2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site

Article abstract of DOI:10.1016/j.bmcl.2014.05.088

2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic benzoxazole system as a replacement for the core scaffold in previously discovered benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of substituted benzoxazole ligands were investigated.

Full text of DOI:10.1016/j.bmcl.2014.05.088

Accepted Manuscript
2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry
site
Kevin D. Rynearson, Brian Charrette, Christopher Gabriel, Jesus Moreno, Mark
A. Boerneke, Sergey M. Dibrov, Thomas Hermann
PII:
S0960-894X(14)00597-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.088
BMCL 21702
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 January 2014
15 May 2014
16 May 2014
Please cite this article as: Rynearson, K.D., Charrette, B., Gabriel, C., Moreno, J., Boerneke, M.A., Dibrov, S.M.,
Hermann, T., 2-Aminobenzoxazole ligands of the hepatitis C virus internal ribosome entry site, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.088
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2-Aminobenzoxazole ligands of the hepatitis C virus internal
ribosome entry site
Kevin D. Rynearson,1 Brian Charrette, Christopher Gabriel,2 Jesus Moreno,3 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
1current address: Department of Neuroscience, University of California, San Diego
2current address: Department of Chemistry & Biochemistry, University of California, Santa
Barbara
3current address: Department of Chemistry & Biochemistry, University of California, Los
Angeles
*Corresponding author: tch@ucsd.edu
ABSTRACT
2-Aminobenzoxazoles have been synthesized as ligands for the hepatitis C virus (HCV) internal
ribosome entry site (IRES) RNA. The compounds were designed to explore the less basic
benzoxazole system as a replacement for the core scaffold in previously discovered
benzimidazole viral translation inhibitors. Structure-activity relationships in the target binding of
substituted
benzoxazole
ligands
were
investigated.

The highly conserved internal ribosome entry site (IRES) in the 5’ untranslated region of
the hepatitis C virus (HCV) RNA genome has previously been exploited as a target for 2-
aminobenzimidazole translation inhibitors which selectively suppress the synthesis of viral
proteins in infected human host cells (Figure 1).1 These compounds bind to an internal loop in
the IRES subdomain IIa to capture an extended conformation of the RNA and prevent viral
translation initiation.2 Conformational capture of the IIa target had been investigated by a FRET-
based assay which also served as a tool for measuring ligand affinity.3 From crystal structure
determination of the RNA target in complex with benzimidazole 1 a detailed picture emerged of
the interactions involved in ligand binding (Figure 1C).4 The 2-aminobenzimidazole scaffold
plays a key role in target recognition, by engaging in base stacking interactions with the benzene
ring and providing two hydrogen bonds to the Hoogsteen edge of a guanosine residue (G110).
While beneficial for RNA target binding, the 2-amino-imidazole system, whose electronic
structure resembles guanidine, confers high basicity to the benzimidazole translation inhibitors.
The basic pKa value of 2-aminobenzimidazole is around 7.5.5 Therefore, compounds such as 1
carry three positive charges under physiological conditions.
Here, we have explored replacement of the benzimidazole system in IIa RNA-targeted
ligands by the less basic benzoxazole whose pKa value is around 4.5 (Figure 1B).5 Since the
significant reduction in basicity of the core scaffold would affect the ability of benzoxazole
ligands to engage in a key hydrogen bond involving the protonated N3 position (Figure 1C), we
synthesized and evaluated substituted benzoxazole derivatives for IIa RNA target binding.
Introduction of the oxazole ring necessitated relocation of the N1-linked polar side chain, which
engages in hydrogen bonding with the RNA backbone in the compound 1 complex (Figure 1C).

