Discovery of Selective Small-Molecule Inhibitors for the β-Catenin/T-Cell Factor Protein-Protein Interaction through the Optimization of the Acyl Hydrazone Moiety

Article abstract of DOI:10.1021/acs.jmedchem.5b00223

Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the β-catenin/Tcf protein-protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor optimization resulted in chemically stable derivatives that disrupt the β-catenin/Tcf PPI. The binding mode of new inhibitors was characterized by site-directed mutagenesis and structure-activity relationship studies. This series of inhibitors with a new scaffold exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. One derivative of this series suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells. This compound represents a solid starting point for the development of potent and selective β-catenin/Tcf inhibitors (Chemical Equation).

Full text of DOI:10.1021/acs.jmedchem.5b00223

Article
pubs.acs.org/jmc
Discovery of Selective Small-Molecule Inhibitors for the β‑Catenin/T-
Cell Factor Protein−Protein Interaction through the Optimization of
the Acyl Hydrazone Moiety
J. Leon Catrow,† Yongqiang Zhang,† Min Zhang, and Haitao Ji*
Department of Chemistry, Center for Cell and Genome Science, University of Utah, 315 South 1400 East, Salt Lake City, Utah
84112−0850, United States
S
* Supporting Information
ABSTRACT: Acyl hydrazone is an important functional group for the discovery of bioactive small molecules. This functional
group is also recognized as a pan assay interference structure. In this study, a new small-molecule inhibitor for the β-catenin/Tcf
protein−protein interaction (PPI), ZINC02092166, was identified through AlphaScreen and FP assays. This compound contains
an acyl hydrazone group and exhibits higher inhibitory activities in cell-based assays than biochemical assays. Inhibitor
optimization resulted in chemically stable derivatives that disrupt the β-catenin/Tcf PPI. The binding mode of new inhibitors was
characterized by site-directed mutagenesis and structure−activity relationship studies. This series of inhibitors with a new scaffold
exhibits dual selectivity for β-catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs. One derivative of this series
suppresses canonical Wnt signaling, downregulates the expression of Wnt target genes, and inhibits the growth of cancer cells.
This compound represents a solid starting point for the development of potent and selective β-catenin/Tcf inhibitors.
optimization of the inhibitors with this chemotype and the
discovery of a selective inhibitor for the β-catenin/T-cell factor
(Tcf) protein−protein interaction (PPI), an important down-
stream effector of canonical Wnt signaling.
INTRODUCTION
■
The formation of an acyl hydrazone moiety has emerged as an
important strategy in dynamic combinatorial chemistry (DCC)
to discover bioactive small molecules for protein targets.1−3
The acyl hydrazone products from DCC can be directly
isolated and characterized because the acyl hydrazone group is
stable under acidic and physiological conditions. Acyl hydro-
zone-based DCC has been used to discover potent inhibitors
for acetylcholinesterase,4 Bacillus subtilis HPr kinase/phospha-
tase,5 aspartic protease,6 and β-tryptase.7 The crystal structure
of several acyl hydrazones in complexes with their protein
targets have been reported.8−12 On the other hand, the acyl
hydrazone moiety has been recognized as a substructure of pan
assay interference compounds (PAINS)13 or causing false
positives in bioassays.14 Many biological activities have been
associated with acyl hydrazones.15 A literature search resulted in
11490 publications between 1993 and 2014 that discussed the
biological activities of acyl hydrazones (see Figure S1 in the
Supporting Information). However, few compounds were
advanced to clinic studies. Two therapeutic drugs that do
contain this functional group are nitrofurantoin and levosi-
mendan. Therefore, it is urgent to evaluate the relationship
between the biochemical and cell-based activities of acyl
hydrazones and contemplate possible directions for inhibitor
optimization. Herein, we report the biological evaluation and
Canonical Wnt signaling plays a critical role in regulating cell
proliferation, differentiation, and cell−cell communication.16,17
β-Catenin is a key mediator of this signaling pathway. The
hyperactivation of canonical Wnt signaling promotes the
transcription of cell proliferation, migration, and survival
genes, such as cyclin D1,18 c-myc19 and survivin,20 and is
strongly associated with the initiation and progression of many
cancers including colorectal carcinoma and fibroses. Cancer
stem cells, which are resistant to conventional chemo- and
radiotherapies and especially virulent, are also controlled by the
hyperactivation of canonical Wnt signaling.21−23 The formation
of the β-catenin/Tcf complex in the cell nucleus is the
penultimate step of canonical Wnt signaling. The overactivation
of canonical Wnt signaling correlates with the formation of this
complex.24−26 Selective inhibition of the β-catenin/Tcf PPI
represents an appealing therapeutic strategy.
The crystal structures of β-catenin in complexes with human
Tcf4,27−29 mouse Lef1,30 and Xenopus Tcf331 have been
Received: February 8, 2015
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of the reported β-catenin/Tcf inhibitors. Compounds 1, 2, and 4−6 have quinone reactive groups (PAINS
substructures 374, 376, and 377).13 Compound 3 has a reactive redox recycler moiety, toxoflavin.49 Compound 7 has an acyl hdrazone moiety
(PAINS substructures 270 and 323).13 Compounds 9 and 10 have the rhodanine-like groups (PAINS substructures 170 and 171).13,52,53
reported. These structures reveal a large protein−protein
interface between β-catenin and Tcf [3500 Å2 for β-catenin/
Tcf4 PPIs (PDB id, 2GL729) and 3700 Å2 for β-catenin/Lef1
PPIs (PDB id, 3OUX30)]. The crystallographic and bio-
chemical studies also revealed three hot regions on β-catenin
for the β-catenin/Tcf PPI:27,31−35 hot region 1 includes K435
and K508 of β-catenin where D16 and E17 of Tcf4 binds, hot
region 2 is K312 and K345 of β-catenin where E24 and E29 of
Tcf4 bind using two alternative conformations,27−29 and hot
region 3 is a hydrophobic pockets lined with F253, I256, F293,
A295, and I296. V44 and L48 of Tcf4 as well as axin36 bind
with this pocket. The crystal structures of β-catenin in
complexes with cadherin37 and adenomatous polyposis coli
(APC)38−40 were reported. A comparison of these crystal
structures indicated that β-catenin used the same binding
groove to bind cadherin (residues 819−876 of human E-
cadherin) and APC (residues 1477−1519 of human APC 20-
amino acid repeat 3, APC-R3; an APC repeat with the highest
binding affinity). The central binding area of β-catenin was
called the charge-button-recognition region (Supporting
Information Figure S2).31 One charge button is K312, and
the other is K435. A conserved segment of Tcf/Lef, cadherin,
and APC, DxθθxΦx2−7E, binds with this area. The biological
assays confirm that the binding mode of Tcf/Lef, cadherin, and
APC with β-catenin is mutually exclusive.41 The β-catenin/
cadherin PPI is essential for the integrity of epithelial junctions
and the β-catenin/APC PPI is critical for β-catenin degradation
in normal cells. The inhibitor that disrupts the β-catenin/
cadherin PPI alters cell−cell adhesion, while the β-catenin/
APC inhibitor overactivates Wnt signaling in normal cells and
promotes new cancer formation. However, it is challenging to
achieve inhibitor selectivity for β-catenin/Tcf over β-catenin/
cadherin or β-catenin/APC PPIs because the dissociation
constant (Kd) of the β-catenin/Tcf PPI is 7−10 nM32,42,43 and
much lower than those of the β-catenin/cadherin (Kd = 41−82
nM)39,42,44 and β-catenin/APC (Kd = 0.6−3.1 μM for APC-
R339,40,42,45,46) PPIs.
