Targeting promoter G-quadruplex DNAs by indenopyrimidine-based ligands

Article abstract of DOI:10.1002/cmdc.201402394

The formation of G-quadruplex structures can regulate telomerase activity and the expression of oncogenes at the transcriptional and translational levels. Therefore, stabilization of G-quadruplex DNA structures by small molecules has been recognized as a promising strategy for anticancer drug therapy. One of the major challenges in this field is to impart stabilizing molecules with selectivity toward quadruplex structures over duplex DNAs, and to maintain specificity toward a particular quadruplex topology. Herein we report the synthesis and binding interactions of indenopyrimidine derivatives, endowed with drug-like properties, with oncogenic promoters of c-myc and c-kit, telomeric and duplex DNAs. The results show specific stabilization of promoter over telomeric quadruplexes and duplex DNAs. Molecular modeling studies support the experimental observations by unraveling the dual binding mode of ligands by exploiting the top and bottom quartets of a G-quadruplex structure. This study underscores the potential of the indenopyrimidine scaffold, which can be used to achieve specific G-quadruplex-mediated anticancer activity.

Full text of DOI:10.1002/cmdc.201402394

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DOI: 10.1002/cmdc.201402394
Targeting Promoter G-Quadruplex DNAs by
Indenopyrimidine-Based Ligands
K. V. Diveshkumar, Saaz Sakrikar, S. Harikrishna, V. Dhamodharan, and P. I. Pradeepkumar*[a]
The formation of G-quadruplex structures can regulate telo-
merase activity and the expression of oncogenes at the tran-
scriptional and translational levels. Therefore, stabilization of
G-quadruplex DNA structures by small molecules has been rec-
ognized as a promising strategy for anticancer drug therapy.
One of the major challenges in this field is to impart stabilizing
molecules with selectivity toward quadruplex structures over
duplex DNAs, and to maintain specificity toward a particular
quadruplex topology. Herein we report the synthesis and bind-
ing interactions of indenopyrimidine derivatives, endowed
with drug-like properties, with oncogenic promoters of c-myc
and c-kit, telomeric and duplex DNAs. The results show specific
stabilization of promoter over telomeric quadruplexes and
duplex DNAs. Molecular modeling studies support the experi-
mental observations by unraveling the dual binding mode of
ligands by exploiting the top and bottom quartets of a
G-quadruplex structure. This study underscores the potential
of the indenopyrimidine scaffold, which can be used to ach-
ieve specific G-quadruplex-mediated anticancer activity.
Introduction
G-quadruplexes are four-stranded secondary nucleic acid struc-
tures formed by guanine-rich sequences. Potential quadruplex-
forming sequences are found in telomeres (chromosomal
ends), promoter regions of oncogenes, introns and UTRs of
mRNAs.[1–3] Human telomeric repeat sequences are able to fold
into quadruplex structures with various topologies such as an-
tiparallel, hybrid, and parallel under physiological conditions
(Figure 1).[2] Stabilization of these quadruplex structures by
small molecules has been shown to inhibit telomerase activity,
thereby halting cancer progression.[2] The promoter regions of
important proto-oncogenes such as c-myc, c-kit, vegf, bcl-2,
and hif-1a contain G-rich sequences, which have a propensity
to form parallel quadruplex structures.[4–6] Unlike telomeric
DNA, promoter quadruplex-forming sequences, being present
in the double-stranded form, must compete energetically with
duplex DNA to form quadruplex structures. However, molecu-
lar crowding conditions and dynamic forces derived from neg-
ative superhelicity aid in the formation of promoter quadru-
plex structures.[7] Proto-oncogenes such as c-myc and c-kit are
overexpressed up to 80% in various tumors such as gastro-
intestinal, ovarian, and breast cancers.[6] The promoter region
of c-myc DNA contains seven nuclease-hypersensitive elements
(NHEs), in which NHE III1, located from À142 to À115 base
pairs upstream of the P1 promoter, contains quadruplex-form-
ing sequences. Since the NHE III1 region controls 90% of c-myc
expression, stabilization of G-quadruplex structures by small
molecules in this region can reg-
ulate gene expression.[8,9] Anoth-
er widely studied proto-onco-
gene is c-kit, which plays an im-
portant role in cell survival, pro-
liferation, and differentiation.[6]
Two quadruplex-forming se-
quences, namely c-kit1 and c-
kit2, are present in the same
core promoter: c-kit1 DNA be-
Figure 1. Schematic representation of a G-quartet and various quadruplex structural motifs. The G-quartet is
formed by self-association of guanine bases through Hoogsteen hydrogen bonding; formation of the G-quadru-
plex structure occurs through stacking of G-quartets stabilized by monovalent metal ions.
tween À87 and À109 base pairs,
and c-kit2 DNA between À140
and À160 base pairs from the
transcription start site.[10] The use
of small molecules to stabilize G-quadruplex structures present
in c-kit DNAs can regulate gene expression and thus inhibit
a wide range of cancerous growths.[5]
[a] K. V. Diveshkumar, S. Sakrikar, S. Harikrishna, V. Dhamodharan,
Prof. P. I. Pradeepkumar
Department of Chemistry
Indian Institute of Technology Bombay, Powai, Mumbai 400076 (India)
E-mail: pradeep@chem.iitb.ac.in
The structure, stability, and topology of G-quadruplex DNAs
depend on various factors such as the nature of metal ions
present, as well as the sequence and length of the loops.[1–3]
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402394.
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Monovalent cations such as K+ and Na+ can stabilize the
quadruplex structure by neutralizing the negative potential
created by oxygen atoms of guanine bases.[11] G-quadruplex
structures can be formed from a single strand (intramolecular)
or multiple strands (intermolecular), and the topology can be
parallel, antiparallel, or mixed-hybrid type, depending on the
nature of the loops (Figure 1).[12] Therefore, the major challenge
in targeting G-quadruplexes lies
cific stabilization of such parallel promoter quadruplexes can
be achieved by small molecules, which are designed to interact
with grooves or loops along with top and bottom quartets of
G-quadruplex structures. Along these lines, we report herein
three derivatives of indenopyrimidine that are capable of spe-
cifically stabilizing the promoter quadruplex DNAs over human
telomeric and duplex DNAs (Figure 2). A detailed evaluation of
in the design of small molecules
that can selectively stabilize
quadruplexes over various DNA
structures and achieve specificity
among various quadruplex top-
ologies.
A large number of molecules
have been explored for their se-
lective stabilization of quadru- Figure 2. Structures of DNA G-quadruplex-stabilizing ligands. Ligands InPy1 and InPy2 contain pyrrolidine side
chains of varying length, whereas InPy3 has an N,N-dimethylethane side chain; all three contain the same aryl
plex
DNAs
over
duplex
indenopyrimidine core.
DNAs.[13–18] TMPyP4, a porphyrin
derivative, is well-studied
a
quadruplex stabilizer both in vitro and in vivo and shows anti-
cancer activity.[19] The ligand was found to be nonselective, as
it showed affinity for other DNA structures including duplex
DNAs. However, the selectivity toward quadruplex DNAs was
later improved through various TMPyP4 derivatives such as
metallated and phenol quaternary ammonium porphyrins.[20,21]
Telomestatin and its derivatives,[22] acridine derivatives,[23] pery-
lene,[24] quarfloxin,[25] and corolone derivatives[26] were reported
the specific stabilization properties toward promoter quadru-
plex DNAs by these molecules was carried out using various
biophysical methods (CD titration, CD melting, fluorimetric ti-
trations, Job plot analysis) biochemical assays (Taq DNA poly-
merase stop assays), molecular modeling, and dynamics stud-
ies.
for their selective stabilization of telomeric quadruplexes over Results and Discussion
duplex DNAs. Triazole-linked acridine derivatives have been re-
Ligand design and synthesis
ported for their selective binding of telomeric quadruplex
DNAs over duplex and c-kit quadruplex DNAs.[27] An acyclic te-
lomestatin heptamer derivative, TOxaPy, was reported for its
potency in recognizing antiparallel topology (under Na+ condi-
tions) over the mixed-hybrid topology (under K+ conditions)
of telomeric quadruplex DNAs.[28] Although the molecule
showed selectivity over duplex DNAs, moderate stabilization
was obtained with c-myc and c-kit promoter quadruplex DNAs
having parallel topology.[28] Conversely, N-methyl mesoporphyr-
in IX (NMM) was reported for its ability to induce and stabilize
a parallel topology of telomeric quadruplex DNAs under K+
conditions over antiparallel (under Na+ conditions) telomeric
quadruplex DNAs.[29,30] Ellipticine derivatives,[31] piperazinyl-sub-
stituted quindolines,[32] bisindole carboxamide derivatives,[33] di-
ethynyl pyridine derivatives,[34] and furan-based oligopepti-
des[35] were also reported for their preferential affinity for pro-
moter quadruplexes over duplex DNAs. A recent study from
our research group has reported bisquinolium and bispyridini-
um derivatives of naphthyridine, which can selectively stabilize
telomeric and promoter quadruplex DNAs over duplex
DNAs.[36,37] Notably, most of the small molecules reported thus
far have limited preference for a given topology, or lack drug-
like properties.
