Small molecule ligands for bulged RNA secondary structures

Article abstract of DOI:10.1021/ol901478x

A class of wedge-shaped small molecules has been designed, synthesized, and shown to bind bulged RNA secondary structures. These minimally cationic ligands exhibit good affinity and selectivity for certain RNA bulges as demonstrated in a fluorescent inter

Full text of DOI:10.1021/ol901478x

ORGANIC
LETTERS
2009
Vol. 11, No. 18
4052-4055
Small Molecule Ligands for Bulged RNA
Secondary Structures
S. Todd Meyer and Paul J. Hergenrother*
Department of Chemistry, Roger Adams Laboratory, UniVersity of Illinois,
Urbana, Illinois 61801
hergenro@uiuc.edu
Received June 29, 2009
ABSTRACT
A class of wedge-shaped small molecules has been designed, synthesized, and shown to bind bulged RNA secondary structures. These
minimally cationic ligands exhibit good affinity and selectivity for certain RNA bulges as demonstrated in a fluorescent intercalator displacement
assay.
While the vast majority of drugs are ligands for proteins,
the targeting of nucleic acids could emerge as a comple-
mentary approach. In particular, cellular RNA is an attractive
and underexploited target for small molecule therapeutics.1
The broad utility of RNA interference and the clinical success
of ribosome-binding antibiotics hints at the potential uses
for compounds that bind selectively to RNA.
The propensity of RNA to adopt hairpin loops, internal loops,
and bulged secondary structures provides a unique opportunity
for specific recognition by small molecule ligands.1a Thus, one
method to target RNA is to identify individual small molecule
“modules” that bind to specific RNA secondary structural
elements and combine them for selective RNA targeting. As a
complement to compounds that bind selectively to RNA hairpin
loops,2 herein we disclose a novel class of ligands selective for
bulged RNA secondary structures.
gb, Figure 1)3 as well as by synthetic analogues developed
by Goldberg and co-workers, such as DDI (Figure 1).4
Derivatives of NCSi-gb and DDI possess a unique geometry
that enables strong and reasonably selective binding to bulged
regions of DNA, and one report shows that some DDI
derivatives have moderate affinity for certain RNA bulges.4e
We initially sought to derivatize the DDI spirocyclic alcohol
in the preparation of a collection of RNA-binding com-
pounds. However, certain DDI derivatives are susceptible
to decomposition via a proposed retro-aldol pathway,4f,5 and
indeed, we found that 1 (Figure 1) was highly sensitive to
strong bases. Certain acylated derivatives of 1 were also
unstable in aqueous environments. We thus sought a more
hydrolytically stable scaffold that would be amenable to
broad-scale derivatization.
(3) Stassinopoulos, A.; Ji, J.; Gao, X.; Goldberg, I. H. Science 1996,
272, 1943.
Our investigations in this area were inspired by the thiol-
independent breakdown product of neocarzinostatin (NCSi-
(4) (a) Xi, Z.; Hwang, G.-S.; Goldberg, I. H.; Harris, J. L.; Pennington,
W. T.; Fouad, F. S.; Qabaja, G.; Wright, J. M.; Jones, G. B. Chem. Biol.
2002, 9, 925. (b) Ma, D.; Lin, Y.; Xiao, Z.; Kappen, L.; Goldberg, I. H.;
Kallmerten, A. E.; Jones, G. B. Bioorg. Med. Chem. 2009, 17, 2428. (c)
Kappen, L. S.; Lin, Y.; Jones, G. B.; Goldberg, I. H. Biochemistry 2007,
46, 561. (d) Hwang, G.-S.; Jones, G. B.; Goldberg, I. H. Biochemistry 2003,
42, 8472. (e) Xiao, Z.; Zhang, N.; Lin, Y.; Jones, G. B.; Goldberg, I. H.
Chem. Commun. 2006, 4431. For a review, see: (f) Jones, G. B.; Lin, Y.;
Ma, D.; Xiao, Z.; Hwang, G.-S.; Kappen, L.; Goldberg, I. H. Curr. Top.
Med. Chem. 2008, 8, 436.
(1) For reviews of small molecule-RNA binding, see: (a) Thomas, J. R.;
Hergenrother, P. J. Chem. ReV. 2008, 108, 1171. (b) Tor, Y. ChemBioChem
2003, 4, 998. (c) Gallego, J.; Varani, G. Acc. Chem. Res. 2001, 34, 836.
(d) Sucheck, S. J.; Wong, C.-H. Curr. Opin. Chem. Biol. 2000, 4, 678. (e)
Chow, C. S.; Bogdan, F. M. Chem. ReV. 1997, 97, 1489.
(2) (a) Liu, X.; Thomas, J. R.; Hergenrother, P. J. J. Am. Chem. Soc.
2004, 126, 9196. (b) Thomas, J. R.; Liu, X.; Hergenrother, P. J. J. Am.
Chem. Soc. 2005, 127, 12434. (c) Thomas, J. R.; Liu, X.; Hergenrother,
P. J. Biochemistry 2006, 45, 10928.
(5) Gaikwad, N. W.; Hwang, G.-S.; Goldberg, I. H. Org. Lett. 2004, 6,
4833
.
10.1021/ol901478x CCC: $40.75
Published on Web 08/13/2009
 2009 American Chemical Society

