Pyrimidinone nicotinamide mimetics as selective tankyrase and Wnt pathway inhibitors suitable for in vivo pharmacology

Article abstract of DOI:10.1021/ml5003663

The canonical Wnt pathway plays an important role in embryonic development, adult tissue homeostasis, and cancer. Germline mutations of several Wnt pathway components, such as Axin, APC, and ?-catenin, can lead to oncogenesis. Inhibition of the poly(ADP-r

Full text of DOI:10.1021/ml5003663

Letter
pubs.acs.org/acsmedchemlett
Pyrimidinone Nicotinamide Mimetics as Selective Tankyrase and Wnt
Pathway Inhibitors Suitable for in Vivo Pharmacology
Jeffrey W. Johannes,*,† Lynsie Almeida,† Bernard Barlaam,‡ P. Ann Boriack-Sjodin,† Robert Casella,†
Rosemary A. Croft,‡ Allan P. Dishington,‡ Lakshmaiah Gingipalli,† Chungang Gu,† Janet L. Hawkins,‡
Jane L. Holmes,‡ Tina Howard,‡ Jian Huang,† Stephanos Ioannidis,† Steven Kazmirski,†
Michelle L. Lamb,† Thomas M. McGuire,‡ Jane E. Moore,‡ Derek Ogg,‡ Anil Patel,‡ Kurt G. Pike,‡
Timothy Pontz,† Graeme R. Robb,‡ Nancy Su,† Haiyun Wang,† Xiaoyun Wu,† Hai-Jun Zhang,†
Yue Zhang,† Xiaolan Zheng,† and Tao Wang†
†AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡AstraZeneca R&D Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
S
* Supporting Information
ABSTRACT: The canonical Wnt pathway plays an important
role in embryonic development, adult tissue homeostasis, and
cancer. Germline mutations of several Wnt pathway
components, such as Axin, APC, and ß-catenin, can lead to
oncogenesis. Inhibition of the poly(ADP-ribose) polymerase
(PARP) catalytic domain of the tankyrases (TNKS1 and
TNKS2) is known to inhibit the Wnt pathway via increased
stabilization of Axin. In order to explore the consequences of
tankyrase and Wnt pathway inhibition in preclinical models of
cancer and its impact on normal tissue, we sought a small molecule inhibitor of TNKS1/2 with suitable physicochemical
properties and pharmacokinetics for hypothesis testing in vivo. Starting from a 2-phenyl quinazolinone hit (compound 1), we
discovered the pyrrolopyrimidinone compound 25 (AZ6102), which is a potent TNKS1/2 inhibitor that has 100-fold selectivity
against other PARP family enzymes and shows 5 nM Wnt pathway inhibition in DLD-1 cells. Moreover, compound 25 can be
formulated well in a clinically relevant intravenous solution at 20 mg/mL, has demonstrated good pharmacokinetics in preclinical
species, and shows low Caco2 efflux to avoid possible tumor resistance mechanisms.
KEYWORDS: Wnt, oncogenesis, pharmacokinetics, Caco2, tankyrase, PARP
he discovery and development of small molecule
inhibitors of the canonical Wnt pathway has been a
with and PARsylates Axin. Tankyrase inhibition prevents
PARsylation of Axin, which in turn leads to increased Axin
protein levels in the cell. Higher levels of Axin lead to
sequestration and/or destruction of ß-catenin, preventing
translocation of ß-catenin to the nucleus and a reduction in
transcription of Wnt pathway genes. Moreover, they showed
evidence that XAV939 can inhibit soft agar colony formation of
DLD-1 cells grown under low serum conditions.
