Discovery of Novel Spiroindoline Derivatives as Selective Tankyrase Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.8b01888

The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/β-catenin signal by preventing poly ADP-ribosylation-

Full text of DOI:10.1021/acs.jmedchem.8b01888

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Article
Discovery of Novel Spiroindoline Derivatives as Selective Tankyrase Inhibitors
Fumiyuki Shirai, Takeshi Tsumura, Yoko Yashiroda, Hitomi Yuki, Hideaki Niwa, Shin Sato, Tsubasa
Chikada, Yasuko Koda, Kenichi Washizuka, Nobuko Yoshimoto, Masako Abe, Tetsuo Onuki, Yui Mazaki,
Chizuko Hirama, Takehiro Fukami, Hirofumi Watanabe, Teruki Honma, Takashi Umehara, Mikako
Shirouzu, Masayuki Okue, Yuko Kano, Takashi Watanabe, Kouichi Kitamura, Eiki Shitara, Yukiko
Muramatsu, Haruka Yoshida, Anna Mizutani, Hiroyuki Seimiya, Minoru Yoshida, and Hiroo Koyama
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01888 • Publication Date (Web): 18 Mar 2019
Downloaded from http://pubs.acs.org on March 18, 2019
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Watanabe, Takashi; Meiji Seika Pharma Co., Ltd., Medicinal Chemistry
Laboratory
Kitamura, Kouichi; Meiji Seika Pharma Co., Ltd., Pharmacology Research
Laboratory
Shitara, Eiki; Meiji Seika Pharma Co., Ltd., Pharmaceutical Research
Division, Pharmaceutical Research Center
Muramatsu, Yukiko; Japanese Foundation for Cancer Research, Division
of Molecular Biotherapy, Cancer Chemotherapy Center,
Yoshida, Haruka; Japanese Foundation for Cancer Research, Division of
Molecular Biotherapy, Cancer Chemotherapy Center
Mizutani, Anna; Japanese Foundation for Cancer Research, Division of
Molecular Biotherapy, Cancer Chemotherapy Center
Seimiya, Hiroyuki; Japanese Foundation for Cancer Research, Division
ofMolecular Biotherapy, Cancer Chemotherapy Center
Yoshida, Minoru; RIKEN Center for Sustainable Resource Science,
Chemical Genomics Research Group
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Koyama, Hiroo; RIKEN Center for Sustainable Resource Science, Drug
Discovery Chemistry Platform Unit
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Discovery of Novel Spiroindoline Derivatives as
Selective Tankyrase Inhibitors
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†,*
§§
§
††
‡‡
Fumiyuki Shirai , Takeshi Tsumura , Yoko Yashiroda , Hitomi Yuki , Hideaki Niwa ,
‡‡
¶¶
†
†
Shin Sato , Tsubasa Chikada , Yasuko Koda , Kenichi Washizuka , Nobuko
†
‡
‡
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§
Yoshimoto , Masako Abe , Tetsuo Onuki , Yui Mazaki , Chizuko Hirama , Takehiro
¶
††
††
‡‡
Fukami , Hirofumi Watanabe , Teruki Honma , Takashi Umehara , Mikako
‡‡
‡§
§§
§§
†‡
Shirouzu , Masayuki Okue , Yuko Kano , Takashi Watanabe , Kouichi Kitamura ,
†§
†¶
†¶
†¶
Eiki Shitara , Yukiko Muramatsu , Haruka Yoshida , Anna Mizutani , Hiroyuki
Seimiya , Minoru Yoshida§, ‡, ‡¶, Hiroo Koyama†,*.
†¶
†
‡
Drug Discovery Chemistry Platform Unit; Drug Discovery Seed Compounds
§
Exploratory Unit; Chemical Genomics Research Group, RIKEN Center for Sustainable
¶
Resource Science; RIKEN Program for Drug Discovery and Medical Technology
Platforms, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
1
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†
†
‡‡
Drug Discovery Computational Chemistry Platform Unit; Drug Discovery Structural
Biology Platform Unit; RIKEN Center for Biosystems Dynamic Research, 1-7-22
Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
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Medicinal Chemistry Laboratory; Pharmacokinetics and Analysis Laboratory;
†
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†§
Pharmacology Research Laboratory; Pharmaceutical Research Division,
‡§
Pharmaceutical Research Center; Process Chemistry Laboratory, Manufacturing &
Control Research Labs, Meiji Seika Pharma Co., Ltd., 760 Morooka-cho, Kohoku-ku,
Yokohama, Kanagawa 222-8567, Japan
†¶Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese
Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8850, Japan
‡¶Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo
113-8657, Japan
KEYWORDS: tankyrase, Wnt/β-catenin signaling, AXIN, β-catenin, mouse xenograft
model, spiroindoline, P-glycoprotein
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ABSTRACT: The canonical WNT pathway plays an important role in cancer pathogenesis.
Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2)
has been reported to reduce the Wnt/β-catenin signal by preventing poly ADP-ribosylation
dependent degradation of AXIN, a negative regulator of Wnt/β-catenin signaling. With the goal
of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out
to find small molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting
from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was
discovered as a potent TNKS/TNKS2 inhibitor with >7,000-fold selectivity against the PARP1
enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human
colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor
growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a
promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.
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
INTRODUCTION
The discovery and development of small molecule inhibitors of the canonical WNT pathway,
which plays a key role in embryonic development and self-renewal of tissues including the
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intestinal epithelium, has been a challenging task in cancer drug discovery. Human tumors
frequently harbor mutations in genes that encode important proteins such as adenomatous
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polyposis coli (APC), AXIN, and -catenin along the WNT pathway. These mutations lead to
constitutive activation of WNT signaling, transcription of the genes independent of the WNT
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ligand stimulation, and aberrant proliferation. It has been reported that a majority (~85%) of
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colorectal cancers (CRC) carry APC mutations in the WNT signaling pathway. While a number
of small molecules have been reported in the literature that can modulate this pathway by
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targeting various proteins (e.g. Porcupine and β-catenin kinases ), no new chemical entity has
yet entered the market for the treatment of cancer.8
Tankyrases [tankyrase-1: Telomeric repeat binding factor1 (TRF1) interacting ankyrin-related
ADP-ribose polymerase-1/ TNKS/ TNKS1/ ARTD5/ PARP-5a; tankyrase-2: TNKS2/ ARTD6/
PARP-5b] constitute a class of the poly(ADP-ribose) polymerase (PARP) enzyme superfamily.
All PARP enzymes contain a PARP catalytic domain that can catalyze the post-translational
+
addition of many ADP-riboses successively onto target proteins (PARsylation) using β-NAD as
a substrate.9
In 2009, a group at Novartis reported that inhibition of the catalytic activity of tankyrases can
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attenuate the WNT pathway signaling via AXIN stabilization. A small molecule tankyrase
inhibitor XAV939 demonstrated that inhibition of the ubiquitination of AXIN through
PARsylation causes accumulation of AXIN. Since AXIN is a concentration-limiting factor in the
destruction of the AXIN--catenin complex, an increased concentration prevents translocation of
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-catenin to the nucleus and attenuates the transcription of the WNT pathway genes. XAV939
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inhibited colony formation of -catenin–dependent DLD-1 cells. These findings gave important
insights to the therapeutic potential of tankyrase inhibitors for the treatment of cancer.
Because of the highly divergent functions of tankyrases including regulation of the WNT
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signaling pathway through modification of β-catenin levels, regulation of telomere length and
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Hippo pathway , control of the mitotic checkpoint and vesicle transport modulation , the
tankyrases have attracted significant interests from pharmaceutical companies as targets of
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therapeutic intervention. Even though several tankyrase inhibitors in diverse structure classes
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have been reported and different types of binding motifs have been identified, there have been
only a few reports of feasibility studies of tankyrase inhibitors as potential anticancer agents
(Fig. 1).
The earliest preclinical study results were reported by Waaler et al. in 2011, where JW67 and
JW74 were tested in both xenograft and ApcMin mouse models. JW67 and JW74 have unique
18
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structures as compared with XAV939 and bind to an induced adenosine pocket of the enzyme.
