Development of novel dual binders as potent, selective, and orally bioavailable tankyrase inhibitors

Article abstract of DOI:10.1021/jm401317z

Tankyrases (TNKS1 and TNKS2) are proteins in the poly ADP-ribose polymerase (PARP) family. They have been shown to directly bind to axin proteins, which negatively regulate the Wnt pathway by promoting β-catenin degradation. Inhibition of tankyrases may offer a novel approach to the treatment of APC-mutant colorectal cancer. Hit compound 8 was identified as an inhibitor of tankyrases through a combination of substructure searching of the Amgen compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds for further in vivo validation studies.

Full text of DOI:10.1021/jm401317z

Article
pubs.acs.org/jmc
Development of Novel Dual Binders as Potent, Selective, and Orally
Bioavailable Tankyrase Inhibitors
Zihao Hua,*,† Howard Bregman,† John L. Buchanan,† Nagasree Chakka,† Angel Guzman-Perez,†
Hakan Gunaydin,∥ Xin Huang,∥ Yan Gu,∥ Virginia Berry,‡ Jingzhou Liu,‡ Yohannes Teffera,‡
Liyue Huang,‡ Bryan Egge,⊥ Renee Emkey,⊥ Erin L. Mullady,⊥ Steve Schneider,⊥ Paul S. Andrews,⊥
Lisa Acquaviva,§ Jennifer Dovey,§ Ankita Mishra,§ John Newcomb,§ Douglas Saffran,§ Randy Serafino,§
Craig A. Strathdee,§ Susan M. Turci,§ Mary Stanton,# Cindy Wilson,§ and Erin F. DiMauro*,†
‡
§
†Department of Chemistry Research and Discovery, Department of Pharmacokinetics and Drug Metabolism, Oncology Research,
∥Department of Molecular Structure, Bioassay and Profiling, and Pharmaceutics, Amgen Inc., 360 Binney Street, Cambridge,
⊥
#
Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Tankyrases (TNKS1 and TNKS2) are proteins
in the poly ADP-ribose polymerase (PARP) family. They have
been shown to directly bind to axin proteins, which negatively
regulate the Wnt pathway by promoting β-catenin degradation.
Inhibition of tankyrases may offer a novel approach to the
treatment of APC-mutant colorectal cancer. Hit compound 8
was identified as an inhibitor of tankyrases through a
combination of substructure searching of the Amgen
compound collection based on a minimal binding pharmacophore hypothesis and high-throughput screening. Herein we
report the structure- and property-based optimization of compound 8 leading to the identification of more potent and selective
tankyrase inhibitors 22 and 49 with improved pharmacokinetic properties in rodents, which are well suited as tool compounds
for further in vivo validation studies.
target proteins. This post-translational modification is termed
PARsylation. PARP1 and PARP2, the two most characterized
family members, have been pursued as cancer drug targets for
more than a decade, although the specific cellular functions of
many PARP proteins remain to be determined.3 Tankyrases
were first discovered as factors that regulate telomere
homeostasis by modifying the negative regulator of telomere
length, TRF1.4 Recently, tankyrases have been shown to
directly bind to and PARSylate axin proteins and have therefore
gained increased attention as potential drug targets.5 Axin, a
negative regulator of the Wnt pathway, has been reported to be
the concentration-limiting factor in regulating the efficiency of
the β-catenin destruction complex. Overexpression of axin
induces β-catenin degradation even in cell lines expressing
truncated APC.6 These findings suggest that a small molecule
inhibitor of tankyrase activity would antagonize the Wnt/β-
catenin signaling transduction pathway by stabilizing axin and
promoting β-catenin degradation and might therefore be a
useful therapeutic agent for suppressing the growth and survival
of APC mutant CRC cells.
INTRODUCTION
■
The evolutionarily conserved Wnt/β-catenin (canonical)
signaling transduction pathway plays a critical role in embryonic
development and maintenance of homeostasis in mature
tissues.1 A key feature of the canonical pathway is the regulated
proteolysis of the downstream effector β-catenin by the β-
catenin destruction complex, which consists of adenomatous
polyposis coli (APC), axis inhibition protein (axin), glycogen
synthase kinase 3β (GSK3β), and casein kinase 1α (CK1α).
The activity of the canonical pathway is dependent on the
amount of β-catenin in the cytoplasm. In the absence of Wnt
activation, the cytoplasmic β-catenin level is kept low through
phosphorylation, mediated by the destruction complex, and
subsequent ubiquitination and proteosomal degradation. Upon
Wnt stimulation, the destruction complex dissociates, resulting
in accumulation and translocation of active β-catenin to the
nucleus and transcription of Wnt pathway responsive genes.
Aberrant activation of the Wnt/β-catenin signaling pathway has
been observed in many cancers.2 Notably, most colorectal
cancers (CRCs) are initiated by mutations of APC, leading to
increased β-catenin mediated signaling.
The first reports of potent and selective small molecule
tankyrase inhibitors described two structurally distinct chemo-
types, XAV9397 and inhibitors of Wnt response (IWRs)8
Tankyrase 1 (TNKS1, PARP-5a) and tankyrase 2 (TNKS2,
PARP-5b) are members of the poly ADP-ribose polymerase
(PARP) family of 17 proteins that share a catalytic PARP
domain. PARP proteins use nicotinamide adenine dinucleotide
(NAD+) as a substrate to transfer ADP-ribose polymers onto
Received: August 26, 2013
Published: December 2, 2013
© 2013 American Chemical Society
10003
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
Figure 1. Structures of selected tankyrase inhibitors and their binding modes to TNKS1 or TNKS2 (indicated in parentheses).
Figure 2. Identification of compound 8 as a tankyrase inhibitor via substructure search and screening.
(Figure 1). XAV939 was originally developed as a weak
inhibitor of PARP1/2 (IC50 of 2.2 and 0.11 μM) but was
demonstrated to be a more potent inhibitor of TNKS1 and
TNKS2 with IC50 values of 11 and 4 nM, respectively. IWR2
inhibits TNKS1 and TNKS2 with IC50 values of 160 and 350
nM, respectively. The reported cocrystal structure of TNKS2
with XAV939 reveals ligand binding to the nicotinamide pocket
through similar interactions as observed for other PARP1/2
inhibitor complexes, with three conserved hydrogen bonds
between the ligand and the protein, via the carbonyl and NH
group of the quinazolinone moiety.9 A novel binding mode of
IWR compounds to tankyrase was discovered both internally10
different analogues of JW74 (413 and G007-LK14) were
independently disclosed as potent and selective inhibitors of
TNKS1/2. The cocrystal structures of 4 and G007-LK
complexed to TNKS1 or TNKS2 catalytic domains demon-
strate ligand binding to the induced pocket similar to the IWR
compounds, and also to a hydrophobic nook with the phenyl
ring on triazole nitrogen. Further, a recent Genentech
publication disclosed proof-of-concept antitumor efficacy with
compound G007-LK in two APC mutant CRC models in
mice.15
Toward identifying a potent and selective small molecule
inhibitor of tankyrases,16 we initiated a substructure search
based on the novel binding motif derived from the IWR2/
TNKS1 cocrystal structure. Approximately 1000 diverse
compounds possessing the hypothetical minimal binding
pharmacophore (7) were selected from the Amgen compound
collection (Figure 2). High-throughput screening of these
compounds with an enzymatic TNKS1 autoparsylation assay
identified N-(2-methoxyphenyl)-4-(3-(4-oxo-3,4-dihydroquina-
zolin-2-yl)propanamido)benzamide (8) as the most potent
inhibitor of tankyrase among them, with an IC50 value of 10
nM. Compound 8 also demonstrated potent cellular activity
(IC50 = 160 nM) in a total β-catenin (TBC) degradation assay
in SW480 cells.17
and at Akademi University, Finland, by Lehtio and co-
̈
workers11 through high-resolution cocrystal structures of the
human TNKS1 or TNKS2 catalytic domain in complex with
IWR2 or IWR1. The TNKS1/IWR2 cocrystal structure reveals
that IWR2 does not bind to the nicotinamide pocket but to an
induced pocket that becomes available only upon binding of the
IWR2 molecule. There are three hydrogen bonds between
IWR2 and TNKS1: one between a carbonyl oxygen of the
pyrrolidine dione group and the main chain NH of Tyr1213,
another one between the carbonyl oxygen of the amide and the
main chain NH of Asp1198, and one C−H···OC hydrogen
bond between the C−H at the 6-position of the quinoline and
the main chain carbonyl oxygen of Gly1196. During the course
of our investigations into the binding mode of IWRs, triazole
compound JW74 (3) was reported as an inhibitor of Wnt
signaling in a HEK293-STF assay.12 More recently, two
A cocrystal structure of compound 8 bound to TNKS1
revealed key interactions in both the induced pocket and the
nicotinamide pocket. Structural comparison suggests that 8
overlays well with both XAV939 and IWR2. This “dual-
10004
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
Figure 3. Overlay of cocrystal structures of XAV939 (PDB code 3UH4, yellow), IWR2 (PDB code 4DVI, green), and compound 8 (PDB code 4I9I,
magenta) bound to TNKS1.
binding” mode, with the quinazolinone of 8 engaging the
nicotinamide pocket and the rest of the molecule occupying the
induced pocket (Figure 3), is presumably important for both
affinity and specificity. The carbonyl oxygen of the
quinazolinone is hydrogen-bonded to the side chain OH of
Ser1221 and the main chain NH of Gly1185, while the NH of
the quinazolinone is hydrogen-bonded to the main chain
oxygen of Gly1185. The remainder of molecule 8 binds to the
induced pocket that is accessed by the IWRs (2a, 2b) and JW
type (3−5) compounds, making an additional three hydrogen
bonds to tankyrase: one between the left-hand-side amide
oxygen and the main chain NH of Tyr1213, another one
between the right-hand-side amide oxygen and the main chain
NH of Asp1198, and a final one between the terminal phenyl
group and the main chain oxygen of Gly1196 (C−H···OC
hydrogen bond). Both the methoxyphenyl group of 8 and the
quinoline group of IWR2 engage in a π−π stacking interaction
with the side chain of His1201 of the D-loop. We spectulated
that this novel “dual-binding” tankyrase inhibitor chemotype
that accesses both the nicotinamide pocket and the induced
pocket may provide advantages in terms of potency and
selectivity over similar members of the PARP family and other
NAD+-utilizing enzymes. Very recently, a tankyrase inhibitor
(6a) that binds simultaneously to the nicotinamide pocket,
induced pocket, and “nook” pocket has demonstrated in vivo
acitivty as an antagonist of Wnt/β-catenin pathway activity.
Interestingly, a truncated derivative 6b that also binds to the
nicotinamide pocket and the induced pocket demonstrated
tankyrase inhibitory activity comparable to that of compound
8.16b
the exploration of structure−activity relationships (SARs)
within this new class of tankyrase dual binders, with the goals
of improving the enzymatic and cellular potency and
physicochemical and rodent PK properties in order to achieve
adequate exposure for in vivo studies. During the course of
these studies, we utilized rational structure- and property-based
design.
CHEMISTRY
■
Our general strategy for the construction of the tankyrase
inhibitors described herein involved amide coupling of
carboxylic acid 21 with various trans-4-substituted cyclohexan-
amines. The synthesis of trans-4-(5-phenyloxadiazolyl)-
cyclohexanamine 12 is shown in Scheme 1. A solution of
trans-4-Boc-aminocyclohexanecarboxylic acid (9) was treated
with benzhydrazide in the presence of DIPEA and HATU to
Scheme 1. Synthesis of trans-4-(5-Phenyl-1,3,4-oxadiazol-2-
a
yl)cyclohexanamine (12)
Lead compound 8 suffered from poor plasma stability in
mouse (t1/2 = 40 min),18 low solubility (6.6 μg/mL in fasted
state simulated intestinal fluid (SIF) at pH 6.8), and high
clearance (CL = 10.9 L h−1 kg−1) in a rat iv pharmacokinetic
(PK) study.19 We initiated medicinal chemistry efforts toward
a
Reagents and conditions: (a) benzhydrazide, DIPEA, HATU,
CH3CN; (b) TsCl, DIPEA; (c) TFA, CH2Cl2, 88% overall yield for
three steps.
10005
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
afford the corresponding diacylhydrazide 10, which was
subsequently treated with p-toluenesulfonyl chloride to afford
the corresponding 1,3,4-oxadiazole 11. Removal of the Boc-
protecting group with trifluoroacetic acid afforded the desired
trans-4-(5-phenyloxadiazolyl)cyclohexanamine 12.
