Synthesis and evaluation of quinazolone derivatives as a new class of c-KIT G-quadruplex binding ligands

Article abstract of DOI:10.1021/ml400271y

The c-KIT G-quadruplex structures are a novel class of attractive targets for the treatment of gastrointestinal stromal tumor (GIST). Herein, a series of new quinazolone derivatives with the expansion of unfused aromatic ring system were designed and synt

Full text of DOI:10.1021/ml400271y

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Letter
Synthesis and Evaluation of Quinazolone Derivatives
as a New Class of c-KIT G-Quadruplex Binding Ligands
Xiaoxiao Wang, Chen-Xi Zhou, Jin-Wu Yan, Jin-Qiang Hou, Shuo-Bin
Chen, Tian-Miao Ou, Lian-Quan Gu, Zhi-Shu Huang, and Jia-Heng Tan
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400271y • Publication Date (Web): 13 Aug 2013
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Synthesis and Evaluation of Quinazolone Derivatives as a
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New Class of c-KIT G-Quadruplex Binding Ligands
Xiaoxiao Wang,‡ Chen-Xi Zhou,‡ Jin-Wu Yan, Jin-Qiang Hou, Shuo-Bin Chen, Tian-Miao Ou,
Lian-Quan Gu, Zhi-Shu Huang, and Jia-Heng Tan*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
KEYWORDS c-KIT G-quadruplex, quinazolone derivatives, adaptive scaffold, biophysical study, cellular study
ABSTRACT: The c-KIT G-quadruplex structures are a novel class of attractive targets for the treatment of gastrointestinal
stromal tumor (GIST). Herein, a series of new quinazolone derivatives with the expansion of unfused aromatic ring
system were designed and synthesized. Subsequent biophysical studies demonstrated that the derivatives with adaptive
scaffold could effectively bind to and stabilize c-KIT G-quadruplexes with good selectivity against duplex DNA. More
importantly, these ligands further inhibited the transcription and expression of c-KIT gene and exhibited significant
cytotoxicity on the GIST cell line HGC-27. Overall, these quinazolone derivatives represent a new class of promising c-KIT
G-quadruplex ligands. The experiment results have also reinforced the idea of inhibition of c-KIT expression through
targeting c-KIT G-quadruplex DNA.
In the presence of certain cations, guanine-rich single
strands can form higher-order structures defined as
G-quadruplexes. The primary building block of these
structures is the π-stacked G-quartet, in which four
guanine bases are connected by a network of eight
Hoogsteen hydrogen bonds.1 G-quadruplex motifs are
widely dispersed in the eukaryotic genomes and
concentrate in close vicinity to transcriptional start sites.2,
approved by FDA for use of the treatment of GIST.12
However, new patterns of drug resistance owing to
mutations in c-KIT protein have reduced its efficacy.13
Recently it has been shown that treatment of cells with
c-KIT G-quadruplex binding ligands can reduce their
c-KIT expression levels thus exhibiting an alternative
pathway for the design of small molecules inhibiting GIST
cell growth.14-16 Therefore, development of small
molecules as c-KIT G-quadruplex ligands has been
considered a promising strategy to overcome the c-KIT
protein mutation related resistance. Inspiringly, the
structure of c-KIT G-quadruplex DNA has been
determined by the NMR and crystallographic studies.17, 18
Such well-resolved structure exhibit unprecedented
conformation as compared with other G-quadruplex DNA
and provides an important platform for further
structure-based drug design.