Indenopyrimidine derivatives were previously reported as an-
tagonists of dual A2A/A1 adenosine receptors for the potential
treatment of Parkinson’s disease.[38] Several derivatives of in-
denopyrimidine were demonstrated to have potent activity in
animal models of Parkinson’s disease.[38] We hypothesized that
aryl indenopyrimidine derivatives can be tuned as efficient G-
quadruplex-stabilizing ligands owing to their simplicity in
structure, synthesis, and drug-like properties. We designed and
synthesized three derivatives of indenopyrimidine that can sat-
isfy the structural features of G-quadruplex-stabilizing agents
(Figure 2). These compounds contain an aromatic core that
can stack on the G-quartet, and ethyl/propyl side chains termi-
nated with an amino functionality, which will aid interaction
with the negatively charged phosphate backbone of the
grooves and loops of quadruplex structures. Moreover, low
molecular weight and a minimal number (less than five) of hy-
drogen bond acceptors and donors contribute to the drug-like
properties of these molecules.
Synthetic access to the indenopyrimidine derivatives is de-
picted in Scheme 1. All three derivatives (InPy1–3) were syn-
thesized from commercially available starting materials 6-me-
thoxyindanone (1) and benzaldehyde (2). Preparation of the in-
termediate compound 5 was carried out by following reported
procedures with slight modifications.[38] Compound 1 was con-
densed with 2 under acidic conditions to obtain 3 in 85%
yield. Dehydrated aldol product 3 was first refluxed with free
The wealth of structural information on promoter quadru-
plexes shows that most of the promoter quadruplexes adopt
parallel topology and have unique grooves and loops
(medium-sized grooves and propeller loops).[12] Therefore, spe-
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Scheme 1. Synthesis of indenopyrimidine derivatives InPy1, InPy2, and InPy3. Reagents and conditions: a) AcOH, conc HCl, 908C, 6 h; b) 1. guanidine·HCl,
NaOH, EtOH, 808C, 18 h, 2. air, NaOH, DMF, 808C, 16 h; c) LiCl, H2O, NMP, 1808C, 62 h; d) 1,2-dibromoethane, K2CO3, CH3CN, 758C, 18 h; e) 1,3-dibromopro-
pane, K2CO3, CH3CN, 758C, 18 h; f) pyrrolidine, CH3CN, reflux, 3 h; g) N,N-dimethylethane-1,2-diamine, CH3CN, reflux, 3 h.
guanidine base (obtained by treating guanidine hydrochloride
with sodium hydroxide), which, upon further oxidation in air,
furnished the cyclized product 4 in 38% yield. Hydrolysis of 4
with lithium chloride and water in N-methyl-2-pyrrolidone af-
forded compound 5 in 94% yield.[38] Treatment of compound
5 with 1,2-dibromoethane and 1,3-dibromopropane under
basic conditions delivered compounds 6 and 7 in respective
yields of 80 and 83%.[39] Finally, 6 was refluxed with pyrrolidine
and N,N-dimethylethane-1,2-diamine to afford the final com-
pounds InPy1 and InPy3 in 86 and 83% yields, respectively.
Similarly, compound 7 was refluxed with pyrrolidine to furnish
InPy2 in 95% yield.
a negative peak at 240 nm reflect parallel quadruplex topolo-
gy.[34,41,42]
The interactions of ligands with telomeric and promoter
quadruplex DNAs, c-myc and c-kit1, in the presence and ab-
sence of added metal ions were studied by CD titration experi-
ments. Titration of the human telomeric DNA with InPy1 in
the absence of added monovalent cations was carried out to
explore the capacity of the ligand to induce a particular quad-
ruplex topology. CD spectra of telomeric DNA without ligands
exhibited a small positive peak at 255 and a negative peak at
234 nm, which do not account for any particular quadruplex
topology (Figure 3A). However, titration with InPy1 resulted in
decreased ellipticity for the peaks at 255 and 234 nm. At
higher ligand concentration, the appearance of a new peak
around 295 nm was observed, which cannot be attributed to
any quadruplex topology. These results clearly show that
InPy1 is incapable of inducing any particular quadruplex topol-
ogy in telomeric DNA. However, in the case of InPy2 and
InPy3, weak enhancement of peaks at 290 and 260 nm were
observed at higher ligand concentrations, indicating weak in-
duction of antiparallel topology for telomeric DNA (Figure S1,
Supporting Information). Titration of telomeric DNA under
added K+ conditions showed a strong peak around 290 nm
along with a shoulder peak around 255 nm and a small peak
around 235 nm that are characteristic peaks for a (3+1) hybrid
quadruplex structure (Figure S1, Supporting Information).[41]
Circular dichroism studies
Circular dichroism (CD) spectroscopy is a practical tool to gain
insight into various topologies, the effects of metal ions, and
the stability G-quadruplex structures.[40] Although CD spectros-
copy does not account for the molecularity (intra versus inter)
of the DNA strands involved in forming a quadruplex structure,
the potential of the ligands to induce and stabilize various
quadruplex topologies can be well studied by CD titration ex-
periments. In CD spectra, positive peaks at 290 and 240 nm,
and a negative peak at 260 nm represent an antiparallel quad-
ruplex structure, whereas a dominant positive peak at 260 and
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CD melting studies
Thermal stabilization properties of ligands with quadruplex
and duplex DNAs can be studied with the aid of CD spectros-
copy by comparing the melting temperatures of DNAs at a suit-
able wavelength in the presence or absence of ligands.[43] For
telomeric DNA, melting studies were performed under K+
(mixed-hybrid topology) and under Na+ conditions (antiparal-
lel topology) by monitoring ellipticity at 295 nm. Telomeric
DNA in the absence of ligands showed Tm values of 54 and
458C under K+ and Na+ conditions, respectively (Figure 4A,
Figure 3. CD titration spectra of A) telomeric and B) wild-type c-myc DNA
with InPy1 in the absence of added metal ions (12.5 mm DNA in 50 mm Tris,
pH 7.2).
There was no further significant change in the spectra ob-
served upon titration with increasing concentrations of InPy1
(0–5 equiv), indicating the absence of any conformational
changes and retention of the pre-folded quadruplex structure.
Additionally, no significant changes in the CD spectra were ob-
served by titrating telomeric DNA with InPy1 in the presence
of added Na+ ions, indicating retention of the existing anti-
parallel topology (Figure S1, Supporting Information).[42]
Promoter quadruplex DNAs were reported for their ability to
form a strong parallel topology even in the absence of added
monovalent cations.[34] CD titration experiments for c-myc and
c-kit1 in the presence and absence of added monovalent ions
were performed with increasing concentrations of ligands to
study their ability to induce or enhance conformational
changes in the existing quadruplex topology. CD spectra of
c-myc and c-kit1 in the absence of added K+ ions showed an
increase in ellipticity of peaks at 260 and 240 nm, clearly indi-
cating further induction of existing parallel quadruplex topolo-
gy (Figure 3B and Figures S2 and S3, Supporting Information).
CD spectra for c-myc and c-kit1 DNAs in the presence of added
K+ ions clearly showed the presence of parallel topology, and
upon titration with ligands InPy1, InPy2, and InPy3, retention
of the existing parallel topologies was observed (Figures S2
and S3, Supporting Information). It was evident from the CD ti-
tration experiments that all ligands are able to further induce
and stabilize existing parallel topology in promoter quadruplex
DNAs.
Figure 4. CD melting curves for A) telomeric [10 mm KCl, 90 mm LiCl] and
B) c-myc [1 mm KCl and 99 mm LiCl] DNAs (10 mm DNA, 10 mm lithium caco-
dylate buffer, pH 7.2) in the absence or presence of ligands (5 equiv).
and Figure S4, Supporting Information). Strikingly, there was
no significant change (maximum DTm =1.58C) in the melting
temperature under both salt conditions (K+ and Na+) for the
telomeric quadruplex DNA upon addition of 5 equivalents of
the ligands (Table 1). This further validates that ligands do not
stabilize telomeric quadruplex DNAs. To determine the selectiv-
ity of ligands over duplex DNAs, CD melting studies for duplex
DNAs were carried out at 242 nm under similar salt and buffer
conditions. Thermal stabilization was very low in the case of
duplex DNAs (maximum DTm =0.68C) with any of the ligands,
even at higher concentrations (Figure S4, Supporting Informa-
tion).
The stabilization properties of ligands with promoter c-myc
and c-kit1 quadruplex DNAs were monitored at 263 nm under
K+ conditions (Figure 4B, and Figure S4, Supporting Informa-
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Table 1. Thermal stability of quadruplex and duplex DNAs with ligands
measured by CD melting experiments.
