A novel 3-arylethynyl-substituted pyrido[2,3,-b]pyrazine derivatives and pharmacophore model as Wnt2/β-catenin pathway inhibitors in non-small-cell lung cancer cell lines

Article abstract of DOI:10.1016/j.bmc.2011.07.028

We developed Wnt/β-catenin inhibitors by identifying 13 number of 3-arylethynyl-substituted pyrido[2,3,-b]pyrazine derivatives that were able to inhibit the Wnt/β-catenin signal pathway and cancer cell proliferation. In the optimization process, a series of 2,3,6-trisubstituted pyrido[2,3,-b] pyrazine core skeletons showed were shown to higher activity than 2,3,6-trisubstituted quinoxaline's and thus hold promise for use as potential small-molecule inhibitors of the Wnt/β-catenin signal pathway in non-small-cell lung cancer cell (NSCLC) lines. And we have studied the pharmacophore mapping for compound 954, which presented the highest activity with a fit value of 2.81. The pharmacophore mapping for the compounds including 954, pyrido[2,3,-b]pyrazine core had hydrogen-bond acceptor site and hydrophobic center roles.

Full text of DOI:10.1016/j.bmc.2011.07.028

Bioorganic & Medicinal Chemistry 19 (2011) 5639–5647
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A novel 3-arylethynyl-substituted pyrido[2,3,-b]pyrazine derivatives
and pharmacophore model as Wnt2/b-catenin pathway inhibitors
in non-small-cell lung cancer cell lines
Young-Dae Gong a, , Mi-Sook Dong b, , Sang-Bum Lee b, Nayeon Kim a, Mi-Seon Bae a, Nam-Sook Kang c
⇑
,
⇑
,
a Center for Innovative Drug Library Research, Dongguk University, Pil-dong 3-ga, Jung-gu, Seoul 100-715, Republic of Korea
b Graduate School of Biotechnology, Korea University, Seoul 136-701, Republic of Korea
c Center for Drug Discovery Platform Technology, Korea Research Institute of Chemical Technology, PO Box 107, Singseongno, Yuseong-gu, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We developed Wnt/b-catenin inhibitors by identifying 13 number of 3-arylethynyl-substituted pyr-
ido[2,3,-b]pyrazine derivatives that were able to inhibit the Wnt/b-catenin signal pathway and cancer cell
proliferation. In the optimization process, a series of 2,3,6-trisubstituted pyrido[2,3,-b]pyrazine core skel-
etons showed were shown to higher activity than 2,3,6-trisubstituted quinoxaline’s and thus hold prom-
ise for use as potential small-molecule inhibitors of the Wnt/b-catenin signal pathway in non-small-cell
lung cancer cell (NSCLC) lines. And we have studied the pharmacophore mapping for compound 954,
which presented the highest activity with a fit value of 2.81. The pharmacophore mapping for the com-
pounds including 954, pyrido[2,3,-b]pyrazine core had hydrogen-bond acceptor site and hydrophobic
center roles.
Received 11 May 2011
Revised 14 July 2011
Accepted 15 July 2011
Available online 23 July 2011
Keywords:
Wnt2
b-Catenin
Pyrido[2,3,-b]pyrazines
Inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Anti-cancer
Pharmacophore
1. Introduction
Lung cancer is the leading cause of cancer-related mortality
worldwide. Studies have shown that APC or b-catenin mutations
Wnt signaling is one of the key signaling pathways that regulate
cell proliferation, differentiation, and morphogenesis.1,2 Wnt pro-
teins constitute a family of highly conserved secreted glycoproteins
that play multiple roles in the development and progression of dis-
eases. Aberrant activation of the canonical Wnt signaling pathway
is related to many human cancers, including colon carcinoma and
melanoma.3,4 b-Catenin is an essential component of cell to cell
interaction and in the Wnt signal pathway plays an important role
as a transcriptional activator with T-cell factor (TCF)/lymphoid
enhancer factor (LEF). In the absence of Wnt protein, the level of
cytosolic b-catenin is maintained low through the degradation of
b-catenin by machinery of destruction complex. Activation of Wnt
signaling inhibits GSK3b activity and accumulates cytosolic b-
catenin level. The elevated level of b-catenin leads to translocation
b-catenin in the nucleus and subsequently the complex formation
with TCF/LEF that induces target gene expression, such as cyclin
D1, c-Myc and survivin.
lead to the accumulation of nuclear b-catenin, which is associated
with greater than 80% of familial colorectal cancers. However,
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.5
The mRNA of human Wnt2 is highly expressed in fetal lung and
weakly expressed in the placenta.6 Inappropriate activation of the
Wnt2/b-catenin pathway has been reported in many human can-
cers, including colorectal, gastric, breast, and cervical cancers.7–9
You10 demonstrated that the Wnt2 protein was overexpressed in
freshly resected human NSCLC tissues and that inhibition of
Wnt2-mediated signaling by siRNA or a monoclonal antibody in-
duced programmed cell death in the NSCLC line A549. Moreover,
the anti-Wnt2 antibody was shown to inhibit cell growth of pri-
mary cultures obtained from patients suffering from NSCLC. The
importance of Wnt2 in the mediation of normal and pathological
processes has motivated considerable efforts to identify b-catenin
inhibitors. Although a wealth of inhibitory compounds is available,
the generation of b-catenin inhibitors with selectivity toward indi-
vidual Wnt isoforms has proven to be challenging. In particular,
good drug-like small organic molecule for Wnt inhibitors are
rare11 though several small molecules were reported as hit or lead
⇑
Corresponding authors. Tel.: +82 2 2260 3206; fax: +82 2 2290 1349 (Y.-D.G.);
tel.: +82 2 3290 4146; fax: +82 2 3290 3951 (M.-S.D.).
E-mail addresses: ydgong@dongguk.edu (Y.-D. Gong), msdong@korea.ac.kr
(M.-S. Dong).