3
A series of G-quadruplex motifs in gene promoter
regions have been investigated, including c-MYC,4 BCL-2,5
VEGF,6 KRAS and two motifs in the c-KIT promoter
(c-KIT1 and c-KIT2).7-9 The unique G-quadruplex
structures of these promoters and their potential
biological functions in gene regulation make them
attractive targets for drug design.3
The proto-oncogene c-KIT, which encodes
a
membrane-bound glycoprotein of the family of growth
factor receptors with tyrosine kinase activity, constitutes
Several small molecules including trisubstituted
isoalloxazines,19 naphthalene diimide derivatives,14
a
cell signaling system that can stimulate cell
proliferation, differentiation, migration, and survival.10
The excessive activation of c-KIT is considered to be the
primary pathogenic event in gastrointestinal stromal
tumor (GIST) and the c-KIT protein has become a major
molecular target for GIST therapy.11 As we know the
kinase inhibitor Gleevec (imatinib mesylate) has been
substituted
indenoisoquinolines
and
benzo[a]phenoxazines have been identified to be c-KIT
G-quadruplex binding ligands and subsequently inhibit
c-KIT transcription and expression levels in cellular
16
studies.15,
These rigid ligands comprise a planar,
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G-quartet. Some flexible ligands with unfused aromatic
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aromatic core with the capability of stacking on the
Scheme 1. Synthesis of Quinazolone Derivatives a
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a
Reagents and conditions: (a) Ac2O (reflux); (b) Aniline (room temperature); (c) N-Methylpiperazine, DMF (reflux); (d)
4-Nitrobenzaldehyde, glacial acetic acid, NaOAc (reflux); Na2S•9H2O, NaOH, EtOH/H2O (reflux); (e) 2-Chloracetyl
chloride/3-Chloropropionyl chloride (reflux); (f) R1NH, NaI, EtOH (reflux); (g) 4-Carboxylbenzaldehyde, glacial acetic
acid, NaOAc (reflux); (h) R2NH, BOP, DMF (reflux).
scaffolds, such as bis-indole carboxamides and
diarylethynyl amides,20, 21 are also found to bind to the
c-KIT G-quadruplex DNA in vitro. However, the
inhibitory effects of unfused aromatic ligands on the
c-KIT transcription and expression levels have not been
explored. Whether they could bind to the G-quadruplex
in c-KIT oncogene at the transcription level still needs to
be clarified. Moreover, further efforts in appropriate drug
design are also needed to control the c-KIT G-quadruplex
selectivity over duplex DNA because ligand interaction
with duplex DNA leads to acute toxic and intolerable side
effects on normal tissues. Unlike rigid aromatic
compounds, unfused aromatic molecules with adaptive
structural feature could prevent themselves from
prevents from intercalating into the duplex DNAs. The
two cationic amino side chains were attached to the
scaffold for improving the G-quadruplex DNA binding
potency and selectivity of the derivatives as well as the
solubility in aqueous medium. Furthermore, we also
interconverted the amido bond of the derivatives to
uncover its influence on the c-KIT G-quadruplex
recognition.
The facile and efficient synthetic route for quinazolone
derivatives (QDs) is shown in Scheme 1. Treatment of
2-amino-4,5-difluorobenzoic acid (1) in acetic anhydride
produced compound 2, which was stirred at room
temperature in aniline to give compound 3. Regioselective
nucleophilic substitution of the fluorine atom of 3 with
N-methylpiperazine gave compound 4. The structure of 4
was further confirmed by HMBC and NOESY experiments
(Supporting Information). Claisen-Schmidt condensation
between 4 and 4-nitrobenzaldehyde yielded the nitro
intermediate product. Without further purification, the
product was reduced to the corresponding amino 5,
acylation of which were then performed through reaction
with the required acid chloride. The target compounds
7a-7f were prepared by substitution of 5 with appropriate
secondary amines. On the other hand, Claisen-Schmidt
condensation between 4 and 4-carboxylbenzaldehyde
yielded the compound 6. The condensation of 6 with
various linear aliphatic amines was catalyzed by BOP in
DMF to give compounds 7g-7j. The coupling constant
across the double bond is ~16 Hz, suggesting these final
intercalating into the duplex DNAs. This may be more
promising to achieve the desirable selectivity.21,
To
22
further explore these stories and to discover drug-like
lead compounds for GIST therapy, we designed a new
class of unfused aromatic molecules targeting c-KIT
G-quadruplex DNA.