iodide, afforded tertiary alcohol 4. Treatment of 4 with
concentrated hydrochloric acid in glacial acetic acid cleanly
effected its dehydration with concomitant cleavage of the
ketal, affording ketone 5. Dehydrogenation of 5 to enone 6
was readily accomplished through a modification of the
Nicolaou method.7 The naphthyl ring system was introduced
through a Diels-Alder cyclization-aromatization sequence,8
which produced ketone 7. Finally, Lewis acid-mediated
cycloisomerization of 7 proceeded smoothly to afford the
scaffold compound 2. The structures of 2 and 7 were verified
through single-crystal X-ray analysis. Gratifyingly, 2 was
shown to adopt the desired wedge shape: the angle between
the noncoplanar aromatic ring systems was determined to
be 38.43 ( 0.12° (Figure 2). The overall synthetic sequence
outlined in Scheme 1 is facile and permits rapid access to
appreciable quantities of 2, a critical factor that enabled the
preparation of multiple lead compounds based on this
scaffold.
Figure 1. Spirocyclic compounds for RNA-binding studies.
The key feature of NCSi-gb and DDI that imbue them with
their affinity for nucleic acid bulges was determined by
Goldberg and co-workers to be the disposition of the aromatic
rings, which form a wedge-like structure. This conformation is
governed by the geometry of the benzobicyclo[3.3.0]octane
core, which confers on 1 and its derivatives a twist angle of
approximately 35°.4a,c,d,f We therefore designed ketone 2
(Figure 1), which was suggested by computational modeling
to have a geometry similar to that of 1. The limited
oxygenation of 2, as compared to 1, was expected to lead to
enhanced stability of the core scaffold.
Scheme 1. Synthesis of Spirocyclic Ketone 2
Figure 2. X-ray structures of ketones 2 and 7.
Given that most RNA-ligand interactions are governed
by electrostatic contacts between the small molecule and the
phosphate backbone of the oligonucleotide,1 it was hypoth-
esized that these synthetic ligands would require some
cationic functionality in order to facilitate binding. A two-
step reductive amination method was therefore developed
to prepare a series of amine derivatives (8-13, Scheme 2).
Ketone 2 was treated with various amines in the presence
of excess Ti(O-i-Pr)4 and 5 Å molecular sieves to afford the
corresponding imines, which were reduced through the action
of sodium borohydride. This reductive amination approach
enabled the introduction of structural diversity through
reaction with dissimilar amines. In an effort to assess the
importance of the spirocycle, several similarly substituted
(6) Cramer, N.; Buchweitz, M.; Laschat, S.; Frey, W.; Baro, A.; Mathieu,
D.; Richter, C.; Schwalbe, H. Chem.sEur. J. 2006, 12, 2488.
(7) Nicolaou, K. C.; Montagnon, T.; Baran, P. S. Angew. Chem., Int.
Ed. 2002, 41, 1386.
The synthesis of 2 commenced from the known ketone 3
(Scheme 1),6 which was used as the racemate. The reaction
of 3 with 2-lithiobiphenyl, prepared from the corresponding
(8) Morris, J. L.; Becker, C. L.; Fronczek, F. R.; Daly, W. H.;
McLaughlin, M. L. J. Org. Chem. 1994, 59, 6484.
Org. Lett., Vol. 11, No. 18, 2009
4053