The synthesis of trans-4-phenoxycyclohexanamines is exem-
plified with trans-4-(4-fluorophenoxy)cyclohexanamine (17)
and described in Scheme 2. Treatment of phthalimide (13)
acid anhydride (T3P) and triethylamine afforded the elaborated
compound 22.
RESULTS AND DISCUSSION
■
Structure−Activity Relationship (SAR). Since a main
goal of this program is to test the tankyrase mechanism in a
mouse pharmacology model, we initially sought to improve the
mouse plasma stability of our lead compound 8 (Table 1). A
Scheme 2. Synthesis of trans-4-(4-
Fluorophenoxy)cyclohexanamine (17)
Table 1. SAR of the Right-Hand-Side Amide and Central
Phenyl Ring toward Improving Mouse Plasma Stability
a
a
Reagents and conditions: (a) ethyl chloroformate, Et3N, DMF, 0 °C
to room temperature, 50%; (b) cis-4-hydroxycyclohexamine·HCl salt,
K2CO3, H2O, 67%; (c) 4-fluorophenol, DIAD, PPh3, THF, 51%; (d)
NH2NH2, H2O, EtOH, 95%.
with ethyl chloroformate in the presence of triethylamine
followed by displacement with cis-4-hydroxycyclohexanamine
afforded the phthalimide-protected hydroxycyclohexanamine
15. Mitsunobu reaction of 15 with 4-fluorophenol using DIAD
and triphenylphosphine afforded the corresponding phenyl
ether 16. Subsequent removal of the phthalimide protecting
group with hydrazine gave the desired trans-4-(4-
fluorophenoxy)cyclohexanamine (17).
The final assembly in the synthesis of tankyrase inhibitors is
exemplified in Scheme 3 with trans-4-(5-phenyl-1,3,4-oxadiazol-
a
Scheme 3. Synthesis of Dual Binder Analogues
a,b
c
See Experimental Section for details of the assay. See ref 18 for
d
e
details of the mouse plasma stability assay. NA = not measured. Und
= undefined (greater than 50% inhibition was achieved; however,
curve fits could not be attained with acceptable confidence intervals).
a
metabolite identification (MetID) study of 8 in fresh mouse
plasma indicated that hydrolysis of the two amides contributed
to the plasma instability, with the right-hand-side amide as the
major hydrolytic liability. We first introduced a methyl group to
the ortho position of each of the two aryl rings flanking the
right-hand-side amide in an attempt to sterically block the
amide hydrolysis. A methyl group at the ortho position of the
terminal aryl ring (23) improved the plasma stability
(increasing the half-life in mouse plasma by 8-fold), but the
enzymatic potency on tankyrase decreased by 175-fold. A
methyl group at the ortho position of the central aryl ring (24)
led to a 21-fold decrease in tankyrase potency and comparable
plasma instability to des-methyl lead 8. Methylation of the
Reagents and conditions: (a) K2CO3, acetone, 60 °C, 34%; (b) TFA,
CH2Cl2, 95%; (c) trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
cyclohexanamine (12), T3P, Et3N, DMF, room temperature, 51%.
2-yl)cyclohexanamine (12) as the right-hand-side motif. A
mixture of 2-mercaptoquinazolinone (18) and tert-butyl 3-
bromopropanoate (19) in acetone was refluxed in the presence
of potassium carbonate to afford the desired quinazolinone 20.
Removal of the tert-butyl group with trifluoroacetic acid gave
the corresponding carboxylic acid 21. Treatment of carboxylic
acid 21 with trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
cyclohexanamine (12) in the presence of propylphosphonic
10006
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
right-hand-side amide nitrogen resulted in compound (25)
with 40-fold loss of potency on tankyrase and no gain in plasma
stability. Replacement of this right-hand-side amide with a
sulfonamide (26) resulted in a dramatic loss of potency on
tankyrase. We next examined a number of five-membered
heteroaryl groups, directly fused to phenyl, as potential
replacements of the right-hand-side amide functionality.
Analogues with indazole (27), benzimidazole (28), and
benzoxazole (29) offered excellent stability in mouse plasma
but significantly decreased potency on tankyrase with IC50
values ranging from 0.40 to 1.09 μM. In order to maintain the
excellent plasma stability demonstrated by these heteroaryla-
mide replacements and improve the tankyrase activity, we
explored analogues with a direct bond to the terminal phenyl
group rather than a ring fusion to the phenyl group. Indeed,
molecular modeling suggested that a terminal phenyl group
bound directly to a five-membered heteroaryl ring might be
appropriately positioned to engage in a π−π stacking,
hydrophobic interaction with the side chain of His1201 of
the D-loop. Among the five-membered heteroaryls examined
(imidazoles 30 and 31, thiazole 32, 1,2,4-oxadiazole 33, and
1,3,4-oxadiazole 34), only 1,3,4-oxadiazole 34 demonstrated
good potency on tankyrase (IC50 = 63 nM) and showed
improved stability in mouse plasma (t1/2 = 122 min). The
cocrystal structure of 34 bound to TNKS1 revealed a similar
“dual-binding” mode as observed for compound 8 (Figure 4a).
Structural comparison of 8/TNKS1 and 34/TNKS1 revealed
that 34 and 8 overlay very well, with the quinazolinone of both
34 and 8 occupying the nicotinamide pocket and the rest of the
molecules occupying the induced pocket. One of the nitrogen
atoms from the oxadiazole of 34 forms a hydrogen bond to the
main chain NH of Asp1198, similar to the right-hand-side
amide oxygen of 8. The terminal phenyl ring of 34 also engages
in a π−π stacking interaction with the side chain of His1201 of
the D-loop, as hypothesized with modeling.
Moving next to address the mouse plasma instability by
modulating the electronic property of the central phenyl group,
we discovered that simple saturation of the central phenyl ring
of compound 8 significantly improved the stability in mouse
plasma (35, 36). The resulting trans-cyclohexyl compound 35
showed only slightly decreased potency relative to lead
compound 8 and 240-fold greater potency than the
corresponding cis-cyclohexyl stereoisomer 36. The combination
of trans-cyclohexyl as the central ring and phenyl 1,3,4-
oxadiazole as a right-hand-side amide surrogate resulted in
compound 37, which exhibited good enzymatic potency on
tankyrase (IC50 = 50 nM) and significantly improved stability in
mouse plasma (t1/2 > 500 min) relative to 34. Furthermore, it
was devoid of the aniline toxicophore present in 35. This
compound also demonstrated good cellular potency, inhibiting
total β-catenin levels in SW480 cells with an IC50 value of 80
nM.
With the good biological potency and plasma stability
demonstrated by compound 37 having a trans-cyclohexyl group
as the central ring and a phenyl-1,3,4-oxadiazole on the right-
hand side, subsequent SAR studies were focused on
modification of the left-hand-side quinazolinone moiety,
which binds in the nicotinamide pocket and the ethylene
linker between the quinazolinone (nicotinamide pocket binding
motif) and the left-hand-side amide (induced pocket binding
motif) (Table 2). Modification of this region was expected to
improve the potency by aligning the left-hand side of the ligand
to make improved binding contacts in and around the
Figure 4. Overlays of cocrystal structures of TNKS1/TNKS inhibitor
complexes: (a) structure overlay of 8 (magenta) and 34 (PDB code
4MSK, cyan); (b) structure overlay of 34 (cyan) and 22 (PDB code
4MSG, gray); (c) structure overlay of 22 (gray) and 49 (PDB code
4MT9, peach).
nicotinamide pocket. Replacing the phenyl group of the
quinazolinone with a pyridine (38) resulted in a 15-fold loss
of activity in both enzyme and cell assays. Saturation of the
pyridine ring gave a compound (39) that is similarly active on
tankyrase relative to 37 but much less potent in the cellular
assay. Although the introduction of a polar piperidine (40) or
pyrrolidine (41) ring to replace the linear ethylene linker
showed slightly improved enzymatic activity and increased
solubility relative to 37, these analogues suffered a 5- to 10-fold
decrease in cellular potency and rat liver microsomal (RLM)
stability.20 Further fine-tuning of the linear linker to better
access both pockets was anticipated to afford increased
biological activity. Replacing the benzylic carbon with a sulfur
atom (42) showed 3-fold increased tankyrase activity and
10007
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
D-loop, consistent with the observed increase in potency. The
conformation of the sulfur ethylene linker on 22 is quite
different from the close structural analogue 43 (three-carbon
linker). The C−S−C bond angle in 22 is 104° while the C−C−
C bond angle in 43 is 116°, which allows better hydrophobic
interaction for 22 with the side chain of Phe1208 of the D-loop.
In addition, the sulfur atom of 22 engages in a favorable S···
OC interaction22 with the backbone carbonyl of Gly1185
and positions the amide carbonyl and the oxadiazole groups of
22 for engaging in more favorable H-bonding interactions with
the backbone NHs of Tyr1213 and Asp1198, respectively.
These two distances are approximately 2.8 and 2.9 Å for 22 and
2.9 and 3.1 Å for 43. Furthermore, a dihedral scan calculation
suggested that the three-atom linker with sulfur at the benzylic
position has a strong preference for planar geometry and 22
binds with a geometry (6° away from planarity) that is very
close to the preferred geometry.23
Table 2. SAR of the Nicotinamide Pocket Binding Motif and
Linker
Although compound 22 demonstrated excellent enzymatic
and cellular potency, we sought to develop a compound with
similar biochemical potency and improved physicochemical and
pharmacokinetic properties in rodents for extensive in vivo
studies. Toward this end, we focused on replacing the right-
hand-side phenyl-1,3,4-oxadiazole moiety with a smaller group
that maintained the key hydrogen bonding and hydrophobic
interactions with the induced pocket. Molecular modeling
suggested that a simple phenoxy group might be sufficient to
replace the phenyl-1,3,4-oxadiazole moiety with maintenance of
the π−π stacking interaction with the side chain of His1201 of
the D-loop. Thus, a diverse series of phenoxy and pyridinyloxy
compounds were prepared and tested in both enzymatic and
cellular assays (Table 3). Not surprisingly, all the phenoxy and
a
b
NA = not measured. Rat liver microsomal intrinsic clearance.21 CLint
in microsomal stability was measured using the substrate depletion
method. Metabolism was initiated by addition of test compounds to
the prewarmed (37 °C) incubation mixture (0.25 mg of microsomal
protein/mL, 1 mM NADPH, 2 mM magnesium chloride in 50 mM
potassium phosphate buffer, pH 7.4) to achieve a final concentration
of 1 μM. Disappearance of parent was determined after 30 min of
incubation at 37 °C.
Table 3. SAR of Right-Hand-Side Phenoxy Group To
Replace the Phenyl-1,3,4-oxadiazole
similar potency in the cellular assay relative to 37.
Homologation of the linker by one methylene resulted in
compound 43 showing a 8-fold increase of tankyrase activity
relative to 42 and an IC50 of 17 nM in the cellular assay.
Further elongation of the linker by replacing the benzylic
carbon with a sulfur atom gave compound 22, with excellent
potency in both tankyrase (IC50 = 0.1 nM) and cellular (TBC
IC50 = 4 nM) assays. Further homologation of the linker
resulted in compound 44, which exhibited significantly
decreased potency in enzyme and cell, lower solubility, and
reduced microsomal stability. Partial saturation of the
quinazolinone of 22 afforded compound 45 with similar
enzymatic potency, aqueous solubility, and microsomal stability
but slightly decreased cellular potency relative to 22. Removal
of the quinazolinone phenyl ring resulted in compound 46,
leading to a substantial loss in tankyrase activity and cellular
potency.
The cocrystal structure of 22 and TNKS1 revealed binding to
both the nicotinamide pocket and the induced pocket, similar
to 34 (Figure 4b). The central cyclohexyl ring of 22 and the
central phenyl ring of 34 occupy the same spatial pocket but are
oriented almost perpendicular to each other. More importantly,
the sulfur ethylene linker of 22 is engaging in additional
hydrophobic interactions with the side chain of Phe1208 of the
pyridinyloxy compounds showed excellent inhibitory potency
on tankyrase, similar to 22. Unfortunately, most of the phenoxy
compounds suffered from a 4- to 30-fold loss in cellular
potency relative to 22, with the exceptions of 49, 50, and 55,
which emerged as new leads.
