Identification of novel triazole inhibitors of Wnt/β-catenin signaling based on the Niclosamide chemotype

Article abstract of DOI:10.1016/j.bmcl.2018.11.022

Dysregulation of the Wnt signaling pathway is an underlying mechanism in multiple diseases, particularly in cancer. Until recently, identifying agents that target this pathway has been difficult and as a result, no approved drugs exist that specifically t

Full text of DOI:10.1016/j.bmcl.2018.11.022

Accepted Manuscript
Identification of novel triazole inhibitors of Wnt/β-catenin signaling based on
the Niclosamide chemotype
Robert A Mook Jr., Jiangbo Wang, Xiu-Rong Ren, Hailan Piao, H. Kim Lyerly,
Wei Chen
PII:
S0960-894X(18)30888-6
https://doi.org/10.1016/j.bmcl.2018.11.022
BMCL 26133
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 June 2018
2 November 2018
10 November 2018
Please cite this article as: Mook, R.A. Jr., Wang, J., Ren, X-R., Piao, H., Kim Lyerly, H., Chen, W., Identification
of novel triazole inhibitors of Wnt/β-catenin signaling based on the Niclosamide chemotype, Bioorganic &
Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.11.022
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Graphical abstract
NO2
N
R1
N
O
Cl
Cl
N
N
H
H
Cl
OH
OH
1

Identification of novel triazole inhibitors of Wnt/β-catenin signaling based on the Niclosamide chemotype
Robert A. Mook, Jr.1*, Jiangbo Wang1, Xiu-Rong Ren1, Hailan Piao1, H. Kim Lyerly2, and Wei Chen1*
1Department of Medicine, Duke University Medical Center, Durham, NC 27710
2Department of Surgery, Duke University Medical Center, Durham, NC 27710
* Corresponding authors: +01 919 684-6635. E-mail address: robert.mook@duke.edu (R. Mook). Tel.: +01 919
684-4433. E-mail address: w.chen@dm.duke.edu (W. Chen).
Keywords:
Niclosamide
Wnt signaling inhibitor
Small molecule
Triazole derivatives
β-catenin
Cancer
Abbreviations:
APC, Adenomatous Polyposis Coli; CRC, colorectal cancer; Dvl, Dishevelled; LEF/TCF, Lymphoid enhancer
factor/T cell factor; Fzd1, Frizzled1; GFP, green fluorescent protein; SAR, structure-activity relationships
2

Abstract
Dysregulation of the Wnt signaling pathway is an underlying mechanism in multiple diseases, particularly
in cancer. Until recently, identifying agents that target this pathway has been difficult and as a result, no
approved drugs exist that specifically target this pathway. We reported previously that the anthelmintic drug
Niclosamide inhibits the Wnt/β-catenin signaling pathway and suppresses colorectal cancer cell growth in vitro
and in vivo. In an effort to build on this finding, we sought to discover new Wnt/β-catenin inhibitors that
expanded the chemotype structural diversity. Here, we asked a specific SAR question unresolved in previous
SAR studies of Niclosamide’s inhibition of Wnt/β-catenin signaling to identify a new structural class of Wnt/β-
catenin signaling inhibitors based on a triazole motif. Similar to Niclosamide, we found that the new triazole
derivatives internalized Frizzled-1 GFP receptors, inhibited Wnt/β-catenin signaling in the TOPflash assay and
reduced Wnt/β-catenin target gene levels in CRC cells harboring mutations in the Wnt pathway. Moreover, in
pilot SAR studies, we found the Wnt/β-catenin SAR trends in the anilide region were generally similar between
the two chemical classes of inhibitors. Overall, these studies demonstrate the ability to use the SAR of the
Niclosamide salicylanilide chemical class to expand the structural diversity of Wnt/β-catenin inhibitors.
3

