Identification of 2,3,6-trisubstituted quinoxaline derivatives as a Wnt2/β-catenin pathway inhibitor in non-small-cell lung cancer cell lines

Article abstract of DOI:10.1016/j.bmcl.2010.07.088

We screened 1434 small heterocyclic molecules and identified thirteen 2,3,6-trisubstituted quinoxaline derivatives that were able to inhibit the Wnt/β-catenin signal pathway and cell proliferation. In the screen, some of the hit compounds such as the ethylene group-coupled quinoxaline derivatives were shown to hold promise for use as potential small-molecule inhibitors of the Wnt/β-catenin signal pathway in non-small-cell lung cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2010.07.088

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5900–5904
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of 2,3,6-trisubstituted quinoxaline derivatives as a
Wnt2/b-catenin pathway inhibitor in non-small-cell lung cancer cell lines
b,
Sang-Bum Lee a, Young In Park a, Mi-Sook Dong a, , Young-Dae Gong
*
*
a Graduate School of Biotechnology, Korea University, Seoul 136701, Republic of Korea
b Department of Chemistry, Dongguk-University-Seoul, Pil-dong 3-ga, Jung-gu, Seoul 100715, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 February 2010
Revised 4 July 2010
Accepted 21 July 2010
Available online 29 July 2010
We screened 1434 small heterocyclic molecules and identified thirteen 2,3,6-trisubstituted quinoxaline
derivatives that were able to inhibit the Wnt/b-catenin signal pathway and cell proliferation. In the
screen, some of the hit compounds such as the ethylene group-coupled quinoxaline derivatives were
shown to hold promise for use as potential small-molecule inhibitors of the Wnt/b-catenin signal path-
way in non-small-cell lung cancer cell lines.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Wnt protein
Small-molecule inhibitors
Anticancer agents
Drug-like library
Wnt proteins constitute a family of highly conserved secreted
glycoproteins that play multiple roles in development and the pro-
gression of diseases. Wnt signaling is one of the key signaling path-
ways that regulate cell proliferation, differentiation, and
morphogenesis.1,2 Aberrant activation of the canonical Wnt signal-
ing pathway is related to many human cancers including colon car-
cinoma and melanoma.3,4 Up to now, 19 different Wnt proteins
and 10 different Frizzled receptors have been identified in the
mouse and human.
Wnt signaling is initiated by the secreted Wnt ligands. Wnt li-
gands bind to a class of seven-pass transmembrane receptors en-
coded by the frizzled genes and LRP5/6 coreceptors. Activation of
the receptor leads to the phosphorylation of the dishevelled pro-
tein and through its association with axin, prevents glycogen syn-
these mutations are found rarely in lung cancers. Although exten-
sive research on the Wnt pathway and cancer has been performed
in colon tumors, more recent work has demonstrated that the Wnt
pathway may play a significant role in lung cancer.8 Several Wnt
proteins have been differentially expressed in non-small-cell lung
cancers (NSCLC), including Wnt 1, -2 and -7a.9
The mRNA of human Wnt2 is highly expressed in fetal lung and
weakly expressed in the placenta.10 Inappropriate activation of the
Wnt2/b-catenin pathway has been reported in many human can-
cers including colorectal, gastric, breast, and cervical cancers.10–13
You14 demonstrated that the Wnt2 protein was overexpressed in
freshly resected human non-small-cell lung cancer (NSCLC) tissues
and inhibition of Wnt2-mediated signaling by siRNA or a monoclo-
nal antibody induced programmed cell death in the NSCLC cell line
A549. Moreover, the anti-Wnt2 antibody was shown to inhibit cell
growth of primary cultures obtained from patients suffering from
NSCLC.14
The importance of Wnt2 in the mediation of normal and path-
ological processes has motivated considerable efforts to identify
b-catenin inhibitors. Although a wealth of inhibitory compounds
are available, the generation of b-catenin inhibitors with selectivity
toward individual Wnt isoforms has proven to be challenging. In
particular, drug-like small organic molecule Wnt inhibitors are
rare.15 Here, we report initial evidence showing that 2,3,6-trisub-
stituted quinoxaline derivatives16 are highly active b-catenin/
Wnt pathway inhibitors and hold promise for treating NSCLC.
To identify small-molecule inhibitors of b-catenin/Wnt path-
way, we screened 1434 compounds using a cell proliferation assay
to select the first hits and then checked the transcriptional activity
thase kinase
3 beta (GSK3b) from phosphorylating crucial
substrates. The GSK3b substrates include the negative regulators
axin and APC, as well as b-catenin itself. Unphosphorylated b-cate-
nin avoids recognition by b-TrCP, a component of an E3 ubiquitin
ligase, and translocates to the nucleus where it interacts with tran-
scription factors such as TCF, LEF, Pygo and Bcl-9.5–7
Lung cancer is the leading cause of cancer-related mortality
worldwide. Studies have shown that APC or b-catenin mutations
lead to the accumulation of nuclear b-catenin, which is associated
with greater than 80% of familial colorectal cancers. However,
* Corresponding authors. Tel.: +82 2 3290 4146; fax: +82 2 3290 3951 (M.-S.D);
tel.: +82 2 2260 3206; fax: +82 2 2268 8204 (Y.-D.G.).
E-mail addresses: msdong@korea.ac.kr (M.-S. Dong), ydgong@dongguk.edu (Y.-
D. Gong).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.088