Co-corresponding authors were same contributed for this paper.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.028

5640
Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
R1
R1
Bioisostere
Core Skeleton Design
N
N
N
N
N
N
O
O
N
DGG-720 (R1=H)
Mw: 317.38, IC50: 758 nm
Core skeleton AlogP: 1.29
DGG-954 (R1=H)
Mw: 352.39, IC50: 120 nm
Core skeleton AlogP: 0.6
Figure 1. Optimization and design strategies for the Wnt2/b-catenin pathway inhibitors of the pyrido[2,3,-b]pyrazine core skeleton based on the bioisostere concept from the
previous quinoxaline hit compound.
N
N
N
N
N
N
N
N
N
N
N
N
O
N
N
O
O
921
922
923
O
O
O
O
O
N
N
N
N
N
N
N
Cl
O
O
N
N
927
928
929
N
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
O
930
953
954
O
F
F
N
N
N
N
N
N
N
N
N
N
O
O
O
966
965
967
F
N
N
N
N
O
968
Figure 2. Structure of the Wnt2/b-catenin pathway inhibitors of the pyrido[2,3,-b]pyrazine core skeleton identified in the primary screening (the code name, DGG, was
omitted before the numbers).
compounds toward Wnt/b-catenin pathway for new drug discov-
ery area.12
activity of the Wnt signaling pathway.16 The inhibition of cell pro-
liferation of A549/Wnt2 cells by the 1434 compounds was
Therefore, we have concentrated our attention on searching for
novel small organic b-catenin inhibitors. We have previously found
initial evidence showing that 2,3,4-trisubstituted quinoxaline
derivatives are highly active b-catenin inhibitors that hold promise
in lung cancer treatment.13 In our previous report, that aimed to
identify small molecule inhibitors of b-catenin, we first screened
1434 compounds14,15 using a cell-based reporter assay that mea-
sures the transcriptional activity of b-catenin-TCF/LEF. It was
dependent on Wnt signaling and could be used to monitor the
screened at a compound concentration of 5 lM and compounds
that reproducibly inhibited growth by over 50% were selected.17
Thirteen number of 2,3,4-trisubstituted quinoxaline derivatives
were shown reproducibly to have an IC50 below 5 lM. One of the
best hit compound, DGG-720, exhibited the essential functional
of a high activity due to the presence of the 3-ethynyl group. How-
ever, in our previous paper we could not find out good druggable
lead compound via the introduction of 3-ethynyl series building
blocks. Thus, based on our previous results, we have tested

Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
5641
Figure 3. IC50 values of the 13 compounds selected from the 1st screening using the cytotoxicity assay with A549/Wnt2 cells. Cal C, calphostin C, the positive control.20
Figure 4. The effect of compounds chosen from the first screening on the cell proliferation at 15% and 30% of IC50 in A549/Wnt2 cells.
whether development of a new core skeleton based on the bioiso-
stere concept compared with that of quinoxaline may improve the
physicochemical properties as well as the cell proliferation of
A549/Wnt2 cells.
2. Result and discussion
We designed a scaffold combining the quinoxaline moiety
found in pyrido[2,3,-b]pyrazine with the pyridine ring system

5642
Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
Figure 5. The effect of compounds chosen from the first screening on the TCF/b-catenin transcriptional activity at the IC50 of cell proliferation in A549 cells. Cal C, Calphostin
C, the positive control.20
Figure 6. b-Catenin protein levels in total, cytosolic and nuclear fraction prepared from the A549/Wnt2 cells treated with the 13 first ‘hit’ compounds at the IC50 of
cytotoxicity. (A) b-Catenin protein levels in total cell fraction, cytosol and nuclear fraction and (B) the band ratio of total b-catenin/GAPDH, cytosol b-catenin/GAPDH and
nuclear b-catenin/PARP. GAPDH and PARP are reference proteins for the cytosolic and nuclear fractions, respectively.
(Fig. 1), and synthesized one hundred 3-arylethynyl-substituted
pyrido[2,3,-b]pyrazine derivatives16 designed via the bioisostere
concept, as shown in Figure 1.18 In the first round of screening,
13 pyrido[2,3,-b]pyrazine derivatives reproducibly exhibited an
decreased cell proliferation about 15% and 30% of cytotoxicity IC50
values at 24 and 48 h, respectively using CellTiter 96 non-radioac-
tive cell proliferation assay kit (Promega, USA) (Fig. 4). The differ-
ences of assays between cytotoxicity and cell proliferation were
the cell seeding number and culture time as described in Section
4. Among 13 compounds, compounds 953, 954, 965 and 966 sig-
nificantly inhibited the cell proliferation in a dose-dependent
manner with 208, 43, 830, and 535 nM of IC50 values at 72 h,
respectively. Their ratios of IC50 values in cell proliferation to cyto-
toxicity were 3.07–4.64.
Next, we examined the inhibitory effect of the 13 pyrido[2,3,-b]
pyrazine hit compounds on the Wnt/b-catenin signal pathway at
the cytotoxicity IC50 level determined in Figure 3. For the Topflash
assay, A549/Wnt2 cells were transiently transfected with pSuper-
Topflash with pRL/TK-which coded the renilla luciferase for the
determination of the plasmid transfection efficiency- and treated
IC50 of cytotoxicity below 5 lM in A549/Wnt2 cells and were sub-
jected to further screening,19 as shown in Figures 2 and 3. In this
study, we revealed that pyrido[2,3,-b]pyrazine structure (DGG-
954) showed better physicochemical properties and stronger activ-
ity than quinoxaline (DGG-720). Especially, A log P (0.6) of the 954
compound having pyrido[2,3,-b]pyrazine structure showed much
lower value than that of the quinoxaline core skeleton’s 720 com-
pound (1.29), as shown in Figure 1. We convinced this clue can
make the possibility of much higher for developing new drug
candidate.
To study the relationship between the cytotoxicity and the
inhibition of cell proliferation, the effect of 13 selected compounds

Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
5643
Figure 7. Effect of the 13 compounds on the mRNA expression of cyclin D1 in A549/Wnt2 cells: (A) RT-PCR band and (B) the band ratio of cyclin D1/Actin.
