Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site

Article abstract of DOI:10.1016/j.bmcl.2014.05.009

We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands were established that were consistent with the crystal structure of the translation inhibitor complex.

Full text of DOI:10.1016/j.bmcl.2014.05.009

Accepted Manuscript
Aryl-substituted aminobenzimidazoles targeting the hepatitis C virus internal
ribosome entry site
Kejia Ding, Annie Wang, Mark A. Boerneke, Sergey M. Dibrov, Thomas
Hermann
PII:
S0960-894X(14)00496-X
http://dx.doi.org/10.1016/j.bmcl.2014.05.009
BMCL 21622
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
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Accepted Date:
7 January 2014
1 May 2014
5 May 2014
Please cite this article as: Ding, K., Wang, A., Boerneke, M.A., Dibrov, S.M., Hermann, T., Aryl-substituted
aminobenzimidazoles targeting the hepatitis C virus internal ribosome entry site, Bioorganic & Medicinal Chemistry
Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.009
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Aryl-substituted aminobenzimidazoles targeting the
hepatitis C virus internal ribosome entry site
Kejia Ding, Annie Wang, Mark A. Boerneke, Sergey M. Dibrov and Thomas Hermann*
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman
Drive, La Jolla, California 92093, United States
*Corresponding author: tch@ucsd.edu
ABSTRACT
We describe the exploration of N1-aryl-substituted benzimidazoles as ligands for the hepatitis C
virus (HCV) internal ribosome entry site (IRES) RNA. The design of the compounds was guided
by the co-crystal structure of a benzimidazole viral translation inhibitor in complex with the
RNA target. Structure-binding activity relationships of aryl-substituted benzimidazole ligands
were established that were consistent with the crystal structure of the translation inhibitor
complex.

Infection with hepatitis C virus (HCV), which affects over 180 million individuals
worldwide, is a major cause of hepatocellular carcinoma and the leading indication for liver
transplantation.1 While two classes of direct antiviral drugs are now available, including protease
and polymerase inhibitors,2, 3 the high genetic variability of HCV poses a constant threat of
resistance development and requires the exploration of additional targets for antiviral therapy.4-6
Since the HCV RNA genome contains several highly conserved cis-acting elements that adopt
defined structures, the repertoire for antiviral intervention may be expanded to RNA targets. The
internal ribosome entry site (IRES) in the 5’ untranslated region (UTR) of the viral genome
stands out for its high sequence conservation in clinical isolates and serves an essential function
for ribosome recruitment and initiation of HCV protein synthesis in infected host cells. We have
recently established7 the subdomain IIa RNA of the HCV IRES as the target for 2-
aminobenzimidazole viral translation inhibitors8 (Figure 1). Benzimidazole scaffolds have been
recognized as useful platforms for the synthesis of biologically active ligands for other RNA
targets, including RNA loops,9, 10 the HIV TAR element,11 microRNAs12 and RNA repeat
motifs.13, 14 The 2-aminobenzimidazoles inhibit the IRES function by capturing an extended
conformation of the IIa RNA target which has been proposed to block translation initiation.15 A
Förster resonance energy transfer (FRET) assay has been developed to monitor the selective
conformational capture by ligands and measure their target affinity for the IIa RNA.7, 16
Crystal structure analysis of the IIa RNA in complex with the 2-aminobenzimidazole 1
revealed the key interactions responsible for target recognition by the translation inhibitors.17
Since previous structure-binding activity relationship studies on the 2-aminobenzimidazoles had
revealed the importance of a rigid core scaffold for target affinity,8 we explored N1-aryl-

substituted benzimidazoles (2) which also lack the synthetically cumbersome chroman scaffold.
The compounds designed here retain the amino-imidazole core required for recognition of the IIa
RNA and provide a rigid aryl linker for a dimethylamino substituent that participates in
interactions with a phosphate group while avoiding an entropic penalty for pre-organization of
the dimethylaminopropyl chain in ligands such as 1 (Figure 1B,C). Modeling studies suggested
that the twist conformation between the imidazole and aryl ring along with an appropriate meta-
substitution position for the dimethylamino group would provide a rigid vector for interaction
with the phosphate group of residue A109 (Figure 1C).
Here, we describe the synthesis and target affinity testing of several chemical series of
N1-aryl-substituted 2-aminobenzimidazoles (Figure 2), including the compounds 3 and the 2-
amide derivatives 4. To evaluate the importance of the 2-amino substituent, we prepared 2-
hydroxybenzimidazole analogs 5. A small number of dimethylaminopropyl-substituted
compounds 6 which carried aryl substituents at the benzene ring were included as well.
Synthesis of phenylether derivatives in the series 3, 4 and 5 (R1 = R4O, Scheme 1 and
Table 1) was planned to progress through an N-aryl-substituted o-phenylenediamine common
intermediate (12) which was cyclized to the benzimidazole by reaction with isothiocyanates or
phosgene. The intermediate 12 was prepared over three steps from halogen-substituted
nitrophenols 7. Alkylation of 7 yielded phenylethers 9. The parent phenol 9 (R4 = OH) was
obtained as the tert-butyldimethylsilyl (TBDMS) ether derivative. Coupling with anilines 10
followed by reduction of the nitro group furnished the common intermediate 12. Cyclization of
12 with acetyl or benzoyl isothiocyanates gave the 2-amide derivatives 4 which were further
hydrolyzed to the parent 2-aminobenzimidazoles 3. Treatment of 12 with phosgene yielded 2-
hydroxybenzimidazole analogs 5. Two aniline derivatives in the 1-aryl-2-aminobenzimidazole

