Benzo(h)quinoline derivatives as G-quadruplex binding agents

Article abstract of DOI:10.1016/j.bmcl.2009.02.016

G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadruplexes have been postulated to play important roles in a number of biological systems including gene regulation and the inhibition of enzyme function. Recen

Full text of DOI:10.1016/j.bmcl.2009.02.016

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1584–1587
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Benzo(h)quinoline derivatives as G-quadruplex binding agents
*
Hanumantharao Paritala, Steven M. Firestine
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
G-quadruplexes are unusual structures formed from guanine-rich sequences of nucleic acids. G-quadru-
plexes have been postulated to play important roles in a number of biological systems including gene reg-
ulation and the inhibition of enzyme function. Recently, our laboratory reported on the synthesis and
evaluation of a triaza-cyclopentaphenanthrene compound which bound to G-quadruplexes with good
affinity and selectivity. This compound contains a 4-pyridone group which has not been previously uti-
lized in other quadruplex binding agents. In this Letter, we describe the synthesis and evaluation of 4-
pyridone containing 2- and 3-carboxy-benzoquinolines as G-quadruplex binding agents. We find that
these compounds are capable of binding G-quadruplexes with a Ka in the range of 3 ꢀ 105 Mꢁ1 and with
a 10-fold selectivity for quadruplex over duplex DNA.
Received 21 November 2008
Revised 4 February 2009
Accepted 5 February 2009
Available online 8 February 2009
Keywords:
G-quadruplex
Quadruplex binding agents
Nucleic acids
Ó 2009 Elsevier Ltd. All rights reserved.
G-quadruplexes are four stranded nucleic acid structures
formed from guanine-rich sequences in which sets of four guanine
bases interact with each other via hydrogen bonds.1,2 Quadruplex-
es can be found in either DNA and RNA and can be formed both in-
ter- and intramolecularly.1 One of the earliest known examples of
G-quadruplexes are the telomere sequences where quadruplexes
are thought to play a role in maintenance of the chromosomal
ends.3 Since this time, putative quadruplex forming sequences
have been found in well over 300,000 different places in the human
genome4 and biochemical studies have shown that quadruplexes
can form from sequences derived from telomers,5,6 the immuno-
globulin switch regions,7 the insulin linked polymorphic region,8,9
and the c-myc oncogene.10
To create simple analogs of 1, we eliminated the N-methylpyr-
role region of the molecule and converted the nitroquinoline into a
naphthalene. After the eliminations described above,
a
benzo(h)quinolinone core remained (Fig. 2). To enhance quadru-
plex binding affinity, we anticipated that we would need to add
either a cationic group, which could form favorable electrostatic
interactions with the phosphate groups of the quadruplex, or aro-
matic groups which could enhance binding by forming additional
-stacking interactions with the guanine bases. To determine
where these substituents should be added, molecule modeling
was conducted on both the 2-carboxy and 3 carboxy derivatives
p
Small molecules that bind to quadruplexes have been exten-
sively studied as potential anticancer agents and gene regulators.11
Numerous chemical classes have been shown to possess quadru-
plex affinity with some examples being acridines,12,13 cationic por-
phyrins,14 ethidium bromide derivatives,15 anthraquinones,16
perylenes,17 macrocyclic compounds,18 and steroids.19 Recently,
our laboratory discovered that compound 1 (Fig. 1) was a selective
G-quadruplex binding agent.20 The compound possessed a Kapp of
2.7 ꢀ 104 Mꢁ1 and bound approximately 20-fold better to quadru-
plex DNA than to dsDNA.20 Compound 1 contains a 4-pyridone
group which has not been previously utilized in G-quadruplex
binding agents and thus we chose to investigate whether simple
4-pyridone derivatives would be selective quadruplex DNA bind-
ing agents. To examine this, we have prepared a number of 4-pyri-
done compounds and determined their ability to bind to G-
quadruplex DNA.
Figure 1. Triaza-cyclopenta(b)phenanthrene based quadruplex binding agent.
* Corresponding author. Tel.: +1 313 577 0455; fax: +1 313 577 2033.
E-mail address: sfirestine@wayne.edu (S.M. Firestine).
Figure 2. Targeted molecules: 3-carboxy-4-hydroxybenzo(h)quinoline derivatives
(2) and 2-Carboxy-4-hydroxybenzo(h)quinoline derivatives (3).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.02.016

