Discovery of potent, orally bioavailable, small-molecule inhibitors of WNT signaling from a cell-based pathway screen

Article abstract of DOI:10.1021/jm501436m

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target.

Full text of DOI:10.1021/jm501436m

Article
pubs.acs.org/jmc
Discovery of Potent, Orally Bioavailable, Small-Molecule Inhibitors of
WNT Signaling from a Cell-Based Pathway Screen
Aurelie Mallinger,† Simon Crumpler,† Mark Pichowicz,† Dennis Waalboer,† Mark Stubbs,†
́
Olajumoke Adeniji-Popoola,† Bozena Wood,‡ Elizabeth Smith,† Ching Thai,† Alan T. Henley,†
Katrin Georgi,§ William Court,† Steve Hobbs,† Gary Box,† Maria-Jesus Ortiz-Ruiz,† Melanie Valenti,†
Alexis De Haven Brandon,† Robert TePoele,† Birgitta Leuthner,§ Paul Workman,† Wynne Aherne,†
Oliver Poeschke,§ Trevor Dale,‡ Dirk Wienke,§ Christina Esdar,§ Felix Rohdich,§ Florence Raynaud,†
Paul A. Clarke,† Suzanne A. Eccles,† Frank Stieber,§ Kai Schiemann,§ and Julian Blagg*,†
†Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London SW7 3RP, U.K.
‡School of Bioscience, Cardiff University, Cardiff CF10 3XQ, U.K.
§Merck KGaA, Merck Serono, 64293 Darmstadt, Germany
S
* Supporting Information
ABSTRACT: WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the
generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9
using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the
pyridine−piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this
twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545),
a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT
pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This
work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-
based pathway assay without knowledge of the biochemical target.
catenin degradation by the destruction complex limits β-
catenin-mediated gene transcription. Key components of the β-
catenin destruction complex include Axin, APC (the protein
product of the adenomatous polyposis coli tumor suppressor
gene), and glycogen synthase kinase-3-β (GSK3-β), which
phosphorylates β-catenin and thereby renders β-catenin a
substrate for ubiquitination and degradation by the 26S
proteasome.5
Overexpression of WNT-regulated genes can cause trans-
formation of mammalian epithelial cells.6 Clinically, 80% of
colon cancers have defects in the APC gene, leading to high
levels of β-catenin.7,8 A subset of colon cancers and melanomas
harbor β-catenin mutations that prevent its phosphorylation
INTRODUCTION
■
The WNT signaling network is a major regulator of mammalian
development through control of cellular functions such as
proliferation and differentiation.1 WNT signaling is frequently
deregulated in malignancy,2 especially in colon cancer.3 Binding
of WNT ligands, a family of secreted glycosylated proteins, to
the Frizzled and LRP families of cell surface receptors initiates a
series of signaling events via the cytoplasmic protein Dish-
evelled (DSH). Upon activation, DSH recruits Axin and
destabilizes the destruction complex that normally acts to
degrade the transcriptional cofactor β-catenin. In the absence of
degradation, β-catenin accumulates and translocates to the
nucleus, where it recruits coactivators and associates with the
LEF/TCF family of DNA-binding proteins, thereby altering
expression of a variety of genes, including cyclin D1 and c-
MYC.4 In cells not subject to stimulation by WNT ligands, β-
Received: September 18, 2014
© XXXX American Chemical Society
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Figure 1. Small-molecule inhibitors of the WNT pathway: 1,19 2,22 3,22 4,23 5,25 6,26 7,27 and 8.28
and subsequent degradation.9 Many other cancers also show
evidence of inappropriate WNT pathway activation, including
raised levels of β-catenin.10−13 In addition, defective WNT
signaling plays a key role in the generation and maintenance of
cancer stem cells.14 Validation of a pivotal role for WNT
signaling in cancer is therefore very strong, and compounds
that dampen WNT pathway activity could provide useful
molecularly targeted therapeutics for the treatment of
cancer;6,15,16 indeed, the fully humanized monoclonal antibody
OMP-18R5 that targets Frizzled receptors is efficacious in
patient-derived mouse tumor models17 and is currently in a
phase I clinical trial.18 Despite the importance of the WNT
pathway, it is only recently that small-molecule inhibitors have
been identified and progressed toward clinical trial.16
Compound 1 (ICG001) blocks the interaction between β-
catenin and the transcriptional coactivating protein CREB
binding protein (CBP), leading to a reduction of colon
adenoma formation in mouse models.19 The β-catenin−CBP
interaction was shown to promote stem/progenitor marker
expression, while the related β-catenin−p300 interaction (not
inhibited by 1) promoted expression of genes involved in
proliferative responses such as c-Myc.20 PRI-724 (structure
undisclosed), an inhibitor of the β-catenin−CBP interaction,
has now been progressed to clinical trial.21 Compounds 2
(XAV939) and 3 (IWR-1) have been disclosed as inhibitors of
WNT signaling via inhibition of the tankyrase activity required
for degradation of Axin; 2 was discovered by target
deconvolution from a cell-based pathway screen.22,23 4 (IWP-
2), a small-molecule inhibitor of porcupine, the acyltransferase
essential for secretion of functional WNT ligands, was also
discovered by cell-based pathway screening (Figure 1).23
Subsequent to the identification of tankyrase and porcupine
as validated molecular targets, many small-molecule inhibitors
have been reported.24 Examplar tankyrase inhibitors include 5
(NVP-TNKS656),25 6 (G007-LK),26 and compound 727 from
Amgen; the porcupine inhibitor 8 (LGK974)28 is currently in a
phase I clinical trial.
Many WNT pathway mutations occur at, or upstream of, β-
catenin. Therefore, if the pathway is blocked at or below this
point, an inhibitor should be active against multiple tumors
driven by a WNT-activating mutation. With this in mind, we set
out to discover small-molecule inhibitors of WNT signaling and
employed a cell-based pathway screening strategy to identify
compounds that block WNT signaling at, or downstream of, β-
catenin. While our work was in progress a number of reports
appeared describing successful cell-based pathway screens
against the WNT pathway22,23 and, for example, BMP
signaling29 revealing hitherto undiscovered regulatory mecha-
nisms, increasing our confidence in such an approach. We have
previously reported our screening strategy30 and describe here
the medicinal chemistry optimization of a 3,4,5-trisubstituted
pyridine hit (9) to give potent and orally bioavailable small-
molecule inhibitors of WNT signaling. We demonstrate a 350-
fold potency enhancement in a cell-based assay of WNT
B
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Scheme 1. General Synthetic Route
pathway activity through hypothesis-driven medicinal chemistry
design respectful of physicochemical properties likely to elicit
good cell permeability.
protein expression was induced using 10 μM estradiol. We
screened 63 040 compounds at an assay concentration of 20
μM and selected compounds that reproducibly gave >50%
inhibition in the 7dF3 luciferase reporter assay (n = 2) and did
not inhibit luciferase enzymatic activity nor lead to >50% cell
death at 20 μM.30 A control cell line incorporating a thymidine
kinase promoter-driven renilla luciferase reporter construct was
also utilized to deselect compounds that demonstrated potent
and efficacious inhibition of non-WNT-mediated transcrip-
tion.30
CHEMISTRY
■
Our synthetic strategy to most desired 3,4,5-trisubstituted
pyridines involved SNAr displacement of the 4-chloro
substituent in either 3,4,5-trichloropyridine (10) or 3-bromo-
4,5-dichloropyridine (11) using the appropriate N-boc-
protected pyrrolidine 12, piperidine 13, or NH-piperidine 14
to give the 4-substituted analogues 9, 15−22, 39, 41, 43, 45,
47, 49, 51, 53, 57, and 84 according to our previously
published methodology.31 Subsequent palladium-mediated
cross-coupling of appropriate aryl boronic acids or esters gave
the corresponding 3,4,5-trisubstituted pyridines 26, 28−38, 40,
42, 44, 46, 48, 50, 52, 54, and 65−81 (Scheme 1). Compounds
59, 60, and 62 described in Table 5 were prepared by
sequential cross-coupling at the 3- and 5-positions of 8-(3-
bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one
(57). Compounds 63 and 64 were prepared directly from 8-(3-
chloro-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (44), while compound 56 was
prepared by SNAr displacement of the 4-chloro substituent in 3-
bromo-4-chloro-5-methylpyridine, followed by palladium-medi-
ated cross-coupling at the 3-position. Preparations of non-
commercial aryl boronic acids or esters are described in the
Experimental Section and the Supporting Information.
Hit compound 9 (IC50 = 1.76 μM) was the only exemplar of
a 3,4,5-trisubstituted pyridine in our screening library at that
time; however, despite being a singleton, it was among the most
attractive chemicals, with a low molecular weight and
acceptable physicochemical properties (Table 1 and Figure
2). Compound 9 blocked activation of the pathway by the
GSK-3β inhibitor bromoindirubin-3′-oxime (BIO) (IC50 = 2.60
μM), indicative of action at or below GSK-3β-mediated
phosphorylation of β-catenin. We then further characterized
the locus of interaction for compound 9 using a cell-based assay
cascade in which WNT signaling is activated at distinct levels by
inducible expression of cDNA-encoded activating components
of the canonical WNT pathway; a more robust development of
our previously reported cell-based assay cascade.30 This cell-
based assay cascade included activation of WNT signaling
through expression of either ΔN-LRP6 (a constitutively
activating component of the WNT receptor), Dvl-2 (a
Disheveled homologue and activator of WNT signaling),
Axin-GID (Axin-GSK3β Interaction Domain, a dominant
negative form of Axin that binds to and inhibits GSK-3β),
ΔN-β-catenin (a stabilized form of β-catenin resistant to
degradation), or VP16-TCF (a TCF transcription factor that is
constitutively active in the absence of β-catenin). Compound 9
demonstrated potency and efficacy in all these cell-based assays,
similar to the potency observed in the 7dF3 primary assay and
RESULTS AND DISCUSSION
■
To discover inhibitors of WNT signaling, we used an inducible
luciferase reporter assay in human embryonic kidney cells
(HEK293) that contained both estrogen receptor-DSH and
TCF-luciferase-IRES-GFP constructs.30 GFP fluorescence was
used to sort cells by flow cytometry that were highly induced by
estradiol. Clone 7dF3 was selected for use in HTS format and
gave a 40-fold induction of luciferase activity when DSH
C
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Table 1. Modification of the Piperidine-4-carboxamide
MLM and HLM values indicate percent turnover after 30 min of incubation.
consistent with intracellular inhibition of WNT signaling at or
below the TCF locale (IC50’s = 0.9−7.6 μM, Table 2).