Extensive efforts have been made to discover β-catenin/Tcf
inhibitors, but few were successful. A high-throughput screen-
ing (HTS) on 52000 compounds identified six natural products
as β-catenin/Tcf inhibitors, 1 (PKF115−584), 2
(CGP049090), 3 (PKF118−310), 4 (PKF118−744), 5
(ZTM000990), and 6 (PKF222−815) in Figure 1.47 Com-
pound 4 was used as a template for inhibitor optimization.48
Nonetheless, all of these compounds have PAINS substructures
(quinone13 or toxoflavin49) and are frequent hits in biochemical
assays. A combination of virtual and biophysical screening
identified 7 (PNU-74654).50 No further study was report for
this compound. A compound screening by knocking down axin
identified three inhibitors, 8 (iCRT3), 9 (iCRT5), and 10
(iCRT14) in Figure 1 for the Wnt effectors downstream of the
axin-mediated degradation complex.51 The pulldown assay
revealed that 8 and 9 disrupted the β-catenin/Tcf PPI at high
micromolar concentrations. Compounds 8−10 were reported
to suppress the transcriptional activity of canonical Wnt
signaling, downregulate Wnt/β-catenin-induced target genes,
and inhibit the growth of colorectal cancer cells in vitro and in
vivo. Compounds 9 and 10 contain a rhodanine PAINS
substructure and are frequent hits in biochemical assays.13,52,53
Compound 8 was reported to inhibit the β-catenin/androgen
receptor interaction and androgen signaling.54 A virtual
screening using the Tcf4 D16ELISF21 binding site of β-catenin
B
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Figure 2. Results of compound screening. (A) Chemical structures of five compounds. Dianion 19 (ZINC20828864) was not further evaluated. (B)
FP and AlphaScreen assay results. Each set of data is expressed as mean standard deviation (n = 3). Details are in Supporting Information Figure
S3. (C) Wnt-responsive TOPFlash luciferase reporter assay results using pcDNA3.1−β-catenin transfected HEK293 cells and colorectal cancer
SW480 cells. Each set of data is expressed as mean standard deviation (n = 3). Details are in Supporting Information Figure S4. (D) FOPFlash
luciferase reporter assay results of 18 using pcDNA3.1−β-catenin transfected HEK293 cells and colorectal cancer SW480 cells. Each set of data is
expressed as mean standard deviation (n = 3). (E) MTs assay results of β-catenin/Tcf inhibitors on the growth of colorectal cancer cells, SW480,
HCT116, and HT29. Each set of data is expressed as mean standard deviation (n = 3). n.d., not determined. n.a., not applicable because 1
interferes with the AlphaScreen assay.61
identified an organocopper compound, 11 (BC21), that can
disrupt the β-catenin/Tcf PPI, inhibit the transactivation of
canonical Wnt signaling, down-regulate the expression of
canonical Wnt target genes, and inhibit the growth of colorectal
cancer cells.55 The inhibitory selectivity of 11 for β-catenin/Tcf
over β-catenin/cadherin and β-catenin/APC PPIs has not yet
been reported. Hydrocarbon-stapled axin α-helix, 12 (aStAx-
35) in Figure 1, was designed to bind hot region 3 of β-catenin
and inhibit the β-catenin/Tcf PPI.56 Axin is a scaffolding
protein for β-catenin phosphorylation. The use of 12 might
impair the degradation of β-catenin in normal cells. Recently,
we reported targeting the Tcf4 G13ANDE17 binding site to
selectively inhibit the β-catenin/Tcf PPI.57 A bioisosteric
replacement technique was used to design new small organic
molecules (fragments) to mimic the binding mode of Tcf4 D16
and E17, and 13 (UU-T01) was discovered.33 In another series,
human Tcf4 D16 and E17 were maintained in inhibitor
scaffold. On the basis of the structure of Tcf4 peptide
G13ANDE17, a peptidomimetic inhibitor, 14 (UU-T02) in
Figure 1, was reported.57 The diethyl ester of 14 was found to
inhibit canonical Wnt signaling and the growth of colorectal
cancer cells. This compound also exhibited cell-based
selectivities for β-catenin/Tcf over β-catenin/cadherin and β-
catenin/APC PPIs. Further structural optimization of 13 and
14 is under way.
RESULTS
■
Compound Screening to Discover Small-Molecule
Inhibitors for the β-Catenin/Tcf Interaction. The homoge-
neous fluorescence polarization (FP) and AlphaScreen assays
have been established to evaluate β-catenin/Tcf inhibitors.58 In
the FP assay, C-terminally fluorescein-labeled human Tcf4
(residues 7−51) and human β-catenin (residues 138−686)
were used. After β-catenin and fluorescein-Tcf4 form a
protein−protein complex, the fluorophore is attached to a
high-molecular-weight protein complex (≥60 kDa) and has a
low rotational speed when it is excited by plane-polarized light.
The resulting emitted light is significantly polarized. When an
C
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Figure 3. Cell-based characterization of 18. (A) Quantitative real-time PCR study to determine the changes of mRNA expression of AXIN2, cyclin
D1, c-myc, and HPRT in response to different concentrations of 18. Each set of data is expressed as mean standard deviation (n = 3). (B) Western
blot analysis to monitor the changes of protein expression of c-myc, cyclin D1, and β-catenin in response to different concentrations of 18. β-Tubulin
was used as an internal reference. (C) Coimmunoprecipitation experiment to evaluate the inhibitory effect of 18 on the β-catenin/Tcf association.
IP, immunoprecipitation; IB, immunoblotting.
inhibitor completely disrupts the β-catenin/fluorescein-Tcf4
PPI, the fluorophore is only attached to Tcf4 (≤6 kDa), and
the emitted light after excitation will be largely depolarized due
to a high rotational speed of the fluorophore. AlphaScreen is a
bead-based assay. The streptavidin-coated donor beads and the
nickel-chelate acceptor beads are brought together through the
interaction between C-terminally biotinylated human Tcf4
(residues 7−51) and N-terminally His6-tagged human β-catenin
(residues 138−686). On laser excitation at 680 nm, the photo
sensitizers inside the donor beads convert ambient oxygen to a
singlet oxygen state. Only when the donor and acceptor beads
are brought within 200 nm can the singlet oxygen molecules
diffuse to the acceptor beads, resulting in extensive emission at
570 nm. If the inhibitor completely disrupts the β-catenin/Tcf
PPI, no luminescence signal will be observed. In this study, 269
Sigma-Aldrich carboxylic acids and sulfonamides, 90 LO-
PACPfizer synthetic compounds, 24 compounds from the
ZINC library, 117 natural products, and 1593 Diversity Set V
compounds from the Developmental Therapeutics Program of
NCI/NIH were screened. In the screening, carboxylic acids and
acidic sulfonamides were prioritized because the acid-rich Tcf4
D16ELISFKDE24 segment has been recognized as a key binding
motif for the β-catenin/Tcf PPI. The compounds that displayed
a Ki value ≤25 μM in either assay were kept for further
evaluation. As shown in Figure 2A, four compounds exhibited
reproducible and dose-dependent inhibitory activities in both
assays. None of them were false positives in the AlphaScreen
counter screen or aggregated as examined by the spin-down
counter screen59 and the dynamic light scattering experiments.
None of these compounds had fluorescence interference in
either assay. Figure 2B shows the results of the FP and
AlphaScreen assays. Among them, compound 18
(ZINC02092166) is the most potent inhibitor. It is more
potent than known inhibitors 1, 2, and 7−10 and comparable
with 3, 13, and 14 in the parallel assays.33,57
luciferase reporter assays were performed.64 As shown in Figure
2C, compound 16 inhibited the TOPFlash luciferase (a
luciferase reporter with eight wild-type Tcf binding sites)
activity in Wnt-activated HEK293 cells. Compound 18
inhibited the TOPFlash luciferase activity in both tested cell
lines, pcDNA3.1−β-catenin transfected HEK293, and color-
ectal cancer SW480 by dose-dependent manners with the IC50
values of 0.86 0.51 and 0.71 0.45 μM. Compounds 8 and
9, which were identified by the highly sensitive luciferase
reporter (Super 16× TOPFlash) assay,51 were used as the
reference inhibitors for comparison. Compound 18 also
inhibited the FOPFlash luciferase (a luciferase reporter with
eight mutant Tcf binding sites) activity at the concentrations
≥5 μM (Figure 2D), indicating off-target effects at high
concentrations. It was worth noting that 8 and 9 also inhibited
the FOPFlash luciferase reporter activity, as shown in
Supporting Information Figure S4.
The MTs cell viability assay was performed to assess the
effects of these compounds on the growth of colorectal cancer
cell lines, SW480, HCT116, and HT29. These cancer cell lines
were chosen based on their known dependence on the β-
catenin/Tcf PPI for growth and survival. SW480 and HT29
cells harbor the deletions of APC, whereas HCT116 cells
harbor a mutation on β-catenin that blocks β-catenin
phosphorylation and ubiquitination.65,66 The MTs assay results
indicated that 15 inhibited the growth of SW480, HCT116, and
HT29 cells with the IC50 values of 38 1.1, 22 5.9, and 41
1.0 μM. Compound 18 inhibited the growth of SW480,
HCT116, and HT29 cells with the IC50 values of 0.85 0.10,
0.99 0.13, and 1.1 0.093 μM, respectively (Figure 2E).
Effects of 18 on the Expression of Wnt/β-Catenin
Target Genes and the Co-immunoprecipitation Study.
AXIN2 is a specific target gene for the canonical Wnt signaling
pathway.67 Cyclin D1 and c-myc are two Wnt target genes that
drive cancer progression including in SW480 cells.18,19 The
quantitative real-time PCR study indicated that 18 down-
regulated the expression of AXIN2, cyclin D1, and c-myc in a
To determine whether the above compounds inhibit the
transactivation of canonical Wnt signaling, Wnt-responsive
D
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Figure 4. Structural analysis of 18. (A) Potential reactive sites of 18. The intramolecular H-bond between the oxadiazolopyrazine moiety and acyl
hydrazone is shown blue. (B) Chemical structures of 19−25. (C) FP assay results of 19−25. Each set of data is expressed as mean standard
deviation (n = 3). Details are in Supporting Information Figure S5.
dose-dependent manner in SW480 cells, as shown in Figure 3A.
More than 50% of mRNA expression was inhibited at a dose of
2 μM. Compound 18 did not affect the expression of house-
keeping gene HPRT. The protein expression levels of cyclin
D1, c-myc, and β-catenin in SW480 cells were examined by the
Western blot analysis (Figure 3B). The expression of proteins
cyclin D1 and c-myc were significantly reduced after the
treatment with 18. Compound 18 did not reduce the
expression level of β-catenin, indicating that it does not inhibit
the β-catenin degradation pathways. A coimmunoprecipitation
experiment was performed to evaluate the inhibitory potency of
18 in a cellular context. As shown in Figure 3C, compound 18
inhibits the β-catenin/Tcf PPI in a dose-dependent manner in
SW480 cells.
nitrogen atom of the oxadiazolopyrazine moiety and the NH
group of acyl hydrazone may stabilize the structure of 18, as
shown in Figure 4A.
