Mitoxantrone analogues as ligands for a stem-loop structure of tau Pre-mRNA

Article abstract of DOI:10.1021/jm9013407

A series of mitoxantrone (MTX) analogues have been designed, synthesized, and evaluated for binding to and stabilizing a stem-loop structure that serves as a splicing regulatory element in the pre-mRNA of tau, which is involved in Alzheimer's and other ne

Full text of DOI:10.1021/jm9013407

J. Med. Chem. 2009, 52, 6523–6526 6523
DOI: 10.1021/jm9013407
binding. Most recently, we reported the structure of MTX
bound to the tau stem-loop as determined by NMR and
found that MTX interacts at the base of the stem (Figure 1),
intercalating between two G-C base pairs that flank an
unpaired (“bulged”) adenosine (Figure 2).10 (Previous bio-
physical studies have demonstrated that MTX and analogues
can also intercalate DNA.11,12) The structure also suggested
an important role for hydrogen bonding and electrostatic
interactions between the side chains and the major groove.
Herein, we report MTX analogues that are varied in the
aromatic core and the number and type of side chains to
better understand the nature of the interaction of MTX with
the tau stem-loop, comparing the findings with expectations
based on the NMR structure of the complex. These results
provide initial structure-activity relationships to direct the
further design of this promising class of ligands.
Mitoxantrone Analogues as Ligands for a
Stem-Loop Structure of Tau Pre-mRNA
Yang Liu,† Eleanor Peacey,† John Dickson,† Christine
P. Donahue,†, Suxin Zheng,‡ Gabriele Varani,‡,§ and
Michael S. Wolfe*,†
†Center for Neurologic Diseases, Brigham and Women’s Hospital and
Harvard Medical School, 77 Avenue Louis Pasteur, H.I.M. 754,
Boston, Massachusetts 02115, ‡Department of Chemistry and
§Department of Biochemistry, 109 Bagley Hall, Box 351700,
University of Washington, Seattle, Washington 98195. Present
address: Archemix Corp., 300 Third Street,
Cambridge, Massachusetts 02142.
Received September 9, 2009
We carried out systematic dissection of or substitutions
within MTX to modified compounds that consist of the
anthraquinone scaffold with no side chains (1-3), one side
chain (4, 5), two side chains (6-13), three side chains (14), and
four side chains (15) (Figure 3). These changes also included
specific heteroatom replacement (e.g., 7, 8) and altered poly-
amine length (e.g., 10, 12). Straightforward amination13,14 of
difluoro-substituted anthraquinone 16 was applied to prepare
these various MTX analogues (Scheme 1). Treatment of 16
with an excess of various amines assembled 1, 20, and 21. Boc
deprotection of 20 and 21 provided 11 and 12, respectively.
Successive aminations of 16 were carried out to give 5, 9,
and 19. Compound 7 was synthesized via a similar method
(see Supporting Information). Deprotection of 9 and 19
Abstract: A series of mitoxantrone (MTX) analogues have been
designed, synthesized, and evaluated for binding to and stabilizing a
stem-loop structure that serves as a splicing regulatory element in
the pre-mRNA of tau, which is involved in Alzheimer’s and other
neurodegenerative diseases. Several compounds showed signifi-
cantly improved binding activity relative to the original screening
hit mitoxantrone. These findings establish essential structure-
activity relationships to further optimize the activity of this promis-
ing class of compounds.
Neuronal filaments of the microtubule-associated protein
tau are pathological features in Alzheimer’s and other neuro-
degenerative diseases, and evidence supports a role for aber-
rant tau in pathogenesis.1,2 Dominant mutations in the tau
gene are associated with familial frontotemporal dementia
displaying typical tau pathology.3 Many of these mutations
are silent or intronic, occur near the exon 10-intron 10 border
of the tau gene, and increase the inclusion of exon 10 during
pre-mRNA splicing to increase the proportion of tau protein
containing four microtubule-binding domain repeats (4R tau)
to that containing three repeats (3R tau). These mutations
were originally postulated to destabilize a stem-loop struc-
ture atthis exon-intronboundary toregulate exon 10splicing
(Figure 1).4,5 Our laboratory previously validated this
stem-loop as a bona fide structure that can regulate the
alternative splicing of exon 10 in cells.6 Therefore, the tau
stem-loop could represent a therapeutic target for treating
“tauopathies” such as frontotemporal dementia and
Alzheimer’s disease: small molecules that could bind to and
stabilize the tau pre-mRNA stem-loop would have the
opposite effect of disease-causing tau mutations.
afforded
8 and 10, respectively. 2-(2-Aminoethylami-
no)ethanol was treated with 16 in aqueous butanol to mainly
give 6 or mixture 26/27 depending on the solvent butanol/
water ratio. Mixture 26/27 was transformed to 14, while
further aminationof 6 afforded 15. Toward MTXregioisomer
13 (Scheme 2), isopropyl protection and regioselective bromi-
nation15 provided key intermediate 24, which was followed by
amination16,17 and AlCl3 deprotection.
The capacity for the synthesized MTX analogues to bind the
stem-loop was assessed in two ways: a competitive binding
assay and an RNA stability assay. The data from these experi-
ments are summarized in Table 1. The EC50 values represent
the half-maximal effective concentrations for the previously
reported competitive binding assay; active compounds increase
fluorescence by increasing the level of unbound fluorescent
aminoglycoside probe.7 The IC50 values represent the half
maximal inhibitory concentrations for the stability assay; active
compounds decrease fluorescence by preventing the unfolding
of the RNA stem-loop, keeping the fluorophore and quencher
on the oligonucleotide termini in proximity. These two com-
plementary assays gave similar overall results: compounds that
show higher binding affinity also result in better stabilization of
the stem-loop secondary structure. Consistently higher EC50
values from the binding assay compared to IC50 data from the
stability assay likely represent the higher concentration of
MTX analogues required to compete with aminoglycoside
probe to effectively intercalate into RNA.
Via high-throughput screening, our laboratory recently
identified the anticancer drug mitoxantrone as a small mole-
cule that binds to and stabilizes the tau stem-loop (EC50
=
0.71 μM).7 Aminoglycosides such as neomycin are known to
bind to duplex RNA and to the tau splicing regulatory
element,8,9 and MTX could effectively compete with fluores-
cence-labeled aminoglycosides for binding to an oligonucleo-
tide representing the stem-loop RNA. Moreover, MTX
substantially increases the melting temperature of the
stem-loop RNA, demonstrating its stabilizing effect upon
No binding or stabilization activity was observed for
aliphatic polyamine side chains alone (data not shown; see
Supporting Information for structures of side chains tested).
*To whom correspondence should be addressed. Phone: (617) 525-
5511. Fax: (617) 525-5252. E-mail: mwolfe@rics.bwh.harvard.edu.
r
2009 American Chemical Society
Published on Web 10/19/2009
pubs.acs.org/jmc

