Triazole-Based Inhibitors of the Wnt/β-Catenin Signaling Pathway Improve Glucose and Lipid Metabolisms in Diet-Induced Obese Mice

Article abstract of DOI:10.1021/acs.jmedchem.8b01408

Wnt/β-catenin signaling pathway is implicated in the etiology and progression of metabolic disorders. Although lines of genetic evidence suggest that blockage of this pathway yields favorable outcomes in treating such ailments, few inhibitors have been used to validate the promising genetic findings. Here, we synthesized and characterized a novel class of triazole-based Wnt/β-catenin signaling inhibitors and assessed their effects on energy metabolism. One of the top inhibitors, compound 3a, promoted Axin stabilization, which led to the proteasome degradation of β-catenin and subsequent inhibition of the Wnt/β-catenin signaling in cells. Treatment of hepatocytes and high fat diet-fed mice with compound 3a resulted in significantly decreased hepatic lipid accumulation. Moreover, compound 3a improved glucose tolerance of high fat diet-fed mice without noticeable toxicity, while downregulating the genes involved in the glucose and fatty acid anabolisms. The new inhibitors are expected to be further developed for the treatment of metabolic disorders.

Full text of DOI:10.1021/acs.jmedchem.8b01408

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Article
Triazole-Based Inhibitors of the Wnt/#-Catenin Signaling Pathway
Improve Glucose and Lipid Metabolism in Diet-Induced Obese Mice
Obinna N. Obianom, Yong Ai, yingjun li, Wei Yang, Dong Guo, Hong
Yang, Srilatha Sakamuru, Menghang Xia, Fengtian Xue, and Yan Shu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01408 • Publication Date (Web): 03 Jan 2019
Downloaded from http://pubs.acs.org on January 7, 2019
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Triazole-Based Inhibitors of the Wnt/β-Catenin Signaling Pathway Improve
Glucose and Lipid Metabolism in Diet-Induced Obese Mice
Obinna N. Obianom,#,1 Yong Ai,#,1 Yingjun Li, Wei Yang, Dong Guo, Hong Yang,
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≠,1
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*,1
*,1,3
Srilatha Sakamuru, Menghang Xia, Fengtian Xue and Yan Shu
1Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland 21201, USA
2National Center for Advancing Translational Sciences, National Institutes of Health,
Bethesda, MD 20892-3375, USA
3School and Hospital of Stomatology, Guangzhou Medical University, Guangzhou
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10140, China
#These authors contributed equally to this work.
≠Current address: School of Pharmaceutical Engineering, Jiangsu Food & Pharmaceutical
Science College, Huaian, Jiangsu, 223005, China
*
Correspondence to:
Dr. Fengtian Xue at the Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, 20 Penn Street, Baltimore, Maryland 21201, USA; Phone: 410-706-
8
521; Email: fxue@rx.umaryland.edu
Dr. Yan Shu at the Department of Pharmaceutical Sciences, University of Maryland
School of Pharmacy, 20 Penn Street, Baltimore, Maryland 21201, USA; Phone: 410-706-
7
358; Email: yshu@rx.umaryland.edu
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ABSTRACT
Wnt/β-catenin signaling pathway is implicated in the etiology and progression of metabolic
disorders. While lines of genetic evidence suggest that blockage of this pathway yields
favorable outcomes in treating such ailments, few inhibitors have been used to validate the
promising genetic findings. Here, we synthesized and characterized a novel class of
triazole-based Wnt/β-catenin signaling inhibitors, and assessed their effects on energy
metabolism. One of the top inhibitors, compound 3a, promoted Axin stabilization, which
led to the proteasome degradation of β-catenin and subsequent inhibition of the Wnt/β-
catenin signaling in cells. Treatment of hepatocytes and high fat diet-fed mice with
compound 3a resulted in significantly decreased hepatic lipid accumulation. Moreover,
compound 3a improved glucose tolerance of high fat diet-fed mice without noticeable
toxicity, while downregulating the genes involved in the glucose and fatty acid anabolism.
The new inhibitors are expected to be further developed for the treatment of metabolic
disorders.
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INTRODUCTION
The Wnt/β-catenin pathway plays a pivotal role in cell proliferation, differentiation and
1
growth. It regulates the expression of target genes through the transcriptional factor β-
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catenin that forms a cytoplasmic “destruction complex” with other proteins including Axin
and adenomatous polyposis coli (APC). This complex facilitates the phosphorylation of
β-catenin by casein kinase 1α (CK1α) and glycogen synthase kinase 3β (GSK3β), leading
to proteasome degradation of β-catenin during the “off state” of the pathway. The “on-
state”, on the other hand, involves enhanced stability and accumulation of β-catenin in the
cytoplasm, resulting in its increased translocation to the nucleus where it binds to LEF/TCF
transcriptional factors and activates the expression of target genes. Wnt/β-catenin pathway
crosstalks with many other pathways via key proteins such as β-catenin, Axin, and
GSK3β.1,2 Given the crucial function of Wnt/β-catenin pathway, it is not surprising that a
strong link between this pathway and metabolic disorders has been recognized in recent
years.3-5
The association of the Wnt/β-catenin pathway to metabolic disorders was first
established by the identification of a human polymorphism (rs7903146) in the TCF7L2
gene, which encodes a major nuclear partner protein of β-catenin, as a strong risk factor
for type-2 diabetes.6 This polymorphism has been known to enhance TCF7L2
transcription.7-9 Later, Savic et al showed that partial knockdown of Tcf7l2 results in
metabolic phenotypes of smaller body weights, decreased fasting glucose, and improved
glucose tolerance in mice.10 We have published similar observations in mice with the
genetic haploinsufficiency of Tcf7l2.11 However, the role of TCF7L2 in maintaining
metabolic homeostasis in specific tissues remains controversial. TCF7L2 knockdown was
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reported to increase glucose production and gluconeogenic gene expression in cultured
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hepatocytes, and the transgenic mice overexpressing a dominant negative Tcf7l2 mutant
in the proglucagon gene-expressing cells exhibited defective glucose homeostasis.13
However, Boj et al reported that liver-specific Tcf7l2 knockout led to reduced hepatic
glucose production during fasting and improved glucose homeostasis in adult mice on a
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high-fat diet. Recently, Thompson et al reported that liver-specific knockout of β-catenin
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led to a striking protection from fibrosis and liver injury in mice, while Popov et al
showed that the hepatic downregulation of β-catenin by anti-sense oligonucleotides could
improve insulin sensitivity and glucose tolerance in mice.16 Therefore, although the exact
role of the Wnt/β-catenin pathway in metabolism is yet to be illustrated, overall evidence
indicates that downregulation of Wnt/β-catenin signaling may provide a new therapeutic
strategy for the treatment of metabolic disorders such as fatty liver diseases and diabetes.