Ligand
DTm [8C][a]
Telomeric
(K+)
c-myc
c-kit1
Telomeric
(Na+)
Duplex
(17 mer)
InPy1
InPy2
InPy3
7.8Æ0.5
8.2Æ0.5
6.6Æ0.5
9.6Æ0.5
10.1Æ0.2
9.5Æ0.2
1.5Æ0.2
1.2Æ0.2
0.8Æ0.3
1.2Æ0.1
1.1Æ0.2
0.2Æ0.2
0.6Æ0.2
0
0
[a] DTm values are the difference in thermal melting [DTm =Tm (DNA+
5 mol. equiv ligand)ÀTm (DNA)] and are reported as the average ÆSD of
three independent experiments.
tion). For c-myc and c-kit1 quadruplex DNAs in the absence of
ligands, the respective melting temperatures were 58 and
448C. Interestingly, all three ligands showed higher thermal
stabilization with promoter quadruplex DNAs (Table 1). InPy1
and InPy2 elicited an increase in the melting temperature
(DTm =7.8–8.28C), whereas InPy3 showed only a moderate in-
crease in melting temperature (DTm =6.68C). Similarly, for
c-kit1 quadruplex DNA, all ligands were able to impart higher
stabilization, as evident from their melting temperature (DTm =
108C). Thus the results from CD melting studies validate the
specific stabilization of promoter quadruplex DNAs over telo-
meric and duplex DNAs by the ligands.
Figure 5. Fluorescence quenching curves and Stern–Volmer plots for InPy1
with increasing concentrations of c-myc DNA. A) InPy1 (100 mm in 100 mm
KCl, 10 mm lithium cacodylate buffer, pH 7.2) with c-myc DNA (0–30 mm
under identical salt and buffer conditions). B) Stern–Volmer plot for InPy1
with c-myc DNA.
Fluorescence titration experiments
Fluorescence spectroscopy was used to explore the binding
properties of ligands with various quadruplex and duplex
DNAs.[44,45] Interaction of ligands with quadruplex DNAs results
in either enhancement or quenching of the fluorescence inten-
sity of the ligands. A concentration-dependent increase or de-
crease in fluorescence intensity can be used to derive the bind-
ing constant and stoichiometry for the ligand–quadruplex in-
teraction. Emission spectra for ligand InPy1 was characterized
by an intense peak at 536 nm and a less-intense peak at
414 nm after excitation at 350 nm (Figure 5A). Fluorimetric ti-
tration with increasing concentration of pre-annealed c-myc
DNA resulted in the quenching of ligand fluorescence together
with a blue shift by 20 nm for the peak at 536 nm (Figure 5A).
Quenching of fluorescence upon titration and the blue shift in-
dicate strong binding interactions between the ligand and
DNA. Fluorescence intensity was plotted against DNA concen-
tration, and was fitted by using the standard Stern–Volmer
equation to derive the binding constant: Ka =1.0ꢁ105 mÀ1 for
the ligand InPy1 (Figure 5B).[46] Similar titration experiments
performed with telomeric and duplex DNAs yield binding con-
stants Ka =(2.8Æ0.3)ꢁ104 mÀ1 and Ka =(3.6Æ0.1)ꢁ104 mÀ1,
respectively (Figure S5, Supporting Information).
the binding constants observed between c-myc and telomeric/
duplex DNAs further explains the specificity and selectivity of
the ligands.
To gain further insight into the binding stoichiometry for the
ligand–quadruplex interaction, continuous variation analysis
(Job plot) was performed by using the fluorescence properties
of InPy1.[50] The plot of fluorescence intensity versus mole frac-
tion of the ligand showed a stoichiometry of ~2:1 (ligand/
DNA) with c-myc quadruplex DNA (Figure S7, Supporting Infor-
mation). This dual binding mode was further validated by mo-
lecular modeling and dynamics studies (see below).
Taq DNA polymerase stop assays
Selective stabilization of promoter quadruplex DNAs by ligands
was further explored by concentration-dependent Taq DNA
polymerase stop assays using c-myc and telomeric quadruplex-
forming sequences. This assay was carried out by following re-
ported procedures with slight modifications.[51] Templates con-
taining wild-type c-myc, mutated c-myc, and telomeric quadru-
plex-forming sequences were used for the primer-extension re-
action. Reactions were carried out at 508C for c-myc and at
408C for telomeric DNA to unwind the preformed quadruplex
structure. In the case of c-myc, a control experiment with tem-
plate containing a mutated c-myc sequence that cannot form
Similarly, binding constants for c-myc (Ka =1.6ꢁ105 mÀ1), telo-
meric DNA [Ka =(4.5Æ0.4)ꢁ104 mÀ1], and duplex DNA [Ka =
(4.5Æ0.1)ꢁ104 mÀ1] were obtained with the ligand InPy2 (Fig-
ure S6, Supporting Information). Binding constant values ob-
tained for c-myc quadruplex DNA, together with the observed
spectral changes are similar to the reported values for strong
ligand–quadruplex interactions.[47–49] The threefold difference in
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a quadruplex structure was also carried out. If ligands can sta-
bilize the quadruplex structure in the template sequence,
primer extension by Taq polymerase will be stopped at the
quadruplex-forming site. The concentration of ligand required
for the formation of 50% stop product would yield an IC50
value for the ligands; lower IC50 values reflect greater ligand
potency. Because the ligands InPy1 and InPy2 showed higher
thermal stabilization with c-myc DNA in CD melting studies,
these ligands were selected for the study. In the case of the
c-myc template, formation of stop products was observed
upon increasing ligand concentration (Figure 6A, and Fig-
ure S8, Supporting Information). Ligand InPy1 exhibited 50%
inhibition at a concentration of ~46 mm and InPy2 showed
50% stop product at ~31 mm (Figure S8, Supporting Informa-
tion). However, stop products were not observed for the mu-
tated c-myc template under identical reaction conditions, sup-
porting the fact that stabilization of a quadruplex structure is
responsible for the formation of stop products (Figure 6A). In
the case of telomeric DNA, a much smaller quantity of stop
products (~10%) was obtained, even at the higher ligand con-
centrations used (up to 160 mm; Figure 6B). The results ob-
tained from the Taq DNA polymerase stop assays support the
observations from the CD melting experiments, that the li-
gands are able to stabilize the parallel quadruplex structures
present in the promoter DNAs and are unable to stabilize the
quadruplex topologies of telomeric DNA.
Molecular modeling and dynamics studies
To acquire insight into the binding mode and interactions of
InPy1 with c-myc and c-kit G-quadruplex DNA at the atomic
level, molecular docking and dynamics studies were per-
formed. Initially, energy-optimized InPy1 (Figure S9, Support-
ing Information) at HF/6-31G* level in Gaussian09[52] was
docked with c-myc (PDB entry: 2L7V)[53] and c-kit1 (PDB entry:
2O3M)[54] G-quadruplex DNAs using AutoDock4.2.[55] The re-
sults reveal that InPy1 stacks at both the top (top-InPy1) and
bottom (bottom-InPy1) G-quartets of c-myc and c-kit1 G-quad-
ruplex DNAs (Figures S10 and S11, Supporting Information).
This is in line with the experimentally observed 2:1 stoichiome-
try for the ligand–quadruplex complex. On the basis of dock-
ing results, 50 ns of unrestrained MD simulations were per-
formed using AMBER 12.[56]
The binding free energy (DG) of the ligand and G-quadru-
plex was estimated by using the MM-PB/GBSA module in
AMBER 12.[57] For the dual binding mode of InPy1 with c-myc
G-quadruplex DNA, the free energy was found to be À45.12Æ
7.43 kcalmolÀ1 (Table S1, Supporting Information). The pre-
ferred binding site of InPy1 was the top quartet (DG=
À22.56Æ4.13 kcalmolÀ1) over the bottom one (DG=À19.15Æ
7.07 kcalmolÀ1; Table S1, Supporting Information). For the
binding of InPy1 with c-kit1 G-quadruplex DNA, the free
energy was found to be À43.78Æ5.15 kcalmolÀ1 (Table S2,
Supporting Information). Here, the preferred binding site was
the bottom quartet (DG=À22.96Æ3.85 kcalmolÀ1) over the
top (DG=À13.81Æ3.22 kcalmolÀ1). Similar to these results,
a dual binding mode was also observed for derivatives of the
natural product quindoline with c-myc G-quadruplex DNA.[53]
To examine the structural stability of the G-quadruplex–ligand
complex, the root mean square deviations (RMSDs) of the
heavy atoms of the DNA backbone, G-quartet, and the top-
and bottom-InPy1 ligands were calculated with respect to the
initial structure at each ps during 50 ns of MD simulations. The
RMSD graphs (Figures S12 and S13, Supporting Information)
and the average values (Table S3, Supporting Information) sig-
nify that G-quartet was stable during the MD simulation owing
to the stacking interactions of two ligands at the bottom and
top quartets of the G-quadruplex. Consequently, the percent-
age of Hoogsteen hydrogen bond occupancy (Table S4, Sup-
porting Information) in the entire quartet was found to be
>96% during MD simulation for both c-myc and c-kit1 quadru-
plex structures.
