Synthesis and anti-hepatitis C virus activity of novel ethyl 1H-indole-3-carboxylates in vitro

Article abstract of DOI:10.1016/j.bmc.2010.06.058

A series of ethyl 1H-indole-3-carboxylates 9a_{1- 6} and 9b_1-2 were prepared and evaluated in Huh-7.5 cells. Most of the compounds exhibited anti-hepatitis C virus (HCV) activities at low concentration. The selectivity indices of inhibition on entry and replication of compounds 9a₂ (>10; >16.7) and 9b₁ (>6.25; >16.7) were higher than those of the other evaluated compounds, including the lead compound Arbidol (ARB, 6; 15). Moreover, the selective index of inhibition on entry of compound 9a₃ (>6.25) was higher than that of ARB (6). Of these three initial hits, compound 9a₂ was the most potent.

Full text of DOI:10.1016/j.bmc.2010.06.058

Bioorganic & Medicinal Chemistry 18 (2010) 6143–6148
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and anti-hepatitis C virus activity of novel ethyl
1H-indole-3-carboxylates in vitro
a
a
b
b
Grazia Sellitto a, , Aurora Faruolo , Paolo de Caprariis , Sergio Altamura , Giacomo Paonessa ,
*
Gennaro Ciliberto c
a Dipartimento di Scienze Farmaceutiche,Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Italy
b Istituto di Ricerche di Biologia Molecolare, Via Pontina km 30,600, Pomezia, Roma, Italy
c Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Università degli Studi ‘Magna Graecia’ di Catanzaro, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 January 2010
Revised 4 June 2010
Accepted 16 June 2010
Available online 22 June 2010
A series of ethyl 1H-indole-3-carboxylates 9a1–6 and 9b1–2 were prepared and evaluated in Huh-7.5 cells.
Most of the compounds exhibited anti-hepatitis C virus (HCV) activities at low concentration. The selectiv-
ity indices of inhibition on entry and replication of compounds 9a2 (>10; >16.7) and 9b1 (>6.25; >16.7) were
higher than those of the other evaluated compounds, including the lead compound Arbidol (ARB, 6; 15).
Moreover, the selective index of inhibition on entry of compound 9a3 (>6.25) was higher than that of
ARB (6). Of these three initial hits, compound 9a2 was the most potent.
Keywords:
Hepatitis C virus
Arbidol
Ó 2010 Elsevier Ltd. All rights reserved.
Ethyl 1H-indole-3-carboxylates
Synthesis
1. Introduction
cycle, where each step is directly related to membrane activity.
ARB inhibits steps of the HCV life cycle that are dependent on cell
Hepatitis C virus (HCV) infection currently affects approxi-
mately 170 million people worldwide and is resolved in only a
minority of patients.1 The chronic viral infection frequently pro-
gresses to end-stage liver disease, cirrhosis and in some cases, to
the development of hepatocellular carcinoma.2,3 Therefore, hepati-
tis C is now the most frequent indication for liver transplantation.
There is no therapeutic or prophylactic vaccine available for HCV,
and the only effective antiviral therapy, pegylated recombinant
membranes. It displays inhibitory effects on HCV entry, fusion, and
replication, thanks to its tropism for membranes. For example,
ARB-induced inhibition of HCV nonstructural proteins interactions
with endoplasmic reticulum membranes might also contribute to
suppression of HCV replication.9
The biological properties of ethyl 6-bromo-5-hydroxy-1H-in-
dole-3-carboxylates led us to focus on their derivatives as potential
anti-HCV agents.
interferon
a
(IFN-
a
) and ribavirin, produces sustained viral clear-
We designed and synthesised several novel ethyl 1H-indole-3-
carboxylate derivatives in which the hydroxy and bromo groups
at the 5- and 6-positions of the indole ring of Arbidol were
removed and the amino group was introduced in the 5-position
instead of the 4-position. Other modifications were mainly focused
on positions 2 and 5 on the indole ring (Fig. 2). We are interested in
exploring the effects of these changes on the anti-HCV activity of
the resulting compounds.
ance in less than 50% of treated patients.4 New anti-HCV drugs
with novel mechanisms of action are needed.
Ethyl 6-bromo-5-hydroxy-1H-indole-3-carboxylate derivatives
display a variety of biological effects, such as antiviral effects,
immunostimulative effects, and interferon-induced activity. A rep-
resentative agent is Arbidol (ARB, Fig. 1). Launched in Russia for the
prophylaxis and treatment of acute respiratory viral infections,
Arbidol acts as an inhibitor of virus entry and membrane fu-
sion.5–8 ARB is a broad-spectrum antiviral agent that inhibits acute
and chronic HCV infection.
The anti-HCV effect appears to be due to the interaction of ARB
with membranes and to subsequent ARB-induced membrane alter-
ations. Lipids and membranes are clearly central to the HCV life
* Corresponding author. Tel.: +39 089969749.
E-mail address: gsellitto@unisa.it (G. Sellitto).
Figure 1. Structure of Arbidol.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.058

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G. Sellitto et al. / Bioorg. Med. Chem. 18 (2010) 6143–6148
Table 1
Structures of compounds 9a1–6 and 9b1–2
Compound
R1
R2
Figure 2. General structure of target compounds.