Quinazolone and their derivatives are building blocks
for approximately 150 naturally alkaloids, and have been
reported to have wide-ranging biological activities
including antibacterial, antidiabetic, anti-inflamatory,
anti-tumor and several other useful properties.23-25 Several
quinazolone derivatives have been determined to
selectively bind and stabilize the G-quadruplex DNA.25, 26
On the basis of this pharmacophore, we designed and
synthesized a new class of quinazolone derivatives (7a –
7j) targeting c-KIT G-quadruplex DNA. These small
molecules contain an expanded aromatic system via the
introduction of a benzene ring and a benzylidene group
into the quinazolone moiety and two cationic amino side
chains (Scheme 1). The unfused aromatic scaffold not only
maximizes the stacking interaction of the derivatives with
G-quartet, but incorporates features of flexibility that
compounds adopted
Information).
E
configuration (Supporting
To evaluate the stabilizing ability and selectivity of QDs
for c-KIT G-quadruplex DNA, FRET-melting experiments
were carried out. Two dual-labeled c-KIT G-quadruplex
forming
sequences,
F-c-KIT1-T
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(5’-FAM-d[AG3AG3CGCTG3AGGAG3]-TAMRA-3’)
F-c-KIT2-T (5’-FAM-d[G3CG3CGCGAG3AG4]-TAMRA-3’),
and
and a self-complementary duplex control F10T (5’-FAM-
1
2
3
Table 1. Stabilization Temperatures (ΔTm) Determined by FRET-Melting Experiment and Equilibrium
Dissociation Constant (KD) Determined by SPR Assay
4
5
FRET ΔTm (°C) a
SPR KD (M)
6
7
8
9
F-c-KIT1-T
6.4 ± 0.4
5.5 ± 0.4
4.2 ± 0.4
11.3 ± 0.3
11.1 ± 0.3
10.7 ± 0.3
15.1 ± 0.3
13.4 ± 0.3
12.3 ± 0.3
15.9 ± 0.3
F-c-KIT2-T
9.4 ± 0.2
7.8 ± 0.2
7.5 ± 0.2
12.4 ± 0.1
13.6 ± 0.1
12.6 ± 0.1
15.9 ± 0.1
14.3 ± 0.1
13.4 ± 0.1
16.8 ± 0.1
F10T
c-KIT1
duplex
c
b
b
c
c
c
c
c
c
c
7.84 × 10-7
7a
7b
7c
7d
7e
7f
0 ± 0.4
—
—
—
—
—
—
—
—
—
—
b
0 ± 0.4
—
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b
0.1 ± 0.4
0 ± 0.4
—
1.88 × 10-6
6.12 × 10-7
9.16 × 10-7
1.16 × 10-6
1.37 × 10-6
15.7 × 10-6
1.99 × 10-6
0 ± 0.5
0 ± 0.4
7g
7h
7i
0.5 ± 0.4
0.4 ± 0.4
0.4 ± 0.4
0.4 ± 0.4
7j
a ΔTm = Tm (DNA + ligand) - Tm (DNA). The concentrations of F-c-KIT1-T, F-c-KIT2-T and F10T were all 1 μM. The
concentrations of the compounds were 5 μM. In the absence of ligand, Tm values of annealed F-c-KIT1-T, F-c-KIT2-T and
F10T are 56.4, 63.4 and 61.9 °C, respectively. b Not determined. c No significant binding was found for addition of up to 20
ꢀM ligand, which might indicate no specific interactions between the ligand and the DNA.
d[TATAGCTATA-HEG-TATAGCTATA]-TAMRA-3’) were
employed in the experiments. The melting temperatures
(ꢁTm) of all tested strands were determined and are listed
in Table 1.