Corresponding substituents were moved to the exocyclic 2-amino group of the benzoxazoles 2.
In the following, we describe the synthesis and target affinity testing of several chemical series
of substituted 2-aminobenzoxazoles 2 which were designed to explore the impact of various
polar substituents attached at either the benzene or oxazole rings.
The impact of structural variations in the substitution pattern of the exocyclic 2-amino
group was investigated in compounds 2-1 to 2-13 (Table 1) which were prepared as outlined in
Scheme 1. The benzoxazole scaffold was obtained from base-catalyzed condensation of amino-
nitrophenols 3 with carbon disulfide, furnishing nitrothiobenzoxazoles 4 as a common
precursor.6-8 Installation of alkylamino substituents at the 2-position proceeded through
conversion of 4 with oxalyl chloride to the 2-chloroxazole and in situ reaction with the desired
primary amine 5. Dilute reaction conditions were used to disfavor the formation of benzoxazole
dimers. Higher yields were obtained for coupling of the aminopropyl reagents likely due to less
sterical hindrance as compared to the aminoethyl group. Finally, 2-amino substituted aniline
products 2-1 to 2-7, 2-9, 2-10 and 2-12 were prepared through nitro reduction, with best yields
achieved by using Adam’s catalyst.9
For the exploration of modifications of the benzene ring, compounds substituted at the 6-
or 7-position of the benzoxazole scaffold (2-14 to 2-27) were synthesized as outlined in Schemes
2 and 3 (Tables 2, 3). Installation of secondary and tertiary amino functionalities commenced
with aromatic substitution of 5-fluoro-2-nitrophenol 6 with amine reagents 7. Nearly quantitative
yields were achieved in most cases, facilitated by the activating nitro group in the substrate.10
Nitrophenols 8 were then reduced to the diaminophenols which were sensitive to oxidation.11
Since the electron-rich substituted aminophenol scaffold readily decomposes when exposed to
air, the intermediates were immediately cyclized with di(imidazole-1-yl)methanimine12 which

provided expedient access to the desired 6-substituted aminobenzoxazoles 2-14 to 2-21 (Scheme
2).
Benzoxazoles carrying benzylic tertiary amine modifications at the 7-position were
synthesized starting from reductive amination of 3-nitrosalicylaldehyde 9 with various amines 10
to furnish near quantitative yields of nitrophenols 11 (Scheme 3).13-15 The use of primary amines
10 in the reductive amination resulted in secondary amines which required the installation of a
methyl group to obtain the desired tertiary amines (Table 3). This was accomplished by a second
reductive amination step with formaldehyde.16 Reduction of nitrophenols 11 to air-sensitive
aminophenols followed by immediate in situ cyclization was performed as outlined before to
furnish the substituted 7-methylene-2-aminobenzoxazole products 2-22 to 2-27.
Combinations of polar substituents at both the exocyclic 2-amino group and the benzene
ring were explored preliminarily by syntheses of two representative compounds including 2-28
and 2-29 (Table 4). The disubstituted 2-aminobenzoxazole 2-28 was obtained from 2-14 (Table
2) by nucleophilic substitution with N,N-dimethylaminopropyl chloride which proceeded in the
presence of potassium bicarbonate at 75°C. Similarly, 2-23 was reacted by nucleophilic
substitution with the same reagent in the presence of cesium carbonate at 60°C to furnish
compound 2-29.
The identity of the synthesized benzoxazole derivatives 2 was established after column
chromatographic purification by mass- and NMR spectra. See the Supporting Information for
experimental procedures and spectra. Crystal structures were determined for selected derivatives.
The activity of compounds was assessed by testing binding affinity for the IRES IIa RNA in a
FRET assay as previously described.3 Target affinity expressed as EC50 value was determined

from fitting single-site binding dose response curves to data obtained by averaging triplicate
compound titration experiments (Tables 1-4).
Substitution at the excocylic 2-position of the amino-benzoxazole scaffold installed
propyl- or ethyl-linked tertiary amines to furnish compounds that in addition carried an amino
group at the benzene ring (Table 1). A few nitro derivatives (2-8, 2-13) and one unsubstituted
representative (2-11) were synthesized as well. In general, propyl-linked substituents conferred
higher binding affinity to the IIa RNA target than ethyl-linked homologues (2-6, 2-7). Among
compounds carrying the N,N-dimethylaminopropyl group, which is found in the original
benzimidazole inhibitor 1, derivatives with 5- and 7-amino substituents (2-9, 2-10, EC50=52µM,
31µM) were two- to fourfold more active than the 6-amino analog (2-1, EC50=120µM). While an
N,N-dimethylaminopropyl-substituted compound without an amino group at the benzene ring
retained binding (2-11, EC50=110µM), absence of the 2-amino modification led to complete loss
of activity (2-12). Similarly, a 6-nitro substituent abolished binding whether or not a N,N-
dimethylaminopropyl modification was present at the 2-position (2-8, 2-13). Apparently, the
electron withdrawing effect of the nitro group further reduces the basicity of the benzoxazole N3
position which is detrimental for hydrogen bonding to the RNA target (Figure 1C).
Similar affinity as for the N,N-dimethylaminopropyl derivatives was observed with six-
membered ring substituents in 2-3 (EC50=47µM) and 2-4 (EC50=43µM), however, surprisingly
not with the smaller pyrrolidine modification in 2-2 or the bulkier methylpiperazine in 2-5
neither of which bound the IIa RNA target. In combination with the weaker affinity measured for
derivatives which carried shorter ethyl-linked amines (2-6, 2-7, EC50=500µM, 88µM), these
observations suggest that benzoxazole ligand binding may benefit from the proper occupancy by
the 2-amino-substituent of a spatially restricted site of the IIa RNA target. In agreement with this