S.-B. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5900–5904
5901
of the first hits using cell-based reporter gene assay that measures
the transcriptional activity of b-catenin-TCF/LEF in A549/Wnt2
cells. b-catenin-TCF/LEF transcriptional activity was dependent
on Wnt signaling and could be used to monitor the activity of
the Wnt/b-catenin signaling pathway. The 1434 compounds that
were screened represent 20,000 structurally diverse and drugga-
ble, heterocyclic compound libraries, including benzopyrans,17a
oxazoles,17b pyrazoles,17c oxadiazoles,17d thiadiazoles,17d various
thiazoles,17e,17f and pyrimidinediones.17g Calphostin C, a non-spe-
cific Wnt/b-catenin pathway inhibitor, was used in the assay as a
positive control. Inhibition of cell proliferation of A549/Wnt2 cells
by the 1434 compounds were screened at a compound concentra-
Figure 2. IC50 values of the 13 compounds selected from the 1st screen using the
cell proliferation assay with A439 cells. Cal C, calphostin C, the positive control.
tion of 5
by over 50% were selected.18 In the first round of screening, 28
compounds were shown to reproducibly have a IC50 below 5 M.
lM and compounds that reproducibly inhibited growth
Among them, 13 compounds that had a 2,3,4-trisubstituted quin-
oxaline structure were selected (Fig. 1) for further screening. De-
l
N
N
N
O
NH
H
N
N
N
HN
N
N
O
N
O
N
O
702
725
731
N
N
H2N
N
N
N
N
N
N
O
N
O
O
N
744
755
759
O
O
F
N
N
F
N
N
N
O
N
N
N
O
N
O
O
761
783
798
F
O
O
N
N
N
NH
N
N
O
HN
O
N
N
N
N
O
N
813
814
O
F
O
N
N
NH
N
N
O
N
N
O
NH
O
N
823
825
Figure 1. Structure of the Wnt2/b-catenin pathway inhibitors identified in the primary screen.

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S.-B. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5900–5904
spite their structural similarity to the quinoxaline core scaffold, the
IC50 values significantly varied (Fig. 2).
And then we also confirmed that the cell growth inhibition of
selected 13 compounds with low concentration, 15% and 30% of
IC50 (Fig. 3). Cell growth was carried out at 24 and 48 h after treat-
ment by using MTS assay system. These compounds were signifi-
cantly inhibited the cell proliferation without cytotoxicity at 15%
and 30% of IC50 (data not shown). Compounds 755, 761, 783 and
814 were shown the impediment of cell proliferation in a dose
dependent manner.
Next, we examined the inhibitory effect of the 13 derivatives of
2,3,4-trisubstituted quinoxaline, which were chosen from the first
Figure 3. The effect of compounds chosen from the first screen on the cell proliferation at the 15% and 30% of IC50 in A549/Wnt2 cells.

S.-B. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5900–5904
5903
Figure 4. The effect of compounds chosen from the first screen on the Tcf/b-catenin
transcriptional activity at the IC50 of cell proliferation in A549 cells. Cal C, calphostin
C, the positive control.
screen, on the Wnt/b-catenin signal pathway at the IC50 deter-
mined above. For the Topflash assay, A549/Wnt2 cells were tran-
siently transfected with pSuperTopflash and treated with the 13
compounds at IC50 (Fig. 4).19
pFopflash, which has eight mutated TCF/LEF-binding sites and
has no response to the Wnt/b-catenin signal pathway, was used
as a negative control. As shown in Figure 3, the 13 compounds
inhibited the reporter gene activity with a reduction of approxi-
mately 30–90% compared to the non-treated cells without signifi-
cantly affecting the mutant reporter Fopflash. Although three
compounds (759, 761 and 825) among them dramatically de-
creased Topflash reporter activity about below 20%, we could not
figure the relationship between the inhibition of Wnt/b-catenin
signal pathway and the structure of side chain.
The regulation of target genes in Wnt/b-catenin pathway is
dependent on the level of b-catenin and nuclear b-catenin is the
hallmark of activated Wnt signaling. To compare the inhibition of
Wnt/b-catenin pathway with the change of b-catenin levels by
these 13 compounds, the effect of the 13 compounds on the b-cate-
nin protein level in whole cells, cytosol and nuclear fractions was
evaluated in A549/Wnt2 cells using an immunoblot assay
(Fig. 4).20 Although the 13 compounds had structural similarity
to the quinoxaline core scaffold, the levels of b-catenin in whole
cells, cytosol, and nuclear fractions were diversely changed. The
activity of Wnt/b-catenin pathway was proportional to the level
of nuclear b-catenin. Compounds 759, 761 and 825 inhibited the
Figure 6. Effect of the 13 compounds on inhibiting cell proliferation and Tcf/b-
catenin transcriptional activity at the IC50 of A549 cell proliferation. (A) Inhibition of
cell proliferation in H460 cells treated with the 13 compounds using the MTS assay.
(B) Topflash assay of the Tcf/b-catenin dependent transcriptional activity in H460/
Wnt2 cells.
activity of Topflash blow 20% (Fig. 5) and reduced significantly
the nuclear b-catenin level compared with control.
Finally, the inhibitory effect of the 13 compounds at IC50 in
H460, a non-small-lung cancer cell line, was confirmed. The re-
sults, shown in Figure 6A, were confirmed using the MTS assay:
treatment of the cells with the 13 compounds for 24 h significantly
decreased cell proliferation, as was observed in the A549/Wnt2
cells. To test whether the 13 compounds at the IC50 concentration
reduced the b-catenin/Tcf dependent transcriptional activity in
Figure 5. b-Catenine protein levels in whole cells, cytosolic and nuclear fraction prepared from the A549/Wnt2 cells treated with the 13 first ‘hit’ compounds at the IC50 of cell
proliferation.