by global fitting of the kinetic data from various
concentrations of the derivatives using 1:1 Langmuir
binding. As shown in Table 1, KD values of QDs for c-KIT1
G-quadruplex DNA were mostly less than 2 ꢀM, while no
obvious binding of the derivatives to duplex DNA was
observed. This was consistent with the FRET-melting
study. The KD values of 7b and 7c were not determined
because they were precipitated after dilution to the
working concentration for SPR experiments. The binding
of 7a, 7e and 7f to c-KIT1 G-quadruplex DNA was slightly
stronger (lower KD values) than that of 7g-7j. For example,
Compound 7e (KD=0.612 ꢀM) showed a clear preference
to bind c-KIT1 G-quadruplex compared with 7h (KD=1.37
ꢀM). This indicated that the QD-NH-CO- arrangement
performed better in binding interaction than the
QD-CO-NH- arrangement. Also, Compound 7a with a
short side chain has better binding affinity (KD=0.784 µM)
than compound 7d (KD=1.88 µM). These data showed
some discrepancy with the results from FRET-melting
assay. It may be because the affinity indicated by
equilibrium binding in SPR studies and stability from Tm
measurements in FRET-melting cannot be simply
compared.28
As shown in Table 1, the derivatives generally exhibited
stabilization effects on the c-KIT1 and c-KIT2
G-quadruplexes by the enhanced melting temperatures.
Compounds 7a-7c with short side chains showed
relatively modest quadruplex stabilizing ability (4.2-9.4
°C), while 7d-7f with longer chain apparently increased
the melting temperatures (10.7-13.6 °C). This suggested
that chain length could be an important factor for
G-quadruplex stabilization. On the other hand, it has
been shown that the amido bond would enlarge ligand’s
aromatic plane and/or participate in water-relayed
hydrogen bonds with G-quadruplex.27 This may be
important for G-quadruplex stabilization and selectivity.
In this study, we interconverted the position of in-chain
amido bond to study its influences on G-quadruplex
stabilization and selectivity. Comparing the melting
temperatures of 7g-7j with those of 7d-7f, the compounds
with QD-CO-NH- arrangement had stronger quadruplex
stabilization by 2-4 °C. Besides, as expected, all the
derivatives
have
excellent
selectivity
between
Furthermore, circular dichroism (CD) spectroscopy was
applied to investigate the binding properties of QDs to
c-KIT1 and c-KIT2 G-quadruplex DNA. Compound 7e
with greatest binding affinity and competitive ꢁTm was
chose in this study. To explore the influence of amido
bonds on c-KIT G-quadruplex recognition, 7h with an
interconverted amido bond was also selected. In the
presence of 100 mM K+, the CD spectrum of c-KIT1
without ligands showed a major positive band at 263 nm,
a shoulder at about 293 nm and a negative band near 240
G-quadruplex and duplex DNA. None of them were found
to significantly increase the melting temperature of
FRET-tagged duplex DNA F10T. This suggested their poor
ability of binding to duplex DNA.
To evaluate the kinetic constants of QDs for c-KIT
G-quadruplex DNA, surface plasmon resonance (SPR)
experiments were carried out. The c-KIT1 G-quadruplex
DNA and duplex DNA were attached to
streptavidin-coated sensor chip. KD values were calculated
a
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nm. As shown in Figure 1A, upon addition of excess
compounds 7e and 7h to the solution, remarkable
enhancement of the bands near 263 nm was observed.
Under the same condition, the CD spectrum of c-KIT2
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2
3
Table2. IC50 (ꢀM) Values of Qunazolone Derivatives against HGC-27 Cell Line.
4
5
6
MTT IC50 (ꢀM)
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
imatinib
2.3
18.4
1.3
13.4
6.8
8.2
21.2
8.6
17.2
12.3
3.8
7
8
9
without ligands had a major positive band at 263 nm and
a negative band near 240 nm. As shown in Figure 1B, the
addition of excess 7e and 7h to the solution induced a
moderate enhancement of the bands near 263 nm.
Overall, 7e and 7h could induce or enhance the
G-quadruplex formation. Meanwhile, the quadruplex
topology is retained with the binding of 7e and 7h,
indicating that none of these ligands would induce major
changes in c-KIT1 and c-KIT2 quadruplex topology.
of the assay with imatinib as control. As shown in Figure
2A, all the derivatives showed obvious inhibitory effects
on luciferase activity at the concentration of 2.5 ꢀM for wild
promoter construct instead of mutant promoter construct.
No significant inhibitory activity was observed in control
experiments using imatinib. Compounds 7a-7c with short
side chains showed relatively modest inhibitory activities
as compared with compounds 7d-7j containing longer
side chains .
