Investigation of 3-aryl-pyrimido[5,4-e][1,2,4]triazine-5,7-diones as small molecule antagonists of β-catenin/TCF transcription

Article abstract of DOI:10.1016/j.bmcl.2013.08.111

Nearly all colorectal cancers (CRCs) and varied subsets of other cancers have somatic mutations leading to β-catenin stabilization and increased β-catenin/TCF transcriptional activity. Inhibition of stabilized β-catenin in CRC cell lines arrests their growth and highlights the potential of this mechanism for novel cancer therapeutics. We have pursued efforts to develop small molecules that inhibit β-catenin/TCF transcriptional activity. We used xanthothricin, a known β-catenin/TCF antagonist of microbial origin, as a lead compound to synthesize related analogues with drug-like features such as low molecular weight and good metabolic stability. We studied a panel of six candidate Wnt/β-catenin/Tcf- regulated genes and found that two of them (Axin2, Lgr5) were reproducibly activated (9-10 fold) in rat intestinal epithelial cells (IEC-6) following β-catenin stabilization by Wnt-3a ligand treatment. Two previously reported β-catenin/TCF antagonists (calphostin C, xanthothricin) and XAV939 (tankyrase antagonist) inhibited Wnt-activated genes in a dose-dependent fashion. We found that four of our compounds also potently inhibited Wnt-mediated activation in the panel of target genes. We investigated the mechanism of action for one of these (8c) and demonstrated these novel small molecules inhibit β-catenin transcriptional activity by degrading β-catenin via a proteasome-dependent, but GSK3β-, APC-, AXIN2- and βTrCP-independent, pathway. The data indicate the compounds act at the level of β-catenin to inhibit Wnt/β-catenin/TCF function and highlight a robust strategy for assessing the activity of β-catenin/TCF antagonists.

Full text of DOI:10.1016/j.bmcl.2013.08.111

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Investigation of 3-aryl-pyrimido[5,4-e][1,2,4]triazine-5,7-diones
as small molecule antagonists of b-catenin/TCF transcription
Jörg Zeller a, Anjanette J. Turbiak b, Ian A. Powelson a,b, Surin Lee a, Duxin Sun c,g
,
H. D. Hollis Showalter b,g,h, , Eric R. Fearon a,d,e,f,i
⇑
a Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
b Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
c Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, USA
d Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA
e Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA
f Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
g College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
h Vahlteich Medicinal Chemistry Core, University of Michigan, Ann Arbor, MI 48109, USA
i The Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Nearly all colorectal cancers (CRCs) and varied subsets of other cancers have somatic mutations leading
to b-catenin stabilization and increased b-catenin/TCF transcriptional activity. Inhibition of stabilized
b-catenin in CRC cell lines arrests their growth and highlights the potential of this mechanism for novel
cancer therapeutics. We have pursued efforts to develop small molecules that inhibit b-catenin/TCF tran-
scriptional activity. We used xanthothricin, a known b-catenin/TCF antagonist of microbial origin, as a
lead compound to synthesize related analogues with drug-like features such as low molecular weight
and good metabolic stability. We studied a panel of six candidate Wnt/b-catenin/Tcf-regulated genes
and found that two of them (Axin2, Lgr5) were reproducibly activated (9–10 fold) in rat intestinal
epithelial cells (IEC-6) following b-catenin stabilization by Wnt-3a ligand treatment. Two previously
reported b-catenin/TCF antagonists (calphostin C, xanthothricin) and XAV939 (tankyrase antagonist)
inhibited Wnt-activated genes in a dose-dependent fashion. We found that four of our compounds also
potently inhibited Wnt-mediated activation in the panel of target genes. We investigated the mechanism
of action for one of these (8c) and demonstrated these novel small molecules inhibit b-catenin transcrip-
tional activity by degrading b-catenin via a proteasome-dependent, but GSK3b-, APC-, AXIN2- and
bTrCP-independent, pathway. The data indicate the compounds act at the level of b-catenin to inhibit
Wnt/b-catenin/TCF function and highlight a robust strategy for assessing the activity of b-catenin/TCF
antagonists.
Received 22 June 2013
Revised 26 August 2013
Accepted 29 August 2013
Available online xxxx
Keywords:
Wnt signaling
b-Catenin
T-cell factor (TCF)
Small molecule antagonists
Ó 2013 Elsevier Ltd. All rights reserved.