Because the nucleophilic imine carbon atom of 18 was
recognized problematic, a fragmentation of 18 was conducted.
As shown in Figure 4C, compounds 19−21, 23, and 25
exhibited some inhibitory activities for the disruption of the β-
catenin/Tcf PPI but were much lower than 18. The synthesis of
19 and 20 is shown in Scheme 1. The condensation reaction
Scheme 1
Fragmentation of Compound 18. Compound 18
exhibited a higher inhibitory potency in cell-based studies
than in the FP and AlphaScreen assays and inhibited the
FOPFlash luciferase activity. This brought the concern that 18
had off-target effects. An analysis of the structure of 18
indicated two functional groups that might be problematic
(Figure 4A). One is the acyl hydrazone moiety, which has been
identified as a PAINS substructure. Out of three vulnerable sites
in acyl hydrazone, site A, the nucleophilic imine carbon atom, is
most reactive and susceptible to nucleophilic attack. A similar
site in hydrazones has been reported causing frequent hits in
biochemical assays.68,69 The N−N single bond is an
“undesirable” substructure due to the reactivity to nucleo-
philes.70 In any case, site B of 18 is located in an electron-
deficient environment and unlikely to be reactive. Site C, the
amide carbon atom, is more stable than sites A and B. The
second is the oxadiazolopyrazine ring. No false positive
reactivity is associated with this functional group. A related
substructure, benzafurazan, has been reported as a PAINS
substructure.13,71−73 However, the reactivity of benzafurazan
with singlet oxygen74 and nucleophiles75 is caused by the fused
benzene ring, which is absent in 18. The hydrophilic nature of
the oxadiazolopyrazine moiety and the low polarizability of the
tetracyclic ring make 18 unlikely to be a DNA intercalator.76,77
It is worth noting that an intramolecular H-bond between the
between ninhydrin and 1,2,5-oxadiazole-3,4-diamine generated
19, which was converted to 20 through a condensation with
hydrazine hydrate in glacial acid.
Structure−Activity Relationship of the Derivatives of
18. Further optimization started with the resynthesis of 18 to
examine the chemical structure. As shown in Scheme 2, 4-
amino-1,2,5-oxadiazole-3-carboxylic acid was refluxed in meth-
anol under acidic conditions that afforded methyl ester 39,
which was converted to 40 by hydrazinolysis. Compound 18
was obtained through a condensation reaction between 19 and
40. The FP and AlphaScreen assays indicated that the
resynthesized 18 had a similar biological activity as that
observed in commercially available 18 (Figure 2B and Figure
5E). The AutoDock Vina79 blind docking study was performed
to explore the possible binding pockets in β-catenin for 18. The
result predicted that 18 preferentially bound to hot region 1
(AutoDock Vina scores were −8.4, −4.6, and −3.7 kcal/mol for
hot regions 1, 2, and 3, respectively). The Glide,80 AutoDock,81
and AutoDock Vina docking studies predicted one recurring
binding conformation of 18 in β-catenin, as shown in Figure
E
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R469, V511, and I569 did not interfere with the β-catenin/Tcf
PPI. Three mutants, R469A, V511S, and V511S/I569S, were
made.33,57 Native gel electrophoresis, thermal shift, and CD
experiments confirmed the homogeneity, the thermal stability,
and the secondary structure integrity of all purified proteins.
The FP saturation binding assays demonstrated that V511S,
V511S/I516S, and R469A of β-catenin had the same apparent
Kd values as wild-type β-catenin when binding with wild-type
Tcf4, as shown in Supporting Information Figure S7. The FP
competitive inhibition assay was performed to evaluate the
roles of these three residues in inhibitor binding. The Ki value
of the resynthesized 18 for wild-type β-catenin/Tcf4
Scheme 2
interactions was 7.0
4.1 μM. The Ki values of the
resynthesized 18 for β-catenin V511S/Tcf4 and β-catenin
V511S/I569S double mutant/Tcf4 interactions were 2.0 × 101
1.7 and 36 3.1 μM, respectively. It suggests that a change
of pocket B from hydrophobic to hydrophilic greatly reduces
the potency of 18. The Ki value of the resynthesized 18 for β-
catenin R469A/Tcf4 interactions was 46 4.3 μM, indicating
the role of R469 in inhibitor binding.
As shown in Figure 5A, the tetracylic ring of 18 is not
aromatic. Instead, it contains two aromatic substructures,
oxadiazolopyrazine and benzene. The imine carbon atom is
located at the dibenzylic position and potentially reactive. A
5B: the 4-amino-1,2,5-oxadiazole ring was predicted to form H-
bonds with the side chain amino group of K435 and the
backbone NH group of N430, the amide carbonyl group of 18
was predicted to form H-bonds with the side chain amide
group of N516 and the hydroxyl group of S473, the tetracyclic
ring was predicted to form a cation−π interaction with the
positively charged gaunidino group of R469, two nitrogen
atoms of the oxadiazolopyrazine ring formed H-bonds with
K508, and the benzene ring was predicted to locate in
hydrophobic pocket B. Crystallographic analyses indicated that
Figure 5. Inhibitor optimization for 18. (A) Strategy for inhibitor optimization. The numbering of the heteroatoms in the tetracyclic ring is shown.
(B) Glide docking model of 18 with β-catenin. A stick model is shown in Supporting Information Figure S6. (C) Results of the site-directed
mutagenesis studies of the resynthesized 18 and 26. Each set of data is expressed as mean standard deviation (n = 3). Details are in Supporting
Information Figures S7 and S10. (D) Chemical structures of 27−38. (E) FP and AlphaScreen assay results for the resynthesized 18 and 26−38. Each
set of data is expressed as mean standard deviation (n = 3). Details are in Supporting Information Figures S8 and S9. (F) FP selectivity assay
results for the resynthesized 18, 26, 29, 31, 33, and 37. Each set of data is expressed as mean standard deviation (n = 3). Details are in Supporting
Information Figure S11.
F
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Scheme 3
nitrogen atom was used to replace this carbon atom. This
modification converts the tetracyclic ring into an aromatic
system, increases its chemical stability, and maintains the
cation−π interaction with R469. The hydrazide substructure of
18 was changed to an amide, and 26 was designed. The
synthesis of 26 is shown in Scheme 3. Condensation of isatin
with 1,2,5-oxadiazole-3,4-diamine afforded 27 in low yield
(17%). In agreement with the results described previously,82,83
the alkylation in an alkaline medium gave N5-substituted
derivative 28 as the major product. No N4 or N10-alkylated
byproducts were isolated. The ester hydrolysis/peptide bond
coupling sequence generated 26 with moderate yield. The FP
and AlphaScreen assays indicated that the inhibitory activity of
26 was similar to that of 18 (Figure 5E). The site-directed
mutagenesis study indicated that V511, I569, and R469 are
important for the inhibitory activity of 26 (Figure 5C).
Compounds 27−38 were designed to evaluate the
contribution of the 4-amino-1,2,5-oxadiazole ring to inhibitor
potency (Figure 5D). The synthesis is also shown in Scheme 3.
An aza-Michael addition reaction afforded 30. The nucleophilic
substitution reactions in alkaline medium generated 32. The
hydrolysis of the methyl or ethyl ester afforded 29, 31, and 33.
The nucleophilic substitution of 27 with brominated
alkylnitriles or the aza-Michael addition reaction with
acrylonitrile generated 34−36. The [2 + 3] cycloaddition
reaction of 35 and 36 produced 37 and 38 with moderate yield.
Without a side chain, compound 27 showed poor inhibitory
potency. Compounds 29, 31, and 33, in which carboxylic acid
side chains were predicted to form charge−charge interactions
with charge button K435, exhibited a much higher inhibitory
potency. Their methyl or ethyl esters (compounds 28, 30, and
32) were poor inhibitors. The nitrile derivatives, 34−36, also
exhibited poor inhibitory activity. When the nitrile group was
converted to a tetrazole moiety (compounds 37 and 38), a
bioisostere of carboxylic acid, the inhibitory potency was
improved. Among these inhibitors, compound 33 exhibited the
highest inhibitory potency for the β-catenin/Tcf PPI in the
biochemical assays (Figure 5E).
The FP selectivity assay61 was used to quantify inhibitor
selectivities between β-catenin/Tcf, β-catenin/cadherin, and β-
catenin/APC PPIs. As shown in Figure 5F, compounds 26, 29,
31, 33, and 37 show dual selectivity for β-catenin/Tcf over β-
catenin/cadherin and β-catenin/APC PPIs. Compound 18
exhibits marginal selectivity among these three PPIs, again
implying off-target effects. The selectivity of known β-catenin/
Tcf inhibitors were also listed for comparison.57,61 It is worth
noting that the data in Figure 5F reflect inhibitory potency
under the assay conditions used. More analysis will be required
to fully understand the selectivity of the inhibitors for β-
catenin/Tcf over β-catenin/cadherin and β-catenin/APC PPIs.