By contrast, 4-(piperidin-1-yl)pyridine adopts a planar
conformation about the piperidine−pyridine bond in two
reported small-molecule X-ray crystal structures.36 Thus, the
3,5-dichloro substitution pattern in compound 9 reduces the
pKa of the pyridine nitrogen (inductive effect) and induces a
twisted conformation about the piperidine−pyridine bond
(steric effect), which may further reduce the pKa. The measured
pKa of compound 9 is consistent with an observed lack of in
vitro ion channel pharmacology (hERG IC50 > 10 μM) and
absence of CYP450 enzyme inhibition apart from weak
inhibition of CYP3A4 (IC50 = 7 μM, Supplementary Table
S1, SI); these observations are in marked contrast to classical 4-
aminopyridine-based ion channel modulators.37
In the absence of information about the precise biochemical
target of compound 9, we followed a classical ligand-based
design approach. We hypothesized, first, that the H-bond donor
(HBD) and acceptor (HBA) properties of the primary amide
moiety would be essential for activity; second, that the pyridine
nitrogen would be an essential component of activity; and,
third, that it would be important to maintain the observed
twisted conformation about the pyridine−piperidine bond. We
initially set out to test these three hypotheses, cognisant in our
In addition to promising potency in the 7dF3 luciferase cell-
based reporter assay (IC50 = 1.76 μM), compound 9
demonstrated moderate in vitro metabolic stability in human
and mouse liver microsomes (Figure 2). However, we were
concerned that 9 may be a nonspecific pharmacophore for ion
channels by virtue of the 4-aminopyridine motif. A measured
pKa of the pyridine nitrogen (pKa = 3.5)32 indicated that 9 is
essentially nonbasic, in contrast to 4-aminopyridine (pKa =
9.2)33 and 4-(piperidin-1-yl)pyridine (measured pKa = 9.42).32
The inductive electron-withdrawing effect of two m-chloro
substituents is likely to significantly reduce the pKa (pyridine
pKa = 5.24; 3-chloropyridine pKa = 2.84).34,35 We hypothesized
that a twisted conformation about the pyridine−piperidine
bond, driven by the steric bulk of the flanking chlorine atoms,
may also reduce the basicity of compound 9 by abrogating
electron donation from the 4-amino substituent. Indeed, a
twisted conformation was evident in the small molecule X-ray
crystal structure of 9 with a torsion angle of 54.4° observed
between the plane of the piperidine and pyridine rings [Figure
3 and Supplementary Figure S4, Supporting Information (SI)].
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Figure 3. Small-molecule X-ray crystal structure of 9 (panel A)
depicting a twisted conformation about the pyridine−piperidine bond
with the plane of piperidine ring tilted 54.4° against the plane of the
aromatic ring (panel B and Supplementary Figure S4, SI). Data has
been deposited with the Cambridge Crystallographic Data Centre
(deposition number CCDC 1020858).
Methylation of the primary amide in compound 9 to give the
secondary amide 15 or tertiary amide 16 led to 7- and 15-fold
loss in activity, respectively, suggesting that at least one of the
H-bond donors is required for activity. The greater loss of
activity with more sterically demanding amides 17−19
suggested a lack of space adjacent to the amide moiety.
Removal of the carboxamide completely (compound 20) or
removal of the carbonyl moiety (compound 21) led to a
reduction in potency compared with compound 9, consistent
with the hypothesis that both H-bond donor and acceptor
functions of the carboxamide are important components of the
pharmacophore (Table 1).
To explore the extent to which the pyridine nitrogen is
essential for activity, we prepared compound 22, where the
pyridine nitrogen was replaced with a CH (Table 1, entry 9). A
significant potency drop was observed, suggesting that the
hydrogen bond acceptor capability of this nonbasic nitrogen is
indeed crucial for activity.
Figure 2. Attributes of hit compound 9.
Table 2. Assessment of Compounds 9 and 33 in Cell-Based
Assays in Which WNT Signaling Is Activated at Distinct
Levels by Inducible Expression of cDNAs Encoding
We next turned our attention to the requirement for a
twisted conformation about the pyridine−piperidine bond
while the piperidine 4-carboxamide was kept constant.
Replacement of chlorine atoms with protons at both the 3-
and 5-positions on the pyridine ring led to loss of activity, while
replacement of one chloride led to a 13-fold loss in potency
(Table 3, entries 1 and 2). By contrast, replacement of both
chlorine atoms with bulkier bromines led to a 5-fold potency
increase (Table 3, entry 3). Pleasingly, replacement of one
chlorine atom with a phenyl ring in compound 26 (Table 3,
entry 4) resulted in a 20-fold increase in potency over
compound 9; however, the combination of a 3-phenyl, 5-H
(Table 3, entry 5) abrogated activity by 37-fold in comparison
with 26 (Table 3, entry 4), consistent with a requirement for
substitution at both the 3- and 5-positions of the pyridine ring.
The increased potency of compound 26 was of particular
interest, although in vitro microsomal stability was suboptimal.
We set out to explore alternatives to the phenyl ring with
greater stability to oxidative metabolism. Replacement with 4-
substituted pyrazoles led to a loss of activity (Table 3, entries 6
and 7), although some activity loss exhibited by compound 28
(Table 3, entry 6) may result from the presence of an additional
H-bond donor and consequent impaired cell penetration with
respect to its matched pair (Table 3, entry 7). The thiophene
analogue 30, while among the most potent replacements,
exhibited unsurprising metabolic instability,38 and pyridine
replacements had decreased potency (Table 3, entries 9 and
10). Substitution at the para-position of the phenyl ring proved
more fruitful, with both electron-donating and -withdrawing
substituents tolerated (entries 11−13); however, the strongly
electron withdrawing sulfone was not tolerated (entry 14).
a
Activating Components of the Canonical WNT Pathway
IC50 (μM)
WNT pathway activation level
reporter
9
33
Dvl-ER (7dF3 primary assay)
Tet-O-ΔN-LRP6
TCF-luc
TCF-luc
TCF-luc
TCF-luc
TCF-luc
TCF-luc
1.76
0.91
1.20
1.50
2.73
7.61
0.037
0.020
0.020
0.024
0.070
0.23
Tet-O-Dvl2
Tet-O-Axin-GID
Tet-O-ΔN-β-catenin
Tet-O-VP16-TCF4
a
Activating components of the canonical WNT pathway: ΔN-LRP6 (a
constitutively activating component of the WNT receptor), Dvl-2 (a
Disheveled homologue and activator of WNT signaling), Axin-GID
(Axin-GSK3β Interaction Domain, a dominant negative form of Axin
that binds to and inhibits GSK-3β), ΔN-β-catenin (a stabilized form of
β-catenin resistant to degradation), or VP16-TCF (a TCF tran-
scription factor that is constitutively active in the absence of β-
catenin). See the Supplementary Information for assay protocols.
compound design of the cell-based primary assay whereby true
SAR versus an intracellular biochemical target may be masked
by poor cell penetration. Hit compound 9 (HBD count = 2;
HBA count = 4) has excellent passive cell permeability (Caco-
2: Papp = 36 × 10−6 cm s−1, no efflux) and compounds with
equivalent or lower HBD/HBA count were prioritized to avoid
masking of SAR trends against an intracellular target by poor
cell permeability.
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Table 3. Modification of the Pyridine 3- and 5-Substituents
MLM and HLM values indicate percent turnover after 30 min of incubation.
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Increased potency of the p-methoxy analogue 33 in the 7dF3
cell-based luciferase reporter assay (IC50 = 0.037 μM)
compared to 9 (IC50 = 1.76 μM) was mirrored by increased
potency and overall profile in cell-based assays incorporating
different activating components of WNT signaling, suggesting
that the locus of action on the WNT pathway had been
maintained (Table 2).
Table 4. Optimization of the Piperidine-4-carboxamide
Although compound 33 was among the most potent, its
metabolic stability was poor; however, the p-morpholine
derivative 38 (Table 3, entry 16) proved equipotent, with
acceptable metabolic stability, while the corresponding meta-
derivative 37 was 8-fold less potent (Table 3, entry 15). Despite
acceptable metabolic stability, compound 38 demonstrated in
vivo clearance greater than liver blood flow upon iv dosing in
mouse pharmacokinetic studies; we suspected amidase-
mediated cleavage of the primary amide, although this proved
difficult to definitively confirm.
With compound 38 in hand, we were keen to further explore
the piperidine carboxamide in order to understand the optimal
geometry for the H-bond acceptor and donor motifs and to
reduce the potential for putative amidase-mediated cleavage of
the primary carboxamide. Replacement of the 4-carboxamide by
nitrile, a highly directional H-bond acceptor, maintained activity
in the reporter assay and reinforced the notion that an
appropriately positioned H-bond acceptor motif was the
dominant feature (Table 4, entry 1). Introduction of a geminal
methyl group at the 4-position of the piperidine ring also
maintained potency (Table 4, entry 2) and suggested that
spirocyclic carboxamides might be tolerated. With this result in
hand, a range of 6,5-spirocyclic ring scaffolds was explored and,
pleasingly, resulted in compounds with good potency and
metabolic stability. The 2,8-diazaspiro[4.5]decan-1-one 44
exhibited 25-fold improved potency over the isomeric spiro-
cycle 46, consistent with a specific interaction from the amide
motif (Table 4, entries 3 and 4). Methylation of the amide
(entry 5) reduced potency 24-fold, indicating that, in this
spirocyclic scaffold, both H-bond donor and acceptor motifs are
important for activity. An isomeric spirocyclic ring system at the
3-position of the piperidine ring lost significant potency (entry
6), although potency could be restored with the corresponding
5,5-spirocyclic ring system incorporated as a racemate (entry
7). Improving the H-bond donor ability of the spirocyclic NH
by incorporation of an additional carbonyl functionality to give
the acidic imide 54 resulted in a very potent and metabolically
stable cell-based inhibitor of WNT pathway activity; however,
poor solubility (0.003 mg/mL in phosphate buffer at pH 7.4)
was an unfavorable attribute and below our criteria for
compound progression. Considering all the data together,
compound 44 presented the most promising profile, and we
were keen to explore the extent to which the chloro substituent
could be varied (Table 5). Replacement with small alkyl,
cycloalkyl, aryl, or heteroaryl groups (Table 5, entries 2−7), all
of which were designed to maintain cell penetration, resulted in
maintenance of potency for the prop-1-en-2-yl substituent in 59
(Table 5, entry 3) but a loss of potency for other exemplars,
which we ascribed to a lack of toleration of steric bulk in this
region of the putative binding site. In order to avoid potential
metabolic liabilities with the presence of an alkene in
compound 59, we henceforth adopted chloride as the preferred
substituent.
MLM and HLM values indicate percentage parent remaining after
30 min of incubation.
however, clearance remained unacceptably high with corre-
sponding low oral bioavailability (Table 6). Despite an
unfavorable pharmacokinetic profile, other properties of
compound 44 were attractive: it was inactive when tested at
10 μM across a panel of 55 receptors, ion channels, and
enzymes and demonstrated no significant activity at 1 μM
To benchmark the chemical series, we conducted mouse in
vivo pharmacokinetic studies on compound 44, which revealed
improved in vivo clearance compared to compound 38;
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morpholinophenyl (Table 8, entries 11−13). Removal of either
morpholine heteroatom reduced potency versus the parent
compound 44 (Table 8, entries 1 and 2). Incorporation of an
unsubstituted piperazine proved detrimental, whereas the N-
methylpiperazine 68 restored potency with some improvement
in metabolic stability; however, an unfavorable Caco-2
permeability profile (efflux ratio >500) precluded further
progression of this compound.
Table 5. Variation of the 3-Chloro Substituent
Pleasingly, 5- or 6-membered heterocyclic replacements for
the morpholine provided compounds with good potency and
equivalent or improved metabolic stability. The electron-
withdrawing oxadiazole 70 proved the least potent modification
in the set, consistent with the poor activity of the electron-
withdrawing sulfone 36 (Table 3, entry 14). The N-
methylpyrazole 74 proved more potent than its desmethyl
matched pair 73, possibly due to poorer cell penetration of the
latter compound, while the regioisomeric pyrazole 71 also
displayed lower potency. Replacement of the 4-morpholino-
phenyl moiety with fused 6,6- or 5,6-heterocycles (Table 8,
entries 11−13) retained potency but at the expense of higher
metabolic clearance compared to compound 74, which
provided the optimal balance of potency and metabolic stability
in this subseries.