To understand the structural basis for the excellent
enzymatic potency observed with these phenoxy compounds,
a cocrystal structure of compound 49 with TNKS1 was
10008
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
obtained and compared with the cocrystal structure of 22 with
TNKS1. As shown in Figure 4c, the quinazolinone ring of 49
binds in the nicotinamide pocket and overlays very well with
the quinazolinone ring of 22. The phenoxy oxygen of 49 forms
a hydrogen bond with Asp1198, similar to the hydrogen bond
formed by one of the nitrogen atoms from the oxadiazole of 22.
The terminal phenyl ring of 49 also engages in a π−π stacking
interaction with the side chain of His1201 of the D-loop.
To identify a compound for in vivo studies in rodents,
selected phenyloxadiazole compounds (22, 45) and phenoxy
compounds (49, 50, and 55) were further characterized with
enzymatic TNKS2 autoparsylation assay and additional func-
tional cellular assays: axin2 accumulation in SW480 cells, to
evaluate the compound’s ability to cause stabilization and
accumulation of axin protein levels, and Super-Topflash (STF)
reporter transcription in DLD-1 cells, a second colorectal
cancer cell line with truncated APC, to evaluate the compound’s
downstream inhibitory activity on Wnt-associated gene tran-
scription (Table 4). All of the selected compounds showed
and 55). The PK data for intravenous (iv) dosing in rat (0.5
mg/kg) and oral (po) dosing in mouse (30 mg/kg) are shown
in Table 5. Phenyloxadiazole compound 22 and phenoxy
compounds 49 and 50 all displayed low to moderate clearance,
small volume of distributions, and short (50) to moderate (22,
49) half-lives in rats.24 Upon oral administration of a single
dose of 30 mg/kg to mice, phenyloxadiazole compound 22
exhibited significantly higher oral total and free exposure than
the close structural analogue 45. Among the three phenoxy
compounds, 49 showed higher oral free exposure in mice than
50 and 55. In light of their good oral exposure and excellent
cellular activity, we decided to progress phenyloxadiazole
compound 22 and phenoxy compound 49 to further in vitro
selectivity profiling and in vivo pharmacodynamic profiling in
tumor-bearing mice.
Selectivity Profile. Prior to in vivo testing, compounds 22
and 49 were screened for inhibitory activity against PARP1/2
and an extensive panel of kinases. Both compounds showed
excellent selectivity (>10 000-fold) over PARP1/2.26 Com-
pounds 22 and 49 showed less than 50% inhibition when tested
at 1.0 μM across a panel of 100 kinases which were chosen to
cover the diversity of the kinome.27
Table 4. Profile of Selected Compounds in Additional
Functional Cellular Assays
Pharmacodynamic Profile. Both 22 and 49 were
evaluated for Wnt-pathway specific pharmacological activity in
mouse tumor pharmacodynamic (PD) models. Upon once
daily oral administration (at 10 and 50 mg/kg) to mice (n = 4)
bearing human DLD-1 tumors for 3 days, both compounds
exhibited statistically significant, dose-dependent axin2 accu-
mulation (2.1- to 3.5-fold) and inhibition of STF (51−79%) at
day 3 (24 h after the last dose) (Figures 5 and 6). Terminal
TNKS1
IC50
TNKS2
IC50
SW480-TBC SW480-axin2 DLD1-STF
compd
(nM)
(nM)
IC50 (nM)
EC50 (nM)
IC50 (nM)
a
34
37
22
45
49
50
55
63
13
410
80
2450
640
3.9
NA
a
50
14
NA
0.1
0.2
0.1
0.2
0.2
4.1
2.5
7.6
6.2
0.4
3.7
11
0.6
1.3
0.3
0.7
1.1
14
1.9
6.9
8.1
4.0
6.8
8.4
a
NA = not measured.
good potency in the TNKS2 enzymatic assay, which is desirable
because of possible redundancy between these isoforms. Early
lead compounds (34 and 37) with a two-carbon linker, being
less potent in the enzyme assay, consistently displayed only
modest potency in all the cellular assays. Lead-optimized
phenyloxadiazole compounds (22 and 45) and phenoxy
compounds (49, 50, and 55) having good enzymatic potency
and cellular potency in the SW480-TBC cellular assay
demonstrated excellent potencies in TNKS2 autoparsylation
assay and the two additional functional cellular assays.
Pharmacokinetic Profile. Having identified leads showing
excellent in vitro tankyrase cellular inhibitory activity, we
carried out in vivo PK evaluations of phenyloxadiazole
compounds (22 and 45) and phenoxy compounds (49, 50,
Figure 5. Compounds 22 and 49 stabilize axin2 in a DLD-1 mouse
tumor PD model (3 days of q.d. dosing at 10 and 50 mg/kg).
a
b
Table 5. Mean PK Parameters after Intravenous Dose (iv) in Rat or Oral Dose (po) in Mouse
rat iv (0.5 mg/kg)
Vss (L kg−1
0.70
mouse po (30 mg/kg)
d
compd
CL (L h−1 kg−1
0.83
)
)
t1/2 (h)
AUC0−∞ (μM h)
Cmax (μM)
tmax (h)
mouse fu
22
45
49
50
55
1.8
49.5
5.3
18.7
3.6
0.58
3.23
1.50
0.67
1.58
0.017
0.012
0.006
0.032
0.002
c
c
c
NA
NA
NA
0.12
0.66
0.32
0.40
2.8
0.7
141.0
11.5
100.0
24.2
7.0
c
c
c
NA
NA
NA
15.1
a
b
Male rat. Male CD-1 mice; 10% Pluronic F68, 30% HPBCD, 60% water; pH adjusted to 10.0 with sodium hydroxide for 22, 45, 49, and 50, pH
c
d
adjusted to 2.5 with methanesulfonic acid for 55. NA = not measured. Fraction unbound in mouse plasma was determined in duplicate by
equilibrium dialysis using the rapid equilibrium dialysis device. Plasma was spiked with test compounds at a final concentration of 5 μM and dialyzed
against plasma water (ultrafiltrate) for 5 h in a CO2 incubator at 37 °C.25
10009
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
temperature. The assay was then stopped with the above assay buffer
containing a 0.6 μM inhibitor and the following detection
components: 0.05 μg/mL monoclonal anti-PAR antibody (Trevigen
catalog no. 4335-MC-01K-AC) prebound for 60 min with 0.63 μg/mL
protein G AlphaLisa acceptor bead (Perkin-Elmer catalog no.
AL102M) and 5 μg/mL AlphaLisa nickel chelate donor bead
(Perkin-Elmer catalog no. AS101M). The sample was incubated for
16 h at room temperature in the dark, and results were read on a
Perkin-Elmer Envision multilabel reader using the default program set
with laser excitation at 680 nm and emission at 615 nm. Assay
standards were the following: 66 runs with standard which yielded a
mean of 0.0086 μM with a standard deviation of 0.00038; 42 runs with
a different standard which yielded a mean of 0.0013 μM with a
standard deviation of 0.000 64.
Total β-Catenin (TBC) Assay. TNKS inhibition results in
degradation of the total pool of β-catenin in SW-480 cells colorectal
cells. SW480 cells do not express E-cadherin and thus do not have
membrane associated (“non-signaling”) β-catenin which interferes
with Wnt pathway activity analysis. Cells were seeded at 10 000/well
in CellBIND 96-well in 60 μL of normal growth medium (MEMα
supplemented with 10% heat inactivated FBS, GlutaMAX, pyruvate,
and 10 mM HEPES). A 10-point, 3-fold dilution series for each TNKS
inhibitor was constructed, and 20 μL of each diluted compound was
transferred to the plated SW-480 cells (resulting in a final vehicle
(DMSO) concentration of 0.1%). The plates were incubated at 37 °C
for 40−48 h, after which the medium was removed and the cells were
lysed with 75 μL/well MSD lysis buffer. A goat anti-rabbit MSD plate
(catalog no. L41RA-1) was coated with 25 μL of 5 μg/mL of Cell
Signaling antitotal β-catenin polyclonal (catalog no. 9562, lyophilized,
carrier-free special order) and incubated overnight in a cold room with
gentle shaking. The plate was then blocked with 150 μL of blocker “A”
per well and washed 4 times with 150 μL/well TBS-T wash buffer
(150 mM NaCl, 50 mM Tris, pH 7.5, 0.02% Tween-20). Cell lysates
(75 μL) were transferred to prepared MSD plates and incubated at 4
°C overnight with gentle shaking. The next day MSD plates were
washed with TBS-T wash buffer and the antitotal β-catenin mAb
(catalog no. 610153) detection antibody conjugated to SULFO-TAG
was added. The detection antibody was incubated for 1 h at room
temperature with vigorous shaking, after which plates were washed and
processed for analysis by the addition of 150 μL/well MSD read 4×
buffer T with surfactant. The plates were read, and the data were
captured on the SECTOR imager 6000. Assay standards were the
following: 54 runs with a standard which yielded a mean of 0.157 μM
with a standard deviation of 0.116; 38 runs with another standard
which yielded a mean of 0.063 μM with a standard deviation of 0.041.
Axin2 Accumulation Assay. TNKS inhibition results in axin2
accumulation into distinct cytoplasmic foci which were visualized and
quantified using a high-content imaging system (Cellomics Array-
Scan). SW480 cells were grown under normal culture conditions
(RPMI 1640, 10% HI FBS, and 1× sodium pyruvate). On the day of
the assay, cells were plated at 2500 cells per well in 60 μL of assay
medium in Perkin-Elmer Black 384 ViewPlates (Fisher, no.
509052489). TNKS compounds were diluted to generate a 22-point
dose titration in medium and incubated with the cells for 24 h at 37
°C, 5% CO2. The next day the cells were fixed for 15 min in 4%
paraformaldehyde and 0.1% Triton, washed in PBS, and blocked in
PBS with 0.1% Tween-20 and 1% normal goat serum. The cells were
stained with an axin2 primary antibody (Sigma, no. SAB1100677-
200UL) at 1:10000 overnight at 4 °C and an Alexa 488-labeled
secondary antibody (Invitrogen, A11008). The nuclei were visualized
with Hoechst dye. The axin2 foci were quantified on the Cellomics
ArrayScan (a variation of the compartmental analysis protocol was
optimized, and data were analyzed using MEAN_RingSpotAvgIn-
tenCh2), and EC50 values were calculated. Assay standards were the
following: 18 runs with a standard which yielded a mean of 0.311 μM
with a standard deviation of 0.173. Eighteen runs with another
standard which yielded a mean of 0.114 μM with a standard deviation
of 0.065.
Figure 6. Compounds 22 and 49 inhibit STF in a DLD-1 mouse
tumor PD model (3 days of q.d. dosing at 10 and 50 mg/kg).
plasma samples (24 h after last dose) were collected to estimate
compound exposure. For the 50 mg/kg dose group, mean
unbound plasma concentrations were 13 and 9 nM for 22 and
49, respectively. These concentrations are 2- to 3-fold over the
respective in vitro axin2 EC50 values (4 nM for both
compounds) and 22- and 30-fold over the respective STF
inhibition IC50 values (0.6 nM for 22, 0.3 nM for 49).
SUMMARY AND CONCLUSION
■
Substructure searching of the Amgen collection, based on a
minimal binding pharmacophore hypothesis, resulted in the
identification of hit compound 8, which was determined by X-
ray crystallography to interact with tankyrase in a novel “dual-
binding” fashion, occupying both the nicotinamide pocket
(similar to XAV939) and the induced pocket (similar to IWRs
and JWs). Our investigation of SAR made use of both
structure-based and physicochemical-property-based optimiza-
tion, with guiding data from X-ray crystallography and
pharmacokinetic analysis. These efforts led to the identification
of oxadiazole 22 and phenyloxy 49, which showed excellent in
vitro potency as measured by the inhibition of tankyrase 1 and
2, total β-catenin reduction or degradation, STF gene
transcription, and axin accumulation in SW480 and DLD-1
cells. Furthermore, leads 22 and 49 demonstrated oral activity
in a mouse DLD-1 tumor PD model of axin accumulation and
ex vivo STF inhibition.
The leads presented herein are expected to be of
considerable utility in future studies aimed at elucidating the
potential of selective pharmacological tankyrase inhibition in
cancer therapy. Specifically, several analogues from this novel
dual-binder class are extremely potent and selective and
therefore anticipated to be of value in the search for
antiproliferative activity in various cancer cell lines. Orally
bioavailable leads 22 and 49 are particularly suited for the
exploration of extended dosing studies in rodents, for
understanding the suppression of growth and survival of
tumors and overall toxicological profiles.
