the compounds was
evaluated by using a primer-dependent elongation assay as reported earlier [28]. In brief, the
reaction buffer containing 20 mM Tris-HCl (pH 7.0), 100 mM Na-glutamate, 100 mM NaCl, 0.01%
BSA, 0.01% Tween-20, 0.1 mM DTT, 5% glycerol, 20 U/mL of RNasin, 20 ꢀM UTP, 1 ꢀCi [α-
3
2
P]UTP, 0.25 ꢀM polyrA/U , 100 ng NS5BCꢁ21 was incubated with compounds and the
1
2
polymerase reaction was started by addition of 1 mM MnCl in a final volume of 20 µl. The
2
reactions were incubated at 30 °C for 60 minutes, and then stopped by adding 5% trichloroacetic
acid containing 0.5 mM sodium pyrophosphate, filtered through GF-B filters, and successively
washed with water and ethanol. The amount of radiolabeled RNA was quantified using liquid
scintillation counter. The activity of NS5B in the presence of an equal amount of DMSO was set at
100% and that in the presence of the compounds was determined relative to this control.
4.3. Huh7/Rep-Feo1b, Huh7.5-FGR-JC1-Rluc2A reporter system and cellular viability assay
The anti-HCV activity of compounds was measured using the Huh7/Rep-Feo1b and Huh7.5-
FGR-JC1-Rluc2A replicon reporter cells as described earlier [29, 30]. In short, approximately 1 X
4
1
0 cells were plated in 96 well plates and treated with compounds or DMSO for 48 h. The
concentration of DMSO in cell culture was kept constant at 1.0%. The luciferase activities were
measured by following the manufacturer’s protocol (Promega Inc, USA). The activity of the
1
1

ACCEPTED MANUSCRIPT
compounds was evaluated as the comparative levels of the luciferase signals in compound-treated
cells versus DMSO-treated controls. The cellular cytotoxicity assays were conducted in 96 well
plate format using parental Huh7.5 cells. Briefly, cells treated at 6-8 doses of compounds for 48 h
were evaluated employing the CellTiter 96® AQueous One Solution Cell Proliferation kit (Promega
Inc, USA). The luciferase activities of the cells treated with an equal amount of DMSO served as
control.
4.4. Target identification reporter assays
The effect of compound 39 on HCV IRES mediated translation was studied using a dual
luciferase reporter construct (pClneo-Rluc-IRES-Fluc) in which Rluc was translated in a cap-
dependent manner and Fluc was translated via HCV IRES-mediated initiation, as described
previously [29] . Transfections were carried our using LipoD293 reagent in Huh7.5 cells. Sixteen h
post-transfection, the cells were treated with compound or DMSO and Luciferase activity assay was
performed using Dual-Glo Luciferase Assay Kit.
For investigation host-factors as potential targets, hepatoma cells carrying HCV subgenomic
replicons (MH-14) were transfected with 300 ng of gene specific reporter plasmid pCOX-2-FLuc
[
31-34], pHO-1-Luc [35], pISRE-Luc [36], or p3xARE-Luc [37]. Sixteen h post-transfection, cells
were treated with compound 39 or DMSO (control) for 48 h and luciferase activities were measured
as described above. Transfection efficiencies were normalized by Renilla luciferase expression.
4
.5. RT-PCR
Total RNA was isolated using an RNeasy mini kit (Qiagen) and quantified using NanoDrop
ND1000, NanoDrop Technologies). Approximately 500 ng of RNA was reverse transcribed using
M-MLV reverse transcriptase (Life Technologies) and either oligo dT or HCV specific primers in
(
1
8
a final volume of 20 ꢀl. Approximately 50 ng of synthesized cDNA’s were used for PCR
applications using gene specific primers and Power SYBR green PCR master mix (Applied
Biosystems) in a final volume of 25 ꢀl. The PCR was performed on Applied Biosystems 7500 Fast
Dx Real-Time PCR Instrument. The forward and reverse primer sequence for β-Actin was 5’-
AGCGAGCATCCCCCAAAGTT-3’ and 5’-GGGCACGAAGGCTCATCATT-3’, respectively.