Scheme 2. Synthesis of Spirocyclic Amines
Scheme 4. Synthesis of 2-Aminobenzindanes
(Scheme 5). The reductive amination procedure was carried out
on ketone 17 using monoprotected diamine 219 to give 22.
Following Boc deprotection, the free amine was treated with
Goodman’s guanidinylating reagent 2310 in the presence of
triethylamine to afford 24, with subsequent deprotection reveal-
ing the guanidinium compound 25.
derivatives of 7 were also prepared under these conditions.
These compounds (14-16, Scheme 3) retain the overall
wedge shape exhibited by 2, as evidenced by the crystal
structure of 7 in Figure 2, but lack the spirocyclic junction.
Amines derived from 2 and 7 were isolated as single,
racemic diastereomers (shown in Schemes 2 and 3), with
stereochemical assignments made based on coupling constant
analysis and NOESY studies. These compounds were found
to be soluble to at least 50 µM in buffer with 5% DMSO
and stable at room temperature in assay buffer for greater
than 24 h, as determined by HPLC.
Scheme 5. Synthesis of Guanidinylated Compound
Scheme 3. Synthesis of Nonspirocyclic Amines
The synthetic compounds were then assessed against a
panel of RNA constructs containing one-, two-, or three-
base bulges (I-III, Figure 3) to determine their binding
properties. Duplex RNA IV was used as a control to identify
ligands that were nonspecific RNA binding compounds.
Ligand binding to RNA was determined using a fluorescent
intercalator displacement assay.11 Each RNA construct was
incubated with ethidium bromide to form a fluorescent
complex. Subsequent treatment with ligand led either to a
reduction in the fluorescence of the complex, indicating
We also sought to investigate the importance of the wedge
shape in the RNA-ligand binding interaction. As this aspect
of the molecular architecture is controlled by the geometric
constraints of the bicyclo[3.3.0]octane core, ketone 178 was used
to prepare an additional subset of amines (18-20, Scheme 4)
lacking this structural motif. A compound containing a guani-
dinium moiety was also prepared to assess the influence of
cationic character on binding relative to molecular geometry
(9) Callahan, J. F.; Ashton-Shue, D.; Bryan, H. G.; Bryan, W. M.;
Heckman, G. D.; Kinter, L. B.; McDonald, J. E.; Moore, M. L.; Schmidt,
D. B. J. Med. Chem. 1989, 32, 391.
(10) (a) Feichtinger, K.; Zapf, C.; Sings, H. L.; Goodman, M. J. Org.
Chem. 1998, 63, 3804. (b) Feichtinger, K.; Sings, H. L.; Baker, T. J.;
Matthews, K.; Goodman, M. J. Org. Chem. 1998, 63, 8432.
(11) (a) Boger, D. L.; Fink, B. E.; Brunette, S. R.; Tse, W. C.; Hedrick,
M. P. J. Am. Chem. Soc. 2001, 123, 5878. (b) Luedtke, N. W.; Liu, Q.;
Tor, Y. Biochemistry 2003, 42, 11391.
4054
Org. Lett., Vol. 11, No. 18, 2009

respectively) exhibit similar bulge-binding activities, sug-
gesting that the spirocyclic junction is not required to lock
the compounds in the appropriate binding conformation, but
that the compounds may nevertheless adopt a similar
conformation in the presence of RNA. Indeed, the X-ray
crystal structures of 2 and 7 are strikingly similar. However,
the lack of any binding by compounds 18-20 implicates
the wedge shape, or at least the additional aromatic unit, as
important to governing the binding conformation. Interest-
ingly, not even the guanidinium group of 25 is sufficient to
permit binding to the RNA constructs. Thus, while the nature
of the substituent on the amine does appear to exert some
influence on the ability of these compounds to bind RNA
bulges, the overall shape of the molecule is a key factor
enabling binding.
Figure 3. Oligonucleotides for binding studies.
binding of the ligand to the RNA construct, or no response.
Compounds that displaced ethidium bromide from an RNA
construct were subsequently evaluated for dose-dependent
fluorescence quenching. These data were fit to a single-site
binding model by curve-fitting analysis to determine the
dissociation constant (Kd) of the interaction.
Several of the amine compounds bind to RNA bulges with
good affinity and selectivity, while none of the ketones (2,
7, 17) display any binding affinity (Table 1). Among the
The compounds described herein constitute a new class
of easily synthesized RNA-binding agents that are stable in
aqueous buffer. Perhaps most importantly, these compounds
are minimally cationic yet exhibit clear RNA-binding activ-
ity. Through evaluation of these compounds, we have
demonstrated that the spirocycle of 2 is not required for RNA
bulge binding per se, as nonspirocyclic versions that retain
the twist geometry (14 and 15) are still RNA ligands.
Most small molecule ligands for RNA are polycationic
(often containing amino sugars), with increasing cationic
character typically leading to attenuated target selectivity.1
The minimally charged nature of the compounds described
in this work demonstrate the potential advantage of shape-
based binding to RNA secondary structures. With a facile
synthetic route to 2 now in place, multiple derivatives can
be created in an effort to probe the sequence-specificity of
these compounds and to find compounds with even stronger
affinities and selectivities for one-, two-, and three-nucleotide
RNA bulges.
Table 1. Binding Constants (µM) for RNA-Ligand Interactions
I
II
III
IV
2
7
17
8
9
10
11
12
13
14
15
16
18
19
20
25
>20
>20
>20
>20
>20
>20
12.1
9.7
7.8
12.4
6.4
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
5.9
>20
9.4
2.3
>20
>20
>20
>20
>20
>20
>20
6.1
>20
>20
11.5
18.1
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
Acknowledgment. We thank Jason R. Thomas (UIUC)
for helpful conversations and Dr. Scott R. Wilson (UIUC)
for assistance with X-ray crystallography. This work was
supported by the University of Illinois and the National
Institutes of Health (R01AR058361).
>20
>20
>20
>20
Supporting Information Available: Experimental pro-
cedures, binding data, and spectral data for all new com-
pounds, as well as X-ray structural data for 2 and 7. This
material is available free of charge via the Internet at
http://pubs.acs.org.
spirocyclic series, 11, 12 and 13 exhibit low-micromolar
affinity for certain bulge sizes, with 13 having an apparent
preference for the single-base bulge of construct I. Interest-
ingly, the nonspirocyclic isomers of 11 and 12 (14 and 15,
OL901478X
Org. Lett., Vol. 11, No. 18, 2009
4055