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Similar observations have been reported with JW55 in tamoxifen-induced polyposis formation
in conditional Apc truncation mice, where significant reduction of the total tumor area was
observed without measurable effects on body weight after 100mg/kg once daily injection for 21
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days. In 2013, Lau et al. reported G007-LK ,which showed improved antitumor effects with
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COLO-320DM and SW403 (APC-mutant CRC cell lines) mouse xenograft models. In 2015, a
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group at Novartis reported TNKS656 , which binds to both nicotinamide and adenosine pockets
and exhibited impressive tumor growth suppression in a patient-derived PDX-P30 CRC
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xenograft model after 100 mg/kg twice daily subcutaneous injection. In 2016, a group at
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AstraZeneca reported AZ1366 , which binds only to a nicotinamide subsite and exhibited
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significant tumor growth suppression with CRC040 explants after once daily 50mg/kg oral
administration for 28 days, where no data on weight loss and/or intestinal toxicity was
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reported. Since AZ1366 inhibits PARP1 as well, interpretation of the observed efficacy can be
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complicated. Importantly, a report from Genentech indicated that a tankyrase inhibitor G-631, a
nicotinamide pocket binder, caused dose-dependent intestinal toxicity in mice with weak
efficacy (100 mg/kg daily oral administration with a SW403 model) and concluded the clinical
utility of tankyrase inhibitors by oral administration is likely to be limited because of the “on-
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target” intestinal toxicity. However, given the availability of structurally diverse tankyrase
inhibitors and factors such as tissue distribution variability, we decided to further assess the
therapeutic potential of tankyrase inhibitors.28, 29
The primary objective of this study was to discover TNKS/TNKS2 dual enzyme inhibitors
that disrupt WNT pathway signaling and demonstrate cell growth inhibition of COLO-320DM,
which is sensitive to inhibition of Wnt/β-catenin signaling. With the hope of eventually
establishing a proof-of-concept that tankyrase inhibitors can suppress tumor growth effectively,
we intended to optimize the following characteristics of our compounds: 1) Pharmacokinetic
profiles in order to maintain sufficient drug levels during xenograft studies; and 2) PARP
selectivity to ensure that the observed efficacy is due to the inhibition of tankyrases, but not of
other PARPs, in particular PARP1.30
Herein, we describe the discovery of a structurally novel, potent and orally bioavailable
TNKS/TNKS2 dual inhibitor, that demonstrated dose-dependent tumor growth inhibition in a
mouse COLO-320DM xenograft model when administered orally or intraperitoneally.
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
RESULTS AND DISCUSSION
Identification of HTS hit compound 1a. A high-throughput screening (HTS) of The University
of Tokyo Drug Discovery Initiative (DDI) chemical library led to the identification of 1a, which
had IC values of 20.5 nM and 19.4 nM with the TNKS and TNKS2 enzymes, respectively.
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Compound 1a exhibited inhibition of WNT signaling (IC = 233 nM) in the TCF reporter assay
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and cell proliferation (GI = 3.2 μM) in the COLO-320DM CellTiter-Glo luminescence cell
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viability assay (Fig. 2; Supporting Information Fig. S1).
Table 1 summarizes the in vitro activities of 1a, XAV939, and G007-LK. As compared with
the structurally related XAV939, 1a was less potent than XAV939 across the enzymes studied.
When 1a was compared with G007-LK, belonging to a different structure class, 1a had more
potent enzyme activities and a comparable level of activity in the cellular assays. While the
enzyme activities of 1a were respectable as an HTS hit, low aqueous solubility and mouse liver
microsomal stability of 1a needed to be improved for further development. As encouraged by the
relatively small molecular size and potential for structural diversification, and available X-ray
co-crystal data, we set out to develop the structure-activity relationship (SAR) of 1a.
Table 1. Comparison of 1a with XAV939 and G007-LK
Compound
1a
2
XAV939
7.5
G007-LK
58.1
[
a]
0.5
TNKS IC50 (nM)
[a]
1
9.4
3.3
85.5
TNKS2 IC50 (nM)
[
a]
>100,000
33
.2
.13
34,924
351
>100,000
26.7
PARP1 IC50 (nM)
[a]
2
TCF reporter assay (nM)
[a]
3
17.0
1.5
COLO-320DM GI50 (M) (CellTiter-Glo)
[
b]
3
4.28
14.34
1.5
AXIN2 up-regulation@0.33 M
Aq.Sol. (pH7.4) (g/mL)
10.0
0.34
0.4
-6
PAMPA (x10 cm/s)
3.24
11.4
Hu/Mo PB (% free)[c]
Hu/Mo Mics.Stab. (% remaining@30 min.)[d]
4.3 / 2.9
84.1 / 0
0.6 / 8.6
78.4 / 0
2.8 / 3.5
100 / 95.8
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[
a] Mean values of duplicate or triplicate experiments results. [b] Single experiment results. [c]
Human (Hu) /Mouse (Mo) serum protein binding, % value of free fraction. [d] Liver
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microsomal stability was determined by remaining % of compounds after 30 minutes incubation
with human and mouse liver microsome according to the protocol described in the experimental
section.
Chemistry
Synthetic modification of compound 1a was accomplished by the substitution reaction
between 2-methylthioquinazolinone analogues (4a-h) prepared from ketoesters (3a-h) and o-,
m-, or p-methoxyphenylpiperazines (o-5, m-5, and p-5) following the published procedure
3
1
(Scheme 1). Compound 1j was synthesized by oxidation of 1b, and 1i was synthesized by
reductive amination of 1h.
