Inhibition of hepatitis C virus NS5B polymerase by S-trityl-l-cysteine derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.01.010

Structure-based studies led to the identification of a constrained derivative of S-trityl-l-cysteine (STLC) scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives were synthesized and investigated for the

Full text of DOI:10.1016/j.ejmech.2012.01.010

European Journal of Medicinal Chemistry 49 (2012) 191e199
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Inhibition of hepatitis C virus NS5B polymerase by S-trityl- -cysteine derivatives
L
Daniel B. Nichols a, Guy Fournet b, K.R. Gurukumar a, Amartya Basu a, Jin-Ching Lee c, Naoya Sakamoto d,
,
f
,
a,
*
Frank Kozielski e, Ira Musmuca f, Benoît Joseph b , Rino Ragno
, Neerja Kaushik-Basu
**
***
a Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103, USA
b Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Université de Lyon, Université Claude Bernard e Lyon 1, Bâtiment Curien,
43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne, France
c Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
d Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
e The Beatson Institute for Cancer Research, Molecular Motors Laboratory, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK
f Rome Center for Molecular Design, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienzà Universita id Roma, P.le A. Moro 5, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure-based studies led to the identification of a constrained derivative of S-trityl-L-cysteine (STLC)
Received 20 December 2011
Received in revised form
3 January 2012
Accepted 5 January 2012
Available online 12 January 2012
scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives
were synthesized and investigated for their activity against HCV NS5B. Three STLC derivatives, 9, F-3070,
and F-3065, were identified as modest HCV NS5B inhibitors with IC50 values between 22.3 and 39.7 mM.
F-3070 and F-3065 displayed potent inhibition of intracellular NS5B activity in the BHK-NS5B-FRLuc
reporter and also inhibited HCV RNA replication in the Huh7/Rep-Feo1b reporter system. Binding
mode investigations suggested that the STLC scaffold can be used to develop new NS5B inhibitors by
further chemical modification at one of the trityl phenyl group.
Keywords:
Antiviral agents
Hepatitis C
Ó 2012 Elsevier Masson SAS. All rights reserved.
HCV NS5B polymerase
Inhibitors
STLC derivatives
1. Introduction
of additional, efficacious and more cost effective HCV antiviral
therapies that target viral proteins and have limited effects on host
Hepatitis C virus (HCV) infection represents a major public-
health concern. It is estimated that over 200 million people, w3%
of the world population, are chronically infected with the virus
[1e3]. HCV has an array of immune evasion strategies and can
persist in the host for years. Individuals with chronic HCV infection
are at increased risk of developing cirrhosis and hepatocellular
carcinoma [3e7]. Currently, HCV infections are treated by
a combination of pegylated-interferon, the nucleoside analog
ribavirin, and one of two recently approved HCV protease inhibi-
tors, Boceprevir or Telaprevir [8e13]. However, this therapy is
limited in efficacy against the various HCV genotypes. Furthermore,
in addition to its high cost, the current treatment is associated with
severe side effects and a complicated dosing regimen that may limit
patient compliance [11,12]. Also the possibility of selecting drug
resistant HCV variants remains [12,13]. Therefore, the development
biological processes is a priority.
HCV is a member of the Flaviviridae family. The positive sense,
9.6 kb RNA genome is translated into a single 3000 amino acid
polyprotein via an IRES sequence located within the 50 non-
translated region (NTR) of the viral genome [14,15]. The viral pol-
yprotein is processed by both host and viral proteases into indi-
vidual viral proteins consisting of four structural (core, E1, E2, and
p7) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A,
and NS5B) [16]. HCV replicates exclusively in the cytoplasm of host
cells. Replication of the viral RNA genome is mediated by the RNA-
dependent RNA polymerase (RdRp) activity of the HCV nonstruc-
tural protein NS5B [17e19]. Because of the absolute requirement of
NS5B to synthesize nascent HCV RNA, NS5B represents an attractive
target for the development of anti-HCV inhibitors [20,21].
Furthermore, host cells lack RdRp. Therefore, an inhibitor that
blocks RdRp activity should, in theory, have minimal or no effect on
host biological processes. Though, a number of NIs and NNIs with
potent in vitro anti-NS5B activity have been identified in recent
years, they have presented challenges of toxicity and selection of
resistant viruses, thus necessitating identification of better NS5B
inhibitor scaffolds.
* Corresponding author. Tel.: þ1 973 972 8653; fax: þ1 973 972 5594.
** Corresponding author. Tel.: þ33 4 72448135; fax: þ33 4 72431214.
*** Corresponding author. Tel.: þ396 4991 3937; fax: þ396 4991 3627.
E-mail addresses: benoit.joseph@univ-lyon1.fr (B. Joseph), rino.ragno@
uniroma1.it (R. Ragno), kaushik@umdnj.edu (N. Kaushik-Basu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.010

192
D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
The structure of NS5B has been extensively characterized. The
2. Results and discussion
66 kDa viral polymerase resembles a “right hand” with the active
site contained in the palm domain and the RNA interacting region
in the finger and thumb domains [22e25]. Current NS5B inhibitors
can be divided into two classes, nucleoside inhibitors (NI) and non-
nucleoside inhibitors (NNI). Once converted by host proteins into
nucleotides, NIs cause RNA-chain termination upon incorporation
by NS5B into the nascent RNA chains. NNIs bind to one of the five
allosteric sites on NS5B and inhibit the initiation step of RNA
synthesis.
Recently, we reported on the utility of three-dimensional
quantitative structure-activity relationship (3D-QSAR) in combi-
nation with ligand-based and structure-based alignment proce-
dures for in silico screening of new HCV NS5B polymerase
inhibitors [26]. This investigation identified four new NS5B inhib-
itors from forty candidates examined from the NCI diversity set
[26]. The most interesting hit, NSC123526 (Fig. 1), has been re-
ported to be active against other viruses [27] and can be simply
2.1. Molecular modeling
Recently, we utilized structure-based 3-D QSAR modeling to
identify NS5B thumb-binding inhibitors and reported on the
identification of NSC123526 as a modest HCV NS5B inhibitor [26].