series (3h and 3i, Table 1) were synthesized through isothiocyanate cyclization of an
intermediate that was obtained by two consecutive Buchwald-Hartwig aminations of 2,4-
dichloro-1-nitrobenzene 13 (Scheme 2). The N,N-dimethylaminopropyl-substituted
benzimidazoles 6 (Table 2) were synthesized following the route outlined in Scheme 3.
Dichloronitrobenzene precursors were alkylated to install the dimethylaminopropyl chain.
Coupling with aromatic amines or boronic acids led to nitro-aniline (22) and -biphenyl (23)
intermediates which, after reduction, were cyclized with isothiocyanate to furnish the amides 24
whose hydrolysis gave the final products 6.
The identity of all synthesized compounds from the chemical series 3, 4, 5 and 6 was
established after column chromatographic purification by mass- and NMR. Crystal structures
were determined for selected derivatives, including the 2-aminobenzimidazole 3e. Compounds
were tested for binding to the IRES IIa RNA by triplicate titration in the IIa FRET assay as
previously described.16 Target affinity expressed as EC50 value was determined from fitting of
single-site binding dose response curves to the titration data (Tables 1 and 2). See the Supporting
Information for experimental procedures, spectra, crystal structure and FRET titration curves.
Of the nine 1-aryl-2-aminobenzimidazoles 3 synthesized, all but two showed binding to
the IIa RNA target with affinities between 74–250 µM. Exceptions were the unsubstituted phenol
3a and the picolyl ether 3d. Inspection of the binding pocket around 1 in the co-crystal structure
suggested that while the parent phenol 3a is unable to form interactions beyond the amino-
imidazole scaffold, in the derivative 3d the bulky picolyl ether substituent off the 5-position
might interfere with ligand docking at the G110 base of the RNA target (Figure 1C). In contrast,
the more flexible aliphatic dimethylaminoalkyl ether chain was tolerated at the same position, as
attested by the binding of compounds 3b and 3c (250 and 210 µM, respectively). Improvement

of the affinity by 2-fold was seen in derivative 3e (95 µM) which carried a 5-
dimethylaminopropyl ether substituent at the benzimidazole and an additional p-dimethyalmino
group on the imidazole-linked phenyl ring. The same combination of functional groups,
however, with the dimethylaminoalkyl ether chain moved to the 6-position reduced binding of
compound 3f (170 µM). Relocation of the dimethylamino substituent from the para- to the meta-
position at the imidazole-linked phenyl ring increased binding in derivative 3g (120 µM), yet not
to the level of 3e. Finally, aromatic substituents were tolerated at the benzimidazole 6-position as
indicated by the affinity of compounds 3h and 3i, which were among the most active ligands
synthesized here (74 and 100 µM, respectively). In comparison, compounds 6h and 6i which
carried the same aromatic pyridine substituents at the 6-position but had the m-
dimethyalminophenyl ring at the imidazole replaced with a flexible dimethylaminopropyl chain
showed weaker target affinity (both, 160 µM). Attachment of a m-dimethyalminophenyl group at
the 7-position of the benzimidazole retained binding in compound 6j albeit only at 290 µM.
The crystal structure of compound 3e (see Supporting Information) shows a twist angle
of 67° between the imidazole and the phenyl ring attached at the 1-position. As a consequence, a
good fit of the 1-phenyl ring in 3e and the dimethylaminopropyl chain in 1 bound to the IIa RNA
target was observed in a superposition of the two compounds (Figure 3). The meta position of
the phenyl ring in 3e is pointing at the location of the dimethylamino group in 1 which forms a
key hydrogen bond with the phosphate of A109 in the RNA target (Figures 1C and 3B).
Comparison of the derivatives 3f and 3g suggests that higher affinity is achieved by substitution
of the phenyl ring with a dimethylamino group at the meta position compared to the para
position. In the superposition of 3e on the 1 target complex, the dimethylaminopropyl ether chain
at the benzimidazole 5-position is directed towards a cleft lined by the sugar phosphate backbone