Figure 6. Denaturing PAGE (15%, 7m urea) of the Taq DNA polymerase stop
assay in the presence of c-myc, mutated c-myc, and telomeric DNA with in-
creasing ligand concentrations. A) InPy1 (0–160 mm) with c-myc and mutat-
ed c-myc DNA template; B) InPy1 and InPy2 (0–160 mm) with telomeric DNA
template. Primer extension reaction at 508C. Conditions: 100 nm template,
50 nm primer, 0.2 mm dNTs, 5 mm KCl for c-myc template, 10 mm KCl for te-
lomeric template, and 0.5 U Taq polymerase in enzyme buffer (50 mm Tris,
0.5 mm DTT, 0.1 mm EDTA, 5 mm MgCl2, 5 mm KCl for c-myc template, and
10 mm KCl for telomeric template). P: primer, S: stop product, F: full-length
product.
The structure shown in the Figure 7 is the averaged MD
snapshot of c-myc G-quadruplex DNA and InPy1 from the last
15 ns of MD simulations. During the course of MD simulations,
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tionally, in the case of top-InPy1,
the formation of a hydrogen
bond between the nitrogen
atom in the pyrimidine ring of
the ligand and the NH2 group of
the 5’- flanking nucleotide (dA3)
was observed at a distance of
2.5Æ0.4 ꢂ (Figure 7B). As illus-
trated in Figure 7C, the oxygen
atom of the keto group in
bottom-InPy1 forms a hydrogen
bond with the NH group of the
3’-flanking nucleotide (dT20)
atom at a distance of 2.1Æ0.5 ꢂ.
Above-mentioned electrostatic
interactions
and
hydrogen
bonds were observed for ~80%
of the 50 ns simulation time.
The structure shown in the
Figure 8A is the snapshot taken
at the final step of MD simula-
tion (50 ns) of c-kit1 G-quadru-
plex DNA and InPy1. As appar-
ent in Figure 8D, the two ligands
are oriented perpendicular to
each other. For both top- and
bottom-InPy1, the amine group
faces a flanking nucleotide (dT11
for top-InPy1 and dA15 for
bottom-InPy1), whereas the keto
group faces the opposite side.
Neither the top nor bottom
ligand maintains the benzene
ring in the plane of the indeno-
Figure 7. Averaged MD snapshot of InPy1 with c-myc G-quadruplex DNA from the last 15 ns of 50 ns MD simula-
tions. A) InPy1 stacks at both the top and bottom G-quartet of c-myc G-quadruplex (2:1). B) InPy1 and top quar-
tet (axial view), showing hydrogen bonding and electrostatic interactions with the 5’-flanking nucleotide (dA3)
and propeller loop, respectively. C) InPy1 and bottom quartet (axial view), showing hydrogen bonding and elec-
trostatic interactions with the 3’-flanking nucleotide (dT20) and propeller loop, respectively. D),E) Side view of top
and bottom G-quartets with InPy1; the f angle in the ligand and the stacking distance between InPy1 and the
G-quartet are indicated with red lines. Dashed magenta lines indicate the electrostatic interactions and hydrogen
bond distances between the atoms in the ligands and G-quadruplex DNA; all distances are given in ꢂ.
bottom-InPy1 undergoes a significant re-orientation, which is
evident by comparing the docked structure and the averaged
MD structure (Figure 7C,E, and Figure S10, Supporting Informa-
tion). This re-orientation occurs in order to avoid the steric
clash between the benzene ring attached to the indenopyrimi-
dine ring and 3’-flanking nucleotides. Moreover, the orienta-
tions of ligand at the top and the bottom quartet are quite dif-
ferent. At the bottom quartet, the keto group in the indeno-
pyrimidine ring is oriented toward the 3’-flanking nucleotide.
In contrast, at the top quartet, the amine group in the indeno-
pyrimidine ring is directed toward the 5’-flanking nucleotide
(Figure 7B,C).
pyrimidine ring. For bottom-InPy1, the f value was found to
be in the range between 36 and 518. For top-InPy1, many fluc-
tuations were observed, with the f value varying from 20.8 to
54.1. It could be speculated that this out-of-plane orientation
of the benzene ring pushes the entire ligand into a particular
orientation (Figure 8D), with the benzene ring lying outside
the quartets. Furthermore, in this orientation, very specific hy-
drogen bonds and other noncovalent interactions were ob-
served between the quadruplex and InPy1 (Figure 7 and
Figure 8). The non-stacking nature of the benzene ring in
InPy1 with G-quadruplex DNA might contribute to the experi-
mentally reported specificity of these ligands for promoter
quadruplex DNA over telomeric DNA.
For both top-InPy1 and bottom-InPy1, the benzene ring at-
tached to the pyrimidine ring is not in plane with respect to
the indenopyrimidine ring of the ligand. The angle between
the two rings (f) was found to be in the range of 15–288
during the course of simulation. As a result, the benzene ring
does not stack well on the G-quartet surface, and only the in-
denopyrimidine aromatic core stacks optimally on two guanine
bases of the G-quartet (Figure 7D,E). The positively charged
protonated pyrrolidine side chain makes electrostatic interac-
tions with the negatively charged backbone in the propeller
loop (loop-1) of c-myc G-quadruplex DNA (Figure 7B,C). Addi-
For bottom-InPy1, two hydrogen bonds were found to be
present throughout the simulation (100% of the 50 ns run).
The ether oxygen atom of the ligand and the amine of the
flanking nucleotide (dG19) form a hydrogen bond at a distance
of 2.38Æ0.38 ꢂ (Figure 8B). The hydrogen of the amine group
in bottom-InPy1 and the N3 atom of the flanking adenine
(dA15) also make a hydrogen bond, with a distance of 2.04Æ
0.16 ꢂ (Figure 8B). For top-InPy1, hydrogen bonds are visible
only after ~10.75 ns of the MD run and are retained until the
end of the simulation (78.5% of the simulation time). The two
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and promoter regions of onco-
genes have been known to form
quadruplex structures of diverse
topologies in in vitro and in vivo
settings. Stabilizing such quadru-
plex structures with small mole-
cules offers promise in the de-
velopment of anticancer drugs.
However, to develop a therapeu-
tic agent armored with high pre-
cision, the molecules should be
able to specifically bind and sta-
bilize
a particular quadruplex
topology. In this direction, we
have designed and synthesized
a set of indenopyrimidine deriva-
tives that contain an aromatic
core containing side chains of
varying length, terminated with
amine functionalities. These mol-
ecules were screened for their
binding interactions with quad-
ruplex and duplex DNAs. CD ti-
tration experiments showed the
property of ligands to induce
a parallel topology for the c-myc
and c-kit promoter quadruplex
DNAs. CD melting studies vali-
dated the specific stabilization of
promoter quadruplex DNAs over
telomeric and duplex DNAs. The
dual binding mode of the li-
gands with c-myc quadruplex
DNA was confirmed by fluores-
cence titration as well as molec-
Figure 8. Final MD snapshot of InPy1 with c-kit1 G-quadruplex DNA after 50 ns MD simulation. A) InPy1 and c-kit1
G-quadruplex DNA (2:1): stacking occurs at both the top and bottom G-quartets of the G-quadruplex. B) InPy1
and bottom quartet (axial view), showing the hydrogen bonding with flanking nucleotides (dG19 and dA15).
C) InPy1 and top quartet (axial view), showing the hydrogen bonding with the flanking nucleotide (dT11) and
bottom quartet residues (dG9 and dG12). D) Side view of top and bottom InPy1 with G-quartets, with red lines in-
dicating the stacking distance between InPy1 and G-quartets. Dashed magenta lines indicate the hydrogen bond
distance between the atoms in ligand and G-quadruplex DNA; all distances are given in ꢂ.
hydrogen atoms of the top-InPy1 amine group form hydrogen
bonds with the keto oxygen atom of the quartet guanines
(dG12 and dG9), with average distances of 2.33Æ0.52 and
2.52Æ0.56 ꢂ (Figure 8C). The nitrogen atom in the amine
group of top-InPy1 is hydrogen bonded with the hydrogen of
NH in the flanking thymine (dT11), with an average distance of
1.97Æ0.28 ꢂ (Figure 8C). These studies demonstrate that the
accurate positioning of the functional groups and side chains
of InPy1 can specifically identify the potential site in the loops
and in the flanking nucleotides of promoter G-quadruplex
DNAs to achieve optimal target recognition.
ular modeling studies. In summary, these indenopyrimidine de-
rivatives have emerged as specific G-quadruplex modulators
and can be further engineered for potential therapeutic appli-
cations.
Experimental Section
Dry solvents (MeOH, DMF) were obtained from Merck and Spectro-
chem (India), and CH3CN and CH2Cl2 were dried using CaH. Thin-
layer chromatography (TLC) was performed on silica gel plates
(Merck India) pre-coated with fluorescent indicator, with visualiza-
tion by UV light (260 nm). Silica gel (100–200 mesh) was used for
The unique ability of InPy1 to make specific contacts with
flanking nucleotides and the propeller loops present in parallel
promoter G-quadruplex DNAs explains the specificity observed
for this ligand. Overall, the MD simulations and binding free
energy analysis rationalize the preferred 2:1 binding mode of
InPy1 with c-myc and c-kit1 G-quadruplex structures.