Biological Characterization of the Derivatives of 18 as
β-Catenin/Tcf Inhibitors. The Glide docking mode of 33 and
37 with β-catenin is shown in Figure 6A,B. The tetracyclic ring
of 33 and 37 was predicted to form a cation−π interaction with
the positively charged gaunidino group of R469. The benzene
moiety of the tetracylic ring was predicted to locate in
hydrophobic pocket B. Two nitrogen atoms of the
oxadiazolopyrazine ring of 33 and 37 were predicted to form
H-bonds with K508. The carboxylic group of 33 and the
tetrazole ring of 37 were predicted to form a salt bridge with
K435 and H-bonds with the backbone NH group of N470 and
the hydroxyl group of S473. Site-directed mutagenesis was
performed to evaluate the contribution of three residues, R469,
V511, and I569, to inhibitor potency. As shown in Figure 6C,
the Ki value of 33 for the wild-type β-catenin/wild-type Tcf4
interaction was 3.4 3.3 μM. The Ki values of 33 for β-catenin
V511S mutant/wild-type Tcf4, β-catenin V511S/I569S double
mutant/wild-typeTcf4, and β-catenin R469A mutant/wild-type
Tcf4 interactions were 17 1.4, 59 4.2, and 42 3.5 μM,
respectively. The same trend was also observed for 37. These
results suggest the importance of the hydrophobicity in pocket
B and the positively charged side chain of β-catenin R469 for
the inhibitory potency of 33 and 37.
The Wnt-responsive luciferase reporter assay was performed
with pcDNA3.1−β-catenin transfected HEK293 cells for 26,
30, 32, 33, and 37. Compounds 26, 30, 32, and 33 did not
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for the inhibition of two other target genes, cyclin D1 and c-myc.
This compound did not affect the expression of house-keeping
gene HPRT in the parallel assay. The MTs cell viability assay
was performed to assess the inhibitory effects of 26, 30, 32, 33,
35, and 37 on the growth of colorectal cell lines SW480 and
HCT116. Compounds 26, 30, 32, and 33 did not exhibit the
inhibitory activities. Compound 35 inhibited the growth of
SW480 but not HCT116 cells. Compound 37 inhibited the
growth of Wnt-activated cancer cells with the IC50 values of 2.0
× 101 4.1 μM and 31 6.0 μM for SW480 and HCT116
cells, respectively, as shown in Figure 7D.
DISCUSSION
■
Dynamic combinatorial chemistry is a powerful in situ fragment
assembly technique for the discovery of bioactive small
molecules.84 The reaction to form an acyl hydrazone moiety
has been preferred in DCC because it offers a favorable kinetic
and thermodynamic balance for product formation. The acyl
hydrazone structure has both H-bond donor and acceptor sites
for molecular recognition and improves the success rate of the
DCC experiments. Along with the application of the acyl
hydrazone moiety in DCC, many biologically active com-
pounds that contain an acyl hydrazone moiety have been
reported, but few were advanced to the late stage of drug
development. Hence, it is necessary to evaluate the biochemical
and cell-based activities of acyl hydrazones in parallel and
design possible strategies for inhibitor optimization.
Figure 6. Biochemical characterization of 33 and 37 as β-catenin/Tcf
inhibitors. (A,B) Glide docking models of 33 and 37 with β-catenin.
The stick models are shown in Supporting Information Figure S12.
(C) Results of the FP competitive inhibition assay to determine the
effects of site-directed mutagenesis studies for 33 and 37. Each set of
data is expressed as mean standard deviation (n = 3). Details are in
Supporting Information Figure S13.
inhibit the transactivation of canonical Wnt signaling
presumably due to low cell permeability. However, compound
37 suppressed the TOPFlash luciferase activity in a dose-
dependent manner, as shown in Figures 7A. This compound
did not affect the FOPFlash luciferase activity even at 200 μM
(Figure 7B). Compound 37 also down-regulated the expression
of Wnt-specific target gene AXIN2 in a dose-dependent manner
in SW480 cells (Figure 7C). The same trend was also observed
The hyperactivation of the canonical Wnt signaling pathway
causes the growth and progression of many cancers and
fibroses. Human cancers also have a subpopulation of cancer
stem cells that drive tumor growth, seed metastasis, and induce
cancer recurrence after remission. They must be eradicated to
achieve a durable cancer cure. Canonical Wnt signaling is highly
activated in cancer stem cells. The formation of the β-catenin/
Tcf complex in the cell nucleus is the key downstream effector
Figure 7. Cell-based characterization of 37 as a β-catenin/Tcf inhibitor. (A,B) TOPFlash and FOPFlash luciferase reporter assay results of 37 using
pcDNA3.1−β-catenin transfected HEK293 cells. The data are expressed as mean standard deviation (n = 3). (C) Quantitative real time PCR study
to determine the changes of mRNA expression of AXIN2 and HPRT in response to different concentrations of 37. Each set of data is expressed as
mean standard deviation (n = 3). (D) MTs assay to monitor the inhibitory effects of 35 and 37 on the growth of colorectal cancer cells SW480
and HCT116. Each set of data is expressed as mean standard deviation (n = 3).
H
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of canonical Wnt signaling. The selective inhibition of the β-
catenin/Tcf PPI represents an appealing therapeutic target. β-
Catenin and Tcf have a large contacting surface area and a tight
binding affinity. This PPI target has been challenging for
compound screening campaigns. In our screening using the
homogeneous AlphaScreen and FP assays, compound 18 that
contains an acyl hydrazone group was identified as a β-catenin/
Tcf inhibitor. Its inhibitory activity was further confirmed by
the cell-based coimmunoprecipitation study. This compound
blocks the transactivation of canonical Wnt signaling,
suppresses the expression of Wnt/β-catenin target genes, and
inhibits the growth of colorectal cancer cells in dose-dependent
manners. The cell-based activities of 18 were higher than that
from the biochemical assays and 18 inhibited the FOPFlash
luciferase activity, suggesting off-target effects.
The blind docking study predicted that 18 bound the Tcf4
G13ANDE17 binding site of β-catenin. The site-directed
mutagenesis experiments indicated that the residues that are
lined with the Tcf4 G13ANDE17 binding site were sensitive to
inhibitor binding. The previous crystallographic and biochem-
ical studies have demonstrated that β-catenin used the same
binding groove to interact with Tcf, cadherin, and APC with
the central binding area called the charge-button-recognition
region. Our study concluded that three hot regions of β-catenin
contributed differently to the β-catenin/Tcf, β-catenin/
cadherin, and β-catenin/APC PPIs.33,57 Hot region 1 of β-
catenin is more important for the β-catenin/Tcf PPI than for
the other two PPIs. The peptidomimetic inhibitor of Tcf4
G13ANDE17 exhibits dual selectivity for β-catenin/Tcf over β-
catenin/cadherin and β-catenin/APC PPIs.57 The result that
the Tcf4 G13ANDE17 binding site of β-catenin was sensitive to
the inhibitory activity of 18 intrigued us to conduct a structure-
based optimization to replace the potential reactive moiety in
18, albeit that 18 exhibited a low selectivity between these three
protein−protein complexes. Through the replacement of the
acyl hydrazone moiety with a chemically stable substructure,
compounds 26 and 33 were discovered to exhibit comparable
inhibitory activities as 18, however, they showed much higher
selectivity for β-catenin/Tcf over β-catenin/cadherin and β-
catenin/APC PPIs. The results of the site-directed mutagenesis
study and the SAR analysis were in consistent with the
proposed binding modes. Compounds 26, 32, and 33 did not
exhibit cell-based activity. Further optimization resulted in 37
that was active in both biochemical and cell-based assays. This
compound selectively inhibited the transactivation of canonical
Wnt signaling.
catenin/Tcf PPI in both biochemical and cell-based assays,
inhibit the transactivation of canonical Wnt signaling, and
down-regulate the expression of the target genes that are
specific for the canonical Wnt signaling pathway. However, this
compound exhibits a higher inhibitory activity in cell-based
assays than biochemical assays and inhibits the FOPFlash
luciferase activity. A close examination identified that the acyl
hydrazone moiety in 18 is reactive and might cause off-target
effects. On the basis of the proposed binding mode, compound
18 was optimized into chemically stable derivatives. Com-
pounds 26, 29, 31, 33, and 37 were found to exhibit dual
inhibitory selectivity. The binding mode of these inhibitors with
β-catenin was characterized by the site-directed mutagenesis
study and the SAR analysis. These results again indicated that
targeting the Tcf4 G13ANDE17 binding site of β-catenin with small
molecules can selectively inhibit the β-catenin/Tcf PPI.57
Compound 37 was found to block the transactivation of
canonical Wnt signaling, suppress the expression of Wnt-
specific target genes, and inhibit the growth of cancer cells. Its
cell-based inhibitory activities are aligned with that from the
biochemical assays.