The Wnt/b-catenin signaling pathway is a key regulator of cell
proliferation, differentiation, migration and apoptosis.1 Activation
of the canonical Wnt-signaling pathway by Wnt-ligands leads to
an increase in the ‘free’ pool of b-catenin and b-catenin/T-cell fac-
tor (TCF) transcriptional activity.2 In the absence of Wnt-ligands,
b-catenin forms complexes with other proteins in the cytoplasm
including the b-catenin destruction complex consisting of APC
(adenomatous polyposis coli), GSK3b (glycogen synthase kinase
an APC-dependent fashion by SIAH (seven in absentia homolog)
leading to proteasome dependent degradation of b-catenin.4,5
Nearly all colorectal cancers (CRCs) and varying subsets of other
cancer types have somatic gain-of-function mutations in b-catenin
or loss-of-function mutations in key antagonists of b-catenin,
namely the APC and AXIN tumor suppressor proteins.6 These de-
fects lead to constitutive b-catenin stabilization resulting in altered
transcription of downstream b-catenin/T cell factor (TCF)-regu-
lated target genes including Cyclin D1, Lgr5 and Axin2.7
Inhibition of b-catenin in colon cancer cell lines arrests their
growth and highlights the potential of this mechanism for novel
cancer therapeutics.8,9 The search for inhibitors of Wnt/b-catenin
signaling has led to the identification of antagonists acting at dif-
ferent levels of the pathway.10,11 These have derived from both
high throughput screening and synthetic campaigns. Calphostin C
3b), CK1
by GSK3b and CK1
a
(casein kinase 1
, b-catenin is mainly ubiquinated by bTrCP
a
) and AXIN2.3 Upon phosphorylation
a
(b transducin repeat containing protein) and to a minor degree in
⇑
Corresponding author.
E-mail address: showalh@umich.edu (H.D.H. Showalter).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.111
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

2
J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
xanthothricin inhibited b-catenin-dependent Wnt signaling in cells
OH
OH
O
O
and in xenopus axis duplication assay.12 A limitation with xantho-
thricin is that the apparent therapeutic window is small between
b-catenin/TCF inhibition and general cytotoxic effects.12 Based on
its structural simplicity and low molecular weight, we were inter-
ested in utilizing xanthothricin (3) as a lead chemotype to probe
SAR expansion. The heterocyclic core of 3 is made up of the pyrim-
ido[5,4-e]-1,2,4-triazine-5,7(1H,6H)-dione ring system. In earlier
work, we reported on a novel and efficient route to 3-aryl deriva-
tives of 3 that allows for incorporation of aryl and more elaborate
alkyl substituents at the N1 position, thus broadening the scope of
accessible analogues.20 We also reported on the generation of com-
pounds within the isomeric fervenulin series (4, Fig. 1).21
OMe
Me
OH
MeO
MeO
O
O
O
O
S
N
N
Me
OMe
O
OH
CF3
calphostin C
XAV939
1
( )
2
( )
O
O
6
6
N
1
N
N
3
3
NMe
NMe
8
N
N
N
O
N
N
O
Me
Me
Here, we report on the utilization of this chemistry toward
investigating a small series of 3-aryl analogues of 3 and 4 and from
these we have identified four novel Wnt/b-catenin pathway inhib-
itors within the xanthothricin series. We show that these ana-
logues inhibit Wnt/b-catenin/TCF-regulated genes (Axin2, Lgr5) in
rat intestinal epithelial cells (IEC-6) following b-catenin stabiliza-
tion by Wnt-3a ligand treatment. One of these compounds (com-
pound 8c, Table 1) preferentially affects the growth of colon
cancer cell lines (DLD-1 and SW480) with dysregulated b-catenin
signaling. We demonstrate that compound 8c inhibits b-catenin
transcriptional activity by reducing stabilized b-catenin via a
GSK3b-, APC-, AXIN2- and bTrCP-independent but proteasome-
dependent pathway. This strongly suggests compound 8c acts at
the level of b-catenin to inhibit Wnt/b-catenin/TCF function.
The synthesis of N1-substituted pyrimidotriazinedione deriva-
tives within the xanthothricin series was straightforward and fol-
lowed previously established procedures.22–24 As shown in
Scheme 1, condensation of readily available 3-methyl-6-(1-meth-
ylhydrazinyl)pyrimidine-2,4(1H,3H)-dione (5)17 with requisite
4-substituted benzaldehydes led to hydrazone adducts 6a–c in good
yields. Each hydrazone was then cyclized by treatment of NaNO2
in acetic acid to a ꢀ1:1 mixture of 3-substituted-5,7-dioxo-
1,5,6,7-tetrahydropyrimido[5,4-e][1,2,4]triazine 4-oxide (7) and
pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (8). Each mixture
of 7a–c was easily converted to desired product 8a–c, respectively,
by mild treatment with dithiothreitol (DTT). Additional target
derivatives 10a,b and 11c were derived from 6-chloro-3-methyl-
5-nitrouracil (9) by our recently published methodology to make
compounds with more elaborate N1 substituents.20 The synthesis
xanthothricin (toxoflavin)
fervenulin
3
( )
4
( )
Figure 1. Small molecule inhibitors of b-catenin/TCF transcriptional activity and
pyrimido[5,4-e][1,2,4]triazine-5,7-dione chemotypes
(1; PKF115–584, Fig. 1) and xanthothricin (3; toxoflavin, PKF118–
310, Fig. 1) have been reported to disrupt b-catenin/TCF4 binding
leading to an inhibition of b-catenin transcriptional activity.12 Cal-
phostin C can also inhibit b-catenin signaling through PKC (protein
kinase C) in a GSK3b-dependent fashion.13,14 In contrast XAV939
(2, Fig. 1) antagonizes b-catenin levels in a destruction complex
dependent fashion by stabilizing AXIN protein levels.15,16
Antagonists, such as xanthothricin, that act at the level of or
downstream of stabilized b-catenin, and thus circumvent the activ-
ity of the frequently mutated destruction complex in cancer cells,
hold the promise of broader efficacy. Xanthotricin, which was orig-
inally isolated from Pseudomonas cocovenenans, was first synthe-
sized in 1961.17 Since then, a number of analogues have been
prepared in order to explore the potent antibiotic and pharmaco-
logical properties represented by this natural product.18 Many of
these analogues have the same methyl substituent found at the
N1 position of xanthothricin, in large part due to the ease with
which a methyl group can be incorporated via the use of meth-
ylhydrazine early in the synthesis.19
Lepourcelet et al. reported that xanthothricin potently antago-
nizes b-catenin’s binding to TCF4 and b-catenin/TCF-dependent
transcription (IC50 of 0.3 lM in cells). They further showed that
Table 1
Summary of IC50 values determined by Lgr5 and Axin2 gene expression
R1
R1
O
N
O
N
N
N
NMe
O
NMe
N
N
N
N
R
O
R
A
B
No.