1
column chromatography. 13C NMR (100 and 125 MHz) and H NMR
(400 and 500 MHz) data were recorded on 400 and 500 MHz instru-
ments, respectively. Chemical shifts (d, in parts per million) were
referenced to the residual signal of deuterium solvents or TMS:
TMS (0 ppm), CD3OD (3.31 ppm), [D6]DMSO (2.5 ppm) for 1H NMR
spectra; and CDCl3 (77.2 ppm), CD3OD (49.1 ppm), [D6]DMSO
1
(39.5 ppm) for 13C NMR spectra. Multiplicities of H NMR spin cou-
plings are reported as s (singlet), d (doublet), t (triplet), q (quartet),
dd (doublet of doublets), (q) quintet or m (multiplet and overlap-
ping spin systems). Values for apparent coupling constants (J) are
reported in Hz. High-resolution mass spectra (HRMS) were
Conclusions
Potential G-quadruplex-forming sequences present in the spe-
cific segments of the human genome such as telomeric ends
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1
obtained in positive-ion electrospray ionization (ESI) mode using a
Q-TOF (micro, micromass) analyzer.
(PE/EtOAc 7:3); H NMR (500 MHz, [D6]DMSO): d=3.86 (t, J=5.1 Hz,
2H), 4.51 (t, J=5.0 Hz, 2H), 7.17 (dd, J=8.2, 2.1 Hz, 1H), 7.26 (d,
J=2.1 Hz, 1H), 7.46–7.57 (m, 3H), 7.61 (d, J=8.2 Hz, 1H), 7.90 (brs,
2H), 8.00 ppm (d, J=7.0 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO):
d=31.6, 68.9, 107.1, 112.0, 119.1, 125.5, 128.1, 129.7, 130.0, 131.2,
136.1, 142.7, 163.3, 164.7, 165.4, 175.3, 187.0 ppm; HRMS: m/z
[M+H]+ calcd for C19H15N3O2Br: 396.0348, found: 396.0350.
(Z)-2-Benzylidene-6-methoxy-2,3-dihydro-1H-inden-1-one (3).[38]
6-methoxyindanone
1 (1 g, 6.1 mmol) and benzaldehyde 2
(0.62 mL, 6.1 mmol) were dissolved in acetic acid (15 mL) with vig-
orous stirring. To this solution, 4–5 drops of concentrated HCl was
added and held at reflux for 6 h. After completion, the reaction
mixture was poured into 50 mL H2O and was extracted with EtOAc
(3ꢁ100 mL). The organic phase was dried over anhydrous Na2SO4,
evaporated under reduced pressure, recrystallized from Et2O and
dried to give compound 3 as a light-yellow solid (1.29 g, 84%):
Rf =0.8 (PE/EtOAc 8:2); 1H NMR (400 MHz, [D6]DMSO): d=3.81 (s,
3H), 3.98 (s, 2H), 7.22 (d, J=2.5 Hz, 1H), 7.27 (dd, J=5.8, 2.5 Hz,
1H), 7.42–7.55 (m, 4H), 7.56 (d, J=8 Hz, 1H), 7.75 ppm (d, J=8 Hz,
2H); 13C NMR (100 MHz, [D6]DMSO): d=31.2, 55.4, 105.5, 123.4,
127.5, 129.0, 129.8, 130.7, 132.7, 134.8, 135.8, 138.4, 142.6, 159.1,
193.1 ppm; HRMS: m/z [M+H]+ calcd for C17H15O2: 251.1072,
found: 251.1079.
2-Amino-8-(3-bromopropoxy)-4-phenyl-5H-indeno[1,2-d]pyrimi-
din-5-one (7). To a solution of 5 (85 mg, 0.3 mmol) in dry CH3CN
(3 mL), anhydrous K2CO3 (124 mg, 1.2 mmol) and 1,2-dibromopro-
pane (0.12 mL, 1.2 mmol) were added, and the reaction mixture
was heated at 758C for 18 h. The solvent was evaporated, and the
crude mixture was purified by column chromatography (20%
EtOAc in hexane) to give compound 7 as yellow solid (102 mg,
83%): Rf =0.7 (PE/EtOAc 7:3); 1H NMR (400 MHz, [D6]DMSO): d=
2.29 (q, J=6.0 Hz, 2H), 3.69 (t, J=6.6 Hz, 2H), 4.23 (t, J=6.0 Hz,
2H), 7.13 (dd, J=6.0, 2.2 Hz, 1H), 7.24 (d, J=2.2 Hz, 1H), 7.46–7.59
(m, 4H), 7.92 (brs, 2H), 7.99 ppm (d, J=6.6 Hz, 2H); 13C NMR
(100 MHz, [D6]DMSO): d=31.0, 31.6, 66.3, 106.5, 111.5, 118.3, 125.0,
127.6, 128.9, 129.5, 130.7, 135.6, 142.2, 163.3, 164.2, 164.9, 174.8,
186.6 ppm; HRMS: m/z [M+H]+ calcd for C20H17N3O2Br: 410.0504,
found: 410.0484.
2-Amino-8-methoxy-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(4).[38] Powdered NaOH (1.75 g, 43.8 mmol) was added to an EtOH
solution (10 mL) of guanidine·HCl (4.18 g, 43.8 mmol) and was
stirred for 0.5 h. The resulting solution was filtered to remove the
precipitated NaCl, and the filtrate containing free guanine base
was added to an EtOH suspension (8 mL) of compound 3 (730 mg,
2.92 mmol). The reaction mixture was held at reflux for 16 h,
cooled for 30 min, filtered, and the precipitate was dried. The dried
residue (450 mg) without purification was dissolved in DMF
(10 mL), and powdered NaOH (140 mg, 3.5 mmol) was added. Air
was bubbled through the reaction mixture using a needle, and
was heated at 808C. After 16 h, the reaction mixture was cooled,
and H2O was added. The resulting precipitate was filtered, washed
with H2O and cold EtOH. The precipitate was dried to afford com-
pound 4 as a dark-yellow solid (335 mg, 38%): Rf =0.4 (PE/EtOAc
7:3); 1H NMR (400 MHz, [D6]DMSO): d=3.93 (s, 3H), 7.13 (dd, J=
6.0, 2.2 Hz, 1H), 7.25 (d, J=2.2 Hz, 1H), 7.47–7.62 (m, 4H), 7.94
(brs, 2H), 8.00 ppm (d, J=6.7 Hz, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=55.9, 106.2, 111.5, 117.8, 125.0, 127.6, 130.7, 128.8,
129.5, 130.7, 135.7, 142.2, 164.2, 164.2, 164.9, 174.8, 186.6 ppm;
HRMS: m/z [M+H]+ calcd for C18H14N3O2: 304.1086, found:
304.1078.
2-Amino-4-phenyl-8-(2-(pyrrolidin-1-yl)ethoxy)-5H-indeno[1,2-d]-
pyrimidin-5-one (InPy1). To a solution of 6 (50 mg, 0.09 mmol) in
dry CH3CN (2 mL), pyrrolidine (0.02 mL, 0.28 mmol) was added, and
the resulting reaction mixture was held at reflux for 12 h. The sol-
vent was evaporated, and the crude product was purified by
column chromatography using basic alumina as a stationary phase
(3% MeOH in CH2Cl2) to afford compound InPy1 as a yellow solid
(30 mg, 86%): Rf =0.3 (CH2Cl2/MeOH 9.6:0.4); 1H NMR (400 MHz,
CDCl3): d=1.83 (brs, 4H), 2.67 (brs, 4H), 2.97 (t, J=5.7 Hz, 2H),
4.25 (t, J=5.7 Hz, 2H), 5.81 (brs, 2H), 7.02 (dd, J=8.2, 2.2 Hz, 1H),
7.35 (d, J=2.2 Hz, 1H), 7.45–7.54 (m, 3H), 7.65 (d, J=8.2 Hz, 1H),
8.05 ppm (dd, J=7.6, 1.8 Hz, 2H); 13C NMR (100 MHz, CDCl3): d=
23.6, 54.9, 54.9, 67.9, 107.1, 114.0, 118.8, 125.5, 128.1, 129.4, 129.8,
131.2, 135.6, 142.6, 164.1, 164.7, 164.2, 176.0, 187.7 ppm; HRMS:
m/z [M+H]+ calcd for C23H23N4O2: 387.1821, found: 387.1813.
2-Amino-8-(3-cyclopentylpropoxy)-4-phenyl-5H-indeno[1,2-d]-
pyrimidin-5-one (InPy2). To a solution of compound 7 (50 mg,
0.12 mmol) in dry CH3CN (2 mL), pyrrolidine (0.03 mL, 0.37 mmol)
was added, and the reaction mixture was held at reflux for 12 h.