of residues A54-U56 (Figure 3B). This region is located in a flexible internal loop of the IIa
RNA target (Figure 1A) and undergoes a large conformational change upon binding of the ligand
1. Such adaptive binding might allow the accommodation of flexible substituents at the
benzimidazole 5-position, for example, the dimethylaminopropyl ether chain in the 3e derivative.
However, as indicated by the inactivity of compound 3d, which carries a picolyl ether
substituent, bulky and more rigid groups at the 5-position may not be reconciled with target
adaptation.
Acylation of the 2-amino group abolished binding as indicated by compounds in the
series 4. The acetylated derivative 4ba was an exception for which a target affinity of 100 µM
was measured, surpassing that of the parent compound 3b (250 µM). This observation is not
readily reconciled with the crystal structure of the ligand 1 RNA complex, perhaps suggesting
that compound 4ba accesses an alternative binding mode at the target. Nonspecific binding
interactions might also be responsible for the strong precipitating properties of the
hydroxybenzimidazoles 5. All tested compounds in this series led to rapid fluorescence
quenching in the FRET assay at micromolar concentration, apparently due to precipitation of the
dye labeled RNA target.
Here, we have explored N1-aryl-substituted 2-amino-benzimidazoles (2) as ligands for
the IIa RNA target in the HCV IRES element. Synthesis and affinity testing of several chemical
series allowed us to establish structure-binding relationships of the ligands (Figure 4). The
observed pattern of compound activity in the FRET target binding assay was readily explained in
the context of an earlier determined crystal structure of benzimidazole 1 bound to the IIa RNA.
Ligands with the highest affinity showed binding at EC50 values around 74–100 µM which is
inferior to the previously studied tricyclic benzimidazoles such as 1. Given the more facile

synthetic accessibility of the chemical series 2, in combination with guidance from the clear
structure-binding activity relationship established in the current work, we will explore further
optimization of the N1-aryl-substituted benzimidazoles in the future.
Acknowledgments
This work was supported by the National Institutes of Health (grant No. AI72012). Support of
the NMR facility by the National Science Foundation is acknowledged (CRIF grant CHE-
0741968).
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Figure Legends
Figure 1. Ligand design for the internal ribosome entry site (IRES) target of hepatitis C virus
(HCV). A) The IRES element comprises the first 342 nucleotides in the 5’ untranslated region
(UTR) of the HCV RNA genome. The boxed region highlights the subdomain IIa target whose
secondary structure is shown in on the right. B) Structures of a 2-aminobenzimidazole
translation inhibitor 1 which was previously co-crystallized in complex with the subdomain IIa
RNA, and aryl-substituted 2-aminobenzimidazoles 2 which were designed using guidance by the
co-crystal structure of the IIa RNA in complex with 1. C) Model of the 1-phenyl-2-
aminobenzimidazole core of 2 (light blue) docked at the binding site of 1 (yellow) in the
subdomain IIa RNA co-crystal structure.17 Hydrogen bonds and nucleotides participating in
intramolecular contacts are indicated.
Figure 2. Benzimidazole derivatives for targeting the HCV IRES subdomain IIa RNA: 1-aryl-2-
aminobenzimidazoles 3, 1-aryl-2-acylaminobenzimidazoles 4, 1-aryl-2-hydroxybenzimidazoles
5, N,N-dimethylaminopropyl-substituted benzimidazoles 6. Compounds made in each series are
shown in Tables 1 and 2.
Figure 3. A) Crystal structure of compound 3e (see Supporting Information, CCDC deposit
number 978337). Hydrogen atoms are not shown. The dihedral twist angle between the
imidazole and dimethylaniline rings is indicated. B) Model of the compound 3e crystal structure
(light blue) superimposed on the HCV translation inhibitor 1 (yellow) in the subdomain IIa RNA
co-crystal structure.17 Hydrogen bonds and nucleotides participating in intramolecular contacts