Core
R
R1
Lgr5 IC50
(
lM) (Mean SEM)
Axin2 IC50 (lM) (Mean SEM)
1
2
3
8a
8b
Calphostin C
XAV939
Xanthothricin
0.39 0.19
2.9
0.55 0.18
0.64
0.33 0.03
2.67 0.62
NI up to 0.7
1.77 0.28
NI up to 1.2
2.67 0.33
0.30 0.11
NI up to 69
PI up to 179
0.29 0.02
1.53 0.37
NI up to 0.7
1.53 0.42
NI up to 1.2
2.1 0.32
0.26 0.11
NI up to 69
164.7
A
A
A
A
A
A
B
B
Me
Me
Me
CH2CH2OH
Ph
Ph
Me
Ph
OMe
OCH2CH2NEt2
Cl
OMe
OMe
OCH2CH2NEt2
OCH2CH2NEt2
OCH2CH2NEt2
8c
10a
10b
11c
20a
20b
NI, no inhibition
PI, partial inhibition
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
X
X
target genes in rat intestinal epithelial cells (IEC-6 cell line), we
treated IEC-6 cells for 12 h with 50 ng/mL recombinant mouse
Wnt-3a. We then determined the expression levels of the selected
Wnt/b-catenin/Tcf-regulated target genes relative to U6 gene
expression levels by quantitative PCR using gene-specific primers.
The specific candidate Wnt/b-catenin/Tcf-regulated genes studied
were Axin2, Lgr5, Bmp4, Nkd1, Edn1, and Irs1 (Table S1, Supplemen-
tal materials). These genes have previously been highlighted as
candidate targets of the canonical (b-catenin) Wnt signaling path-
way,29–34 and we found evidence that the expression of all six
genes was induced in IEC-6 cells by recombinant Wnt3a.
O
O
O
N
O
n
NMe
(ii)
(i)
NMe
O
NMe
O
H2N
N
N
N
N
H
O
N
N
H
N
N
Me
6a-c
Me
Me
5
7a-c: n = 1
(N-oxide)
8a-c: n = 0
(amine)
a: X = OMe
(iii)
b: X = OCH2CH2NEt2
c: X = Cl
MeO
R2O
(v)
O
O
N
O
O2N
Cl
N
NMe
O
NMe
O
NMe
(iv)
N
N
N
H
N
Ph
N
N
N
O
We chose the two most potently Wnt-3a-induced target genes,
R1
namely Axin2 (10.9-fold 0.9 induction) and Lgr5 (9.5-fold
1
9
10a: R1 = CH2CH2OH
10b: R1 = Ph
11a: R2 = H
induction), to determine how previously described Wnt/b-cate-
nin/TCF pathway antagonists as well as our newly designed small
molecules affected target gene expression. We treated IEC-6 cells
with small molecules at various concentrations for 6 h followed
by the addition and incubation of the cells with 50 ng/mL recombi-
nant mouse Wnt-3a for 12 h. The base-line relative levels of Lgr5
(Fig. 2B) and Axin2 (Fig. 2C) expression, relative to U6 expression
after Wnt-3a stimulation was set to 100% and the relative target
gene expression for each compound at various concentrations
was determined. As expected, we found that previously identified
antagonists of b-catenin signaling (calphostin C, XAV939 and xan-
thothricin) decreased the expression of Lgr5 and Axin2 (Fig. 2B and
C and Table 1). We also found that four of our synthesized pyrim-
idotriazinedione analogues (8a, 8c, 10b and 11c) inhibited the
expression of Lgr5 and Axin2 with varying potencies (Table 1).
Within our small series of synthesized compounds, it was clear
that inhibition of canonical Wnt-dependent activation of down-
stream transcriptional targets was associated almost exclusively
with the N1-substutited core (xanthothricin series). Addition of
solubilizing moieties at R (e.g., 10a) and R1 (e.g., 8b) abolished
activity. For the two isomeric compounds (20a and 20b) having
N8 substituents (fervenulin series), activity was poor or absent.