The tankyrases are members of the PARP enzyme
superfamily of proteins, all of which contain a PARP catalytic
domain that can catalyze the attachment of ADP ribose to the
surface of proteins using the cofactor nicotinamide adenine
dinucleotide (NAD+).9 As a consequence of their linkage to the
Wnt pathway, association with telomeres,10 and spindle
structure and function,11 the tankyrases have attracted a high
level of interest from the drug discovery community. Numerous
research groups have published reports of tankyrase inhibitors
T
long-standing pursuit within oncology.1 Under normal circum-
stances, the Wnt signaling pathway plays a key role in
embryonic development and the regeneration of adult tissue
such as intestinal epithelial cells.2 Human tumors frequently
harbor gene mutations that impact important proteins along
the pathway such as APC, Axin, and ß-catenin.3 In most cases,
these mutations lead to constitutive activation of Wnt signaling,
which results in a Wnt ligand independent increase in the
transcription of Wnt genes and leads to heightened
proliferation.4 Approximately 85% of colorectal cancer (CRC)
harbors APC mutations.5 A plethora of small molecules has
been discovered that can modulate this pathway by targeting
various proteins (e.g., casein kinases and porcupine).6,7 Despite
this, there are currently no commercial drugs that target the
Wnt pathway in oncology.
In 2009, Huang et al. reported that inhibition of the
poly(ADP-ribose) polymerase (PARP) catalytic domain of
tankyrases (TNKS1 and TNKS2) can inhibit the Wnt pathway
via Axin stabilization.8 Using the small molecule tankyrase
inhibitor XAV939, they demonstrated that tankyrase interacts
Received: September 9, 2014
Accepted: January 13, 2015
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
with varying degrees of potency and PARP enzyme family
selectivity.12−14
The goal of our medicinal chemistry effort was to discover a
potent dual TNKS1 and TNKS2 enzyme inhibitor that
demonstrated potent inhibition of the Wnt pathway as
demonstrated in a TCF TOPflash reporter assay in DLD-1
cells and that had sufficient DMPK properties for hypothesis
testing in preclinical in vivo xenograft models. In order to have
confidence that the biological effects of these compounds were
due to TNKS inhibition and not other PARPs, selectivity was
desired against other PARP family members, primarily PARP1.9
To avoid transporter mediated resistance mechanisms in CRC,
we sought compounds with low efflux potential as measured in
a standard Caco2 permeability assay at pH 7.4. Finally, we
strove to optimize this series for IV dosing, to allow greater
control over compound exposure and possibly mitigate
expected on-target intestinal toxicity.15
Figure 1. Compound 2a (yellow) bound to TNKS1 (gray, PDB ID:
4W5S) overlaid with TNKS1 apo structure (cyan, PDB ID: 2RF5).
A high-throughput screen (HTS) of the AstraZeneca
screening collection identified the 2-phenyl quinazolinone 1
(Table 1), which had a 7 nM IC50 as a TNKS1 enzyme
On the basis of the crystal structure, we explored small
substituents on the heteroaryl terminal ring that would bias the
dihedral angle of the terminal biaryl system to 90° and fill space
vacated by Tyr1203. These efforts led to compound 3 (Table
2) featuring a terminal 4-methyl-3-pyridyl ring. The next phase
Table 1. Optimization of HTS Hit Compound 1 to Lead
Optimization Starting Compound 2a
Table 2. 4-Methyl-3-pyridyl Moiety with π-Donating Groups
at the 6-Position Improves Cell Potency
Cmpd
R1
TNKS1 enz IC50 (μM)
Wnt cell IC50 (μM)
1
H
0.007
0.006
0.007
0.004
2.0
1a
2
CH2OH
H
0.145
1.25
0.027
TNKS1
PARP1
Wnt cell
IC50
(μM)
Aq. Sol.
pH 7.4
(μM)
enz IC50 enz IC50
LogD
pH 7.4
2a
CH2OH
Cmpd
R1
(μM)
(μM)
3
4
5
6
7
8
H
F
0.003
0.002
0.006
0.017
0.006
0.019
2.77
0.024
0.032
0.019
0.004
0.004
<0.003
2.1
3.4
3.9
2.9
2.8
>3.5
11
<1
<1
<1
<1
4
3.74
inhibitor16 and was 2 μM in our DLD-1 Wnt reporter assay. 2-
Phenyl quinazolinones have been previously described in the
literature as tankyrase inhibitors.17,18 The potency, small size,
and synthetic accessibility of this compound class encouraged
us to explore the series further. Modeling and systematic
exploration of substituents around the quinazolinone ring
revealed that the 8-position could accommodate small groups.