Therapeutics targeting the Wnt/β-catenin signaling pathway is still in a state of
infancy. One reason is that this pathway is bewilderingly complex, with crosstalks to
numerous others.17-19 In addition, because of the severe phenotypes observed in genetic
2
0
knockout animal models, safety concern is historically present. Approximately one dozen
of Wnt/β-catenin pathway inhibitors with distinct mechanisms have been discovered.21-33
For example, porcupine inhibitors (e.g., LGK-974, Figure 1) decrease the secretion of the
Wnt ligands.34 At the plasma membrane, the inhibition of LRP5/6 binding to Wnt proteins
by inhibitors such as niclosamide and salinomycin can curb the amount of active β-
catenin.22,35 In the cytoplasm, stabilization of the destruction complex (e.g., tankyrase
25
36
inhibitor XAV939 and CK1α activator pyrvinium ) can also attenuate β-catenin levels.
Of note, the safety concern about Wnt inhibitors has not been borne out either preclinically
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or clinically, and recently the β-catenin/CBP inhibitor PRI-724 has entered clinical trials
as a potential new treatment of various cancers.17 Several FDA-approved drugs, such as
glucocorticoids, retinoids, and celecoxib, are also found to be Wnt/β-catenin pathway
inhibitors.17,19 Interestingly, Wnt/β-catenin pathway inhibitors have emerged to replicate
the metabolic outcomes of the above-mentioned genetic manipulation in mice. The CK2
inhibitor CX-4945 has been shown to cause mice to be resistant to high fat diet-induced
obesity and metabolic disorders.37,38 Treatment of mice with a selective tankyrase inhibitor
G007-LK has resulted in profound improvement of glucose tolerance and insulin
sensitivity.39 However, further studies are needed to fully establish the applicability of
these small molecule inhibitors in the treatment of metabolic disorders.
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Herein we report a series of novel Wnt/β-catenin pathway inhibitors based upon a
triazole scaffold (Figure 1). New compounds were designed by modifying the chemical
structure of pyrvinium, an FDA approved anthelmintic effective for pinworm infection.
3
6
The drug has been reported to inhibit Wnt signaling with a high potency . However,
further development of pyrvinium as a therapeutic agent for metabolic disorders is
prohibited by several reasons related to its chemical structure. Pyrvinium possesses a
permanently charged N-methylquinolone group that causes extremely low bioavailability
of the compound.40 The double bond connecting the two ring systems leads to poor
41
solubility. Moreover, the 2,5-dimethyl-1-phenyl-1H-pyrrole is prone to oxidation and
known as one of the pan assay interference compounds (PAINs).42 Herein we describe the
synthesis of new inhibitors 3a-3u employing a neutral aromatic amino group, an amide
linker, and a substituted triazole core. Several of the new compounds showed excellent
inhibitory potency against Wnt signaling. One of the new compounds, 3a, was further
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characterized by various in vitro and in vivo biological assays. Compound 3a showed
improved bioavailability, promising efficacy against diet-induced metabolic disorders in
mice. In addition, compound 3a was well tolerated in mice without any notable toxicity.
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Figure 1. Structures of known Wnt/β-catenin pathway inhibitors LGK974, PRI724,
XAV939, pyrvinium, and the design of new triazole-based inhibitors 3a-3u.
RESULTS AND DISCUSSION
Synthesis. The synthesis of compounds 3a-3u is detailed in Scheme 1. Diazotization of
the amino group of anilines 1a-1n using sodium nitrite (NaNO
2
) and aqueous HCl,
) gave azides, which
followed by the treatment of the intermediate with sodium azide (NaN
3
underwent cyclization with β-ketoester in EtONa/EtOH yielded trizaole-3-carboxylic acids
2a-2p. Next, PyCIU-mediated coupling of compounds 2a-2p with various aromatic
amines in dichloroethane (DCE) provided target compounds 3a-3r in moderate to good
yields. Finally, Pd-catalyzed cross-coupling of compound 3r with various potassium
trifluoroborate derivatives yielded products 3s-3u in good yields.
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Scheme 1. Synthesis of compounds 3a-3ua
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aReagents and conditions: (a) (i) NaNO
, HCl, NaN
, H O, 0 C; (ii) ethyl 3-oxobutanoate
o
2
3
2
for 2a-2n, ethyl 3-oxopentanoate for 2o, ethyl 4-methyl-3-oxopentanoate for 2p, EtONa,
o
EtOH, 80 C; (b) 2-aminoquinoline for 3a-3p, naphthalen-2-amine for 3q, 6-
o
bromoquinolin-2-amine for 3r, PyCIU, DIPEA, DCE, 80 C; (c) potassium trifluoroborate
o
derivatives, Pd(OAc)
2
, XPhos, Cs
2
CO
3
, THF/H
2
O, 80 C, 24-48 h.
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Structure-Activity Relationship. Compound 3a indicated an excellent IC50 value of 4.1
nM in the luciferase gene reporter assay for Wnt signaling activity, which is over 1,000-
fold higher than that of 3b (3-methyl group) and 3c (4-methyl group), and 180-fold higher
than the parent compound pyrvinium (Table 1). Additional methyl group at the 3- (3l) or
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-position (3m) of the phenyl ring led to new inhibitors with 34- and 66-fold decreased
potencies, respectively. The naphthyl analog 3n was a weak inhibitor with an IC50 value
of 5.5 µM. These results indicated that single ortho-substitution is preferred on the phenyl
ring. Substitution of the 2-methyl group with various functionalities generated new
inhibitors 3d-3k. Among them, the F-analog (3d) demonstrated excellent potency with an
IC50 value in the sub-nanomolar range. The Br- (3e), NC- (3f), and MeO- (3g) analogs
also showed low nM potencies. However, large substituents such as amide (3h), ketone
(3i), morpholine (3j) and Ph (3k) turned out to be detrimental to the inhibitory activity.