EXPERIMENTAL SECTION
■
Tankyrase 1 and 2 Assays. The tankyrase 1 biochemical activity
of the compounds was assayed in the following assay buffer (50 mM
MOPS, pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20,
0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6× HIS-tankyrase
1 (no. 1091-1325) was incubated in the presence of compound
(DMSO 1.85% final) in a Perkin-Elmer 384-well Proxiplate Plus
(catalog no. 6008289) with 400 nM NAD for 60 min at ambient
DLD-1 STF Assay. Constitutively Activated Wnt Pathway Reporter
Assay (APC Mutant Cancer Cell Context). DLD-1 colorectal cells
10010
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
engineered with an 8× TCF promoter-driven firefly (FF) luciferase
gene (Wnt reporter) along with an EF1a promoter-driven Renilla
(RN) luciferase gene (control reporter) were used to measure the
potency of tankyrase compounds in the context of the constitutively
activated Wnt pathway due to mutated APC in colorectal cancer cells.
The engineered DLD-1 cells were plated at a density of 10 000 cells/
well in black, clear-bottom, 96-well View plates (PerkinElmer) in
normal growth medium (RPMI with 10% FBS with no antibiotics).
Tankyrase inhibitors were transferred to cells from a 3-fold serially
diluted compound plate. A 10-point dilution series was tested starting
at 10 μM. The plates were incubated at 37 °C for 40−48 h. The Dual-
Glo reagents (Promega) were added as directed by the manufacturer
to assess the firefly (FF) and Renilla (RN) luciferase activity. Luciferase
activity was measured using the EnVision multilabel plate reader
(PerkinElmer).
DLD-1 Tumor PD in Mouse. Subcutaneous tumors were formed
in athymic nude mice using DLD-1 human colon cancer cell line that
had been engineered to express firefly luciferase under a Wnt-
responsive SuperTopFlash (STF) promoter and Renilla luciferase
under a constitutive promoter. Once tumors reached approximately
250 mm3, tumor bearing animals were randomized into groups of 4
and then animals were dosed for 3 days with either compound or
vehicle orally q.d. Tumors for protein analysis and plasma for PK were
collected 14 h after the last dose. Quantitation of axin2 protein was
done using an MSD assay (MesoScale Discovery) using an axin2
antibody from Genetex for capture and a sulfotagged antibody from
R&D Systems for detection. STF transcription assay was done using
the Dual-Glo luciferase assay from Promega, and firefly luciferase
values were normalized using the Renilla luciferase values.
Chemistry. General. Unless otherwise noted, all materials were
obtained from commercial suppliers and used without further
purification. Anhydrous solvents were obtained from Aldrich and
used directly. All reactions involving air- or moisture-sensitive reagents
were performed under a nitrogen or argon atmosphere. Purity (3 min
methods only) and reaction analyses were measured using Agilent
1100 series high performance liquid chromatography (HPLC) systems
with UV detection at 254 and 215 nm (system A consisting of Agilent
Zorbax SB-C18 3.0 mm × 50 mm, 3.5 μm, 5−95% CH3CN in H2O
with 0.1% TFA for 3.6 min at 1.5 mL/min or Halo Phenyl-Hexyl, 3
mm × 50 mm, 2.7 μm, 5−95% CH3CN in H2O with 0.1% TFA for
1.01 min at 2.0 mL/min; system B consisting of Waters Xbridge C18,
3 mm × 50 mm, 3.5 μm, 5−95% CH3CN in H2O with 0.1% formic
acid for 3.6 min at 1.5 mL/min). Silica gel chromatography was
generally performed with prepacked silica gel cartridges (Biotage,
Teledyne-Isco or Interchim). Library purification methods were as
follows: preparative LC/MS, Waters autopurification system; liquid
transfer system, Tecan; drying system, Genevac; preparative column,
Xbridge (19 mm × 100 mm, C18, 10 μm); flow rate, 40 mL/min;
general gradient, 5−95% B, 0.1% additive in both A and B; 10 min
gradient; mobile phase A, water; mobile phase B, acetonitrile; additive,
TFA or NH4OH. 1H NMR spectra were recorded on a Bruker AV-400
(400 MHz) spectrometer or a Varian 400 MHz spectrometer at
ambient temperature. All observed protons are reported as parts per
million (ppm) downfield from tetramethylsilane (TMS) or other
internal reference in the appropriate solvent indicated. Data are
reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling
constants, and number of protons. Low-resolution mass spectrometry
(MS) data were determined on an Agilent 1100 series LCMS with UV
detection at 254 and 215 nm and a low resonance electrospray mode
(ESI). All the compounds were determined to be ≥95% purity by
HPLC.
and the mixture was stirred at room temperature for 24 h. The
reaction mixture was then poured into 14% aqueous NH3 (20 mL),
stirred for 15 min, then transferred to a separatory funnel and diluted
with DCM and water. The organic layer was separated, and the
aqueous layer was extracted with DCM. The combined organic layers
were washed with brine, dried over anhydrous sodium sulfate, filtered,
and concentrated. The crude reaction mixture was purified (ISCO 12 g
column, 0−85% EtOAc−heptanes). The product-containing fractions
were concentrated and loaded onto a 70 mL SCX-2 column with
MeOH−DCM and eluted with 2.0 M NH3 in MeOH. The crude tert-
butyl (trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)carbamate
(11) was taken directly onto the next step without further purification.
To a solution of crude tert-butyl trans-4-(5-phenyl-1,3,4-oxadiazol-2-
yl)cyclohexylcarbamate (11) in DCM (10 mL) at room temperature
was added trifluoroacetic acid (0.98 mL, 12.7 mmol). The resulting
mixture was stirred at room temperature for 19 h. The mixture was
concentrated and loaded onto a 70 mL SCX-2 column with MeOH−
DCM and eluted with 2.0 M NH3 in MeOH to afford trans-4-(5-
1
phenyl-1,3,4-oxadiazol-2-yl)cyclohexanamine (0.45 g, 88%). H NMR
(400 MHz, DMSO-d6) δ 7.95−8.05 (m, 2 H), 7.53−7.68 (m, 5 H),
2.94−3.15 (m, 2 H), 2.15−2.29 (m, 2 H), 2.02−2.10 (m, 2 H), 1.67
(dq, J = 3.2, 12.9 Hz, 2 H), 1.47 (dq, J = 3.3, 12.5 Hz, 2 H); m/z (ESI)
244.2 (M + H)+.
Ethyl 1,3-Dioxoisoindoline-2-carboxylate (14). To a solution
of phthalimide (2.00 g, 13.6 mmol) and triethylamine (2.75 g, 27.2
mmol) in DMF (10 mL) at 0 °C was added ethyl chloroformate (1.30
mL, 13.6 mmol) dropwise over a period of 0.5 h. The reaction mixture
was allowed to warm to ambient temperature and further stirred for 2
h. After completion of the reaction, the reaction mixture was treated
with ice−water, resulting in precipitation of a white solid. The
precipitate was filtered and washed with cold water to obtain crude
ethyl 1,3-dioxoisoindoline-2-carboxylate (1.50 g, 50%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 7.86−7.99 (m, 4 H), 4.53 (q, J = 7.2
Hz, 2 H), 1.45 (t, J = 7.2 Hz, 3 H).
2-(trans-4-(4-Fluorophenoxy)cyclohexyl)isoindoline-1,3-
dione (16). To a solution of crude ethyl 1,3-dioxoisoindoline-2-
carboxylate (0.40 g, 1.8 mmol) and cis-4-aminocyclohexanol hydro-
chloride (0.28 g, 1.8 mmol) in water (4 mL) at 0 °C was added
potassium carbonate (0.76 g, 5.5 mmol) portionwise. After 0.5 h, the
reaction mixture was allowed to warm to ambient temperature and
stirred further for 1 h. The resulting precipitate was filtered and dried
in air to obtain crude 2-(cis-4-hydroxycyclohexyl)isoindoline-1,3-dione
(15) (0.30 g, 67%) as a white solid.
To a mixture of 2-(cis-4-hydroxycyclohexyl)isoindoline-1,3-dione
(15) (4.0 g, 16.3 mmol), 4-fluorophenol (2.2 g, 19.5 mmol), and
triphenylphosphine (6.9 g, 26.1 mmol) in anhydrous THF (50 mL was
added DIAD (5.3 g, 26.1 mmol) dropwise, and the reaction mixture
was stirred at ambient temperature for 12 h. The reaction was
quenched with water, and the aqueous portion was extracted with
ethyl acetate. The combined organic layers were washed with brine
and dried over anhydrous sodium sulfate. The solid was filtered and
the solution was concentrated under reduced pressure. The crude
material was purified by chromatography with 60−120 mesh silica
column chromatography using 6% EtOAc in petroleum ether as eluent
to obtain 2-(trans-4-(4-fluorophenoxy)cyclohexyl)isoindoline-1,3-
1
dione (2.8 g, 51%) as a yellow thick liquid. H NMR (400 MHz,
CDCl3) δ 7.84 (dd, J = 3.2, 5.7 Hz, 2 H), 7.72 (dd, J = 3.2, 5.7 Hz, 2
H), 6.99−6.95 (m, 2 H), 6.89−6.85 (m, 2 H), 4.23−4.20 (m, 2 H),
2.40−2.37 (m, 2 H), 2.28−2.24 (m, 2 H), 1.85−1.81 (m, 2 H), 1.58−
1.55 (m, 2 H); m/z (ESI) 340.2 (M + H)+.
trans-4-(4-Fluorophenoxy)cyclohexanamine (17). To a sol-
ution of 2-(trans-4-(4-fluorophenoxy)cyclohexyl)isoindoline-1,3-dione
(2.8 g, 8.2 mmol) in ethanol (20 mL), was added hydrazine hydrate
(1.2 mL, 24.6 mmol), and the reaction mixture was stirred at ambient
temperature for 12 h. After completion of the reaction, the resulting
precipitate was removed by filtration and the filtrate was dissolved in
5% aqueous HCl (10 mL) and treated with DCM (10 mL). The layers
were separated, and the aqueous layer was further basified to pH ≈ 8−
9 by using 10% NaHCO3 solution. After basification, the aqueous layer
was extracted with DCM and the combined extracts were concentrated
trans-4-(5-Phenyl-1,3,4-oxadiazol-2-yl)cyclohexanamine
(12). To a mixture of trans-4-((tert-butoxycarbonyl)amino)-
cyclohexanecarboxylic acid (0.52 g, 2.12 mmol) and benzhydrazide
(0.29 g, 2.1 mmol) in acetonitrile (10 mL) at room temperature was
added N,N-diisopropylethylamine (1.1 mL, 6.4 mmol), followed by
HATU (0.89 g, 2.3 mmol). The resulting mixture was stirred at room
temperature for 19 h. Then extra N,N-diisopropylethylamine (0.4 mL)
was added followed by p-toluenesulfonyl chloride (1.21 g, 6.4 mmol),
10011
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
to give trans-4-(4-fluorophenoxy)cyclohexanamine (1.5 g, 95%) as a
9.0 Hz, 2H), 7.61 (d, J = 8.9 Hz, 2H), 7.52 (d, J = 7.8 Hz, 1H), 7.45 (t,
J = 7.6 Hz, 1H), 7.19 (dd, J = 1.6, 7.8 Hz, 1H), 7.00−7.12 (m, 1H),
6.77−6.94 (m, 2H), 3.50 (s, 3H), 2.94 (d, J = 5.7 Hz, 2H), 2.90 (d, J =
6.1 Hz, 2H); m/z (ESI) 479.0 (M + H)+.
1
colorless oil. H NMR (400 MHz, CDCl3) δ 6.92−7.01 (m, 2H),
6.79−6.89 (m, 2H), 4.09 (tt, J = 4.1, 10.4 Hz, 1H), 2.82 (tt, J = 4.0,
10.6 Hz, 1H), 2.04−2.19 (m, 2H), 1.90−2.04 (m, 2H), 1.41−1.58 (m,
2H), 1.10−1.35 (m, 3H); m/z (ESI) 210.2 (M + H)+.