We soon found that a hydroxymethyl moiety greatly improved
DLD-1 Wnt reporter potency. Further optimization of the 2-
phenyl ring suggested that the isopropyl group could be
replaced with 5- and 6-membered aromatic rings. Exploration
of various aryl rings gave compound 2a (Table 1), which had
improved cell potency (27 nM Wnt reporter).
Intrigued by the cell potency of compound 2a, we obtained a
crystal structure of this molecule bound to TNKS1 (Figure 1).
Compound 2a (yellow) binds in the nicotinamide pocket of
TNKS1 (gray), making critical H-bonds to the backbone NH
and carbonyl of Gly1185. The quinazolinone ring is sandwiched
between Tyr1224 and Tyr1213 making π-stacking interactions.
The hydroxymethyl moiety points toward Glu1291. Upon
binding, the compound displaces Tyr1203 (from apo structure
in cyan),19 which then reorganizes around the 2-phenyl linker,
while the pyrazole ring partially fills the newly vacated space.
The pyrazole sits at a ∼90° dihedral angle to the linking phenyl,
while its methyl group is solvent exposed.
Cl
0.017
0.206
0.035
NH2
OCH3
N(CH3)2
of optimization of the terminal aryl ring focused on exploring
electron donating and withdrawing substituents at the 6-
position of the 3-pyridyl ring. Introduction of σ-withdrawing
groups, such as F and Cl in compounds 4 and 5, gave no
significant improvement in cell potency but was accompanied
by a large increase in lipophilicity as measured by logD. In
contrast, π-donating groups gave an improvement in cell
potency, as exemplified by 6-NH2 compound 6 and 6-OCH3
compound 7. Dimethylation of the amine was also tolerated to
give compound 8, which had an IC50 < 3 nM in our cell assay,
albeit with high logD and low solubility.
Assuming that dimethylamine 8 adopts a similar binding
mode to compound 2a, we sought to improve the solubility and
lower the logD by adding hydrophilic groups to the amine at
the 6-position of the pyridyl ring, which was predicted to be
solvent exposed (Table 3). Nucleophilic aromatic substitution
of aryl-chloride 5 with methoxyethylamine led to compound 9,
which had high Caco2 permeability and low clearance in rat but
a very low volume of distribution. Hydroxyethyl compound 10
showed improved logD and solubility, but it suffered from high
B
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Table 3. Optimization of the Solvent Group Improves Solubility and Metabolic Stability while Maintaining Permeability and
Potency
a
b
c
d
Unit: 10−6 cm/s. Unit: mL/min·kg. Unit: L/kg. Unit: h.
intrinsic clearance in rat hepatocytes due to oxidation of the
terminal OH to the carboxylic acid, confirmed by metabolite
identification using LC−MS/MS. The intrinsic clearance in rat
hepatocytes was lowered in compound 11 by introducing
geminal methyl groups alpha to the OH; however, logD,
solubility, and PK parameters were no different relative to 9. A
number of cyclic amines were also added to aryl-chloride 5 to
yield morpholine analogue 12, which showed little difference
compared to 9. A path forward was found in the isosteric, basic
piperazine 13, which gave a significant improvement in
solubility and a one unit drop in logD, although the Caco2
permeability of this compound was then less than 1 and the
efflux ratio was above 20.