NSC123526 can be considered as a constrained STLC derivative
(Fig. 1). Since STLC derivative NSC123139 (Fig. 1) was found to be
most potent in inhibiting HMKEg, we performed cross-docking
experiments to investigate whether it could also bind the HCV-
NS5B thumb domain [26,29]. Fig. 2 depicts the docked conforma-
tion of NSC123139 in HCV-NS5B and HMKEg.
The activity of the docked NSC123139 (Predicted pIC50 ¼ 5.64)
was predicted by our 3-D QSAR model in the same range of
NSC123526 (Experimental pIC50 ¼ 4.33, predicted pIC50 ¼ 5.4) [26].
However, NSC123139 exhibited a much weaker inhibition of NS5B
RdRp activity in vitro (Table 1), compared to NSC123526, as
previously reported [26].
viewed as a constrained derivative of the S-trityl-L-cysteine (STLC)
scaffold. STLC derivatives are versatile compounds endowed with
antileukemic activity [28] and are also reported to inhibit the
human mitotic kinesin Eg5 (HMKEg) by a non-competitive mech-
anism [29].
Herein, we describe molecular modeling studies that led us to
explore the potential of STLC and its derivatives to inhibit HCV
NS5B RdRp activity in vitro. Further, we examined the effect of STLC
derivatives on intracellular HCV NS5B RdRp activity and on HCV
RNA replication. Among the tested STLC derivatives, we identified
three compounds as novel HCV NS5B inhibitor leads. These
compounds merit further optimization through classical medicinal
chemistry and virtual screening.
Based on the above partial results, we tested a series of STLC
derivatives for their ability to inhibit NS5B, with the objective of
identifying new lead scaffolds. While our investigations with
additional STLC derivatives were still ongoing, the co-crystal
structures of HMKEg with NSC123139 (pdb entry code 2wog and
2xae) and other STLCs (2xr2 and 3ken) were released [30]. Never-
theless, docking calculations performed through Autodock Vina,
were in good agreement with the experimental results
(rmsd ¼ 0.44) and with similar docking calculations previously
reported [31], thus supporting our protocol.
The above docking protocol was also applied to the other STLCs.
In addition, we analyzed the Autodock Vina proposed binding
Fig. 1. Structures of NSC123526, STLC, STDC and STLC derivatives.

D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
193
Fig. 2. Molecular docking of STLC derivatives in NS5B. Panel A: Docked conformations of NSC123139 (red-colored carbon atoms) and NSC123526 (green-colored carbon atoms)
within NS5B thumb domain. Panel B: HMKEg (PDB entry code 2fme) with docked conformation of NSC123139 (red-colored carbon atoms) and the experimental bound NSC123526
(green-colored carbon atoms). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
mode of the two most active compounds, F-3065 and F-3070, cross-
docked into the 15 NS5B-NNI co-crystal structures as previously
described by us [26]. As expected, docked conformations of F-3065
and F-3070 ((R) and (S) enantiomers of the same compound,
respectively) exhibited the lowest binding energy in the PDB entry
2d3u. Further, the bound conformations of F-3065 and F-3070, in
agreement with the biological data, revealed that the cysteine
stereocenter does not affect the overall binding mode, wherein the
terminal amino acid group is involved in a hydrogen bonding
network, as shown in the ligplot diagrams in Fig. 3. In particular the
2.2. Chemistry
A total of 35 STLC derivatives were utilized in this study (Fig. 1
and Scheme 1). STLC and 14 derivatives (1e14) have been re-
ported previously [29,31]. Compounds F-3070 and F-3065 were
purchased from Bachem, while STDC (NSC123676), NSC123139,
NSC136870, NSC140909, NSC123529, NSC123138, and NSC126217
were procured from NCI/NIH. In accordance with published liter-
ature, 12 STLC derivatives were newly synthesized for this inves-
tigation (Scheme 1). Starting aryldiphenylmethanol compounds 15
were prepared in good yields from appropriate esters (ArCO2Me)
and phenylmagnesium chloride (data not shown) [32]. Condensa-
tion of cysteamine.HCl (16) with Ar(Ph)2COH (Ar ¼ 4-MeePh, 4-
EtePh, 4-n-PrePh, 4-MeSePh, 4-IePh, 4-(Ph)ePh and 2-
naphthyl) 15aeg in TFA gave final compounds 17aeg in 29e47%
a
-amino acid portion of F-3065 makes two hydrogen bonds, one
between its amino group and the carbonyl group of Trp528 (N/O
distance ¼ 3.01 Å) and the other between a carboxy oxygen and the
ε-amino group of Lys533 (O/N distance ¼ 2.98 Å) (Fig. 3A). The
ligplot diagram of the (S) enantiomer F-3070, that forms two
hydrogen bonds with its two carboxy oxygens, one with the gua-
nidinic nitrogen of Arg501 (O/N distance ¼ 3.21 Å) and the other
with ε-amino group of Lys533 (O/N distance 3.05e3.22 Å), is
shown in Fig. 3B. This type of hydrogen bonding network was
observed in all other STLC derivatives (Fig. 5) suggesting that
hydrogen bonds are the leading interactions.
yield (Scheme 1). Treatment of
L-cysteine (18) or L-penicillamine
(19) with Ar(Ph)2COH (Ar ¼ 4-(n-C5H11)-Ph, 4-(n-C6H13)-Ph, 4-PrO-
Ph and 4-n-Bu-Ph) 15hek in the presence of BF3$Et2O afforded
target compounds 17hel in 30e55% yield (Scheme 1).
2.3. Biological studies
Other notable interactions are hydrophobic in nature, and the
trityl moieties are buried in the thumb allosteric binding side
(Fig. 4). For both F-3065 and F-3070, one phenyl is placed in
a pocket formed by Leu419, Arg422, Met423 and Trp528, while the
other two benzenes fill-up two depressions on the enzyme surface.