H. Paritala, S. M. Firestine / Bioorg. Med. Chem. Lett. 19 (2009) 1584–1587
1585
of the core as shown in Figure 2. These models (not shown) sug-
gested that in both analogs the ring system could interact with
the tetrad while placing the cationic tail into one of the grooves
of the quadruplex. This binding orientation allowed for favorable
hydrophobic interactions as well as good electrostatic interactions
with the negatively charged phosphate groups located in the
grooves.
Synthesis of derivatives of 2 and 3 were accomplished according
to the methodology outlined in Schemes 1 and 2. Synthesis of com-
pounds 7–13 was done by amide bond formation from the com-
The synthesized compounds were evaluated for binding and
selectivity to quadruplex DNA using a combination of fluorescence
and SPR methods. We first conducted fluorescence melting studies
using a commercially available G-quadruplex oligonucleotide (G-
Quartet FRET Substrate, Calbiochem) containing a human telomer-
ic repeat sequence.23,24 The 50 and 30 ends of the oligonucleotide
are labeled with a fluorescein–rhodamine FRET pair. Formation of
a quadruplex results in energy transfer from the fluorescein to
the rhodamine and upon heating, the quadruplex melts leading
to a decrease in the FRET signal. If an agent binds to and stabilizes
the quadruplex, an increase in melting temperature is observed.
FRET melting studies were conducted for all compounds at a
mon carboxylic acid,
6 (Scheme 1). Synthesis of 6 was
accomplished from 1-napthylamine by slight modification of the
reported method.21 Reaction of 1-napthylamine with diethyl eth-
oxymalonate under vacuum resulted in the formation of 4. We
found that stirring this reaction under vacuum resulted in substan-
tially higher yields in the reaction due to the efficient removal of
ethanol. Cyclization of 4 to yield 5 was done by refluxing (240–
250 °C) the starting material in diphenyl ether. Upon cooling, the
desired material precipitated as a creamy solid which was col-
lected and washed with petroleum ether. Hydrolysis of 5 in the
presence of refluxing sodium hydroxide generated the acid in
excellent yield. Synthesis of the derivatives was done using stan-
dard peptide coupling conditions under dry reaction conditions.
Unreacted starting material was removed by a simple aqueous
extraction and the desired product was obtained via silica gel chro-
matography. Overall, 7–13 were obtained in a 40–80% yield from 6.
Synthesis of compounds 17–21 was accomplished using the
common intermediate 16.22 Compound 14 was prepared by reac-
tion of diethyl acetylenedicarboxylate with 1-napthylamine and
then heated to reflux in diphenyl ether to afford cyclization to give
15. Compound 15 was then treated with NaOH to provide 16 in
good yields. Synthesis of compounds 17–21 was done using the
same peptide coupling procedure described for 7–13.
concentration of 0.5 lM in 10 mM cacodylate, 100 mM NaCl buffer
(pH 7.4). The concentration of GQ-FRET substrate was half that of
the compound. Melting curves were determined by slowly heating
each sample from 20 to 80 °C at a rate of 1 °C per minute while
measuring the fluorescence at 518 nm at each temperature inter-
val. During the assay, a two minute incubation time at each tem-
perature interval was done prior to fluorescence measurement to
ensure temperature equilibrium. The melting temperature (Tm)
was determined using the first derivative of the obtained melting
curves. As shown in Table 1, several of the compounds showed
an increase in the melting temperature when compared to the oli-
gonucleotide alone indicating binding to the quadruplex. Com-
pounds 8, 10, 11 and 13 displayed the greatest changes in the
Tm, although these were modest, ranging from 3.7 to 5.3 °C. All of
these compounds are 3-substitiuted derivatives of the core
hydroxybenzo(h)quinoline.
To gain additional information on quadruplex binding as well as
to assess the quadruplex selectivity of these compounds, we uti-
lized a fluorescence displacement assay.25–27 This assay has been
extensively used to measure binding of small molecules to
DNA.25–30 In this assay, the binding of the test compound is mon-
Scheme 1.