To mitigate the efflux within this more soluble amine
containing subseries, we sought to tune the amine basicity,
steric environment, and H-bond potential through the addition
of nearby methyl groups. The addition of a single methyl group
α to the amine gave (S)-2-methylpiperazine 14, which had
improved permeability but also improved intrinsic clearance,
presumably due to reduced metabolism of the amine or CH2 α
to the amine. Rat PK of 14 revealed that this compound had an
increased volume of distribution and terminal half-life relative
to 9. Permeability was improved further by N-methylation to
give 15, but the drawback was a compound with poor metabolic
stability in vitro due to rapid N-demethylation and N-oxidation,
as confirmed by metabolite identification using LC−MS/MS.
Further blocking the NH group led to the cis-2,6-dimethyl
piperazine analogue 16, which had a good balance of improved
solubility, low intrinsic clearance, acceptable efflux, and good rat
pharmacokinetics. Interestingly, rigidification of this structure
by linking the methyl groups to form the ethylene bridged
piperazine in 17 gave a high efflux compound. The origin of the
approximately 10-fold difference in efflux between the two
compounds may lie in the accessibility of the NH group. The
piperazine methyl groups in 16 are bis-equatorial, providing
steric hindrance around the NH. In contrast, the alkyl groups
are locked in a bis-axial position in bicyclic compound 17,
further exposing the NH toward H-bonding with efflux
transporters.
A TNKS1-bound crystal structure was obtained for 15
(Figure 2). The terminal pyridine ring sits roughly
perpendicular to the linker phenyl ring, placing the methyl
group below the plane of the core quinazolinone and filling the
hydrophobic space near Tyr1203. The pyridine lone pair makes
a hydrogen bond with an ordered water molecule. The (S)-1,2-
dimethyl piperazine moiety is coplanar with the pyridine ring.
This group is not completely solvent exposed as initially
hypothesized; there are hydrophobic contacts between
Phe1188 and the inner half of the piperazine ring. The
piperazine adopts a conformation that places the 2-methyl
C
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ACS Medicinal Chemistry Letters
Letter
pairs were made, with either a piperidine or piperazine linker
and either the cis-2,6-dimethyl piperazine or the (S)-1,2-
dimethyl piperazine solvent group. All four compounds showed
a significant improvement in solubility, lower logD, reduced
human plasma protein binding, and reduced PARP1 enzyme
potency relative to phenyl. The piperidine linked compounds
18 and 20 had better selectivity against PARP1, lower Caco2
efflux ratios, and lower rat clearance than the piperazine linked
counterparts. Conversely, it was the terminal solvent group that
drove the logD, plasma protein binding, and intrinsic clearances
for these saturated linked compounds. Compounds 20 and 21
bearing the (S)-1,2-dimethyl piperazine feature an N-Me group,
which increased logD and protein binding and reduced
microsomal and hepatocyte stability. Overall, compound 18
had the best properties and rat PK of this set, but the
improvement came at the expense of cell potency. Alternatively,
compound 21 maintained cell potency comparable to phenyl-
linked 16 with improved solubility, but the in vivo PK showed
higher CL and shorter terminal half-life.
Figure 2. Crystal structure of compound 15 bound to TNKS1 (PDB
ID: 4W6E).
group axial to avoid A1,2 strain with the N-Me group. We also
observed an H-bond interaction network between the core
hydroxymethyl group, a water molecule, and Glu1291. In
published quinazolinone−TNKS2 structures,19 a water at this
position is highly conserved even in the absence of an 8-
position substituent (15/18 structures); however, with 15, the
Glu1291 carboxylate shifts ∼2.6 Å to replace an additional
water molecule observed in 11 of these structures.
Finally, we investigated the optimization of the nicotinamide
mimetic core (Table 5). We were intrigued by the possibility of
Table 5. 5,6-Heterocyclic Cores Improve PARP1 Selectivity
and Caco2 Efflux
We next turned our attention to the linker phenyl group.