We were keen to explore the combination of the preferred 1-
methyl-4-phenyl-1H-pyrazole with selected spirocyclic carbox-
amides from Table 4. In comparison with compound 74, we
noted that the corresponding 5,5-spirocyclic carboxamide 78
was significantly less metabolically stable as a racemate (Table
9). The most potent enantiomer 80 (absolute stereochemistry
not assigned) displayed the highest metabolic stability of the
pair; however, this was still inferior to the parent molecule 74
and halted further investigation of this compound. Combining a
spirocyclic imide with our favored 1-methyl-4-phenyl-1H-
pyrazole substituent gave the potent and stable inhibitor 81;
however, in common with other imides prepared in this series,
aqueous solubility proved to be very poor (<0.001 mg/mL in
phosphate buffer at pH 7.4), which precluded further
progression of this compound. 8-(3-Chloro-5-(4-(1-methyl-
1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-
1-one (CCT251545, 74) was therefore selected for progression
to in vivo studies.
In vivo pharmacokinetic studies on compound 74 revealed
moderate clearance in both mouse and rat with moderate to
high oral bioavailability; the volume of distribution in both
species was low, consistent with the pharmacokinetic profile of
a neutral compound (Table 10). Compound 74 displayed high
flux (Papp = 24 × 10−6 cm s−1), acceptable efflux (ER = 2.5),
and minimal hERG inhibition (IC50 > 10 μM); aqueous kinetic
and thermodynamic solubilities were also sufficient for further
progression (94 μM and 0.006 mg/mL, respectively, in pH 7.4
phosphate buffer). Compound 74 demonstrated minimal
activity when tested across a panel of 55 receptors, ion
channels, and enzymes at 1 μM (Table S4, SI); weak inhibition
of CYPs 2C8, 2C9, and 3A4 was observed (see Supplementary
Table S1, SI); however, this was not considered sufficient to
preclude further investigation. Testing versus a panel of 291
MLM and HLM values indicate percentage parent remaining after
30 min of incubation.
across a broad panel of 209 nonmutated kinases (see
Supplementary Tables S2 and S3, SI); in addition, 44 did not
strongly inhibit the known intracellular WNT pathway target
tankyrase (TNKS1 IC50 = 19.0 0 μM, TNKS2 IC50 = 16.0
1.41 μM). Permeability, as assessed by Caco-2 flux, was high
(Papp = 37 × 10−6 cm s−1) with no efflux, while kinetic and
thermodynamic aqueous solubilities were acceptable (164 μM
and 0.017 mg/mL in phosphate buffer at pH 7.4, respectively)
and no plasma instability was observed. We concluded that the
poor oral bioavailability was largely the result of high first pass
clearance and adopted a more accurate measure of intrinsic
metabolic clearance (Clint) across three species to better
inform our selections for in vivo pharmacokinetic studies. In
these in vitro metabolism studies, compound 44 showed high
intrinsic clearance in the mouse consistent with our in vivo
observations (Table 7).
Compound 44 also exhibited increased inhibition of CYP
enzymes (CYP2C9, IC50 = 2.9 μM; CYP3A4, IC50 = 2.1 μM) in
comparison with the original hit 9 and generated a glutathione
adduct in an in vitro reactive metabolite assay (data not
shown); we were therefore keen to replace the morpholine
moiety that has been implicated in reactive metabolite
formation.39 A diverse set of compounds was prepared bearing
saturated rings or aromatic heterocycles as morpholine
replacements (Table 8, entries 1−4 and 5−10, respectively)
as well as 6,6- and 6,5-fused ring systems as isosteres for the 4-
Table 6. Mouse Pharmacokinetics of Compound 44
dose (mg/kg)
route
Cl (mL/min/kg)
65
Vss (L/kg)
1.44
Tmax (h)
AUC (h ng/mL)
bioavailability (%)
T1/2 (h)
0.2
0.5
iv
0.10
0.25
50.8
7.4
NA
6
0.31
po
H
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Journal of Medicinal Chemistry
Article
Table 7. In Vitro Metabolic Stability of Key Spirocyclic Compounds
MLM and HLM figures indicate percent turnover after 30 min of incubation.
kinases at 1 μM revealed weak inhibition of GSK3α and -β as
the only hits (IC50 = 0.462 and 0.690 μM, respectively; see
Supplementary Table S5, SI), insufficiently potent to account
for the observed effects on cell-based WNT pathway activity;
moreover, inhibition of GSK3β may be expected to activate
rather than inhibit the WNT signaling. Compound 74
demonstrated weak inhibition of tankyrase enzymes (TNKS1
IC50 > 10 μM, TNKS2 IC50 = 15.0 0 μM).
To confirm activity on the WNT pathway, a number of
studies were undertaken with compound 74. RT-PCR of 7dF3
cells treated with 74 demonstrated a 12-fold inhibition of
endogenous cMYC transcription (IC50 = 2.3 nM, Supple-
mentary Figure S1, SI), a commonly studied WNT target
gene.40 Compound 74 was also tested in human colorectal
cancer cell lines LS174T (β-catenin mutant) and SW480 (APC
mutant) that have constitutively activated WNT signaling or
PA-1 human teratocarcinoma cells that are WNT ligand
dependent; 74 potently inhibited reporter-based readouts of
basal WNT pathway activity in LS174T and SW480 cells in the
absence of WNT ligand or other stimulants (IC50 = 0.023
S2, SI). Taken together, these data confirm that suppression of
the WNT pathway activity by 74 is not unique to a specific cell
line, is independent of exogenous WNT pathway stimulation,
does not result from destabilization of TCF1 or TCF4, and is
not due to interference with the luciferase-based reporter
readout.
Extension of WNT pathway inhibition to the whole animal
setting was achieved using the APC mutant human colorectal
cancer cell line COLO205-F1756 clone 4. Mice bearing
established COLO205-F1756 clone 4 tumor xenografts were
treated with compound 74 (70 mg/kg po bid for 9 days), at
which point WNT reporter activity was measured by whole
body bioluminescent imaging and therapeutic response by
excised tumor weights. Significant inhibition of WNT signaling
was observed, and treated tumors were smaller than controls:
flux normalized to tumor weight was reduced by 80% (P =
0.0003) and tumor weights by 37.5% (P = 0.0379, Mann−
Whitney U test; Figure 4A,B). Plasma and tumor levels of
compound 74 were determined 2 and 6 h after the last dose,
and at these time points, total plasma and tumor levels
significantly exceeded those required for inhibition of luciferase
reporter activity in a cell-based assay. Taken together, these
data demonstrate inhibition of WNT pathway activity in a
human colorectal cancer xenograft model and some indication
of tumor growth inhibition following oral dosing at exposures
consistent with inhibition of WNT pathway activity.
0.011 μM and 0.190
0.030 μM, respectively) and
demonstrated potent inhibition of WNT3a ligand-dependent
reporter readout in PA-1 cells (IC50 = 0.007 μM). No activity
was observed in RKO-F1522 cells bearing a firefly luciferase
reporter downstream of the EF1-α promoter, a commonly
used, ubiquitously active, house-keeping promoter that has high
activity in most mammalian cells, irrespective of their WNT
dependence (IC50 > 10 μM). We developed an APC mutant
human colorectal cancer cell line (COLO205-F1756 clone 4)
engineered to express a modified luciferase-based WNT
pathway reporter. Compound 74 demonstrated potent
CONCLUSIONS
■
We have described the optimization of a small-molecule
inhibitor of the WNT pathway identified as a singleton from an
HTS of a diverse 63 040 compound library versus a cell-based
luciferase reporter assay. Application of classical hypothesis-
driven, ligand-based SAR enabled the discovery of highly
potent cell-based WNT pathway inhibitors, with compound 74
demonstrating good oral pharmacokinetics in mouse and rat.
With knowledge from our cell-based assay cascade, we
inhibition of WNT pathway activity in this clone (IC50
=
0.035 0.003 μM, n = 8); in addition, we were able to
demonstrate that compound 74 did not affect endogenous
levels of TCF1 or TCF4 at concentrations that reduce
luciferase expression in this cell line (Supplementary Figure
I
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Table 8. Optimization of the Pyridine 5-Substituent
hypothesized that this series of compounds acts at an
intracellular target. Therefore, our medicinal chemistry
optimization necessitated design criteria likely to maintain cell
permeability to avoid masking true WNT-pathway SAR, and
with this in mind, attention to H-bond donor and acceptor
count was critical. The SAR is consistent with the requirement
for a distinct twisted conformation about the pyridine−
piperidine bond, as observed in the small-molecule X-ray
J
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Article
Table 9. Combination of Optimal Pyridine 4- and 5-Substituents
1
Absolute stereochemistry undefined.
Table 10. Pharmacokinetics of Compound 74
species
dose (mg/kg)
route
Cl (mL/min/kg)
Vss (L/kg)
Tmax (h)
AUC (h ng/mL)
bioavailability (%)
t1/2 (h)
mouse
mouse
rat
0.2
0.5
0.2
0.5
iv
31
1.1
NA
1.8
NA
0.10
0.25
0.1
106.8
143.0
129.6
287.5
NA
54
0.55
NA
po
iv
NA
26
NA
88
0.97
NA
rat
po
NA
0.25
crystal structure of the original hit compound 9, and also is
consistent with the lower pKa of the pyridine nitrogen and the
lack of significant ion channel pharmacology across exemplars
of the chemical series. Enhanced potency was achieved through
maintenance of this twisted conformation and replacement of
one of the chlorine atoms with a para-substituted aryl ring.
Methylpyrazole was found to be the optimal para-substituent in
terms of potency, metabolic stability, and membrane
permeability. Combination of the favored methylpyrazole
substituent with a 6,5-spirocyclic carboxamide, in place of the
original piperidine carboxamide in hit compound 9, led to 74, a
potent small-molecule inhibitor of the WNT pathway with
good oral pharmacokinetics and in vivo activity against the
WNT pathway. Compound 74 also demonstrated a lack of
transporter-mediated efflux and a lack of activity across broad in
vitro panels of enzyme, receptor, and ion channel targets.
Further details of the in vivo pharmacological profile of this
chemical tool, including studies to determine its molecular
target, will be reported in due course.
The canonical WNT pathway is of significant interest in
oncology, and the paucity of druggable targets on the pathway
has hampered drug discovery efforts. Pathway-based screening
approaches are becoming increasingly widespread as greater
appreciation of the benefits of this strategy become
apparent.22,23,29 In particular, hits from such screens, by
necessity, have good cell permeability, and components of
protein complexes are more likely to be in a relevant
endogenous conformation, which may reveal new druggable
binding sites or alternative targetable conformations of known
sites; moreover, key signal-determining pathway nodes are
more likely to be discovered as a result of cell-based readouts at
the transcriptional level. Here we describe a successful example
of hypothesis-driven medicinal chemistry optimization from a
singleton hit against a cell-based assay readout for the WNT
pathway in the absence of knowledge of the biochemical target.