O-
N+
O
O
S
O
N
H
H
N
O
N
Cl
O
O
NH
HN
O
N
O
O
N
N
H
O
O
HN
O
O
O
O
O
28
R1
29
R2
30
R3
HN
N
O
F
O
N
N
H
N
N
H
O
N
F
31
R4
32
R5
Figure 8. The reference compounds in the training set obtained from integrity.thomson-pharma.com site as Wnt inhibitors for building the pharmacophore model.
with the 13 compounds at IC50 (Fig. 5).21 pFopflash, which has
eight mutated TCF/LEF-binding sites and no response to the Wnt/
b-catenin signal pathway, was used as the negative control. As
shown in Figure 5, the 13 compounds inhibited the TCF/b-catenin
reporter gene activity with a reduction of approximately 30–90%
compared to the non-treated cells without significantly affecting
the mutant reporter Fopflash. Compound 966 more strongly de-
creased the Topflash reporter activity to 30% compared to the cyto-
toxicity. Compounds 927, 953, 954 and 968 were similarly
inhibited around 50% between the Topflash reporter gene activity
and the cytotoxicity. Although the 13 compounds had structural
similarity to the pyrido[2,3,-b]pyrazine core scaffold, the relation-
ship between the inhibition rate of the Wnt/b-catenin signal path-
way and the structure of the side chain could not be figured out.
The regulation of target genes in the 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
the Wnt/b-catenin pathway with the change of b-catenin levels in-
duced by these 13 compounds, their effect on the b-catenin protein
level in cytosol and nuclear fractions was evaluated in A549/Wnt2
cells in using an immunoblot assay (Fig. 6).22 These 13 compounds
down-regulated the expression of the b-catenin protein all of three
fractions, and compound 966 was the most effective inhibitor.
Compounds 953, 954 and 968 were decreased particularly in the
nucleus b-catenin protein and these results accorded with the Top-
Flash assay. Collectively, our results suggested that the side chain
structure with pyrido[2,3,-b]pyrazine core is important to deter-
mine the inhibition rate of the Wnt/b-catenin pathway.

5644
Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
Table 1
The fit values for 13 hit compounds shown in Figure 11
Inhibitor
Activity
Fit values
954
928
929
921
922
953
923
930
966
967
968
965
927
0.14
0.28
0.38
0.56
0.61
0.81
1.61
1.94
2.00
2.45
2.89
3.54
4.03
2.81
2.63
2.49
1.65
1.51
2.56
2.33
1.50
1.38
1.04
0.78
1.29
1.25
Figure 9. Pharmacophore mapping for reference compound R4, having fit value of
4.00. The green and cyan spheres represent the hydrogen-bond acceptor site and
the hydrophobic sites, respectively.
charges and conjugated gradient methods. The common pharma-
cophores were generated using the HipHop algorithm.24 Prede-
fined pharmacophore features were used to automatically create
the pharmacophore hypothesis model. The list of features of the
minimum and maximum values was as follows: H-bond acceptor
(Hba) 0 and 5, H-bond donor (Hbd) 0 and 5, Hydrophobic (Hy) 0
and 5. The compounds in the training set showed fit values ranging
from 1.05 to 5.99. We have previously reported this model.25
The pharmacophore mapping for reference compound R4 hav-
ing fit value of 4.0 is shown in Figure 9. The green and cyan spheres
represent the hydrogen-bond acceptor site and the hydrophobic
sites, respectively. The pharmacophore mapping for compound
954, which presented the highest activity with a fit value of 2.81,
is shown in Figure 10. The pharmacophore mapping for the com-
pounds including 954 shown in Figure 10, pyrido[2,3,-b]pyrazine
core had hydrogen-bond acceptor site and hydrophobic center
roles. The distance for two hydrophobic sites in branch chains of
pyrido[2,3,-b]pyrazine core is about 5.7 Å. The longer hydrophobic
substituents have, better activity are obtained, such as 954, 928
and 929. And also, we could find out the correlation plot between
pharmacophore mapping and activity for 13 hit compounds as
shown in Figure 11 and Table 1.
Figure 10. Pharmacophore mapping for compound 954, having a fit value of 2.81.
R² = 0.578
4.03
4
3.54
3
2.89
2.45
2
1
0
2
1.94
0.61
1.61
3. Conclusion
0.81
0.56
In conclusion, we developed a novel 3-arylethynyl-substituted
pyrido[2,3,-b]pyrazine as a first lead compound 954 by synthesiz-
ing one hundred pyrido[2,3,-b]pyrazine derivatives, of which 13 3-
arylethynyl-substitutedpyrido[2,3,-b]pyrazine derivatives were
identified as being able to inhibit the Wnt/b-catenin signal path-
way and cell proliferation. And we have studied the pharmaco-
phore mapping for compound 954, which presented the highest
activity with a fit value of 2.81. The pharmacophore mapping for
the compounds including 954, pyrido[2,3,-b]pyrazine core had
hydrogen-bond acceptor site and hydrophobic center roles. Further
studies are currently underway to optimize the potency and selec-
tivity of these 13 derivatives and address their in vivo efficacy and
therapeutic potential. These molecules may serve as useful mech-
anistic probes of the cellular function of the Wnt/b-catenin signal
pathway and anticancer mechanism.
0.38
0.28
0.14
0.50
1.50
2.50
Figure 11. The correlation plot between pharmacophore mapping (fit values in X-
axis) and activity (IC50 in Y-axis) for 13 hit compounds. Each dot is depicted as
activity values.
Since the selected compounds inhibited the TCF/b-catenin tran-
scriptional activity and cell proliferation, we further examined the
expression of cyclin D1, which is related to cell progression and cell
cycle progression and is a well-known Wnt/b-catenin signal target
gene, in A549/Wnt2 cells. The gene expression level of cyclin D1
was inhibited by 24 h-treatment of compounds 953, 954, 966
and 968 (Fig. 7) and these results consistently agreed with results
of Topflash assay (Fig. 5) and b-catenin levels in nucleus (Fig. 6).
