Pyrazole-4-Carboxamide (YW2065): A Therapeutic Candidate for Colorectal Cancer via Dual Activities of Wnt/β-Catenin Signaling Inhibition and AMP-Activated Protein Kinase (AMPK) Activation

Article abstract of DOI:10.1021/acs.jmedchem.9b01252

Dysregulation of the Wnt/β-catenin signaling pathway has been widely recognized as a pathogenic mechanism for colorectal cancer (CRC). Although numerous Wnt inhibitors have been developed, they commonly suffer from toxicity and unintended effects. Moreover, concerns have been raised in targeting this pathway because of its critical roles in maintaining stem cells and regenerating tissues and organs. On the basis of the anthelmintic drug pyrvinium and previous lead FX1128, we have developed a compound YW2065 (1c) which demonstrated excellent anti-CRC effects in vitro and in vivo. YW2065 achieves its inhibitory activity for Wnt signaling by stabilizing Axin-1, a scaffolding protein that regulates proteasome degradation of β-catenin. Simultaneously, YW2065 also led to the activation of the tumor suppressor AMPK, providing an additional anticancer mechanism. In addition, YW2065 showed favorable pharmacokinetic properties without obvious toxicity. The anti-CRC effect of YW2065 was highlighted by its promising efficacy in a mice xenograft model.

Full text of DOI:10.1021/acs.jmedchem.9b01252

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Article
Pyrazole-4-Carboxamide (YW2065): A Therapeutic Candidate for
Colorectal Cancer via Dual Activities of Wnt/#-Catenin Signaling
Inhibition and AMP-Activated Protein Kinase (AMPK) Activation
Wei Yang, yingjun li, Yong Ai, Obinna N. Obianom, Dong Guo, Hong
Yang, Srilatha Sakamuru, Menghang Xia, Yan Shu, and Fengtian Xue
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01252 • Publication Date (Web): 26 Nov 2019
Downloaded from pubs.acs.org on November 26, 2019
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Pyrazole-4-Carboxamide (YW2065): A Therapeutic Candidate for Colorectal
Cancer via Dual Activities of Wnt/β-Catenin Signaling Inhibition and AMP-
Activated Protein Kinase (AMPK) Activation
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Wei Yang,#,1, Yingjun Li,#,1 Yong Ai, Obinna N Obianom, Dong Guo, Hong Yang,
1
1
1
1
⊥
Srilatha Sakamuru, Menghang Xia, Yan Shu,* and Fengtian Xue*,1
2
2
,1,3
1Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland 21201, United States
29
800 Medical Center Drive, National Center for Advancing Translational Sciences,
National Institutes of Health, Bethesda, Maryland 20892, United States
3School and Hospital of Stomatology, Guangzhou Medical University, Guangzhou 510140,
China
#These authors contributed equally to this work.
Corresponding Authors
*
*
Y.S.: phone, 410-706-7493; fax, 410-706-0886; e-mail, yshu@rx.umaryland.edu
F.X.: phone, 410-706-8512; fax, 410-706-0886; e-mail, fxue@ rx.umaryland.edu
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ABSTRACT
Dysregulation of the Wnt/-catenin signaling pathway has been widely recognized as a
pathogenic mechanism for colorectal cancer (CRC). Although numerous Wnt inhibitors
have been developed, they commonly suffer from toxicity and unintended effects.
Moreover, concerns have been raised in targeting this pathway because of its critical roles
in maintaining stem cells and regenerating tissues and organs. Based on the anthelmintic
drug pyrvinium and previous lead FX1128, we have developed a compound YW2065 (1c),
which demonstrated excellent anti-CRC effects in vitro and in vivo. YW2065 achieves
its inhibitory activity for Wnt signaling by stabilizing Axin-1, a scaffolding protein that
regulates proteasome degradation of -catenin. Simultaneously, YW2065 also led to the
activation of the tumor suppressor AMPK, providing an additional anti-cancer mechanism.
In addition, YW2065 showed favorable pharmacokinetic properties without obvious
toxicity. The anti-CRC effect of YW2065 was highlighted by its promising efficacy in a
mice xenograft model.
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INTRODUCTION
Colorectal cancer (CRC) is the third most frequently occurring cancer and the second
leading cause of cancer-related death in the United States.1,2 Although patients with
early-stage CRC can be treated with surgical resection followed by adjuvant therapy,
approximately 50% of CRC patients will develop metastases, which require systemic
medical treatment and lead to high mortality rates.3 Available drugs for metastatic CRC
(mCRC) are limited to chemotherapies such as the thymidylate synthase inhibitors (e.g., 5-
FU and folinic acid) and DNA-alkylating agents (e.g., oxaliplatin and irinotecan).
Recently, biologics targeting the vascular endothelial growth factor (VEGF; bevacizumab)
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and epidermal growth factor receptor (EGFR; cetuximab and panitumumab) started to
emerge,4-6 however, they fail to improve patients’survival rate significantly.2 Thus, novel
therapeutics for the treatment of mCRC is still urgently needed.
Over 90% of mCRC patients carry genetic mutations associated with upregulated
Wnt signaling, which makes this pathway an attractive target against mCRC.8 The
Wnt/β-catenin signaling pathway regulates the expression of its target genes through the
Wnt ligands and intracellular signal transducer β-catenin (Figure 1). During the “off state”
of the pathway when Wnt proteins are not interacting with the LRP6 and Fz receptors, a
cytoplasmic “destruction complex” is formed by the scaffolding protein 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
the proteasomal degradation of β-catenin. During the “on-state”, the formation of the
destruction complex is prohibited. As a result, β-catenin is accumulated and translocated
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into the nucleus, where it binds to the transcriptional factor LEF/TCF along with other
transcriptional coactivators (CBP, P300, PYGO and BCL9), and activates the expression
of its target genes to promote cell proliferation, differentiation and growth.
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Figure 1. The Wnt/β-catenin signaling pathway and examples of known inhibitors.