K
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Article
Figure 4. (A) Inhibition of WNT-driven luciferase reporter activity. (B) Reduced tumor growth with compound 74 in COLO205 human colon
cancer xenografts after oral dosing (70 mg/kg bid). (C) Plasma and tumor total concentrations of 74 at 2 and 6 h after the last dose.
methods. For method A, analytical separation was carried out at 40 °C
on a Merck Purospher STAR column (RP-18e, 30 × 4 mm) using a
flow rate of 3 mL/min in a 2 min gradient elution with detection at
254 nm. The mobile phase was a mixture of methanol (solvent A) and
water containing formic acid at 0.1% (solvent B). Gradient elution was
as follows: 1:9 (A/B) to 9:1 (A/B) over 1.25 min, 9:1 (A/B) for 0.5
min, and then reversion back to 1:9 (A/B) over 0.15 min, and finally
1:9 (A/B) for 0.1 min. For method B, analytical separation was carried
out at 30 °C on a Merck Chromolith SpeedROD column (RP-18e, 50
× 4.6 mm) using a flow rate of 2 mL/min in a 4 min gradient elution
with detection at 254 nm. The mobile phase was a mixture of
methanol (solvent A) and water containing formic acid at 0.1%
(solvent B). Gradient elution was as follows: 1:9 (A/B) to 9:1 (A/B)
over 2.5 min, 9:1 (A/B) for 1 min, and then reversion back to 1:9 (A/
B) over 0.3 min, and finally 1:9 (A/B) for 0.2 min. For method C,
analytical separation was carried out at 30 °C on a Merck Purospher
STAR column (RP-18e, 30 × 4 mm) using a flow rate of 1.5 mL/min
in a 4 min gradient elution with detection at 254 nm. The following
reference masses were used for HRMS analysis: caffeine [M + H]+
195.087652; hexakis(1H,1H,3H-tetrafluoropentoxy)phosphazene [M
+ H]+ 922.009798 and hexakis(2,2-difluoroethoxy)phosphazene [M +
H]+ 622.02896 or reserpine [M + H]+ 609.280657. All compounds
submitted for biological testing were determined to be >95% pure by
method A, B, or C.
Preparation of Compounds in Table 1 Exemplified by
Compound 9. 1-(3,5-Dichloropyridin-4-yl)piperidine-4-carboxa-
mide (9).41 To a stirred suspension of 3,4,5-trichloropyridine (10)
(500 mg, 2.74 mmol) and piperidine-4-carboxamide (350 mg, 2.73
mmol) in NMP (15 mL) at ambient temperature was added
triethylamine (770 μL, 5.48 mmol) in one portion. The reaction
was heated to 220 °C for 60 min under microwave irradiation. Once
cooled, the reaction mixture was partitioned between EtOAc and sat.
NaHCO3. The separated aqueous layer was extracted with EtOAc (2 ×
100 mL), and the combined organic extracts were washed with water
(50 mL) and brine (50 mL), dried over magnesium sulfate, filtered,
and concentrated in vacuo to give a crude oily solid. Purification was
accomplished by flash chromatography on silica gel eluting with
CH2Cl2/1 M methanolic ammonia (95:5 to 92:8) to yield impure title
EXPERIMENTAL SECTION
■
Chemistry. Commercially available starting materials, reagents, and
dry solvents were used as supplied. Flash column chromatography was
performed using Merck silica gel 60 (0.025−0.04 mm). Column
chromatography was also performed on a Biotage SP1 purification
system using Thomson or Biotage Flash silica cartridges. Preparative
TLC was performed on Merck plates. Ion exchange chromatography
was performed using acidic Isolute Flash SCX-II columns or basic
Isolute Flash NH2 columns. Preparative HPLC was conducted using a
Phenomenex Luna column (5 μm, 250 × 21.2 mm, C18, Phenomenex,
Torrance, CA) using a Gilson GX-281 Liquid Handler system
combined with a Gilson 322 HPLC pump (Gilson, Middleton, WI),
over a 15 min gradient elution from 10:90 to 100:0 MeOH:water
(both modified with 0.1% formic acid) at a flow rate of 20 mL/min or
over a 15 min gradient elution from 40:60 to 100:0 MeOH:water
(both modified with 0.1% formic acid) at a flow rate of 20 mL/min.
UV−vis spectra were acquired at 254 nm on a Gilson 156 UV−vis
detector (Gilson, Middleton, WI). Collection was triggered by UV
signal and collected using a Gilson GX-281 Liquid Handler system
(Gilson, Middleton, WI). Raw data was processed using Gilson
Trilution Software. 1H NMR spectra were recorded on a Bruker
Avance-500 or Bruker Avance-400. Samples were prepared as solutions
in a deuterated solvent and referenced to the appropriate internal
nondeuterated solvent peak. 13C NMR spectra were recorded at 126
MHz using an internal deuterium lock. The following internal
references were used: CDCl3 (δC 77.0), CD3OD (δC 49.0), and
DMSO-d6 (δC 39.5). Unobserved resonances for quaternary carbon
atoms are denoted by “Cq not observed”. LC−MS and HRMS
analyses were performed on an Agilent 1200 series HPLC and diode
array detector coupled to a 6210 time-of-flight mass spectrometer with
dual multimode atmospheric pressure CI/ESI source or coupled to an
Agilent 6520 quadrupole-time-of-flight mass spectrometer with dual
multimode atmospheric pressure CI/ESI source. LC−MS analysis was
also performed on a Waters Alliance 2795 separations module and
Waters 2487 dual wavelength absorbance detector coupled to a
Waters/Micromass LCt time-of-flight mass spectrometer with ESI
source. Analytical separation was carried out according to the following
L
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compound (963 mg) as a white solid. The solid was triturated in hot
Et2O (25 mL) to give the title compound 9 as a white solid (519 mg,
69% yield): 1H NMR (500 MHz, CDCl3) δ 1.89−1.97 (m, 4H), 2.33−
2.40 (m, 1H), 3.27−3.33 (m, 2H), 3.38−3.44 (m, 2H), 5.62 (br s,
1H), 5.79 (br s, 1H), 8.32 (s, 2H); 13C NMR (126 MHz, CDCl3) δ
29.4, 42.4, 50.1, 128.6, 149.1, 151.7, 176.9; LC−MS (method A, ESI,
m/z) tR = 1.16 min, 274/276/278 (M + H)+; ESI-HRMS calcd for
C11H1435Cl2N3O (M + H)+ 274.0508, found 274.0509.
dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (43) (70 mg,
0.23 mmol), 4-morpholinophenylboronic acid (60 mg, 0.29 mmol),
and tetrakis(triphenylphosphine)palladium(0) (14 mg, 0.012 mmol)
in MeCN (3 mL), at ambient temperature, was added 0.5 M aqueous
sodium carbonate solution (650 μL, 0.326 mmol). The reaction
mixture was heated to 150 °C under argon in the microwave for 60
min. Once cooled, the reaction mixture was concentrated in vacuo and
then dry-loaded onto silica. Purification was accomplished by flash
chromatography on silica gel eluting with CH2Cl2/MeOH (98:2 to
96:5) to give the title compound 44 (19 mg, 19% yield) as a white
Preparation of Compounds in Table 3 Exemplified by
Compounds 26 and 38. 1-(3-Chloro-5-phenylpyridin-4-yl)-
piperidine-4-carboxamide (26).41 To a stirred suspension of 1-(3,5-
dichloropyridin-4-yl)piperidine-4-carboxamide (9) (100 mg, 0.365
mmol), phenylboronic acid (49 mg, 0.401 mmol), and tetrakis-
(triphenylphosphine)palladium(0) (21 mg, 0.018 mmol) in MeCN (4
mL) was added 0.5 M aqueous sodium carbonate solution (1.0 mL,
0.50 mmol). The reaction was heated to 150 °C under microwave
irradiation for 30 min. Once cooled, the reaction was concentrated in
vacuo and azeotroped with toluene (2 × 50 mL) to give a gray, oily
solid. Purification was accomplished by flash chromatography on silica
gel eluting with CH2Cl2/MeOH (97/3) and by semipreparitive HPLC
(gemini column 250 × 10, 15 min gradient, MeCN/H2O 10:90 to
90:10) to give the title compound 26 as a white solid (41 mg, 36%
yield): 1H NMR (500 MHz, CDCl3) δ 1.73−1.80 (m, 4H), 2.12−2.20
(m, 1H), 2.62 (dt, J = 13.4, 7.1 Hz, 2H), 3.15−3.21 (m, 2H), 5.40 (s,
2H), 7.24−7.30 (m, 2H), 7.38−7.51 (m, 3H), 8.21 (s, 1H), 8.44 (s,
1H); 13C NMR (126 MHz, CDCl3) δ 29.1, 42.4, 50.9, 127.8, 128.3,
128.6, 129.3, 134.1, 137.6, 149.6, 150.7, 152.8, 176.8; LC−MS
(method A, ESI, m/z) tR = 0.99 min, 316/318 (M + H)+; ESI-HRMS
calcd for C17H1935ClN3O (M + H)+ 316.1211, found 316.1206.
1-(3-Chloro-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)piperidine-4-
carboxamide (38). To a stirred suspension of 1-(3,5-dichloropyridin-
4-yl)piperidine-4-carboxamide (9) (70 mg, 0.26 mmol), 4-(4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)morpholine (92 mg, 0.32
mmol), and tetrakis(triphenylphosphine)palladium(0) (15 mg, 5 mol
%) in MeCN (3 mL), at ambient temperature, was added 0.5 M
aqueous sodium carbonate solution (720 μL, 0.360 mmol). The
reaction was heated to 150 °C for 45 min under microwave irradiation.
Once cooled, the reaction was concentrated in vacuo and then dry-
loaded onto silica. Purification was accomplished by flash chromatog-
raphy on silica gel (CH2Cl2/EtOH 98:2 to 93:7) to yield the title
1
solid: H NMR (500 MHz, CDCl3) δ 1.34−1.39 (m, 2H), 1.92−2.01
(m, 4H), 2.74−2.81 (m, 2H), 3.17 (dt, J = 13.0, 4.0 Hz, 2H), 3.21−
3.25 (m, 4H), 3.29 (t, J = 6.9 Hz, 2H), 3.87−3.90 (m, 4H), 5.93 (br s,
1H), 6.98 (d, J = 8.7 Hz, 2H), 7.20 (d, J = 8.7 Hz 2H), 8.17 (br s, 1H),
8.39 (br s, 1H); 13C NMR (126 MHz, CDCl3) δ 31.8, 32.5, 38.7, 41.8,
47.7, 49.1, 67.0, 115.4, 128.3, 128.9, 130.2, 134.0, 149.2, 150.8, 151.0,
152.9, 181.7; LC−MS (method B, ESI, m/z) tR = 2.20 min, 427/429
(M + H)+; ESI-HRMS calcd for C23H2835ClN4O2 (M + H)+ 427.1895,
found 427.1903.