Next, substitution effects of the triazole core (R ) were studied using compounds
2
3o and 3p. Compared to inhibitor 3d, the ethyl analog 3o was slightly less potent. However
with the branched isopropyl group, compound 3p turned out to be over 20-fold less potent
than compound 3d. These results indicated that small group was preferred at the R
2
position.
In addition, removal of the quinoline nitrogen yielded compound 3q that totally lost
inhibitory activity. This result highlighted the importance of the quinoline nitrogen in
maintaining high potency. Finally, we sought to explore the possibility of expanding the
quinoline ring system. The Br-substituted compound 3r indicated similar potency as that
of the parent compound pyrvinium. Similarly, potent inhibitors were obtained when
morpholine (3s), piperidine (3t), or thiomorpholine (3u) were included at the same R
3
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position. These results indicated that further expansion of the quinoline ring could impair
the activity of the inhibitor.
Table 1. Inhibition of Wnt/β-Catenin Signaling Pathway by Compounds 3a-3u
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1
1
1
1
1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
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4
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4
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
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9
0
1
2
3
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5
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9
0
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IC50 (nM)a
Cmpds
R
1
R
2
R
3
X
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
CH
N
3
a
2-Me
3-Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Br
4.1 ± 0.4
>10,000
3b
3
c
4-Me
>10,000
3
d
2-F
1.2 ± 0.2
3
e
2-Br
7.6 ± 0.3
3f
2-CN
2-OMe
2-CONH
34 ± 4.3
3
g
18 ± 3.3
3
h
2
>10,000
3i
2-COMe
2-morpholine
2-Ph
8,800 ± 1322
1,900 ± 5.4
390 ± 68.1
140 ± 5.8
270 ± 23.8
5,500 ± 1186
4.7 ± 0.7
3
j
3
k
3
l
2,3-diMe
2,4-diMe
2,3-Ph (fused)
2-F
3m
3
n
3
o
3
3
p
q
2-F
i-Pr
Me
Me
25 ± 0.5
2-Me
4,900 ± 743
830 ± 98.2
3
r
2-Me
3s
2-Me
2-Me
Me
Me
N
N
1,300 ± 169
1,200 ± 168
3
t
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3
u
2-Me
Me
N
230 ± 28
pyrvinium
750 ± 137
aThe values of IC50 for each compound to inhibit the Wnt signaling activity, as determined
from the luciferase reporter gene assay, were calculated and data are expressed as mean
IC50 (nM) ± SE of each compound from three independent experiments. Note that
compounds 3a-3u did not present any apparent cytotoxicity during the short treatment
duration used for the luciferase gene reporter assay (Table S2).
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Compound 3a, a Potent Inhibitor of the Wnt/β-Catenin Signaling Pathway. As model
compounds, we next chose compounds 3a and 3n and further evaluated them in various
biological assays. We chose compound 3a over the more potent compound 3d (Table 1)
because in the assessment of their potential cytotoxicity in unstimulated normal HEK293
cells with a longer incubation time of 72 h relative to that for the above reporter assay, we
found that 3d was more cytotoxic than 3a (Figure S1). While compound 3d was more
potent than 3a, their potencies remained in the same magnitude. Compound 3a showed
superior potency in HEK293 cells in the presence of Wnt signaling activators LiCl and
Wnt3a (Figure 2A, B). Compound 3a inhibited the Wnt/β-catenin signaling pathway by
stabilization of Axin and subsequent β-catenin degradation (Figure 2C-E and Figure S2A-
B). Stabilization of Axin was confirmed by the increased cytoplasmic punctas as has been
demonstrated previously by other Axin stabilizers.43 The expression of the pathway target
genes including CycD1 and Axin2 was repressed at the messenger RNA (mRNA) levels.
In the presence of LiCl, which activates Wnt/β-catenin pathway via the inhibition of
GSK3β, compound 3a decreased the cellular level of β-catenin while the inactive analog
3
n had little effect (Figure 2F).
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The downregulation of β-catenin levels and the consistent efficacy of compound 3a
in both Wnt3a- and LiCl-conditioned medium suggested a mechanism different from that
of the parent compound pyrvinium, whose effect is diminished in the presence of LiCl.36
We performed additional studies with varying concentrations of LiCl, and observed no
effect by LiCl in changing the inhibitory potency of compound 3a towards Wnt/β-catenin
signaling pathway (Figure S2).
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Figure 2. Characterization of compound 3a as an inhibitor of Wnt/β-catenin signaling
pathway. (A) TCF/LEF responsive luciferase reporter assay in HEK293 cells with varying
concentration of compound 3a and (B) compound 3n in Wnt3a- or LiCl-conditioned
medium. The data were fitted to determine the IC50 of inhibition of LiCl- and Wnt3a-
induced activation of the Wnt/β-catenin signaling pathway (mean ± S.D, n = 4). (C)
Confocal microscope images of 3a-treated HEK293 cells for detection of Axin (Axin1).
The cells were incubated with 1 µM of compound 3a for 14 h. The nucleus was stained by
DAPI. (D) Western blot analysis showing the effect of compound 3a treatment on the
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levels of β-catenin and Axin1 proteins in HEK293 cells in the presence of WNT3a. The
cells were treated with the compound for 2 h. (E) The mRNA expression of the target
genes of Wnt/β-catenin signaling pathway. The cells were treated for 48 h and harvested
with TriZol reagent. *p < 0.05. (F) Western blot analysis of HEK293 cells treated with
compounds 3a and 3n in the presence of lithium chloride for 24 h. RLU- Relative light
units.