Several classes of Wnt inhibitors are known by targeting different components of
the pathway (Figure 1).9-13
14
The Novartis clinical candidate LGK974 and its analogs15,16
(
e.g., IWP-2) are porcupine inhibitors, which function by blocking the secretion of the Wnt
ligands. In addition, inhibitors have been developed to target key protein-protein
17
interactions (PPI) involving β-catenin in the nucleus. For example, CGP049090 and
1
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iCRT3 analogs inhibit the PPI between β-catenin and LEF/TCF, while the Prism/Eisai
pharmaceuticals clinical candidate PRI-724 selectively blocks the PPI of β-catenin with
CBP.19 Although targeting the downstream PPIs demonstrated unique specificities, these
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compounds have suffered from varying degrees of toxicity and unintended effects on tissue
homeostasis. Moreover, XAV939,20 IWR-1,16 G007-LK,21 and NVP-TNKS65622 are
inhibitors of tankyrase (TNKS), a member of poly(ADP-ribose) polymerases (PARPs).
However, competitive inhibitors of TNKS turned out to be insufficient because
polymerization of TNKS, via SAM domains, promotes Wnt signaling independently of the
catalytic activity.23 In addition, TNKS recruits and PARylates numerous protein
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partners24-26 via an ankyrin (ANK)-repeat domain, for various biological purposes,
therefore, targeting the active site may also lead to potential toxicities.27,28 Finally, the
anthelmintic drug pyrvinium has been reported as a Wnt inhibitor by activating CK1α.29
However, pyrvinium has extremely low oral bioavailability and high toxicity, which
prohibits its further application as an anti-cancer therapeutic agent.30 Using pyrvinium as
a template, our laboratory recently developed a compound FX1128 (Figure 2), which
demonstrated excellent inhibitory potency for Wnt signaling, although the anti-
proliferation efficacy of FX1128 was limited (Figure S1).31
In addition to genetic alteration, reprogramming of cellular metabolic pathways is
also commonly associated with mCRC.32,33 The adenosine monophosphate (AMP)-
activated protein kinase (AMPK), as a metabolic switch, plays a crucial role in metabolic
disorders and carcinogenesis in the intestine.34 Activated AMPK phosphorylates the
serine and threonine residues on its substrates, resulting in energy restoration and tumor
suppression.35 For instance, AMPK activation was reported to be a prognostic factor for
mCRC patients receiving chemotherapy in combination with the VEGF-targeting
monoclonal antibody drug bevacizumab.36 It was also known thatAMPK activators, such
as metformin and aspirin, induced anti-proliferative cytotoxicity against CRC cells.37-40
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The complexity of mCRC usually requires combination therapies that attack multiple
anticancer machineries. Therefore, drugs that target dual or multiple pathways often
result in improved anticancer efficacy and delayed drug resistance.40,41 We hypothesized
that compounds that simultaneously inhibit Wnt signaling and activate AMPK pathway can
be promising therapeutics for the treatment of mCRC. 41,42
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Herein, we report the synthesis and characterization of a new series of anti-mCRC
compounds (1a-s and 2a-e) with dual activities of Wnt inhibition and AMPK activation
(Figure 2). The chemical structures of new inhibitors were based on our previous lead
FX1128 by replacing its triazole core, which caused weak anti-cancer activity and poor
aqueous solubility, with a pyrazole ring, a widely used drug-like group in small molecule
drug design. One new compound of the series, YW2065, not only maintained excellent
inhibitory potency for the Wnt signaling, but also potently activated the AMPK pathway.
The dual functional compound YW2065 demonstrated promising anti-mCRC effects in
vitro and in vivo.
Figure 2. The design strategy of pyrazole-based compounds 1a-s and 2a-e.
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RESULTS AND DISCUSSION
Synthesis. The syntheses of compounds 1a-s are detailed in Scheme 1. Condensation
of ethyl acetoacetate (3) with N,N-dimethylformamide dimethyl acetal in EtOH provided
compound 4 in good yields. Next, pyrazole formation using compound 4 and a
substituted hydrazine in the presence of DIPEA led to the generation of the ester
intermediate, which was submitted to a hydrolysis using aqueous NaOH to afford
carboxylic acids 5a-p in good yields. Finally, PyCIU-mediated coupling of acids 5a-p
with various aromatic amines yielded the final products 1a-s in moderate to good yields.
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Scheme 1. Synthesis of compounds 1a-sa
a
o
Reagents and conditions: (a) DMF-DMA, EtOH, 60 C, 97%; (b) R
3
-NHNH , DIPEA,
2
o
EtOH, 60 C; (c) NaOH, EtOH/H
2
O, reflux, 40-98% for two steps; (d) aromatic amines,
o
PyCIU, DIPEA, DCE, 80 C, 24-88%.
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The synthesis of compounds 2a-e began with anilines 6a-c (Scheme 2).
Diazotization of the amino group of compounds 6a-c using NaNO in HCl (aq.), followed
2
by condensation of the diazotized anilines with ethyl 2-chloro-3-oxobutanoate in the
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presence of NaOAc yielded hydrazonoyl chlorides 7a-c. Next, reaction of compounds
7
a-c with either diethyl (2-oxopropyl) phosphonate, 4-(cyclopent-1-en-1-yl)morpholine,
or 4-(cyclohex-1-en-1-yl)morpholine triggered a cyclization reaction to give the ethyl
esters. Then, saponification of the ethyl esters in aqueous NaOH provided pyrazole-3-
carboxylic acids 8a-e in good yields. Finally, the PyCIU-mediated coupling of acids 8a-
e with 2-aminoquinoline yielded compounds 2a-e in moderate to good yields.