The HCV primer sequence was 5’-CGGGAGAGCCATAGTGG-3’ for forward and 5’-
AGTACCACAAGGCCTTTCG-3’ for the reverse primer.
1
2

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4
4
.6. NS3 Helicase assay
.6.1. Chemicals and reagents. Truncated C-terminally His-tagged NS3 protein lacking the N-
terminal protease (NS3h) from the con1 strain of genotype 1b [Genbank accession AB114136], was
expressed and purified as previously described [38, 39].
4.6.2. Molecular beacon based helicase assays. Molecular beacon-based NS3 helicase assays were
performed as described by Hanson et al. [49] Reactions contained 25mM MOPS pH 6.5, 1.25 mM
MgCl , 5% DMSO, 5 µg/ml BSA, 0.01% (v/v) Tween20, 0.05 mM DTT, 5 nM florescent DNA
2
substrate, 12.5 nM NS3h, and 1 mM ATP.
4.6.3. ATP hydrolysis (ATPase) assays. A modified malachite green-based assay was used to
measure helicase-catalyzed ATP hydrolysis (Sweeney et al., 2013). The colorimetric reagent was
prepared fresh by mixing 3 volumes of 0.045% (w/v) malachite green, with 1 volume 4.2%
ammonium molybdate in 4N HCl, and 0.05 volumes of 20% Tween 20. Reactions (30 µL) were
initiated by adding ATP, incubated for 15 minutes at 37 °C, and terminated by adding 200 µL of the
malachite green reagent, followed by 30 µL of 35% sodium citrate. The color was allowed to
develop for 30 minutes and an absorbance at 630 nm was observed.
HCV Helicase-catalyzed ATP hydrolysis in the absence of RNA was monitored in reactions
containing 50 nM HCV NS3h, 25 mM MOPS pH 6.5, 1.25 mM MgCl , 1 mM ATP, 33 µg/ml BSA,
2
0.07% (v/v) Tween 20, 0.3 mM DTT, and 10 % v/v DMSO. Reactions in the presence of polyU
RNA were performed with 4 nM HCV NS3h in the same buffer with 1 µM PolyU (Sigma,
expressed and nucleotide concentration) was added to each reaction.
To determine the compound concentration, it was necessary to reduce helicase-catalyzed ATP
hydrolysis by 50 % (IC ). Reactions were performed in duplicate through a two-fold dilution series
5
0
so that final compound concentrations ranged from 0.5 mM to 0.78 µM. Data obtained from all
reactions within the linear range of the colorimetric assay as determined with a phosphate standard
curve were normalized to controls lacking an inhibitor (100%) and controls lacking an enzyme (0%),
and fitted to a normalized dose response equation with a variable Hill slope using GraphPad Prism
(v. 6). Reactions were performed in duplicate and each titration conformed to the above
concentration response equation. Average IC50 values ± standard deviations were reported. In
another set of controls, 100 µM of inorganic phosphate was titrated with each compound, followed
by the addition of a malachite green reagent. None of the compounds affected the absorbance of the
colorimetric reaction products in these controls.
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3

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Acknowledgements
We thank Drs. Naoya Sakamoto and Hengli Tang for providing the Huh7/Rep-Feo1b and
Huh7.5-FGR-JC1-Rluc2A replicon reporter cells. Plasmids pCIneo-Rluc-IRES-Fluc, pHO-1-Luc
and p3xARE-Luc, were generously shared by Drs. Naoya Sakamoto, Anupam Agarwal, and Dr.
Being-Sun Wung, respectively. We acknowledge and appreciate grant support from the New Jersey
Health Foundation to Neerja Kaushik-Basu and the Russian Foundation for Basic Research (RFBR),
Russia (Projects No. 14-03-31685, 14-03-31709, 14-03-01114).
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Figure and Scheme Captions
Figure 1. DAAs - FDA approved drugs for the treatment of Hepatitis C.
Figure 2. Chemical structures and internal EDASA Scientific codes of the first set of compounds
that underwent biological evaluation.