N-(4-(1-Methyl-1H-indazol-3-yl)phenyl)-3-(4-oxo-3,4-dihy-
droquinazolin-2-yl)propanamide (27). 1H NMR (400 MHz,
DMSO-d6) δ 12.27 (s, 1H), 10.22 (s, 1H), 8.02−8.16 (m, 2H), 7.91
(d, J = 8.9 Hz, 2H), 7.71−7.81 (m, 3H), 7.68 (d, J = 8.5 Hz, 1H), 7.58
(d, J = 7.7 Hz, 1H), 7.40−7.51 (m, 2H), 7.17−7.27 (m, 1H), 4.09 (s,
3H), 2.95 (dd, J = 6.2, 17.8 Hz, 4H); m/z (ESI) 424.5 (M + H)+.
N-(4-(1-Methyl-1H-benzo[d]imidazol-2-yl)phenyl)-3-(4-oxo-
3,4-dihydroquinazolin-2-yl)propanamide (28). 1H NMR (400
MHz, DMSO-d6) δ 10.59 (s, 1H), 8.04−8.18 (m, 1H), 8.00 (d, J = 7.1
Hz, 1H), 7.87−7.97 (m, 4H), 7.71−7.87 (m, 2H), 7.52−7.68 (m, 3H),
7.48 (ddd, J = 1.2, 7.1, 8.0 Hz, 1H), 4.03 (s, 3H), 3.00 (t, J = 3.3 Hz,
4H); m/z (ESI) 424.5 (M + H)+.
N-(4-(Benzo[d]oxazol-2-yl)phenyl)-3-(4-oxo-3,4-dihydroqui-
nazolin-2-yl)propanamide (29). 1H NMR (400 MHz, DMSO-d6) δ
8.09−8.18 (m, 2H), 7.94−8.07 (m, 3H), 7.78 (dtd, J = 1.7, 3.1, 4.6 Hz,
2H), 7.52 (t, J = 7.5 Hz, 1H), 7.34−7.46 (m, 3H), 7.15−7.26 (m, 1H),
2.84−2.94 (m, 2H), 2.73−2.84 (m, 2H); m/z (ESI) 411.3 (M + H)+.
3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N-(4-(2-phenyl-1H-
imidazol-5-yl)phenyl)propanamide (30). 1H NMR (400 MHz,
DMSO-d6) δ 12.29 (br s, 1H), 10.29 (s, 1H), 8.02−8.14 (m, 4H),
7.81−7.88 (m, 2H), 7.78 (ddd, J = 1.6, 7.0, 8.3 Hz, 1H), 7.70−7.75
(m, J = 8.6 Hz, 2H), 7.63 (d, J = 7.5 Hz, 3H), 7.57 (d, J = 7.6 Hz, 1H),
7.43−7.51 (m, 1H), 2.95 (dd, J = 5.3, 13.4 Hz, 4H); m/z (ESI) 437.1
(M + H)+.
3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N-(4-(2-phenyl-1H-
imidazol-5-yl)phenyl)propanamide (31). 1H NMR (400 MHz,
DMSO-d6) δ 10.21 (s, 1H), 8.10 (dd, J = 1.2, 7.9 Hz, 1H), 7.89−7.94
(m, 2H), 7.85 (dd, J = 1.2, 8.3 Hz, 2H), 7.75−7.79 (m, 1H), 7.72 (d, J
= 2.1 Hz, 1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.57 (d, J = 7.6 Hz, 1H),
7.44−7.48 (m, 1H), 7.34−7.40 (m, 2H), 7.16−7.24 (m, 1H), 2.95 (dd,
J = 6.1, 16.6 Hz, 4H); m/z (ESI) 437.4 (M + H)+.
3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N-(4-(2-phenylthiazol-
4-yl)phenyl)propanamide (32). 1H NMR (400 MHz, DMSO-d6) δ
10.23 (s, 1H), 8.08−8.13 (m, 1H), 8.07 (s, 1H), 7.95−8.05 (m, 4H),
7.78 (ddd, J = 1.6, 7.0, 8.3 Hz, 1H), 7.67−7.74 (m, 2H), 7.56−7.60
(m, 1H), 7.50−7.56 (m, 3H), 7.47 (ddd, J = 1.2, 7.1, 8.0 Hz, 1H), 2.95
(dd, J = 6.3, 17.1 Hz, 4H); m/z (ESI) 453.3 (M + H)+.
tert-Butyl 3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-
propanoate (20). To a mixture of 2-mercaptoquinazolin-4(3H)-
one (2.50 g, 14.0 mmol) and potassium carbonate (3.88 g, 28.1 mmol)
in acetone (70 mL) was added tert-butyl 3-bromopropionate (3.52 g,
16.8 mmol). The mixture was stirred at 60 °C for 6 h. The solid was
filtered off, and the solution was concentrated. The crude material was
purified by chromatography through a Redi-Sep prepacked silica gel
column (80 g, eluting with a gradient of 0−40% EtOAc in hexane) to
provide tert-butyl 3-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)-
propanoate (1.18 g, 28% yield) as a white solid. 1H NMR (400
MHz, DMSO-d6) δ 12.53 (br s, 1 H), 8.04 (dd, J = 7.9, 1.3 Hz, 1 H),
7.77 (ddd, J = 8.3, 7.0, 1.7 Hz, 1 H), 7.53 (d, J = 7.9 Hz, 1 H), 7.43 (td,
J = 7.5, 1.2 Hz, 1 H), 3.39 (t, J = 6.8 Hz, 2 H), 2.73 (t, J = 6.8 Hz, 2
H), 1.42 (s, 9 H); m/z (ESI) 307.2 (M + H)+.
3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)propanoic Acid
(21). To a mixture of tert-butyl 3-((4-oxo-3,4-dihydroquinazolin-2-
yl)thio)propanoate (0.49 g, 1.6 mmol) in DCM (8.0 mL) was added
trifluoroacetic acid (0.36 mL, 4.8 mmol). The mixture was stirred at
room temperature for 6 h. The solvent and extra trifluoroacetic acid
were evaporated to give 3-((4-oxo-3,4-dihydroquinazolin-2-yl)thio)-
propanoic acid (0.39 g, 97% yield) as an off-white solid. 1H NMR (400
MHz, DMSO-d6) δ 7.87−8.05 (m, 1 H), 7.58−7.76 (m, 1 H), 7.38−
7.52 (m, 1 H), 7.34 (ddd, J = 8.0, 7.1, 1.2 Hz, 1 H), 3.29 (t, J = 6.9 Hz,
2 H), 2.66 (t, J = 6.9 Hz, 2 H); m/z (ESI) 251.2 (M + H)+.
General Procedure A for the Synthesis of Tankyrase
Inhibitors. To a vial charged with carboxylic acid (0.9 mmol) and
amine (0.9 mmol) in DMF (1.0 mL) was added HATU (0.9 mmol).
The reaction mixture was stirred at 60 °C overnight. The crude
mixture was purified by chromatography through a Redi-Sep
prepacked silica gel column (12 g, eluting with a gradient of 0−60%
DCM−MeOH−NH4OH (90:10:1)−DCM) to provide the desired
product.
N-(2-Methoxyphenyl)-4-(3-(4-oxo-3,4-dihydroquinazolin-2-
1
yl)propanamido)benzamide (8). H NMR (400 MHz, DMSO-d6)
δ 12.27 (br s, 1 H), 10.38 (s, 1 H), 9.28 (s, 1 H), 8.09 (dd, J = 8.0, 1.1
Hz, 1 H), 7.92 (d, J = 8.8 Hz, 2 H), 7.69−7.83 (m, 4 H), 7.56 (d, J =
7.73 Hz, 1 H), 7.43−7.51 (m, 1 H), 7.17 (td, J = 7.7, 1.7 Hz, 1 H),
7.09 (dd, J = 8.4, 1.3 Hz, 1 H), 6.96 (td, J = 7.7, 1.4 Hz, 1 H), 3.84 (s,
3 H), 2.96 (dq, J = 10.8, 5.2 Hz, 4 H); m/z (ESI) 443.2 (M + H)+.
N-(2-Methoxy-6-methylphenyl)-4-(3-(4-oxo-3,4-dihydroqui-
nazolin-2-yl)propanamido)benzamide 2,2,2-Trifluoroacetate
3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N-(4-(3-phenyl-1,2,4-
1
oxadiazol-5-yl)phenyl)propanamide (33). H NMR (400 MHz,
DMSO-d6) δ 12.28 (s, 1H), 10.55 (s, 1H), 8.12−8.17 (m, 2H), 8.06−
8.12 (m, 3H), 7.85−7.92 (m, 2H), 7.77 (ddd, J = 1.6, 7.0, 8.3 Hz, 1H),
7.57−7.65 (m, 3H), 7.54−7.57 (m, 1H), 7.47 (ddd, J = 1.2, 7.1, 8.0
Hz, 1H), 2.98 (t, J = 4.2 Hz, 4H); m/z (ESI) 438.3 (M + H)+.
3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N-(4-(5-phenyl-1,3,4-
1
(23). H NMR (500 MHz, DMSO-d6) δ 12.22 (br s, 1 H), 10.29
(s, 1 H), 9.38 (s, 1 H), 8.09 (dd, J = 1.2, 7.9 Hz, 1 H), 7.94 (d, J = 8.3
Hz, 2 H), 7.80−7.73 (m, 1 H), 7.70 (d, J = 8.7 Hz, 2 H), 7.56 (d, J =
8.0 Hz, 1 H), 7.48−7.43 (m, 1 H), 7.17 (t, J = 7.9 Hz, 1 H), 6.90 (d, J
= 8.1 Hz, 1 H), 6.86 (d, J = 7.7 Hz, 1 H), 3.73 (s, 3 H), 3.01−2.90 (m,
4 H), 2.14 (s, 3 H); m/z (ESI) 457.2 (M + H)+.
1
oxadiazol-2-yl)phenyl)propanamide (34). H NMR (400 MHz,
DMSO-d6) δ 12.29 (s, 1 H), 10.48 (s, 1 H), 8.05−8.18 (m, 5 H),
7.82−7.88 (m, 2 H), 7.77 (ddd, J = 8.3, 7.0, 1.6 Hz, 1 H), 7.60−7.68
(m, 3 H), 7.56 (d, J = 7.6 Hz, 1 H), 7.44−7.50 (m, 1 H), 2.97 (dd, J =
7.3, 4.3 Hz, 4 H); m/z (ESI) 438.3 (M + H)+.
N-(2-Methoxyphenyl)-2-methyl-4-((3-(4-oxo-3,4-dihydro-2-
1
trans-N-(2-Methoxyphenyl)-4-(3-(4-oxo-3,4-dihydroquinazo-
quinazolinyl)propanoyl)amino)benzamide (24). H NMR (400
1
lin-2-yl)propanamido)cyclohexanecarboxamide (35). H NMR
MHz, DMSO-d6) δ 12.26 (s, 1 H), 10.19 (s, 1 H), 9.09 (s, 1 H), 8.09
(dd, J = 1.1, 7.9 Hz, 1 H), 7.85 (d, J = 6.7 Hz, 1 H), 7.77 (ddd, J = 1.6,
7.0, 8.3 Hz, 1 H), 7.55 (d, J = 7.6 Hz, 1 H), 7.52−7.42 (m, 4 H), 7.17−
7.11 (m, 1 H), 7.09−7.04 (m, 1 H), 6.98−6.91 (m, 1 H), 3.81 (s, 3 H),
3.00−2.86 (m, 4 H), 2.39 (s, 3 H); m/z (ESI) 457.2 (M + H)+.
N-(2-Methoxyphenyl)-N-methyl-4-(3-(4-oxo-3,4-dihydroqui-
nazolin-2-yl)propanamido)benzamide 2,2,2-Trifluoroacetate
(400 MHz, DMSO-d6) δ 12.18 (br s, 1 H), 8.95 (s, 1 H), 8.08 (dd, J =
1.1, 8.0 Hz, 1 H), 7.97−7.90 (m, 1 H), 7.86 (d, J = 7.9 Hz, 1 H), 7.77
(ddd, J = 1.6, 7.0, 8.3 Hz, 1 H), 7.58−7.53 (m, 1 H), 7.46 (ddd, J =
1.2, 7.1, 8.0 Hz, 1 H), 7.08−6.97 (m, 2 H), 6.91−6.83 (m, 1 H), 3.82
(s, 3 H), 3.56−3.43 (m, 1 H), 2.87−2.81 (m, 2 H), 2.58 (t, J = 7.4 Hz,
2 H), 2.49−2.42 (m, 1 H), 1.82 (d, J = 11.2 Hz, 4 H), 1.51−1.36 (m, 2
H), 1.27−1.12 (m, 2 H). m/z (ESI) 449.2 (M + H)+.