The Wnt signaling pathway is a fundamental growth control pathway that is essential to the regulation of
many biological process and is critical to stem cell development and the proper growth and maintenance of
tissues 1. As a result, dysregulation of the pathway is an underlying factor in many diseases, particularly cancer,
where mutations are found frequently in various components of the pathway 1-3. In colorectal cancer (CRC),
more than 93% of tumors had alteration in the pathway, ~80% of which were mutations in adenomatous
polyposis coli (APC) or -catenin that result in hyperactivation of Wnt signaling 1, 4. Given the importance of
the Wnt signaling pathway, there has been an intensive effort for more than three decades to understand the
mechanistic details of Wnt signaling and to develop therapies that target the pathway 1, 5
.
Our understanding of the mechanistic complexities of the signaling pathway has grown considerably during
this time 1, 3, 6. In the canonical Wnt pathway, a key component is the cytosolic protein β-catenin, whose levels
are controlled by a destruction complex consisting of Axin, APC, and two serine-theonine kinases, CK1α/δ and
GSK3α/β. Signaling is activated by secreted Wnt proteins that bind the seven transmembrane receptor Frizzled
and the single transmembrane receptors LRP5/6 resulting in activation of cytosolic proteins called Dishevelled
(Dvl), internalization of the Frizzled receptor, and stabilization and translocation of cytosolic β-catenin proteins
into the nucleus. In the nucleus, β-catenin binds and activates the transcription factor LEF/TCF to initiate the
transcription of Wnt/-catenin target genes. In CRC, most of the mutations observed in the pathway result in an
increase in the level of β-catenin protein and activation of the pathway. Thus, agents that decrease the level of
β-catenin could be useful therapeutic agents. Overall, the Wnt signaling pathway consists of many intracellular
protein-protein interactions for which traditional drug discovery approaches have been difficult 5, 7. As a result,
no drugs have been approved that specifically target the pathway 8.
Recent efforts, however, have provided opportunities to inhibit the pathway 1, 9-18. Our contribution to this
field was the discovery that the anthelmintic drug Niclosamide (Figure 1), inhibits Wnt/β-catenin signaling 19, a
discovery that has since been confirmed by others 20-22. Mechanistically, we found that Niclosamide promoted
Frizzled internalization, downregulated Dvl and β-catenin protein levels and inhibited colorectal cancer cell
4

growth in vitro and in vivo 19, 23. Initial SAR studies indicated that Niclosamide inhibition of Wnt signaling
appeared unique among structurally-related anthelmintic agents, and that the potency and Wnt/β-catenin
functional response was dependent on small changes in the chemical structure of Niclosamide 24.
Niclosamide was approved by the FDA in 1982 for use in humans to treat tapeworm infections 25 and has
been identified as an essential medicine by the World Health Organization 26. Niclosamide is a salicylic acid
derivative that belongs to the salicylanilide class of anthelmintic agents. Similar to other members of the
salicylanilide class, Niclosamide has been used widely as an anthelmintic agent in livestock 27. Its anthelmintic
activity reported involves uncoupling of oxidative phosphorylation 27-30. Niclosamide has protonophore activity
and contains an aryl β-hydroxy carbonyl pharmacophore that is present in a large number of natural products
and drugs that have a broad range of biological activity 31. Thus, it is perhaps not surprising that since
Niclosamide’s discovery more than 50 years ago, additional biological activity has been discovered that clearly
indicate Niclosamide is a multi-functional drug 31.
Niclosamide’s multi-functional activity has generated considerable interest in its use to treat cancer,
rheumatoid arthritis, diabetes, and bacterial and viral infections 31-38. In addition to our clinical trial in colorectal
cancer (NCT02687009), there are now four additional clinical trials of Niclosamide listed in ClinicalTrials.gov
in cancer (NCT02519582, NCT02532114, NCT02807805), and in rheumatoid arthritis (NCT03160001).
Particular relevant to cancer therapy, Niclosamide inhibits the growth of cancer cells from multiple tumor types
and is active against drug resistant cancers 23, 31, 39-41. In addition to its ability to inhibit the Wnt signaling
pathway, Niclosamide inhibits other key oncogenic signaling pathways such as mTOR , NF-kB, Notch and
STAT-3 and has effects on metabolism, including activation of AMPK 31. Niclosamide is poorly absorbed upon
oral administration and is cleared rapidly from systemic distribution 23, 42, 43. Although Niclosamide’s
pharmacokinetic properties are appropriate for its anthelmintic indication, its pharmacokinetic and solubility
properties can limit its use to treat diseases where systemic exposure is required.
5