We next studied a structure–activity relationship of pyrido[2,3,-
b]pyrazine core skeleton hit compounds. To clearly investigate the
structure–activity relationship studies of pyrido[2,3,-b]pyrazine
core skeleton hit compounds, we selected 5 known compounds
(R1–R5) from the Prous site (http://prous.integrity.com) as Wnt
inhibitors and used them in the training set, as shown in Figure
8. After the 3D structures of these compounds were generated
using CONCORD,23 they were minimized using Gasteiger–Huckel
4. Experimental
4.1. Chemistry
4.1.1. General for synthesis
All chemicals were reagent grade and used as purchased. Reac-
tions were monitored by thin layer chromatography (TLC) analysis
using Merck Silica Gel 60 F-254 thin layer plates or attenuated

Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
5645
total reflection Fourier transform infrared (ATR-FTIR) analysis
using TravelIR™ (SensIR Technology). Flash column chromatogra-
phy was carried out on Merck Silica Gel 60 (230–400 mesh). The
crude products were purified by parallel chromatography using
Quad3™. 1H NMR and 13C NMR spectra were recorded in d units
relative to deuterated solvent as an internal reference using a Bru-
ker 500 MHz NMR instrument. Liquid chromatography–mass spec-
trometry (LC–MS) analysis was performed on an electrospray
ionization (ESI) mass spectrometer with photodiode-array detector
(PDA) detection. LC–MS area percentage purities of all products
were determined by LC peak area analysis (XTerraMS C18 column,
4.6 mm  100 mm; PDA detector at 200–400 nm; gradient, 5–95%
CH3CN/H2O). High-resolution mass spectrometry fast-atom bom-
bardment (HRMS-FAB) spectra were obtained using API 4000Q
TRAP LC/MS/MS system (Applied Biosystems).
centrated in vacuum to remove the solvent and then water was
added. The mixture was extracted with ethyl acetate and the or-
ganic layer was washed with water and dried over MgSO4. After re-
moval of solvent in vacuum, the residue was purified by SiO2
column chromatography (CH2Cl2/n-hexane = 3:2) to yield the de-
sired compound 4, 2-chloro-3-(phenylethynyl)pyrido[3,2-b]pyra-
zine (83%, 4.6 g). 1H NMR (500 MHz, CDCl3) d 7.46 (m, 6H), 7.73
(m, 6H), 8.34 (d, J = 8.3 Hz, 2H), 9.19 (dd, J = 2.31, 1.8 Hz, 2H); MS
(ESI) m/z 266 ([M+H]+).
4.2.4. Synthesis of 3-(phenylethynyl)-2-(2-(pyridin-2-yl)ethoxy)
pyrido[3,2-b]pyrazine (Hit compound, 954)
To
a
stirred solution of 2-(pyridin-2-yl)ethanol (1.4 g,
11.6 mmol) in tetrahydrofuran (THF; 10 ml) solution was added
sodium hydride dispersion (60%) in mineral oil (743 mg,
18.6 mmol) at room temperature for 20 min, after which THF
(10 ml) solution of the prepared compound 4, 2-chloro-3-(phen-
ylethynyl)pyrido[3,2-b]pyrazine (2.47 g, 9.3 mmol), was dropped
4.2. Synthetic procedures for the preparation of 3-(phenylethy
nyl)-2-(2-(pyridin-2-yl)ethoxy)pyrido[3,2-b]pyrazine (Lead com
pound 954)
N
NH2
NH2
Oxalic acid
3 N aq. HCl
reflux
N
N
N
OH
OH
N
N
N
Cl
Cl
SOCl2, DMF(cat.)
CHCl3, reflux
1
2
3
HO
N
5
6
N
N
N
N
N
Pd(OAc)2, CuI, PPh3, TEA
60% NaH
THF, rt
CH3CN, 60 o
C
N
N
Cl
O
4
Lead compound 954
for 1 h. Stirring was continued at room temperature for 8 h.
4.2.1. Synthesis of pyrido[3,2-b]pyrazine-2,3-diol (2)
The resulting mixture was concentrated in vacuo to remove
the solvent and then water was added. The mixture was ex-
tracted with ethylacetate and the organic layer was washed with
water and dried over MgSO4. After removal of solvent in vacuo,
the residue was purified by SiO2 column chromatography
(CH2Cl2/ethanol = 9:1) to yield the desired compound 954, (79%,
2.84 g). 1H NMR (500 MHz, CDCl3) d 3.42 (t, J = 6, 6.5 Hz, 2H),
4.96 (t, J = 6.5, 6.4 Hz, 2H), 7.13 (s, 1H), 7.37 (m, 4H), 7.55 (dd,
J = 1.2, 4.2 Hz, 2H), 7.61 (t, J = 0.9, 7.1 Hz, 2H), 8.14 (d,
J = 0.9 Hz, 1H), 8.51 (dd, J = 1.8, 6.5 Hz, 1H), 8.95 (dd, J = 1.1,
0.6 Hz, 1H); 13C NMR d 158.3, 157.0, 150.7, 149.6, 148.3, 136.4,
136.1, 135.7, 135.0, 132.6, 132.2, 132.1, 130.0, 128.6, 128.5,
125.2, 124.0, 121.8, 121.7, 97.6, 84.9, 77.4, 77.1, 76.9, 66.9,
37.6; MS (ESI) m/z 353 ([M+H]+); HRMS-ESI (m/z): [M+H]+ calcd
for C22H16N4O1 352.1324; found 353.143.