Figure 3. Structures of EDASA analogues of 2-phenyl-4,5,6,7-tetrahydro-1H-indole 31.
Figure 4. Dose-dependent response of compound 39 assayed in MH-14 cells. **p<0.01
Scheme 1. Synthesis of 2-aryl-4,5,6,7-1H-tetrahydroindoles. The explicit structures of compounds
31 and 34-44 are reported in Figure 3.
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Table 1: Anti-HCV activities and cytotoxicity of the first 33 EDASA Scientific compounds evaluated on gt
1
b and 2a.
Huh7/Rep-Feo1b
Huh7.5-FGR-JC1-Rluc2A
a
CC50
µM)
Cpd
b
c
b
c
(
Inhibition,
%
EC50
d
Inhibition,
%
EC50
d
SI
SI
(µM)
(µM)
1.1 ±
.9
8.5 ±
.9
1
1
2
>200
>200
19 ± 6
22 ± 9
ND
ND
ND
80 ± 5
51 ± 8
>18
>4
0
4
2
ND
3
3
4
>200
>200
NI
NI
ND
ND
ND
ND
49 ± 6
ND
ND
ND
36 ± 10
ND
3.6 ±
5
6
7
>200
ND
49 ± 12
26 ± 1
NI
ND
ND
ND
ND
ND
ND
66 ± 7
54 ± 4
78 ± 3
>8
ND
>17
4.0
ND
1
1
1
1
2
1.7 ±
>200
0
.9
4.1 ±
.9
5.6 ±
.7
7.3 ±
.2
1.1 ±
.4
8
9
>200
>200
>200
>200
21 ± 2
19 ± 10
21 ± 2
NI
ND
ND
ND
ND
ND
ND
ND
ND
77 ± 7
72 ± 3
77 ± 7
67 ± 5
>14
>13
>12
>9
1
3
1
1
0
1
3
4
1
1
2
3
>200
>200
19 ± 8
NI
ND
ND
ND
ND
NI
ND
ND
ND
14 ± 8
ND
2
0.6 ±
1
1
1
4
5
6
85.6 ± 5.9
<25
17 ± 3
88 ± 2
39 ± 4
ND
ND
ND
ND
ND
ND
65 ± 5
99 ± 1
60 ± 6
4
2.9
ND
ND
>9
2
2
2.5 ±
>200
3
.8
8.1 ±
.8
1
7
>200
NI
ND
ND
62 ± 1
>7
4
1
1
2
2
2
8
9
0
1
2
>200
>200
>200
<25
NI
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
55 ± 9
46 ± 8
29 ± 8
99 ± 1
44 ± 9
ND
ND
ND
ND
ND
ND
ND
ND
ND
NI
54 ± 8
92 ± 1
f
>200
NI
ND
2
4.8 ±
4
2
2
2
3
4
5
>200
>200
>200
45 ± 6
19 ± 9
NI
ND
ND
ND
ND
ND
ND
61 ± 9
85 ± 9
73 ± 3
>8
.5
.3 ±
.5
.9 ±
.4
7.4 ±
.8
ND
7
>27
>41
0
4
0
1
2
2
2
2
6
7
8
9
>200
>200
NI
NI
ND
ND
ND
ND
5
76 ± 7
43 ± 10
98 ± 2
NI
>11
ND
>19
ND
0
2
4.3 ±
.2
ND
6.0 ±
1.0
114.7± 14.6
>200
73 ± 9
NI
1
ND
ND

ACCEPTED MANUSCRIPT
3
3
0
1
>200
NI
ND
ND
9
28 ± 4
88 ± 8
ND
ND
13
1
2.4 ±
8.7 ±
1.9
109.9 ± 2.9
66 ± 9
1.0
3
3
2
3
>200
>200
50 ± 4
45 ± 2
ND
ND
ND
25 ± 5
ND
ND
ND
ND
48 ± 11
ND
a
CC50 values were determined in Huh7.5 parental cells by the MTS assay. CC50 = is the concentration required to reduce
the bioreduction of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
into formazan by 50%. The reported value represents the means ± SD of data derived from three independent experiments.
b
c
Anti-HCV activity of the compounds were carried out at 50 µM in preliminary screening. The inhibition data from 8-12
quarter log dilutions were used to generate the dose response curves. EC50 = the effective concentration required to inhibit
virus induced cytopathic effect by 50%. The reported values represent the means ± SD of data derived from three
d
independent experiments. SI: selectivity index ratio of CC to EC . ND: not determined. NI: no inhibition.