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Mechanism of Wnt/β-catenin Pathway Inhibition by Compound 3a. Sequential
phosphorylation of β-catenin on residues Ser33, Ser37 and Thr41 by GSK3β is a critical
step in β-catenin degradation. By causing increase in the phosphorylation at these sites,
many small molecule Wnt inhibitors facilitate the degradation of β-catenin. To further
ascertain if our new inhibitors function through a similar mechanism, we examined the
inhibitory potency of compound 3a in HEK293 cells overexpressed with wild-type and
mutant (S33Y) β-catenin (Figure S3). Overexpression of both exogenous β-catenin
plasmids led to significant increases in Wnt signaling activities as reflected by the
luciferase reporter gene assay, with the S33Y mutant giving a larger increase than the wild
type. Nonetheless, compound 3a inhibited Wnt signaling with a similar potency
irrespective of overexpression of either β-catenin, suggesting that the inhibitory effects of
compound 3a may be independent of GSK3β phosphorylation of at least Ser33 on β-
catenin. Note that aside from Ser33 phosphorylation, GSK3β also phosphorylates Ser37
and Thr41 of β-catenin. Thus the mutated β-catenin construct used in our assay may not
be sufficient to impair the function of GSK3β, as immunoprecipitation of β-catenin showed
that compound 3a strengthened the binding of GSK3β and Axin to β-catenin (Figure 3A).
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Thus, the results suggest that compound 3a treatment may lead to fortification of the
destruction complex to propagate the degradation of β-catenin.
We next silenced major components of the β-catenin destruction complex, CK1α,
GSK3β and Axin1, to assess their contribution to the effect of inhibitor 3a. Only the
knockdown of CK1 and GSK3β partially abolished the Wnt inhibitory effect while Axin1
silencing had no effect on the gene reporter assay of inhibitor 3a (Figure 3B). Next, we
performed surface plasmon resonance (SPR) analysis using recombinant CK1α, GSK3β,
and Axin to determine the binding affinity of compound 3a to these proteins. We found
that compound 3a could not bind to CK1α and Axin (data not shown), while exhibiting a
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weak binding (K = 4.3 µM) for recombinant GST-tagged GSK3β (Figure S4A-C).
D
Further, we knocked down GSK3β and performed the reporter assay for compound 3a.
The results show that exclusion of GSK3β only decreased the effect of compound 3a by
1
.7 fold (IC50 of si-control = 20 nM vs. si-GSK3β = 34 nM) (Figure S4D). The non-
correlation of the IC50 value from the reporter assay to the K of weak binding to GSK3β
D
suggests that GSK3β is probably not the main target of compound 3a, although it may play
a direct role in the inhibitory effect of compound 3a on Wnt/β-catenin signaling pathway.
In order to determine the inhibitory effect of compound 3a in other cells bearing
mutations that lead to an activated Wnt/β-catenin pathway, we overexpressed the luciferase
reporter constructs in SW480 and HepG2 cell lines. The SW480 cells have inactivating
mutations in APC, which lead to ineffective tethering of β-catenin to the destruction
complex and subsequent increase in cytoplasmic and nuclear β-catenin levels.44 In
contrast, HepG2 cells contain a deletion of the amino acids 25-140 of the CTNNB1
(encoding β-catenin) gene, which includes the binding sites of GSK3β and CK1α (Figure
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C).45 Inhibition of the reporter gene activity by compound 3a was well observed in
SW480 cells, but insignificant in HepG2 cells (Figure 3D). These results suggest the
necessity of the full length of β-catenin with intact GSK3β and CK1α sites for the inhibitory
activity of compound 3a.
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Figure 3. Role of Wnt/β-catenin pathway effector proteins in the inhibition by compound
3
3
a. (A) Immunoprecipitation of β-catenin in HEK293 to determine the effect of compound
a treatment (12 h) on the interaction of β-catenin with other pathway effector proteins.
(B) TCF/LEF gene reporter assay of compound 3a in the presence of knockdown of
GSK3β, Axin and CK1α. The HEK293 cells were treated with or without compound 3a
(20 nM) for 24 h. (C) Western blot analysis of protein expression of β-catenin in SW480
and HepG2 cells. (D) TCF/LEF gene reporter assay of SW480 and HepG2 cells treated
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with compound 3a. Data are represented as mean ± S.D, n = 3. RLU- relative light units.
*
** p < 0.001.
Compound 3a Decreased Lipid Accumulation and Altered the Expression of
Lipogenic and Gluconeogenic Genes in Hepatocytes. It has been reported that the
Wnt/β-catenin pathway may play a role in cellular metabolism. Specifically, Axin has been
implicated in lipid metabolism. In mice, knockdown of Axin results in significantly
increased hepatic lipid accumulation.46 Because compound 3a stabilized the cellular level
of Axin, we performed a Nile red staining assay to examine the effect of compound 3a on
lipid accumulation in human hepatic Huh7 cells (Figure 4A). Treatment of the cells with
compound 3a dramatically decreased lipid accumulation. Similar effects were observed
by a known Axin stabilizer XAV939. Next, we performed a quantitative PCR to determine
the effect of compound 3a on the gene expression of lipogenesis and gluconeogenesis in
mouse hepatocytes. Our results showed that compound 3a downregulated the mRNA
levels of the gluconeogenic (PEPCK and G6PASE) and lipogenic genes (FASN, ACAA1A,
ACOT4, and SCD1) (Figure 4B). These results suggested that compound 3a might
decrease the accumulation of lipids in hepatocytes by downregulating gluconeogenic and
lipogenic pathways as a consequence of Axin stabilization.
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Figure 4. Compound 3a decreased lipid accumulation and the expression of lipogenic and
gluconeogenic genes in hepatocytes. (A) Nile red staining assay of the Huh7 cells treated
with 5 µM of compounds XAV939 or 3a for 36 h and together with 200 µM oleate for 16
h. TD, transmitted light differential interference contrast image. (B) The mRNA expression
of various lipogenic and gluconeogenic genes in normal mouse hepatocytes treated with 5
µM of compound 3a. Data represents mean ± S.D. of triplicates.