To determine if compounds of the xanthothricin series have good
biopharmaceutical properties such as long biological half-life, we
carried out a metabolic stability study. We incubated compound
11c, the most potent inhibitor of Lgr5 and Axin2 gene expression
11b: R2= CH2CH2Br
(vi)
(vii) 11c: R2 = CH2CH2NMe2
Scheme 1. Synthesis of analogues in the xanthothricin series. (i) 4-Substituted
benzaldehyde, EtOH, reflux (31–87% yield); (ii) NaNO2, aq AcOH, 0–25 °C (ꢀ80%
yield); (iii) DTT, EtOH (47–87% yield); (iv) see Ref.20; (v) for 10b, BBr3, DCM (63%
yield); (vi) BrCH2CH2Br, Cs2CO3, DMF (47% yield); (vii) NHMe2, ACN, 80 °C (33%
yield). See Supplemental materials for experimental details.
of N8-substituted pyrimidotriazinedione derivatives (fervenulin
series) was achieved by two reaction pathways and is shown in
Scheme 2. Heating compound 8a or the 7a/8a mixture in DMF
led to a well precedented N1 demethylation reaction to give
12.25,26 Selective N8 methylation then was carried out with Cs2CO3
as base to give 13. Ether demethylation with BBr3 provided phenol
14a to which was appended an aqueous solubilizing side chain to
afford 20a. A series of reactions starting from 1-phenylpyrimidine-
2,4,6-trione (15)24 led to known intermediate 18,27 which was then
taken to N8 phenyl congener 20b by reactions similar to those de-
scribed above. Full experimental procedures for compounds out-
lined in Schemes 1 and 2 are given in Supplemental materials.
We also developed a simple process to make xanthothricin (3;
Scheme S3, Supplemental materials). While not as efficient overall
as the best reported synthesis,28 its brevity makes it preferable
when smaller quantities of compound are required.
All compounds reported in this Letter were rigorously purified,
and their structural assignments are supported by 1H NMR spec-
troscopy and mass spectrometry.
in IEC-6 cells, at 1
lM at 37 °C with mouse liver microsomes. Under
these conditions compound 11c has
a
half-life of 34.7 min
Identification of small molecule inhibitors of canonical Wnt—sig-
naling. To establish the induction of Wnt/b-catenin/Tcf-regulated
(Table S2, Supplemental materials) consistent with good metabolic
stability.
MeO
(i)
MeO
(ii)
HO
O
N
O
N
O
7a/8a
or
N
N
N
N
N
(iii)
NMe
O
NMe
O
NMe
O
8a
N
N
N
N
H
N
R
12
Me
14a:
R = Me
13
14b: R = Ph
O
(vii)
O
O
N
O
N
O
NMe
(ii)
NPh
O
NH
NMe
(vi)
(iv)
Cl
(v)
NMe
N
N
N
O
H2N
N
O
O
N
H
15
O
Me Ph
Cl
N
O
Ph
Me Ph
Ph
19
16
17
18
+ N-3 Ph isomer
OH
O
Et2N
O
N
N
NMe
O
(viii)
14a,b
20a:
R = Me
N
N
R
20b: R = Ph
Scheme 2. Synthesis of analogues in the fervenulin series. (i) DMF, 90 °C (65–73% yield); (ii) for 12 and 16, dimethyl sulfate, Cs2CO3, acetone, rt À50 °C (84–97% yield); (iii) for
14a, BBr3, DCM (65% yield); (iv) POCl3 (48% yield); (v) methylhydrazine, EtOH, reflux (88% yield); (vi) 4-OH–benzaldehyde, EtOH, reflux (64% yield); (vii) NaNO2, aq AcOH, 0–
25 °C, then DTT, EtOH (44% yield); (viii) ClCH2CH2NEt2. HCl, Cs2CO3, acetone, rt À50 °C (44–63% yield). See Supplemental materials for experimental details.
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

4
J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
compound 8c, and surviving colonies present at two weeks after
plating were visualized (Fig. 3B and D). Xanthothricin inhibited
the growth of colonies in both the RKO and DLD-1 cell lines
(Fig. 3C), with greater effects on the RKO cell line (i.e., a line which
does not display b-catenin dysregulation). In contrast, compound
8c preferentially inhibited the growth of colonies in DLD-1 cells
relative to RKO cells (Fig. 3E). Hence, the data are consistent with
the notion that compound 8c may interfere with Wnt/b-catenin/
TCF function in CRC cells.