50
50

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Table 2: Anti-HCV activities and cytotoxicity of analogues of 31 evaluated on gt 1b and 2a.
a
Huh7/Rep-Feo1b
Huh7.5-FGR-JC1-Rluc2A
CC50
µM)
Cpd
(
b
c
d
b
c
d
Inhibition, % EC50 (µM) SI
Inhibition, % EC50 (µM) SI
3
3
3
3
3
3
4
4
4
4
4
4
5
6
7
8
9
0
1
2
3
4
>200
45.6 ± 6.1
<25
71 ± 6
81 ± 3
92 ± 1
50 ± 8
37 ± 18
95 ± 4
75 ± 7
35 ± 6
99 ± 1
96 ± 2
74 ± 4
29.2 ± 1.2
35.8 ± 3.4
ND
>7
66 ± 10
96 ± 3
99 ± 1
83 ± 3
48 ± 11
99 ± 1
98 ± 2
69 ± 2
96 ± 2
99 ± 1
95 ± 3
12.3 ± 1.0
9.9 ± 1.6
ND
>16
>5
ND
>10
ND
32
27
4
>1
ND
ND
ND
10
155.6± 11
>200
ND
15.4 ± 3.9
ND
ND
80.8 ± 3.1
>200
7.9 ± 0.5
15.0 ± 1.3
ND
2.6 ± 0.4
7.3 ± 1.4
32.1 ± 4.1
4.9 ± 0.3
6.5 ± 0.6
13.7 ± 2.1
13
118.8 ± 2.8
137.4 ± 1.0
48.9 ± 1.7
84.3 ± 1.3
ND
12
11.8 ± 0.6
9.2 ± 0.6
13.2 ± 1.4
28
8
5
6
6
a
CC50 values were determined in Huh7.5 parental cells by the MTS assay. CC50 = is the concentration required to reduce
the bioreduction of MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
into formazan by 50%. The reported value represents the means ± SD of data derived from three independent experiments.
b
c
Anti-HCV activity of the compounds were carried out at 50 µM in preliminary screening. The inhibition data from 8-12
quarter log dilutions were used to generate the dose response curves. EC50 = the effective concentration required to inhibit
virus induced cytopathic effect by 50%. The reported values represent the means ± SD of data derived from three
d
independent experiments. SI: selectivity index ratio of CC to EC . ND: not determined.
50
50

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•
•
•
The one-pot synthesis and anti-HCV evaluation of a series of 2-phenyl-4,5,6,7-tetrahydro-
1
H-indoles is reported
Compound 39 is the most potent derivative, displaying EC values of 7.9 µM (SI = 10) and
5
0
2
.6 µM (SI = 32) in genotype 1b and 2a, respectively.
Biochemical assays show that derivative 39 has no effect on HCV NS5B polymerase, NS3
helicase, IRES mediated translation and selected host factors.

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Supporting Information
Discovery of the 2-Phenyl-4,5,6,7-Tetrahydro-1H-indole as
a Novel Anti-Hepatitis C Virus Targeting Scaffold.
a,d,§
b,§
c,*
a,d
Ivan A. Andreev , Dinesh Manvar , Maria Letizia Barreca , Dmitry S. Belov , Amartya
b
e
d
a,d
Basu , Noreena L. Sweeney , Nina K. Ratmanova , Evgeny R. Lukyanenko , Giuseppe
c
c
e
a,*
b,*
Manfroni , Violetta Cecchetti , David N. Frick , Andrea Altieri , Neerja Kaushik-Basu ,
d
Alexander V. Kurkin
a
EDASA Scientific srls., Via Stingi, 37, 66050 San Salvo (CH), Italy
Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers, The State
b
University of New Jersey, New Jersey Medical School, New Jersey 07103, USA
c
Department of Pharmaceutical Sciences, University of Perugia, Via A. Fabretti, 48, 06123
Perugia, Italy
d
Chemistry Department of Lomonosov Moscow State University, Moscow, 119991, GSP-2,
Leninskie gory, 1/3
e
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N.