9a1
9a2
9a3
9a4
9a5
9a6
9b1
9b2
Phenylsulfonyl
Phenylsulfonyl
Phenylsulfonyl
Phenylsulfonyl
Phenylsulfonyl
Phenylsulfonyl
Hydrogen
Dimethylamino
Pyrrolidinyl
4-Methyl piperazinyl
Morpholino
Homopiperazinyl
Imidazolyl
Dimethylamino
Pyrrolidinyl
2. Results and discussion
2.1. Design of derivatives
The general design of ARB analogs was guided by the modular
composition of this synthetic product.
Hydrogen
ARB antiviral activity toward HCV is due probably to a direct ef-
fect of ARB on virus-cell membrane interactions where ARB inter-
calates into membranes and adopts a consistent orientation with
the formation of an ‘ARB cage’. This lead to excessive stabilization
of cell membranes, which become resistant to HCV fusion.
Its tropism for membranes or membrane like environments is
due to ARB’s indole-derived structure and to its interaction with lip-
ids via other hydrophobic and aromatic moieties on the molecule.
In particular, the indole ring exhibits a preference for mem-
brane interfaces, due to its flat rigid structure and to its aromaticity
azine (9a1–6). Previously, it was demonstrated that the oxidation of
sulfide reduced the cellular toxicities and kept up or increased the
antiviral activities5; in this regard, we introduced phenylsulfo-
nylmethyl groups at the 2-position.
Subsequently, for investigating the influence of changes to the
steric environment and the role of phenylsulfonylmethyl group
on the antiviral activity, we introduced the methyl group at the
2-position (9b1–2).
and establishes cation–p interactions with the positively charged
2.2. Synthetic approach
quaternary ammonium lipid headgroups. The S-phenyl group
could interact with the hydrophobic fatty acid chains of phospho-
lipids inside the bilayer. The amino groups could bind the phos-
The compounds 9a1–6 and 9b1–2 were synthesised, and their
structures are listed in Table 1.
The synthesis of target compounds 9a1–5 and 9b1–2 was achieved
using a convenient five-step procedure starting from 4-bromo-2-
iodoaniline and b-keto esters 3a and 3b, respectively, as depicted
in Scheme 1.
phate moieties of phospholipids, establishing
a salt bridge
between two adjacent phospholipid molecules as an ion pair com-
plex. At low pH, these interactions increase for the protonation of
the amino groups. The ester group could be a substrate for hydro-
lysis in vivo, leading to intracellular accumulation of the cleaved
compound. In light of the above considerations, ARB may be con-
sidered a pro-drug which is chemically converted into an active
drug by cellular metabolic processes.9,10
In order to maintain anti-HCV activity we preserved the groups
responsible of Arbidol interaction with membranes: indole ring, S-
phenyl group, ester group and amino group. In particular, the first
series of compounds contains analogs whose structures are closely
related to ARB. The indole building block was simplified by elimi-
nation of hydroxy and bromo groups at the 5- and 6-positions of
the indole ring and by shift of amino group form 4-position to 5-
position (Fig. 2) to evaluate the influence of the polar group.
In order to examine whether the nature of the amino group
could have an effect on the potential anti-HCV activity, we intro-
Moreover, the thioether 3a was prepared via etherification of
ethyl 4-chloroacetoacetate 2 with thiophenol 1 (Scheme 2).11,12
The compound 3a and the commercially available compound 3b
were treated with 4-bromo-2-iodoaniline 4 in a copper-catalyzed
Ullmann-type coupling reaction to give the key intermediates
5a–b. The N-alkylation of compounds 5a–b with iodomethane
using the procedure of Kikugawa afforded in high yields the com-
pounds 6a–b.
These compounds were subsequently subjected to a Suzuki–
Miyaura cross-coupling reaction with potassium vinyltrifluo-
roborate, affording the intermediates 7a–b in good yields. The
combination of osmium tetroxide and sodium periodate (Lemi-
eux–Johnson oxidation) efficiently achieved both the oxidative
cleavage of the vinyl group to an aldehyde group and the oxidation
of the sulfide group of compound 7a to sulfonyl group, thereby
yielding the compounds 8a–b.
duced
a variety of amines, including imidazole, pyrrolidine,
dimethylamine, 4-methyl piperazine, morpholine and homopiper-
Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) CuI, BINOL, Cs2CO3, DMSO, 50 °C, 5–7 h, 48–56%; (b) ICH3, KOH, DMF, 0 °C, 1–2 h, 92–97%; (c)
potassium vinyltrifluoroborate, 6 mol% PdCl2, 18 mol % PPh3, Cs2CO3, THF/H2O 9:1; 85 °C, 20–22 h, 80–86%; (d) OsO4, 2,6-lutidine, dioxane, NaIO4 in H2O, rt, 20 min, 90–95%;
(e) aliphatic amine, acetic acid, THF, NaB(OAc)3H, MW, 100–130 °C, 20 min, 75–95%.