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Figure 2. Effects of the derivatives on c-KIT promoter
activity. MCF-7 cells were transient transfected with plasmid
containing full-length wild promoter of c-KIT or its mutant
and pRL-TK and then treated with different compounds at
the concentration of 2.5 ꢀM (A) and compounds 7e and 7h at
different concentrations (B) for 48 h. The efficiency of
transfection was normalized by pRL-TK. Luciferase activity
was plotted relative to the untreated group, which was
assigned a value of 100%. The error bars represent the
standard error from triplicates of three independent
experiments.
Figure 1. CD spectra of 5 ꢀM c-KIT1 (A) and c-KIT2 (B) in 10
mM Tris–HCl buffer, pH 7.2, 100 mM KCl, without ligand (■),
with 25 ꢀM compound 7e (●), and 25 ꢀM compound 7h (▲).
Beyond the biophysical studies, it is more important to
evaluate the cellular effects of these compounds and see
whether they could also bind to the c-KIT G-quadruplexes
in cellular conditions and accordingly reduce the gene
transcriptional and expression level. Thus, the short-term
cell growth inhibitory activity of the derivatives 7a-7j was
firstly evaluated using MTT assay in HGC-27 cell, a
human gastric carcinoma cell line with c-KIT
overexpression. All the derivatives showed cytotoxicity on
the HGC-27 cells, ranging from 2.3 to 21.2 ꢀM (Table 2).
The activities of compounds 7a, 7c and 7e were
comparable with that of imatinib.
Considering the similar inhibitory activities displayed
by 7d-7j, compounds 7e and 7h with interconverted
amido bonds and best inhibitory effects on luciferase
activity for wild promoter construct were chosen for
further study. The effects of 7e and 7h at different
concentrations on c-KIT promoter activity are shown in
Figure 2B. Obviously, they both could dose-dependently
decrease the luciferase activity for wild promoter
construct. The addition of 7e (5 μM) resulted in about
45% reduction of luciferase activity for wild promoter
construct, two times stronger than its reduction for
mutant promoter construct (22%). At the same
concentration, 7h could also exert inhibitory effects on
To further explore the effect of QDs on c-KIT promoter
activity, luciferase activity assay was employed. Two
luciferase constructs were used in the assay. One of them
contained a full-length wild promoter of c-KIT with native
c-KIT1 and c-KIT2 sequences while the other had the
mutant promoter with mutated c-KIT1 and c-KIT2
sequences that could not form G-quadruplex structures.
All the derivatives (7a-7j) were included in the screening
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DNA. In addition, although there were correlations
luciferase activity of wild and mutant construct (37% vs
1
2
3
4
5
6
20%). All these results tell us that QDs could inhibit the
activity of c-KIT gene through their effective interaction
with the promoter G-quadruplex structures, and in
parallel with SPR and MTT results, compound 7e showed
stronger inhibitory effects on c-KIT promoter activity
than its analog 7h.
between biophysical data and cellular results to some
extent, it was also noteworthy that several compounds
with similar binding affinities for c-KIT G-quadruplex
showed different cytotoxicity on the HGC-27 cells and
there was no significant correlation between cell
proliferation and luciferase inhibition data. It has been
7
8
reported that the cellular effects of G-quadruplex binding
29
ligands were complex.3,
Thus, the cellular effects of
9
these quinazolone derivatives could not be simply
explained by c-KIT G-quadruplex interactions. Other
possible genomic G-quadruplex targets (telomere, c-MYC,
BCL-2, etc.) could be involved in drug action. To
overcome the problem of drug selectivity, structural
modification was also needed.
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Further progress on the development of quinazolone
derivatives with high stabilizing ability, binding affinity
and specificity for c-KIT G-quadruplex DNA could come
from the application of structure-based design methods
based on the unprecedented NMR or crystallographic
c-KIT G-quadruplex structures.17, 18 In order to understand
the nature of the ligand–DNA interactions and help
rationalize the experimental results, we did an initial
molecular docking study using compounds 7e and 7h.