A solution of pyridine-2,3-diamine (1) (5.0 g, 45.8 mmol) and
oxalic acid (4.8 g, 53.3 mmol) in 3 N aq HCl (100 ml) was stirred
at reflux condition for 24 h. The resulting mixture was filtered
and then washed with cold water and dried in a vacuum oven at
50 °C. The desired product 2, pyrido[3,2-b]pyrazine-2,3-diol, was
obtained in good yield (89%, 6.7 g). 1H NMR (500 MHz, DMSO) d
7.13 (m, 1H), 7.46 (dd, J = 1.4, 6.3 Hz, 1H), 8.07 (m, 1H), 11.98 (s,
1H), 12.33 (s, 1H); MS (ESI) m/z 164 ([M+H]+).
4.2.2. Synthesis of 2,3-dichloropyrido[3,2-b]pyrazine (3)
To a stirred solution of pyrido[3,2-b]pyrazine-2,3-dione (2)
(4.2 g, 26.0 mmol) in chloroform (CHCl3, 100 ml) was added thionyl
chloride (9.3 g, 78.0 mmol) and N,N-dimethylformamide (DMF,
0.5 ml) at reflux condition for 24 h. The resulting mixture was con-
centrated in vacuo to remove the solvent and then water was
added. The desired product was filtered and washed with water
and dried in a vacuum oven at 50 °C. The desired product 3, 2,3-
dichloropyrido[3,2-b]pyrazine, was obtained in good yield (78%,
4.8 g). 1H NMR (500 MHz, DMSO) d 7.81 (m, 1H), 8.43 (dd, J = 6.6,
1.7 Hz, 1H), 9.19 (dd, J = 3.1, 1.5 Hz, 1H); MS (ESI) m/z 200 ([M+H]+).
4.2.5. Synthesis of 3-(phenylethynyl)-2-(2-(pyrrolidin-1-yl)eth
oxy)pyrido[3,2-b]pyrazine 921
1H NMR (500 MHz, CDCl3) d 1.81 (s, 4H), 2.77 (s, 4H), 3.05 (t,
J = 5.7 Hz, 2H), 4.72 (t, J = 5.7 Hz, 2H), 7.40–7.42 (m, 3H), 7.56 (t,
J = 4.2 Hz, 1H), 7.65 (dd, J = 1.8, 6.3 Hz, 2H), 8.95 (dd, J = 1.8,
2.4 Hz, 1H); MS (ESI) m/z 345 ([M+H]+).
4.2.3. Synthesis of 2-chloro-3-(phenylethynyl)pyrido[3,2-b]pyra
zine (4)
To a stirred solution of 2,3-dichloropyrido[3,2-b]pyrazine (3)
(4.76 g, 18.5 mmol) in dimethylsulfoxide (DMSO, 2 ml) solution
was added phenylacetylene (2.3 ml, 21.3 mmol), triethylamine
(18.0 ml, 129.6 mmol), palladium(II) acetate (290 mg, 1.3 mmol),
copper(I) iodide (437 mg, 1.7 mmol) and triphenylphosphine
(388 mg, 2.0 mmol) at 80 °C for 2 h. The resulting mixture was con-
4.2.6. Synthesis of 2-(1-methylpiperidin-3-yloxy)-3-(phenylethy
nyl)pyrido[3,2-b]pyrazine 922
1H NMR (500 MHz, CDCl3) d 1.62 (d, J = 2.05 Hz, 1H), 1.72 (dd,
J = 3.5, 7.0 Hz, 1H), 1.90 (m, 1H), 2.17 (t, J = 10.3 Hz, 2H), 2.31 (s,
3H), 2.35 (s, 1H), 2.64 (d, J = 11.21 Hz, 1H), 3.09 (d, J = 9.0 Hz,
1H), 5.36 (m, 1H) 7.39 (t. J = 4.9 Hz, 3H) 7.56 (dd, J = 4.2, 4.0 Hz,

5646
Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
1H), 7.63 (dd, J = 1.6, 6.0 Hz, 2H), 8.10 (dd, J = 1.8, 6.5 Hz, 1H), 8.88
(dd, J = 1.8, 2,4 Hz, 1H); MS (ESI) m/z 345 ([M+H]+).
4.2.15. Synthesis of 3-((3-fluorophenyl)ethynyl)-2-(2-(pyrrolid
in-1-yl)ethoxy) pyrido[3,2-b] pyrazine 967
1H NMR (500 MHz, CDCl3) d 3.27 (s, 2H), 4.97 (s, 2H), 7.17 (s,
1H), 7.26 (s, 1H), 7.38 (t, J = 14.0, 5.7 Hz, 1H), 7.46 (d, J = 7.85 Hz,
1H), 7.59 (dd, J = 4.3, 3.8 Hz, 1H), 8.37 (dd, J = 6.6, 1.4 Hz, 1H),
8.99 (dd, J = 1.5, 2.5 Hz, 1H); MS (ESI) m/z 362([M+H]+).
4.2.7. Synthesis of N,N-dimethyl-3-((3-(phenylethynyl)pyrido[3,
2-b]pyrazin-2-yloxy)methyl) aniline 923
1H NMR (500 MHz, CDCl3) d 2.96 (d, J = 5.74 Hz, 6H), 5.63 (s,
2H), 6.75 (s, 1H), 6.99 (d, J = 2.2 Hz, 2H), 7.40 (d, J = 1.52 Hz, 1H),
7.42 (d, J = 8.1 Hz, 3H), 7.59 (dd, J = 4.3, 4.0 Hz, 1H), 7.67 (dd,
J = 1.5, 6.7 Hz, 2H), 8.19 (dd, J = 1.8, 6.4 Hz, 1H), 8.98 (dd, J = 1.8,
2.4 Hz, 1H); MS (ESI) m/z 381 ([M+H]+).
4.2.16. Synthesis of N-(2-(3-((3-fluorophenyl)ethynyl)pyrido[3,
2-b]pyrazin-2-yloxy)thyl)-N-isopropylpropan-2-amine 968
1H NMR (500 MHz, CDCl3) d 1.08 (d, J = 6.5 Hz, 12H), 2.95 (t,
J = 6.9 Hz, 2H), 3.08–3.14 (m, 2H), 4.51 (t, J = 6.9 Hz, 2H), 7.15 (t,
J = 8.3 Hz, 1H), 7.36–7.41 (m, 2H), 7.47 (d, J = 7.7 Hz, 1H), 7.59
(dd, J = 4.1, 8.3 Hz, 1H), 8.14 (dd, J = 1.7, 8.3 Hz, 1H), 8.96 (dd,
J = 1.7, 4.1 Hz, 1H); MS (ESI) m/z 393 ([M+H]+).