For many PPI targets, it is difficult to discover PAINS-free
inhibitors by either high-throughput screening or fragment-
based screening. This study provides a new path to approaching
chemically stable hits through the optimization of the acyl
hydrazone moiety after the initial hit is discovered. The new
chemically stable and selective β-catenin/Tcf inhibitors with a
new scaffold reported in this study provide an excellent starting
point to generate more potent and more selective inhibitors
specific for the β-catenin/Tcf PPI. Indeed, the inhibitory
selectivities of new derivatives for β-catenin/Tcf over β-
catenin/cadherin and β-catenin/APC PPIs are still relatively
low. However, in consideration that the Kd value of the β-
catenin/Tcf PPI is much lower than that for the β-catenin/
cadherin and β-catenin/APC PPIs, the inhibitory selectivities of
26, 29, 31, 33, and 37 are significant. Further, the selectivity
profile of the inhibitors for the β-catenin/Tcf PPI over the β-
catenin/cadherin and β-catenin/APC PPIs can be optimized.
Pockets B and C in parts A and B of Figure 6 are only
important for the β-catenin/Tcf PPI. The modification of the
inhibitors by better occupying pockets B and C would lead to
new inhibitors with better potency and selectivity.
EXPERIMENTAL SECTION
■
Protein Expression and Purification. β-Catenin V511S, V511S/
I569S, and R469A mutants have been made previously.33,57 Wild-type
β-catenin and its mutants (residues 138−686) were cloned into a pET-
28b vector carrying a C-terminal 6× histidine (Novagen) or a
pEHISTEV vector carrying an N-terminal 6× histidine (from Dr.
Hanting Liu, St. Andrew University, UK) and transformed into
Escherichia coli BL21 DE3 (Novagen). Cells were cultured in LB
medium with 30 μg/mL kanamycin until the OD600 was approximately
0.8, and then protein expression was induced with 400 μM of IPTG at
20 °C overnight. Cells were lysed by sonication. The proteins were
purified by Ni-NTA affinity chromatography (30210, Qiagen) and
dialyzed against a buffer containing 20 mM of Tris (pH 8.8), 100 mM
NaCl, 10% glycerol, and 3 mM DTT. The purity of β-catenin was set
to >95% as determined by SDS-PAGE gel analysis. Native non-
denaturing gel electrophoresis experiment and the thermal-shift assay
on an iCycler iQ real-time detection system (Bio-Rad) were
performed for each purified protein. In the thermal shift assay, protein
unfolding was evaluated through measuring the fluorescence changes
of fluorescent dye Sypro Orange with purified β-catenin proteins. A
temperature increment of 1 °C/min was applied to monitor protein
stability and detect protein aggregation. CD spectra were measured on
CONCLUSION
■
Most PPIs for which potent small-molecule inhibitors are
discovered have a contacting surface area of <2500 Å2. The
discovery of small-molecule inhibitors for the PPI that has a
larger contacting surface areas is challenging.85 It is even more
challenging when the same protein interface is used to bind
multiple different protein partners. The β-catenin/Tcf PPI
represents such a case. β-Catenin/Tcf, β-catenin/cadherin, and
β-catenin/APC PPIs share the same surface area of β-catenin.
Furthermore, β-catenin and Tcf have a large contacting surface
area (3500 Å2 for β-catenin/Tcf4 PPIs, and 3700 Å2 for β-
catenin/Lef1 PPIs) and a strong binding affinity (Kd = 7−10
nM for β-catenin/Tcf4 PPIs). Low hit rates have been
associated with compound screening efforts for this target.
Our screening based on the homogeneous FP and AlphaScreen
assays identified a small molecule, 18, that can disrupt the β-
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a J-815 spectropolarimeter (Jasco). All spectra were recorded using a 1
mm path-length quartz cell. The CD spectra were averaged over three
scans, and the wavelength was scanned from 260 to 190 nm in steps of
1 nm. All spectra were recorded at room temperature, and the baseline
was corrected by subtracting the CD spectra of a blank control
containing all of the substances except protein. Samples were prepared
at a concentration around 1−5 μM in a buffer of 10 mM potassium
phosphate and 100 mM potassium fluoride at pH 7.0 to ensure that
the transmission of light through the sample was not restricted. All
proteins were stable, and no aggregation was observed under storage
or assay conditions. Proteins were aliquoted and stored at −80 °C. C-
Terminally biotinylated human Tcf4 (residues 7−51), C-terminally
fluorescein-labeled human Tcf4 (residues 7−51), C-terminally
fluorescein-labeled human E-cadherin (residues 819−873), and C-
terminally fluorescein-labeled human APC-R3 (residues 1477−1519)
were synthesized by InnoPep, Inc. (http://www.innopep.com/) and
HPLC purified with purity >95%. The structures were validated by
LC/MS (liquid chromatography/mass spectrometry). The peptide
sequences are shown in Supporting Information Figure S14.
FP and AlphaScreen Assays. The procedures for the FP and
AlphaScreen competitive inhibition assays have been described
previously58,61 Briefly, all of the tested compounds were prepared as
10 mM DMSO stocks. In the primary screen, the concentrations of the
compounds and DMSO were set to 50 μM and 1% (v/v). Only the
compounds which AlphaScreen or FP signal decreases were greater
than 50% in the single-point β-catenin/Tcf assay were evaluated by
counter screen. Compounds that were confirmed active in the
competitive inhibition assay and inactive in the counter screen were
further evaluated with the dose−response relationship. In the FP
competitive inhibition assay, 10 nM human β-catenin and 2.5 nM of
C-terminally fluorescein-labeled human Tcf4 were incubated in an
assay buffer of 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM
KH2PO4, 100 μg/mL of bovine γ-globulin, and 0.01% Triton-X100 for
15 min at 4 °C. Bovine γ-globulin and Triton-X100 were included in
the assay buffer to decrease the likelihood of compound acting by
aggregate formation. Different concentrations of the tested com-
pounds in the assay buffer were added to each test plate to make a final
volume of 100 μL. Each assay plate was covered black and gently
mixed on an orbital shaker at 4 °C for 1.5 h to reach equilibrium
before the polarization values were read. The IC50 value was
determined by nonlinear least-squares analysis of GraphPad Prism
5.0. The Ki values were derived.86 Experiments were performed in
triplicate and carried out in the presence of 1% DMSO. For the β-
catenin/E-cadherin inhibition assay, 150 nM human β-catenin was
incubated with 5 nM of C-terminally fluorescein-labeled human E-
cadherin in the assay buffer for 30 min at 4 °C. Different
concentrations of the tested compounds in the assay buffer were
added to each test plates to make a final volume of 100 μL. For the β-
catenin/APC-R3 inhibition assay, 1440 nM human β-catenin was
incubated with 5 nM of C-terminally fluorescein-labeled human APC-
R3 in the assay buffer for 30 min at 4 °C. Different concentrations of
the tested compounds in the assay buffer were added to each test plate
to make a final volume of 100 μL. In the AlphaScreen competitive
inhibition assay for the β-catenin/Tcf4 PPI, 5 nM of C-terminally
biotinylated human Tcf4 and 20 nM of N-terminally His6-tagged
human β-catenin were incubated in an assay buffer of 20 mM HEPES
(pH 7.4), 100 mM NaCl, 0.1% BSA, and 0.001% Triton X-100 at 4 °C
for 15 min. Different concentrations of the tested compounds (10−12
compound concentrations typically for each compound) were added
in 20 μL of assay buffer. The assay plates were covered black and
gently mixed on an orbital shaker at 4 °C for 45 min. The streptavidin-
coated donor beads and the nickel chelate acceptor beads were added
to a final concentration of 10 μg/mL in 25 μL assay buffer. The
mixture was incubated for 2 h at 4 °C before detection. The IC50 value
was then determined by nonlinear least-squares analysis of GraphPad
Prism 5.0, and the Ki values were derived.86
Compound 19 was purchased from InterBioScreen (Moscow, Russia)
with the catalogue number of STOCK1S-52622. Compounds 1 and 2
were purchased from Santa Cruz Biotechnology (Santa Cruz,
California, USA) with the catalogue numbers of SC-3545 and SC-
255013. Compound 3 was purchased from Calbiochem (Billerica,
Massachusetts, USA) with the catalogue number of 219331.
Compounds 8 and 9 were resynthesized in our lab.
MTs Cell Viability Assay. Colorectal cancer cell lines, SW480,
HT29, an HCT116, and human normal cell line HEK293 were seeded
in 96-well plates at 4 × 103 cells/well, maintained overnight at 37 °C
and incubated with the tested compounds at various concentrations.
Cell viability was monitored after 72 h using a freshly prepared mixture
of 1 part phenazine methosulfate (PMS, Sigma) solution (0.92 mg/
mL) and 19 parts 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-
yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTs, Promega) solution
(2 mg/mL). Cells were incubated in 10 μL of this solution at 37 °C for
3 h, and A490 was measured. The effect of each compound is expressed
as the concentration required to reduce A490 by 50% (IC50) relative to
DMSO-treated cells. Experiments were performed in triplicate.
Cell Transfection and Luciferase Assay. FuGENE 6 (E2962,
Promega) in a 96-well plate format was used for the transfection of
HEK293 and SW480 cells according to the manufacturer’s
instructions. HEK293 cells were cotransfected with 45 ng of the
TOPFlash or FOPFlash reporter gene, 135 ng of pcDNA3.1−β-
catenin and 20 ng of pCMV-RL normalization reporter gene. SW480
cells were cotransfected with 60 ng of the TOPFlash or FOPFlash
reporter gene and 40 ng of pCMV-RL normalization reporter. Cells
were cultured in DMEM and 10% fatal bovine serum at 37 °C for 24 h,
and different concentrations of inhibitors were then added. After 24 h,
the luciferase reporter activity was measured using the Dual-Glo
system (E2940, Promega). Normalized luciferase activity in response
to the treatment with the inhibitors was compared with that obtained
from the cells treated with DMSO. Experiments were performed in
triplicate.