1
(25). H NMR (500 MHz, DMSO-d6) δ 10.04 (s, 1 H), 8.07 (dd, J
= 1.1, 8.0 Hz, 1 H), 7.78−7.72 (m, 1 H), 7.52 (d, J = 8.0 Hz, 1 H),
7.49−7.42 (m, 1 H), 7.36 (d, J = 8.3 Hz, 2 H), 7.20−7.09 (m, 4 H),
6.94 (d, J = 8.0 Hz, 1 H), 6.83 (t, J = 7.2 Hz, 1 H), 3.68 (s, 3 H), 3.19
(s, 3 H), 2.95−2.88 (m, 2 H), 2.86−2.78 (m, 2 H); m/z (ESI) 457.2
(M + H)+.
cis-N-(2-Methoxyphenyl)-4-(3-(4-oxo-3,4-dihydroquinazo-
1
lin-2-yl)propanamido)cyclohexanecarboxamide (36). H NMR
(500 MHz, DMSO-d6) δ 12.13 (s, 1 H), 8.83 (s, 1 H), 8.07 (d, J = 8.4
Hz, 1 H), 7.94 (d, J = 7.3 Hz, 1 H), 7.89 (d, J = 7.4 Hz, 1 H), 7.77−
7.72 (m, 1 H), 7.56 (d, J = 8.3 Hz, 1 H), 7.44 (t, J = 7.6 Hz, 1 H),
7.08−7.00 (m, 1 H), 6.93−6.87 (m, 1 H), 3.82 (s, 3 H), 2.88−2.82
(m, 2 H), 2.68−2.62 (m, 2 H), 2.55 (br s, 2 H), 1.83 (d, J = 10.6 Hz, 2
H), 1.72−1.63 (m, 2 H), 1.63−1.47 (m, 4 H); m/z (ESI) 449.2 (M +
H)+.
N-(4-(N-(2-Methoxyphenyl)sulfamoyl)phenyl)-3-(4-oxo-3,4-
1
dihydroquinazolin-2-yl)propanamide (26). H NMR (500 MHz,
DMSO-d6) δ 12.19 (br s, 1H), 10.37 (s, 1H), 9.21 (dd, J = 1.23, 9.6
Hz, 1H), 8.08 (d, J = 8.9 Hz, 1H), 7.75 (t, J = 7.0 Hz, 1H), 7.68 (d, J =
10012
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
3-(4-Oxo-3,4-dihydro-2-quinazolinyl)-N-(trans-4-(5-phenyl-
1,3,4-oxadiazol-2-yl)cyclohexyl)propanamide (37). 1H NMR
(400 MHz, DMSO-d6) δ 12.19 (s, 1 H), 8.09 (dd, J = 1.2, 8.0 Hz,
1 H), 7.96−8.03 (m, 2 H), 7.93 (d, J = 7.7 Hz, 1 H), 7.78 (ddd, J =
1.6, 7.0, 8.3 Hz, 1 H), 7.54−7.66 (m, 4 H), 7.43−7.51 (m, 1 H), 3.53−
3.68 (m, 1 H), 3.00 (tt, J = 3.5, 11.9 Hz, 1 H), 2.86 (t, J = 7.3 Hz, 2
H), 2.61 (t, J = 7.4 Hz, 2 H), 2.17 (d, J = 11.4 Hz, 2 H), 1.91 (dd, J =
3.1, 13.0 Hz, 2 H), 1.64 (dq, J = 2. 9, 12.8 Hz, 2 H), 1.26−1.44 (m, 2
H); m/z (ESI) 444.2 (M + H)+.
General Procedure B for the Synthesis of Tankyrase
Inhibitors. To a vial charged with amine (0.28 mmol) in DMF
(1.0 mL) were added triethylamine (0.12 mL, 0.84 mmol), carboxylic
acid (0.28 mmol), and 1-propanephosphonic acid cyclic anhydride
(50+% solution in DMF) (0.18 mL, 0.28 mmol). The reaction mixture
was stirred at room temperature overnight. The crude mixture was
purified by chromatography through a Redi-Sep prepacked silica gel
column (12 g, eluting with a gradient of 0−60% DCM−MeOH−
NH4OH (90:10:1)−DCM) to provide the desired product.
3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(5-
phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)propanamide (22). 1H
NMR (400 MHz, DMSO-d6) δ 12.50 (br s, 1 H), 8.02−8.09 (m, 1 H),
7.96−8.02 (m, 2 H), 7.90 (d, J = 7.6 Hz, 1 H), 7.72−7.83 (m, 1 H),
7.57−7.66 (m, 3 H), 7.50−7.57 (m, 1 H), 7.43 (ddd, J = 8.0, 7.1, 1.2
Hz, 1 H), 3.55−3.71 (m, 1 H), 3.41 (t, J = 6.8 Hz, 2 H), 3.00 (tt, J =
11.9, 3.6 Hz, 1 H), 2.56−2.63 (m, 2 H), 2.12−2.24 (m, 2 H), 1.85−
2.02 (m, 2 H), 1.56−1.75 (m, 2 H), 1.22−1.44 (m, 2 H); m/z (ESI)
476.2 (M + H)+.
3-(4-Oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)-N-(trans-
4-(5-phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)propanamide
(38). 1H NMR (400 MHz, DMSO-d6) δ 12.22 (br s, 1 H), 8.66 (dd, J
= 4.6, 1.9 Hz, 1 H), 8.22 (dd, J = 7.9, 2.0 Hz, 1 H), 7.72−7.79 (m, 2
H), 7.69 (d, J = 7.7 Hz, 1 H), 7.30−7.43 (m, 3 H), 7.24 (dd, J = 7.8,
4.6 Hz, 1 H), 3.82 (q, J = 5.3 Hz, 1 H), 3.36 (dtd, J = 11.4, 7.5, 7.5, 4.2
Hz, 1 H), 2.66 (t, J = 7.3 Hz, 2 H), 2.41 (t, J = 7.4 Hz, 2 H), 1.81−2.00
(m, 2 H), 1.67 (dd, J = 13.4, 3.6 Hz, 2 H), 1.29−1.49 (m, 2 H), 1.00−
1.21 (m, 2 H); m/z (ESI) 445.2 (M + H) .
3-(4-Oxo-3,4,5,6,7,8-hexahydropyrido[2,3-d]pyrimidin-2-yl)-
N-(trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)-
propanamide (39). 1H NMR (400 MHz, DMSO-d6) δ 11.23 (br s, 1
H), 8.00 (dd, J = 7. 8, 1.7 Hz, 2 H), 7.82 (d, J = 7.6 Hz, 1 H), 7.56−
7.68 (m, 3 H), 6.55 (br s, 1 H), 3.52−3.69 (m, 1 H), 3.17 (br s, 2 H),
2.93−3.06 (m, 1 H), 2.55−2.64 (m, 2 H), 2.39−2.49 (m, 2 H), 2.29 (t,
J = 6.1 Hz, 2 H), 2.18 (d, J = 12.1 Hz, 2 H), 1.92 (d, J = 10.1 Hz, 2 H),
1.56−1.75 (m, 4 H), 1.30−1.44 (m, 2 H); m/z (ESI) 449.2 (M + H)+.
(S)-1-((4-Oxo-3,4-dihydroquinazolin-2-yl)methyl)-N-(trans-4-
(5-phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)piperidine-3-car-
boxamide (40). 1H NMR (500 MHz, DMSO-d6) δ 11.22 (br s, 1 H),
8.11 (d, J = 7.9 Hz, 1 H), 7.94−8.02 (m, 2 H), 7.75−7.87 (m, 2 H),
7.55− 7.70 (m, 4 H), 7.50 (t, J = 7.5 Hz, 1 H), 3.51−3.61 (m, 1 H),
3.45 (s, 2 H), 2.95 (t, J = 12.0 Hz, 1 H), 2.77 (d, J = 11.2 Hz, 2 H),
2.37 (br s, 1 H), 2.26−2.35 (m, 1 H), 2.18−2.26 (m, 1 H), 2.13 (d, J =
15.4 Hz, 2 H), 1.86 (d, J = 13.6 Hz, 2 H), 1.55−1.73 (m, 4 H), 1.51
(d, J = 11.1 Hz, 1 H), 1.35−1.46 (m, 1 H), 1.23−1.35 (m, 2 H); m/z
(ESI) 513.3 (M + H)+.
2-((R)-1-((4-Oxo-3,4-dihydroquinazolin-2-yl)methyl)-
pyrrolidin-2-yl)-N-(trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)-
cyclohexyl)acetamide (41). 1H NMR (500 MHz, DMSO-d6) δ
11.75 (br s, 1 H), 8.08−8.16 (m, 1 H), 7.94−8.04 (m, 3 H), 7.75−7.85
(m, 1 H), 7.55−7.66 (m, 4 H), 7.49 (t, J = 7.6 Hz, 1 H), 3.94 (d, J =
14.6 Hz, 1 H), 3.55−3.69 (m, 1 H), 3.39 (d, J = 14.6 Hz, 1 H), 2.89−
3.04 (m, 3 H), 2.33−2.44 (m, 2 H), 2.22 (dd, J = 13.7, 6.9 Hz, 1 H),
2.15 (t, J = 12.7 Hz, 2 H), 1.81−1.98 (m, 3 H), 1.51−1.74 (m, 5 H),
1.23−1.45 (m, 2 H); m/z (ESI) 513.3 (M + H)+.
4-(4-Oxo-3,4-dihydro-2-quinazolinyl)-N-(trans-4-(5-phenyl-
1,3,4-oxadiazol-2-yl)cyclohexyl)butanamide (43). 1H NMR (400
MHz, DMSO-d6) δ 12.16 (br s, 1 H), 8.08 (dd, J = 7.92, 1.4 Hz, 1 H),
7.95−8.03 (m, 2 H), 7.78 (td, J = 7.7, 1.6 Hz, 2 H), 7.54−7.68 (m, 4
H), 7.41−7.51 (m, 1 H), 3.51−3.66 (m, 1 H), 2.90−3.05 (m, 1 H),
2.61 (t, J = 7.5 Hz, 2 H), 2.15 (t, J = 7.34 Hz, 4 H), 1.85−2.03 (m, 4
H), 1.54−1.72 (m, 2 H), 1.22−1.41 (m, 2 H); m/z (ESI) 458.2 (M +
H)+.
4-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(5-
phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)butanamide (44). 1H
NMR (400 MHz, DMSO-d6) δ 12.52 (br s, 1 H), 7.93−8.08 (m, 3
H), 7.82 (d, J = 7.6 Hz, 1 H), 7.76 (t, J = 7.5 Hz, 1 H), 7.55−7.66 (m,
3 H), 7.52 (d, J = 8.0 Hz, 1 H), 7.41 (t, J = 7.5 Hz, 1 H), 3.61 (d, J =
7.8 Hz, 1 H), 3.19−3.27 (m, 2 H), 2.90−3.05 (m, 1 H), 2.23 (t, J = 7.1
Hz, 2 H), 2.16 (d, J = 12.5 Hz, 2 H), 1.84−2.01 (m, 4 H), 1.55−1.73
(m, 2 H), 1.26−1.43 (m, 2 H); m/z (ESI) 490.2 (M + H)+.
3-((4-Oxo-3,4,5,6,7,8-hexahydroquinazolin-2-yl)thio)-N-
(trans-4-(5-phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)-
propanamide (45). 1H NMR (400 MHz, DMSO-d6) δ 12.40 (br s, 1
H), 8.00 (dd, J = 7. 8, 1.7 Hz, 2 H), 7.86 (d, J = 7.5 Hz, 1 H), 7.52−
7.67 (m, 3 H), 3.62 (d, J = 7.8 Hz, 1 H), 3.21−3.29 (m, 2 H), 3.00 (t, J
= 11.8 Hz, 1 H), 2.29 (br s, 2 H), 2.18 (d, J = 12.6 Hz, 2 H), 1.93 (d, J
= 10.4 Hz, 2 H), 1.54−1.77 (m, 6 H), 1.24−1.43 (m, 2 H); m/z (ESI)
480.2 (M + H)+.