Scheme 2. Synthesis of compounds 2a-ea
aReagents and conditions: (a) i) NaNO
, HCl (aq.), 0 C; ii) ethyl 2-chloroacetoacetate
o
2
o
NaOAc, EtOH/H
2
O, 0-25 C, 88-95%; (b) i) for 8a-c, diethyl (2-oxopropyl)phosphonate,
DME, LiOH, rt, 10 h; for 8d-e, ii) 4-(cyclopent-1-en-1-yl)morpholine or 4-(cyclohex-1-
3
en-1-yl)morpholine, Et N, 12 h, rt; (c) NaOH, MeOH, reflux, 70-85% for two steps; (d) 2-
o
aminoquinoline, PyCIU, DIPEA, DCE, 80 C, 63-71%.
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Structure-Activity Relationship (SAR). The inhibitory potency of the synthesized
compounds against the Wnt signaling pathway was determined using the Wnt-HEK293
luciferase gene reporter assay that was described previously (Table 1).31 Compound 5-
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methyl-1-phenyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1a) had an IC50 value of
2
.2 nM, which was similar to that of FX1128, while >300-fold and >200-fold more potent
than pyrvinium and TNKS inhibitor XAV939, respectively. We then studied substitution
effects on the phenyl ring. Replacement of the methyl group with an F atom resulted in
a new inhibitor (1b) with excellent potency (IC50 = 0.92 nM), while with Br (1c) and Et
(1d) groups at the same position yielded compounds with similarly good potencies. A
second F (1e) or Br (1f) substituent at the ortho-position was well-tolerated. However,
an additional halogen at the para- (1g) or meta-position (1h) was detrimental to the potency.
The 2,5- and 2,6-dimethyl analogs (1i-1j) indicated IC50 values of 14 and 26 nM,
respectively. These results indicated that single substituent at the ortho-position of the
ring is preferred. Interestingly, inclusion of a nitrogen atom to the phenyl ring (1k)
restored the high potency of the inhibitor, indicating that polar aromatic characteristics can
be tolerated.
Next, the effect of R on the pyrazole ring was studied. Replacing the Me group
1
of 1a with a Cl (1l) decreased the potency by 13 folds, while substitution of the Me group
with larger Br (1m) or Et (1n) group retained high potency. However, further increase of
the size to an i-Pr resulted in an analog (1o) with significantly decreased potency, although
the similar sized N,N-dimethyl amino (1p) restored the potency of the compound.
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Then we sought out to substitute the 2-aminoquinoline moiety using compound 1d
as a template. The naphthyl analog 1q and its regio-isomer 1r showed no obvious
inhibitory activity at concentrations up to 1 µM. These results highlighted the importance
of the presence and location of the quinoline nitrogen atom in maintaining high potency.
Truncation of the quinoline yielded a new compound 1s, which was >300-fold less potent
than compound 1d. This result demonstrated that the phenyl ring of the quinoline
fragment was also critical.
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Table 1. Inhibition of the Wnt/β-Catenin Signaling Pathway by Compounds 1a-s
compound
R
1
R
2
R
3
IC50 (nM)a
2.2 ± 0.3
1
a
Me
Me
Me
Me
Me
Me
1b
0.92 ± 0.1
2.3 ± 0.4
1.2 ± 0.3
1.6 ± 0.3
3.2 ± 0.6
1
c (YW2065)
1d
1
e
1
f
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g
Me
Me
Me
Me
Me
Cl
16 ± 1
14 ± 3.1
14 ± 1.3
26 ± 0.9
2.2 ± 0.3
29 ± 5
1h
1i
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0
1
j
1k
1
l
1
m
Br
3.7 ± 0.3
1.7 ± 0.3
15 ± 1
1n
Et
1
o
p
q
i-Pr
1
N(CH
3
)
2
6.0 ± 1.5
>1,000
1
Me
1r
1s
Me
>1,000
Me
380 ± 5
750 ± 137
pyrvinium
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XAV939
450 ± 130
aThe values of IC50 for each compound were calculated and data are expressed as mean
IC50 (nM) ± SD from three independent experiments.
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We have also synthesized and tested the regioisomers of the pyrazole core (2a-e).
As shown in Table 2, compounds 2a-e indicated significantly decreased potency compared
to the ones in Table 1. These results indicated that the position of the amide substitution
on the pyrazole core plays a critical role in the inhibitory activity of inhibitors.
Table 2. Inhibition of the Wnt/β-Catenin Signaling Pathway by Compounds 2a-e
compound
R
H
n
-
IC50 (nM)a
637 ± 67
250 ± 13
67 ± 9
2a
2b
Me
Et
H
-
2
c
-
2d
1
2
140 ± 4
2
e
H
260 ± 42
aThe IC50 values were calculated and data are expressed as mean IC50 (nM) ± SD from
three independent experiments.
Next, compounds 1a-1p, which showed promising potencies in the luciferase gene
reporter assays, were further tested for their activities in killing human CRC SW480 cells
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(Figure 3A). Nine compounds that showed over 50% growth inhibition at 50 μM were
then tested in full dose responses to obtain their GI50 values (Figure 3B). Compounds 1c
(
YW2065) and 1f demonstrated good potencies with GI50 values of 2.4 μM and 3.9 μM,
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respectively. The TANK inhibitor XAV939 indicated a GI50 value of >50 μM. Note that
GI50 values of our new inhibitors in this CRC cell killing assays were significantly higher
than the corresponding IC50 values obtained from the gene reporter assays. We reasoned
that the reporter assay was designed with high sensitivity and respond rapidly to
perturbations of the Wnt signaling pathway. However, when the compounds were tested
in killing the much more complex cancer cells, the effects usually took a longer time to
take place. Therefore, we observed significantly decreased sensitivities of inhibitors in
killing the SW480 cells. Moreover, the effects of Wnt inhibition can be alleviated by the
function of other biological pathways, which might also contribute to the delayed cell death.
Figure 3. (A) Anti-proliferation activity of selected compounds on the growth of SW480
cells. (B) The GI50 values were calculated and data was expressed as mean GI50 (μM) ±
SD from three independent experiments.