By comparing the binding mode of the most active STLCs with that
of the experimental co-crystallized compound found in 2d3u and
considering the conserved binding modes shown in Fig. 5, we
believe the STLC can be used as a starting scaffold, whose activity
could be improved by inserting a side chain in one of the two
surface bound benzene rings to better fill the binding cleft formed
by Leu419, Met423, Ile482, Val485, Ala486, Leu489 and Leu497
(Fig. 6) and occupied by a 2-(4-cyanophenyl)thiophene group in the
original complex (PDB ID 2d3u). As expected and within the limit of
any predictive model, the application of our 3-D QSAR to all the
new STLCs, predicted these compounds to have activities between
With the objective of identifying novel HCV NS5B inhibitors, we
investigated STLC and its derivatives employing the in vitro NS5B
RdRp inhibition assay as described previously [33e35]. Recombi-
nant HCV NS5B (genotype 1b) carrying an N-terminal His-tag and
C-terminal 21-amino acid truncation (NS5BCΔ21) was purified to
homogeneity by Ni-NTA chromatography and used as a source of
enzyme [33e35]. Wedelolactone, a documented NS5B inhibitor,
was employed as an internal reference standard, and yielded an IC50
value of 36.0 mM (data not shown), consistent with our previously
reported value [34]. In order to identify a wider range of NS5B
inhibitor candidates, preliminary screening of STLC and its deriva-
tives was conducted at 100 mM compound concentration. While the
parent STLC molecule yielded only w12% inhibition of NS5B RdRp
activity during preliminary screening, its thirty-five derivatives,
with the exception of 17l, exhibited a much higher inhibition
ranging from 14 to 83% (Table 1). Of these, three compounds 9, F-
the 10e100
mM range (data not shown).

194
D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
Table 1
Anti-HCV NS5B RdRp activity of STLC derivatives.
Compound
% Inhibitiona
IC50 (m
M)b
STLC
STDC
NSC123139
NSC136870
NSC140909
NSC123529
NSC123138
NSC126217
1
2
3
4
5
6
7
8
12.6 ꢂ 2.3
17.0 ꢂ 0.6
23.1 ꢂ 1.6
23.4 ꢂ 2.9
30.9 ꢂ 3.7
14.7 ꢂ 1.5
28.3 ꢂ 5.9
20.2 ꢂ 2.5
22.4 ꢂ 5.4
22.6 ꢂ 2.0
36.8 ꢂ 2.4
31.2 ꢂ 1.5
20.5 ꢂ 1.1
36.9 ꢂ 2.5
43.5 ꢂ 0.8
44.3 ꢂ 3.0
60.0 ꢂ 3.4
17.2 ꢂ 2.9
19.1 ꢂ 1.7
22.5 ꢂ 2.2
34.0 ꢂ 1.1
33.1 ꢂ 0.7
31.7 ꢂ 1.8
28.7 ꢂ 2.1
27.4 ꢂ 4.2
24.0 ꢂ 4.5
36.7 ꢂ 2.1
36.0 ꢂ 1.0
33.3 ꢂ 2.3
28.3 ꢂ 5.9
16.1 ꢂ 3.0
14.0 ꢂ 3.3
22.0 ꢂ 1.4
n.i.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
9
10
11
12
13
14
39.7 ꢂ 0.9
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
17l
F-3070
E-3205
F-3065
82.8 ꢂ 1.3
40.2 ꢂ 0.7
76.7 ꢂ 2.4
22.3 ꢂ 5.9
n.d.
24.6 ꢂ 6.0
n.d., not determined.
n.i., no inhibition.
a
Percent inhibition was determined at 100
m
M concentration of the indicated
compound and represents an average of at least two independent measurements in
duplicate.
b
The IC50 values of the compounds were determined from dose-response curves
employing 8e12 concentrations of each compound in duplicate in two independent
experiments. Curves were fitted to data points using nonlinear regression analysis
and IC50 values were interpolated from the resulting curves using GraphPad Prism
3.03 software.
Fig. 3. Panel A: Ligplot diagram for F-3065 docked in NS5B (PDB ID 2d3u). Panel B:
Ligplot diagram for F-3070 docked in NS5B (PDB ID 2d3u).
3070, and F-3065 having ꢀ60% anti-NS5B activity at 100
m
M were
reporter as an indicator of HCV RNA replication, and has beenwidely
employed to identify inhibitors of HCV RNA replication [37].
Only three STLC derivatives F-3070, F-3065, and E-3205
inhibited intracellular NS5B RdRp activity in the BHK-NS5B-FRLuc
further pursued for their IC50 value determination. This analysis
resulted in the identification of F-3070 and F-3065 with near
similar IC50 values, as the two most potent of the 36 STLC deriva-
tives examined in this investigation, while 9 exhibited w1.6e1.8-
fold higher IC50 value compared to the two afore-mentioned
compounds. Together, these data suggest that STLC scaffold may
offer further scope for improvement of its anti-NS5B activity.
To evaluate the anti-HCV activity of STLC compounds in a more
biologically relevant setting, we employed the BHK-NS5B-FRLuc
reporter and the Huh7/Rep-Feo1b reporter systems [36,37]. The
former reporter system carries stably transfected NS5B and a bicis-
tronic reporter gene, (þ)FLuc-(ꢁ)UTR-RLuc for cell based investi-
gations of HCV NS5B RdRp inhibitors [36]. The advantage of this
system is that it can simultaneously measure intracellular HCV
NS5B RdRp activity as reflected by the ratio of Renilla to firefly
luciferase luminescence and cellular viability which is reflected by
the firefly luciferase luminescence, thus enabling the identification
of potent non-toxic inhibitors. The Huh7/Rep-Feo1b reporter
system, on the other hand, autonomously replicates the subgenomic
HCV genotype 1b replicon RNA carrying the firefly luciferase
reporter at 100 mM concentration (Table 2). The two more potent
of these, F-3070 and F-3065 exhibited ꢀ84% inhibition while E-
3205 displayed only w44% inhibition of NS5B RdRp activity,
consistent with the in vitro data. In terms of their cytotoxicity
parameters, F-3070 and F-3065 did not affect cell viability at
100 mM, as was evident from equivalent levels of firefly luciferase
luminescence in compound treated cells versus DMSO controls.