The solvent was evaporated, and the crude product was purified
by column chromatography using basic alumina as a stationary
phase (3% MeOH in CH2Cl2) to afford compound InPy2 as a yellow
solid (40 mg, 95%): Rf =0.3 (CH2Cl2/MeOH 9.6:0.4); 1H NMR
(400 MHz, CD3OD): d=1.87 (brs, 4H), 2.06 (q, J=6.4 Hz, 2H), 2.69
(brs, 4H), 2.75 (t, J=6.9 Hz, 2H), 4.14 (t, J=5.9 Hz, 2H), 7.03 (d, J=
8.0 Hz, 1H), 7.33 (brs, 1H), 7.43–7.60 (m, 4H), 8.02 ppm (d, J=
7.0 Hz, 2H); 13C NMR (100 MHz, CD3OD): d=24.2, 30.8, 55.2, 67.4,
68.8, 108.4, 109.7, 113.6, 119.1, 120.2, 126.2, 128.9, 131.0, 132.0,
137.2, 143.7, 165.4, 166.5, 176.9, 189.0 ppm; HRMS: m/z [M+H]+
calcd for C24H25N4O2: 401.1978, found: 401.1968.
2-Amino-8-hydroxy-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one
(5).[38] Compound 4 (90 mg, 0.3 mmol) was dissolved in N-methyl-
2-pyrrolidone together with LiCl (254 mg, 6 mmol) and H2O
(0.2 mL). The reaction mixture was heated at 1808C for 2–3 days
and was diluted with THF and EtOAc, washed with H2O and brine.
The organic phase was dried over anhydrous Na2SO4, evaporated
under reduced pressure and dried to give compound 5 as a yellow
solid (80 mg, 94%): Rf =0.5 (PE/EtOAc 6:4); 1H NMR (400 MHz,
[D6]DMSO): d=6.94 (dd, J=6.0, 2.5 Hz, 1H), 7.15 (d, J=2.0 Hz, 1H),
7.46–7.54 (m, 4H), 7.88 (brs, 2H), 7.99 (d, J=6.5 Hz, 2H),
10.79 ppm (brs, 1H); 13C NMR (100 MHz, [D6]DMSO): d=107.9,
111.7, 118.9, 125.4, 127.5, 127.7, 129.6, 130.7, 135.8, 142.4, 163.3,
164.0, 164.9, 175.1, 186.8 ppm; HRMS: m/z [M+H]+ calcd for
C17H12N3O2: 290.0930, found: 290.0938.
2-Amino-8-(2-(2-(dimethylamino)ethylamino)ethoxy)-4-phenyl-
5H-indeno[1,2-d]pyrimidin-5-one (InPy3). To a solution of com-
pound 6 (40 mg, 0.09 mmol) in dry CH3CN (2 mL), N,N-dimethyl-
ethane-1,2-diamine (0.03 mL, 0.29 mmol) was added, and the reac-
tion mixture was held at reflux for 12 h. The solvent was evaporat-
ed, and the crude product was purified by column chromatogra-
phy using basic alumina as a stationary phase (5% MeOH in
CH2Cl2) to afford InPy3 as a dark-yellow solid (30 mg, 83%): Rf =0.4
(CH2Cl2/MeOH 9.5:0.5); 1H NMR (400 MHz, CDCl3): d=1.99 (s, 1H),
2-Amino-8-(2-bromoethoxy)-4-phenyl-5H-indeno[1,2-d]pyrimi-
din-5-one (6). Compound 5 (85 mg, 0.3 mmol) was dissolved in
dry CH3CN (3 mL), anhydrous K2CO3 (124 mg, 0.9 mmol) and 1,2-di-
bromoethane (0.11 mL, 1.2 mmol) were added, and the mixture
was heated at 758C for 18 h. The solvent was evaporated, and the
crude compound was purified by column chromatography (20%
EtOAc in hexane) to give 6 as a yellow solid (94 mg, 80%): Rf =0.5
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2.25 (s, 6H), 2.53 (t, J=6.0 Hz, 2H), 2.82 (t, J=6.0 Hz, 2H), 3.07 (t,
J=4.7 Hz, 2H), 4.19 (t, J=4.7 Hz, 2H), 6.15 (brs, 2H), 6.98 (d, J=
8.0 Hz, 1H), 7.30 (brs, 1H), 7.44–7.54 (m, 3H), 7.61 (d, J=8.2 Hz,
1H), 8.03 ppm (dd, J=7.4, 1.9 Hz, 2H); 13C NMR (125 MHz, CDCl3):
d=45.1, 46.6, 48.4, 58.4, 67.9, 107.0, 113.6, 118.5, 125.4, 128.0,
129.3, 129.7, 131.1, 135.4, 142.4, 163.9, 164.5, 165.0, 175.7,
187.5 ppm; HRMS: m/z [M+H]+ calcd for C23H26N5O2: 404.2087,
found: 404.2080.
wavelength range of 362–700 nm after an excitation at 352 nm.
DNA was pre-annealed in the presence of 100 mm KCl and 10 mm
lithium cacodylate buffer by heating at 958C and gradual cooling
to room temperature over 4–5 h. The ligand concentration used
was 100 mm and was titrated with increasing concentrations of
DNA (0–30 mm). Fluorescence intensity (F/Fo) was plotted against
DNA concentration, and binding constants were calculated by
curve fitting using the Stern–Volmer equation, F/Fo =1+Ka[DNA],
in which F and Fo are the fluorescence intensities of the ligand in
the presence and absence of DNA, respectively, [DNA] is the con-
centration of DNA, and Ka is the binding constant. Job plot analysis
for InPy1 with c-myc was performed with an excitation wavelength
of 350 nm and emission spectra were recorded in the range of
360–700 nm. The total concentration of ligand+DNA complex was
kept constant at 5 mm. Samples were prepared by varying the
mole fractions (0–1) of InPy1 in the sample. Mole fractions of
InPy1 were plotted against the fluorescence intensities to obtain
the stoichiometry for ligand–DNA complexes. All curves were ana-
lyzed by Origin 8.0 software.
Ligand stock solution preparation. Stock solutions for all ligands
were made to 10 mm in DMSO. They were further diluted to 1 mm
by using 10 mm HCl for all experiments.
Oligonucleotides. All oligonucleotides were synthesized at 1 mm
scales using appropriate controlled pore glass (CPG) as a 3’-solid
support in a Mermade-4 DNA/RNA synthesizer and were purified
by 20% PAGE. The concentration of all oligonucleotides was mea-
sured at 260 nm in a UV/Vis spectrophotometer using appropriate
molar extinction coefficients. The following oligonucleotides were
used for CD titration, melting, and fluorescence experiments: telo-
meric DNA (5’-AGG GTT AGG GTT AGG GTT AGG G-3’), wild-type c-
myc DNA (5’-TGA GGG TGG GGA GGG TGG GGA A-3’), c-kit1 DNA
(5’-GGG AGG GCG CTG GGA GGA GGG-3’), and duplex DNA (5’-CCA
GTT CGT AGT AAC CC-3’ and the complementary sequence 5’-GGG
TTA CTA CGA ACT GG-3’). For Taq DNA polymerase stop assays, the
primer sequence (5’-ACG ACT CAC TAT AGC AAT TGC G-3’), tem-
plate containing wild-type c-myc sequence (5’-TGA GGG TGG GGA
GGG TGG GGA AGC CAC CGC AAT TGC TAT AGT GAG TCG T-3’),
mutated c-myc sequence (5’-TGA GGG TGG GTA GAG TGG GTA
AGC CAC CGC AAT TGC TAT AGT GAG TCG T-3’), and template con-
taining human telomeric sequence (5’-AGG GTT AGG GTT AGG GTT
AGG GGC CAC CGC AAT TGC TAT AGT GAG TCG T-3’) were used.
5’-End radiolabeling of oligonucleotides. DNA (10 pmol) was 5’-
end labeled by T4 polynucleotide kinase (PNK; 5 U) in 1ꢁPNK
buffer for forward reaction [50 mm Tris·HCl pH 7.6, 10 mm MgCl2,
5 mm DTT, 0.1 mm each spermidine and 0.1 mm EDTA] and
[g-32P]ATP (370 CimmolÀ1 in 3 mL) in a total volume of 10 mL for 1 h
at 378C followed by deactivation of the enzyme by heating at
708C for 3 min. The end-labeled DNA was then purified using
a QIAquick Nucleotide removal kit protocol provided by the manu-
facturer (Qiagen).