are indicated. Residues G52 and A53, which stack below and above the ligand are shown
transparent.
Figure 4. Structure-binding activity relationships discovered for 1-aryl-2-aminobenzimidazoles
targeting the HCV IRES subdomain IIa RNA.
Scheme 1. Synthesis of 1-aryl-2-aminobenzimidazoles 3, 1-aryl-2-acylaminobenzimidazoles 4
and 1-aryl-2-hydroxybenzimidazoles 5 from halo-nitrophenols 6. Preparation of 5-substituted
benzimidazoles commenced from 4-chloro-3-nitrophenol (X=Cl), that of 6-substituted products
from 3-fluoro-4-nitrophenol (X=F). Reagents and conditions: a) (for R4=H, 8=TBDMSCl)
K2CO3, ACN, reflux, 2.5-28 h, 52-94 % yield; b) Cs2CO3, Pd2(dba)3, ( )BINAP, 19-28 h, 31-91
% yield; c) K2CO3, ACN, reflux, 21-41 h, 34-36 % yield; d) H2 (1 atm), Pd/C or PtO2, MeOH,
RT, 1.5-25 h; e) acylNCS (acyl=acetyl or benzoyl), DIPC, DIPEA, ACN, 22-48 h, 20-66 % yield
over two steps; f) phosgene (20 % in toluene), pyridine, DCM, RT, 3-24 h, 50-64 % yield two
steps; g) 1N HCl, H2O, dioxane, reflux, 18-28 h, 27-96 % yield. Abbreviations: TBDMSCl =
tert-butyldimethylsilyl chloride; ACN = acetonitrile; dba = dibenzylideneacetone; BINAP = 2,2′-
bis(diphenylphosphino)-1,1′-binaphthalene; RT
=
room temperature; DIPC
=
N,N′-
diisopropylcarbodiimide; DIPEA = N,N-diisopropylethylamine; DCM = dichloromethane.
Scheme 2. Synthesis of 1-aryl-2-aminobenzimidazoles 3h and 3i. Reagents and conditions: ba)
Cs2CO3, Pd2(dba)3, ( )BINAP, toluene, reflux, 21 h, 43 % yield, two steps; 14 was synthesized
from N,N-dimethyl-3-nitroaniline by reduction with H2 (1 atm), Pd/C in MeOH, 20 hr; bb)
Cs2CO3, Pd2(dba)3, ( )BINAP, dioxane, reflux, 20-44 h, 34-43 % yield d) H2 (1 atm), Pd/C or
PtO2, MeOH, RT, 21-23 h h; e) benzoylNCS, DIPC, DIPEA, ACN, 24-26 h, 22-32 % yield over
two steps; g) 1N HCl, H2O, dioxane, reflux, 23-26 h, 27-56 % yield.

Scheme 3. Synthesis of N,N-dimethylaminopropyl-substituted benzimidazoles 6 from dichloro-
nitrobenzenes 13. Preparation of 6-substituted benzimidazoles commenced from 1,2-dichloro-4-
nitrobenzene, that of 7-substituted products from 1,2-dichloro-3-nitrobenzene. Reagents and
conditions: a) K2CO3, ACN, reflux, 24 h, 96-97 % yield; b) Cs2CO3, Pd2(dba)3, ( )BINAP, 19-
23 h, 37-80 % yield range; d) H2 (1 atm), Pd/C or PtO2, MeOH, RT, 21-25 h; e) benzoylNCS,
DIPC, DIPEA, ACN, 19-21 h, 29-33 % yield over two steps; g) 1N HCl, H2O, dioxane, reflux,
17-21 h, 22-46 % yield.

Table 1. Activity of 1-aryl-benzimidazole derivatives 3, 4 and 5 in the FRET assay.
R1
R2
H
R3
H
EC50 [ M]a
n.a.
Compound
3a
5-OH
5-
3b / 5b
250 ± 66
210 ± 39
H
H
H
H
N
O
5-
3c / 5c
3d
N
O
O
n.a.
H
H
5-
5-
6-
6-
N
N
3e
N
N
N
O
O
O
95 ± 6
170 ± 16
120 ± 5
H
H
H
p-
p-
N
N
3f / 5f
3g
m-
H
N
N
N
3h
3i
74 ± 2
H
H
m-
m-
6-
6-
N
H
N
100 ± 12
N
4ba
4ca
4ab
4bb
4cb
4eb
4fb
100 ± 13
> 500
n.a.
H
H
H
H
H
CH3
CH3
N
5-
5-
O
N
O
5-OH
5-
C6H5
C6H5
C6H5
> 500
> 500
precip.
precip.
N
O
5-
5-
6-
N
O
O
O
N
N
N
N
C6H5
C6H5
p-
p-
aEC50 values (
aminobenzimdiazoles 3 and acyl derivates 4. All tested 2-hydroxybenzimidazoles 5 precipitated
RNA at concentration between 50-100 µM.
standard error from triplicate experiments) are shown for 2-
a

Table 2. Activity of N,N-dimethylaminopropyl-substituted benzimidazoles 6 in the FRET assay.
R1
EC50 [ M]a
Compound
H
N
6h
6i
160 ± 15
6-
6-
N
H
N
160 ± 29
N
N
6j
290 ± 100
7-
aEC50 values are shown standard error from triplicate experiments.