Treatment with E-3205 however, decreased cell viability by w70%
at 100
tives as well as the parent molecule, exhibited ꢀ50% reduction in
cell viability at 100 M, with only a marginal 15e30% decrease in
mM concentration. The remaining thirty-three STLC deriva-
m
intracellular NS5B activity (data not shown), consistent with the
in vitro RdRp data.
In the Huh7/Rep-Feo1b reporter system, compounds F-3070
and F-3065 exhibited an overall similar pattern of cell viability and
HCV RNA replication inhibition, corresponding to w73e74% and
w89e91%, respectively at 100 mM concentration (Table 2). E-3205,

D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
195
Fig. 6. F-3065 (orange) and F-3070 (cyan) overlapped on the 2d3u co-crystallized
ligand (green). HCV NS5B (PDB ID 2d3u, in pink ribbon) and the thumb allosteric
surface (in atom type color) are also shown. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Docked conformation of F-3065 (orange) and F-3070 (cyan) in NS5B (PDB ID
2d3u). The enzyme surface is shown in atom type color. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
this article.)
This study validates structure-based molecular modeling coupled
with 3-D QSAR prediction, as a viable strategy for identification of
new structural scaffolds targeting NS5B. STLC binding mode anal-
ysis revealed a common way by which STLCs bind to the HCV NS5B
thumb allosteric site and further suggested that improved STLC
derivatives may be achieved by chemical modification at one of the
trityl phenyl ring.
however, exhibited decreased cell viability (44%) compared to the
other two compounds, though its inhibition of HCV RNA replication
(w89%) was similar. It is worth noting here that the inhibition
observed in this system may be partly attributed to the cellular
toxicity effects of these compounds.
The results in this present study suggest that STLC derivatives
inhibit HCV RNA replication by targeting the NS5B polymerase. It is
possible that other host factors such as HMKEg are also targeted by
STLCs in the HCV replicase complex and needs to be elucidated.
These studies provide a platform to optimize the STLC scaffold as
a potent anti-NS5B inhibitor. An extensive focused virtual screening
approach is ongoing on a database constituted of more than 500 K
trityl cysteine analogs to optimize the newly reported lead
compounds.
4. Experimental section
4.1. Molecular modeling
All molecules were generated by means of molecular mechanics
of Chemaxon Marvin software (http://www.chemaxon.com/).
Molecular graphics images were produced using UCSF Chimera
package from the Resource for Biocomputing, Visualization, and
Informatics at the University of California, San Francisco on a 3 GHz
AMD CPU equipped IBM-compatible workstation with the Debian
5.0 version of the Linux operating system. Different from the
previous protocol, the faster Autodock Vina [38] docking program
was used in place of Autodock for all docking studies. Docking
assessment was conducted via re-docking, re-docking modeled,
cross-docking and cross-docking modeled as previously reported
[26]. Autodock Vina proved to be as good as Autodock (data not
shown), but much faster in calculations. The compounds were then
submitted for structure-based molecular alignment through cross-
docking protocols as previously reported [26]. For activity predic-
tions, the previously developed SB 3-D QSAR model was applied
without any modification [26]. The program ligplot v. 4.0 was used
[39] to generate the ligand/NS5B interaction maps.
3. Conclusion
In summary, STLC derivatives were identified as novel inhibitors
of HCV NS5B polymerase activity in vitro and in cell based assays.
4.2. Chemistry
General methods: Melting points were determined using a Büchi
capillary instrument and are uncorrected. Optical rotations were
measured at the sodium D line (589 nm) at 25 ꢃC with a Per-
kineElmer 241 polarimeter using a 1 dm path length cell. 1H and
13C NMR spectra were recorded on a Bruker 300, 400 or 500 MHz
spectrometers. Chemical shifts (d) are in parts per million. The
following abbreviations were used to designate the multiplicities:
s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet,
br ¼ broad. Mass spectra were recorded with a PerkineElmer SCIEX
Fig. 5. STLC analogues docked within the HCV NS5B (in pink ribbon) thumb allosteric
surface. The compounds overlap in this pocket. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)

196
D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
R
NH2
NH3.Cl
a
S
R
OH
+
HS
16
15a R- = Et
15d R- = MeS
17a R- = Et
17d R- = MeS
15b R- = Ph 15e R- = I
17b R- = Ph 17e R- = I
15c R- = n-Pr 15f R- = Me
17c R- = n-Pr 17f R- = Me
NH2
NH3.Cl
a
b
S
R
OH
+
+
HS
16
15g
17g
R
CO2H
NH2
CO2H
S
R
OH
NH2
HS
18
15h R- = n-C5H11
15i R- = n-C6H13
15j R- = n-PrO
17h R- = n-C5H11
17i R- = n-C6H13
17j R- = n-PrO
R
CO2H
NH2
CO2H
NH2
b
S
R
OH
+
HS
19
15k R- = n-Bu
15j R- = n-PrO
17k R- = n-Bu
17l R- = n-PrO
Scheme 1. Synthesis of STLC derivatives 17ael. Reagents and conditions: (a) TFA, rt, 3 h; (b) BF3$Et2O, AcOH, rt, 2 h.
API spectrometer. Elemental analyses were performed on a Ther-
moquest Flash 1112 series EA analyzer. Elemental analyses were
found to be within ꢂ0.4 of the theoritical values. Purity of tested
compounds was >95%. All commercially available reagents and
solvents were used without further purification. STLC and deriva-
tives 1e14 have been previously described [29]. E-3205, F-3070 and
F-3065 were purchased from Bachem. STDC (NSC124676),
NSC123139, NSC136870, NSC140909, NSC123529, NSC123138, and
NSC126217 were procured from NCI/NIH.
