Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective, and Orally Bioavailable Porcupine Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.6b00038

Blockade of aberrant Wnt signaling is an attractive therapeutic approach in multiple cancers. We developed and performed a cellular high-throughput screen for inhibitors of Wnt secretion and pathway activation. A lead structure (GNF-1331) was identified from the screen. Further studies identified the molecular target of GNF-1331 as Porcupine, a membrane bound O-acyl transferase. Structure-activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound 19, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1 xenograft tumor model.

Full text of DOI:10.1021/acsmedchemlett.6b00038

Letter
pubs.acs.org/acsmedchemlett
Discovery of Pyridinyl Acetamide Derivatives as Potent, Selective,
and Orally Bioavailable Porcupine Inhibitors
Dai Cheng, Jun Liu, Dong Han, Guobao Zhang, Wenqi Gao, Mindy H. Hsieh, Nicholas Ng,
Shailaja Kasibhatla, Celin Tompkins, Jie Li, Auzon Steffy, Fangxian Sun, Chun Li, H. Martin Seidel,
Jennifer L. Harris, and Shifeng Pan*
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, United States
S
* Supporting Information
ABSTRACT: Blockade of aberrant Wnt signaling is an
attractive therapeutic approach in multiple cancers. We
developed and performed a cellular high-throughput screen
for inhibitors of Wnt secretion and pathway activation. A lead
structure (GNF-1331) was identified from the screen. Further
studies identified the molecular target of GNF-1331 as
Porcupine, a membrane bound O-acyl transferase. Struc-
ture−activity relationship studies led to the discovery of a novel series of potent and selective Porcupine inhibitors. Compound
19, GNF-6231, demonstrated excellent pathway inhibition and induced robust antitumor efficacy in a mouse MMTV-WNT1
xenograft tumor model.
KEYWORDS: Wnt signaling, Porcupine inhibitor
As part of our effort to target this important oncogenic
pathway, we are interested in searching for small molecules that
specifically block Wnt ligand secretion.13 To do this, we
performed a high-throughput cell-based screen of a 2.4-million
compound library, using a two-cell coculture assay system that
includes a Wnt ligand secreting cell, a stable L-cell line
overexpressing Wnt3A, and a Wnt reporter cell, a TM3 cell line
stably transfected with a Wnt-responsive luciferase reporter
gene. To confirm the specificity for inhibiting Wnt secretion,
the screening hits were triaged through two counter assays, a
Hedgehog (Hh) coculture assay and a Wnt reporter gene assay
(RGA) with the addition of Wnt3A conditioned medium. A
specific Wnt secretion inhibitor should be inactive in the two
counter assays. From this screening approach, we identified
several chemical classes that specifically block Wnt ligand
secretion. One of these is exemplified in GNF-1331 (Figure 1).
Through further target elucidation efforts, we determined
Porcupine (PORCN) is its molecular target.13 At the time of
this discovery, an inhibitor of Wnt signaling, IWP2, was also
reported to function through inhibiting PORCN by Lum and
nt signaling is tightly controlled during cellular
Wproliferation, differentiation, and embryonic morpho-
genesis.1,2 Aberrant activation of this pathway plays a critical
role in a variety of cancers, such as cutaneous squamous cell
carcinoma (SCC), breast cancer, and colorectal cancer.3−6 Wnt
signaling can be stimulated upon the binding of Wnt ligands to
their receptors LRP5/6 and Frizzled at the plasma membrane,
or be activated by downstream activating mutations.3−6
Activated Wnt signaling results in the accumulation of β-
catenin in the cytoplasm through the disruption of β-catenin
degradation machinery that consists of AXIN2, GSK3β, APC,
and others. Increased β-catenin then translocates into the
nucleus to form a complex with LEF/TCF and subsequently
induces downstream gene expression. Up-regulation of Wnt
signaling due to mutations in downstream components, such as
APC and β-catenin, is well documented in colorectal cancer. In
addition, increased pathway activity has been reported in
various cancers in association with overexpression of Wnt
ligands or costimulants such as R-Spondin 2/3 (RSPO2/3), or
silencing of Wnt inhibitor genes.7 More recently, mutations of
the RSPO coreceptors RNF43/ZNFR3 have been shown to
play critical roles in several cancers, including pancreatic,
colorectal, gastric cancer, and cholangiocarcinoma.8−10 Thus,
inhibition of Wnt signaling is an attractive approach for
anticancer therapy. Despite intensive research efforts, ther-
apeutic intervention of activated Wnt signaling in cancers
harboring downstream mutations has had limited success due
to lack of tractable targets. In contrast, there have been some
successes in developing therapeutics targeting the upstream
components of the Wnt pathway, including LRP6 and Frizzled
antibodies.11,12
Figure 1. Structure of GNF-1331, a screening hit.