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Encouraged by the activity of YW2065 in both luciferase gene reporter assay and
CRC cell killing assay, we next studied the mechanism of action of this compound (Figure
4
). Similar to pyrvinium and FX1128, YW2065 induced β-catenin degradation in
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HEK293 cells (Figure 4B). Interestingly, YW2065 was found to enhance the expression
of Axin-1 in fluorescent imaging (Figure 4A) and western blot analysis (Figure 4B).
Axin-1 is not only a master scaffolding protein for the destruction complex of β-catenin,
but also a key regulator of multiple pathways. It was reported that Axin-1 enhances
AMPK activation through the Axin-LKB1-AMPK complex, which facilitates the Thr172
phosphorylation and subsequent activation of AMPK.43 To verify the relationship
between Axin-1 and AMPK, we overexpressed Axin-1 in HEK293 cells and found that the
overexpression of Axin-1 led to increased AMPK phosphorylation (Figure 4C). Similar
activation of AMPK was also observed when HEK293 cells were exposed to compound
YW2065 and several analogs, while the effect was abolished byAxin-1 knockdown (Figure
4D, Figure S2). These results suggested that YW2065 achieved its dual activities of Wnt
inhibition and AMPK activation through the mechanism of Axin-1 stabilization. Note
that TNKS inhibitors (e.g., XAV939) also function by stabilizing Axin. However, our
previous results showed that the YW2065 analog FX1128 did not bind to TNKS in SPR
31
binding assay. More importantly, TNKS inhibitor XAV939 failed to activate AMPK.31,44
These data highlighted that YW2065 achieved dual activities of Wnt inhibition and AMPK
activation through Axin-1 stabilization via a novel mechanism different from TNKS
inhibitors. The mechanism for YW2065 to activate AMPK is also likely different from
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those of other known AMPK activators such as metformin, AICAR and salicylates.
Whereas metformin and AICAR activate AMPK via the disruption of the adenylate charge
in the cell, salicylates directly act on the AMPK β1-subunit.45,46 Currently additional
investigations are undergoing to elucidate whether YW2065 stabilized Axin-1 through
direct binding or other indirect mechanism.
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Figure 4. Dual function of YW2065 for Wnt signaling inhibition and AMPK activation.
(A) Enhanced Axin-1 protein expression by YW2065 (1 µM) treatment. HEK293 cells
were stained with anti-Axin-1 antibody and DAPI, and the images were taken under
fluorescence microscopy. (B) Enhanced Axin-1 expression and increased β-catenin
degradation by YW2065 or its analog FX1128 in HEK293 cells. (C) Increased AMPK
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phosphorylation by Axin-1 overexpression (empty pcDNA 3 vs. pcDNA3-Axin-1) in
HEK293 cells. (D) Axin-1 knockdown abolished the effect of AMPK activation by
YW2065 in HEK293 cells.
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Next, the activity of YW2065 was further investigated using human CRC SW480
and SW620 cells (Figure 5). SW480 and SW620 cells are derived from the primary and
metastatic sites of the same patient, respectively. Both cells contain hyperactivated Wnt
signaling pathway. Similar to the results from gene reporter assays using HEK293 cells
(
Table 1), YW2065 demonstrated excellent inhibitory potencies (IC50 = 1.15 nM and 0.19
nM, respectively) in the TOPflash reporter assays using both SW480 and SW620 cells
Figure 5A). Because the lymph node metastatic site derived SW620 cells have been
(
widely used as a mCRC cellular model, additional Western blot and RT-PCR analyses were
performed using this cell line. Upon treatment of YW2065, the protein level of Axin-1
was increased dose-dependently, while the protein level of β-catenin was significantly
decreased (Figure 5B). We also confirmed that the mRNA levels of Wnt target genes,
including c-Myc, cyclin D1, MMP7, and S100A4, were significantly decreased upon the
treatment of YW2065 (Figure 5C). Moreover, YW2065 activated AMPK through
phosphorylation, which in turn, led to the phosphorylation of the downstream substrate
acetyl-CoA carboxylase (ACC) in a dose-dependent manner (Figure 5D). These results
confirmed YW2065’s dual activities of Wnt inhibition and AMPK activation in CRC cells.
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Figure 5. Inhibition of Wnt/β-catenin pathway and activation of AMPK by YW2065 in
CRC cells. (A) TOPflash reporter assay was performed in SW480 and SW620 cells.
(
B) Dose response of β-catenin and Axin-1 levels upon treatment of YW2065. (C)
Decreased mRNA levels of Wnt target genes in SW620 cells upon exposure to YW2065
for 24 h. (D) Dose response of AMPK and ACC phosphorylation upon the treatment of
YW2065.
To characterize the in vitro anti-CRC activities of YW2065, we performed the
colony formation assay using SW480, SW620, and an additional human CRC HT29 cell
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lines (Figure 6A). The results showed that compound YW2065 dose-dependently
inhibited the colony formation of all three cell lines. In addition, as analyzed by flow
cytometry, treatment of YW2065 dose-dependently caused apoptosis in all three CRC cells
(Figure 6B and data not shown). Importantly, SW620 cells became resistant to the
cytotoxicity of YW2065 when constitutively active β-catenin mutant (S33Y) was
overexpressed, or the Axin-1 gene was knocked down (Figure 6C). These results were in
consistent with the mechanistic engagement of Wnt signaling as a critical part of the action
of YW2065.
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Figure 6. YW2065 inhibited CRC cell proliferation and growth in vitro. (A) Inhibition
of colony formation of CRC cells by YW2065 treatment. Cells (500 cells/well in 6-well
plate) were treated with vehicle or YW2065 until visible colonies formed. (B) Induction
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of apoptosis by YW2065 treatment in SW620 cells. SW620 cells were treated with
YW2065 for 48 h, then stained with AAD-7 and annexin V, then analyzed by flow
cytometry. (C) SW620 cells became resistant to YW2065 cytotoxicity when Axin-1 was
knocked down by lentivrus encoding shRNA against Axin-1 or a constitutively active β-
catenin mutant (S33Y) was overexpressed. Cells were treated with YW2065 or vehicle
for 72 h.