Table 2
Anti-HCV effects of STLC derivatives in cell based reporter assay.
Compound
BHK-NS5B-FR Luca
Huh7/Rep-Feo1bb
Viability (%)
Inhibition (%)
Viability (%)
Inhibition (%
F-3070
E-3205
F-3065
100.0
30.2
100.0
85.4
44.4
84.3
72.6
44.2
74.1
89.5
88.6
91.2
aBHK-NS5B-FRLuc and bHuh7/Rep-Feo1b reporter cells were treated with the
indicated compounds at 100 M concentration for 42 h. Cell viability in the BHK-
4.2.1. General procedure for preparation of compounds 17aeg
At 0 ꢃC and under argon atmosphere, a solution of cysteamine
HCl (16) (1.33 mmol) was added dropwise to a solution of appro-
priate alcohol 15 (1.33 mmol) in TFA (5 mL). The reaction mixture
was stirred at room temperature for 1 h, evaporated and extracted
with a saturated solution of NaHCO3(aq) and EtOAc. The organic
m
NS5B-FRLuc reportera was estimated as the relative levels of Firefly luciferase in
compound treated cells versus DMSO controls, while that in the Huh7/Rep-Feo1b
cellsb was evaluated by the MTS assay. The inhibitory effect of the compounds on
NS5B RdRp activitya and HCV RNA replicationb is presented as percent of DMSO
treated controls. Data represents an average of three independent experiments in
duplicate.

D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
197
phase was dried over MgSO4, filtered, and evaporated under
vacuum. The oil was crystallized from Et2O or Et2O/pentane 1:1. The
desired compounds 17aeg were obtained by filtration in the range
of 29e47% yield.
H
Ar), 7.24e7.34 (m, 8H, HAr), 7.41e7.45 (m, 4H, HAr); 13C NMR
(100 MHz, DMSO-d6): 20.5 (CH3), 29.0 (CH2), 37.9 (CH2), 66.2 (Cq),
d
126.9 (2ꢄ CH), 128.2 (4ꢄ CH), 128.7 (2ꢄ CH), 129.0 (6ꢄ CH), 136.1
(Cq), 141.1 (Cq), 144.2 (2ꢄ Cq); MS (ESI): m/z 356 [M þ Na]þ; Anal.
Calcd for C22H23NS: C 79.23, H 6.95, N 4.20, found: C 78.88, H 7.03,
N 4.19.
4.2.1.1. 2-[1-(4-Ethylphenyl)-1,1-diphenylmethylthio]ethanamine
(17a). Starting alcohol ¼ 1-(4-ethylphenyl)-1,1-diphenylmethanol
(15a). Yield: 31%; mp 138e140 ꢃC; 1H NMR (300 MHz,
4.2.1.7. 2-[1-(2-Naphthyl)-1,1-(diphenyl)methylthio]ethanamine
CD3OD þ D2O):
d
1.23 (t, 3H, J ¼ 7.5 Hz, CH3), 2.45e2.59 (m, 4H, 2
(17g). Starting alcohol
(15g) [32,40]. Yield: 32%; mp 126e128 ꢃC; 1H NMR (300 MHz,
CD3OD þ D2O): 2.34e2.47 (s, 4H, 2 CH2), 7.21e7.34 (m, 6H, HAr),
7.43e7.49 (m, 6H, HAr), 7.55 (dd, 1H, J ¼ 1.9, 8.9 Hz, HAr), 7.70e7.83
(m, 4H, HAr); 13C NMR (100 MHz, DMSO-d6):
35.6 (CH2), 40.8
(CH2), 66.0 (Cq), 126.4 (CH), 126.5 (CH), 126.8 (2ꢄ CH), 127.2 (CH),
127.3 (CH), 127.5 (CH), 128.1 (5ꢄ CH), 128.2 (CH), 129.1 (4ꢄ CH),
131.6 (Cq), 132.3 (Cq), 141.9 (Cq), 144.5 (2ꢄ Cq); MS (ESI): m/z 392
[M þ Na]þ; Anal. Calcd for C25H23NS: C 81.26, H 6.27, N 3.79, found:
C 81.38, H 6.31, N 3.89.
¼
1-(2-naphthyl)-1,1-diphenylmethanol
CH2), 2.58 (q, 2H, J ¼ 7.5 Hz, CH2), 7.17 (d, 2H, J ¼ 8.5 Hz, HAr),
7.22e7.35 (m, 8H, HAr), 7.43e7.46 (m, 4H, HAr); 13C NMR (100 MHz,
d
DMSO-d6):
d 15.3 (CH3), 27.6 (CH2), 28.6 (CH2), 37.7 (CH2), 66.2
(Cq), 126.9 (2ꢄ CH), 127.5 (2ꢄ CH), 128.2 (4ꢄ CH), 129.0 (6ꢄ CH),
141.3 (Cq), 142.3 (Cq), 144.2 (2ꢄ Cq); MS (ESI): m/z 370 [M þ Na]þ;
Anal. Calcd for C23H25NS: C 79.49, H 7.25, N 4.03, found: C, 79.47, H
7.20, N 3.97.
d
4.2.1.2. 2-[1,1-Diphenyl-4-(phenyl)phenylmethylthio]ethanamine
(17b). Starting alcohol
thanol (15b). Yield: 30%; mp 160e162 ꢃC; 1H NMR (300 MHz,
CD3OD þ D2O): 2.50e2.62 (s, 4H, 2 CH2), 7.27e7.63 (m, 19H, HAr);
13C NMR (125 MHz, DMSO-d6):
28.7 (CH2), 37.8 (CH2), 66.2 (Cq),
¼
1-(4-phenylphenyl)-1,1-diphenylme-
4.2.2. General procedure for preparation of compounds 17hel
At 0 ꢃC and under argon atmosphere, a solution of BF3$Et2O
(1.33 mmol) was added dropwise to a solution of appropriate
d
d
126.4 (2ꢄ CH), 126.6 (2ꢄ CH), 127.0 (2ꢄ CH), 127.6 (CH), 128.3 (4ꢄ
CH), 128.9 (2ꢄ CH), 129.0 (4ꢄ CH), 129.6 (2ꢄ CH), 138.6 (Cq), 139.2
(Cq), 143.2 (Cq), 143.9 (2ꢄ Cq); MS (ESI): m/z 418 [M þ Na]þ; Anal.