Received: January 26, 2016
Accepted: May 10, 2016
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00038
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co-workers.14,15 PORCN is the enzyme responsible for post-
translational palmitoylation of Wnt proteins that is required for
both Wnt secretion as well as binding of Wnts to their
receptors.16,17 In our recent report, we described in vitro and in
vivo properties of our first-in-class PORCN inhibitor WNT974,
which is currently undergoing clinical trials.13 Several other
groups have also recently reported Porcupine inhibitors to
inhibit Wnt signaling activity.18−21 Herein, we report the hit to
lead and lead optimization efforts, which led to the discovery of
WNT974 (15) and GNF-6231 (19), a molecule with further
improved physicochemical properties, as a potent, selective, and
orally bioavailable Porcupine inhibitor that blocks Wnt
signaling in vitro and in vivo.
Our initial focus was to develop a tool compound that is
suitable for in vivo target validation work. GNF-1331 shows
good potency in the Wnt secretion coculture assay with an IC50
of 12 nM. It binds to PORCN in a radioligand binding assay
with an IC50 of 8 nM. However, it exhibits poor
pharmacokinetic properties with rapid clearance and low
systemic exposure after oral administration in mice (dose
normalized Cmax and AUC are 0.03 μM·h and 0.014 μM,
respectively), which prevent it from being used in vivo. From
the initial structure−activity relationship (SAR) analysis of
existing analogues in our compound collection, the pyridine
nitrogen appears to be important for activity. We envisioned
the thioether moiety can be one of the key metabolically labile
spots that result in poor pharmacokinetic properties. And we
further hypothesized that the thioether moiety together with
the N-propyl triazole functions as a linker between the two key
binding motifs on both sides of the molecule. To test this
hypothesis, we replaced the entire moiety with a simple phenyl
group as shown in 1 (Figure 2) in our first attempt. This
It is well-known that pyridine nitrogen with no adjacent
substitution, as in 1, can cause strong inhibition of CYP
enzymes.22 One of the tactics to address this issue is to
introduce a substituent at the adjacent carbon next to the
nitrogen. Indeed, introduction of a methyl group at the 2-
position as shown in 2 ameliorated the CYP inhibitory activity,
up to 10 μM testing concentration. More encouragingly, this
modification led to a 4-fold increase in the in vitro potency.
However, significant loss of activity was observed when moving
the 4-pyridine nitrogen to the 3-position as shown in 3.
Methylation of the amide nitrogen in 4 or reversal of the amide
configuration in 5 was also less tolerated. Interestingly, the
replacement of the fused benzothiazole ring with phenyl
thiazole in 6 led to only a slight decrease in potency. This loss
of activity can be completely compensated by the further
replacement of thiazole with either a pyridine or a phenyl as
shown in 7 or 8, respectively. The amide linkage appears
essential, since either urea 9 or carbamate 10 result in complete
loss of activity, up to 10 μM.