Initial SAR during the hit-to-lead phase indicated that even
small substituents around the phenyl, such as F or CH3, were
deleterious to TNKS1 enzyme potency. However, we
postulated that solubility could be improved by introducing
saturated linkers that would maintain a similar geometry.20
A
number of phenyl isosteres were employed, with piperazine and
piperidine distinguishing themselves (Table 4). Four matched
Table 4. Replacement of the Phenyl Linker with a Piperazine
or Piperidine Improves Solubility
Cmpd
22
23
24
25
Wnt cell IC50 (μM)
TNKS1 enz IC50 (μM)
PARP1 enz IC50 (μM)
LogD pH 7.4
0.012
0.004
>30
2.1
38
<0.003
0.024
0.005
>30
2.1
32
0.005
0.006
>30
2.5
30
0.008
5
3.6
8
Aq. Sol. pH 7.4 (μM)
Hu PPB (% free)
1.6
14
<1
4
12
7.2
13
a
Hu Mics CLint
6
b
Rat Heps CLint
5
4
4
8
Caco2 AB (10−6 cm/s)
0.1
194
5
4.3
3.8
4
5.5
2.7
11
Cmpd
18
19
20
21
Caco2 efflux ratio
A
CH
CH3
H
N
CH
H
N
Rat IV CL (mL/min·kg)
Rat IV Vdss (L/kg)
4
R2
R3
CH3
H
H
1.6
5.0
2.5
8.2
3.0
11
4.4
5.7
CH3
0.012
0.003
11
CH3
0.004
0.004
3
Rat IV t1/2 (h)
Wnt cell IC50 (μM)
TNKS1 enz IC50 (μM)
PARP1 enz IC50 (μM)
LogD pH 7.4
0.038
0.004
11
0.037
0.004
3
a
b
Unit: μL/min·mg. Unit: μL/min·106.
morphing the quinazolinone core into a 5,6-heterocyclic ring
system, but we were cognizant of the significant cell potency
obtained by inclusion of the CH2OH group. We designed
compound 22, featuring a pyrrolotriazinone core, which
allowed stable attachment of the CH2OH group to the 7-
position of a 5,6-fused ring system. The 7-CH2OH heterocycle
22 was less potent in our cell assay than 16, but it had excellent
PARP1 selectivity and maintained the good DMPK properties
of the quinazolinone analogue 16. However, Caco2 perme-
ability and efflux were worse for compound 22 compared to 16.
We also made the corresponding 7-CH3 pyrrolotriazinone
compound 23 as a control. Encouragingly, the 7-CH3
1.4
651
8
1.6
151
8
2.4
205
2
2.3
530
3
Aq. Sol. pH 7.4 (μM)
Hu PPB (% free)
a
Hu Mics CLint
11
12
25
32
b
Rat Heps CLint
Caco2 AB (10−6 cm/s)
5
21
12
23
0.8
17
0.3
41
3.5
10
0.7
44
Caco2 efflux ratio
Rat IV CL (mL/min·kg)
Rat IV Vdss (L/kg)
Rat IV t1/2 (h)
22
35
17
28
6.5
4.4
3.2
1.7
2.1
1.8
1.9
1.0
a
b
Unit: μL/min·mg. Unit: μL/min·106.
D
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ACS Medicinal Chemistry Letters
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compound was <3 nM in our cell assay with good selectivity
against PARP1 and similar rat PK to 16, but it had high logD
and correspondingly low solubility. Next, we synthesized 24
and 25, which were predicted to be less lipophilic (ClogP 2.5
and 3.3, respectively) relative to 23 (ClogP 3.5). The
pyrazolopyrimidinone 24 was 10-fold less potent in our Wnt
cell reporter assay compared to 23 but had improved efflux
versus 16. In contrast, the pyrrolopyrimidinone 25 maintained
single digit nanomolar potency in the cell while lowering logD
to 2.5. Compound 25 had much improved PARP1 selectivity
compared to core analogue 16 while maintaining good rat in
vivo PK properties. Moreover, compound 25 had similarly
improved Caco2 permeability and efflux ratio versus 16.