Calcd for C27H25NS: C 81.98, H 6.37; N 3.54, found: C 82.26, H 6.44,
N 3.73.
alcohol 15 (0.86 mmol),
L
-cysteine (18) or -penicillamine (19)
L
(0.77 mmol) in AcOH (1 mL). The reaction mixture was stirred at
room temperature for 3 h. Addition of 10% solution of NaOAc (2 mL),
then H2O (2 mL) led to the formation of a gum. After elimination of
the supernatant, the final compound was precipitated by addition
of pentane or Et2O. The desired compounds 17hel were obtained
by filtration in the range of 30e55% yield.
4.2.1.3. 2-[1,1-Diphenyl-4-(propyl)phenylmethylthio]ethanamine
(17c). Starting
methanol (15c). Yield: 45%; mp 133e135 ꢃC; 1H NMR (300 MHz,
CD3OD þ D2O): 0.94 (t, 3H, J ¼ 7.3 Hz, CH3), 1.58e1.70 (m, 2H,
alcohol
¼
1,1-diphenyl-1-(4-propylphenyl)
4.2.2.1. S-[1-(4-Pentylphenyl)-1,1-diphenylmethyl]-
L
-cysteine
d
(17h). Starting alcohol
¼
1-(4-pentylphenyl)-1,1-diphenylme-
25
CH2), 2.45e2.49 (m, 2H, CH2), 2.50e2.60 (m, 4H, CH2), 7.14 (d, 2H,
thanol (15h). Yield: 55%; mp 127e129 ꢃC; ½
a
ꢅ
¼ þ61 (c ¼ 0.52
589
J ¼ 8.3 Hz, HAr), 7.22e7.34 (m, 8H, HAr), 7.42e7.45 (m, 4H, HAr); 13
C
in MeOH); 1H NMR (300 MHz, CD3OD þ D2O):
d 0.91 (t, 3H,
NMR (100 MHz, DMSO-d6):
d
13.8 (CH3), 23.9 (CH2), 28.6 (CH2), 36.7
J ¼ 6.7 Hz, CH3), 1.32e1.39 (m, 4H, 2 CH2), 1.56e1.66 (m, 2H, CH2),
2.59 (broad t, 2H, J ¼ 7.9 Hz, CH2), 2.70 (dd, 1H, J ¼ 9.2,13.5 Hz, CH2),
2.82 (dd, 1H, J ¼ 4.2, 13.5 Hz, CH2), 3.04 (dd, 1H, J ¼ 4.2, 9.2 Hz, CH),
7.13 (d, 2H, J ¼ 8.5 Hz, HAr), 7.20e7.35 (m, 8H, HAr), 7.43e7.46 (m,
(CH2), 37.7 (CH2), 66.2 (Cq), 126.9 (2ꢄ CH), 128.1 (2ꢄ CH), 128.2 (4ꢄ
CH), 128.9 (2ꢄ CH), 129.0 (4ꢄ CH), 140.8 (Cq), 141.3 (Cq), 144.2 (2ꢄ
Cq); MS (ESI): m/z 384 [M þ Na]þ; Anal. Calcd for C24H27NS: C,
79.73, H 7.53, N 3.87, found: C 79.55, H 7.40, N 3.82.
4H, HAr); 13C NMR (100 MHz, CD3OD):
d 14.4 (CH3), 23.6 (CH2), 32.3
(CH2), 32.7 (CH2), 34.0 (CH2), 36.4 (CH2), 54.6 (CH), 68.0 (Cq), 128.0
(2ꢄ CH), 129.1 (6ꢄ CH), 130.6 (2ꢄ CH), 130.7 (4ꢄ CH), 142.8 (Cq),
143.0 (Cq), 145.8 (2ꢄ Cq), 172.0 (CO); MS (ESI): m/z 456 [M þ Na]þ;
Anal. Calcd for C27H31NO2S: C 74.79, H 7.21, N 3.23, found: C 74.79, H
7.17, N 3.25.
4.2.1.4. 2-[1,1-Diphenyl-4-(methylthio)phenylmethylthio]ethan-
amine (17d). Starting alcohol ¼ 1,1-diphenyl-1-(4-methylthioph-
enyl)methanol (15d). Yield: 40%; mp 142e144 ꢃC; 1H NMR
(300 MHz, CD3OD þ D2O):
CH2), 7.20e7.37 (m, 10H, HAr), 7.43e7.46 (m, 4H, HAr); 13C NMR
(125 MHz, DMSO-d6): 14.3 (CH3), 28.6 (CH2), 37.7 (CH2), 66.0 (Cq),
d 2.47 (s, 3H, CH3), 2.50e2.59 (m, 4H,
d
4.2.2.2. S-[1-(4-Hexylphenyl)-1,1-diphenylmethyl]-L-cysteine (17i).
125.3 (2ꢄ CH), 127.0 (2ꢄ CH), 128.2 (4ꢄ CH), 128.9 (4ꢄ CH), 129.6
(2ꢄ CH), 137.0 (Cq), 140.3 (Cq), 143.9 (2ꢄ Cq); MS (ESI): m/z 388
[M þ Na]þ; Anal. Calcd for C22H23NS2: C 72.28, H 6.34, N 3.83,
found: C 72.00, H 6.35, N 3.77.
Starting alcohol ¼ 1-(4-hexylphenyl)-1,1-diphenylmethanol (15i).