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S.-B. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5900–5904
11. Park, J. K.; Song, J. H.; He, T. C.; Nam, S. W.; Lee, J. Y.; Park, W. S. Neoplasma
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H460/Wnt2, the cells were transiently transfected with SuperTop-
flash and treated with the 13 compounds at the IC50, as described
in the materials and methods. As illustrated in Figure 6B, these
compounds decreased the b-catenin/Tcf dependent transcriptional
activity in H460/Wnt2. Compounds 759 and 761 significantly de-
creased the Tcf/b-catenin dependent transcriptional activity by
more than 80% in both cell lines, A549/Wnt2 and H460/Wnt2.
In summary, we screened 1434 small heterocyclic molecules
and identified 13 number of the 2,3,6-trisubstituted quinoxaline
derivatives that were able to inhibit the Wnt/b-catenin signal path-
way and cell proliferation.
Among the 13 number of quinoxaline derivatives, some com-
pounds, including compounds 759, 761 and 825, hold promise
for use as potential small-molecule inhibitors of the Wnt/b-catenin
signal pathway. Further studies are currently underway to opti-
mize the potency and selectivity of the 2,3,6-trisubstituted quinox-
aline derivatives and address their in vivo efficacy and therapeutic
potential including the selectivity to cancer cells. These molecules
may serve as useful mechanistic probes of the cellular function of
the Wnt/b-catenin signal pathway and anticancer mechanism.
13. Watanabe, O.; Imamura, H.; Shimizu, T.; Kinoshita, J.; Okabe, T.; Hirano, A.;
Yoshimatsu, K.; Konno, S.; Aiba, M.; Ogawa, K. Anticancer Res. 2004, 24, 3851.
14. You, L.; He, B.; Xu, Z.; Uematsu, K.; Mazieres, J.; Mikami, I.; Reguart, N.; Moody,
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quinoxaline derivatives, Korea Pat. No. 0889389.; (b) Gong, Y.-D.; Dong, M. S.;
Hwang, S. H.; Lee, T. I.; Lee, S. B. 2-Substituted aminoalkylenyloxy-3-
substituted phenylethynyl-6-aminoquinoxaline, Korea Pat. Appl. No. 2008-
0076221.; (c) Synthetic procedure was mentioned in Supplementary data.
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18. Cell proliferation assay: Cells were seeded in 96-well plates (5 Â 103 cells/well)
and allowed to attach for about 24 h. The MTS assay (3-(4,5-dimethylthiazol-2-
yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was used
to measure the inhibition effect of the chemical library on cell proliferation.
Cells were seeded onto 96-well microplates and treated for 24 h with test the
compounds at various concentrations. Cell proliferation was determined using
a CellTiter 96 non-radioactive cell proliferation assay kit according to the
manufacturer’s protocol (Promega, USA). The plate was incubated at 37 °C in a
CO2 incubator for 30 min and the absorbance was measured on a molecular
dynamic plate reader (Bio-Rad, Germany) at 490 nm.
Acknowledgments
We are grateful to the Seoul Research and Business Develop-
ment Program (grant number 10574), and a basic research pro-
gram (grant number 2010-0004128) of National Research
Foundation of Korea for financial support of this research.
19. Luciferase reporter gene assay: Cells were transfected with the luciferase
reporter constructs pSuperTopflash containing eight Tcf consensus binding
sites upstream of the firefly luciferase cDNA, or pSuperFopflash, a plasmid
containing mutated Tcf binding sites (kindly provided by professor Ja-Hyun
Baek at the School of Life Sciences & Biotechnology at Korea University, Seoul,
Korea). Cells (4 Â 104) were transfected using the Transfast transfection
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.088.
reagent (Promega, USA) with
1 lg reporter construct and 15 ng pRL-TK
plasmid and incubated with various concentrations of selected compounds
at 37 °C. After 24 h, the cells were lysed in 50 l passive lysis buffer (Promega,
l
USA). Firefly luciferase and Renilla luciferase activity were determined using
the Dual-Glo Luciferase Assay System (promega, USA). Results are expressed as
the mean SEM of normalized ratios of firefly luciferase activity and renilla
luciferase activity measurements for each triplicate set.
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