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To further study target specificity of YW2065, it was tested against a panel of 273
kinases. The results indicated that YW2065 inhibited none of the tested kinases with over
40% at the concentration of 1000 nM. It displayed a moderate inhibitory activity (39%
inhibition) for RPS6KA3, and weak activities against 10 other kinases including CDK17,
GSK3A, MAP4K4, MAPKAPK3, MELK, NEK6, NTRK1, PAK3, PRKCQ, and SRC
(
Table S1).
Next, the pharmacokinetic (PK) profile of compound YW2065 was determined
(Table 3). Compound YW2065 was administrated as a single dose at 5 mg/kg, either
intravenously (I.V.) or orally (P.O.). The maximum serum concentration (Cmax) was
determined to be approximately 5 µg/mL, and the Tmax was measured to be around 18 min.
Compared to the extremely low bioavailability of pyrvinium, the bioavailability (F%) of
YW2065 was improved to be about 23%. The area under the curve (AUC) values of I.V.
and P.O. were calculated to be 57.5 µg∙h/ mL and 13.2 µg∙h/ mL, respectively. Finally,
the half-life (T1/2) of compound YW2065 was determined to be about 3 h.
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Table 3. Single Dose Pharmacokinetics of YW2065.a
Route of Administration
Dose (mg/kg)
I.V. (n = 3)
P.O. (n = 5)
5.0
5.0
C
max (µg/mL)
5.2 ± 2.6
18.0 ± 6.7
22.9 ± 9.3
13.2 ± 3.8
2.9 ± 1.6
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T
max (min)
F (%)
AUC(0-inf) (µg∙h/mL)
1/2 (h)
57.5 ± 16.1
3.1 ± 1.5
T
aPharmacokinetic parameters of YW2065 derived from Phoenix WinNonlin 7.3 using the
non-compartmental analysis. Data is presented as estimates average ± standard deviation.
Considering the favorable PK properties of YW2065, we proceeded to evaluate its
anti-CRC efficacy in vivo using an SW620 tumor xenograft mice model (Figure 7).47
SW620 cells were implanted subcutaneously in the female nude mice on the right flank.
2
After tumors reached a volume of 100-200 mm , mice (n = 6/group) received YW2065 (10
mg/kg or 50 mg/kg) or vehicle intraperitoneally (I.P.). Compound YW2065 reduced both
tumor volume (Figure 7A) and final tumor weight (Figure 7B). The tumor volume in the
high dosage group (50 mg/kg/day) was decreased by 49% compared to the vehicle group
in the last measurement. In addition, YW2065 was also well tolerated based on body
weight and liver/kidney function tests (Figures S3-S4). Mouse body weight gain showed
no significant difference in the control and YW2065 treated groups. In a 21-day toxicity
test at a dose of 100 mg/kg/day (I.P.), minimal system toxicity of compound YW2065 was
observed. To assess β-catenin inhibition and AMPK activation, the tumor tissues were
harvested and subjected to western blot and RT-PCR analysis. Compared to the samples
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from vehicle group, the phosphorylation of AMPK and ACC was increased in those from
the YW2065-treated group (Figure 7C). In addition, the transcripts of Wnt target genes
Axin-2, MMP-7 and S100A4 were reduced upon treatment of compound YW2065 (Figure
7D). It should be noted that the tolerability and efficacy of YW2065 has yet to be fully
elucidated in the context of systemic exposure for specific routes of dosing. Nevertheless,
as proof of concepts, we have demonstrated that YW2065 has exhibited an improved
pharmacokinetic properties as compared to the parent pyrvinium and importantly, the
therapeutic effects against in vivo colon cancer growth.
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Figure 7. YW2065 suppressed SW620 tumor growth in vivo. Six-week old nude mice
6
were injected with 2 × 10 SW620 cells subcutaneous. After tumors reach a volume of
2
1
00-200 mm , mice were received I.P. injection of vehicle or 10 mg/kg, 50 mg/kg of
YW2065 each day (n = 6 each group). The tumor growth (A) was measured every other
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day. *P < 0.05 as compared to the vehicle control in t test. The mice were euthanized at
day 32, and tumors were weighed (B). The phosphorylation of AMPK and ACC in the
tumor tissues was detected by western blot (C). The expression of Wnt pathway target
genes (Axin-2, MMP-7, and S100A4) in tumor tissues were detected by RT-PCR (D).
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CONCLUSIONS
The mCRC, with complex genetic profiles and poor prognoses, remains a significant cause
of cancer related morbidity worldwide. We reported a series of pyrazole-based small
molecules that displayed potent anti-CRC activities in vitro and in vivo. One of the
compounds, YW2065, displayed dual activities of Wnt inhibition and AMPK activation,
which was convergent by Axin-1 stabilization. Moreover, YW2065 demonstrated
favorable PK properties and suppressed tumor growth in a xenograft mouse model.
Therefore, YW2065 represents a promising new lead for further development of anti-CRC
therapeutics.
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EXPERIMENTAL SECTION
Chemical Analysis. All chemicals were obtained from commercial suppliers and used
without further purification. Analytical thin layer chromatography was visualized by
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1
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
13
(
J/Hz). C 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
3
or
DMSO-d , respectively. The purities of title compounds were determined by analytic
6
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% acetonitrile/55%
water (containing 0.1% TFA) over 20 mins with a detection at 260 nM and a flow rate at
1.0 mL/min. All tested compounds were > 95% pure. Yields were not optimized. 4b-f was
commercially available. Pyrazole compounds 5e, 5f, 5l-p and 8e were prepared as reported
previously. 48,49
General Procedure A: Synthesis of Compounds 1a-s. To a solution of 5 (1.2 mmol),
aromatic amines (1.0 mmol) in dichloroethane (5.0 mL) was added 1-(chloro-1-
pyrrolidinylmethylene)pyrrolidinium hexafluorophosphate (398 mg, 1.2 mmol) and
o
DIPEA (516 mg, 4 mmol). The mixture was allowed to stir at 80 C overnight. The solvent
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was removed under vacuum and the resulting residue was redissolved in ethyl acetate (15
mL). The resulting solution was washed with H
2
O and brine, dried over Na
2
SO and
4
concentrated. The crude material was purified by column chromatography to give
0
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compounds 1a-s (24-88%).