25
589
Yield: 40%; mp 129e131 ꢃC; ½
a
ꢅ
¼ þ53 (c ¼ 0.54 in MeOH). 1H
NMR (300 MHz, CD3OD þ D2O):
d
0.90 (t, 3H, J ¼ 6.7 Hz, CH3),
1.30e1.42 (m, 6H, 3 CH2), 1.54e1.66 (m, 2H, CH2), 2.59 (broad t, 2H,
J ¼ 7.9 Hz, CH2), 2.70 (dd, 1H, J ¼ 9.2, 13.5 Hz, CH2), 2.82 (dd, 1H,
J ¼ 4.2, 13.5 Hz, CH2), 3.03 (dd, 1H, J ¼ 4.2, 9.2 Hz, CH), 7.12 (d, 2H,
4.2.1.5. 2-[1-(4-Iodophenyl)-1,1-diphenylmethylthio]ethanamine (17e).
Starting alcohol
¼
1-(4-iodophenyl)-1,1-diphenylmethanol (15e).
J ¼ 8.3 Hz, HAr), 7.21e7.35 (m, 8H, HAr), 7.43e7.46 (m, 4H, HAr); 13
C
Yield: 47%; mp 150e152 ꢃC; 1H NMR (300 MHz, CD3OD þ D2O):
d
2.54
NMR (100 MHz, CD3OD): d 14.4 (CH3), 23.7 (CH2), 30.1 (CH2), 32.5
(s, 4H, 2 CH2), 7.21e7.36 (m, 8H, HAr), 7.41e7.44 (m, 4H, HAr), 7.68 (d,
(CH2), 32.8 (CH2), 34.0 (CH2), 36.4 (CH2), 54.7 (CH), 68.0 (Cq), 128.0
(2ꢄ CH), 129.1 (6ꢄ CH), 130.6 (2ꢄ CH), 130.7 (4ꢄ CH), 142.8 (Cq),
143.0 (Cq), 145.8 (2ꢄ Cq), 172.0 (CO); MS (ESI): m/z 470 [M þ Na]þ;
Anal. Calcd for C28H33NO2S: C 75.13, H 7.43, N 3.13, found: C 74.87, H
7.30, N 3.02.
2H, J ¼ 10.8 Hz, HAr); 13C NMR (125 MHz, DMSO-d6):
d 28.8 (CH2), 37.7
(CH2), 66.0 (Cq), 93.4 (Cq),127.1 (2ꢄ CH),128.3 (4ꢄ CH),128.9 (4ꢄ CH),
131.4 (2ꢄ CH), 137.0 (2ꢄ CH), 143.5 (2ꢄ Cq), 143.8 (Cq); MS (ESI): m/z
468 [M þ Na]þ; Anal. Calcd for C21H20INS: C 56.63, H 4.53, N 3.15,
found: C 56.60, H 4.61, N 3.22.
4.2.2.3. S-[1,1-Diphenyl-1-(4-propoxyphenyl)methyl]- -cysteine (17j).
L
4.2.1.6. 2-[1-(4-Methylphenyl)-1,1-diphenylmethylthio]ethanamine
(17f). Startingalcohol ¼ 1-(4-methylphenyl)-1,1-diphenylmethanol
(15f). Yield: 29%;mp138e140 ꢃC; 1HNMR (300 MHz, CD3ODþ D2O):
Starting alcohol ¼ 1,1-diphenyl-1-(4-propoxyphenyl)methanol (15j)
25
589
[41]. Yield: 43%; mp 144e146 ꢃC; ½
a
ꢅ
¼ þ60 (c ¼ 0.51 in MeOH); 1H
NMR (300 MHz, CD3OD þ D2O):
d
1.03 (t, 3H, J ¼ 7.3 Hz, CH3),
d
2.32 (s, 3H, CH3), 2.48e2.55 (m, 4H, 2 CH2), 7.13 (d, 2H, J ¼ 8.1 Hz,
1.72e1.84 (m, 2H, CH2), 2.68 (dd, 1H, J ¼ 9.2, 13.4 Hz, CH2), 2.83 (dd,

198
D.B. Nichols et al. / European Journal of Medicinal Chemistry 49 (2012) 191e199
1H, J ¼ 4.0, 13.4 Hz, CH2), 3.04 (dd,1H, J ¼ 4.0, 9.2 Hz, CH), 3.92 (t, 2H,
J ¼ 6.4 Hz, CH2), 6.92 (d, 2H, J ¼ 8.8 Hz, HAr), 7.22e7.33 (m, 8H, HAr),
using nonlinear regression analysis and IC50 values were interpo-
lated from the dose-response curves using GraphPad Prism 3.03
software.
7.43e7.45 (m, 4H, HAr); 13C NMR (100 MHz, CD3OD):
d 10.8 (CH3), 23.6
(CH2), 34.2 (CH2), 55.0 (CH), 67.7 (Cq), 70.5 (CH2), 114.9 (2ꢄ CH), 127.9
(2ꢄ CH) 129.1 (4ꢄ CH), 130.5 (4ꢄ CH),132.0 (2ꢄ CH), 137.2 (Cq), 146.0
(Cq), 146.1 (Cq), 159.3 (Cq), 172.3 (CO); MS (ESI): m/z 444 [M þ Na]þ;
Anal. Calcd for C25H27NO3S: C 71.23, H 6.46, N 3.32, found: C 71.44, H
6.54, N 3.30.
4.3.2. Cell culture
BHK-NS5B-FRLuc reporter cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal
bovine serum, 5% antibiotic-antimycotic, 5% nonessential amino
acid, 1 mg/mL G418 and 10
mg/mL blasticidin. Huh7/Rep-Feo1b
4.2.2.4. S-[1-(4-Butylphenyl)-1,1-diphenylmethyl]-
L
-penicillamine
replicon reporter cells were cultivated in DMEM containing 10%
fetal calf serum, 5% antibiotic and 0.5 mg/mL G418. All cell lines
were incubated at 37 ꢃC in the presence of 5% CO2 supplement.