G. Sellitto et al. / Bioorg. Med. Chem. 18 (2010) 6143–6148
6145
in vitro anti-HCV activity. Its IC50 (5 and 3
l
M) values in both
entry and replication assays were highly comparable to those of
Arbidol (2 and 1 M, respectively). Interestingly, compound 9a2
did not display cytotoxicity at 50 M concentration, while at
100 M concentration an inhibition of cell viability of 95% and 80%
on entry and replication, respectively, was detected. Therefore com-
pound 9a2 selectivity indices (>10, >16.7) were higher than those of
Arbidol (6, 15).
l
l
l
Scheme 2. Synthesis of ethyl 3-oxo-4-(phenylthio)butanoate 3a. Reagents and
condition: (a) KOH, methanol, rt, 94%.
The analogs 9a3 and 9b1 also exhibited significant efficacy
against HCV (structures were listed in Fig. 3). Their IC50 values
on entry were 8 and 8 lM and on replication were 4 and 3 lM,
respectively, which were higher than the positive control Arbidol.
Also in these cases, the selectivity indices of 9a3 and 9b1 on HCV
entry (>6.3) and on HCV replication (>12.5, >16.7) were higher
than or comparable to those of ARB.
Compounds 9a1–6, with the same groups in position 1–3 but
different ammines in 5-position, showed an IC50, on entry and
Scheme 3. Synthesis of N-Boc homopiperazine 13. Reagent and condition: (a)
dichloromethane, 0 °C, 1 h, 88%.
Finally, we synthesised the target compounds 9a1–5 and 9b1–2
via a rapid microwave-assisted reductive amination procedure that
involves the formation of an imine and subsequent reduction with
NaB(OAc)3H in a one-pot reaction. To produce compound 9a5, we
used N-Boc protected homopiperazine, which was synthesised
according to Scheme 3.
Imidazole could not be introduced into the 5-position by direct
reductive amination with 8a. Instead, it was realized through a
two-step sequence beginning with the reduction of the aldehyde
group of compound 8a to an alcohol group to obtain the interme-
diate 10a. This was followed by treatment of 10a with imidazole
and 1,1-carbonyldiimidazole in acetonitrile to generate the deriva-
tive 9a6 in good yield (Scheme 4).
replication, in the range from 3 to 21 lM concentration. These
results suggested that the different amines introduced at the 5-
position had a relatively minor influence on the antiviral activi-
ties. Moreover, the imidazolyl group in compound 9a6 seemed
to shift the selectivity toward inhibition of HCV entry, to reduce
the potency against HCV replication and to enhance the
cytotoxicity.
Compounds 9b1 and 9b2 exhibited strong anti-HCV effects. This
revealed that the elimination of the phenylsulfonyl moiety pre-
served the anti-HCV activity.
These preliminary results demonstrated that the removal of the
6-bromo and 5-hydroxy groups from the indole ring of Arbidol did
not have an influence on its anti-HCV efficacy. This implies that
these groups might not be the antiviral pharmacophores.
2.3. Anti-HCV analysis
All of the target compounds 9a1–6, 9b1–2 and Arbidol were tested
in vitro in Huh-7.5 cells for cytotoxicity and anti-HCV activity, spe-
cifically, the ability to inhibit the entry and replication of HCV. The
properties of these compounds are summarized in Table 2.
3. Conclusion
In summary, we synthesised a series of new ethyl 1H-indole-3-
carboxylate derivatives and examined their anti-HCV activities and
Several compounds exhibited inhibitory effects on HCV
comparable to Arbidol. Compound 9a2 showed the most potent
Scheme 4. Synthesis of compound 9a6. Reagents and conditions: (a) NaBH4 in methanol, THF, rt, 1 h, 91%; (b) imidazole, 1,1-carbonyldiimidazole, acetonitrile, reflux, 16 h,
67%.
Table 2
Anti-HCV activity and cytotoxicity of target compounds in vitro
Compound
HCV entry (genotype 2a)
HCV replication (genotype 2a)
a
b
a
b
IC50
(l
M)
TC50
(l
M)
SIc
>2.6
>10
IC50
(l
M)
TC50
(l
M)
SIc
9a1
9a2
19
5
>50
>50
10
3
>50
>50
>5
>16.7
N.A. (50)d
N.A. (50)d
95% Inh. (100)e
80% Inh. (100)e
9a3
9a4
9a5
9a6
9b1
9b2
Arbidol
8
14
21
5
8
13
2
>50
>50
>50
23
>50
>50
12
>6.3
4
4
12
15
3
>50
>50
>50
18
>50
>50
15
>12.5
>12.5
>4.2
1.2
>16.7
>10
>3.6
>2.4
4.6
>6.3
>3.8
6
5
1
15
a
b
c
IC50 is 50% inhibitory concentration in Huh-7.5 cells.
TC50 is 50% cytotoxic concentration.
Selectivity index (SI: TC50/IC50).
d
e
No cytotoxic activity at 50
lM concentration.