5
-Methyl-1-phenyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1a). The title
1
compound was synthesized according to General Procedure A (81%): H-NMR (400 MHz,
CDCl
3
) δ 8.74 (s, 1H), 8.55-8.53 (d, J = 9.6 Hz, 1H), 8.20-8.18 (d, J = 9.6 Hz, 1H), 8.09
(
s, 1H), 7.84-7.82 (d, J = 8.8 Hz, 1H), 7.79-.77 (d, J = 8.0 Hz, 1H), 7.68-7.64 (t, J = 8.0
13
Hz, 1H), 7.53-7.49 (m, 2H), 7.47-7.43 (m, 4H), 2.66 (s, 3H); C-NMR (100 MHz, CDCl )
3
δ 162.2, 151.2, 146.4, 143.6, 138.6, 138.4, 130.1, 129.3, 128.8, 127.6, 127.0, 126.3, 125.5,
+
125.1, 115.4, 114.5, 12.1. HRMS [M + H] (ESI-TOF) calcd for C20
H
17
4
N O 329.1402,
found 329.1409. HPLC: t = 5.45 min, 99.9%.
R
1
-(2-Fluorophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1b). The
1
title compound was synthesized according to General Procedure A(82%, a white solid) H-
NMR (400 MHz, DMSO-d
8.0 Hz, 1H), 7.90-7.88 (d, J = 8.0 Hz, 1H), 7.55-7.71 (t, J = 8.0 Hz, 1H), 7.65-7.61 (m, 2H),
.57-7.50 (m, 2H), 7.46-7.42 (t, J = 8.0 Hz, 1H), 2.46 (s, 3H); 13C-NMR (100 MHz,
DMSO-d ) δ 167.5, 162.7, 160.2, 157.2, 151.6, 150.1, 145.8, 145.6, 143.1, 136.9, 136.8,
6
) δ 10.93 (s, 1H), 8.64 (s, 1H), 8.39 (m, 2H). 7.96-7.94 (d, J =
7
6
135.1, 134.6, 132.9, 132.1, 130.9, 130.6, 130.1, 122.0, 121.9, 120.6, 119.8, 16.2. HRMS
+
[
M + H] (ESI-TOF) calcd for C20
H16FN
4
O 347.1308, found 347.1300. HPLC: t = 5.83
R
min, 99.7%.
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1-(2-Bromophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide
(1c,
YW2065). The title compound was synthesized according to General Procedure A (65%,
1
a yellow solid): H-NMR (400 MHz, CDCl
3
) δ 8.74 (s, 1H), 8.55-8.53 (d, J = 8.0 Hz, 1H),
1
1
1
1
1
1
1
1
1
1
2
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2
3
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8
9
0
8.20-8.18 (d, J = 8.8 Hz, 1H), 8.11 (s, 1H), 7.84-7.82 (d, J = 8.8 Hz, 1H), 7.79-7.77 (d, J =
.8 Hz, 1H), 7.74-7.72 (d, J = 8.8 Hz, 1H), 7.68-7.64 (t, J = 8.0 Hz, 1H), 7.49-7.44 (m, 2H),
8
1
3
7.42-7.37 (m, 2H), 2.50 (s, 3H); C-NMR (100 MHz, CDCl
3
) δ 162.1, 151.2, 146.6, 145.2,
1
38.8, 138.5, 137.8, 133.6, 131.4, 130.0, 129.6, 128.4, 127.6, 127.2, 126.3, 125.1, 122.3,
+
1
14.8, 114.5, 11.4. HRMS [M + H] (ESI-TOF) calcd for C20
407.0513. HPLC: t = 10.8 min, 100%.
-(2-Ethylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1d). The
title compound was synthesized according to General Procedure A (84%, a brown solid):
1H-NMR (400 MHz, DMSO-d
) δ 10.85 (s, 1H), 8.62 (s, 1H), 8.39 (m, 2H), 7.96-7.94 (d,
H15BrN
4
O 407.0507, found
R
1
6
J = 8.0 Hz, 1H), 7.90-7.88 (d, J = 8.0 Hz, 1H), 7.55-7.71 (t, J = 8.0 Hz, 1H), 7.53-7.50 (m,
3H), 7.42-7.38 (t, J = 8.0 Hz, 1H), 7.34-7.32 (d, J = 8.0 Hz, 1H), 2.36-2.31 (m, 5H), 1.03-
13
1
.0 (q, J = 8.0 Hz, 3H); C-NMR (100 MHz, DMSO-d
6
) δ 167.7, 157.2, 151.6, 149.4,
146.5, 144.7, 143.1, 142.1, 135.2, 135.1, 134.8, 133.0, 132.9, 132.1, 131.9, 130.8, 130.1,
+
1
20.6, 119.1, 28.6, 19.7, 16.5. HRMS [M + H] (ESI-TOF) calcd for C22
H
21
4
N O 357.1715,
found 357.1715. HPLC: t = 9.03 min, 99.2%.
R
1-(2,6-Difluorophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1e).