Given the need to identify drugs that inhibit Wnt/β-catenin signaling and the limitations of Niclosamide’s
properties, we sought to expand the chemical diversity of inhibitors available to us via a classical “scaffold-
hopping” strategy 44. In our previous SAR studies of Niclosamide, we found that a trifluoromethyl group was a
suitable replacement of the nitro group and resulted in an inhibitor of Wnt/β-catenin signaling (2) (IC50 = 0.29 ±
0.06 μM) with similar potency to Niclosamide (IC50 = 0.34 ± 0.08μM) (Figure 1) 9, 45. However, upon insertion
4'
NO2
O
CF3
O
Cl
5
Cl
N
2'
N
H
Cl
H
OH
2
OH
2
1 Niclosamide
N
O
R1
N
Cl
4
Cl
N
H
OH
N
H
2
CF3
OH
3
Triazole series
Figure 1. Chemical structures of Niclosamide, Niclosamide analogs, and the triazole class of Wnt/β-
catenin inhibitors.
of one carbon atom between the anilide ring and the amide NH group of 2, the resulting salicylic amide
derivative 3 had much poorer inhibitory activity (IC50 = 15.10 ± 1.40 μM) 45. To better understand if loss of
activity was due to an increase in the steric demands of extending the inhibitor’s structure by 1 atom and
changing the position of the anilide ring or if the loss of activity resulted from the loss of conjugation of the N-
H bond into the aromatic ring and a decrease in the N-H hydrogen-bond donating properties, we designed a
series of triazole compounds in which the salicylic scaffold of Niclosamide was simultaneously converted into a
triazole scaffold (Figure 1). This change inserts one carbon atom between the anilide ring and the amide NH
group and changes the anilide ring position in a similar fashion to the less-active derivative 3 while allowing the
N-H bond to maintain conjugation with the aromatic ring.
6

Based on SAR data indicating Niclosamide derivatives without the 2’-chloro substituent in the anilide
inhibited Wnt/β-catenin signaling 45 and the ready availability of the commercially available starting materials
in our lab, we first prepared triazole 4 in three steps using published procedures (Figure 2) 46.
O
O
O
O
Cl
Cl
1. DBU
+
NH2
OH
R
N
R
Cl
H
(60-76%)
OH
2. Melt
3. NH2NH2
(3-10%)
N
N
R
Cl
N
H
OH
Figure 2. Synthesis of triazole derivatives.
We then evaluated the Wnt/β-catenin inhibitory activity of triazole 4 using assays and protocols previously
reported. Upon testing this compound in the Fzd1-GFP internalization and the TOPflash Wnt3A-stimulated β-
catenin gene transcription assay 9, 24, 45, we were delighted to find it induced a robust punctate pattern in the
Fzd1-GFP assay indicative of internalization of the Fzd1 receptor and inhibited Wnt3A-stimulated β-catenin
gene transcription with an IC50 of 1.41 ± 0.34 µM (Table 1 and Figure S1 in supplemental information). To
enable better comparison of structure and inhibitory activity, we also prepared salicylamide 10, and tested it and
the imide intermediate 11 for Wnt/β-catenin inhibitory activity. Consistent with previous results obtained with
the trifluoromethyl-substituted salicylamide derivative 3, the nitro-substituted salicylamide derivative 10 was
considerably less active in both assays than the similarly substituted triazole 4. The imide intermediate 11
however, had a different profile. Whereas its activity in the Fzd1-GFP assay was similar to DMSO control, it
increased Wnt3A-stimulated signaling in the TOPFlash assay. The reasons for this result are not understood at
this time. Based on the data obtained with triazole 4, we postulate that conjugation of the N-H group and/or its
hydrogen bonding donating ability was the important SAR feature for inhibitory activity.
Having identified the inhibitory activity of triazole 4, we then conducted pilot SAR studies that varied the
nitro substituent at the 4-position of the pendent aryl ring to understand if the SAR at this position, a key
7

position in the Niclosamide series, tracked into the new series. Similar to the SAR studies conducted in the
Niclosamide series, we selected substituents with a range of electron-donating and electron-withdrawing
8