Taq DNA polymerase stop assays. These assays were performed
using reported procedures.[51] Appropriate amounts of labeled
primer oligonucleotide (~20000 cpm) were mixed with cold primer
(50 nm) and template (100 nm), and they were annealed in an an-
nealing buffer [5 mm Tris (pH 8), 15 mm NaCl, 0.1 mm EDTA] by
heating at 958C for 5 min and then gradual cooling to room tem-
perature over 4–5 h. The annealed primer–template was mixed
with 1ꢁ polymerase buffer [50 mm Tris, 0.5 mm DTT, 0.1 mm EDTA,
5 mm MgCl2, 5 mm KCl for c-myc template and 10 mm KCl for telo-
meric template], 1 mgmLÀ1 BSA, and 0.2 mm dNTPs. The ligands in
appropriate concentrations were added to the reaction mixture
(10 mL total volume) and incubated for 30 min at room tempera-
ture. Finally, the primer-extension reaction was initiated by adding
Taq DNA polymerase (0.5 U) and incubated at 508C for c-myc and
at 408C for telomeric DNA for 30 min. The extension reaction was
stopped by adding 10 mL 2ꢁ stop buffer (10 mm EDTA, 10 mm
NaOH, 0.1% each bromophenol blue (w/v) and xylene cyanol (w/v)
in formamide). Samples were analyzed in 15% denaturing PAGE in
which 1ꢁ TBE (89 mm each of Tris and boric acid and 2 mm EDTA,
pH ~8.3) was used as running buffer, and gels were imaged by au-
toradiography with a PhosphorImager; quantification of gels was
performed using ImageQuant 5.2 software.
CD titration studies. CD spectra were recorded on a Jasco 815 CD
spectrophotometer in the wavelength range of 220–320 nm using
a quartz cuvette with 1.0 mm path length. The scanning speed of
the instrument was set at 100 nmminÀ1, and the response time
was 2 s. Baseline was measured using 50 mm Tris buffer (pH 7.2)
for experiments without salts, and with 100 mm KCl for the experi-
ments in the presence of salts. The strand concentration of oligo-
nucleotide used was 12.5 mm. Each spectrum is an average of three
measurements at 258C. All spectra were analyzed using Origin 8.0
software.
CD melting studies. For melting studies, 10–15 mm strand concen-
tration of oligonucleotide in 10 mm lithium cacodylate (pH 7.2),
the required amount of monovalent salts (LiCl and KCl) and 5 mo-
l equiv ligands (50–75 mm) were used. Telomeric DNA (10 mm DNA
in 10 mm KCl for K+ conditions, and 10 mm NaCl, 10 mm sodium
cacodylate for Na+ conditions (pH 7.2) and 90 mm LiCl), c-kit1 DNA
(10 mm DNA in 10 mm KCl and 90 mm LiCl), c-myc DNA (10 mm in
1 mm KCl and 99 mm LiCl) and duplex DNA (15 mm in 10 mm KCl
and 90 mm LiCl) were annealed by heating at 958C for 5 min, fol-
lowed by gradual cooling to room temperature. Ligands (5 equiv)
were added to the annealed DNAs and were kept at 48C over-
night. Thermal melting was monitored at 295, 263, and 242 nm for
telomeric, promoter, and duplex DNAs, respectively, at a heating
rate of 18CminÀ1. The melting temperatures were determined
from sigmoidal curve fit using the Boltzmann function in Origin 8.0
software.
Molecular modeling. The coordinates of c-myc (PDB entry: 2L7V)
and c-kit1 (PDB entry: 2O3M) G-quadruplex DNA structures were
retrieved and prepared for docking; the InPy1 structure was opti-
mized using Gaussian 09[52] (HF/6-31G* level). Using Auto-
Dock 4.2,[55] docking studies were carried out with the Lamarckian
genetic algorithm following the procedure developed for G-quad-
ruplex DNA and ligand docking. To facilitate the docking to c-kit1
DNA, terminal 5’-nucleotide dA1 was removed from the PDB file.
Subsequent to the docking studies, MD simulations were carried
out using AMBER 12.[56] The procedure for MD simulations was
used from the protocol developed by Haider and Neidle.[58] Briefly,
RESP[59] charge fitted InPy1 was complexed with c-myc and c-kit1
Fluorescence titration experiments. Fluorimetric titrations were
performed on a Cary Eclipse spectrofluorimeter by using a 1 mL
quartz cuvette with a 1 cm path length. Emission spectra for InPy1
(100 mm) were recorded in the wavelength range of 360–700 nm
with an excitation wavelength of 350 nm, and for InPy2 in the
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 2754 – 2765 2763

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[18] S. A. Ohnmacht, S. Neidle, Bioorg. Med. Chem. Lett. 2014, 24, 2602–
2612.
[19] R. T. Wheelhouse, D. Sun, H. Han, F. X. Han, L. H. Hurley, J. Am. Chem.
Soc. 1998, 120, 3261–3262.
[20] C. Romera, O. Bombarde, R. Bonnet, D. Gomez, P. Dumy, P. Calsou, J. F.
Gwan, J. H. Lin, E. Defrancq, G. Pratviel, Biochimie 2011, 93, 1310–1317.
[21] P. Wang, L. Ren, H. He, F. Liang, X. Zhou, Z. Tan, ChemBioChem 2006, 7,
1155–1159.
[22] M. Y. Kim, H. Vankayalapati, K. Shin-Ya, K. Wierzba, L. H. Hurley, J. Am.
Chem. Soc. 2002, 124, 2098–2099.
G-quadruplex DNA (2:1) using Generalized AMBER force field
(GAFF)[60] and AMBER FF10SB, respectively. The system was then
solvated using TIP3P water molecules extended up to 12 ꢂ in an
octahedral box. The neutrality of the system was achieved by
adding K+ ions. The solvated system was then subjected to equili-
bration followed by 50 ns of MD simulation with coordinates saved
for each ps. Binding free energy of ligands were estimated using
MM-PB/GBSA methods,[57] RMSDs of the heavy atoms, Hoogsteen
hydrogen bonding occupancies, and dihedral angles were calculat-
ed using the ptraj module. Figures were rendered using PyMOL
(http://www.pymol.org).
[23] M. Read, R. J. Harrison, B. Romagnoli, F. A. Tanious, S. H. Gowan, A. P.
Reszka, W. D. Wilson, L. R. Kelland, S. Neidle, Proc. Natl. Acad. Sci. USA
2001, 98, 4844–4849.
[24] C. Sissi, L. Lucatello, A. P. Krapcho, D. J. Maloney, M. B. Boxer, M. V. Ca-
marasa, G. Pezzoni, E. Menta, M. Palumbo, Bioorg. Med. Chem. 2007, 15,
555–562.
[25] W. Duan, A. Rangan, H. Vankayalapati, M. Y. Kim, Q. Zeng, D. Sun, H.
Han, O. Y. Fedoroff, D. Nishioka, S. Young Rha, E. Izbicka, D. D. Von Hoff,
L. H. Hurley, Mol. Cancer Ther. 2001, 1, 103–120.
[26] M. Franceschin, A. Alvino, V. Casagrande, C. Mauriello, E. Pascucci, M.
Savino, G. Ortaggi, A. Bianco, Bioorg. Med. Chem. 2007, 15, 1848–1858.
[27] S. Sparapani, S. M. Haider, F. Doria, M. Gunaratnam, S. Neidle, J. Am.
Chem. Soc. 2010, 132, 12263–12272.
[28] F. Hamon, E. Largy, A. Gudin-Beaurepaire, M. Rouchon-Dagois, A. Sidibe,
D. Monchaud, J. L. Mergny, J. F. Riou, C. H. Nguyen, M. P. Teulade-Fichou,
Angew. Chem. Int. Ed. 2011, 50, 8745–8749; Angew. Chem. 2011, 123,
8904–8908.
[29] J. M. Nicoludis, S. T. Miller, P. D. Jeffrey, S. P. Barrett, P. R. Rablen, T. J.
Lawton, L. A. Yatsunyk, J. Am. Chem. Soc. 2012, 134, 20446–20456.
[30] N. C. Sabharwal, V. Savikhin, J. R. Turek-Herman, J. M. Nicoludis, V. A.
Szalai, L. A. Yatsunyk, FEBS J. 2014, 281, 1726–1737.
[31] R. V. Brown, F. L. Danford, V. Gokhale, L. H. Hurley, T. A. Brooks, J. Biol.
Chem. 2011, 286, 41018–41027.
Acknowledgements
We are thankful to the Computer Centre, IIT Bombay, for use of
the HPC facility; to Prof. David A. Case (Rutgers University) for
waiving the licensing fee for AMBER 12; and to Prof. Ruchi Anand
(IIT Bombay) for providing access to her laboratory facilities. This
work is supported financially by a grant from the Department of
Atomic Energy—Board of Research in Nuclear Sciences (DAE-
BRNS; grant no: 2012/37C/4/BRNS-1063). K.V.D. and S.H. thank
the Council of Scientific and Industrial Research (CSIR) and DAE-
BRNS, respectively, for their fellowships.
Keywords: DNA
· G-quadruplexes · indenopyrimidines ·
promoters · specificity
[32] P. V. L. Boddupally, S. Hahn, C. Beman, B. De, T. A. Brooks, V. Gokhale,
L. H. Hurley, J. Med. Chem. 2012, 55, 6076–6086.
[33] J. Dash, R. N. Das, N. Hegde, G. D. Pantos, P. S. Shirude, S. Balasubrama-
nian, Chem. Eur. J. 2012, 18, 554–564.