The propeller-type c-KIT G-quadruplex NMR structure
(PDB ID: 2O3M) was applied as a template.17 The binding
free energy for 7e and 7h were estimated to be -12.67 and
-10.77 kcal·mol-1, respectively (Supporting Information).
This trend is consistent with the results of SPR, MTT,
luciferase and RT-PCR assays. The docking utilizing the
NMR structure will be used to provide additional
information for the better design of more effective ligands
targeting c-KIT G-quadruplex DNA. For example, more
planar aromatic moiety (indole, benzimidazole, etc.)
could be introduced instead of 3-phenyl group to control
the planarity and aromaticity of the compounds. Other
methodologies such as the “in situ” click chemistry could
be employed to explore appropriate side chains of the
quinazolone and/or benzylidene moieties.30 Detailed
investigations on the structural modification of these
small molecules are now underway.
Figure 3. Effects of compound 7e and 7h at the
concentration of 1.25, 2.5 and 5 ꢀM on c-KIT transcription.
On the basis of results from luciferase activity assay, a
study on the effects of compounds 7e and 7h on c-KIT
transcription was carried out using HGC-27 cell line. We
evaluated 7e and 7h at the final concentration of 1.25, 2.5,
5 ꢀM through quantitation of mRNA using reverse
transcription polymerase chain reaction (RT-PCR). Upon
the treatment of 7e at three different concentrations for
24 hours, transcription of c-KIT was reduced by 4.9%,
22.8%, 52.5%, respectively, related to a control gene
β-actin (Figure 3). For compound 7h, 5.7%, 10.4%, 35.7%
were observed. Obviously, 7e and 7h showed good
dose-dependent inhibition on the transcription of c-KIT
gene in HGC-27 cells. It was also found that 7e, compared
with 7h, exhibited superior inhibitory effects on c-KIT
transcription levels in most cases. This is in agreement
with the results from SPR, MTT and luciferase assays. It
has been further confirmed that QDs are c-KIT
G-quadruplex binding ligands acting both biophysical and
cellular conditions.
In summary, a novel series of quinazolone derivatives
with an unfused aromatic scaffold were designed,
synthesized and evaluated. These small molecules showed
promising binding to G-quadruplex DNA in c-KIT
oncogene instead of duplex DNA. Moreover, the
derivatives effectively inhibited the transcription and
expression of c-KIT gene and exhibited significant
cytotoxicity on the GIST cell line HGC-27. All these
results provided enlightenment for the design of flexible
and adaptive c-KIT G-quadruplex ligands. The
experimental results have also proved and reinforced the
idea of inhibition of c-KIT expression through targeting
c-KIT G-quadruplex DNA.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures for
synthesis and characterization of quinazolone derivatives,
biophycial assays, cellular assays and docking experiments
are included. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
On the other hand, structures of these existing
derivatives need to be further modified because their
stabilizing ability for c-KIT G-quadruplex DNA and
inhibitory effects on c-KIT transcription levels were
* Tel: 8620-39943053. E-mail: tanjiah@mail.sysu.edu.cn
Author Contributions
‡These authors contributed equally.
apparently less potent than those of reported planar fused
15
aromatic compounds.14,
The unfused aromatic ring
Notes
system seems too feasible and nonplanar to effectively
stack on the G-quartet and stabilize the G-quadurplex
The authors declare no competing financial interest.
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Targeting the c-Kit promoter G-quadruplexes with 6-substituted
indenoisoquinolines. ACS Med. Chem. Lett.s 2010, 1, 306-310.
(16) McLuckie, K. I. E.; Waller, Z. A. E.; Sanders, D. A.;
Alves, D.; Rodriguez, R.; Dash, J.; McKenzie, G. J.; Venkitaraman,
A. R.; Balasubramanian, S. G-quadruplex-binding
benzo[a]phenoxazines down-regulate c-KIT expression in
human gastric carcinoma cells. J. Am. Chem. Soc. 2011, 133,
2658-2663.
ACKNOWLEDGMENT
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This work was supported by Natural Science Foundation of
China (Nos. 91213302, 21272291, and 81001400).
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