Fzd1-GFP Internalization Inhibition Wnt/β-catenin transcription
at 12.5 µM a
Compound Number
Structure
TopFlash IC50 (µM) ± SE b
NO2
O
Cl
Cl
0.34 ± 0.07
1
5
N
H
Cl
OH
N
N
NO2
N
1.41 ± 0.34
2.88 ± 0.84
4
5
4
2
0
1
0
5
0
0
H
OH
N
N
Cl
Cl
CF3
N
H
OH
N
N
60% inhibition at 10 μM,
14% inhibition at 5 μM
OCH3
6
N
H
OH
N
N
Cl
Cl
F
1.91 ± 0.60
4.02 ± 0.98
1.84 ± 0.60
7
N
H
OH
N
N
N
8
H
OH
Cl
N
N
Cl
Cl
9
NO2
N
H
OH
O
43% inhibition at 10µM,
3% inhibition at 5 µM
N
10
11
H
OH
NO2
O
O
increased signal,
153 % of control at 10µM
Cl
N
H
OH
NO2
a
Table 1. SAR of Wnt/β-catenin signaling.
Fzd1-GFP assay: Internalization of Frizzled1-GFP stably expressed in
U2OS cells was determined by confocal microscopy. Cells were treated with compounds for 6 hours, fixed, and scored
visually by the amount of punctate observed versus DMSO control. Punctate similar to control = 0, trace amount of
punctate greater than control = 1, moderate = 3, strong = 5. Images in Supplemental Information. b Inhibition of Wnt3A-
stimulated Wnt/β-catenin transcription was determined by TOPFlash assay using HEK293 cells stably expressing a
TOPFlash luciferase reporter and Renilla luciferase reporter control. Cells were stimulated with Wnt3A-conditioned
medium in the presence of DMSO or compounds from 0.04 to 10µM for 8 hrs. The TOPFlash reporter activity of Wnt3A
conditioned media with DMSO treatment was set as 100%. Data were fit using GraphPad Prism (mean ± SEM, n ≥3).
Percent inhibition at the two highest concentrations tested is reported for weak inhibitors that do not give a full dose
response over the inhibitor concentration assayed.
9

properties. Consistent with results obtained in the Niclosamide chemotype 24, 45, a trifluoromethyl substituent
was active (5) while substitution at the para position with an electron-donating methoxyl substituent 47 (6), an
electron-withdrawing fluorine atom (7), or a hydrogen atom at this position (8) showed a trend toward less
activity in both the Fzd1-GFP and the TOPFlash assays, although the magnitude of the difference was not as
large as in the Niclosamide series. In the Niclosamide series, we noted earlier that a 2’-chloro substituent
increased potency when there was a nitro group at the 4’-position 24, 45. To test whether the addition of a
corresponding 2’chloro-substituent in the aryl ring of the triazole would produce a similar increase in potency,
we prepared triazole 9. Unfortunately, a similar increase in potency in the triazole series (4 vs 9) was not
observed by the addition of the chloro-substituent. Overall, however, the trend in inhibitory activity of the
triazole derivatives appeared consistent with the inhibitory activity of similarly substituted Niclosamide
derivatives 24, 45
.
To further confirm the Wnt/β-catenin inhibitory activity of this new chemical series, compounds with
significant activity in the Fzd1-GFP internalization and the Wnt3A-TOPflash assays were evaluated by
immunoblot in two CRC cell lines with aberrant Wnt signaling (HCT-116, β-catenin mutant; SW480, APC
mutant) for their ability to reduce the levels of Wnt/β-catenin target gene proteins (Figure 3). Consistent with
the results of the Frizzled1-GFP and the Wnt3A-stimulated TOPflash assays, Niclosamide (1), and triazole
derivatives 4, 5 and 9 each reduced the levels of the Wnt/β-catenin target genes: β-catenin, Axin2, c-Myc,
Survivin and Cyclin D1 relative to controls in both cell lines, thereby demonstrating their ability to inhibit
Wnt/β-catenin signaling in CRC cells with aberrant Wnt pathway signaling.
In general, Niclosamide derivatives with IC50 values in the Wnt3A-stimulated TOPflash assay in the single
digit micromolar or less range evaluated to date also typically produce a robust punctate pattern in the Fzd1-
GFP internalization assay. However during the course of our studies, we have identified a few compounds that
do not produce a robust response in the Fzd1-GFP internalization assay yet inhibit Wnt/β-catenin signaling with
10