Detailed PARP family enzyme data and pharmacokinetic data
for compounds 9 and 25 are presented in Table 6. The enzyme
blot (see Supporting Information Figure S1). At early time
points (3 and 8 h), 9, 15, and 25 showed similar TNKS1/2
inhibition and Axin2 stabilization as XAV939. At longer times
(24, 48, and 72 h), 9, 15, and 25 showed qualitatively stronger
and longer lasting stabilization of Axin2, TNKS1, and TNKS2
at lower concentrations compared to XAV939.
Compound 9 was synthesized on multigram scale and was
made available to external collaborators as an orally bioavailable
small molecule inhibitor of TNKS1/2 and the Wnt pathway
carrying some activity against PARP1 and 2. This compound (9
= AZ1) has demonstrated efficacy in KRAS wild type patient-
derived xenograft models of CRC in combination with
irinotecan.22 Optimized compound 25 has been used as an
IV probe compound to explore the in vivo effects of TNKS1/2
inhibition on normal tissue and tumor xenografts. The results
of these experiments will be reported in due course.
Table 6. PARP Enzyme Selectivity, DMPK Profiles, and
Formulations for Compounds 9 and 25
ASSOCIATED CONTENT
* Supporting Information
■
S
Cmpd
9
25
TNKS1/2 and Axin2 Western blot data; crystallization
conditions and crystallographic data; TNKS1, PARP1, DLD-1
Wnt reporter, and Caco2 assay protocols; detailed solution
formulations; and experimental details for key compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
TNKS1 IC50 (μM)
TNKS2 IC50 (μM)
PARP1 IC50 (μM)
PARP2 IC50 (μM)
PARP6 IC50 (μM)
mouse IV CL (mL/min·kg)
mouse IV Vss (L/kg)
mouse IV t1/2 (h)
mouse oral F (%)
mouse PPB (% free)
rat oral F (%)
0.003
0.010
0.559
0.232
>3
0.003
0.001
2.0
0.5
>3
53
24
2.6
5.5
4.3
12
AUTHOR INFORMATION
Corresponding Author
*E-mail: jeffrey.johannes@astrazeneca.com.
■
3.4
69
2.2
3.6
18
Notes
59
rat PPB (% free)
0.2
3.7
The authors declare no competing financial interest.
IV formulation
0.4 mg/mL, TEG/40% 20 mg/mL, 20%
SBECD 50/50, pH 5 SBECD, pH 4
ABBREVIATIONS USED
■
TEG, tetraethylene glycol; heps, hepatocytes; CL, clearance;
PK, pharmacokinetics; mics, microsomes; aq, aqueous; sol,
solubility; PPB, plasma protein binding; CLint, intrinsic
clearance; SBECD, sulfobutylether-beta-cyclodextrin sodium
salt; IV, intravenous; A1,2, 1,2-allylic; Hu, human; enz, enzyme;
APC, adenomatous polyposis coli; DMPK, drug metabolism
and pharmacokinetics
assays in this table utilize a different assay technology than the
enzyme assays presented Tables 1−5 (see Supporting
Information for assay details). Oral lead 9 showed potent
dual TNKS1/2 enzyme inhibition with moderate selectivity
against PARP1 and 2 and good selectivity over PARP6.21 While
the bioavailability of 59% for 9 in rat was good, the PK was
characterized by very low volume of distribution and short half-
life. Rat protein binding was quite high at only 0.2% unbound.
Mouse PK for 9 showed good bioavailability of 69% with a
higher volume and longer half-life along with reduced protein
binding. Because of the poor solubility and lack of a strongly
basic nitrogen, methoxyethylamine 9 was difficult to formulate
for IV dosing, only achieving 0.4 mg/mL in an IV solution
useful only for preclinical studies.