8-(3,5-Dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione
(53). To a stirred suspension of 3,4,5-trichloropyridine (10) (215 mg,
1.18 mmol) and 2,8-diazaspiro[4,5]decan-1,3-dione (180 mg, 1.07
mmol) in NMP (4.5 mL), at ambient temperature, was added
triethylamine (450 μL, 3.20 mmol). The reaction mixture was heated
to 220 °C under microwave irradiation for 60 min. Once cooled, the
mixture was diluted with MeOH (20 mL) and placed on an SCX
column (10 g). Elution was carried out with CH2Cl2/MeOH followed
by 1 M methanolic ammonia. Concentration of the ammonia extract in
vacuo gave a light orange solid. Recrystallization from EtOAc gave 8-
(3,5-dichloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione (53)
1
(206 mg, 62% yield) as a cream solid: H NMR (500 MHz, CDCl3)
δ 1.69 (d, J = 13.4 Hz, 2H), 2.28 (ddd, J = 13.4, 10.7, 4.1 Hz, 2H), 2.72
(s, 2H), 3.32 (ddd, J = 13.2, 10.7, 2.6 Hz, 2H), 3.48 (dt, J = 13.2, 4.1
Hz, 2H), 8.26 (br s, 1H), 8.37 (br s, 2H); LC−MS (method B, ESI,
m/z) tR = 2.43 min, 314/316/318 (M + H)+.
8-(3-Chloro-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1,3-dione (54). Ten microwave vials were
prepared as follows: To a stirred suspension of 8-(3,5-dichloropyr-
idin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione (53) (300 mg, 0.764
mmol), 4-morpholinophenylboronic acid (222 mg, 0.963 mmol), and
tetrakis(triphenylphosphine)palladium (0) (100 mg, 0.0850 mmol) in
MeCN (12 mL) at ambient temperature was added 0.5 M aqueous
sodium carbonate solution (3 mL). The reaction was heated to 150 °C
by microwave for 60 min. Once cooled, the reaction mixtures from the
ten vials were combined, concentrated in vacuo, and dry-loaded onto
silica. Purification was accomplished by flash chromatography
(hexane/EtOAc 30:70) to obtain the title compound 54 (950 mg,
25% yield) as a white solid: 1H NMR (500 MHz, CDCl3) δ 1.48−1.54
(m, 2H), 2.07−2.14 (m, 2H), 2.55 (s, 2H), 2.75 (t, J = 11.7 Hz, 2H),
3.18−3.23 (m, 2H), 3.23−3.27 (m, 4H), 3.88−3.92 (m, 4H), 6.97−
7.05 (m, 2H), 7.17−7.24 (m, 2H), 8.10 (s, 1H), 8.25 (s, 1H), 8.47 (s,
1H); 13C NMR (126 MHz, CDCl3) δ 33.3, 40.6, 44.2, 47.2, 48.7, 66.8,
115.2, 127.8, 130.0, 148.1, 150.0, 150.9, 153.0, 174.9, 181.4, (2 Cq not
observed); LC−MS (method B, ESI, m/z) tR = 2.20 min, 441/443 (M
+ H)+; ESI-HRMS calcd for C23H2635ClN4O3 (M + H)+ 441.1688,
found 441.1693.
Preparation of Compounds in Table 5 Exemplified by
Compounds 56, 59, and 60. 8-(3-Bromo-5-methylpyridin-4-yl)-
2,8-diazaspiro[4.5]decan-1-one (55). A solution of 3-bromo-4-
chloro-5-methylpyridine (300 mg, 1.45 mmol), triethylamine (610
μL, 4.36 mmol), and 2,8-diazaspiro[4.5]decan-1-one hydrochloride
(291 mg, 1.53 mmol) in NMP (4.4 mL) was heated to 220 °C under
microwave irradiation for 5 h at 220 °C. Once cooled, the reaction was
partitioned between EtOAc and sat. NaHCO3 (100 mL each), the
separated organic layer was extracted with EtOAc (2 × 75 mL), and
the combined organic extracts were washed with brine (75 mL), dried
over MgSO4, filtered, and concentrated in vacuo to give a crude yellow
solid. The solid was recrystallized in hot EtOAc, filtered, washed with
ether, and dried in vacuo to yield 8-(3-bromo-5-methylpyridin-4-yl)-
2,8-diazaspiro[4.5]decan-1-one (55) (44 mg, 9% yield) as an off white
1
compound 38 (53 mg, 52% yield) as a pale orange/pink solid: H
NMR (500 MHz, DMSO-d6) δ 1.53−1.60 (m, 4H), 2.04−2.11 (m,
1H), 2.49−2.57 (m, 2H), 3.00−3.06 (m, 2H), 3.15−3.20 (m, 4H),
3.73−3.78 (m, 4H), 6.73 (br s, 1H), 7.03 (d, J = 8.7 Hz, 2H), 7.18 (d,
J = 8.7 Hz, 3H), 8.13 (br s, 1H), 8.40 (br s, 1H); 13C NMR (126 MHz,
CDCl3) δ 29.2, 42.4, 48.9, 51.0, 67.0, 115.4, 128.3 130.1, 134.0, 147.9,
149.7, 150.9, 153.6, 176.9, (1 Cq not observed); LC−MS (method B,
ESI, m/z) tR = 1.96 min, 401/403 (M + H)+; ESI-HRMS calcd for
C21H2635ClN4O2 (M + H)+ 401.1739, found 401.1746.
Preparation of Compounds in Table 4 Exemplified by
Compounds 44 and 54. 8-(3,5-Dichloropyridin-4-yl)-2,8-diazaspiro-
[4.5]decan-1-one (43). To a stirred suspension of 3,4,5-trichloropyr-
idine (10) (509 mg, 2.79 mmol) and 2,8-diazaspiro[4,5]decan-1-one
hydrochloride (410 mg, 2.66 mmol) in NMP (14.6 mL), at ambient
temperature, was added triethylamine (1.10 mL, 7.89 mmol). The
reaction mixture was heated to 220 °C under microwave irradiation for
60 min. Once cooled, the reaction mixture was partitioned between
EtOAc and sat. NaHCO3, the separated aqueous layer was extracted
with EtOAc (2 × 100 mL), and the combined organic extracts were
washed with brine (50 mL), dried over magnesium sulfate, filtered, and
concentrated in vacuo to give a crude pale yellow oil. Purification was
accomplished by flash chromatography on silica gel eluting with
CH2Cl2/EtOH (97.5:2.5 to 97:3) to give 8-(3,5-dichloropyridin-4-yl)-
2,8-diazaspiro[4.5]decan-1-one (43) (422 mg, 53% yield) as a white
1
solid: H NMR (500 MHz, CDCl3) δ 1.53 (br d, J = 13.1 Hz, 2H),
2.07−2.18 (m, 4H), 3.33 (dt, J = 11.9, 2.6 Hz, 2H), 3.37−3.45 (m,
4H), 6.66 (br s, 1H), 8.33 (br s, 2H); LC−MS (method B, ESI, m/z)
tR = 2.66 min, 300/302/304 (M + H)+.
8-(3-Chloro-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (44). To a stirred suspension of 8-(3,5-
M
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1
solid: H NMR (500 MHz, CDCl3) δ 1.51−1.58 (m, 2H), 2.06−2.15
yield): 1H NMR (500 MHz, CDCl3) δ 1.50−1.56 (m, 2H), 2.07−2.14
(m, 5H), 2.16 (t, J = 6.9 Hz, 2H), 3.15−3.20 (m, 2H), 3.36−3.42 (m,
4H), 4.97−4.98 (m, 1H), 5.26−5.28 (m, 1H), 5.81 (s, 1H), 8.15 (s,
1H), 8.39 (s, 1H); LC−MS (method C, ESI, m/z) tR = 1.78 min, 306/
308 (M + H)+.
(m, 2H), 2.16 (t, J = 6.8 Hz, 2H), 2.31 (s, 3H), 3.18 (br s, 2H), 3.33
(br s, 2H), 3.39 (t, J = 6.8 Hz, 2H), 6.18 (br s, 1H), 8.21 (br s, 1H),
8.45 (br s, 1H); LC−MS (method C, ESI, m/z) tR = 1.42 min, 324/
326 (M + H)+.
8-(3-Methyl-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (56). To a stirred solution of 8-(3-
bromo-5-methylpyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (55) (36
mg, 0.11 mmol), Pd(dppf)Cl2·CH2Cl2 (4.5 mg, 5.6 μmol), and 4-
morpholinophenylboronic acid (25 mg, 0.12 mmol) in MeCN (830
μL), at ambient temperature, was added 0.5 M aqueous sodium
carbonate solution (310 μL, 0.150 mmol). The reaction mixture was
sealed in a microwave vial under argon and heated to 120 °C for 60
min under microwave irradiation. The reaction was concentrated in
vacuo and then diluted with MeOH/DCM and dry-loaded onto silica.
Purification was accomplished by flash chromatography on silica gel
eluting with DCM/EtOH (99:1 to 90:10) to yield the title compound
56 (20 mg, 44% yield) as a clear colorless oil, which solidified on
8-(3-(4-Morpholin-4-ylphenyl)-5-(prop-1-en-2-yl)pyridin-4-yl)-
2,8-diazaspiro[4.5]decan-1-one (59). 8-(3-Chloro-5-(prop-1-en-2-
yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (58) (40 mg, 0.13
mmol), 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-
morpholine (57 mg, 0.20 mmol), sodium carbonate (35 mg, 0.33
mmol), and dichlorobis(tricyclohexylphosphine)palladium(II) (9.7
mg, 0.013 mmol) were loaded in a microwave vial under argon, and
then acetonitrile (1.0 mL) and water (260 μL) were added. The
reaction mixture was heated at 150 °C for 35 min under microwave
irradiation. The solvent was evaporated and the crude material was
purified via Biotage column chromatography (DCM/EtOH 98:2 to
90:10) to give the title compound 59 as a colorless oil (24.5 mg, 44%
yield): 1H NMR (500 MHz, CDCl3) δ 1.21−1.25 (m, 2H), 1.80−1.88
(m, 2H), 1.96 (t, J = 6.9 Hz, 2H), 2.13 (s, 3H), 2.72−2.79 (m, 2H),
3.11−3.16 (m, 2H), 3.21−3.24 (m, 4H), 3.27 (t, J = 6.9 Hz, 2H),
3.87−3.91 (m, 4H), 5.00−5.02 (m, 1H), 5.24−5.26 (m, 1H), 5.52 (s,
1H), 7.00 (d, J = 8.8 Hz, 2H), 7.30 (d, J = 8.8 Hz, 2H), 8.17 (s, 1H),
8.18 (s, 1H); LC−MS (method C, ESI, m/z) tR = 1.87 min, 433 (M +
H)+; ESI-HRMS calcd for C26H33N4O2 (M + H)+ 433.2598, found
433.2595.
8-(3-Isopropyl-5-(4-morpholin-4-ylphenyl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (60). 8-(3-(4-Morpholinophenyl)-5-
(prop-1-en-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (59) (5
mg, 0.01 mmol) was solubilized in ethanol (0.5 mL) and a few drops
of DCM. Then 1 M HCl (35 μL, 0.035 mmol) and palladium 10% on
carbon (3 mg, 0.01 mmol) were added. The reaction was
hydrogenated under a balloon of hydrogen at rt for 2 days and
filtered over Celite. The crude material was filtered on a flash NH2
column and concentrated to give the title compound 60 as a colorless
oil (4 mg, 80% yield): 1H NMR (500 MHz, CDCl3) δ 1.28−1.33 (m,
8H), 1.88−2.00 (m, 4H), 2.58−2.68 (m, 2H), 2.85−2.92 (m, 2H),
3.21−3.25 (m, 4H), 3.25−3.29 (m, 2H), 3.37−3.45 (m, 1H), 3.88−
3.92 (m, 4H), 5.64 (s, 1H), 6.96−6.99 (m, 2H), 7.20 (d, J = 8.3 Hz,
2H), 8.15 (s, 1H), 8.42 (s, 1H); 13C NMR (126 MHz, CDCl3) δ 24.0,
26.2, 30.5, 32.4, 38.6, 42.0, 48.5, 49.2, 66.9, 115.1, 130.4, 130.6, 134.3,
140.9, 147.9, 150.3, 150.5, 154.5, 181.6; LC−MS (method C, ESI, m/
z) tR = 1.85 min, 435 (M + H)+; ESI-HRMS calcd for C26H35N4O2 (M
+ H)+ 435.2755, found 435.2747.