[34] J. Dash, P. S. Shirude, S.-T. D. Hsu, S. Balasubramanian, J. Am. Chem. Soc.
2008, 130, 15950–15956.
[35] T. Agarwal, S. Roy, T. K. Chakraborty, S. Maiti, Biochemistry 2010, 49,
8388–8397.
[36] V. Dhamodharan, S. Harikrishna, C. Jagadeeswaran, K. Halder, P. I. Pra-
deepkumar, J. Org. Chem. 2012, 77, 229–242.
[37] K. Gracie, V. Dhamodharan, P. I. Pradeepkumar, K. Faulds, D. Graham, An-
alyst 2014, 139, 4458–4465.
[1] S. Burge, G. N. Parkinson, P. Hazel, A. K. Todd, S. Neidle, Nucleic Acids
Res. 2006, 34, 5402–5415.
[2] P. Murat, Y. Singh, E. Defrancq, Chem. Soc. Rev. 2011, 40, 5293–5307.
[3] S. Balasubramanian, S. Neidle, Curr. Opin. Chem. Biol. 2009, 13, 345–
353.
[4] J. L. Huppert, S. Balasubramanian, Nucleic Acids Res. 2007, 35, 406–413.
[5] S. Balasubramanian, L. H. Hurley, S. Neidle, Nat. Rev. Drug Discovery
2011, 10, 261–275.
[6] Y. Qin, L. H. Hurley, Biochimie 2008, 90, 1149–1171.
[7] V. Gonzꢃlez, L. H. Hurley, Annu. Rev. Pharmacol. Toxicol. 2010, 50, 111–
129.
[38] B. C. Shook, S. Rassnick, N. Wallace, J. Crooke, M. Ault, D. Chakravarty,
J. K. Barbay, A. Wang, M. T. Powell, K. Leonard, V. Alford, R. H. Scannevin,
K. Carroll, L. Lampron, L. Westover, H. K. Lim, R. Russell, S. Branum, K. M.
Wells, S. Damon, S. Youells, X. Li, D. A. Beauchamp, K. Rhodes, P. F. Jack-
son, J. Med. Chem. 2012, 55, 1402–1417.
[8] J. Seenisamy, E. M. Rezler, T. J. Powell, D. Tye, V. Gokhale, C. S. Joshi, A.
Siddiqui-Jain, L. H. Hurley, J. Am. Chem. Soc. 2004, 126, 8702–8709.
[9] T. Simonsson, P. Pecinka, M. Kubista, Nucleic Acids Res. 1998, 26, 1167–
1172.
[10] S. Rankin, A. P. Reszka, J. Huppert, M. Zloh, G. N. Parkinson, A. K. Todd, S.
Ladame, S. Balasubramanian, S. Neidle, J. Am. Chem. Soc. 2005, 127,
10584–10589.
[39] F. C. Meng, F. Mao, W. J. Shan, F. Qin, L. Huang, X. S. Li, Bioorg. Med.
Chem. Lett. 2012, 22, 4462–4466.
[40] S. Paramasivan, I. Rujan, H. P. Bolton, Methods 2007, 43, 324–331.
[41] A. Ambrus, D. Chen, J. Dai, T. Bialis, R. A. Jones, D. Yang, Nucleic Acids
Res. 2006, 34, 2723–2735.
[11] D. Sen, W. Gilbert, Nature 1990, 344, 410–414.
[12] a) A. Risitano, K. R. Fox, Nucleic Acids Res. 2004, 32, 2598–2606; b) P.
Hazel, J. Huppert, S. Balasubramanian, S. Neidle, J. Am. Chem. Soc. 2004,
126, 16405–16415; c) M. Cevec, J. Plavec, Biochemistry 2005, 44,
15238–15246; d) P. A. Rachwal, I. S. Findlow, J. M. Werner, T. Brown, K. R.
Fox, Nucleic Acids Res. 2007, 35, 4214–4222; e) P. A. Rachwal, T. Brown,
K. R. Fox, FEBS Lett. 2007, 581, 1657–1660.
[13] T. Qu, Y. Lu, J. Jan, Z. Huang, K. Wong, L. Gu, ChemMedChem 2008, 3,
690–713.
[14] D. Monchaud, M. P. Teulade-Fichou, Org. Biomol. Chem. 2008, 6, 627–
636.
[15] Q. Li, J. F. Xiang, H. Zhang, Y. L. Tang, Curr. Pharm. Des. 2012, 18, 1973–
1983.
[16] D. L. Ma, T. S. Lai, F. Y. Chan, W. H. Chung, R. Abagyan, Y. C. Leung, K. Y.
Wong, ChemMedChem 2008, 3, 881–884.
[17] S. Cosconati, A. Rizzo, R. Trotta, B. Pagano, S. Iachettini, S. D. Tito, I.
Lauri, I. Fotticchia, M. Giustiniano, L. Marinelli, C. Giancola, E. Novellino,
A. Biroccio, A. Randazzo, J. Med. Chem. 2012, 55, 9785–9792.
[42] E. M. Rezler, J. Seenisamy, S. Bashyam, K. Mu-Yong, E. White, W. D.
Wilson, L. H. Hurley, J. Am. Chem. Soc. 2005, 127, 9439–9447.
[43] A. Guꢄdin, L. Lacroix, J. L. Mergny, Methods Mol. Biol. 2010, 613, 25–35.
[44] J. Mohanty, N. Barooh, V. Dhamodharan, S. Harikrishna, P. I. Pradeepku-
mar, A. C. Bhasikuttan, J. Am. Chem. Soc. 2013, 135, 367–376.
[45] Y. P. Kumar, S. Bhowmik, R. N. Das, I. Bessi, S. Paladhi, R. Ghosh, H.
Schwalbe, J. Dash, Chem. Eur. J. 2013, 19, 11502–11506.
[46] S. Shi, J. Zhao, X. Geng, T. Yao, H. Huang, T. Liu, L. Zheng, Z. Li, D. Yang,
L. Ji, Dalton Trans. 2010, 39, 2490–2493.
[47] A. Arora, C. Balasubramanian, N. Kumar, S. Agrawal, R. P. Ojha, S. Maiti,
FEBS J. 2008, 275, 3971–3983.
[48] N. Ranjan, D. P. Arya, Molecules 2013, 18, 14228–14240.
[49] C. Wei, Y. Wang, M. Zhang, Org. Biomol. Chem. 2013, 11, 2355–2364.
[50] A. Cummaro, I. Fotticchia, M. Franceschin, C. Giancola, L. Petraccone,
Biochimie 2011, 93, 1392–1400.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2014, 9, 2754 – 2765 2764

CHEMMEDCHEM
FULL PAPERS
www.chemmedchem.org
[51] a) H. Han, L. H. Hurley, M. Salazar, Nucleic Acids Res. 1999, 27, 537–542;
b) J. Seenisamy, S. Bashyam, V. Gokhale, H. Vankayalpati, C. L. Grand, A.
Siddiqui-Jain, N. Streiner, W. D. Wilson, L. H. Hurley, J. Am. Chem. Soc.
2005, 127, 2944–2959.
[53] J. Dai, M. Carver, L. H. Hurley, D. Yang, J. Am. Chem. Soc. 2011, 133,
17673–17680.
[54] A. T. Phan, V. V. Kuryavyi, S. Burge, S. Neidle, D. J. Patel, J. Am. Chem.
Soc. 2007, 129, 4386–4392.
[55] G. M. Morris, D. S. Goodsell, R. S. Halliday, R. Huey, W. E. Hart, R. K.
Belew, A. J. Olson, J. Comput. Chem. 1998, 19, 1639–1662.
[56] D. A. Case, AMBER 12, University of California, San Francisco, CA (USA),
2012.
[57] P. A. Kollman, I. Massova, C. Reyes, B. Kuhn, S. Huo, L. Chong, M. Lee, T.
Lee, Y. Duan, W. Wang, O. Donini, P. Cieplak, J. Srinivasan, D. A. Case,
T. E. Cheatham, Acc. Chem. Res. 2000, 33, 889–897.
[58] S. Haider, S. Neidle, Methods Mol. Biol. 2010, 608, 17–37.
[59] T. Fox, P. A. Kollman, J. Phys. Chem. B 1998, 102, 8070–8079.
[60] J. Wang, R. M. Wolf, J. W. Caldwell, P. A. Kollman, D. A. Case, J. Comput.
Chem. 2004, 25, 1157–1174.
[52] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R.
Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A. Petersson, H. Na-
katsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G.
Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Ha-
segawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven,
J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. Bearpark, J. J. Heyd, E.
Brothers, K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Ra-
ghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N.
Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin, R.
Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Morokuma, V. G. Zakr-
zewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, A. D. Dan-
iels, ꢅ. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J. Fox, Gaussi-
an 09, Revision A.02, Gaussian Inc., Wallingford, CT (USA), 2009.
Received: September 13, 2014
Published online on October 30, 2014
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ChemMedChem 2014, 9, 2754 – 2765 2765