4.2.8. Synthesis of 2-chloro-3-((3-methoxyphenyl)ethynyl)pyr
ido[3,2-b]pyrazine 927
1H NMR (500 MHz, CDCl3) d 3.91 (t, J = 11.0, 8.4 Hz, 3H), 7.06
(m, 1H), 7.28 (d, J = 16.2 Hz, 1H), 7.37 (d, J = 7.5 Hz, 2H), 7.75 (dd,
J = 4.2, 4.2 Hz, 1H), 8.39 (dd, J = 6.8, 1.5 Hz, 1H), 9.22 (dd, J = 1.8,
2,4 Hz, 1H); MS (ESI) m/z 296 ([M+H]+).
4.3. Biology
4.3.1. General for screening
4.3.1.1. Cell culture. The human lung carcinoma cell line A549
was obtained from American Tissue Type Culture Collection
(Manassas, VA) and cultured in RPMI-1640 media supplemented
with 10% fetal bovine serum (FBS, Gibco-BRL, USA), 100 U/ml pen-
icillin G, and 100 U/ml streptomycin (Gibco-BRL, USA). Cell cul-
tures were incubated at 37 °C in 5% CO2/95% air, and the medium
was replaced every third day.
4.2.9. Synthesis of 3-(3-((3-methoxyphenyl)ethynyl)pyrido[3,2-
b]pyrazin-2-yloxy)-N,N-dimethylpropan-1-amine 928
1H NMR (500 MHz, CDCl3) d 2.15 (m, 2H), 2.32 (d, J = 6.9 Hz, 6H),
2.62 (t, J = 7.3, 7.2 Hz, 2H), 3.84 (t, J = 7.5, 2.0 Hz, 3H), 4.63 (q,
J = 6.7, 6.4, 6.4 Hz, 2H), 7.00 (s, 1H), 7.21 (dd, J = 1.2, 2.5 Hz, 1H),
7.29 (t, J = 5.4, 2.0 Hz, 1H), 7.32 (d, J = 7.9 Hz, 1H), 7.59 (dd,
J = 4.3, 3.9 Hz, 1H), 8.15 (dd, J = 1.8, 6.5 Hz, 1H), 8.96 (dd, J = 1.8,
2.4 Hz, 1H); MS (ESI) m/z 363 ([M+H]+).
4.3.1.2. Preparation of Wnt2 over-expressed cell line. A549/
Wnt2 cells are over-expressed human Wnt2 by stable transfection
of pUSEampWnt2, as described in Lee et al.13 Briefly, A549 cells
with 70% confluence in 60 mm culture were transfected with the
pUSEampWnt2 or the empty vector using Transfast transfection
reagent (Promega, USA). After 24 h of transfection, the medium
was replaced with G418-containing medium which were selected
for the stable transfectants.
4.2.10. Synthesis of 3-(3-((3,5-dimethoxyphenyl)ethynyl)pyrido
[3,2-b]pyrazin-2-yloxy)-N,N-dimethylpropan-1-amine 929
1H NMR (500 MHz, CDCl3) d 2.11 (m, 2H), 2.30 (s, 6H), 2.58(t,
J = 7.3, 7.2 Hz, 2H), 3.81 (d, J = 11.1 Hz, 6H), 4.62 (t, J = 6.45,
6.45 Hz, 2H), 6.55 (t, J = 2.3, 2.2 Hz, 1H), 6.82 (d, J = 2.3 Hz, 2H),
7.57 (dd, J = 4.25, 4.0 Hz, 1H), 8.13 (dd, J = 1.7, 6.5 Hz, 1H), 8.95
(dd, J = 1.7, 2.5 Hz, 1H); MS (ESI) m/z 392([M+H]+).
4.3.1.3. Cytotoxicity andcell proliferation assay. Cells (8 Â 103 for
cytotoxicity or 3 Â 103 cells/well for cell proliferation) were seeded
onto 96-well microplates, allowed to attach for about 24 h and then
treated with various concentrations of test compounds. After 24, 48
and 72 h, cell proliferation was determined by using a CellTiter 96
non-radioactive cell proliferation assay kit [MTS (3-(4,5-dimethylthi-
azol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tet-
razolium) assay] according to the manufacturer’s protocol (Promega,
USA). For cytotoxicity test, cell viability was measured at 24 h after
chemical treatment by MTS assay kit (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, Ger-
many) at 490 nm.
4.2.11. Synthesis of N,N-dimethyl-2-(3-(p-tolylethynyl)pyrido[3,
2-b]pyrazin-2-yloxy) propan-1-amine 930
1H NMR (500 MHz, CDCl3) d 1.49 (d, J = 6.3 Hz, 3H), 2.39 (s, 6H),
2.41 (s, 3H), 2.61 (dd, J = 4.5, 13.3 Hz, 1H), 2.79–2.83 (m, 1H), 7.22,
(d, J = 7.9 Hz, 2H), 7.54–7.57 (m, 3H), 8.12 (dd, J = 1.8, 8.3 Hz, 1H),
8.93 (dd, J = 1.8, 4.2 Hz, 1H); MS (ESI) m/z 347 ([M+H]+).