1
standing: H NMR (500 MHz, CDCl3) δ 1.28−1.34 (m, 2H), 1.88−
1.96 (m, 4H), 2.31 (s, 3H), 2.63 (br t, J = 12.3 Hz, 2H), 2.91−2.97
(m, 2H), 3.20−3.24 (m, 4H), 3.28 (t, J = 6.9 Hz, 2H), 3.87−3.91 (m,
4H), 6.25 (br s, 1H), 6.97 (d, J = 8.7 Hz, 2H), 7.18 (d, J = 8.7 Hz,
2H), 8.17 (br s, 1H), 8.27 (br s, 1H); 13C NMR (126 MHz, CDCl3) δ
16.4, 31.0, 32.5, 38.7, 42.1, 47.6, 49.1, 66.9, 115.1, 130.3, 130.4, 150.4,
150.6, 150.7, 155.3, 181.8; LC−MS (method C, ESI, m/z) tR = 1.70
min, 407 (M + H)+; ESI-HRMS calcd for C24H31N4O2 (M + H)+
407.2442, found 407.2447.
3-Bromo-4,5-dichloropyridine (11).42 n-Butyllithium (1.6 M in
hexane, 7.15 mL, 11.4 mmol) was added to a solution of
diisopropylamine (1.70 mL, 11.9 mmol) in THF (30 mL) at −78
°C. The reaction was stirred for 30 min and then 3-bromo-5-
chloropyridine (2.00 g, 10.4 mmol) in THF (10 mL) was added
dropwise over 7 min. The reaction was stirred for 45 min, resulting in
a yellow/brown suspension. Hexachloroethane (4.92 g, 20.8 mmol) in
THF (7 mL) was added at −78 °C and the dark brown reaction
mixture was stirred at −78 °C for 75 min. The cooling bath was
removed and the brown suspension was allowed to warm to rt (about
30 min). The clear brown solution was quenched with sat. aq NH4Cl
(100 mL) and the aqueous layer was extracted with ether (3 × 70
mL). The combined organic layers were washed with water (2 × 100
mL) and brine (70 mL), dried over MgSO4, and filtered, and the
solvent was evaporated in vacuo. The crude product was purified by
flash chromatography (CH2Cl2/cyclohexane, 1:4 to 1:3) to give the 3-
bromo-4,5-dichloropyridine (11) (2.05 g, 87% yield) as a white solid:
1H NMR (500 MHz, CDCl3) δ 8.54 (s, 1H), 8.64 (s, 1H); LC−MS
Preparation of Compounds in Table 8 Exemplified by
Compounds 68 and 74. 8-(3-Chloro-5-(4-(4-methylpiperazin-1-
yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (68). 8-(3-
Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (57) (35
mg, 0.10 mmol), 1-methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenyl)piperazine (34 mg, 0.11 mmol), and Pd(dppf)Cl2·
CH2Cl2 (3.7 mg, 5.1 μmol) were loaded in a microwave vial under
argon, and then degassed MeCN (1.8 mL) and 0.5 M aqueous sodium
carbonate solution (284 μL, 0.142 mmol) were added. The reaction
mixture was heated under microwave irradiation at 120 °C for 60 min.
The solvent was evaporated and the crude material was dry-loaded and
purified via Biotage column chromatography (CH2Cl2/MeOH 97/3 to
85/15) to give the title compound 68 (38 mg, 85% yield) as a cream
(method B, ESI, m/z) tR = 3.01 min, 225/227/229 (M + H)+.
8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one
(57). 3-Bromo-4,5-dichloropyridine (11) (1.00 g, 4.41 mmol) and boc-
2,8-diazaspiro[4.5]decan-1-one (1.35 g, 5.29 mmol) were introduced
into a microwave vial with 1-methoxy-2-propanol (11 mL) and
triethylamine (1.80 mL, 13.2 mmol). The reaction mixture was heated
under microwave irradiation for 2.5 h at 220 °C. The solvent was
evaporated and the crude material was purified via Biotage column
chromatography (CH2Cl2/EtOH, 99.8:0.2 to 97:3) to give 8-(3-
bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (57)
1
(1.14 g, 75% yield) as a white solid: H NMR (500 MHz, CDCl3)
1
solid: H NMR (500 MHz, CDCl3) δ 1.32−1.38 (m, 2H), 1.94−2.02
δ 1.52−1.58 (m, 2H), 2.11−2.20 (m, 4H), 3.32−3.41 (m, 6H), 6.00
(br s, 1H), 8.35 (s, 1H), 8.49 (s, 1H); LC−MS (method B, ESI, m/z)
tR = 2.68 min, 344/346/348 (M + H)+.
(m, 4H), 2.39 (s, 3H), 2.60−2.65 (m, 4H), 2.70−2.78 (m, 2H), 3.11−
3.17 (m, 2H), 3.27−3.31 (m, 6H), 5.54 (br s, 1H), 7.00 (d, J = 8.7 Hz,
2H), 7.19 (d, J = 8.7 Hz, 2H), 8.18 (s, 1H), 8.39 (s, 1H); 13C NMR
(126 MHz, CDCl3) δ 31.4, 32.4, 38.8, 42.0, 46.1, 47.7, 48.6, 55.0,
115.7, 128.3, 128.7, 130.1, 133.9, 149.1, 150.6, 151.0, 152.9, 181.8;
LC−MS (method C, ESI, m/z) tR = 1.43 min, 440/442 (M + H)+;
ESI-HRMS calcd for C24H3135ClN5O (M + H)+ 440.2212, found
440.2205.
8-(3-Chloro-5-(prop-1-en-2-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]-
decan-1-one (58). 8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro-
[4.5]decan-1-one (57) (200 mg, 0.580 mmol), isopropenylboronic
acid (120 μL, 0.638 mmol), and Pd(dppf)Cl2·CH2Cl2 (24 mg, 0.029
mmol) were loaded in a microwave vial under argon, and then
acetonitrile (10.4 mL) and 0.5 M aqueous sodium carbonate solution
(1.60 mL, 0.812 mmol) were added. The reaction mixture was heated
at 120 °C for 60 min under microwave irradiation, concentrated, and
purified via Biotage column chromatography (DCM/EtOH 99:1 to
95:5) to give 8-(3-chloro-5-(prop-1-en-2-yl)pyridin-4-yl)-2,8-
diazaspiro[4.5]decan-1-one (58) as a colorless oil (122 mg, 69%
8-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-
2,8-diazaspiro[4.5]decan-1-one (74). 8-(3-Bromo-5-chloro-pyridin-4-
yl)-2,8-diaza-spiro[4.5]decan-1-one (57) (250 mg, 0.725 mmol), 1-
methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-
pyrazole (82) (268 mg, 0.943 mmol), and Pd(dppf)Cl2·CH2Cl2 (30
N
DOI: 10.1021/jm501436m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mg, 0.036 mmol) were introduced into a microwave vial, and then
MeCN (13 mL) and aqueous sodium carbonate solution (0.5 M, 2.00
mL, 1.02 mmol) were added. The reaction mixture was heated under
microwave irradiation at 120 °C for 1 h and concentrated. The crude
material was purified via Biotage column chromatography (CH2Cl2/
EtOH, 96:4 to 91:9), and trituration with hot EtOAc gave the title
NH3 in MeOH. The filtrate was concentrated and purified on an
SCX2 cartridge (loading with DCM, byproduct elution with DCM/
MeOH 9/1, elution of product with DCM/MeOH/NH3 9/1/0.01) to
give 2,8-diazaspiro[4.5]decane-1,3-dione (83) (754 mg, 97%) as a
white solid: 1H NMR (500 MHz, DMSO-d6) δ 1.41 (dd, J = 12.5, 1.4
Hz, 2H), 1.65 (td, J = 12.5, 3.9 Hz, 2H), 2.44−2.53 (m, 2H), 2.56 (s,
2H), 2.86 (dt, J = 12.5 Hz, 3.9, 2H); LC−MS (method B, ESI, m/z) tR
= 0.17 min, 169 (M + H)+.
1
compound 74 (700 mg, 76% yield) as a white solid: H NMR (500
MHz, CDCl3) δ 1.33−1.39 (m, 2H), 1.94−2.01 (m, 4H), 2.71−2.79
(m, 2H), 3.14−3.19 (m, 2H), 3.28 (t, J = 6.8 Hz, 2H), 3.97 (s, 3H),
5.58 (br s, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.3 Hz, 2H), 7.68
(s, 1H), 7.82 (s, 1H), 8.22 (s, 1H), 8.44 (s, 1H); 13C NMR (126 MHz,
CDCl3) δ 31.6, 32.4, 38.7, 39.3, 41.8, 47.8, 122.7, 125.6, 127.2, 128.3,
129.8, 132.3, 133.7, 135.7, 136.9, 149.6, 150.9, 152.9, 181.6 ; LC−MS
(method C, ESI, m/z) tR = 2.37 min, 422/424 (M + H)+; ESI-HRMS
calcd for C23H2535ClN5O (M + H)+ 422.1742, found 422.1730.
Preparation of Compounds in Table 9 Exemplified by
Compound 81. 8-(3-Chloro-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)-
pyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-dione (81). Two micro-
wave vials were prepared as follows: 1-methyl-4-(4-(4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-pyrazole (82) (52 mg, 0.18
mmol), 8-(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-
1,3-dione (84) (52 mg, 0.18 mmol), and Pd(dppf)Cl2·CH2Cl2 (5.7
mg, 7.0 μmol) were loaded into a microwave vial, and then 0.5 M
aqueous sodium carbonate solution (390 μL, 0.195 mmol) and MeCN
(2.5 mL) were added. The reaction mixture was heated at 120 °C for
60 min under microwave irradiation. The reaction mixtures from the
two vials were combined and the volatiles were evaporated. The crude
material was purified via Biotage column chromatography (CH2Cl2/
EtOH, 97:3 to 92:8) to give the title compound 81 (70 mg, 58% yield)
8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decane-1,3-
dione (84). 3-Bromo-4,5-dichloropyridine (11) (1.00 g, 4.41 mmol)
and 2,8-diazaspiro[4.5]decane-1,3-dione (83) (1.10 g, 6.61 mmol)
were introduced in a microwave vial, and then NMP (11 mL) and
triethylamine (1.90 mL, 13.2 mmol) were added. The reaction mixture
was heated under microwave irradiation for 1 h at 220 °C. The
reaction mixture was poured into water (110 mL) and the precipitate
was filtered to give 8-(3-bromo-5-chloropyridin-4-yl)-2,8-
diazaspiro[4.5]decane-1,3-dione (84) (1.08 g, 68% yield) as a white
1
solid: H NMR (500 MHz, DMSO-d6) δ 1.64−1.70 (m, 2H), 1.94−
2.01 (m, 2H), 2.69 (s, 2H), 3.24−3.30 (m, 4H), 8.47 (s, 1H), 8.57 (s,
1H), 11.18 (br s, 1H); LC−MS (method B, ESI, m/z) tR = 2.50 min,
358/360/362 (M + H)+.