Based on these initial SAR and pharmacokinetic data, we
chose 7 as a new lead for further optimization with the
emphasis on aqueous solubility and other drug-like properties.
Given the highly lipophilic nature of 7, which has a cLogP of
4.8, one of our strategies to improve solubility was focused on
reducing lipophilicity. The selected SAR is summarized in
Table 1.
Realizing that multiple phenyl rings in the structure of
compound 7 contribute to the high lipophilicity, we focused
our initial attempts on introducing nitrogen into the rings to
lower cLogP and subsequently to improve aqueous solubility.
As seen from the results in the table, noticeable increases in
solubility were achieved using this approach, as shown in 11−
13, and the resulting compounds retained excellent in vitro
activity. Interestingly, the substitution of a methyl group at the
R2 position (14 and 15) led to further increases in solubility,
especially for the latter. This is likely due to the increased
dihedral angle between the two pyridines, which leads to
reduced π stacking. The methyl group at the R1-position can be
replaced by an electron withdrawing fluorine (16) without
losing potency. However, a hydroxymethyl group (17) is less
tolerated at this position. In addition to reducing lipophilicity,
we also tried to increase the sp3 fraction in the molecules to
improve the physicochemical properties. Replacement of the
terminal aromatic ring by piperizine was tolerated (18), but
resulted in a 30-fold decrease in potency. This loss of activity,
however, can be completely restored by the formation of either
acetamides (19−20) or methyl carbamate (21). As expected,
these modifications resulted in further improvement of aqueous
solubility as measured in the high-throughput solubility assay.
To further confirm this improved property, the aqueous
solubility was determined in a neutral pH buffer using
crystalline materials. The free base of 19 showed a good
solubility of 357 μM in contrast to 5 μM observed for the free
base of 15.
Figure 2. Structure−Activity Relationship of 1.
modification led to not only drastically improved in vivo
pharmacokinetic properties but also much improved in vitro
potency. In mice, compound 1 exhibits low clearance (2.49
mL/min/kg) and nearly complete oral bioavailability with dose
normalized AUC and Cmax of 26 μM·h and 4.1 μM,
respectively. It shows a 6-fold improved potency in the
coculture reporter gene assay with an IC50 of 2 nM.
While 1 is a suitable in vivo validation tool, it bears some
liabilities, such as strong inhibition of CYPs (e.g., IC50 of 0.8
μM on CYP3A4). Our initial chemistry effort was thus directed
toward understanding the general SAR of this scaffold in order
to further optimize the drug-like properties. The selected
results are summarized in Figure 2. The Wnt coculture RGA is
used to guide the SAR studies.
Based on favorable potency, specificity, and other drug-like
properties, such as physicochemical properties, in vitro
ADMET, and in vivo pharmacokinetics, compound 19, GNF-
6231, was chosen as one of the advanced compounds for
further in vitro and in vivo testing. Compound 19 exhibits
exquisite specificity for PORCN. In our broad off-target panel
profiling, there are no appreciable activities observed, at least up
to 10 μM testing concentrations, for more than 200 off-targets,
which include GPCRs, kinases, proteases, transporters, ion
B
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Table 1. IC50 Values for Select Compounds in the Wnt3a Co-culture RGA
a
b
c
See Supporting Information for detailed assay descriptions. HT: high throughput. ND: not determined.
channels, and nuclear receptors. Like the other analogues,
compound 19 shows IC50s of greater than 10 μM on all CYP
isoforms tested (2C9, 2D6, 3A4). Compound 19 is moderately
bound to mouse, rat, dog, monkey, and human plasma proteins
(88.0, 83.1, 90.9, 71.2, and 95%, respectively) as determined by
rapid equilibrium dialysis. It shows high permeability in a Caco-
2 human cell permeability assay (PappA-B of 7.5 × 10−6 cm/s)
with a possible efflux (efflux ratio of 3.2). In vitro metabolic
stability studies indicate that the microsomal clearance of 19 is
low across preclinical species and humans. It shows good oral
bioavailability, ranging from 72 to 96% in preclinical species
(mouse, rat, and dog) when dosed in solution formulations
(Table 2). Consistent with the in vitro microsomal clearance
data, the systemic clearance (CL) is low in all species, relative
to respective hepatic blood flow (Table 2). The terminal half-
lives are 2.4, 2.8, and 8.9 h in mouse, rat, and dog, respectively.