In Vivo Efficacy of Compound 3a against Diet-Induced Metabolic Disorders in Mice.
With the promising in vitro effects of compound 3a on metabolism in hepatocytes, we
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further studied its efficacy in vivo against metabolic disorders using a mouse model.
Initially, we briefly assessed the pharmacokinetic and physicochemical properties of
compound 3a. Compared to the reported parameters for pyrvinium, our new analogue
showed improved physicochemical and pharmacokinetic properties with an oral
bioavailability of 21% (pyrvinium has less than 1%). The plasma half-life of compound
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a is 2.8 – 3.3 h, with an oral maximal concentration of 2.2 µg/mL in the plasma after a
single dose of 10 mg/kg (Table S3). We then proceeded to efficacy studies in the high fat
diet-fed mouse model. We performed a pilot dose escalation study to determine the optimal
dose for compound 3a and to ensure little or no toxicity to the mice (data not shown).
While we did not observe any noticeable toxicity at up to 200 mg/kg of compound 3a, we
found promising efficacy and hepatic improvement at 40 mg/kg so we chose this dose to
conduct a more comprehensive study in mice. Wild type C57BL/6J mice were fed with a
high fat diet or normal chow diet for 6 weeks before treatment. The mice were then divided
into four groups: two were fed normal chow and the other two were fed with the high fat
diet. Compound 3a was administered intraperitoneally every 2 days at 40 mg/kg for 11
weeks, a dose selected based on the pilot dose escalation studies. After 11 weeks of
treatment, compound 3a significantly improved glucose tolerance in the high fat diet group
(Figure 5A-B). The inhibition of Wnt signaling and the improvement of glucose tolerance
by compound 3a were confirmed by the decreased hepatic mRNA levels of Wnt target
genes and those of gluconeogenesis and lipogenesis in the mice fed with the high fat diet,
respectively (Figure 6A). Of note, the effects of compound 3a on gene expression were
insignificant in the mice fed with the normal chow diet, suggesting a selectivity of
compound 3a towards metabolic disorders (Figure 6B).
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Figure 5. Improvement of glucose tolerance by compound 3a in C57BL/6J mice fed with
a high fat diet. (A) Intraperitoneal glucose tolerance test (IPGTT) was carried out on the
mice fed with the high fat diet (HFD) and normal chow diet (NC) at the start of treatment
(WK0) and after 11 weeks (WK11) of treatment. The mice received intraperitoneal
injection of 40 mg/kg compound 3a or vehicle (corn oil) every two days. (B) The area
under the curve (AUC, min*mg/dL) for the IPGTT. Data represents mean ± SEM, n = 5
per group. *p < 0.05 as compared to the vehicle group.
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Figure 6. Effects of compound 3a on the hepatic expression of select Wnt/β-catenin
pathway target genes and those involved in energy metabolism in mice. (A) The mRNA
expression of select genes in the mice fed with a high fat diet. (B) The mRNA expression
of select genes in the mice fed with normal chow diet. The mice received i.p. injection of
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0 mg/kg compound 3a or vehicle (corn oil) every two days for 11 weeks. Data represents
mean ± SEM, n = 5 per group. * p < 0.05, ** p < 0.01.
To further assess the efficacy of compound 3a against the metabolic disorders induced by
the high fat diet in mice, we conducted additional measurements. The body weight and the
liver weight adjusted by the body weight were increased in the high fat diet-fed mice.
However, the increases were suppressed by the treatment of compound 3a. Of note, the
treatment caused no effects in the normal chow diet-fed mice (Figure 7). Consistently, in
our hepatic histological examination, compound 3a treatment drastically reduced the
hepatic lipid accumulation induced by the high fat diet. In addition, the hepatic triglyceride
content and the serum cholesterol level were reduced by the treatment of compound 3a in
the high fat diet-fed mice. Importantly, the mice that received compound 3a treatment did
not exhibit any significant toxicity as indicated by the blood chemical measurements of
liver and kidney function and by the histological examination (Table 2, Figure S5). Taken
together, the effects of compound 3a were pronounced in the mice with metabolic disorders
induced by the high fat diet. Inhibition of the Wnt signaling pathway appeared to mediate
the efficacy of compound 3a against the metabolic disorders induced by the high fat diet
because the results phenocopied those seen in genetic knockdown of Axin, Ctnnb1 (β-
catenin) and Tcf7l2 in the mice fed a high fat diet.11,46,47
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Figure 7. Effects of compound 3a treatment on body weight gain and hepatic lipid
accumulation in C57BL/6J mice. (A) Body weight gain in high fat diet and normal chow
group mice treated with 40 mg/kg compound 3a or vehicle (corn oil). (B) Hematoxylin
and eosin staining for liver tissue samples. (C) Hepatic triglyceride content and (D) the
ratio of liver/body weight in high fat diet-fed mice. The mice received i.p. injection of 40
mg/kg compound 3a or vehicle (corn oil) every two days for 11 weeks. V= vehicle, 3a =
Compound 3a. * p < 0.05.
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Table 2. Serum Biochemical Parameters in Mice Received Compound 3a or Vehicle
High fat diet (n=10)
Normal chow (n=10)
Physiologic
parameter
Unit
vehicle
3a
p-value vehicle
NS
3a
p-value
NS
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ALT
U/L
U/L
63.3 ± 26.9
58.3 ± 3.8
21.0 ± 1.9
20.0 ± 5.1
12.8 ± 1.6
57.5 ± 1.1
Alkaline
Phosphatase
AST
51.0 ± 4.6
NS
66.5 ± 1.8
NS
U/L
131 ± 15.6
94.8 ± 17.2
0.20 ± 0.03
223 ± 18.1
0.20 ± 0.02
313 ± 40.8
85.3 ± 7.7
14.8 ± 0.3
4.80 ± 0.6
NS
119 ± 28.3 74.5 ± 6.25
NS
Total bilirubin
Cholesterol
Creatinine Jaffe
LDH
mg/dL 0.20 ± 0.0
mg/dL 316 ± 25.0
mg/dL 0.30 ± 0.02
NS
0.20 ± 0.0
0.20 ± 0.0
NS
0.003
NS
183 ± 16.6 194 ± 13.7
0.20 ± 0.01 0.2 ± 0.01
324 ± 63.5 190 ± 20.2
NS
NS
U/L
469 ± 67
<0.001
NS
<0.001
NS
Triglycerides
BUN
mg/dL 93.5 ± 12.1
mg/dL 17.3 ± 0.5
mg/dL 6.30 ± 0.5
69.0 ± 2.5
19.0 ± 1.8
4.00 ± 0.2
55.0 ± 4.8
21.8 ± 0.5
3.80 ± 0.3
NS
NS
Uric Acid
NS
NS
The mice were fed either a high-fat diet or normal chow diet. Data analysis was performed
using ANOVA and Tukey's post-hoc test. The values are expressed as mean ± SE. NS, not
significant; ALT, Alanine Aminotransferase; AST, Aspartate aminotransferase; LDH,
Lactate dehydrogenase; BUN, Blood urea nitrogen.