The title compound was synthesized according to General Procedure A (64%, a yellow
1
foam): H-NMR (400 MHz, CDCl
3
) δ 8.91 (s, 1H), 8.53-8.51 (d, J = 9.6 Hz, 1H), 8.18 (s,
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1H), 8.18-8.16 (d, J = 8.8 Hz, 1H), 7.82-7.80 (d, J = 8.4 Hz, 1H), 7.77-7.75 (d, J = 8.0 Hz,
1
H), 7.65-7.61 (t, J = 7.2 Hz, 1H), 7.48-7.40 (m, 2H), 7.11-7.07 (t, J = 8.0 Hz, 1H), 2.53
13
(
s, 3H); C-NMR (100 MHz, CDCl
3
) δ 161.9, 159.6, 157.1, 157.0, 151.2, 146.4, 139.9,
0
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138.6, 131.6, 131.5, 131.4, 130.0, 127.6, 127.1, 126.3, 125.1, 115.2, 114.6, 112.5, 112.3,
+
1
0.9. HRMS [M + H] (ESI-TOF) calcd for C20
H
15
F
2
4
N O 365.1214, found 365.1210.
HPLC: t
R
= 7.08 min, 99.5%.
1
-(2-Bromo-6-fluorophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide
(
1f). The title compound was synthesized according to General Procedure A (69%, a white
1
solid): H-NMR (400 MHz, CDCl
3
) δ 8.89 (s, 1H), 8.55-8.53 (d, J = 8.8 Hz, 1H), 8.20-
8
.18 (t, J = 4.8 Hz, 2H), 7.83-7.81 (d, J = 8.0 Hz, 1H), 7.78-7.76 (d, J = 8.8 Hz, 1H), 7.67-
7.63 (t, J = 8.0 Hz, 1H), 7.54-7.52 (d, J = 8.0 Hz, 1H), 7.45-7.41 (t, J = 8.0 Hz, 1H), 7.40-
1
3
7
1
.35 (m, 1H), 7.25-7.21 (t, J = 8.0 Hz, 1H), 2.50 (s, 3H); C-NMR (100 MHz, CDCl ) δ
3
61.9, 160.2, 157.6, 151.2, 146.5, 145.9, 139.7, 138.6, 132.3, 132.2, 130.1, 128.9, 127.6,
+
127.1, 126.5, 126.3, 125.1, 123.9, 115.9, 115.8, 115.1, 114.6, 10.9. HRMS [M + H] (ESI-
TOF) calcd for C20
H
15BrFN
4
O 425.0413, found 425.0416. HPLC: t = 9.03min, 99.9%.
R
1-(2,4-Difluorophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1g).
The title compound was synthesized according to General Procedure A (82%, a brown
1
solid): H-NMR (400 MHz, CDCl
3
) δ 8.75 (s, 1H), 8.54-8.52 (d, J = 8.8 Hz, 1H), 8.22-
8.20 (d, J = 8.8 Hz, 1H), 8.14 (s, 1H), 7.84-7.82 (d, J = 8.8 Hz, 1H), 7.81-7.79 (d, J = 8.8
Hz, 1H), 7.69-7.65 (t, J = 8.8 Hz, 1H), 7.50-7.44 (m, 2H), 7.06-7.02 (m, 2H), 2.55 (s, 3H);
13C-NMR (100 MHz, CDCl
) δ 164.4, 164.3, 161.9, 158.4, 155.7, 151.1, 146.4, 145.6,
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139.4, 138.7, 130.1, 130.0, 127.6, 127.0, 126.3, 125.2, 115.2, 114.5, 112.4, 112.2, 105.4,
+
1
05.2, 104.9, 11.2. HRMS [M + H] (ESI-TOF) calcd for C20
H
15
F
2
4
N O 365.1214, found
3
65.1202. HPLC: t = 6.07 min, 99.8%.
R
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1-(3-Chloro-2-fluorophenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide
(
1h). The title compound was synthesized according to General Procedure A (56%, a white
1
solid): H-NMR (400 MHz, CDCl
3
) δ 8.70 (s, 1H), 8.54-8.52 (d, J = 8.4 Hz, 1H), 8.22-
8
.20 (d, J = 8.8 Hz, 1H), 8.14 (s, 1H), 7.84-7.82 (d, J = 8.8 Hz, 1H), 7.81-7.79 (d, J = 8.0
Hz, 1H), 7.69-7.65 (t, J = 7.2 Hz, 1H), 7.58-7.54 (t, J = 7.2 Hz, 1H), 7.48-7.44 (t, J = 7.2
1
3
Hz, 1H), 7.41-7.37 (t, J = 7.2 Hz, 1H), 7.28-7.24 (t, J = 8.0 Hz, 1H), 2.58 (s, 3H); C-NMR
100 MHz, CDCl ) δ 161.8, 154.2, 151.6, 151.1, 146.5, 145.6, 139.5, 138.6, 131.8, 130.1,
127.6, 127.3, 127.2, 126.3, 125.2, 124.9, 124.8, 122.7, 115.4, 114.5, 11.3. HRMS [M + H]+
ESI-TOF) calcd for C20 O 381.0918, found 381.0928. HPLC: t = 10.21 min,
9.8%.
(
3
(
H
15ClFN
4
R
9
1-(2,5-Dimethylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1i).
The title compound was synthesized according to General Procedure A(56%, a yellow oil):
1H-NMR (400 MHz, CDCl
8.8 Hz, 1H), 8.11 (s, 1H), 7.84-7.82 (d, J = 8.8 Hz, 1H), 7.79-7.77 (d, J = 8.8 Hz, 1H),
.68-7.64 (t, J = 8.0 Hz, 1H), 7.46-7.42 (t, J = 8.0 Hz, 1H), 7.21-7.18 (m, 2H), 7.04 (s, 1H),
) δ 8.84 (s, 1H), 8.56-8.54 (d, J = 8.8 Hz, 1H), 8.20-8.18 (d, J
3
=
7
13
2.45 (s, 3H), 2.35 (s, 3H), 1.98 (s, 3H); C-NMR (100 MHz, CDCl
3
) δ 162.4, 151.3, 146.4,
1
44.4, 138.6, 138.4, 137.3, 136.7, 132.5, 130.9, 130.6, 130.0, 127.6, 127.1, 126.2, 125.1,
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+
114.6, 114.4, 20.7, 16.7, 11.3. HRMS [M + H] (ESI-TOF) calcd for C22
H
21
N O 357.1715,
4
found 357.1728. HPLC: t
R
= 8.39 min, 99.9%.