% of inhibition at 100 M concentration.
l

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G. Sellitto et al. / Bioorg. Med. Chem. 18 (2010) 6143–6148
Figure 3. Chemical structures of typical compounds that showed significant anti-HCV activity.
cytotoxicities in Huh-7.5 cells. According to the above results, the
following conclusions can be drawn:
4.1.1.1. Ethyl 5-bromo-2-((phenylthio)methyl)-1H-indole-3-car-
boxylate (5a). Elution with n-hexane/EtOAc (95:5) afforded 5a
(56%) as yellow powder; mp: 142–144 °C; 1H NMR (CDCl3): d
1.47 (t, J = 7.2 Hz, 3H, –OCH2CH3); 4.42 (q, J = 7.2 Hz, 2H,
–OCH2CH3); 4.80 (s, 2H, –CH2-sulfonyl–); 7.18 (d, 1H, –UH);
7.20–7.5 (m, 6H, –UH and –PhH); 8.20 (s, 1H, –UH); 8.8 (s, 1H, –
NH); MS: m/z 390.0, 392.0 [MH+].
1. The elimination of the phenylsulfonyl group at the 2-position
seemed to preserve the antiviral activities.
2. Compounds with different amines introduced at the 5-position
did not show a wide variation in anti-HCV effects.
3. The 6-bromo and 5-hydroxy groups might not be the pharma-
cophores responsible for the anti-HCV effects.
4.1.1.2. Ethyl 5-bromo-2-methyl-1H-indole-3-carboxylate (5b).
Elution with toluene/EtOAc (95:5) afforded 5b (48%) as gray-green
powder; mp: 163–165 °C; 1H NMR (CDCl3): d 1.5 (t, J = 7.2 Hz, 3H,
–OCH2CH3); 2.75 (s, 3H, –CH3); 4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3);
7.2 (d, 1H, –UH); 7.35 (dd, J = 8.55, 1.75 Hz, 1H, –UH); 8.28 (s, 1H,
–UH); 8.38 (s, 1H, –NH); MS: m/z 282.1, 284.1 [MH+].
Despite several changes made to the Arbidol structure in our
derivatives, the antiviral activity was maintained, and the cytotox-
icity was reduced.
These encouraging results on HCV entry and replication reveal
some promising leads in the search for new and efficient anti-
HCV therapies.
4.1.2. General procedure for the synthesis of compounds 6a–b
To a stirred solution of compound 5a or 5b (9 mmol) in DMF
(40 mL) at 0 °C was added freshly powdered potassium hydroxide
(10 mmol). After stirring the mixture for 30 min at 0 °C, methyl
iodide (13.5 mmol) was added dropwise with vigorous stirring.
After 1 h, the reaction mixture was diluted with diethyl ether
and washed with saturated NaCl solution. The organic layer was
dried (Na2SO4), and the solvent was removed in vacuo to yield
6a–b.
4. Experimental
4.1. Chemistry
Microwave experiments were performed in a CEM Discover
monomode reactor (CEM Corp., Matthews, NC). All reactions were
conducted in a specially adapted cylindrical Pyrex vessel. All re-
agents were analytical grade and purchased from Sigma–Aldrich
(Milano, Italy). Flash chromatography was performed on Carlo Erba
silica gel 60 (230–400 mesh; Carlo Erba, Milan, Italy). TLC was car-
ried out using plates coated with silica gel 60F 254 nm purchased
from Merck (Darmstadt, Germany). Melting points were deter-
mined in open capillary tubes on an Electrothermal 9100 appara-
tus and are uncorrected. 1H NMR spectra were registered on a
Bruker AC 300. Chemical shifts are reported in ppm.
4.1.2.1. Ethyl 5-bromo-1-methyl-2-((phenylthio)methyl)-1H-
indole-3-carboxylate (6a). White powder (97%); mp: 109–
111 °C; 1H NMR (CDCl3): d 1.45 (t, J = 7.2 Hz, 3H, –OCH2CH3);
3.70 (s, 3H, –NCH3); 4.35 (q, J = 7.2 Hz, 2H, –OCH2CH3); 4.78 (s,
2H, –CH2-sulfonyl–); 7.18 (d, 1H, –UH); 7.20–7.5 (m, 6H, –UH
and –PhH); 8.23 (s, 1H, –UH); MS: m/z 404.0, 406.0 [MH+].
4.1.2.2. Ethyl 5-bromo-1,2-dimethyl-1H-indole-3-carboxylate
(6b). Brown powder (92%); mp: 98–100 °C; 1H NMR (CDCl3): d
1.5 (t, J = 7.2 Hz, 3H, –OCH2CH3); 2.83 (s, 3H, –CH3); 3.77 (s, 3H,
–NCH3); 4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3); 7.2 (d, 1H, –UH); 7.35
(dd, J = 8.55, 1.75 Hz, 1H, –UH); 8.28 (s, 1H, –UH); MS: m/z 296.1,
298.1 [MH+].
All target compounds were assessed for purity by analytical
high performance liquid chromatography (HPLC) using an Agilent
1100 series instrument equipped with a UV detector monitoring
at 254 nm, HP Chem Station software, and a Waters C18 RP-col-
umn (5
l
m, 300 mm  3.9 mm). All target compounds were found
to have >95% purity.
Mass spectra (MS) were taken in ESI mode on a Finnigan LCQ
Deca ion trap instrument. Microanalyses were carried out on a Car-
lo Erba 1106 elemental analyzer.