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CONCLUSION
We have designed, synthesized, and characterized a novel class of triazole-based
compounds as novel inhibitors of the Wnt/β-catenin signaling pathway. One inhibitor of
the class, compound 3a, potently inhibits Wnt/β-catenin signaling pathway and elicits
favorable efficacy against the metabolic disorders induced by high fat diet. Our results
have indicated that the new inhibitors stabilized the cellular level of Axin, fortifying the β-
catenin destruction complex to attenuate β-catenin cytoplasmic level and suppress the
transcription of Wnt/β-catenin pathway target genes. The identification of direct protein
target of compound 3a is currently underway in our group using biotinylated chemical
affinity chromatography, proteomics, and other biochemical methods. Based on the parent
scaffold for these new compounds, further modification can be made to generate potent
analogues with even improved drug properties that may be applied to the treatment of
metabolic disorders, such as fatty liver, diabetes, and obesity, and other diseases, such as
cancers, with aberrant regulation of Wnt/β-catenin pathway.
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EXPERIMENTAL SECTION
. Chemical Analysis. All chemicals were obtained from commercial suppliers and used
1
without further purification. Analytical thin layer chromatography was visualized by
0
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ultraviolet light at 256 nM. H NMR spectra were recorded on a Varian (400 MHz)
spectrometer. Data are presented as follows: chemical shift (in ppm on the δ scale relative
to δ = 0.00 ppm for the protons in tetramethylsilane (TMS), integration, multiplicity (s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant
(J/Hz). 13C NMR spectra were recorded at 100 MHz, and all chemical shifts values are
reported in ppm on the δ scale with an internal reference of δ 77.0 or 39.0 for CDCl
DMSO-d , respectively. The purities of title compounds were determined by analytic
HPLC, performed on an Agilent 1100 instrument and a reverse-phase column (Waters
XTerrra RP18, 5 μM, 4.6 × 250 mm). All compounds were eluted with 45% CH CN/55%
O (0.1% TFA) over 20 min with a detection at 260 nm and a flow rate at 1.0 mL/min.
3
or
6
3
H
2
All tested compounds were >95% purity. Yields were not optimized. Compounds 1a-n
and potassium trifluoroborate derivatives were commercially available.
General Procedure A: Synthesis of Compounds 2a-2p. To a mixture of substituted
aniline 1a-1n (20 mmol) in concentrated HCl (8.0 mL) was added a solution of NaNO
2
o
o
(1.4 g, 21 mmol) in H
2
O (6.0 mL) dropwise at 0 C. The mixture was stirred at 0 C for 1
o
h followed by the addition of a solution of NaN
The resulting mixture was stirred for another 1 h, extracted with diethyl ether (Et
three times. The combined organic layers were washed with brine, dried over Na SO
3
(1.3 g, 20 mmol) in H
2
O (6.0 mL) at 0 C.
2
O) for
, and
2
4
concentrated. To the resulting residue was added ethyl 3-oxobutanoate, or ethyl 3-
oxopentanoate, or ethyl 4-methyl-3-oxopentanoate (22 mmol) and a solution of NaOEt
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(
2.04 g, 30 mmol) in EtOH (120 mL) at room temperature. The mixture was stirred at 80
oC overnight, cooled and concentrated. To the resulting residue was added HCl (1 N, 250
mL) and the resulting precipitate was collected by filtration and washed with water to offer
the crude product that was recrystallized from EtOH to give 2a-2p (25-83%).
1
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0
5
-Methyl-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxylic acid (2a). The title compound was
1
synthesized according to General Procedure A (55%): H-NMR (400 MHz, DMSO-d
6
) δ
1
3
1
3.16 (s, 1H), 7.57-7.52 (m, 2H), 7.46-7.44 (m, 2H), 2.32 (s, 3H), 2.00 (s, 3H); C-NMR
(100 MHz, DMSO-d ) δ 163.0, 140.1, 136.5, 135.5, 134.6, 131.7, 131.2, 127.8, 127.6, 17.1,
6
9
.6.
5
-Methyl-1-(m-tolyl)-1H-1,2,3-triazole-4-carboxylic acid (2b). The title compound was
1
synthesized according to General Procedure A (50%): H-NMR (400 MHz, DMSO-d
6
) δ
1
3
13.47 (s, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.78-7.73 (m, 3H), 2.83 (s, 3H), 2.75 (s, 3H); C-
NMR (100 MHz, DMSO-d ) δ 163.0, 140.0, 139.3, 136.9, 135.7, 131.2, 130.0, 126.4,
6
1
23.0, 21.3, 10.3.
5
-Methyl-1-(p-tolyl)-1H-1,2,3-triazole-4-carboxylic acid (2c). The title compound was
1
synthesized according to General Procedure A (46%): H-NMR (400 MHz, DMSO-d
6
) δ
1
3
1
3.08 (s, 1H), 7.42-7.38 (m, 4H), 2.41 (s, 3H), 2.36 (s, 3H); C-NMR (100 MHz, DMSO-
d
6
) δ 163.1, 140.3, 136.8, 133.3, 130.5, 125.7, 123.2, 21.2, 10.1.