1
-(2,6-Dimethylphenyl)-5-methyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1j).
0
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0
The title compound was synthesized according to General Procedure A (74%, a yellow
1
foam): H-NMR (400 MHz, DMSO-d
6
) δ 10.85 (s, 1H), 8.67 (s, 1H), 8.39 (m, 2H), 7.96-
7.94 (d, J = 8.0 Hz, 1H), 7.90-7.88 (d, J = 8.0 Hz, 1H), 7.76-7.72 (t, J = 8.0 Hz, 1H), 7.54-
7
.50 (t, J = 8.0 Hz, 1H), 7.40-7.36 (t, J = 8.0 Hz, 1H), 7.29-7.27 (m, 2H), 2.29 (s, 3H), 1.91
13
(
s, 6H); C-NMR (100 MHz, DMSO-d
6
) δ 167.7, 157.2, 151.6, 149.1, 145.2, 143.1, 141.8,
141.1, 135.1, 134.8, 133.5, 132.9, 132.1, 130.8, 130.1, 120.6, 119.1, 21.9, 15.8. HRMS [M
+
+
H] (ESI-TOF) calcd for C22
H
21
N
4
O 357.1715, found 357.1714. HPLC: t = 7.82 min,
R
99.6%.
-Methyl-1-(3-methylpyridin-2-yl)-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide
5
(
1k). The title compound was synthesized according to General Procedure A (24%, a white
1
solid): H-NMR (400 MHz, CDCl
3
) δ 8.55 (s, 1H), 8.53 (s, 1H), 8.47-8.46 (d, J = 4.0 Hz,
1
H), 8.21-8.19 (d, J = 8.8 Hz, 1H), 8.01 (s, 1H), 7.85-7.83 (d, J = 8.0 Hz, 1H), 7.81-7.79
(d, J = 8.0 Hz, 1H), 7.77-7.75 (d, J = 8.0 Hz, 1H), 7.70-7.66 (t, J = 8.0 Hz, 1H), 7.47-7.43
1
3
(
t, J = 8.0 Hz, 1H), 7.39-7.36 (m, 1H), 2.58 (s, 3H), 2.20 (s, 3H); C-NMR (100 MHz,
CDCl
3
) δ 162.1, 151.1, 150.0, 146.7, 144.7, 140.6, 138.5, 131.0, 130.0, 127.6, 127.3, 126.3,
+
125.1, 124.8, 114.9, 114.5, 17.2, 11.4. HRMS [M + H] (ESI-TOF) calcd for C20
H
18
5
N O
3
44.1511, found 344.1507. HPLC: t = 3.73 min, 99.6%.
R
5-Chloro-1-phenyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1l). The title
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compound was synthesized according to General Procedure A (55%, a white solid): H-
NMR (400 MHz, CDCl ) δ 8.93 (s, 1H), 8.53 (s, 1H), 8.27 (s, 1H), 8.22-8.20 (d, J = 8.0
Hz, 1H), 7.86-7.84 (d, J = 8.0 Hz, 1H), 7.80-7.78 (d, J = 8.0 Hz, 1H), 7.68-7.64 (t, J = 8.0
3
1
1
1
1
1
1
1
1
1
1
2
2
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5
5
5
5
5
5
5
5
6
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
13
Hz, 1H), 7.55-7.45 (m, 5H); C-NMR (100 MHz, CDCl
3
) δ 159.5, 150.8, 141.6, 139.9,
1
38.7, 138.6, 130.0, 129.6, 129.4, 129.2, 127.4, 126.5, 126.1, 125.3, 119.5, 115.3, 114.6.
+
HRMS [M + H] (ESI-TOF) calcd for C19
H14ClN
4
O 349.0856, found 349.0652. HPLC: t
R
=
13.58 min, 99.0%.
5
-Bromo-1-phenyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1m). The title
1
compound was synthesized according to General Procedure A (60%, a white solid): H-
NMR (400 MHz, CDCl
3
) δ 8.94 (s, 1H), 8.56-8.54 (d, J = 8.0 Hz, 1H), 8.29 (s, 1H), 8.22-
8.20 (d, J = 8.0 Hz, 1H), 7.87-7.85 (d, J = 8.4 Hz, 1H), 7.81-7.79 (d, J = 8.0 Hz, 1H), 7.70-
1
3
7
.66 (t, J = 8.0 Hz, 1H), 7.54 (m, 5H), 7.48-7.44 (d, J = 8.0 Hz, 1H); C-NMR (100 MHz,
CDCl
3
) δ 161.0, 150.8, 142.1, 138.7, 138.2, 130.0, 129.5, 129.2, 127.6, 127.4, 126.5, 126.2,
+
125.3, 119.6, 117.9, 114.8, 114.6. HRMS [M + H] (ESI-TOF) calcd for C19
H
14BrN O
4
3
93.0351, found 393.0355. HPLC: t = 13.12 min, 99.2%.
R
5-Ethyl-1-phenyl-N-(quinolin-2-yl)-1H-pyrazole-4-carboxamide (1n). The title
1
compound was synthesized according to General Procedure A (66%, a white solid): H-
NMR (400 MHz, CDCl
9.6 Hz, 1H), 8.06 (s, 1H), 7.85-7.83 (d, J = 8.4 Hz, 1H), 7.80-7.78 (d, J = 8.0 Hz, 1H),
.69-7.65 (t, J = 8.0 Hz, 1H), 7.54-7.43 (m, 6H), 3.09-3.04 (q, J = 8.0 Hz, 2H), 1.25-1.21
3
) δ 8.66 (s, 1H), 8.55-8.53 (d, J = 9.6 Hz, 1H), 8.20-8.18 (d, J =
7
1
3
(t, J = 8.0 Hz, 3H); C-NMR (100 MHz, CDCl ) δ 161.8, 151.2, 149.6, 146.7, 138.8, 138.5,
3
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