hypothesis is the finding that the fourfold loss of affinity determined for the N,N-
dimethylaminoethyl derivative (2-6, EC50=500µM) compared to the propyl analog (2-1,
EC50=120µM) is restored by addition of the bulkier piperidine in 2-7 (EC50=88µM).
Modification at the 6-position furnished active compounds only for primary amine
substituents while secondary amines gave derivatives that did not bind the IIa RNA target (Table
2). The rigidly attached bulky groups in the latter compounds (2-17 to 2-21) might lead to steric
clashes in the binding pocket while more flexible substituents as in 2-14, 2-15 and 2-16 might be
accommodated. Extended groups at the 6-position increase binding affinity as attested by
derivatives 2-14 and 2-16 which were the most potent benzoxazole derivatives discovered in this
study (EC50=25µM, 27µM). Based on the crystal structure of the IIa RNA target in complex with
translation inhibitor 1 and structure-activity relationship data of benzimidazole derivatives,17 we
had previously concluded that extended flexible substituents at the 5- and 6-position are directed
towards a cleft lined by the sugar phosphate backbone of residues A54-U56 in the IIa RNA
internal loop (Figure 1C). Comparison of the crystal structures for the RNA target free and in
complex with 1 shows that this region undergoes a large conformational change upon ligand
binding.4, 18 This adaptive binding might benefit accommodation of flexible substituents at the 6-
position which, in turn, might increase the binding affinity of benzoxazole ligands such as 2-14
and 2-16. Rigid bulky groups attached at the same position, for example in derivatives 2-17 to 2-
21, might not be reconciled with target adaptation.
The 7-position of the benzoxazole scaffold was accessible to modification by reductive
amination of a benzaldehyde precursor 9, furnishing tertiary amino substituents attached at a
benzylic position in 7-methylene-benzoxazoles (Scheme 3 and Table 3). Only two of the
derivatives synthesized in this series showed IIa RNA target binding, albeit with weak affinity

(2-23, 2-25, EC50=190µM, 600µM), while three other compounds were inactive (2-22, 2-24, 2-
26). The lack of an electron donating amino group attached directly to the benzene in the
benzylic amino derivatives, which reduces the basicity and ability of the N3 position to
participate in hydrogen bonding to the RNA target, might explain the lower activity of the 7-
methylene-benzoxazole derivatives. Flexibility of the substituent and polarity of the terminal
functional group were important for binding, as indicated by the inactivity observed for the rigid
piperazine in 2-24 or the less polar nitrile in 2-26. The primary benzylamine itself in 2-27 was
not sufficient to confer RNA target binding, in agreement with the inactivity of the unsubstituted
compound 2-12 which we discussed above.
While the primary goal of the current study was the initial exploration of structure-
binding activity relationship trends of monosubstituted 2-aminobenzoxazoles as ligands of the
IIa RNA target, we included two disubstituted derivatives, 2-28 and 2-29 (Table 4), which were
designed to combine beneficial modification patterns observed in the monosubstituted
compounds. Surprisingly, both disubstituted benzoxazoles showed weaker than expected target
binding. Two N,N-dimethylaminopropyl groups at the 2- and 6-position each were combined in
compound 2-28, which had a target affinity (EC50=63µM) superior to the 2-monosubstituted 2-1
(EC50=120µM) but weaker than the 6-monosubstituted 2-14 (EC50=25µM) derivative. The 2,7-
disubstituted compound 2-29 was a marginal ligand of the IIa RNA target (EC50=310µM),
showing an even lower affinity than the monosubstituted 2-23 (EC50=190µM) and a tenfold loss
in activity compared to the homolog 2-10 (EC50=31µM) which, however, had an electron
donating amino group attached directly at the benzene. As was discussed above, increase of the
electron density in the benzoxazole system strengthens the basicity of the N3 position which is a
key participant in hydrogen bonding to the RNA target. For both disubstituted derivatives, 2-28