1
-(2-Fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2d). The title
1
compound was synthesized according to General Procedure A (60%). H-NMR (400 MHz,
DMSO-d ) δ 13.24 (s, 1H), 7.74-7.68 (m, 2H), 7.64-7.56 (m, 1H), 7.52-7.45 (m, 1H), 2.40
6
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13
(
s, 3H); C-NMR (100 MHz, DMSO-d
6
) δ 162.8, 157.5, 155.0, 140.8, 136.7, 133.5, 133.5,
1
29.5, 126.1, 123.2, 123.0, 117.6, 117.4, 9.5.
1
-(2-Bromophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2e). The title
0
1
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3
4
5
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8
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0
1
2
3
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7
8
9
0
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1
compound was synthesized according to General Procedure A (80%): H-NMR (400 MHz,
1
3
DMSO-d
6
) δ 13.26 (s, 1H), 7.98-7.96 (m, 1H), 7.73-7.62 (m, 3H), 2.34 (s, 3H); C-NMR
(
100 MHz, DMSO-d ) δ 167.6, 145.3, 141.3, 139.3, 138.8, 138.1, 134.9, 134.4, 125.9, 14.4.
6
1
-(2-Cyanophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2f). The title
1
compound was synthesized according to General Procedure A (71%): H-NMR (400 MHz,
DMSO-d
6
) δ 13.37 (s, 1H), 8.23-8.21 (d, J = 6.8 Hz, 1H), 8.05-8.01 (t, J = 7.2 Hz), 7.92-
13
7
1
.87 (m, 2H), 2.47 (s, 3H); C-NMR (100 MHz, DMSO-d
6
) δ 162.7, 140.6, 136.9, 136.8,
35.3, 134.9, 132.0, 128.8, 115.6, 110.5, 9.8.
1
-(2-Methoxyphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2g). The title
1
compound was synthesized according to General Procedure A (80%): H-NMR (400 MHz,
DMSO-d
.35-7.33 (d, J = 8.0 Hz, 1H), 7.20-7.16 (t, J = 8.0 Hz, 1H), 3.80 (s, 3H), 2.31 (s, 3H); C-
NMR (100 MHz, DMSO-d ) δ 163.0, 154.1, 140.8, 136.3, 132.7, 128.9, 123.9, 121.3,
13.3, 56.4, 9.5.
6
) δ 13.14 (s, 1H), 7.66-7.62 (t, J = 8.0 Hz, 1H), 7.49-7.47 (d, J = 8.0 Hz, 1H),
1
3
7
6
1
1-(2-Carbamoylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2h). The title
1
compound was synthesized according to General Procedure A (65%): H-NMR (400 MHz,
DMSO-d
(m, 1H), 7.38 (s, 1H), 2.38 (s, 3H); C-NMR (100 MHz, DMSO-d
36.0, 135.9, 131.4, 131.0, 129.1, 128.5, 129.1, 128.5, 128.3, 9.9.
6
) δ 13.10 (s, 1H), 7.93 (s, 1H), 7.70-7.68 (m, 1H), 7.67-7.62 (m, 2H), 7.55-7.52
1
3
6
) δ 167.6, 163.2, 140.8,
1
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-(2-Acetylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2i). The title
1
compound was synthesized according to General Procedure A (40%): H-NMR (400 MHz,
DMSO-d ) δ 8.02-7.99 (m, 1H), 7.77-7.73 (m, 2H), 7.61-7.59 (m, 1H), 2.32 (s, 3H), 2.26
6
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
5
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5
5
5
5
5
5
5
6
0
1
2
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0
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2
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8
9
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2
3
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8
9
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2
3
4
5
6
7
8
9
0
(s, 3H); C-NMR (100 MHz, DMSO-d ) δ 199.0, 163.0, 140.7, 136.5, 136.3, 133.3, 132.6,
6
1
31.5, 130.4, 128.8, 29.3, 9.8.
5
-Methyl-1-(2-morpholinophenyl)-1H-1,2,3-triazole-4-carboxylic acid (2j). The title
1
compound was synthesized according to General Procedure A (83%): H-NMR (400 MHz,
DMSO-d
6
) δ 13.17 (s, 1H), 7.63-7.59 (t, J = 8.0 Hz, 1H), 7.44-7.42 (d, J =8.0 Hz, 1H),
7
2
.33-7.31 (d, J = 8.0 Hz, 1H), 7.30-7.26 (t, J = 8.0 Hz, 1H), 3.42 (br s, 4H), 2.63 (br s, 4H),
1
3
.38 (s, 3H); C-NMR (100 MHz, DMSO-d ) δ 162.9, 148.0, 140.5, 136.7, 132.3, 129.2,
6
129.1, 124.0, 120.9, 66.6, 51.4, 10.0.
1-([1,1'-Biphenyl]-2-yl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2k). The title
1
compound was synthesized according to General Procedure A (65%): H-NMR (400 MHz,
DMSO-d
6
) δ 7.72-7.68 (m, 1H), 7.63-7.61 (m, 2H), 7.53-7.51 (m, 1H), 7.26-7.21 (m, 3H),
1
3
7.02-6.96 (m, 2H), 1.97 (s, 3H); C-NMR (100 MHz, DMSO-d
6
) δ 164.2, 140.1, 138.9,
1
37.9, 137.5, 133.5, 131.5, 131.4, 129.3, 129.0, 128.8, 128.4, 128.3, 9.5.
1
-(2,3-Dimethylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2l). The title
1
compound was synthesized according to General Procedure A (70%): H-NMR (400 MHz,
DMSO-d
(s, 3H), 2.30 (s, 3H), 1.81 (s, 3H); C-NMR (100 MHz, DMSO-d
41.2, 139.3, 138.9, 137.0, 131.6, 130.1, 24.9, 18.7, 14.3.