Figure 3. Reduction of Wnt/β-catenin target genes by Niclosamide and triazole 4, 5 and 9. Cells were
treated with DMSO or compounds (5 μM) in DMSO for 18 hours, the cytosolic fraction and the whole cell
lysates were probed with antibodies to the proteins indicated. β-actin was used as a loading control. A.
Western blot images B. Quantification of the protein levels of Western blots in A normalized to the β-Actin
loading control and graphed as a percentage of protein level in the DMSO treated control cells.
11

IC50s of 1-4 μM in the TOPflash assay 9, 24, 45. Herein, compound 8 (Table 1) has this profile for which studies
in HCT-116 cells confirmed the reduction in Wnt/β-catenin target genes (Supplemental Figure S2). The
mechanistic underpinnings to account for this observation are not clear at present. Given Niclosamide has
multifunctional activity, one explanation may be that these derivatives have a different mechanism or a different
selectivity profile.
Overall, these studies provide insight into the SAR of the amide region that impact potency and demonstrate
the ability to extend the SAR findings of the Niclosamide chemotype into a novel triazole class of Wnt/β-
catenin inhibitors. Whether the antiviral and antibiotic activity, inhibition of other signaling pathways and the
effects on blood glucose and fatty liver also extend into this chemotype, given the structural relationship of
these triazole derivatives to Niclosamide, is an open question. The compounds herein are less potent inhibitors
of Wnt/β-catenin signaling than Niclosamide. Additional studies are underway to further define the Wnt/β-
catenin inhibitory SAR to improve potency, the activity against pathways modulated by Niclosamide, and the
pharmacokinetic properties of the series to support their evaluation in CRC tumor models and models of other
diseases for which Niclosamide has significant biological activity 31.
Acknowledgements
This work was funded in part by 5 R01 CA172570 (WC), BC123280 (WC), and Clinical Oncology
Research Center Development Grant 5K12-CA100639-08 (RAM). Wei Chen is a V foundation Scholar and an
American Cancer Society Research Scholar. NMR instrumentation in the Duke NMR Spectroscopy Center was
funded by the NIH, NSF, North Carolina Biotechnology Center and Duke University. The authors gratefully
acknowledge this support and the support of Professor Eric Toone and David Gooden of the Duke Small
Molecule Synthesis Facility.
12

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14

Fzd1-GFP
Internalization at 12.5
µM a
Compound
Number
Inhibition Wnt/β-catenin transcription
Structure
TopFlash IC50 (µM) ± SE b
1
4
5
4
2
0
1
0
5
0
0
0.34 ± 0.07
1.41 ± 0.34
2.88 ± 0.84
5
60% inhibition at 10 μM,
14% inhibition at 5 μM
6
7
1.91 ± 0.6
4.02 ± 0.98
1.84 ± 0.60
8
9
43% inhibition at 10µM,
3% inhibition at 5 µM
10
11
increased signal,
153 % of control at 10µM
15

Highlights
 New class of Wnt/β-catenin inhibitors discovered from SAR studies of Niclosamide
 The amide SAR of Niclosamide clarified
 Replacement of Niclosamide’s amide with a triazole provides Wnt inhibitors
16