Synthesis and evaluation of novel bacterial rRNA-binding benzimidazoles by mass spectrometry

Article abstract of DOI:10.1016/j.bmcl.2003.11.031

A series of novel benzimidazoles were efficiently synthesized using both solution- and solid-phase chemistry. These compounds were found to bind to the bacterial 16S ribosomal RNA A-site with micromolar affinities using unique mass spectrometry-based assays.

Full text of DOI:10.1016/j.bmcl.2003.11.031

Bioorganic & Medicinal Chemistry Letters 14 (2004) 695–699
Synthesis and evaluation of novel bacterial rRNA-binding
benzimidazoles by mass spectrometry
Yun He,* Jun Yang, Baogen Wu, Dale Robinson, Kelly Sprankle, Pei-Pei Kung,
Kristin Lowery, V.Mohan, Steve Hofstadler, Eric E.Swayze and Rich Griffey
Ibis Therapeutics, A Division of Isis Pharmaceuticals, Inc., 2292 Faraday Av., Carlsbad, CA 92008, USA
Received 2 October 2003; revised 7 November 2003; accepted 14 November 2003
Abstract—A series of novel benzimidazoles were efficiently synthesized using both solution- and solid-phase chemistry.These
compounds were found to bind to the bacterial 16S ribosomal RNA A-site with micromolar affinities using unique mass spectro-
metry-based assays.
# 2003 Elsevier Ltd.All rights reserved.
The interactions between RNA and biological macro-
molecules are clearly essential for many vital processes
in molecular biology, and the excitement over RNA-
based viruses has fueled an interest in the development
of potential RNA inhibitors.Recently, a considerable
amount of attention has been focused on new RNA-
binding molecules for drug discovery.1À7 RNA offers
several selective advantages over DNA as a therapeutic
agent.First, chromosomal DNA is packaged exten-
sively, significantly limiting its accessibility to small
molecule regents.Second, DNA repair systems are
available in the cell, whereas analogous enzymes for
RNA repair are virtually unknown.Finally, RNA exhi-
bits a high level of diversity in terms of tertiary folding,
and therefore will likely have a greater potential for
selective targeting based on structure rather than
sequence.Historically, however, RNA-based drug dis-
covery has proved to be extremely difficult.The only
successful example is the oxazolidinone antibiotics devel-
oped by Pfizer.8 Few classes of compounds are known to
bind RNA with SAR information.These include ami-
noglycosides and cationic peptides.Discovery of RNA
binders using traditional high throughput assays such as
filter binding, fluorescence, SPA, SPR, etc., has proved to
be equally unsuccessful.We have screened combinatorial
libraries for RNA binders, but the hit rates were essen-
tially zero.Even for the ‘RNA-directed’ libraries, the hit
rates were still very low (a few per 100,000), and most of
these hits were not ‘SAR-able’.Practically, these are not
meaningful discoveries for drug development.
One major reason for this outcome is that most tradi-
tional assays are not sensitive enough to detect the
typically weak RAR binders, and most of the com-
pounds fall out of the detecting range and thus the lim-
ited number of hits wouldn’t be able to give enough
SAR information for further studies.Recently, we have
developed a MS-based high throughput screening
assay.5,6,9À12 This assay is extremely sensitive and could
detect RNA binders with KD ranging from nonamolar
to minimolar.In addition, we could use the MS-based
assay to carry out competition experiments and deter-
mine the binding locations in the target RNA.In this
Keywords: Benzimidazoles; rRNA; Mass spectrometry.
* Corresponding author at present address.Department of Medicinal
Chemistry, Genomics Institute of the Novartis Research Foundation
(GNF), 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA.
Tel.: +1-858-332-4706. Fax: +1-858-812-1651; e-mail: yhe@gnf.org
Figure 1. Sequence of E. coli 16S RNA A-site target and molecular
modeling of 1 bound to the active site.
0960-894X/$ - see front matter # 2003 Elsevier Ltd.All rights reserved.
doi:10.1016/j.bmcl.2003.11.031