6
) δ 13.14 (s, 1H), 7.46-7.45 (m, 1H), 7.36-7.32 (m, 1H), 7.26-7.25 (m, 1H), 2.36
13
6
) δ 167.8, 144.9, 143.9,
1
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2
2
2
2
2
2
2
3
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3
3
3
3
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5
6
1
-(2,4-Dimethylphenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (2m). The title
1
compound was synthesized according to General Procedure A (73%): H-NMR (400 MHz,
DMSO-d ) δ 13.01 (s, 1H), 7.28-7.24 (m, 2H), 7.20-7.18 (m, 1H), 2.34 (s, 3H), 2.26 (s,
6
1
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
1
H), 1.88 (s, 3H); C-NMR (100 MHz, DMSO-d
6
) δ 163.0, 140.9, 140.1, 136.4, 135.1,
32.2, 132.1, 128.0, 127.5, 21.1, 17.0, 9.6.
5
-Methyl-1-(naphthalen-1-yl)-1H-1,2,3-triazole-4-carboxylic acid (2n). The title
1
compound was synthesized according to General Procedure A (72%): H-NMR (400 MHz,
DMSO-d
6
) δ 13.15 (s, 1H), 8.27-8.25 (d, J = 8.0 Hz, 1H), 8.16-8.14 (d, J = 8.0 Hz, 1H),
7
.80-7.30 (m, 2H), 7.69-7.65 (t, J = 8.0 Hz, 1H), 7.62-7.58 (t, J = 8.0 Hz, 1H), 7.14-7.12
13
(d, J = 8.0 Hz, 1H), 2.31 (s, 3H); C-NMR (100 MHz, DMSO-d ) δ 163.0, 141.1, 136.7,
6
134.1, 131.5, 129.2, 128.9, 128.8, 127.9, 127.7, 126.2, 126.0, 122.0, 9.7.
5-Ethyl-1-(2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylic acid (2o). The title
1
compound was synthesized according to General Procedure A (80%): H-NMR (400 MHz,
DMSO-d
J = 8.0 Hz, 1H), 2.84-2.78 (q, J = 7.2 Hz, 2H), 1.02-0.98 (t, J = 7.2 Hz, 3H); C-NMR
100 MHz, DMSO-d ) δ 167.4, 162.6, 160.1, 150.5, 140.9, 138.6, 138.5, 134.6, 130.9,
30.8, 122.3, 122.1, 21.6, 17.6.
6
) δ 13.31 (s, 1H), 7.77-7.74 (m, 2H), 7.64-7.59 (t, J = 8.8 Hz, 1H), 7.52-7.48 (t,
1
3
(
6
1
1
-(2-Fluorophenyl)-5-isopropyl-1H-1,2,3-triazole-4-carboxylic acid (2p). The title
1
compound was synthesized according to General Procedure A (25%): H-NMR (400 MHz,
DMSO-d
6
) δ 13.01 (s, 1H), 7.77-7.74 (m, 2H), 7.64-7.60 (t, J = 8.0 Hz, 1H), 7.52-7.48 (t,
13
J = 8.0 Hz, 1H), 3.24-3.20 (m, 1H), 1.23-1.21 (d, J = 6.8 Hz, 6H); C-NMR (100 MHz,
DMSO-d
6
) δ 167.6, 162.8, 160.3, 153.5, 140.6, 138.7, 138.6, 135.0. 130.8, 122.2, 122.0,
29.5, 24.6.
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General Procedure B: Synthesis of Compounds 3a-3r. To a solution of 2-
aminoquinoline or naphthalen-2-amine or 6-bromoquinolin-2-amine (0.5 mmol) and 2a-
2p (0.675 mmol) in DCE (2 mL) was added PyCIU (0.775 mmol) and DIPEA (2.33 mmol).
o
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
6
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7
8
9
0
The mixture was stirred at 80 C overnight, then cooled and concentrated. To the resulting
residue was added ethyl acetate (30 mL), and the mixture was washed with brine, dried
over Na
2
SO
4
and concentrated. The crude material was purified by column
chromatography (hexane/AcOEt, v/v = 4/1 to 2/1) to give products 3a-3r (47-69%).
5-Methyl-N-(quinolin-2-yl)-1-(o-tolyl)-1H-1,2,3-triazole-4-carboxamide (3a). The title
1
compound was synthesized according to General Procedure B (60%, a white solid): H-
NMR (400 MHz, DMSO-d ) δ 10.22 (s, 1H), 8.49-8.47 (d, J = 8.8 Hz, 1H), 8.42-8.40 (d,
6
J = 8.8 Hz, 1H), 8.00-7.98 (d, J = 8.0 Hz, 1H), 7.91-7.89 (d, J = 8.0 Hz, 1H), 7.69 (t, J =
13
8
.0 Hz, 1H), 7.61-7.56 (m, 3H), 7.51-7.50 (m, 2H), 2.45 (s, 3H), 2.04 (s, 3H); C-NMR
(
100 MHz, DMSO-d ) δ 160.0, 150.8, 146.8, 139.5, 139.1, 137.6, 135.5, 134.4, 131.8,
6
131.4, 130.7, 128.3, 127.8, 127.7, 127.7, 126.4, 125.8, 114.7, 17.2, 9.4. HRMS (ESI): m/z
+
[
M + H] calcd for C20
H
18
N
5
O, 344.1511, found, 344.1510. HPLC: t = 8.75 min, 98.2%.
R
5
-Methyl-N-(quinolin-2-yl)-1-(m-tolyl)-1H-1,2,3-triazole-4-carboxamide (3b). The
title compound was synthesized according to General Procedure B (65%, a white solid):
1H-NMR (400 MHz, CDCl
3
) δ 9.92 (s, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 8.0 Hz,
1H), 7.92 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.70 (t, J = 7.2, 7.6 Hz, 1H), 7.47
(d, J = 7.2 Hz, 2H), 7.38 (d, J = 8.0 Hz, 1H), 7.33 (s, 1H), 7.27 (d, J = 5.6 Hz, 1H), 2.71
1
3
(
s, 3H), 2.48 (s, 3H); C-NMR (100 MHz, CDCl ) δ 160.0, 150.6, 146.9, 140.1, 138.4,
3
138.1, 137.9, 135.3, 130.9, 129.9, 129.4, 127.8, 127.5, 126.4, 125.9, 125.1, 122.2, 114.2,
29
ACS Paragon Plus Environment