6524 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Liu et al.
Scheme 1. Synthesis of MTX Analogues 1, 4-12, 14, and 15 by
Amination
Figure 1. The tau stem-loop and site of MTX binding.
Scheme 2. Synthesis of MTX Analogue 13
Figure 2. Interaction between MTX and the tau stem-loop struc-
ture as deduced by NMR.10 MTX intercalates between two G-C
base pairs that flank the unpaired (“bulged”) A-2. The carbon
atoms of MTX are shown in light-blue.
Table 1. Binding and Stabilizing Activity of MTX Analogues
compd
side chain
EC50 (μM)
IC50 (μM)
MTX
1
2
0
0
0
1
1
2
2
2
2
2
2
2
2
3
4
0.89
>100a
>100a
>100
75
0.46
>100a
>100a
>100
5.7
2
3
4
5
29a
5.8a
6
16a
14a
2.3a
5.7a
7
8
0.31
12a
0.18
2.4a
9
10
11
12
13
14
15
0.38
0.16
0.13
4.4a
0.80
1.2
0.23
0.13
0.13
1.5a
0.42
0.34
a Stock solution of compound in DMSO. The presence of DMSO (2%
final concentration) had no effect on background fluorescence.
Figure 3. Structures of mitoxantrone and analogues 1-15.
This finding indicates an essential role of aromatic stacking
for tau stem-loop binding.
Introduction of only one NHCH2CH2NHCH2CH2OH
substituent into the aromatic core in 4 and 5 results in
measurable activity in the binding and stability assays,
although the potency is much weaker than MTX. Thus, the
secondsidechaininMTX contributes substantiallyto binding
(Figure 2). MTX regioisomer 13 is nearly 5 times less potent
than MTX in the binding assay and 3 times less potent in the
stability assay, indicating that the 5,8 arrangement of the two
side chains in MTX is preferable. The substitution of the 1,4
On the other hand, without any aliphatic amine side chains,
aromatic anthraquinones 1-3 alone cannot bind RNA
either. The interaction between these aromatic rings and the
two GC base pairs (at the bulge region; see Figure 2) is
apparently too weak to allow the aromatic system to effec-
tively intercalate. These data suggest that the aromatic core
must cooperate with the aliphatic side chains for binding to
the RNA.