Quantitative Real Time PCR Analysis. SW480 cells at 1 × 106/
mL were treated with different concentrations of the tested
compounds for 24 h. Total RNAs were extracted with TRIzol
(15596026, Life Technologies), and the cDNA was synthesized with
the superscript III first-strand kit (18080−051, Invitrogen).
Quantitative real-time PCR was performed using the iQ SYBR green
supermix kit (170−8880, BIO-RAD) on an iQ5 multicolor real-time
PCR reaction system (BIO-RAD). The threshold cycle (CT) values
were normalized to that of internal reference GAPDH. The primer
pairs for human GAPDH were forward, 5′-GAAGGTGAAGGTCG-
GAGTC-3′, and reverse, 5′-GAAGATGGTGATGGGATTTC-3′; for
human HPRT, forward, 5′-GCTATAAATTCTTTGCT-
G A C C T G C T G - 3 ′ , a n d r e v e r s e , 5 ′ - A A T T A C T T T -
TATGTCCCCTGTTGACTGG-3′; for human AXIN2, forward, 5′-
AGTGTGAGGTCCACGGAAAC-3′, and reverse, 5′-CTTCA-
CACTGCGATGCATTT-3′; for human c-myc, forward, 5′-
CTTCTCTCCGTCCTCGGATTCT-3′, and reverse, 5′-GAAGGT-
GATCCAGACTCTGACCTT-3′; and for human cyclin D1, forward,
5′-ACAAACAGATCATCCGCAAACAC-3′, and reverse, 5′-
TGTTGGGGCTCCTCAGGTTC-3′. Experiments were performed
in triplicate.
Western Blotting. SW480 cells at 1 × 106 cells/mL were treated
with different concentrations of 18 for 24 h. Cell were lysed in a buffer
containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors. After
centrifugation at 12000 rpm for 20 min at 4 °C, the supernatant was
loaded onto an 8% SDS polyacrylamide gel for electrophoretic
analysis. Separated proteins were transferred onto nitrocellulose
membranes for immunoblot analysis. The antibodies against c-myc
(D84C12, Cell Signaling), cyclin D1 (sc-853, Santa Cruz Biotechnol-
ogy, Inc.), and β-tubulin (sc-55529, Santa Cruz Biotechnology, Inc.)
were used. IRDye 680LT goat antimouse IgG (827−11080, LiCOR)
or IRDye 800CW goat antirabbit IgG (827−08365, LiCOR) was used
as the secondary antibody. The images were detected by the Odyssey
infrared imaging system (LiCOR). The Western blot bands were
Compounds Sources. Compounds 15−17 and 22−25 were
purchased from Sigma-Aldrich. Compounds 18 and 21 were
purchased from Zelinsky Institute, Inc. (Newark, Delaware, USA)
with the catalogue numbers of UZI/7116003 and UZI/2587998.
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quantified by LI-COR Image Studio Lite 4.0. Experiments were
performed in duplicate.
mass spectrometer with an ESI source. Thin-layer chromatography
was carried out on E. Merck precoated silica gel 60 F254 plates with
visualization accomplished with phosphomolybdic acid or ninhydrin
spray reagent or with a UV−visible lamp. Column chromatography
was performed with SiliaFlash F60 (230−400 mesh).
9H-Indeno[1,2-b][1,2,5]oxadiazolo[3,4-e]pyrazin-9-one (19). To
a solution of ninhydrin (0.32 g, 1.80 mmol) in a solvent mixture
(ethanol:glacial acid = 1:1, 10 mL) was added 1,2,5-oxadiazole-3,4-
diamine (0.18 g, 1.80 mmol). After stirring for 18 h at room
temperature, the mixture was heated to gentle reflux for another 6 h. It
was then cooled to room temperature, and the resulting precipitate
was filtered and washed with water to yield 19 as pale-yellow solid
(0.29 g, 72% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm 8.31 (d, J
= 7.2 Hz, 1H), 8.18−8.02 (m, 2H), 7.97 (d, J = 7.2 Hz, 1H). 13C NMR
(125 MHz, DMSO-d6) δ ppm 189.55, 161.70, 140.01, 139.95, 138.69,
136.52, 126.06. HRMS (ESI) calcd for C11H4N4O2 (M + Na)+
247.0232, found 247.0230.
(Z)-9-Hydrazono-9H-indeno[1,2-b][1,2,5]oxadiazolo[3,4-e]-
pyrazine (20). To a solution of 19 (0.15 g, 0.67 mmol) in a mixture of
ethanol and glacial acid (10 mL, 1:1) was added hydrazine hydrate
(0.33 g, 6.70 mmol). The mixture was heated to gentle reflux for 2 h
and then cooled to room temperature. The resulting precipitate was
filtered and washed with water to afford 20 as red solid (0.14 g, 93%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm 10.48 (d, J = 13.8 Hz,
1H), 10.40 (d, J = 13.8 Hz, 1H), 8.07 (d, J = 7.5 Hz, 1H), 7.73−7.66
(m, 2H), 7.46 (t, J = 7.5 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) δ
ppm 161.75, 155.02, 152.69, 152.51, 144.75, 135.86, 129.81, 128.86,
128.48, 124.66, 119.65. HRMS (ESI) calcd for C11H6N6O (M − H)−
237.0525, found 237.0531.
Coimmunoprecipitation Assay. SW480 cells at 1 × 106/mL
were treated with different concentrations of 18 for 24 h. Cells were
lysed in buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1%
Nonidet P-40, 2 mM EDTA, and protease inhibitors. The lysates were
preadsorbed to A/G plus agarose (sc-2003, Santa Cruz Biotechnology,
Inc.) at 4 °C for 1 h. Preadsorbed lysates were incubated with a
specific primary antibody overnight at 4 °C. A/G plus agarose was
then added to the lysates mixture and incubated for 3 h. The beads
were washed 5 times with the lysis buffer at 4 °C. The bound protein
was eluted by boiling in the SDS sample buffer and loaded onto 8%
SDS polyacrylamide gel for electrophoretic analysis. Separated
proteins were transferred onto nitrocellulose membranes for
immunoblot analysis. The primary antibodies were against β-catenin
(610153, BD Biosciences) and Tcf4 (05−511, Millipore). IRDye
680LT goat antimouse IgG (827-11080, LiCOR) was used as the
secondary antibody. The images were detected by the Odyssey
infrared imaging system (LiCOR). Experiments were performed in
duplicate.
Ligand Docking using AutoDock Vina. Autodock Vina was
used for the initial blind docking of 18. Exhaustiveness was increased
to 12 (exhaustiveness = 1 2), and 18 ligand conformations
(num_modes =18) were generated for each binding region. All
other parameters were left as default values.
Ligand Docking using Glide 5.8. The 3-D coordinates of all
ligands were generated by Schrodinger LigPrep with Epik to expand
the protonation and tautomeric states at pH = 7.0. The energy
minimization was then applied to all ligands with the OPLS_2005
force field and the GB/SA water solvation condition. The partial
charges of the ligands were calculated with the OPLS_2005 force field.
The grid box was defined to include all the residues in the Tcf4
G13ANDE17 binding site.57 The default parameters were used in
receptor grid generation. The standard precision mode was used in
ligand docking. The ligand scaling factor was set to 0.5 for the atoms
with the partial charges lower than 0.15. The number of poses per
ligand for the initial phase of docking was increased to 10000. The
1000 best poses per ligand were kept for energy minimization with a
maximum number of the minimization steps of 5000. A maximum of
100000 ligand poses per docking run and 50 poses per ligand were
collected. Up to 100 poses per ligand were kept for the postdocking
minimization. The default settings were used for the remaining
parameters.
Ligand Docking using AutoDock 4.2. The partial atomic
charges were calculated using the Gasteiger−Marsili method. The
rotatable bonds in the ligands were defined using AutoTors, which also
united the nonpolar hydrogens and partial atomic charges to the
bonded carbon atoms. The grid maps were calculated using AutoGrid.
The AutoDock area was defined to include all the residues of the Tcf4
G13ANDE17 binding site, and the grid spacing was set to 0.375 Å.
Docking was performed using the Lamarckian genetic algorithm, and
the pseudo-Solis and Wets method was applied for the local search.
Each docking experiment was performed 100 times, yielding 100
docked conformations. The other settings were the default parameters.
All of the ligands followed the same docking protocol. The results of
the docking experiments were evaluated by the auxiliary clustering
analysis and the visual inspection.