1586
H. Paritala, S. M. Firestine / Bioorg. Med. Chem. Lett. 19 (2009) 1584–1587
Scheme 2.
Table 1
FRET melting and fluorescence displacement studies to examine quadruplex binding by compounds 7–21
Compd no.
Melting studies (°C) Tm
Thiazole orange displacement studies
Duplex DNA dinding constants 17 bp
(105 Mꢁ1
Tm
Change in temperature
Quadruplex DNA binding constants 22G
Ratio 22G/17 bp
(D
Tm)
(105 Mꢁ1
)
)
7
8
9
52.28
56.52
51.42
55.99
56.40
52.42
54.92
51.92
51.64
52.58
52.20
52.50
1.05
5.29
0.19
4.36
5.17
1.19
3.69
0.69
0.41
1.35
1.0
1.4
1.4
0.53
0.53
2.5
6.0
2.5
0.22
0.51
0.71
2.5
10.4
0.4
0.37
0.84
7.4
0.55
0.05
0.21
0.74
0.21
0.24
0.75
0.5
10
11
12
13
17
18
19
20
21
0.3
0.19
0.42
3.9
0.23
0.05
0.53
0.5
0.53
0.41
1.1
1.27
2.5
itored by a change in fluorescence due to the displacement of a
bound fluorescent dye. Recently, it has been shown that thiazole
orange is an excellent dye for these studies since it is safer than
ethidium bromide and binds equally well to both quadruplex and
double stranded DNA.28,29
Thiazole orange (TO) displacement studies were conducted
using oligo 22G (50-AG3T2A-G3T2AG3T2AG3-30), and oligo 17 bp
(50-C2AGT2C-GTAGTA2C3-30;50-G2TCA2GCATCAT2G3-30).29,30 Previ-
ous researchers have shown that 22G folds into a G-quadruplex
while 17 bp adopts a duplex DNA structure. Binding to each oligo-
nucleotide was determined by titrating the test compound into a
by 50% was taken as the IC50 value. Binding constants were calcu-
lated assuming a simple competitive binding model.25,26 Binding
constants and the ratio of quadruplex to duplex affinity is shown
in Table 1.
An examination of the data presented in Table 1 indicate that
there is little correlation between the compounds identified as
quadruplex binders by melting versus fluorescence displacement
studies. For example, compounds 10 and 13 display changes in
melting of between 3 and 4 °C but have modest affinity as mea-
sured by the fluorescence displacement assay. Conversely, some
compounds display good affinities as measured by fluorescence
displacement (i.e., 18) but not by the FRET melting assay. Similar
observations have been made in the literature and these differ-
ences have been attributed to distinct binding modes or sites of
the compounds being tested.29,30 For 10 and 13, it is likely that
these compounds bind to a site distinct from that of thiazole or-
ange and induce displacement by indirect mechanisms (i.e., chang-
solution containing the oligonucleotide (0.25
lM) and TO
(0.25 M) in 10 mM cacodylate buffer (pH 7.4) supplemented with
l
100 mM KCl. After each addition, the fluorescence spectra were ta-
ken and the area of the peak was determined using the instrument
software. The area was plotted as a function of compound concen-
tration and the concentration of compounds that reduced the area

H. Paritala, S. M. Firestine / Bioorg. Med. Chem. Lett. 19 (2009) 1584–1587
1587
ing conformation of the thiazole orange binding site). In contrast,
Acknowledgments
for molecules like 18, there is a direct competition with thiazole or-
ange for binding to the terminal tetrad.29,30 Given this, we predict
that 11 and 18 likely bind to the terminal tetrad of the quadruplex
whereas compounds 8, 10 and 13 probably intercalate or bind to
the grooves of the quadruplex.
The fluorescence displacement assay also provided us with a
method to determine the selectivity of our agents (Table 1).29,30
An examination of the selectivity ratio of these compounds indi-
cates that most agents display a preference for duplex DNA (22G/
17 bp ratio below 1). However, two compounds, 11 and 18 show
marked selectivity for quadruplex DNA. Compound 11 binds to
quadruplex DNA approximately 10-times better than duplex
DNA, while 18 binds 7-times better.
The authors thank Wayne State University and the Juvenile Dia-
betes Research Foundation for funding. We also thank Dr. W. David
Wilson (Georgia State University) and members of his research
group for conducting surface plasmon resonance studies on our
agents.
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