Compound 25 was also profiled against a panel of PARP
family enzymes and showed excellent potency as a dual TNKS1
and TNKS2 inhibitor with good selectivity against PARPs 1, 2,
and 6 (Table 6). In sharp contrast to 9, 25 formulated well in a
clinically relevant IV solution at 20 mg/mL using SBECD as an
excipient at pH 4. Dosed at 25 mg/kg IV to nude mice, 25 had
a CL of 24 mL/min·kg and a half-life of 4 h. The bioavailability
of 25 in mouse and rat was only moderate at 12% and 18%,
respectively.
REFERENCES
■
(1) Anastas, J. N.; Moon, R. T. WNT signalling pathways as
therapeutic targets in cancer. Nat. Rev. Cancer 2013, 13, 11−26.
(2) Pinto, D.; Gregorieff, A.; Begthel, H.; Clevers, H. Canonical Wnt
signals are essential for homeostasis of the intestinal epithelium. Genes
Dev. 2003, 17, 1709−13.
(3) Segditsas, S.; Tomlinson, I. Colorectal cancer and genetic
alterations in the Wnt pathway. Oncogene 2006, 25, 7531−7.
(4) Clevers, H. Wnt/beta-catenin signaling in development and
disease. Cell 2006, 127, 469−80.
(5) Markowitz, S. D.; Bertagnolli, M. M. Molecular origins of cancer:
Molecular basis of colorectal cancer. N. Engl. J. Med. 2009, 361, 2449−
60.
(6) Voronkov, A.; Krauss, S. Wnt/beta-catenin signaling and small
molecule inhibitors. Curr. Pharm. Des. 2013, 19, 634−64.
(7) Kahn, M. Can we safely target the WNT pathway? Nat. Rev. Drug.
Discovery 2014, 13, 513−32.
(8) Huang, S. M.; Mishina, Y. M.; Liu, S.; Cheung, A.; Stegmeier, F.;
Michaud, G. A.; Charlat, O.; Wiellette, E.; Zhang, Y.; Wiessner, S.;
Hild, M.; Shi, X.; Wilson, C. J.; Mickanin, C.; Myer, V.; Fazal, A.;
Tomlinson, R.; Serluca, F.; Shao, W.; Cheng, H.; Shultz, M.; Rau, C.;
Schirle, M.; Schlegl, J.; Ghidelli, S.; Fawell, S.; Lu, C.; Curtis, D.;
Kirschner, M. W.; Lengauer, C.; Finan, P. M.; Tallarico, J. A.;
In order to confirm that the mechanism of action of this
series of tankyrase inhibitors is blocking the Wnt pathway
through Axin, DLD-1 cells were treated with compounds 9 and
25 at 0.3 and 0.8 μM, compound 15 at 0.3 μM, and XAV939 at
1 μM and analyzed for TNKS1, TNKS2, and Axin2 via Western
E
DOI: 10.1021/ml5003663
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Bouwmeester, T.; Porter, J. A.; Bauer, A.; Cong, F. Tankyrase
inhibition stabilizes axin and antagonizes Wnt signalling. Nature 2009,
461, 614−20.
(9) Rouleau, M.; Patel, A.; Hendzel, M. J.; Kaufmann, S. H.; Poirier,
G. G. PARP inhibition: PARP1 and beyond. Nat. Rev. Cancer 2010, 10,
293−301.
(10) Smith, S.; Giriat, I.; Schmitt, A.; de Lange, T. Tankyrase, a
poly(ADP-ribose) polymerase at human telomeres. Science 1998, 282,
1484−7.
(11) Chang, P.; Coughlin, M.; Mitchison, T. J. Tankyrase-1
polymerization of poly(ADP-ribose) is required for spindle structure
and function. Nat. Cell Biol. 2005, 7, 1133−9.
(12) Lehtio, L.; Chi, N. W.; Krauss, S. Tankyrases as drug targets.
̈
FEBS J. 2013, 280, 3576−93.