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6525
aromatic hydroxyl groups in MTX with NHCH2CH2NH-
CH2CH2OH to create structures with three or four side chains
(14 and 15, respectively) might have led to enhanced binding
potency. However, these additional substitutions had little
effect on tau stem-loop binding or stabilization. Apparently,
one or two additional NHCH2CH2NHCH2CH2OH substit-
uents installed on the other side of MTX neither sterically
hinder formation of the ligand-RNA complex nor build
more hydrogen bonds or electrostatic interactions with
RNA to enhance binding. However, the retention of binding
potency by 14 and 15 suggests that substitution of the
aromatic hydroxyl groups at position 1 or 4 of the anthra-
quinone ring system can be well tolerated in the complex
and that attaching additional functionality on MTX in
these positions could be advantageous (e.g., to build in addi-
tional interaction with the unpaired adenosine located
nearby10).
MTX analogues 6-12 were built with two identical or
different side chains. Strengthened by two side chains, 6 shows
2-fold better binding affinity and stabilization activity than 5
(containing one side chain), even though the two electron-
withdrawing fluoro atoms of 6 have considerable negative
effect on intercalation. This result confirms the importance of
having two side chains together interacting with RNA. Com-
pared to MTX, the binding of 6 is decreased ∼18-fold and the
stabilization effect decreased 5-fold because of the change
from two hydroxyls to two fluoro atoms. Besides the
decreased aromatic stacking force, possible loss of the sug-
gested hydrogen bonding of aromatic hydroxyls with the
20 hydroxyl groups of the RNA10 might also offer some
explanation for the low potency of 6. Replacement of one
NHCH2CH2NHCH2CH2OH substituent in MTX with the
ether NHCH2CH2OCH2CH2OH in 7 results in a 15-fold
increasein EC50 and a 12-fold increasein IC50. The substantial
decrease in binding and stabilization of the stem-loop sug-
gests that the substitution of ether for secondary amine may
lead to loss of a critical hydrogen bond or electrostatic
interaction and emphasizes that a suitable side chain is a
critical factor for binding.
structural features as “In-PRiNts” (inhibitor of protein-
ribonucleotide sequences) reported by Hamy and co-workers.18
In summary, a series of MTX analogues were designed,
synthesized, and evaluated in vitro for their ability to bind to
and stabilize the tau pre-mRNA stem-loop structure, and
some of these compounds show substantially higher binding
affinity than MTX. The results are largely consistent with the
recently reported structure of the MTX-RNA complex eluci-
dated by NMR. We propose a highly synergistic binding mode
in which the aromatic ring system and two side chains work
together, through intercalation and major groove interaction,
respectively, to give strong binding to the tau stem-loop near
the bulged adenosine. The structure-activity validation and
improved activity provide a design platform for next-genera-
tion ligands targeting the tau stem loop. Through iterative
design, synthesis, and evaluation, we hope to build in greater
potency and specificity to eliminate the cytotoxicity of MTX
and identify compounds that work in cells to alter tau pre-
mRNA splicing. Such efforts are underway in our laboratory.
Acknowledgment. This work was supported by a Zenith
Fellows Award from the Alzheimer’s Association to M.S.W.
Supporting Information Available: Experimental details and
compound characterization. This material is available free of
charge via the Internet at http://pubs.acs.org.
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