We therefore sought to determine the effects of compound 8c
on the canonical Wnt/b-catenin signaling pathway in DLD-1 cells
and assessed the effects of increasing doses of compound 8c on
the levels of the ‘free’ signaling-competent pool of b-catenin in
DLD-1 cells as well as the total pool of b-catenin. We found that
free b-catenin protein levels, and to a lesser extend total b-catenin
protein levels, were diminished by compound 8c (Fig. 4A). Simi-
larly, in SW480 cells, which have dysregulated b-catenin/TCF tran-
scriptional activity due to APC defects, compound 8c reduced free
b-catenin levels and to a lesser extent total b-catenin levels
(Fig. 4B). To determine if this mechanism is due to a stabilization
of AXIN2 protein, akin to how the XAV939 compound acts, we
studied AXIN2 protein levels and found a dose-dependent decrease
of AXIN2 in response to compound 8c (Fig. 4B), suggesting that
compound 8c acts via a mechanism that does not require AXIN2
protein stabilization. We then sought to determine if compound
8c exerted the observed effects on b-catenin levels via mechanisms
independent of the known activity of GSK3b in regulating the free
pool of b-catenin. To do so, we treated HEK293T cells with 5 lM
SB216763, a previously described GSK3b antagonist,36 and various
concentrations of compound 8c. Interestingly, in HEK293T cells, we
found that in the presence of GSK3b antagonist and elevated levels
of the free pool of b-catenin, compound 8c was able to reduce the
levels of free b-catenin and did not affect total b-catenin protein
levels (Fig. 4C).
We next determined if the reduction of free b-catenin levels by
compound 8c requires proteasome activity. We treated HEK293T
cells simultaneously with compound 8c, the GSK3b-inhibitor
SB216762 and the proteasome inhibitor MG-132.37 As previously
shown compound 8c reduced free b-catenin protein levels in the
presence of SB216763, but this effect was abrogated in the pres-
ence of the proteasome inhibitor MG-132 (Fig. 4D). This finding
demonstrates that compound 8c promotes down-regulation of
b-catenin in a proteasome-dependent fashion. The major ubiquitin
ligase required for b-catenin ubiquitination in cells are the beta-
transducin repeat-containing proteins 1 and 2 (bTrCP1/2). To ad-
dress if compound 8c requires bTrCP1/2 function we transfected
HEK293T cells with dominant negative bTrCP (dnbTrCP) and trea-
ted the cells with compound 8c. As previously shown, compound
8c reduced free b-catenin levels in the presence of SB216763.
When b-catenin was stabilized by SB216763 treatment and
dnbTrCP transfection, compound 8c was still able to reduce the
free pool of b-catenin (Fig. 4E). This suggests that compound 8c
can promote degradation of b-catenin in a bTrCP-independent
fashion. Because the only other defined, albeit minor, pathway
for regulating the free pool of b-catenin, that is, Siah1/2-dependent
regulation of b-catenin, also has been shown to require a SIAH–APC
protein complex, we did not pursue studies to exclude a role for
compound 8c in regulating free b-catenin levels via changes in
SIAH1/2 levels or function since the effect of compound 8c on
b-catenin was independent of APC. In summary, the findings dem-
onstrate that compound 8c is able to promote down-regulation of
the free pool of b-catenin in an APC-, GSK3b-, and bTrCP-indepen-
dent fashion, but in a proteasome-dependent manner.
Figure 2. Wnt-3a activation of presumptive Wnt/b-catenin/TCF-regulated target
genes and inhibition of Wnt-3a activated target genes Axin2 and Lgr5 in rat
intestinal epithelial cells (IEC-6) by small molecules. See Supplemental materials for
experimental details. (A) IEC-6 cells were treated for 12 h with 50 ng/mL
recombinant mouse Wnt-3a. The expression levels of the selected Wnt/b-catenin/
TCF-regulated target genes (Axin2, Lgr5, Bmp4, Nkd1, Edn1, Irs1) relative to U6 gene
expression levels were determined by quantitative PCR using gene specific primers.
Each bar represents the mean with standard error mean (SEM) from three
independent experiments. (B and C) Inhibition of Wnt-3a activated target genes
Lgr5 and Axin2 by calphostin C, XAV939, xanthothricin and xanthothricin analogues.
The base-line relative levels of Lgr5 (B) and Axin2 expression (C) (relative to U6
expression) after Wnt-3a stimulation was set to 100%. The relative target gene
expression for each compound at various concentrations was determined (logM À6
corresponds to 1 lM).
Compound specificity and mechanism of action. We hypothesized
that our synthesized compounds inhibited b-catenin/Tcf transcrip-
tional activity by reducing b-catenin protein levels. To address this
notion, we therefore treated DLD-1 CRC cells for 20 h with the
compounds and analyzed b-catenin protein levels by Western blot.
We found that compounds 8a and 8c reduced total b-catenin pro-
tein levels, as well as that of Cyclin D1, which is expressed from a
presumptive b-catenin/TCF-regulated gene (Fig. 3A).
To determine if these newly identified small molecules prefer-
entially affect the proliferation and survival of CRC cells with
b-catenin/TCF defects, we performed colony formation assays with
selected colon cancer-derived cell lines. Depletion of b-catenin
with shRNA inhibits colony formation of DLD-1 but not RKO cells.15
The DLD-1 cell line, which displays dysregulated b-catenin/TCF
transcriptional activity due to bi-allelic APC mutations and the
RKO cell line, which has no evidence of b-catenin dysregulation35
were seeded in 10% serum containing xanthothricin (3) or
We used a cell-based screening approach with rat intestinal epi-
thelial cells (IEC-6) to identify novel small molecule antagonists of
canonical (b-catenin-dependent) Wnt signaling. We selected one
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
Figure 3. Compound specificity assays. See Supplemental materials for experimental details. (A) Xanthothricin analogues affect total b-catenin protein levels and cyclin D1
protein levels in DLD-1 cells. (B and D) DLD-1 and RKO colon cancer cells were seeded in 10% serum containing xanthothricin or compound 8c and surviving colonies present
at two weeks after plating were visualized (C and E, respectively). Colony counts from two independent experiments at each concentration done in triplicate are shown
(ns = not statistically significant; ⁄p < 0.05; ⁄⁄⁄p < 0.0001).