3-((6-Oxo-1,6-dihydropyrimidin-2-yl)thio)-N-(trans-4-(5-phe-
nyl-1,3,4-oxadiazol-2-yl)cyclohexyl)propanamide (46). 1H
NMR (400 MHz, DMSO-d6) δ 12.65 (br s, 1 H), 8.00 (dd, J = 7.8,
1.7 Hz, 2 H), 7.89 (d, J = 7.5 Hz, 2 H), 7.53−7.69 (m, 4 H), 6.10 (br s,
1 H), 3.62 (dd, J = 7.4, 4.2 Hz, 1 H), 3.00 (t, J = 11.9 Hz, 1 H), 2.18
(d, J = 12.0 Hz, 2 H), 1.84−2.00 (m, 2 H), 1.58−1.74 (m, 2 H), 1.27−
1.45 (m, 2 H); m/z (ESI) 426.2 (M + H)+.
N-(trans-4-(4-Fluorophenoxy)cyclohexyl)-3-((4-oxo-3,4-dihy-
1
droquinazolin-2-yl)thio)propanamide (47). H NMR (400 MHz,
DMSO-d6) δ 12.50 (br s, 1 H), 8.04 (dd, J = 8.0, 1.2 Hz, 1 H), 7.85 (d,
J = 7.6 Hz, 1 H), 7.77 (td, J = 7.68, 1.6 Hz, 1 H), 7.54 (d, J = 8.2 Hz, 1
H), 7.39−7.46 (m, 1 H), 7.05−7.12 (m, 2 H), 6.92−6.99 (m, 2 H),
4.15−4.30 (m, 1 H), 3.54−3.68 (m, 1 H), 3.40 (t, J = 6.8 Hz, 2 H),
2.55−2.62 (m, 2 H), 1.99−2.08 (m, 2 H), 1.79−1.89 (m, 2 H), 1.23−
1.48 (m, 4 H); m/z (ESI) 442.2 (M + H)+.
N-(trans-4-(4-Chlorophenoxy)cyclohexyl)-3-((4-oxo-3,4-di-
hydroquinazolin-2-yl)thio)propanamide (48). 1H NMR (400
MHz, DMSO-d6) δ 12.50 (br s, 1 H), 8.04 (dd, J = 8.0, 1.2 Hz, 1 H),
7.86 (d, J = 7.4 Hz, 1 H), 7.73−7.81 (m, 1 H), 7.54 (d, J = 8.3 Hz, 1
H), 7.38−7.46 (m, 1 H), 7.25−7.33 (m, 2 H), 6.94−7.01 (m, 2 H),
4.28 (t, J = 4.3 Hz, 1 H), 3.53−3.67 (m, 1 H), 3.40 (t, J = 6.8 Hz, 2 H),
2.55−2.62 (m, 2 H), 2.03 (d, J = 9.8 Hz, 2 H), 1.85 (d, J = 9.7 Hz, 2
H), 1.24−1.52 (m, 4 H); m/z (ESI) 458.2 (M + H)+.
N-(trans-4-(4-Cyanophenoxy)cyclohexyl)-3-((4-oxo-3,4-dihy-
1
droquinazolin-2-yl)thio)propanamide (49). H NMR (400 MHz,
DMSO-d6) δ 12.31 (s, 1 H), 7.85 (dd, J = 7 0.9, 1.2 Hz, 1 H), 7.69 (d,
J = 7.5 Hz, 1 H), 7.50−7.62 (m, 3 H), 7.35 (d, J = 8.1 Hz, 1 H), 7.20−
7.27 (m, 1 H), 6.91−6.97 (m, 2 H), 4.21−4.34 (m, 1 H), 3.37−3.51
(m, 1 H), 3.21 (t, J = 6.7 Hz, 2 H), 2.40 (t, J = 6.8 Hz, 2 H), 1.87 (d, J
= 9.7 Hz, 2 H), 1.60−1.74 (m, 2 H), 1.09−1.35 (m, 4 H); m/z (ESI)
449.2 (M + H)+.
3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(pyri-
1
din-4-yloxy)cyclohexyl)propanamide (50). H NMR (400 MHz,
DMSO-d6) δ 12.50 (br s, 1 H), 8.35 (d, J = 6.2 Hz, 2 H), 8.04 (d, J =
6.9 Hz, 1 H), 7.88 (d, J = 7.6 Hz, 1 H), 7.77 (t, J = 6.9 Hz, 1 H), 7.53
(d, J = 7.9 Hz, 1 H), 7.42 (t, J = 7.3 Hz, 1 H), 6.97 (d, J = 6.0 Hz, 2
H), 4.34−4.54 (m, 1 H), 3.63 (d, J = 6.3 Hz, 1 H), 3.40 (t, J = 6.5 Hz,
2 H), 2.59 (t, J = 6.8 Hz, 2 H), 2.06 (d, J = 9.9 Hz, 2 H), 1.86 (d, J =
9.3 Hz, 2 H), 1.29−1.54 (m, 4 H); m/z (ESI) 425.2 (M + H)+.
3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(pyri-
2-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(5-
phenyl-1,3,4-oxadiazol-2-yl)cyclohexyl)acetamide (42). 1H
NMR (400 MHz, DMSO-d6) δ 12.53 (br s, 1 H), 8.14 (d, J = 7.5
Hz, 1 H), 7.80−8.00 (m, 3 H), 7.66 (t, J = 7.0 Hz, 1 H), 7.48 (d, J =
6.9 Hz, 4 H) 7.40 (d, J = 8.12 Hz, 1 H) 7.31 (t, J = 7.2 Hz, 1 H), 3.82
(s, 2 H), 3.51 (d, J = 6.8 Hz, 1 H), 2.80−2.98 (m, 1 H), 2.05 (d, J =
11.2 Hz, 2 H), 1.82 (d, J = 10.8 Hz, 2 H), 1.41−1.62 (m, 2 H), 1.16−
1.40 (m, 2 H); m/z (ESI) 462.2 (M + H)+.
1
din-2-yloxy)cyclohexyl)propanamide (51). H NMR (400 MHz,
DMSO-d6) δ 12.50 (br s, 1 H), 8.10−8.18 (m, 1 H), 8.04 (dd, J = 7.9,
1.2 Hz, 1 H), 7.85 (d, J = 7.7 Hz, 1 H), 7.77 (ddd, J = 8.3, 7.0, 1.6 Hz,
1 H), 7.63−7.73 (m, 1 H), 7.54 (d, J = 7.9 Hz, 1 H), 7.37−7.50 (m, 1
H), 6.93 (ddd, J = 7.09, 5.0, 0.9 Hz, 1 H), 6.75 (dt, J = 8.4, 0.9 Hz, 1
H), 4.80−5.02 (m, 1 H), 3.53−3.71 (m, 1 H), 3.40 (t, J = 6.7 Hz, 2
10013
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
H), 2.54−2.63 (m, 2 H), 2.01−2.13 (m, 2 H), 1.77−1.93 (m, 2 H),
1.39−1.56 (m, 2 H), 1.23−1.39 (m, 2 H); m/z (ESI) 425.2 (M + H)+.
N-(trans-4-(3-Chlorophenoxy)cyclohexyl)-3-((4-oxo-3,4-di-
hydroquinazolin-2-yl)thio)propanamide (52). 1H NMR (400
MHz, DMSO-d6) δ 12.55 (br s, 1 H), 8.09 (dd, J = 7.9, 1.1 Hz, 1 H),
7.91 (d, J = 7.6 Hz, 1 H), 7.82 (ddd, J = 8.3, 7.0, 1.6 Hz, 1 H), 7.59 (d,
J = 8.1 Hz, 1 H), 7.48 (ddd, J = 8.0, 7.1, 1.1 Hz, 1 H), 7.34 (t, J = 8.2
Hz, 1 H), 7.05−7.13 (m, 1 H), 7.00 (ddd, J = 10.9, 8.2, 0.8 Hz, 1 H),
7.00 (ddd, J = 15.3, 8.2, 0.8 Hz, 1 H), 4.41 (s, 1 H), 3.45 (t, J = 6.7 Hz,
2 H), 2.60−2.68 (m, 2 H), 1.99−2.16 (m, 2 H), 1.83−1.99 (m, 2 H),
1.29−1.57 (m, 4 H); m/z (ESI) 458.2 (M + H)+.
3-((4-Oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(trans-4-(3-
cyanophenoxy)cyclohexyl)propanamide (53). 1H NMR (400
MHz, DMSO-d6) δ 12.49 (s, 1 H), 8.03 (dd, J = 1.3, 7.9 Hz, 1 H), 7.86
(d, J = 7.3 Hz, 1 H), 7.76 (dt, J = 1.6, 7.7 Hz, 1 H), 7.53 (d, J = 7.92
Hz, 1 H), 7.38−7.49 (m, 3 H), 7.35 (td, J = 1.2, 7.7 Hz, 1 H), 7.29
(ddd, J = 1.0, 2.6, 8.4 Hz, 1 H), 4.33−4.49 (m, 1 H), 3.55−3.67 (m, 1
H), 3.36−3.42 (m, 2 H), 2.58 (t, J = 6.7 Hz, 2 H), 2.00−2.09 (m, 2
H), 1.79−1.90 (m, 2 H), 1.28−1.50 (m, 4 H); m/z (ESI) 449.2 (M +
H)+.
N-(trans-4-(4-Chloro-3-cyanophenoxy)cyclohexyl)-3-((4-oxo-
3,4-dihydroquinazolin-2-yl)thio)propanamide (54). 1H NMR
(400 MHz, DMSO-d6) δ 12.50 (br s, 1 H), 8.04 (dd, J = 7.9, 1.1
Hz, 1 H), 7.87 (d, J = 7.5 Hz, 1 H), 7.73−7.81 (m, 1 H), 7.64 (d, J =
3.0 Hz, 1 H), 7.60 (d, J = 9.0 Hz, 1 H), 7.54 (d, J = 8.0 Hz, 1 H),
7.38−7.47 (m, 1 H), 7.32 (dd, J = 9.0, 3.0 Hz, 1 H), 4.35−4.50 (m, 1
H), 3.55−3.69 (m, 1 H), 3.40 (t, J = 6.7 Hz, 2 H), 2.59 (t, J = 6.9 Hz, 2
H), 2.04 (d, J = 10.1 Hz, 2 H), 1.85 (d, J = 9.7 Hz, 2 H), 1.28−1.50
(m, 4 H); m/z (ESI) 483.2 (M + H)+.
N-(trans-4-(3-Chloro-4-cyanophenoxy)cyclohexyl)-3-((4-oxo-
3,4-dihydroquinazolin-2-yl)thio)propanamide (55). 1H NMR
(400 MHz, DMSO-d6) δ 12.51 (s, 1 H), 8.04 (dt, J = 7.9, 0.8 Hz, 1
H), 7.81−7.92 (m, 2 H), 7.77 (ddd, J = 8.3, 7.0, 1.6 Hz, 1 H), 7.54 (d,
J = 8.0 Hz, 1 H), 7.43 (ddd, J = 8.0, 7.1, 1.17 Hz, 1 H), 7.37 (d, J = 2.5
Hz, 1 H), 7.13 (dd, J = 8.9, 2.5 Hz, 1 H), 4.43−4.65 (m, 1 H), 3.52−
3.71 (m, 1 H), 3.40 (t, J = 6.7 Hz, 2 H), 2.54−2.63 (m, 2 H), 2.05 (d, J
= 10.2 Hz, 2 H), 1.76−1.92 (m, 2 H), 1.27−1.56 (m, 4 H); m/z (ESI)
483.2 (M + H)+.
Wnt response; TBC, total β-catenin; SIF, simulated intestinal
fluid; DIPEA, diisopropylethylamine; HATU, O-(7-azabenzo-
triazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluoro-
phosphte; DIAD, diisopropyl azodicarboxylate; T3P, propyl-
phosphonic acid anhydride; SAR, structure−activity relation-
ship; LE, ligand efficiency; LipE, lipophilic efficiency; STF,
supertop flash; DMSO, dimethylsulfoxide; TFA, trifluoroacetic
acid; DCM, dichloromethane; DMF, N,N-dimethylformamide
REFERENCES
■
(1) Clevers, H. Wnt/β-catenin signaling in development and disease.
Cell 2006, 127, 469−480.
(2) (a) Reya, T.; Clevers, H. Wnt signaling in stem cells and cancer.
Nature 2005, 434, 843. (b) Barker, N.; Clevers, H. Mining the Wnt
pathway for cancer therapeutics. Nat. Rev. Drug Discovery 2006, 5,
997−1014. (c) Polakis, P. The many ways of Wnt in cancer. Curr.