(17k). Starting alcohol ¼ 1-(4-butylphenyl)-1,1-diphenylmethanol
25
(15k). Yield: 30%; mp 123e125 ꢃC; ½
a
ꢅ
¼ þ171 (c ¼ 0.54 in
589
MeOH); 1H NMR (300 MHz, CD3OD þ D2O):
0.93 (t, 3H, J ¼ 7.3 Hz,
d
CH3), 1.30 (s, 3H, CH3), 1.32e1.40 (m, 2H, CH2), 1.42 (s, 3H, CH3),
1.54e1.64 (m, 2H, CH2), 1.85 (s, 1H, CH), 2.59 (t, 2H, J ¼ 7.5 Hz, CH2),
7.14 (d, 2H, J ¼ 8.3 Hz, HAr), 7.19e7.34 (m, 6H, HAr), 7.56 (d, 2H,
J ¼ 8.5 Hz, HAr), 7.67e7.70 (m, 4H, HAr); 13C NMR (100 MHz,
4.3.3. BHK-NS5B-FRLuc reporter assay
The effect of the compounds on intracellular NS5B RdRp activity
was screened employing the BHK-NS5B-FRLuc reporter system as
previously described [36]. Briefly, BHK-NS5B-FRLuc reporter cells
were plated at a confluence of 1ꢄ104 cells/well in 96 well plates and
CD3OD): d 14.3 (CH3), 23.4 (CH2), 25.9 (CH3), 27.9 (CH3), 34.7 (CH2),
36.1 (CH2), 53.5 (Cq), 61.9 (CH), 69.2 (Cq), 127.9 (2ꢄ CH), 129.0 (6ꢄ
CH) 130.7 (2ꢄ CH), 130.8 (2ꢄ CH), 130.9 (2ꢄ CH), 142.9 (Cq), 143.2
(Cq), 146.0 (Cq),146.1 (Cq),170.6 (CO); MS (ESI): m/z 470 [M þ Na]þ;
Anal. Calcd for C28H33NO2S: C 75.13, H 7.43, N 3.13, found: C 75.45,
H, 7.53, N 3.32.
incubated with DMSO (1%) or the indicated compound (100 mM) for
42 h. Reporter gene expression was measured with a Dual-Glo
Luciferase Assay Kit (Promega, USA) in accordance with the
manufacturer’s instructions. Effect of the compounds on cell
viability was estimated as the relative levels of firefly luciferase in
compound treated cells versus DMSO controls. The inhibitory effect
of the compounds on the intracellular NS5B RdRp activity was
evaluated from the percent reduction in RLuc to FLuc luminescence
signal in compound treated cells versus DMSO controls.
4.2.2.5. S-[1,1-Diphenyl-1-(4-propoxyphenyl)methyl]-L-penicillamine
(17l). Starting alcohol ¼ 1,1-diphenyl-1-(4-propoxyphenyl)meth-
25
anol (15j). Yield: 34%; mp 133e135 ꢃC; ½
a
ꢅ
¼ þ69 (c ¼ 0.15 in
589
MeOH); 1H NMR (300 MHz, CD3OD þ D2O):
1.03 (t, 3H, J ¼ 7.1 Hz,
d
CH3), 1.31 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.74e1.81 (m, 2H, CH2), 1.92
(s, 1H, CH), 3.93 (t, 2H, J ¼ 6.0 Hz, CH2), 6.87 (d, 2H, J ¼ 8.4 Hz, HAr),
7.20e7.30 (m, 6H, HAr), 7.56 (d, 2H, J ¼ 8.4 Hz, HAr), 7.64e7.72 (m,
4.3.4. Huh7/Rep-Feo1b reporter system
The effect of the compounds on HCV RNA replication was
screened employing the Huh7/Rep-Feo1b replicon reporter cells as
previously described [42]. Briefly, 1ꢄ104 Huh7/Rep-Feo1b cells
4H, HAr); 13C NMR (100 MHz, CD3OD):
d 10.8 (CH3), 23.7 (CH2), 25.9
(CH3), 28.4 (CH3), 53.2 (Cq), 62.0 (CH), 69.2 (Cq), 70.5 (CH2), 114.6
(2ꢄ CH), 127.8 (2ꢄ CH) 128.7 (4ꢄ CH), 129.5 (4ꢄ CH), 131.6 (2ꢄ CH),
137.3 (Cq), 146.0 (2ꢄ Cq), 159.6 (Cq), 172.6 (CO); MS (ESI): m/z 472
[M þ Na]þ; Anal. Calcd for C27H31NO3S: C 72.13, H 6.95, N 3.12,
found: C 71.99, H 7.00, N 3.11.
were plated in 96 well plates and treated with 100 mM concentra-
tion of the indicated compound or DMSO for 42 h. The concentra-
tion of DMSO in cell culture was kept constant at 1.0%. Cell viability
was measured by the colorimetric MTS assay employing the Cell-
Titer 96AQueous one solution assay reagent (Promega, USA). Inhib-
itory effect of the compounds on HCV RNA replication was
measured as the relative levels of firefly luciferase signals in
compound treated cells versus DMSO controls.
4.3. Biological studies
4.3.1. NS5B inhibition assay
Recombinant NS5B carrying the N-terminal histidine-tag was
purified from the plasmid pThNS5BCΔ21 expressed in Escherichia
Acknowledgments
coli DH5
a by Ni-NTA chromatography [33,34]. The compounds
This work was supported by the National Institute of Health
Research Grants DK066837 and CA153147 to N.K.-B. We would like
to thank Prof. Garland R. Marshall (Washington University of St.
Louis - MO) for critical reading of the manuscript.
were dissolved in dimethylsulfoxide (DMSO) as a 10 mM stock
solution and stored at ꢁ20 ꢃC. Serial dilutions were made in DMSO
immediately prior to the assay. The activity of the compounds
against HCV NS5B was evaluated by the standard primer dependent
elongation assay as previously described [33,34]. Briefly, prelimi-
nary screening was performed in the presence or absence of
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