4.1.3. General procedure for the synthesis of compounds 7a–b
A solution of potassium vinyltrifluoroborate (8 mmol), PdCl2
(0.2 mmol), PPh3 (0.6 mmol), Cs2CO3 (9.6 mmol), and compound
6a or 6b (3.2 mmol) in THF/H2O (9:1) (24 mL) was heated at
85 °C and stirred for 20–22 h. Then, the reaction mixture was
cooled to room temperature and diluted with H2O, followed by
extraction with dichloromethane. The solvent was removed in va-
cuo, and the crude product was purified by silica gel chromatogra-
phy (n-hexane/EtOAc, 9:1) to give compounds 7a–b.
Compound 3a was synthesised in accordance with the literature
procedures.11,12
4.1.1. General procedure for the synthesis of compounds 5a–b
A mixture of 4-bromo-2-iodoaniline (20 mmol), ethyl acetoace-
tate 3b or 3-oxo-4 (phenylthio)butanoate 3a (22 mmol), CuI
(2 mmol), BINOL (4 mmol) and Cs2CO3 (20 mmol) in DMSO
(35 mL) was stirred at 50 °C for 5–7 h under an atmosphere of
nitrogen. After this time, the mixture was partitioned between ethyl
acetate and saturated NH4Cl. The organic layer was washed with
brine, dried over sodium sulfate and concentrated in vacuo. The
residue was purified by flash chromatography over silica gel to give
5a–b.
4.1.3.1. Ethyl 1-methyl-2-((phenylthio)methyl)-5-vinyl-1H-indole-
3-carboxylate (7a). Yellow powder (97%); mp: 103–105 °C;
1H NMR (CDCl3): d 1.42 (t, J = 7.2 Hz, 3H, –OCH2CH3); 3.70 (s, 3H,
–NCH3); 4.35 (q, J = 7.2 Hz, 2H, –OCH2CH3); 4.78 (s, 2H, –CH2-sulfo-
nyl–); 5.23 (d, J = 10.96 Hz, 1H, CH2@CH–); 5.7 (d, J = 11.54 Hz, 1H,

G. Sellitto et al. / Bioorg. Med. Chem. 18 (2010) 6143–6148
6147
CH2@CH–); 6.87 (dd, J = 17.87, 10.74 Hz, 1H, CH2@CH–); 7.25 (d,
1H, –UH); 7.35–7.5 (m, 6H, –UH and –PhH); 8.20 (s, 1H, –UH);
MS: m/z 352.0 [MH+].
4.05 (q, J = 7.2 Hz, 2H, –OCH2CH3); 5.25 (s, 2H, –CH2-sulfonyl–);
7.3–7.75 (m, 7H, –UH and –PhH); 8.0 (s, 1H, –UH); MS: m/z 441.0
[MH+].
4.1.6. Synthesis of N-Boc homopiperazine 13
To a solution of dichloromethane (45 mL) and homopiperazine
11 (1.84 g, 18 mmol), maintained at 0 °C using an ice bath, was
4.1.3.2. Ethyl 1,2-dimethyl-5-vinyl-1H-indole-3-carboxylate
(7b). Yellow powder (92%); mp: 51–53 °C; 1H NMR (CDCl3): d
1.5 (t, J = 7.2 Hz, 3H, –OCH2CH3); 2.83 (s, 3H, –CH3); 3.77 (s, 3H,
–NCH3); 4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3); 5.23 (d, J = 11.18 Hz,
1H, CH2@CH–); 5.7 (d, J = 17.54, 1H, CH2@CH–); 6.87 (dd,
J = 17.10, 10.74 Hz, 1H, CH2@CH–); 7.25 (d, 1H, –UH); 7.45 (dd,
J = 8.55, 1.75 Hz, 1H, –UH); 8.20 (s, 1H, –UH); MS: m/z 244.0 [MH+].
added dropwise
a di-tert-butyldicarbonate solution 12 (2 g,
9 mmol in 18 mL dichloromethane). The mixture was stirred for
an additional 1 h then was filtered. The filtrate was concentrated
to dryness. Water (27 mL) was added to the resulting oil, and the
mixture was filtered. The filtrate was saturated with potassium
carbonate and extracted with diethyl ether (3 Â 13 mL). The
solvent was dried over sodium sulfate and concentrated to dryness,
yielding 13 (1.58 g, 88%).
4.1.4. General procedure for the synthesis of compounds 8a–b
OsO4 (0.1 mmol) was added to a stirred mixture of 7a or 7b
(1 mmol) and 2,6-lutidine (2 mmol) in dioxane (26 mL). The mix-
ture turned from colorless to black in 1 min. Sodium periodate
(4 mmol) in water (6 mL, warmed to dissolve) was added. A gray
precipitate was immediately formed. The mixture was stirred for
20 min, then extracted with water and dichloromethane. The
organic layers were combined, dried, filtered and concentrated.
The residue was purified by flash chromatography over silica gel
(n-hexane/EtOAc, 7:3) to yield 8a–b.