Opin. Genet. Dev. 2007, 17, 45−51.
(3) (a) Martin, S. A.; Lord, C. J.; Ashworth, A. DNA repairs
deficiency as a therapeutic target in cancer. Curr. Opin. Genet. Dev.
2008, 18, 80−86. (b) Jones, P. Development of poly(ADP-
ribose)polymerase (PARP) inhibitors in oncology. Annu. Rep. Med.
Chem. 2010, 45, 229−243. (c) Ferraris, D. V. Evolution of poly(ADP-
ribose) polymerase-1 (PARP-1) inhibitors. From concept to clinic. J.
Med. Chem. 2010, 53, 4561−4584.
(4) Smith, S.; Giriat, I.; Schmitt, A.; de Lange, T. Tankyrase, a
poly(ADP-ribose) polymerase at human telomeres. Science 1998, 282,
1484−1487.
(5) (a) Riffell, J. L.; Lord, C. J.; Ashworth, A. Tankyrase-targeted
therapeutics: expanding opportunities in the PARP family. Nat. Rev.
Drug Discovery 2012, 11, 923−936. (b) Lehtio, L.; Chi, N.-W.; Krauss,
̈
S. Tankyrase as drug targets. FEBS J. 2013, 280, 3576−3593 and
references herein..
(6) Waaler, J.; Ondrej, M.; Lucie, T.; Huyen, D.; Vladimir, K.; Ray,
W. S.; Erik, P. J.; Marie, P. N.; Tor, J. E.; Olga, M.; Gradl, D.;
Voronkov, A.; von Kries, J. P.; Krauss, S. A novel tankyrase inhibitor
decreases canonical Wnt signaling in colon carcinoma cells and
reduces tumor growth in conditional APC mutant mice. Cancer Res.
2012, 72 (11), 2822−2832 and references herein..
(7) 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.;
Bouwmeester, T.; Porter, J. A.; Bauer, A.; Cong, F. Tankyrase
inhibition stabilizes axin and antagonizes Wnt signaling. Nature 2009,
46, 614−620.
(8) (a) Chen, B.; Dodge, M. E.; Tang, W.; Lu, J.; Ma, Z.; Fan, C.-W.;
Wei, S.; Hao, W.; Kilgore, J.; Williams, N. S.; Roth, M. G.; Amatruda, J.
F.; Chen, C.; Lum, L. Small molecule-mediated disruption of Wnt-
dependent signaling in tissue regeneration and cancer. Nat. Chem. Biol.
2009, 5 (2), 100−107. (b) Lu, J.; Ma, Z.; Hsieh, J.-C.; Fan, C.-W.;
Chen, B.; Longgood, J. C.; Williams, N. S.; Amatruda, J. F.; Lum, L.;
Chen, C. Structure−activity relationship studies of small-molecule
inhibitors of Wnt response. Bioorg. Med. Chem. Lett. 2009, 19 (14),
3825−3827.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of key intermediates leading to compounds 8, 23, 24,
35, 38, 39, 42, and 44−46; Figure S1 showing the dihedral scan
calculation of sulfur ethylene linker and three-carbon linker.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
■
*Z.H.: phone, 617-444-5233; e-mail, zihao.hua@amgen.com.
*E.F.D.: phone, 617-444-5189; e-mail, erin.dimauro@amgen.
com.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(9) Karlberg, T.; Markova, N.; Johansson, I.; Hammarstrom, M.;
Schutz, P.; Weigelt, J.; Schuler, H. Structural basis for the interaction
between tankyrase-2 and potent wnt-signaling inhibitor. J. Med. Chem.
2010, 53, 5352−5355.
(10) Gunaydin, H.; Gu, Y.; Huang, X. Novel binding mode of a
potent and selective tankyrase inhibitor. PLoS One 2012, 7, e333740.
The authors thank Dhan Bagal, Matthew Potter, Paul H.
Krolikowski, Grace Bi, and Shawn Ayube for analytical support,
and Dr. Margaret Chu-Moyer for proof-reading the manuscript.
ABBREVIATIONS USED
■
(11) Narwal, M.; Venkannagari, H.; Lehtio, L. Structural basis of
̈
TNKS, tankyrase; APC, adenomatous polyposis coli; axin, axis
inhibition protein; GSK3β, glycogen synthase kinase 3β; CK1α,
casein kinase 1α; PARP, poly ADP-ribose polymerase; CRC,
colorectal cancer; NAD, nicotinamide adenine dinucleotide;
TRF1, telomere repeating binding factor 1; IWR, inhibitor of
selective inhibition of human tankyrase. J. Med. Chem. 2012, 55,
1360−1367.
(12) Waaler, J.; Machon, O.; von Kries, J. P.; Wilson, S. R.; Lundenes,
E.; Wedlich, D.; Gradl, D.; Paulsen, J. E.; Machonova, O.; Dembinski,
J. L.; Dinh, H.; Krauss, S. Novel synthetic antagonists of canonical Wnt
10014
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015

Journal of Medicinal Chemistry
Article
signaling inhibit colorectal cancer cell growth. Cancer Res. 2011, 71,
197−205.
(20) A high-throughput RLM assay as a surrogate of MLM allowed
us to identify promising compounds for further PK and PD studies in
mice.
(13) Shultz, M. D.; Kirby, C. A.; Stams, T.; Chin, D. N.; Blank, J.;
Charlat, O.; Cheng, H.; Cheung, A.; Cong, F.; Feng, Y.; Fortin, P. D.;
Hood, T.; Tyagi, V.; Xu, M.; Zhang, B.; Shao, W. [1,2,4]Triazol-3-
ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazoles: antagonists of the Wnt
pathway that inhibit tankyrases 1 and 2 via novel adenosine pocket
binding. J. Med. Chem. 2012, 55, 1127−1136.
(21) (a) Floby, E.; Briem, S.; Terelius, Y.; Sohlenius-Sternbeck, A.-K.
The use of a cocktail of probe substrates for drug-metabolizing
enzymes for the assessment of the metabolic capacity of hepatocyte
preparations. Xenobiotica 2004, 34, 949−959. (b) Bissell, D. M.;
Guzelian, P. S. Phenotypic stability of adult rat hepatocytes in primary
monolayer culture. Ann. N.Y. Acad. Sci. 1980, 249, 85−98.
(22) For the first example of S···OC interaction, see the following:
Goldstein, B. M.; Mao, D. T.; Marquez, V. E. Ara-tiazofurin:
conservation of structural features in an unusual thiazole nucleoside.
J. Med. Chem. 1988, 31, 1026−1031.
(14) Voronkov, A.; Holsworth, D. D.; Waaler, J.; Wilson, S. R.;
Ekblad, B.; Perdreau-Dahl, H.; Dinh, G.; Drewes, G.; Hopf, C.; Morth,
J. P.; Krauss, S. Structural basis and SAR for G007-LK, a lead stage
1,2,4-triazole based specific tankyrase 1/2 inhibitor. J. Med. Chem.
2013, 56, 3012−3023.
(23) For more details, see the Supporting Information.
(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, 3132−3144.
(24) Selected compounds 22 and 49 showed relatively low oral
bioavailability at 13% and 21%, respectively, likely due to their
moderate solubility. The oral bioavailability (% F) is based on 2 mg/kg
po PK study in rats.
(25) Huang, L.; Chen, A.; Roberts, J.; Janosky, B.; Be, X.; Berry, L.;
Lin, M.-H. J. Use of uptake intrinsic clearance from attached rat
hepatocytes to predict hepatic clearance for poorly permeable
compounds. Xenobiotica 2012, 42 (9), 830−840.
(26) The PARP1 biochemical assay was purchased as a kit from
Trevigen (catalog no. 4676-096-K) and used per manufacturer’s
recommendations. The PARP2 biochemical assay was purchased as a
kit from BPS (catalog no. 80552) and used per manufacturer’s
recommendations. 22: PARP1 IC50 = undefined, PARP2 IC50 = 2.45
μM. 49: PARP1 IC50 = undefined, PARP2 IC50 = 1.76 μM.
(27) The kinase profiling was done using the KINOMEscan service
provided by DiscoveRx (formerly Ambit Bioseciences).
(16) During the preparation of this manuscript, studies on the
identification of different types of tankyrase inhibitor were reported:
(a) Narwal, M.; Haikarainen, T.; Fallarero, A.; Vuorela, P. M.; Lehtio,
̈
L. Screening and structural analysis of flavones inhibiting tankyrases. J.
Med. Chem. 2013, 56, 3507−3517. (b) Shultz, M. D.; Cheung, A. K.;
Kirby, C. A.; Firestone, B.; Chen, C. H.; Chen, Z.; Chin, D.; DiPietro,
L.; Fazal, A.; Feng, Y.; Fortin, P. D.; Gould, T.; Lagu, B.; Lei, H.;
Lenoir, F.; Majumdar, D.; Ochala, E.; Palermo, M. G.; Pham, L.; Pu,
M.; Smith, T.; Stams, T.; Tomlinson, R. C.; Toure, B. B.; Visser, M.;
Wang, R. M.; Waters, N. J.; Shao, W. Identification of NVP-TNKS656:
the use of structure−efficiency relationships (SER) to generate a
highly potent, selective and orally active tankyrase inhibitor. J. Med.
Chem. 2013, 56, 6495−6511. (c) Shultz, M. D.; Majumdar, D.; Chin,
D.; Fortin, P. D.; Feng, Y.; Gould, T.; Kirby, C. A.; Stams, T.; Waters,
N. J.; Shao, W. Structure−efficiency relationship of [1,2,4]triazol-3-
ylamines as novel nicotinamide isosteres that inhibit tankyrases. J. Med.
Chem. 2013, 56, 7049−7059. (d) Larsson, E. A.; Jansson, A.; Ng, F.
M.; Then, S. W.; Panicker, R.; Liu, B.; Sangthongpitag, K.; Pendharkar,
V.; Tai, S. J.; Hill, J.; Dan, C.; Ho, S. Y.; Cheong, W. W.; Poulsen, A.;
Blanchard, S.; Lin, G. R.; Alam, J.; Keller, T. H.; Nordlund, P.
Fragment-based ligand design of novel potent inhibitors of tankyrases.
J. Med. Chem. 2013, 56, 4497−4508. (e) Bregman, H.; Chakka, N.;
Guzman-Perez, A.; Gunaydin, H.; Gu, Y.; Huang, X.; Berry, V.; Liu, J.;
Teffera, Y.; Huang, L.; Egge, B.; Mullady, E. L.; Schneider, S.;
Andrews, P. S.; Mishra, A.; Newcomb, J.; Serafino, R.; Strathdee, C. A.;
Turci, S. M.; Wilson, C.; DiMauro, E. F. The discovery of novel,
induced-pocket binding oxazolidinones as potent, selective, and orally
bioavailable tankyrase inhibitors. J. Med. Chem. 2013, 56, 4320−4342.
(17) Bregman, H.; Gunaydin, H.; Gu, Y.; Schneider, S.; Wilson, C.;
DiMauro, E. F.; Huang, X. Discovery of a class of novel tankyrase
inhibitors that bind to both the nicotinamide pocket and the induced
pocket. J. Med. Chem. 2013, 56, 1341−1345.
(18) Plasma stability assay: Lithium heparanized, CD-1 mouse
plasma was spiked with 1 μM substrate. The spiked plasma is
incubated at 37 °C for 3 h. Aliquots are collected at 0, 0.167, 1, 2, and
3 h (n = 2). The CD-1, lithium heparanized mouse plasma samples
were extracted using acetonitrile protein precipitation. Tolbutamide
was used as an internal standard. The substrate concentrations were
determined by LC−MS/MS using reversed-phase ultraperformance
liquid chromatography on an ACQUITY UPLC BEH C18 analytical
column (50 mm × 2.1 mm, 1.7 μm) with 0.1% formic acid in water
(mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase
B). A gradient at a flow rate of 0.50 mL/min was utilized, with a total
run time of 2.0 min. Substrates were monitored using electrospray
ionization (ESI) with multiple reaction monitoring (MRM) in the
positive or negative ion mode.
(19) Intravenous dose at 5 mg/kg with male rats: CL = 10.9 L h−1
kg−1; Vss = 4.69 L/kg; T1/2 = 0.37 h; AUCinf = 0.11 μM·h.
10015
dx.doi.org/10.1021/jm401317z | J. Med. Chem. 2013, 56, 10003−10015