(13) Haikarainen, T.; Krauss, S.; Lehtio, L. Tankyrases: structure,
function and therapeutic implications in cancer. Curr. Pharm. Des.
2014, 20, 6472−88.
(14) Liscio, P.; Carotti, A.; Asciutti, S.; Karlberg, T.; Bellocchi, D.;
Llacuna, L.; Macchiarulo, A.; Aaronson, S. A.; Schuler, H.; Pellicciari,
̈
R.; Camaioni, E. Design, synthesis, crystallographic studies, and
preliminary biological appraisal of new substituted triazolo[4,3-
b]pyridazin-8-amine derivatives as tankyrase inhibitors. J. Med. Chem.
2014, 57, 2807−12.
(15) Lau, T.; Chan, E.; Callow, M.; Waaler, J.; Boggs, J.; Blake, R. A.;
Magnuson, S.; Sambrone, A.; Schutten, M.; Firestein, R.; Machon, O.;
Korinek, V.; Choo, E.; Diaz, D.; Merchant, M.; Polakis, P.; Holsworth,
D. D.; Krauss, S.; Costa, M. A novel tankyrase small-molecule inhibitor
suppresses APC mutation-driven colorectal tumor growth. Cancer Res.
2013, 73, 3132−44.
(16) The concentration of TNKS1 in this enzyme assay is 110 nM.
Positive control compounds reproducibly show low IC50s (see
Supporting Information). Since the true active concentration of the
enzyme is not known, these control compounds may give a better
sense of the assay sensitivity than the theoretical limit of 55 nM.
(17) Nathubhai, A.; Wood, P. J.; Lloyd, M. D.; Thompson, A. S.;
Threadgill, M. D. Design and Discovery of 2-Arylquinazolin-4-ones as
Potent and Selective Inhibitors of Tankyrases. ACS Med. Chem. Lett.
2013, 4, 1173−7.
(18) Haikarainen, T.; Koivunen, J.; Narwal, M.; Venkannagari, H.;
Obaji, E.; Joensuu, P.; Pihlajaniemi, T.; Lehtio, L. para-Substituted 2-
̈
phenyl-3,4-dihydroquinazolin-4-ones as potent and selective tankyrase
inhibitors. ChemMedChem 2013, 8, 1978−85.
(19) Lehtio, L.; Collins, R.; van den Berg, S.; Johansson, A.;
̈
Dahlgren, L. G.; Hammarstrom, M.; Helleday, T.; Holmberg-
̈
Schiavone, L.; Karlberg, T.; Weigelt, J. Zinc binding catalytic domain
of human tankyrase 1. J. Mol. Biol. 2008, 379, 136−45.
(20) Lovering, F.; Bikker, J.; Humblet, C. Escape from flatland:
increasing saturation as an approach to improving clinical success. J.
Med. Chem. 2009, 52, 6752−6.
(21) Tuncel, H.; Tanaka, S.; Oka, S.; Nakai, S.; Fukutomi, R.;
Okamoto, M.; Ota, T.; Kaneko, H.; Tatsuka, M.; Shimamoto, F.
PARP6, a mono(ADP-ribosyl) transferase and a negative regulator of
cell proliferation, is involved in colorectal cancer development. Int. J.
Oncol. 2012, 41, 2079−86.
(22) Tai, W.; Quackenbush, K.; Purkey, A.; Bagby, S.; Messersmith,
W.; Song, E.; Pitts, T.; Arcaroli, J. KRAS wild type tumors exhibit
increased efficacy to the combination of AZ1 (a tankyrase inhibitor) +
irinotecan in a patient-derived CRC explant model. In Proceedings of
the AACR-NCI-EORTC International Conference on Molecular Targets
and Cancer Therapeutics, October 19−23, 2013; AACR: Philadelphia,
PA, 2013; Vol.12 (11 Suppl), Abstract nr B30.
F
DOI: 10.1021/ml5003663
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