4.1.7. General procedure for the synthesis of compounds 9a3–5
Compound 8a (0.04 g, 0.1 mmol), 4-methylpiperazine or mor-
pholine or 1-BOC-homopiperazine (0.15 mmol), acetic acid
(0.06 mL, 1.02 mmol) and NaB(OAc)3H (0.055 g, 0.24 mmol) were,
in that order, added to dry THF (4 mL). The mixture was irradiated
with microwaves for 10 min at 130 °C. Additional amine (0.15 mol)
and NaB(OAc)3H (0.055 g, 0.24 mmol) were added, and the mixture
was irradiated at 130 °C for 5 min. This gave 100% conversion to
product. The reaction mixture was filtered and concentrated. The
residue was dissolved in methanol (1.5 mL) and concd HCl
(0.5 mL) and irradiated using microwaves at 100 °C for 5 min.
The mixture was filtered and purified by flash chromatography
4.1.4.1. Ethyl 5-formyl-1-methyl-2-((phenylsulfonyl)methyl)-
1H-indole-3-carboxylate (8a). Gray-white powder (95%); mp:
136–138 °C; 1H NMR (CDCl3): d 1.3 (t, J = 7.2 Hz, 3H, –OCH2CH3);
4.03 (s, 3H, –NCH3); 4.15 (q, J = 7.2 Hz, 2H, –OCH2CH3); 5.33 (s,
2H, –CH2-sulfonyl–); 7.45–7.75 (m, 6H, –UH and –PhH); 7.95 (d,
1H, –UH); 8.56 (s, 1H, –UH); 10.12 (s, 1H, –COH); MS:
m/z 386.1 [MH+].
over silica gel to give the desired compounds 9a3–5
.
4.1.7.1. Ethyl 1-methyl-5-((4-methylpiperazin-1-yl)methyl)-2-
((phenylsulfonyl)methyl)-1H-indole-3-carboxylate (9a3). Elu-
tion with benzene/MeOH (9:1) afforded 9a3 (0.045 g, 94%) as a
brownish-yellow powder; mp: 94–96 °C; 1H NMR (CDCl3): d 1.3
(t, J = 7.2 Hz, 3H, –OCH2CH3); 2.57 (s, 3H, 4-CH3 of piperazinyl);
2.75–3.05 (m, 8H, 8H of piperazinyl); 3.75 (s, 2H, –CH2N<); 3.95
(s, 3H, –NCH3); 4.05 (q, J = 7.2 Hz, 2H, –OCH2CH3); 5.25 (s, 2H,
–CH2-sulfonyl–); 7.3–7.7 (m, 7H, –UH and –PhH); 7.95 (s, 1H,
–UH); MS: m/z 470.1 [MH+].
4.1.4.2. Ethyl 5-formyl-1,2-dimethyl-1H-indole-3-carboxylate
(8b). Beige powder (92%); mp: 118–120 °C; 1H NMR (CDCl3): d 1.5
(t, J = 7.2 Hz, 3H, –OCH2CH3); 2.83 (s, 3H, –CH3); 3.77 (s, 3H,
–NCH3); 4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3); 7.45 (d, 1H, –UH);
7.88 (dd, J = 8.55, 1.75 Hz, 1H, –UH); 8.67 (s, 1H, –UH); 10.12 (s,
1H, –COH); MS: m/z 246.1 [MH+].
4.1.5. General procedure for the synthesis of compounds 9a1–2
Compound 8a (0.04 g, 0.1 mmol), dimethylamine or pyrrolidine
(0.12 mmol), acetic acid (0.06 mL, 1.02 mmol) and NaB(OAc)3H
(0.05 g, 0.21 mmol) were, in that order, added to dry THF (4 mL).
The mixture was irradiated with microwaves for 10 min at
130 °C. Additional amine (0.12 mol) and NaB(OAc)3H (0.05 g,
0.21 mmol) were added, and the mixture was irradiated at 130 °C
for 5 min. This gave 100% conversion to product. The reaction mix-
ture was filtered and concentrated. The residue was dissolved in
methanol (1.5 mL) and concd HCl (0.5 mL) and irradiated using
microwaves at 100 °C for 5 min. The mixture was filtered and puri-
fied by flash chromatography over silica gel (benzene/methanol,
4.1.7.2. Ethyl 1-methyl-5-(morpholinomethyl)-2-((phenylsulfo-
nyl)methyl)-1H-indole-3-carboxylate (9a4). Elution with ben-
zene/MeOH (95:5) afforded 9a4 (0.039 g, 85%) as an rose powder;
mp: 109–111 °C; 1H NMR (CDCl3):
d 1.3 (t, J = 7.2 Hz, 3H,
–OCH2CH3); 2.53 (s, 4H, 4H of morpholino); 3.65 (s, 2H, –CH2N<);
3.75 (s, 4H, 4H of morpholino); 3.95 (s, 3H, –NCH3); 4.05 (q,
J = 7.2 Hz, 2H, –OCH2CH3); 5.25 (s, 2H, –CH2-sulfonyl–); 7.3–7.7
(m, 7H, –UH and –PhH); 7.95 (s, 1H, –UH); MS: m/z 457.0 [MH+].