Mouse Liver Microsomal Stability. Compounds (10 μM) were
incubated with male CD1 mouse liver microsomes (1 mg/mL) protein
in the presence of 1 mM NADPH, 2.5 mM UDP-glucuronic acid
(UDPGA), and 3 mM MgCl2 in 10 mM PBS at 37 °C. Incubations
were conducted for 0 and 30 min. Control incubations were generated
by the omission of NADPH and UDPGA from the incubation
reaction. The percentage compound turned over was determined after
analysis by LC−MS.
Human Liver Microsomal Stability. Compounds (10 μM) were
incubated with mixed-gender, pooled human liver microsomes (1 mg/
mL) protein in the presence of 1 mM NADPH, 2.5 mM UDPGA, and
3 mM MgCl2 in 10 mM PBS at 37 °C. Incubations were conducted for
0 and 30 min. Control incubations were generated by the omission of
NADPH and UDPGA from the incubation reaction. The percentage
compound turned over was determined after analysis by LC−MS.
Mouse, Rat, and Human Clint Determination. Microsomes
(final concentration 0.5 mg/mL), 50 mM phosphate buffer pH 7.4,
and compound (final concentration 1 μM) were added to the assay
plate and allowed to preincubate for 5 min at 37 °C. The reaction was
initiated by the addition of NADPH (final concentration 1.5 mM) and
the plate was shaken at 800 rpm at 37 °C. After 0, 5, 10, 20, and 30
min, aliquots were taken, and the reaction was stopped using cold
acetonitrile. The samples were centrifuged at 4000 rpm for 30 min at 4
°C and analyzed by LC−MS/MS. Four test compounds were pooled
for analysis. The in vitro intrinsic clearance was calculated from the
rate of compound disappearance.
Caco-2 Papp Determination. Caco-2 cells (TC7 clone) were
maintained in DMEM in an atmosphere of 8.5% CO2. For transport
experiments, 0.125 × 106 cells/well were seeded on polycarbonate
filter inserts and allowed to grow and differentiate for 10−14 days
before the cell monolayers were used for experiments. Drug transport
experiments were carried out using a cocktail approach in a 4-
dimensional setting. Apparent permeability coefficients were deter-
mined for A > B and B > A directions with and without the presence
of cyclosporine A as a transporter inhibitor. Up to five test items and
reference compounds were dissolved in Hank’s balanced salt solution
at pH 7.4 to yield a final concentration of 1 μM. The assays were
performed in HBSS containing 25 mM HEPES (pH 7.4) in an
atmosphere of 5% CO2 at 37 °C. Prior to the study, the monolayers
were washed in prewarmed HBSS. At the start of the experiments,
prewarmed HBSS containing the test items was added to the donor
side of the monolayer and HBSS without test items was added to the
receiver side. The plates were shaken at 150 rpm at 37 °C during the
experiment. After 2 h, the transwell insert containing the monolayer
was carefully removed and placed in a new plate, and aliquots of both
the receiver and donor sides were taken and diluted with an equal
volume of ACN containing the internal standard. The mixture was
centrifuged and the supernatant analyzed by LC−MS/MS. The
apparent permeability coefficients (Papp) were calculated using the
1
as a white solid: H NMR (500 MHz, DMSO-d6) δ 1.46−1.52 (m,
2H), 1.77−1.84 (m, 2H), 2.47 (s, 2H), 2.62−2.69 (m, 2H), 3.02−3.08
(m, 2H), 3.89 (s, 3H), 7.34 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz,
2H), 7.95 (d, J = 0.8 Hz, 1H), 8.22 (s, 1H), 8.23 (s, 1H), 8.48 (s, 1H),
11.12 (br s, 1H); 13C NMR (126 MHz, DMSO-d6) δ 32.9, 39.2, 39.7,
44.0, 47.7, 121.8, 125.4, 127.9, 128.5, 130.1, 132.6, 134.1, 134.9, 136.6,
149.2, 151.1, 152.6, 177.4, 183.7; LC−MS (method C, ESI, m/z) tR =
2.30 min, 436/438 (M + H)+; ESI-HRMS calcd for C23H2335ClN5O2
(M + H)+ 436.1535, found 436.1524.
1-Methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)-1H-pyrazole (82). 1-Chloro-4-iodobenzene (6.39 g, 26.8
mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (5.58 g, 26.8 mmol), sodium carbonate (6.25 g, 59.0
mmol), and Pd(dppf)Cl2·CH2Cl2 (2.20 g, 2.68 mmol) were loaded
into a flask, and then a mixture of THF/H2O 3/1 (117 mL) was
added. The reaction mixture was heated in an oil bath at 80 °C
overnight. The solvents were evaporated, and the residue was purified
by column chromatography (cyclohexane/EtOAc 99:1 to 70:30) to
afford 4-(4-chlorophenyl)-1-methyl-1H-pyrazole as a white solid (3.80
g, 74% yield): 1H NMR (500 MHz, CDCl3) δ 3.93 (s, 3H), 7.31 (d, J
= 8.7 Hz, 2H), 7.38 (d, J = 8.7 Hz, 2H), 7.57 (s, 1H), 7.72 (s, 1H);
LC−MS (method B, ESI, m/z) tR = 2.88 min, 193/195 (M + H)+.
4-(4-Chlorophenyl)-1-methyl-1H-pyrazole (3.30 g, 17.1 mmol),
bis(pinacolato)diboron (5.20 g, 20.6 mmol), potassium acetate (5.00
g, 51.4 mmol), Xphos (650 mg, 1.37 mmol), and Pd2dba3 (310 mg,
0.343 mmol) were loaded in a flask, and dioxane (34 mL) was added.
The reaction mixture was stirred in an oil bath at 85 °C overnight. The
solvent was evaporated and the crude product purified by column
chromatography (cyclohexane/EtOAc, 90:10 to 70:30) to afford 1-
methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-
pyrazole (82) as a white solid (3.90 g contaminated by 10% of 1-
1
methyl-4-phenyl-1H-pyrazole, corrected yield 75%): H NMR (500
MHz, CDCl3) δ 1.35 (s, 12H), 3.93 (s, 3H), 7.47 (d, J = 8.3 Hz, 2H),
7.64 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.79 (s, 1H); LC−MS (method
B, ESI, m/z) tR = 3.06 min, 284/285 (M + H)+.
2,8-Diazaspiro[4.5]decane-1,3-dione (83):43 To a suspension of 8-
benzyl-2,8-diazaspiro[4.5]decane-1,3-dione (1.20 g, 4.65 mmol) and
concentrated acetic acid (270 μL, 4.65 mmol) in ethanol (20 mL) was
added palladium hydroxide (20 wt % on carbon, wet, 360 mg, 4.65
mmol) and the mixture stirred under a H2 atmosphere for 24 h at rt.
The mixture was filtered over Celite and washed with EtOH and 1 M
O
DOI: 10.1021/jm501436m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
formula Papp = (Vrec/A × C0donor) × dCrec/dt × 106 with dCrev/dt
being the change in concentration in the receiver compartment with
time, Vrec the volume of the sample in the receiver compartment,
C0donor the concentration in the donor compartment at time 0, and A
the area of the compartment with the cells.
Protein Binding Assays. Protein binding of test compounds was
determined by ultrafiltration using serum from mouse, rat, and human.
The test items (final concentration 5 μM) were incubated in triplicate
with three different serum dilutions (1:2, 1:5, and 1:10) for 30 min at
37 °C using slight agitation. After the incubation, the 96-well filter
plates were centrifuged for 45 min at 3500 rpm and 37 °C; 25 μL
portions of filtrate samples were treated with 50 μL of ethanol and 50
μL of internal standard solution and analyzed by LC−MS/MS. The
fraction unbound was calculated from the drug concentrations in the
filtrate samples.
absence or presence of the test compound (10 dilution concen-
trations) for 90 min at 30 °C. The reaction was stopped by the
addition of 1 μL of 50 mM EDTA solution, and 2 μL of the detection
solution [1.6 μM SA-Xlent (Cisbio, Codolet, France), 7.4 nM Anti-
GST-K (Eu-labeled anti-GST, Cisbio, Codolet, France) in 50 mM
HEPES, 800 mM KF, 0.1% BSA, 20 mM EDTA, 0.1% CHAPS, pH
7.0)] was added. After 1 h of incubation at rt the HTRF was measured
with an Envision multimode reader (PerkinElmer LAS Germany
GmbH) at an excitation wavelength of 340 nm (laser mode) and
emission wavelengths of 615 and 665 nm. The ratio of the emission
signals was determined. The full value used was the inhibitor-free
reaction. The pharmacological zero value used was XAV-939 (Tocris)
in a final concentration of 5 μM. The inhibitory values (IC50) were
determined using either the program Symyx Assay Explorer or
Condosseo from GeneData.
In Vivo Mouse PK in-Life Phase. Female NMRI mice (n = 5)
received either a single intravenous (bolus) injection or a single oral
administration (by gavage) of the compound in a cocktail preparation.
Doses of 0.2 mg/kg (per compound, intravenous) and 0.5 mg/kg (per
compound, orally) were given as solutions in DMSO/PEG 200/water.
Consecutive blood samples were taken retroorbitally under isofluorane
inhalation from n = 3 animals per route of administration after 0.1 (iv
only), 0.25 (po only), 0.5, 1, 2, 4, and 6 h and were further processed
to obtain plasma. In addition, feces of two mice per route of
administration were collected over the time interval from 0 to 24
(pooled for analysis, no blood sampling).
In Vivo Rat PK in-Life Phase. Male Wistar rats (n = 6) received
either a single intravenous (bolus) injection or a single oral
administration (by gavage) of the compound in a cocktail preparation.
Doses of 0.2 mg/kg (per compound, intravenous) and 0.5 mg/kg (per
compound, orally) were given as solutions in DMSO/PEG 200/water.
Consecutive blood samples were taken sublingually under isofluorane
inhalation from n = 3 animals per route of administration after 0.1 (iv
only), 0.25 (po only), 0.5, 1, 2, 4, and 6 h and were further processed
to obtain plasma. In addition, feces and urine of three rats per route of
administration were collected over the time interval from 0 to 24
(pooled for analysis, no blood sampling).
Bioanalytics. The concentrations of compound in plasma and
feces were quantified using an UPLC method with tandem mass
spectrometric detection (LC−MS/MS). The LC−MS system
consisted of a Waters Acquity UPLC coupled to an AB Sciex mass
spectrometer API 5500 Q-trap. The UPLC separation was carried out
on a reversed-phase column (Acquity UPLC BEC C8, 1.7 μM, 2.1 ×
50 mm) using a mobile phase gradient with 0.1% formic acid and
acetonitrile as eluents. The detection of the drug was performed using
multiple reaction monitoring in the positive ionization mode. Plasma
samples were spiked with internal standard, and the analyte was
extracted from the matrix using tert-butyl methyl ether (tBME). The
organic phase was evaporated to dryness under a stream of nitrogen.
The residue was dissolved in acetonitrile/0.1% formic acid for LC−
MS/MS analysis. Feces samples were homogenized with 4 times their
volume of ethanol/water (4:1). Aliquots of the aqueous ethanolic
extracts were further diluted with acetonitrile/0.1% formic acid, spiked
with internal standard, and directly injected into the system.