Compound 19 is expected to have minimal to marginal
distribution to tissues compared to total body water following
intravenous administration to mouse (Vss 0.57 L/kg), rat (Vss
0.70 L/kg), and dog (Vss 0.25 L/kg).
The mouse mammary tumor virus (MMTV)-Wnt1 trans-
genic mice spontaneously develop murine breast tumors. The
xenograft model derived from these tumors has been effectively
used to evaluate the in vivo pharmacodynamics and antitumor
efficacy of Wnt inhibitors.12 Given that 19 shows favorable
potency and a PK profile across preclinical species upon oral
administration, we elected to assess its in vivo activity in the
subcutaneous MMTV-Wnt1 xenograft mouse model.
To determine the inhibition of Wnt signaling by 19 in the
MMTV-Wnt1 tumors, Wnt target gene Axin2 expression
(Axin2 mRNA) was measured by quantitative RT-PCR through
TaqMan assays. In the single dose PK/PD relationship study,
compound 19 was dosed at 3 mg/kg orally in MMTV-Wnt1
tumor bearing mice, and plasma and tumor samples were
collected at various time points (1, 5, 7, 10, and 24 h post-
dosing). The plasma was analyzed for PK measurements, and
the tumor was analyzed for both PK and PD measurements. As
we can see from the results shown in Figure 3, compound 19
inhibited the signaling activity effectively, as shown by the
reduction of Axin2 mRNA levels with the maximal inhibition of
about 90% observed at 5 h post-dosing, a delayed PD response
in relation to the maximal drug concentrations (Cmax). The
Axin2 expression was back to baseline level 24 h after treatment
accompanying decreasing drug concentrations.
a
Table 2. In Vivo Pharmacokinetics in Preclinical Species
CL (mL/min/kg)
Mean SD
Vss (L/kg)
Mean SD
t1/2 (h)
F (%)
Mean SD Mean SD
Mouse
Rat
5.33 0.81
5.35 0.65
0.67 0.14
0.57 0.10
0.70 0.05
0.25 0.06
2.44 0.13
2.77 0.14
8.86 1.76
96 19
84
72 22
3
Dog
To test its in vivo antitumor efficacy, the MMTV-Wnt1
tumor bearing mice were treated with 19 by daily oral
administration for 2 weeks at three different dose levels.
a
The compound was administered as a solution in 20% PEG300/3%
ETPGS for mouse and rat or 25% PEG300/5% Solutol for dog.
C
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Synthetic schemes, procedure, and analytical data for 15
and 19; analytical data of 1−14, 16−18, and 20−21;
general animal study protocols; high-throughput sol-
ubility assay protocols (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: (858) 812-1621. E-mail: span@gnf.org.
■
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
■
ADMET, absorption, distribution, metabolism, excretion,
toxicity; CYP, cytochromes P450; PK/PD, pharmacokinetic/
pharmacodynamics; GPCR, G-protein coupled receptor
Figure 3. PK−PD relationship of 19 in the mouse MMTV-Wnt1
tumor model (n = 3 per time point).
Changes in tumor volume for each of the treated (T) and
control (C) groups were measured, and the T/C ratio was
calculated to express the level of efficacy. As shown in Figure 4,
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Treatment with 19, GNF-6231, in MMTV-Wnt1 subcuta-
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repeat dosing treatment. Porcupine inhibitors may represent a
new therapeutic approach for diseases with aberrant Wnt
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00038.
■
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