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Y. He et al. / Bioorg. Med. Chem. Lett. 14 (2004) 695–699
Figure 2. Competitive displacement of glucosamine from the E. coli 16S A-site RNA by 1.
Scheme 1. Synthesis of piperidine-modified benzimidazoles and their binding affinities (KD) for E. coli 16S A-site.9 (a) EDC (1.2 equiv), DMAP
(cat.), DMF, 25 ꢀC, 4 h; (b) NaOH, H2O, 100 ꢀC, 10 h, 66% over two steps; (c) TFA/CH2Cl2 (1:1), 25 ꢀC, 1 h 95%; (d) Pd/C, H2, MeOH, 25 ꢀC, 6 h,
90%; (e) 6.0 M HCl/dioxane, 25 ꢀC, 1 h; (f) Ac2O, Et3N, CH2Cl2, 25 ꢀC, 2 h, 87% over two steps.

Y. He et al. / Bioorg. Med. Chem. Lett. 14 (2004) 695–699
697
article, we report the discovery of novel benzimidazoles
that bind to bacterial ribosomal RNA using our MS-
based high throughput screening assays, and the appli-
cation of the MS-based assay to carry out SAR studies
on this class of compounds.
believe that 1 binds to the desired RNA decoding region
and could potentially inhibit bacterial translations (Fig.
2).2À4, 38
After establishing the binding of 1 to the correct loca-
tion on the target RNA, we decided to carry out sys-
tematic chemical modifications to study the SAR
around the benzimidazole.The synthesis of compound 1
and piperidine-modified benzimidazoles are shown in
Scheme 1.Treatment of commercially available 5-nitro-
1,2-dianiline (2) and N-Boc-isonipecotic acid (3) with
EDC in the presence of catalytic amount of DMAP led
to the formation of the corresponding amide 4a together
with its regioisomers.The crude mixture was then
refluxed in aqueous sodium hydroxide solution for 8 h
to give the cyclized intermediate 5a.Treatment of com-
pound 5a with 20% TFA in dichloromethane at room
temperature for 30 min led to the formation of com-
pound 6 which was then hydrogenated over Pd/C to
give 1.Our initial effort suggested that the electron
withdrawing nitro group at C5 position (6c) is preferred
over the corresponding amino group (1) for binding to
the target 16S RNA A-site.Thus, a series of piperidine-
modified analogues with a nitro substitution at 5 position
(6a–7b) were synthesized following the same synthetic
route and all these compounds were screened against
16S RNA A-site.Clearly, a basic NH group with the
correct orientation in piperidine region is required to
maintain the affinity, since acetylation (7a), methylation
(7b), removal (6b) of the free NH group and unsatura-
tion of the piperidine ring (6c) all diminished the bind-
ing affinity.The NH group is critical, presumably
because it forms a hydrogen bond with the negatively
charged phosphate in the RNA backbone (Fig.1 ).
It has been established that the 16S A-site is involved in
bacterial translation and the aminoglycosides are
known to bind to this region.13À20 Bacterial 16S A-site
represents a prime target for discovering antibacterial
agents and most of the research work in this field has
been focusing on the modification of the natural
aminoglycosides.14,19,21À35 However, these aminoglyco-
sides are generally associated with many toxic issues, such
as nephro- and oto-toxicity.36,37 For our antibacterial
program, we set out to search for novel small molecules
that would bind to the 16S A-site of Escherichia coli
ribosome RNA, whose sequence is shown in Figure 1.
From our MS-based screening assays we discovered a
novel amino benzimidazole (1 in Figs.1 and 2 ) that
binds to the target RNA with an estimated KD of 500
mM.Although its low affinity is not suitable for most
conventional assays, the amino benzimidazole was a
good starting point for SAR studies using our MS-
based assay.
First, MS-based competition experiments were used to
determine the binding location of 1 to the target RNA.
Glucosamine is the A-ring of paromomycin that is known
to bind to the target RNA and inhibits bacterial transla-
tion.Our studies suggest that 1 and glucosamine compete
for the same binding site on the target RNA.Since glu-
cosamine binds to the target RNA at the same location as
it is in paromomycin binding to the same target, we
Scheme 2. One-pot synthesis of benzimidazoles and their binding affinities (KD) for E. coli 16S A-site.(a) PPA, 100 ꢀC, 2 h, 63–89%.