Biochemical Activity Testing of TNKS1 and -2 (Autoparsy-
lation Assay). The autoparsylation assay was run in two steps: first,
the enzymatic reaction in which GST-tagged TNKS1 or TNKS2
transferred biotinylated ADP-ribose to itself from biotinylated NAD as
the cosubstrate and, second, the detection reaction where a time-
resolved FRET between cryptate-labeled anti-GST bound to the GST
tag of the enzyme and Xlent-labeled streptavidin bound the biotin-
parsylation residue was analyzed. The autoparsylation activity was
detectable directly via the increase in HTRF signal.
Taqman Assays. 7dF3 cells were seeded at 20 000 cells/well into
96-well cell culture plates. After overnight incubation the cells were
treated with 74 at concentrations ranging from 9.1 μM to 0.068 nM.
After 2 or 6 h of incubation, cells were washed with PBS and stored at
−80 °C. Cells were lysed in Cells-to-cDNA II Cell lysis buffer (Life
Technologies Ltd., Paisley, UK) and transferred to PCR plates. Lysis
was completed by heating at 75 °C for 15 min. Lysates were DNase
treated (1 μL/well Life Technologies) at 37 °C for 15 min and heat
inactivated at 75 °C for 5 min. cDNA synthesis was performed using
the High-Capacity cDNA Reverse Transcription Kit (Life Technolo-
gies). Taqman reactions were performed in 384-well plates using 1.5
μL cDNA and 1× FAM-labeled primer probe for the measurement of
target gene and 1× VIC-labeled primer probe for LRP0 and were
multiplexed in each well as the internal reference control with TaqMan
Universal Master Mix (Life Technologies). Plates were run for 40
cycles using an ABI Via7 Sequence Detection System and software. Ct
values for target genes were normalized relative to the LRP0 control
before being expressed relative to the vehicle treated control sample.
In Vitro Cell-Based Reporter Assays. 7Df3 Luciferase Reporter
Assay. 7dF3 cells were seeded at 5000 per well in 50 μL of Dulbecco’s
modified Eagle’s medium (DMEM) with 4 mM supplemental L-
glutamine in 384-well white tissue culture plates (Corning 3570). After
incubating overnight at 37 °C/5% CO2, compounds ranging in final
concentration from 90 μM to 0.3 nM were added in duplicate to the
wells. After 2 h of further incubation as above, β-oestradiol was added
to a final concentration of 10 μM. The cells were incubated for 24 h at
37 °C/5% CO2 and then 25 μL of luciferase reagent (SteadyGlo,
Promega) was added, and the cells were mixed. After leaving the plate
for 60 min at room temperature, luminescence was read on a plate
luminescence reader (TopCount, PerkinElmer). The percentage
inhibition for each compound concentration was calculated using
total counts (no compound) and low counts (no β-oestradiol) as highs
and lows, respectively. IC50 was subsequently determined using a
curve-fitting software package in Excel (Excelfit, IDBS).
Tet-O-WNT Reporter Lines. T-Rex HEK293 Tet-O cells (Life
Technologies) were transfected with the TCF-firefly luciferase-IRES-
GFP (TLIG) reporter, and a highly responsive reporter clone was
derived using an analogous process to that previously used to derive
the HEK293-based 7df3 cell line.30 Expression constructs coding for
WNT inducers that act at different levels of the pathway30 were
introduced downstream of the Tet-O promoter by FLP-FRT-site-
directed recombination. This generated a set of cell lines that contain
an identical TCF-luciferase reporter in combination with Tet-inducible
cDNAs encoding ΔN-LRP6, Dvl2, Axin-GID, ΔN-β-catenin, and
VP16-TCF4.
Assay Protocol for Tet-O-WNT Reporter Cell Lines. Cells were
seeded from frozen at 40 000/well into 96-well plates in 80 μL of
medium. On day 3, eight compound concentrations ranging from 30
μM to 14 nM were prepared at 333× final concentration in DMSO
and then diluted to 5× final concentration in OptiMEM (Life
Technologies) supplemented with 0.05 μg/mL doxycycline hyclate
(Sigma-Aldrich). These solutions were then pipetted at 20 μL/well
into each well of the plate. On day 4, medium was removed, and cells
were lysed in 30 μL/well GLOlysis buffer (Promega) for 30 min.
BrightGLO firefly luciferase substrate (Promega) was pipetted at 30
The autoparsylation assay was performed in 384-well HTRF
(Cisbio, Codolet, France) assay format in Greiner low-volume 384-
well microtiter plates; 250 nM GST-tagged TNKS1 (1023−1327 aa)
or 250 nM GST-tagged TNKS2 (873−1166 aa) and 5 μM bio-NAD
(Biolog, Life science Inst., Bremen, Germany) as cosubstrate were
incubated in a total volume of 5 μL (50 mM HEPES, 4 mM MgCl2,
0.05% Pluronic F-68, 1.4 mM DTT, 0.5% DMSO, pH 7.7) in the
P
DOI: 10.1021/jm501436m
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
μL/well. Firefly luciferase activity was measured on a Fluostar plate
reader (BMG) in luminescence mode.
positive ionization mode. Plasma samples were spiked with internal
standard, and the analyte was extracted from the matrix (plasma and
tumor homogenized with 3 v/w PBS) with 3−4 volumes of methanol
and directly injected into the system.
Generation of Other Cell-Based Reporter Cell Lines. SW480
human colorectal cancer cells were cotransfected with Topflash and
CMV-lacZ reporter plasmids as described previously.30 RKO (ATCC
CRL-2577) cells were transfected with plasmid F1522 encoding
PEST-destabilized firefly luciferase (luc2P, designated Fluc2P;
Promega) and IRES-driven PEST-destabilized renilla (hRlucP;
Promega) downstream of the EF-1-α promoter.44 LS174T (ATCC
CL-188) and COLO205 (ATCC, #CCL-222) human colorectal
cancer cells were transduced with a TCF/LEF lentiviral firefly
luciferase reporter construct. This construct was generated by
synthesizing and cloning 16 repeats of the TCF/LEF responsive
element (5′-AGATCAAAGG-3′) upstream of the minimal pTA
promoter driving PEST-destabilized firefly luciferase Fluc2P (t1/2 < 1
h) from plasmid pGL4.24 (Promega). The TCF/LEF-pTA-Fluc2P
cassette was subcloned into lentiviral vector pCDF1-MCS2-EF1-Puro
(Systems Biosciences) to generate the final reporter construct F1756,
which contained a puromycin resistance gene for selection purposes.
F1756 was cotransfected with pPackF1 packaging mix (Systems
Biosciences) into the HEK293 cell line. F1756 reporter lentivirus was
recovered in cell media after 48 h, purified, and used to transduce cell
lines. After 72 h, successfully transduced cells were selected using 5
μg/mL puromycin and stable colonies left to grow for 2 weeks.
Colonies showing high-level basal luciferase expression on addition of
luciferin to the flask and imaging on an IVIS 200 (PerkinElmer Inc.)
were picked and expanded. Subsequent studies identified clone 4 as a
suitable candidate for further studies. Reporter activity (and its
response to inhibition by compounds) was measured in vitro in 384-
well plates using Steady-Glo luciferase reagent (Promega) for firefly
luciferase. Cells were exposed to compound for 24 h before reporter
assays were conducted. Cell viability was assayed in parallel using
Alamar blue (Promega). PA-1 human teratocarcinoma cells were
stably transfected with a TOPflash TCF reporter plasmid (Merck
Millipore) that was modified by introduction of a neomycin resistance
marker. Single clones were selected and tested for WNT-driven
reporter pathway activation by addition of Wnt-3A supernatant
produced by L Wnt-3A cells (ATCC, CRL-2647). Reporter activity
was measured in 96-well plates using Steady-Glo luciferase reagent
(Promega). Cells were treated with compound for 6 h and
subsequently stimulated with Wnt-3A supernatant for 24 h before
reporter assays were conducted.
COLO205-F1756 Clone 4 Human Tumor Xenograft Model
Expressing a Modified Luciferase-Based Wnt Reporter Con-
struct. Female athymic CrTac:NCr-Foxn1nu mice from Taconic (6−8
weeks of age) were injected subcutaneously in the flank with 3 million
COLO205-F1756 clone 4 cells, and when tumor xenografts reached a
mean diameter of 5−6 mm (equating to a mean volume of about 100
mm3), mice (n = 8 per group) were dosed orally by gavage with
compound 74 (70 mg/kg bid for 8 days) or vehicle (10% dimethyl
sulfoxide; 5% Tween 20 in sterile saline). Luminescent flux as a
measure of WNT pathway activity was quantified by whole body
imaging in cohorts of four animals at 2 and 6 h after the final dose.
This was achieved by sc injection of 100 μL of 30 mg/mL D-Luciferin
firefly substrate (XR-1001, PerkinElmer) into anaesthetized mice and
imaging with a Xenogen IVIS 200 (XR-1001, PerkinElmer), utilizing
the software Living Image (version 4). At these time points,
heparinized blood samples were collected by cardiac puncture under
terminal anesthesia, and plasma was separated by centrifugation.
Tumors were excised and weighed, and both plasma and tumor
samples were snap frozen for PK analyses.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR spectra for all final compounds; preparation of
compounds 15−25, 27−37, 39−42, 45−52, 61−67, 69−73,
and 75−80; CYP450 inhibition for compounds 9 and 74
(Supplementary Table S1), summaries of CEREP and kinase
profiles (Supplementary Tables S2−S5); Supplementary
Figures S1 and S2; and small-molecule X-ray crystallographic
data analysis for compound 9. This material is available free of
charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*E-mail: julian.blagg@icr.ac.uk. Telephone: +44(0)
2087224051.
■
Notes
The authors declare the following competing financial
interest(s): A.M., S.C., M.P., D.W., M.S., O.A-P., E.S., C.T.,
A.T.H., W.C., S.H., G.B., M.-J.O.R., M.V., A.D.H.B., R.T., P.W.,
W.A., F.R., P.A.C., S.A.E., and J.B. are current or former
employees of The Institute of Cancer Research, which has a
commercial interest in the development of WNT pathway
inhibitors. K.G., O.P., D.W., C.E., F.R., F.S., and K.S. are
current employees of Merck Serono.
ACKNOWLEDGMENTS
■
This work was supported by Cancer Research UK (grant
number C309/A11566). We acknowledge NHS funding to the
NIHR Biomedical Research Centre at The Institute of Cancer
Research and The Royal Marsden. We thank Dr. Amin Mirza,
Mr. Meirion Richards, and Dr Maggie Liu for their assistance
with NMR, mass spectrometry, and HPLC. We thank Prof. Dr.
H. Fueß of the TU Darmstadt for the X-ray crystal structure of
compound 9 and Dr. Mark Honey for resynthesis of selected
compounds.
ABBREVIATIONS USED
■
Cl, clearance; Clint, intrinsic clearance; DSH, Dishevelled; ER,
efflux ratio; GSK3β, glycogen synthase kinase 3β; GFP, green
fluorescent protein; GID, GSK3β-interaction domain; HLM,
human liver microsomes; IRES, internal ribosome entry site;
MLM, mouse liver microsomes; Papp, apparent permeability
coefficient; Vss, apparent volume of distribution at steady state
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