4.1.7.3. Ethyl 5-((1,4-diazepan-1yl)methyl)-1-methyl-2-((phen-
ylsulfonyl)methyl)-1H-indole-3-carboxylate (9a5). Elution with
benzene/MeOH/NH4OH (9:1:0.25) afforded 9a5 (0.04 g, 83%) as a
9:1) to give the desired compounds 9a1–2
.
white powder; mp: 98–100 °C; 1H NMR (CD3OD):
d 1.3 (t,
4.1.5.1. Ethyl 5-((dimethylamino)methyl)-1-methyl-2-((phenyl-
sulfonyl)methyl)-1H-indole-3-carboxylate (9a1). Sandy powder
(0.033 g, 78%); mp: 205–208 °C; 1H NMR (CDCl3): d 1.45 (t,
J = 7.2 Hz, 3H, –OCH2CH3); 2.45 (s, 6H, –N(CH3)2); 3.82 (s, 2H,
–CH2N<); 3.95 (s, 3H, –NCH3); 4.05 (q, J = 7.2 Hz, 2H, –OCH2CH3);
5.25 (s, 2H, –CH2-sulfonyl–); 7.3–7.75 (m, 7H, –UH and –PhH);
8.0 (s, 1H, –UH); MS: m/z 415.0 [MH+].
J = 7.2 Hz, 3H, –OCH2CH3); 2.0 (s, 1H, –NH of homopiperazine);
2.1 (m, 2H, 2H of homopiperazine); 2.95 (t, 2H, 2H of homopiper-
azine); 3.1 (t, 2H, 2H of homopiperazine); 3.35 (t, 4H, 4H of homo-
piperazine); 3.90 (s, 3H, –NCH3); 4.0 (s, 2H, –CH2N<); 4.15 (q,
J = 7.2 Hz, 2H, –OCH2CH3); 5.4 (s, 2H, –CH2-sulfonyl–); 7.4–7.75
(m, 7H, –UH and –PhH); 8.05 (s, 1H, –UH); MS: m/z 470.1 [MH+].
4.1.8. General procedure for the synthesis of compounds 9b1–2
Compound 8b (0.04 g, 0.1 mmol), dimethylamine or pyrrolidine
(0.12 mmol), acetic acid (0.095 mL, 1.02 mmol) and NaB(OAc)3H
(0.074 g, 0.21 mmol) were, in that order, added to dry THF
(4 mL). The mixture was irradiated with microwaves for 10 min
at 130 °C. Additional amine (0.12 mmol) and NaB(OAc)3H
4.1.5.2. Ethyl 1-methyl-2-((phenylsulfonyl)methyl)-5-((pyrroli-
din-1yl)methyl)-1H-indole-3-carboxylate (9a2). Beige powder
(0.039 g, 87%); mp: 118–120 °C; 1H NMR (CDCl3):
d 1.3 (t,
J = 7.2 Hz, 3H, –OCH2CH3); 1.87 (s, 4H, –pyrrolidinyl H); 2.73 (s,
4H, –pyrrolidinyl H); 3.82 (s, 2H, –CH2N<); 3.95 (s, 3H, –NCH3);

6148
G. Sellitto et al. / Bioorg. Med. Chem. 18 (2010) 6143–6148
(0.074 g, 0.21 mmol) were added, and the mixture was irradiated
at 130 °C for 5 min. This gave 100% conversion to product. The
reaction mixture was filtered and concentrated. The residue was
dissolved in methanol (1.5 mL) and concd HCl (0.5 mL) and irradi-
ated using microwaves at 100 °C for 5 min. The mixture was fil-
tered and purified by flash chromatography over silica gel
Technologies) supplemented with 2 mM L-glutamine, 100 U/mL
of penicillin, 100 lg/mL of streptomycin, and 10% fetal bovine ser-
um.13 Cells were sub-cultivated twice per week, with a 1:4 split
ratio.
4.2.2. In vitro anti-HCV assay
(benzene/methanol, 95:5) to give the desired compounds 9b1–2
.
The antiviral activities of compounds 9a1–5, 9b1–2 against HCV
were measured in Huh-7.5 hepatoma cells using replication and
entry assays as previously described by Pacini et al.14 For the rep-
lication assay, an RNA transcript of a replicon containing a firefly
luciferase reporter was transfected in Huh-7.5 cells; these were
subsequently plated on 96-well plates in the presence of test
compounds and Arbidol. For the infection assay, viral particles
containing a subgenomic HCV RNA carrying a firefly luciferase re-
porter gene were used to infect naive Huh-7.5 cells; these were
plated in a 96-well format in the presence of test compounds
and Arbidol. After three days of incubation luciferase activity
was measured with a Bright-Glo luciferase assay system (Prome-
ga) following the manufacturer’s instructions. The IC50 and selec-
tivity indices of the evaluated compounds and Arbidol were
calculated.
4.1.8.1. Ethyl 5-((dimethylamino)methyl)-1,2-(dimethyl)-1H-
indole-3-carboxylate (9b1). Bone powder (0.033 g, 75%); mp:
67–69 °C; 1H NMR (CDCl3): d 1.45 (t, J = 7.2 Hz, 3H, –OCH2CH3);
2.65 (s, 6H, –N(CH3)2); 2.83 (s, 3H, –CH3); 3.77 (s, 3H, –NCH3);
4.30 (s, 2H, –CH2– N<); 4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3); 7.45
(d, 1H, –UH); 7.60 (dd, J = 8.55, 1.75 Hz, 1H, –UH); 8.15 (s, 1H,
–UH); MS: m/z 275.0 [MH+].