compound (8c) that appeared to preferentially interfere with the
oncogenic growth potential of colon cancer cells carrying endoge-
nous gene defects in Wnt/b-catenin/TCF signaling relative to a co-
lon cancer cell line known to lack demonstrable defects in
canonical Wnt signaling. We demonstrated that compound 8c
inhibits b-catenin transcriptional activity by reducing the stabi-
lized free pool of b-catenin via a GSK3b-, APC-, AXIN2 and bTrCP-
independent but proteasome-dependent mechanism.
We did not pursue a Tcf/luciferase-reporter cell-based assay,
because our initial studies suggested that some of the xanthothri-
cin analogues we synthesized can nonspecifically inhibit firefly
luciferase reporter genes expressed under control of other consti-
tutive promoter/enhancer elements (J.Z. and E.R.F., unpublished
results).38
Our studies and results here on the effects of some previously
published compounds on cellular genes regulated by the Wnt/
b-catenin/TCF pathway are consistent with previously published
findings in the literature. For instance, we found that calphostin C,
XAV939 and xanthothricin all decreased the expression of Lgr5 and
Axin2 in IEC-6 cells (Fig. 2B and C, and Table 1), with IC50 values sim-
ilar to those previously determined in TCF-reporter gene assays.12,15
Under conditions of UV-light and oxygen, xanthothricin was
previously reported to generate reactive oxygen species.39
Nucleoredoxin (NRX), a thioredoxin family protein, interacts with
dishevelled (Dvl). Increased NRX protein levels suppress Wnt/
b-catenin signaling.40 We can likely exclude that effects of 8c on
b-catenin degradation could occur through generation of reactive
oxygen species. The effects of 8c on stabilized b-catenin that we
observed were independent of APC and GSK3b, as both exert their
function downstream of Dvl. Furthermore we incubated all com-
pounds with cells in the dark.
Considerable efforts have been made to identify drugs and drug
candidates that antagonize Wnt/b-catenin signaling.41 Neverthe-
less, very few antagonists that act in a destruction complex-inde-
pendent fashion to reduce stabilized b-catenin protein levels in
cells are known with only one such class of compounds, to the best
of our knowledge, described in the literature. A recent cell-based
screen for Wnt pathway inhibitors identified a series of acyl-hydra-
zones, such as the FDA-approved drug ciclopiroxoalmine, that
appear to promote degradation of stabilized b-catenin in a destruc-
tion complex-independent fashion.42 Previous studies reported
that xanthothricin inhibits b-catenin/TCF transcriptional activity
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

6
J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Figure 4. Effect of compound 8c on b-catenin levels. See Supplemental materials for experimental details. (A–C) Effects of compound 8c on free and total b-catenin protein
levels in DLD-1 cells (A), SW480 cells (B) and HEK293T cells (C–E). (C) treated with 5 M of the GSK3b antagonist SB216763. Compound 8c degrades b-catenin in a
proteasome dependent fashion (D). Compound 8c degrades b-catenin in a bTrCP independent fashion (E).
l
by disrupting b-catenin/TCF4 binding.12 In contrast to the reported
effects of xanthothricin, we show here that compound 8c reduced
stabilized b-catenin protein levels and we did not find evidence
that it disrupted b-catenin/TCF4 binding (J.Z. and E.R.F., unpub-
lished results). These findings suggest perhaps that through modi-
fication of the C3 position of xanthothricin, compound 8c may have
altered the presumptive b-catenin–TCF4 complex disrupting
activity in some fashion to promote the degradation of stabilized
b-catenin. Because 8c can promote the degradation of stabilized
b-catenin in a destruction complex-independent fashion requiring
proteasomal activity independent of the major ubiqutin ligase
bTrCP, how then does it promote b-catenin degradation? One pos-
sibility is that compound 8c increases the ubiquitination of b-cate-
nin through activation of a still-to-be defined and little used
ubiquitin ligation pathway that specifically regulates b-catenin
ubiquitination and proteasomal degradation. Another possibility
is that compound 8c may increase proteasomal degradation of
b-catenin without ubiquitination. For example, dicoumarol-induced
p53 degradation by the proteasome occurs independently of
ubiquitination.43
Wnt signaling. We profiled one compound (8c) that appears to
preferentially interfere with the oncogenic growth potential of co-
lon cancer cells carrying endogenous gene defects in Wnt/b-cate-
nin/TCF signaling relative to a colon cancer cell line known to
lack demonstrable defects in canonical Wnt signaling. We have
also demonstrated that 8c inhibits b-catenin transcriptional activ-
ity by reducing the stabilized free pool of b-catenin via a GSK3b-
and APC-independent pathway. The findings strongly suggest that
8c acts at the level of b-catenin to inhibit Wnt/b-catenin/TCF func-
tion and highlights the potential of carrying out screens for other
small molecules likely to have inhibitory effects on downstream
Wnt signaling factors and mechanisms in cancer cells. Further
work will be required for a more detailed understanding of the
means by which compound 8c and related compounds promote
b-catenin degradation. Insights into the means by which these pro-
mote b-catenin degradation may have important consequences for
the targeting of cancer cells with mutational defects in b-catenin or
in key destruction components, such as APC and Axin.