Cramer St., Milwaukee, WI 53211, USA
*
corresponding authors
§
equal contribution
S1

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Compounds 1-33 have been procured from the EDASA Scientific public available compound
repertory (http://www.edasascientific.com/page/catalogue). A report of their characterization via
1
13
H NMR, C NMR and m.p. can be found on pages S2 to S11.
The synthetic procedure and compound characterization of compounds 34-44 is reported on
pages S12 to S22.
1
Ethyl 1,2-dimethyl-5-hydroxy-indole-3-carboxylate (1)
m.p. = 208 – 209 °C.
1
H NMR (DMSO-d , 400 MHz): δ = 1.35 (t, J = 7.1 Hz, 3H), 2.66 (s,
6
3
H), 3.63 (s, 3H), 4.26 (q, J = 7.1 Hz, 2H), 6.68 (dd, J = 8.7, 2.4 Hz, 1H),
.26 (d, J = 8.7 Hz, 1H), 7.38 (d, J = 2.2 Hz, 1H), 8.94 (s, 1H).
7
1
3
C NMR: (DMSO-d , 100 MHz): δ = 11.7, 14.5, 29.6, 58.6, 101.9,
6
1
05.5, 110.3, 111.3, 127.1, 130.7, 145.3, 152.6, 165.2.
1
-[2-(1H-Indol-3-yl)ethyl]-5-oxopyrrolidine-3-carboxylic acid (2)
O
m.p. = 217 – 220 °C.
O
1
H NMR (DMSO-d , 400 MHz): δ = 2.40 - 2.55 (m, 2H), 2.87 (t,
6
OH
N
J = 7.5 Hz, 2H), 3.17 (tdd, J = 9.1, 7.3, 6.0 Hz, 1H), 3.47 (dd, J =
8.9, 7.8 Hz, 2H), 3.50-3.61 (m, 2H), 6.99 (ddd, J = 8.0, 7.1, 1.0
Hz, 1H), 7.08 (td, J = 7.5, 1.1 Hz, 1H), 7.16 (d, J = 2.3 Hz, 1H),
N
H
2, BB 0217717
7
.35 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 10.83 (s, 1H), 12.60 (br. s, 1H).
1
3
C NMR: (DMSO-d , 100 MHz): δ = 22.9, 33.7, 35.5, 42.5, 49.7, 111.3, 111.4, 118.2, 118.3,
6
1
2
21.0, 122.7, 127.1, 136.3, 171.8, 174.6.
2
-Pyridin-4-ylquinoline-4-carboxylic acid (3)
m.p. = 310 – 312 °C.
1
H NMR (DMSO-d , 400 MHz): δ = 7.77 (t, J = 7.6 Hz, 1H), 7.90 (t, J =
6
7
.6 Hz, 1H), 8.22 (d, J=8.6 Hz, 1H), 8.26 (d, J = 5.1 Hz, 1H), 8.55 (s, 1H),
.68 (d, J = 8.4 Hz, 1H), 8.79 (d, J = 5.0 Hz, 2H).
8
1
3
C NMR: (DMSO-d , 100 MHz): δ = 119.1, 121.3 (2C), 124.1, 125.5,
6
1
28.7, 130.0, 130.6, 138.3, 144.8, 148.3, 150.5 (2C), 153.6, 167.5.
1
Velezheva, V. S.; Kornienko, A. G.; Topilin, S. V.; Turashev, A. D.; Peregudov, A. S.; Brennan, P. J. Journal of
Heterocyclic Chemistry, 2006, 43, 873 – 879.
2
ASTRAZENECA AB Patents: WO2009/82346 A1,2009; WO 2009/082346 A1
S2