4-Amino-1,2,5-oxadiazole-3-carbohydrazide (40). To a solution
of 4-amino-1,2,5-oxadiazole-3-carboxylic acid (0.50 g, 3.87 mmol) in
methanol (30 mL) was bubbled HCl (gas). The mixture was heated to
gentle reflux for 8 h. Methanol was then removed under vacuum to
give the crude product as pale-yellow oil. To this residue was added
diethyl ether (30 mL) and stirred for 30 min. The resulting precipitate
was filtered to give desired product 39 (0.50 g, 91% yield) as white
solid. It was used directly in next step without further purification. To
a solution of 39 (0.15 g, 1.05 mmol) in methanol (10 mL) was added
hydrazine hydrate (0.08 g, 1.57 mmol), and the mixture was heated to
gentle reflux. After 15 h, the solvent was removed completely under
1
vacuum to give 40 (0.09 g, 60% yield) as white solid. H NMR (500
MHz, acetone-d6) δ ppm 9.50 (brs, 1H), 5.76 (s, 2H), 4.50 (brs, 2H).
13C NMR (-d6) δ ppm 157.02, 156.06, 140.00. HRMS (ESI) calcd for
C3H5N5O2 (M + H)+ 144.0516, found 144.0519.
(Z)-4-Amino-N′-(9H-indeno[1,2-b][1,2,5]oxadiazolo[3,4-e]-
pyrazin-9-ylidene)-1,2,5-oxadiazole-3-carbohydrazide (18). To a
solution of 19 (0.05 g, 0.22 mmol) in ethanol (10 mL) was added
40 (0.03 g, 0.22 mmol) and glacial acid (1 mL). The reaction mixture
was heated to gentle reflux overnight and then poured into ice water.
The resulting precipitate was filtered and washed with water to give 18
as yellow solid (0.06 g, 78% yield). 1H NMR (500 MHz, DMSO-d6) δ
ppm 13.55 (brs, 1H), 8.26 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 7.0 Hz,
1H), 7.92 (t, J = 7.5 Hz, 1H), 7.82 (t, J = 7.0 Hz, 1H), 6.01 (s, 2H).
13C NMR (125 MHz, DMSO-d6) δ ppm 162.75, 158.62, 157.14,
155.38, 153.17, 152.29, 152.04, 144.32, 140.35, 136.76, 134.59, 133.67,
125.23, 122.82. HRMS (ESI) calcd for C14H7N9O3 (M − H)−
348.0594, found 348.0604.
Chemical Synthesis. General Methods, Reagents, and Materi-
als. All reagents were purchased from Aldrich and Acros Organics and
used without further purification unless stated otherwise. 1H NMR and
13C NMR spectra were recorded on a Varian VXR-500 (500 MHz), a
Varian Inova-400 (400 MHz), or a Varian Unity-300 (300 MHz)
spectrometer (125.7, 100, and 75 MHz for 13C NMR spectra,
respectively) in DMSO-d6, acetone-d6, and CDCl3. Chemical shifts
were reported as values in parts per million (ppm), and the reference
resonance peaks were set at 7.26 ppm (CHCl3), 2.50 ppm
[(CD2H)2SO], 2.05 ppm [(CD2H)2CO] for the 1H NMR spectra
and 77.23 ppm (CDCl3), 39.52 ppm (DMSO-d6), and 29.84 ppm
(acetone-d6) for the 13C NMR spectra. Low-resolution and high-
resolution mass spectra were determined on a Micromass Quattro II
5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indole (27). To a
solution of isatin (0.20 g, 1.36 mmol) in glacial acid (5 mL) was added
1,2,5-oxadiazole-3,4-diamine (0.14 g, 1.36 mmol). The resulting
mixture was heated to gentle reflux for 15 h and then poured into
ice water. The resulting precipitate was filtered and washed with water
1
to afford 27 as red solid (0.05 g, 17% yield). H NMR (300 MHz,
DMSO-d6) δ ppm 12.35 (brs, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.73 (t, J
= 7.5 Hz, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H). 13C
NMR (75 MHz, DMSO-d6) δ ppm 153.96, 152.62, 152.50, 151.93,
148.81, 136.37, 125.33, 123.11, 118.86, 113.38. HRMS (ESI) calcd for
C10H5N5O (M − H)− 210.0416, found 210.0427.
Ethyl-2-(5H-[1,2,5]oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-
yl)acetate (28). To a solution of 27 (0.08 g, 0.38 mmol) and ethyl 2-
K
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bromoacetate (0.09 g, 0.57 mmol) in DMSO (10 mL) was added
K2CO3 (0.08 g, 0.57 mmol). The resulting mixture was heated to 80
°C overnight. After 15 h, the reaction mixture was cooled to room
temperature and diluted with ethyl acetate (80 mL), washed with brine
(20 mL × 3), dried over Na2SO4, filtered, and concentrated. The
residue was purified by column chromatography (silica gel,
hexanes:acetone = 5:1) to afford 28 (0.08 g, 73% yield) as red solid.
1H NMR (500 MHz, DMSO-d6) δ ppm 8.28 (d, J = 7.5 Hz, 1H), 7.85
(t, J = 7.0 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H),
5.22 (s 2H), 4.18 (q, J = 7.0 Hz, 2H), 1.21 (t, J = 7.0 Hz, 3H). 13C
NMR (125 MHz, DMSO-d6) δ ppm 167.98, 153.15, 152.32, 152.07,
151.64, 148.92, 136.50, 125.30, 124.05, 118.50, 112.39, 62.25, 43.35,
14.70. HRMS (ESI) calcd for C14H11N5O3 (M + Na)+ 320.0760,
found 320.0759.
2-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)acetic
Acid (29). To a solution of 28 (0.08 g, 0.27 mmol) in a solvent mixture
(14 mL, THF:MeOH:H2O = 4:2:1) was added LiOH (0.05 g, 2.15
mmol). The mixture was stirred for 8 h at room temperature. Then,
the pH value was adjusted to 4−5 with HCl (1 M), diluted with water
(50 mL), and extracted with ethyl acetate (20 mL × 3). The combined
organic phase was dried over Na2SO4 and concentrated to give 29
(0.05g, 68%) as orange solid. 1H NMR (500 MHz, DMSO-d6) δ ppm
13.36 (brs, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.84 (t, J = 8.0 Hz, 1H), 7.69
(d, J = 8.0 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 5.10 (s, 2H). 13C NMR
(125 MHz, DMSO-d6) δ ppm 169.34, 153.20, 153.00, 152.11, 151.63,
149.10, 136.51, 125.27, 123.95, 118.44, 112.38, 43.38. HRMS (ESI)
calcd for C12H7N5O3 (M − H)− 268.0471, found 268.0485.
2-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-N-(4-
amino-1,2,5-oxadiazol-3-yl)acetamide (26). To a solution of 29
(0.18 g, 0.67 mmol) and 4-methylmorpholine (0.14 g, 0.34 mmol) in
THF (15 mL) was added isobutyl chloroformate (0.09 g, 0.67 mmol)
at −15 °C. The resulting mixture was stirred for 1 h at the same
temperature. Then 1,2,5-oxadiazole-3,4-diamine (0.10 g, 1.00 mmol)
was added slowly. The temperature was allowed to rise to room
temperature gradually and stirred for another 1 h. The mixture was
diluted with ethyl acetate (80 mL), washed with brine (20 mL × 3),
dried over Na2SO4, filtrated, and concentrated. The residue was
purified by column chromatography (silica gel, hexanes:acetone = 2:1
to 1:1) to afford 26 (0.12 g, 51% yield) as orange solid. 1H NMR (500
MHz, DMSO-d6) δ ppm 11.13 (brs, 1H), 8.32 (d, J = 7.5 Hz, 1H),
7.87 (t, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.45 (t, J = 7.5 Hz,
1H), 6.04 (s, 2H), 5.29 (s, 2H). 13C NMR (125 MHz, DMSO-d6) δ
ppm 166.66, 153.50, 152.88, 152.36, 152.13, 151.96, 149.26, 144.25,
136.50, 125.26, 124.04, 118.69, 112.57, 44.85. HRMS (ESI) calcd for
C14H9N9O3 (M − H)− 350.0750, found 350.0755.
and dried over Na2SO4, filtrated, and concentrated. The residue was
purified by column chromatography (silica gel, hexanes:acetone = 3:1
to 1:1) to afford 30 as orange solid (0.084 g, yield 60%). H NMR
1
(400 MHz, CDCl3) δ ppm 8.33 (d, J = 7.6 Hz, 1H), 7.79 (t, J = 8.4
Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 4.60 (t, J =
6.8 Hz, 2H), 3.66 (s, 3H), 3.02 (t, J = 6.8 Hz, 2H). 13C NMR (100
MHz, CDCl3) δ ppm 171.27, 151.81, 151.39, 151.16, 150.42, 147.90,
135.48, 125.20, 123.25, 118.42, 110.81, 52.11, 37.90, 21.11. HRMS
(ESI) calcd for C14H11N5O3 (M + Na)+ 320.0760, found 320.0760.
3-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-
1
propanoic Acid (31). Orange solid, and yield 80%. H NMR (400
MHz, DMSO-d6) δ ppm 12.46 (brs, 1H), 8.26 (d, J = 7.6 Hz, 1H),
7.85 (t, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 7.6 Hz,
1H), 4.48 (t, J = 6.8 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H). 13C NMR (100
MHz, DMSO-d6) δ ppm 172.65, 153.56, 152.09, 151.96, 151.49,
148.66, 136.88, 124.99, 123.33, 118.52, 112.25, 32.10, 31.14. HRMS
(ESI) calcd for C13H9 N5O3 (M + Na)+ 306.0603, found 306.0612.