4.1.8.2. Ethyl 5-((dimethylamino)methyl)-1,2-(dimethyl)-1H-
indole-3-carboxylate (9b2). Nut-brown powder (0.04 g, 83%);
mp: 125–127 °C; 1H NMR (CDCl3): d 1.45 (t, J = 7.2 Hz, 3H,
–OCH2CH3); 1.59 (m, 4H, –pyrrolidinyl H); 2.25 (m, 4H, –pyrrolidinyl
H); 2.83 (s, 3H, –CH3); 3.77 (s, 3H, –NCH3); 4.30 (s, 2H, –CH2– N<);
4.45 (q, J = 7.2 Hz, 2H, –OCH2CH3); 7.45 (d, 1H, –UH); 7.78 (dd,
J = 8.55, 1.75 Hz, 1H, –UH); 8.15 (s, 1H, –UH); MS: m/z 301.0 [MH+].
4.2.3. Cytotoxicity assay
Cytotoxicity induced by the test compounds in cultures of cells
was also determined.
4.1.9. Synthesis of ethyl 5-(hydroxymethyl)-1-methyl-2-((phenyl
sulfonyl)methyl)-1H-indole-3-carboxylate (10a)
The toxicity of compounds 9a1–5 and 9b1–2 in Huh-7.5 cells
was evaluated by CellTiter-Blue Viability Assay (Promega) that
is used to estimate the number of viable cells present in multi-
well plates; the reagent is a buffered solution containing highly
purified resazuin. Resazuin is dark blue in color and has little
intrinsic fluorescence. Metabolically active cells can reduce resa-
zuin to resorufin, which is pink and highly fluorescent, with an
excitation maximum of 579 nm and an emission maximum of
584 nm. Nonviable cells rapidly lose metabolic capacity, do not
reduce the indicator dye, and thus do not generate a fluorescent
signal.
To an ice-cooled solution of the corresponding aldehyde 8a
(0.12 g, 0.3 mmol) in THF (1.5 mL) was added NaBH4 (0.029 g,
0.75 mmol) in methanol (3.8 mL). Then, the resulting mixture
was stirred at rt for 1 h. After complete conversion, the solvent
was distilled off under reduced pressure. Then, water was added,
and the resulting mixture was extracted with ethyl acetate. The
combined organic phases were washed with brine, dried over
Na2SO4 and evaporated under reduced pressure. The desired prod-
uct was purified by chromatography on silica gel (hexane/EtOAc
6:4) to give 10a (0.106 g, 91%) as white solid; mp: 172–174 °C;
1H NMR (CDCl3): d 1.3 (t, J = 7.2 Hz, 3H, –OCH2CH3); 2.0 (s, 1H,
–CH2OH); 3.97 (s, 3H, –NCH3); 4.05 (q, J = 7.2 Hz, 2H, –OCH2CH3);
4.82 (s, 2H, –CH2-sulfonyl–); 5.28 (s, 2H, –CH2OH); 7.4–7.75 (m,
7H, –UH and –PhH); 8.05 (s, 1H, –UH); MS: m/z 388.1 [MH+].
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.058.
4.1.10. Synthesis of ethyl 5-((1H-imidazol-1-yl)methyl)-1-methyl-
2-((phenyl sulfonyl)methyl)-1H-indole-3-carboxylate (9a6)
To a solution of compound 10a (0.06 g, 0.15 mmol) in acetoni-
trile (1.5 mL) was added CDI (0.127 g, 0.75 mmol) and imidazole
(0.021 g, 0.3 mmol). The resulting solution was heated to reflux
for 16 h. After cooling to ambient temperature, the reaction mix-
ture was diluted with water and extracted with ethyl acetate.
The combined organic phases were washed with brine, dried over
Na2SO4 and evaporated under reduced pressure. The desired prod-
uct was purified by chromatography on silica gel (dichlorometh-
ane/methanol, 98:2) to obtain 9a6 (0.044 g, 67%) as a pearl-white
powder; mp: 95–97 °C; 1H NMR (CDCl3): d 1.3 (t, J = 7.2 Hz, 3H,
–OCH2CH3); 3.97 (s, 3H, –NCH3); 4.05 (q, J = 7.2 Hz, 2H,
–OCH2CH3); 5.28 (s, 4H, –CH2-sulfonyl– and –CH2N<); 6.95 (s,
1H, 2-1H of imidazolyl) 7.05–7.2 (m, 2H, 4,5-2H of imidazolyl);
7.4–7.75 (m, 7H, –UH and –PhH); 7.9 (s, 1H, –UH); MS: m/z 438.1
[MH+].
References and notes
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4.2. Biological assay
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14. Pacini, L.; Graziani, L. B.; Bartholomew, L.; De Francesco, R.; Paonessa, G. J. Virol.
2009, 83, 9079.
4.2.1. Cell lines and culture conditions
The human hepatoma-derived cell line Huh-7.5 was grown in
high-glucose Dulbecco’s modified Eagles’ medium (DMEM; Life