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Y. He et al. / Bioorg. Med. Chem. Lett. 14 (2004) 695–699
Interestingly, the extended piperidine analogues (6g,h
and i) showed improved affinities, which are likely due
to better orientations of the NH groups to contact the
phosphate backbone.
showed no appreciable binding for the target RNA,
which suggests the crucial role of the nitrogen moiety in
the benzimidazole.Next, we focused our effort on the
benzimidazole analogues with modifications at N1.This
series of compounds were efficiently synthesized by
employing the solid-phase chemistry (Scheme 3).Wang
resin was first converted into imidazole carbonyl deriv-
ative, which was then allowed to react with compound
10g to give common intermediate 11.Compound 11
reacted readily with a variety of alkylating or acylating
reagents to give the corresponding alkyl or acyl pro-
ducts, which after removal of Boc group with TFA in
dichloromethane led to the desired N-1 substituted
analogues in excellent yields and purity (13a–w).
A series of benzimidazole-modified analogues were then
prepared to further establish the SAR in the aromatic
region.A one-pot procedure was employed to quickly
access these compounds (Scheme 2).This procedure
required the simple heating of a suitable 1,2-dianiline (8)
with isonipecotic acid (9) in the presence of polyphos-
phoric acid (PPA).The free benzimidazoles were then
isolated in good to excellent yields after basic work-up.
Using the MS-based screening assay, we established
that (1) electron donating groups such as, NH2 and
OMe reduced the affinities for the 16S RNA (1, 10a); (2)
certain hydrophobic substitutions such as a methyl
(10b), bromo (10c) and chloro (10d) decreased the
potency; (3) insertion of nitrogen atoms into the aro-
matic moiety, particularly at the C5 but not C4 position
(10i) reduced activities; and finally (4) electron-with-
drawing groups enhanced the binding affinities (10e–g).
MS-based binding assays for these compounds suggest
that benzyl substitutions at N-1 are well tolerated.Both
the electron deficient (13a–f) and electron rich (13g–j)
benzyl groups could be incorporated while still retaining
the affinity.The electron deficient pyridinyl analogues
(13k,l) showed low micromolar affinities.Small hydro-
phobic alkyl substitutions (13m,o) are also acceptable.
However, benzothiazole (13q), toluenesulfonamide 13u,
phenol ester 13v analogues completely lost the affinity,
suggesting that certain structural flexibility in the sub-
stituents are required and the proper orientations of
By applying the same protocol, the benzothiazole ana-
logue 10l was also prepared starting from 2-aminothio-
phenol and isonipecotic acid (Scheme 2).Compound 10l
Scheme 3. Solid-phase synthesis of N1 substituted benzimidazoles and their binding affinities (KD) for E. coli 16S A-site.(a) Et 3N, CH2Cl2, 25 ꢀC, 1
h; (b) RX, NaH, or K2CO3; or RCl, Et3N (13u and 13v), DMF; (c) TFA/CH2Cl2 (20:1), 25 ꢀC, 5 h; >80% overall yield, >95% purity.

Y. He et al. / Bioorg. Med. Chem. Lett. 14 (2004) 695–699
699
the substituents are important for good binding to the
target RNA.The polar, negatively charged carboxy
substitution (13w) in this region completely removed the
activities.These data again suggest that this portion of
benzimidazole is situated in the hydrophobic pocket of
the target RNA.
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In summary, a novel series of benzimidazoles was iden-
tified to bind to the bacterial E. coli 16S ribosomal
RNA A-site using a mass spectrometry-based assay.
The same assay was utilized to guide SAR studies.
These benzimidazoles were efficiently synthesized using
both the solution- and solid-phase chemistry and some
of the analogues exhibited low micromolar affinities
towards the E. coli 16S RNA A-site.The MS-based
screening assay allowed the discovery and quick estab-
lishment of a SAR of this series of benzimidazoles that
would be difficult to achieve otherwise.This study also
represents a practical application of our MS technolo-
gies in RNA-based small molecular drug discovery.
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Financial support thanks to USAMRID DAMD717-
02-2-0023.The US..Army Medical Research Acquisi-
tion Activity 820 Chandler Street, Fort Detrick MD
21702-5014, USA is the awarding and administering
office.The content of this manuscript does not necessa-
rily reflect the position or policy of the Government,
and no official endorsement should be inferred.
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