4-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-
butanoic Acid (33). Orange solid, and yield 65%. 1H NMR (400
MHz, DMSO-d6) δ ppm 12.08 (brs, 1H), 8.27 (d, J = 7.6 Hz, 1H),
7.86 (t, J = 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 7.2 Hz,
1H), 4.30 (t, J = 6.8 Hz, 2H), 2.38 (t, J = 7.2 Hz, 2H), 2.07−2.00 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ ppm 174.38, 153.67, 152.21,
151.97, 148.78, 136.10, 125.11, 123.29, 118.60, 111.76, 109.99, 41.07,
31.23, 23.06. HRMS (ESI) calcd for C14H11N5O3 (M + Na)+
320.0760, found 320.0761.
2-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-
acetonitrile (34). To a solution of 27 (0.03 g, 0.14 mmol) in DMF (10
mL) was added NaH (60%) (0.007 g, 0.17 mmol) at 0 °C. The
resulting mixture was stirred for 0.5 h at the same temperature before
2-bromoacetonitrile (0.02 g, 0.17 mmol) was added into it. It was then
allowed to warm to room temperature gradually and stirred for
another 1 h. The reaction mixture was then quenched with water (30
mL) and diluted with ethyl acetate (60 mL). The organic phase was
washed with brine (20 mL × 2), dried over Na2SO4, and concentrated.
The residue was purified by column chromatography (silica gel,
hexanes:acetone = 5:1) to afford 34 (0.025 g, 71% yield) as yellow
1
solid. H NMR (500 MHz, acetone-d6) δ ppm 8.37 (d, J = 8.0 Hz,
1H), 7.97 (t, J = 8.5 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.56 (t, J = 8.0
Hz, 1H), 5.51 (s 2H). 13C NMR (125 MHz, acetone-d6) δ ppm
152.75, 152.33, 151.73, 151.12, 147.17, 135.97, 125.04, 124.21, 119.23,
114.31, 111.31, 29.44. HRMS (ESI) calcd for C12H6N6O (M + Na)+
273.0501, found 273.0508.
4-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-
1
butanenitrile (36). Orange solid, yield 50%. H NMR (400 MHz,
DMSO-d6) δ ppm 7.61−7.64 (m, 2H), 7.16 (t, J = 7.6 Hz, 1H), 6.97
(d, J = 8.0 Hz, 1H), 3.87 (t, J = 7.2 Hz, 2H), 2.48 (t, J = 7.2 Hz, 2H),
2.11−2.07 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ ppm 158.41,
150.19, 138.62, 125.81, 124.16, 118.61, 117.73, 109.77, 38.85, 23.49,
15.02. HRMS (ESI) calcd for C14H10N6O (M + Na)+ 301.0814, found
301.0808.
Ethyl 4-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-
yl)butanoate (32). To a solution of 27 (0.017 g, 0.08 mmol) in
DMF (5 mL) was added NaH (0.0030 g, 0.12 mmol) at 0 °C. The
resulting mixture was stirred for 0.5 h at 0 °C before adding ethyl 4-
bromobutanoate (0.023 g, 0.12 mmol). The reaction solution was
allowed to warm to room temperature gradually then stirred overnight.
The mixture was diluted with ethyl acetate (50 mL), washed with
brine (20 mL × 3), dried over Na2SO4, filtrated, and concentrated.
The residue was purified through column chromatography (silica gel,
hexanes:acetone = 3:1 to 1:1) to afford 32 as orange solid (0.010 g,
3-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-yl)-
propanenitrile (35). To a solution of 27 (0.20 g, 0.95 mmol) in
acetonitrile (10 mL), DBU (0.072 g, 0.47 mmol) was added. The
solution was stirred for 0.5 h. Then, acrylonitrile (0.075 g, 1.4 mmol)
was added. The reaction solution was heated to 50 °C and stirred for
24 h. It was then diluted with ethyl acetate (50 mL), washed with brine
(20 mL × 3), and dried over Na2SO4, filtrated, and concentrated. The
residue was purified by column chromatography (silica gel,
hexanes:acetone = 5:1 to 1:1) to afford 35 as orange solid (yield
1
yield 58%). H NMR (400 MHz, CDCl3) δ ppm 8.20 (d, J = 7.2 Hz,
1H), 7.71 (t, J = 7.2 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.31 (t, J = 7.2
Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H), 4.07 (q, J = 6.0 Hz, 2H), 2.43 (t, J =
6.4 Hz, 2H), 2.20−2.12 (m, 2H) 1.19 (t, J = 6.0 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ ppm 172.54, 151.80, 151.27, 151.19, 150.41,
147.98, 135.56, 125.06, 123.11, 118.23, 110.64, 60.71, 40.93, 30.94,
22.68, 14.12. HRMS (ESI) calcd for C16H15N5O3 (M + Na)+
348.1073, found 348.1081.
Methyl 3-(5H-[1,2,5]Oxadiazolo[3′,4′:5,6]pyrazino[2,3-b]indol-5-
yl)propanoate (30). To a solution of 27 (0.10 g, 0.47 mmol) in
acetonitrile (15 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) (0.24 g, 1.57 mmol). The resulting mixture was stirred for 0.5
h before adding methyl acrylate (0.14 g, 1.57 mmol). The reaction
solution was heated to 50 °C and stirred for another 24 h. It was then
diluted with ethyl acetate (50 mL), washed with brine (20 mL × 3),
1
62%). H NMR (400 MHz, DMSO-d6) δ ppm 8.28 (d, J = 7.6 Hz,
1H), 7.89−7.84 (m, 2H), 7.42 (t, J = 7.2 Hz, 1H), 4.61 (t, J = 6.8 Hz,
2H), 3.10 (t, J = 6.4 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ ppm
153.39, 152.02, 151.94, 151.51, 148.27, 136.17, 125.11, 123.69, 119.10,
118.51, 112.17, 37.66, 16.43. HRMS (ESI) calcd for C13H8N6O (M +
Na)+ 287.0657, found 287.0661.
5-(2-(2H-Tetrazol-5-yl)ethyl)-5H-[1,2,5]oxadiazolo[3′,4′:5,6]-
pyrazino[2,3-b]indole (37). To a solution of 35 (0.02 g, 0.076 mmol)
in toluene (10 mL) was added nBu3SnN3 (0.13 g, 0.38 mmol). The
resulting mixture was heated to reflux for 48 h. It was then cooled to
L
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Journal of Medicinal Chemistry
Article
room temperature, and the pH value was adjusted to 4−5 with HCl (1
M) and diluted with ethyl acetate (60 mL). The organic phase was
washed with brine (20 mL × 2), dried over Na2SO4, and concentrated.
The residue was purified by column chromatography (silica gel,
CH2Cl2:MeOH = 10:1 to 5:1) to afford 37 (15.00 mg, 65% yield) as
red solid. 1H NMR (500 MHz, DMSO-d6) δ ppm 8.28 (d, J = 7.8 Hz,
1H), 7.81 (t, J = 8.1 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 7.8
Hz, 1H), 4.66 (t, J = 6.6 Hz, 2H), 3.46 (t, J = 6.6 Hz, 2H). 13C NMR
(125 MHz, DMSO-d6) δ ppm 154.29, 153.81, 152.22, 152.17, 151.79,
148.66, 136.36, 125.33, 123.69, 118.74, 111.92, 40.36, 22.30. HRMS
(ESI) calcd for C13H9N9O (M − H)− 306.0852, found 306.0871.
5-(3-(2H-Tetrazol-5-yl)propyl)-5H-[1,2,5]oxadiazolo[3′,4′:5,6]-
pyrazino[2,3-b]indole (38). Red solid, yield 57%. 1H NMR (500
MHz, DMSO-d6) δ ppm 8.26 (d, J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz,
1H), 7.72 (d, J = 8.5 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 4.40 (t, J = 6.5
Hz, 2H), 3.02 (t, J = 7.5 Hz, 2H), 2.32−2.26 (m, 2H). 13C NMR (125
MHz, DMSO-d6) δ ppm 153.81, 152.35, 152.18, 151.91, 148.92,
136.35, 125.32, 123.58, 118.82, 112.06, 41.32, 25.55, 21.10. HRMS
(ESI) calcd for C14H11N9O (M + Na)+ 344.0984, found 344.0990.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Assay results, HPLC conditions, and HPLC tracers (PDF,
CSV). The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acs.jmedchem.5b00223.
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AUTHOR INFORMATION
Corresponding Author
■
*Phone: (801) 581-6747. Fax: (801) 581-8433. E-mail:
markji@chem.utah.edu.
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Author Contributions
†J.L.C. and Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was in part supported by the Pulmonary Fibrosis
Foundation I. M. Rosenzweig Young Investigator Award
(award number 235170). We thank David A. Jones at the
Univeristy of Utha for the TOPFlash, FOPFlash, and β-catenin
expression constructs and the Center for High-Performance
Computing at the University of Utah for computer time.
ABBREVIATIONS USED
■
APC, adenomatous polyposis coli; DCC, dynamic combinato-
rial chemistry; FP, fluorescence polarization; Kd, dissociation
constant; LC/MS, liquid chromatography/mass spectrometry;
PPI, protein−protein interaction; PAINS, pan assay interfer-
ence compounds; Tcf, T-cell factor
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