Acknowledgments
In conclusion, our work highlights a robust strategy for assess-
ing the activity of b-catenin/Tcf antagonists. We used a cell-based
screening approach with rat intestinal epithelial cells (IEC-6) to
identify several novel small molecule antagonists of canonical
E.R.F. and H.D.H.S. were supported by a Cancer Center Innova-
tion Grant. A.J.T. gratefully acknowledges financial support of the
NIGMS (Grant number GM007767), the American Chemical Society
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111

J. Zeller et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
7
17. Daves, G. D., Jr.; Robins, R. K.; Cheng, C. C. J. Am. Chem. Soc. 1962, 84, 1724.
18. Levenberg, B.; Linton, S. N. J. Biol. Chem. 1966, 241, 846.
19. Nagamatsu, T.; Yamasaki, H.; Hirota, T.; Yamato, M.; Kido, Y.; Shibata, M.;
Yoneda, F. Chem. Pharm. Bull. 1993, 41, 362.
Division of Medicinal Chemistry, Sheila B. Cresswell, and Fred and
Dee Lyons Graduate Fellowships.
20. Turbiak, A. J.; Showalter, H. D. H. Tetrahedron Lett. 2009, 50, 1996.
21. Turbiak, A. J.; Showalter, H. D. H. Synthesis 2009, 4022.
22. Lacrampe, J. F. A.; Connors, R. W.; Ho, C. Y.; Richardson, A.; Freyne, E. J. E.;
Buijnsters, P. J. J.; Bakker, A. C. WO2004007498A2; 2004, p 71.
23. Yoneda, F.; Nagamatsu, T. Chem. Pharm. Bull. 2001, 1975, 23.
24. Blehaut, H.; Bellamy, F.; Matt, C.; Giraud, S.; Charre, D. WO2010072807A2;
2010, p 53.
Supplementary data
Supplementary data (chemical syntheses and biological assays)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2013.08.111.
25. Yoneda, F.; Nagamatsu, T. J. Heterocycl. Chem. 1974, 11, 271.
26. Nagamatsu, T.; Yamasaki, H. J. Chem. Soc., Perkin Trans. 1 2001, 130.
27. Senda, S.; Hirota, K. Chem. Pharm. Bull. 1974, 22, 1459.
28. Black, T. H. J. Heterocycl. Chem. 1987, 24, 1373.
29. Barker, N.; Van, E. J. H.; Jaks, V.; Kasper, M.; Snippert, H.; Toftgard, R.; Clevers,
H. Cold Spring Harb. Symp. Quant. Biol. 2008, 73, 351.
30. Bommer, G. T.; Feng, Y.; Iura, A.; Giordano, T. J.; Kuick, R.; Kadikoy, H.; Sikorski,
D.; Wu, R.; Cho, K. R.; Fearon, E. R. J. Biol. Chem. 2010, 285, 1928.
31. Kim, J.-S.; Crooks, H.; Dracheva, T.; Nishanian, T. G.; Singh, B.; Jen, J.; Waldman,
T. Cancer Res. 2002, 62, 2744.
32. Kim, T. H.; Xiong, H.; Zhang, Z.; Ren, B. Oncogene 2005, 24, 597.
33. Leung, J. Y.; Kolligs, F. T.; Wu, R.; Zhai, Y.; Kuick, R.; Hanash, S.; Cho, K. R.;
Fearon, E. R. J. Biol. Chem. 2002, 277, 21657.
34. Van Raay, T. J.; Fortino, N. J.; Miller, B. W.; Ma, H.; Lau, G.; Li, C.; Franklin, J. L.;
Attisano, L.; Solnica-Krezel, L.; Coffey, R. J. PLoS One 2011, 6, e18650.
35. Sparks, A. B.; Morin, P. J.; Vogelstein, B.; Kinzler, K. W. Cancer Res. 1998, 58,
1130.
36. Coghlan, M. P.; Culbert, A. A.; Cross, D. A. E.; Corcoran, S. L.; Yates, J. W.; Pearce,
N. J.; Rausch, O. L.; Murphy, G. J.; Carter, P. S.; Cox, L. R.; Mills, D.; Brown, M. J.;
Haigh, D.; Ward, R. W.; Smith, D. G.; Murray, K. J.; Reith, A. D.; Holder, J. C.
Chem. Biol. 2000, 7, 793.
37. Rock, K. L.; Gramm, C.; Rothstein, L.; Clark, K.; Stein, R.; Dick, L.; Hwang, D.;
Goldberg, A. L. Cell 1994, 78, 761. Cambridge, Mass.