4.2.12. Synthesis of N,N-diethyl-2-(3-((4-methoxyphenyl)ethyn
yl)pyrido[2,3-b]pyrazin-2-loxy) ethan amine 953
1H NMR (500 MHz, CDCl3) d 1.14 (t, J = 7.1 Hz, 6H), 2.78 (q,
J = 7.2 Hz, 4H), 3.10 (t, J = 5.9 Hz, 2H), 3.86 (s, 3H), 4.68 (t,
J = 5.9 Hz, 2H), 6.93 (dd, J = 1.9, 7.0 Hz, 2H), 7.57 (q, J = 4.3 Hz,
1H), 7.62 (dd, J = 1.9, 6.9 Hz, 2H), 8.14 (dd, J = 1.8, 8.3 Hz, 1H),
8.95 (dd, J = 1.8, 4.3 Hz, 1H); MS (ESI) m/z 377 ([M+H]+).
4.3.1.4. Preparation of cytoplasmic, nuclear and whole cell extr
act. After the treatment of test chemicals for 24 h, cells were
fractionated to cytoplasm and nuclear parts by the method of
Wong.20 Briefly, cells were washed two times with ice-cold phos-
phate-buffered saline (PBS) and resuspended in 400 ml of buffer A
[10 mM HEPES at pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT,
0.5 mM PMSF] supplemented with a complete protease inhibitor
cocktail (Roche, USA). The cells were allowed to swell on ice for
15 min, lysed gently with 12.5 ml of 10% Nonide P-40, and centri-
fuged at 2000g for 10 min. The supernatant was collected and used
as the cytoplasmic extracts. The nuclei pellet was resuspended in
40 ml of buffer C [20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 450 mM
NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF] sup-
plemented with a complete protease inhibitor cocktail (Roche,
USA), incubated for 30 min in ice, and the nuclear debris was spun
down at 20,000g for 15 min. The supernatant (nuclear extract) was
collected, frozen in liquid nitrogen and stored at À80 °C until ready
4.2.13. Synthesis of 2-(3-((4-methoxy-2-methylphenyl)ethynyl)
pyrido[3,2-b] pyrazin-2-yloxy)-N,N-dimethylethan amine 965
1H NMR (500 MHz, CDCl3) d 2.41 (s, 6H), 2.62 (s, 3H), 2.89 (t,
J = 5.8 Hz, 2H), 3.84 (s, 3H), 4.68 (t, J = 5.8 Hz, 2H), 6.76 (dd,
J = 2.5, 8.5 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 7.54–7.56 (m, 1H),
7.58 (d, J = 8.6 Hz, 1H), 8.14 (dd, J = 1.6, 8.2 Hz, 1H), 8.93 (dd,
J = 1.6, 4.2 Hz, 1H); MS (ESI) m/z 363 ([M+H]+).
4.2.14. Synthesis of 2-(3-((3-fluorophenyl)ethynyl)pyrido[3,2-b]
pyrazin-2-yloxy)-N,N-dimethyl ethan amine 966
1H NMR (500 MHz, CDCl3) d 1.14 (t, J = 7.0 Hz, 6H), 2.76 (q,
J = 7.0 Hz, 4H), 3.07 (t, J = 5.8 Hz, 2H), 4.69 (t, J = 5.8 Hz, 2H), 7.14
(t, J = 8.2 Hz, 1H), 7.36–7.38 (m, 2H), 7.44–7.46 (m, 1H), 7.60 (dd,
J = 4.2, 8.2 Hz, 1H), 8.16 (dd, J = 1.6, 8.2 Hz, 1H), 8.97 (dd, J = 1.6,
4.2 Hz, 1H); MS (ESI) m/z 365 ([M+H]+).

Y.-D. Gong et al. / Bioorg. Med. Chem. 19 (2011) 5639–5647
5647
4. Morin, P. J.; Sparks, A. B.; Korinek, V.; Barker, N.; Clevers, H.; Vogelstein, B.;
Kinzler, K. W. Science 1997, 275, 1787.
5. Van Scoyk, M.; Randall, J.; Sergew, A.; Williams, L. M.; Tennis, M.; Winn, R. A.
Transl. Res. 2008, 151, 175.
for analysis. The protein content was determined using the Brad-
ford Assay (Bio-Rad, Germany).
4.3.1.5. Western blot analysis. Protein samples (20–30 lg of pro-
6. Katoh, M. Int. J. Oncol. 2001, 19, 1003.
7. Park, J. K.; Song, J. H.; He, T. C.; Nam, S. W.; Lee, J. Y.; Park, W. S. Neoplasma
2009, 56, 119.
8. Cheng, X. X.; Wang, Z. C.; Chen, X. Y.; Sun, Y.; Kong, Q. Y.; Liu, J.; Li, H. Cancer
Lett. 2005, 223, 339.
9. 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.
10. You, L.; He, B.; Xu, Z.; Uematsu, K.; Mazieres, J.; Mikami, I.; Reguart, N.; Moody,
T. W.; Kitajewski, J.; McCormick, F.; Jablons, D. M. Oncogene 2004, 23, 6170.
11. (a) Barker, N.; Clevers, H. Nat. Rev. Drug Disc. 2006, 5, 997; (b) McMillan, M.;
Kahn, M. Drug Discovery Today 2005, 10, 1467.
tein) were subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS–PAGE) and transferred to nitrile cellulose
membranes (Whatman, Germany) for Western blotting. The pri-
mary antibodies used were b-catenin, GAPDH and PARP (Santa
Cruz Biotechnology, Santa Cruz, USA). Immunoreactive proteins
were visualized with enhanced chemiluminescence (ECL; Amer-
sham Biosciences, USA) according to the manufacturer’s protocol.
12. (a) Saraswati, S.; Alfaro, M. P.; Thorn, C. A.; Atkinson, J.; Lee, E.; Young, P. P. PLoS
One 2010, 5, e15521; (b) Lu, J.; Ma, Z.; Hsieh, J.-C.; Fan, C.-W.; Chen, B.;
Longgood, J. C.; Williams, N. S.; Amatruda, J. F.; Lum, L.; Chen, C. Bioorg. Med.
Chem. Lett. 2009, 19, 3825; (c) William, J. M.; Jeffrey, C. K.; Ramesh, B.; Peter, V.