and 2-29, the reduced affinity as compared to the monosubstituted analogs could be rooted in the
entropic penalty associated with preorganization of many rotatable bonds of the side chains
disfavoring interaction with the RNA. Arrangement of the side chains for binding to the IIa RNA
target that avoids steric clashes, specifically involving the substituent at the 2-position, could be
further disfavored by electrostatic repulsion of the N,N-dimethylamino terminal groups which are
protonated under the assay conditions.
In the current study, we have studied 2-aminobenzoxazoles (2) as ligands for the IIa RNA
target in the HCV IRES element. The goal was to explore if the benzoxazole scaffold is a viable
replacement for the more basic benzimidazole system found in previously discovered HCV
translation inhibitors. Synthesis and affinity testing of monosubstituted 2-aminobenzoxazole
derivatives allowed us to establish structure-binding activity relationships of the ligands. The
observed pattern of compound activity in the FRET target binding assay were explained in the
context of an earlier determined crystal structure of a benzimidazole translation inhibitor (1)
bound to the IIa RNA. The most active benzoxazole ligands were binding the target at EC50
values around 25–45µM which was inferior to the benzimidazole compounds. While the lower
basicity of the benzoxazole scaffold might confer superior drug-like properties to inhibitors, a
detrimental effect on target affinity was observed in the compounds studied here. Future studies
will focus on benzoxazole derivatives in which the basicity of the N3 position, a key interaction
site for RNA target binding, is modulated by the addition of electron donating substituents at the
benzene ring.
Acknowledgments

We thank Kathleen Sered and Carrie Tan for help with compound synthesis. 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 and Scheme Legends
Figure 1. A) Secondary structure of the HCV IRES subdomain IIa RNA target. B) Structures of
a 2-aminobenzimidazole translation inhibitor 1 which was previously co-crystallized in complex
with the subdomain IIa, and 2-aminobenzoxazoles 2 which were designed here as less basic
ligands for the RNA target. C) Superposition of the benzoxazole scaffold (light blue) on the
benzimidazole inhibitor 1 (yellow) in the crystal structure of the IIa RNA complex.4 Hydrogen
bonds are shown as dashed lines. Residues G52 and A53, which stack below and above the
ligand are shown transparent. Numbering of substitution positions in the benzoxazole is
indicated.
Scheme 1. Synthesis of 2-amino-substituted 2-aminobenzoxazoles 2-1 to 2-13 (R=NO2 or H;
R1=NO2, H or NH2) (Table 1). Reagents and conditions: a) KOH, CS2, H2O, EtOH, 80°C, 77%
yield; b) oxalyl chloride, DCM, DMF, then addition of TEA and 5, 0°C to 23°C, 46–88% yield;
compounds 2-8, 2-11 and 2-13 were isolated after this step; anilines were obtained through
reduction by either method outlined in c or d with generally higher yields obtained using Adam’s
catalyst in d; c) SnCl2, HCl, EtOH, 50°C, 53-64% yield; d) PtO2, H2, 23°C, 68-82% yield.
Scheme 2. Synthesis of 6-substituted 2-aminobenzoxazoles 2-14 to 2-21 (Table 2). Reagents
and conditions: e) 7, ACN, reflux, 80%-quantitative yield; f) 10% Pd/C, H2, MeOH; g)
di(imidazole-1-yl)methanimine, ACN, reflux, 41–86% yield over 2 steps.
Scheme 3. Synthesis of substituted 7-methylene-2-aminobenzoxazoles 2-22 to 2-27 (Table 3).
Reagents and conditions: h) 10, NaBH(OAc)3, THF, 92%-quantitative yield; f) Pd/C, H2, MeOH;
g) di(imidazole-1-yl)methanimine, ACN, reflux, 51–77% yield over 2 steps.

Table 1. Activity of 2-amino-substituted 2-aminobenzoxazoles 2-1 to 2-13 in the FRET assay.

Table 2. Activity of 6-substituted 2-aminobenzoxazoles 2-14 to 2-21 in the FRET assay.

Table 3. Activity of substituted 7-methylene-2-aminobenzoxazoles 2-22 to 2-27 in the FRET
assay.
R1
Compound
EC50 [ M]
N
n.a.
190
2-22
2-23
N
N
N
2-24
2-25
n.a.
600
N
N
N
N
O
N
2-26
2-27
n.a.
n.a.
NH2

Table 4. Activity of disubstituted 2-aminobenzoxazoles 2-28 and 2-29 in the FRET assay.