38. Cong, F.; Cheung, A. K.; Huang, S.-M. A. Annu. Rev. Pharmacol. Toxicol. 2012, 52,
57.
References and notes
1. Clevers, H. Cell 2006, 127, 469. Cambridge, MA, U.S..
2. MacDonald, B. T.; Tamai, K.; He, X. Dev. Cell 2009, 17, 9.
3. Kimelman, D.; Xu, W. Oncogene 2006, 25, 7482.
4. Matsuzawa, S.-I.; Reed, J. C. Mol. Cell 2001, 7, 915.
5. Winston, J. T.; Strack, P.; Beer-Romero, P.; Chu, C. Y.; Elledge, S. J.; Harper, J. W.
Genes Dev. 1999, 13, 270.
6. Polakis, P. Curr. Opin. Genet. Dev. 2007, 17, 45.
7. Fearon, E. R. Annu. Rev. Pathol.: Mech. Dis. 2011, 6, 479.
8. Roh, H.; Green, D. W.; Boswell, C. B.; Pippin, J. A.; Drebin, J. A. Cancer Res. 2001,
61, 6563.
9. van de Wetering, M.; Sancho, E.; Verweij, C.; de Lau, W.; Oving, I.; Hurlstone, A.;
van der Horn, K.; Batlle, E.; Coudreuse, D.; Haramis, A.-P.; Tjon-Pon-Fong, M.;
Moerer, P.; van den Born, M.; Soete, G.; Pals, S.; Eilers, M.; Medema, R.; Clevers,
H. Cell 2002, 111, 241. Cambridge, MA, U.S..
10. Barker, N.; Clevers, H. Nat. Rev. Drug Disc. 2006, 5, 997.
11. Ewan, K.; Pajak, B.; Stubbs, M.; Todd, H.; Barbeau, O.; Quevedo, C.; Botfield, H.;
Young, R.; Ruddle, R.; Samuel, L.; Battersby, A.; Raynaud, F.; Allen, N.; Wilson,
S.; Latinkic, B.; Workman, P.; McDonald, E.; Blagg, J.; Aherne, W.; Dale, T. Cancer
Res. 2010, 70, 5963.
12. Lepourcelet, M.; Chen, Y.-N. P.; France, D. S.; Wang, H.; Crews, P.; Petersen, F.;
Bruseo, C.; Wood, A. W.; Shivdasani, R. A. Cancer Cell 2004, 5, 91.
13. Cook, D.; Fry, M. J.; Hughes, K.; Sumathipala, R.; Woodgett, J. R.; Dale, T. C.
EMBO J. 1996, 15, 4526.
39. Koh, S.; Kim, H.; Kim, J.; Goo, E.; Kim, Y.-J.; Choi, O.; Jwa, N.-S.; Ma, J.;
Nagamatsu, T.; Moon, J. S.; Hwang, I. Plant Biotechnol. J. 2011, 9, 348.
40. Funato, Y.; Michiue, T.; Asashima, M.; Miki, H. Nat. Cell Biol. 2006, 8, 501.
41. Takahashi-Yanaga, F.; Kahn, M. Clin. Cancer Res. 2010, 16, 3153.
42. Song, S.; Christova, T.; Perusini, S.; Alizadeh, S.; Bao, R.-Y.; Miller, B. W.; Hurren,
R.; Jitkova, Y.; Gronda, M.; Isaac, M.; Joseph, B.; Subramaniam, R.; Aman, A.;
Chau, A.; Hogge, D. E.; Weir, S. J.; Kasper, J.; Schimmer, A. D.; Al-awar, R.;
Wrana, J. L.; Attisano, L. Cancer Res. 2011, 71, 7628.
14. Kobayashi, E.; Nakano, H.; Morimoto, M.; Tamaoki, T. Biochem. Biophys. Res.
Commun. 1989, 159, 548.
15. Huang, S.-M. A.; Mishina, Y. M.; Liu, S.; Cheung, A.; Stegmeier, F.; Michaud, G.
A.; Charlat, O.; Wiellette, E.; Zhang, Y.; Wiessner, S.; Hild, M.; Shi, X.; Wilson, C.
J.; Mickanin, C.; Myer, V.; Fazal, A.; Tomlinson, R.; Serluca, F.; Shao, W.; Cheng,
H.; Shultz, M.; Rau, C.; Schirle, M.; Schlegl, J.; Ghidelli, S.; Fawell, S.; Lu, C.;
Curtis, D.; Kirschner, M. W.; Lengauer, C.; Finan, P. M.; Tallarico, J. A.;
Bouwmeester, T.; Porter, J. A.; Bauer, A.; Cong, F. Nature 2009, 461, 614. London,
U.K..
43. Asher, G.; Lotem, J.; Sachs, L.; Kahana, C.; Shaul, Y. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 13125.
16. Karlberg, T.; Markova, N.; Johansson, I.; Hammarstrom, M.; Schutz, P.; Weigelt,
J.; Schuler, H. J. Med. Chem. 2010, 53, 5352.
Please cite this article in press as: Zeller, J.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.08.111