N. B.; Fukuyama, S.; Krishnamurthy, G.; Ronald, L. M.; Keith, P.; Barb, S.;
Eugene, J. T. Bioorg. Med. Chem. 2010, 18, 190; (d) Kouken, K.; Midori, A. A.; Li,
X.; Masami, I. Bioorg. Med. Chem. Lett. 2007, 15, 4254; (e) Wang, H.; Hao, J.;
Hong, C. Chem. Biol. 2011, 6, 192.
4.3.1.6. Transfection and luciferase reporter gene assay. Cells
were transfected with the luciferase reporter constructs pSuper-
Topflash containing eight TCF consensus binding sites upstream
of firefly luciferase cDNA, or pSuperFopflash, a plasmid with mu-
tated TCF binding sites (kindly provided by professor Ja-Hyun Baek
at the School of Life Sciences and Biotechnology at Korea Univer-
sity, Seoul, Korea). Cells were transfected using Lipofectamine
2000 (Invitrogen Co., USA) with reporter construct and pRL-TK
plasmid and incubated with various concentrations of the selected
13. Lee, S.-B.; Park, Y. I.; Dong, M. S.; Gong, Y.-D. Bioorg. Med. Chem. Lett. 2010, 20,
5900.
14. We have synthesized various drug-like heterocyclic libraries15 more than
20,000 small molecules by combinatorial synthetic technology. In the first
stage, we selected 1434 representative compounds by virtual screening based
on drug-like property considering with known Wnt pathway inhibiters.
15. (a) Gong, Y.-D.; Lee, T. J. Comb. Chem. 2010, 12, 393; (b) Hwang, J. Y.; Choi, H.-S.;
Seo, J.-S.; La, H.-J.; Yoo, S.-E.; Gong, Y. D. J. Comb. Chem. 2006, 8, 897; (c) Hwang,
J. Y.; Gong, Y. D. J. Comb. Chem. 2006, 8, 297; (d) Hwang, J. Y.; Choi, H. S.; Lee, D.
H.; Yoo, S. E.; Gong, Y. D. J. Comb. Chem. 2005, 7, 136; (e) Hwang, J. Y.; Choi, H.
S.; Lee, D. H.; Gong, Y. D. J. Comb. Chem. 2005, 7, 816; (f) Lee, I. Y.; Kim, S. Y.; Lee,
J. Y.; Yu, C.-M.; Lee, D. H.; Gong, Y. D. Tetrahedron Lett. 2004, 45, 9319.
16. (a) Gong, Y.-D.; Jeon, M. K.; Hwang, S. H.; Dong, M. S.; Lee, S. B.; Kang, K. H.; Oh,
C. H. Novel 2-Sustituted Aminoalkylenyloxy-3-Substituted Phenyl Ethynyl
Quinoxaline Derivatives, Korea Pat. No. 0889389.; (b) Gong, Y.-D.; Dong, M. S.;
Hwang, S. H.; Lee, T. I.; Lee, S. B. 2-Sustituted Aminoalkylenyloxy-3-Substituted
Phenylethynyl-6-Amino Quinoxaline, Korea Pat. Appl. No. 2008-0076221.
17. Cell proliferation assay: Cell proliferation was determined by using a CellTiter
96 non-radioactive cell proliferation assay kit according to the manufacturer’s
protocol (Promega, USA). The detail procedure was mentioned in biological
experimental part.
compounds at 37 °C. After 24 h, cells were lysed in 50 ll passive ly-
sis buffer (Promega, USA). Firefly luciferase and renilla luciferase
activity were determined by 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 lucifer-
ase activity measurements for each triplicate set.
Acknowledgments
This research was supported by grants from the National R&D
Program for Cancer Control (grant number 1020050), and from
the Seoul Research and Business Development Program (grant
number 10574), and the National Research Foundation of Korea
(2010-0004128) funded by the Ministry of Education, Science
and Technology, and by the Ministry of Knowledge Economy,
Korea.
18. Lee, S. J.; Gong, Y.-D.; Jeon, M. K.; Moon, S. H.; Jun, K. H.; Kim, J. M.; Lee, S. H.;
Lee, H. H.; Kang, M. J. 2-Arylalkylenyloxy-3-Ethynylpyrido[2,3-b] Pyrazine
Derivatives, Korea Pat. App. No. 2006-0063214.
19. We checked cytotoxicity of the 13 compounds in human lung fibroblast cell
line (LL47) and were over 0.5–1.0 lM higher than those in A549 cells.
20. In the early stage of our research, we used Calphostin C as positive control
since we could not obtain other known comparison inhibitors from
commercial market. And then we have synthesized and used the Pfizer lead
compound (Data Monitor Research Store: preclinical stage, pyrinium pamoate)
as positive control.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.028.
21. Wong, B. C. Y.; Jiang, X. H.; Fan, X. M.; Lin, M. C. M.; Jiang, S. H.; Lam, S. K.; Kung,
H. F. Oncogene 2003, 22, 1189.
22. Chalet, T.; Rita, G. N.; Anthony, J. R.; Beata, P.; Fatima, K.; Zhaobin, Z.;
Huanchun, Z.; Quincy, T.; Ainsley, C. N.; Paraskevi, G.; Wei, Z.; Jeffrey, J. O.;
Manuela, M. P.; Nicolaou, K. C.; Erwin, G. V. M. Cancer Res. 2005, 65, 605.
23. Concord; Tripos Inc., 1699 South Hanley Road, St. Louis, MO 63144.
24. Catalyst User Guide, Accelrys Software Inc., San Diego, 2005.
25. Gong, Y.-D.; Kang, N. S. Bull. Korean Chem. Soc. 2010, 31, 2039.
References and notes
1. Moon, R. T.; Brown, J. D.; Torres, M. Trends Genet. 1997, 13, 157.
2. Nusse, R. Trends Genet. 1999, 15, 1.
3. Chesire, D. R.; Ewing, C. M.; Sauvageot, J.; Bova, G. S.; Isaacs, W. B. Prostate 2000,
45, 323.