Syntheses of 7-18 are depicted in Scheme 2. Following the same or modified procedure
depicted in Scheme 1, compounds (10-13, 15, and 16) were synthesized in one or two steps in
moderate to good yields via substitution reactions of 4-(2-methoxyphenyl)piperidine (19), or
substituted spiropiperidines (20-22, 24, and 25) with S-oxide of 4a. Meanwhile, compounds (7-9,
1
4, 17, and 18) were synthesized by a condensation reaction between ethyl 2-
cyclohexanonecarboxylate 3a and corresponding amidine intermediates (e.g. 29).32
Synthesis of substituted spiroindoline/indolinone derivatives was accomplished as outlined in
Scheme 3. Following the same procedure for compound 7, compounds 15a-h and, 16a-h were
synthesized in two steps; introduction of an amidine functional group and cycloaddition with 3a
to form the tetrahydroquinazolinone (THQ) rings from substituted spiroindoline/indolinone
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intermediates (24 and 25). For the synthesis of spiroindoline/indolinone derivatives, preparation
of the substituted piperidinyl spiro intermediate was the key step. A Fischer indole cyclization
reaction was employed between 4-piperidine aldehyde 36 and substituted phenylhydrazines (37)
to construct a bicyclic spiroindole skeleton [F-I intermediate]. Reduction of the intermediate with
sodium triacetoxyborohydride gave the spiroindolines (38), while oxidation by m-CPBA gave
the spiroindolinones (39). Benzyloxycarbonyl (Cbz)-deprotection of 38 or 39 with trifluoroacetic
acid gave spiro intermediates 24 or 25, respectively. Boc-protection of 38 or 39, and deprotection
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of the Cbz-group under a hydrogenation condition (H / Pd-Carbon) gave spiro intermediates
2
Boc-24 or Boc-25, respectively. Regioisomers with regard to the R-group generated in the Fisher
indole synthesis were chromatographically separated at this stage.
Syntheses of analogues with a hydroxyethyl group were accomplished following the approach
outlined in Scheme 4. For the indoline series, reductive amination of the THQ-indoline
derivatives 15, 15c, 15d, and 15f with glycolaldehyde dimer and sodium triacetoxyborohydride
gave hydroxyethyl substituted compounds 40, 40c, 40d, and 40f, respectively. For the
indolinone series, alkylation of indolinone (39) by 2-chloroethyltetrahydropyranyl ether and
sodium hydride, and deprotection of the tetrahydropyranyl and Cbz groups under standard
conditions, gave 1-hydroxyethyl-indolinone (42). Introduction of an amidine group to 42
followed by condensation with keto-ester 3a gave desired compounds (43, 43c, 43d, and 43f).
Whereas the synthesis of 42a and 49a via Fischer indole cyclization generated regioisomers, we
devised a regioselective synthetic route for these compounds (Scheme 4). The spiroindolinone
precursor (42a) was obtained by gem-bis alkylation of commercially available 2-(2,6-
difluorophenyl)acetonitrile 44a with bis(chloroethyl)benzylamine, followed by hydrolysis of the
nitrile group to an amide, and a nucleophilic aromatic substitution reaction to spiroindolinone
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4
6a, introduction of 2-hydroxyethyl group to the 1-NH position, and deprotection of the benzyl
group. The spiroindoline precursor 49a was obtained by reduction of spiroindolinone 46a by
lithium aluminum hydride, introduction of a 2-hydroxyethyl group, and deprotection of the
benzyl group.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Structure-activity relationship
SAR of tetrahydroquinazolinone analogues. First, we generated a series of
tetrahydroquinazolinones with a methoxyphenylpiperidine moiety, which were found to be
1
7
nicotinamide pocket binders by X-ray crystallography, and tested for their ability to inhibit
TNKS/TNKS2 and PARP1 enzyme activities in a biochemical Enzyme-linked immunosorbent
assay (ELISA), TCF reporter activity in the human colon cancer DLD-1 cell line for functional
activity, and cell proliferation of COLO-320DM cells (Table 2).
Systematic optimization of the 6-position of the bicyclic ring structure revealed that the
nicotinamide binding pocket could accommodate methylene, sulfur, oxygen and
difluoromethylene moieties in the ring, whereas bulky substituents were not tolerated because
enzymatic activity decreases (1e-j). We found that a sulfur-containing ring like that of XAV939
greatly improved the TNKS/TNKS2 enzyme, and cellular TCF reporter activities, but not cell
proliferation inhibition activities. This contrast was thought to be due to the vulnerability of the
sulfide moiety to oxidation depending on growth period (1b vs. 1j). For potency and aqueous
solubility, we decided to preserve the tetrahydroquinazolinone ring (THQ) and shifted our focus
to other parts of the molecule.
Analysis of the effects of the substitution pattern of the methoxy group on the phenyl group (o-,
m-, and p-methoxy derivatives of 1) indicated that the ortho substituent gives the most potent
enzyme, TCF reporter and cell growth inhibitory activities. It appeared that the ortho-methoxy
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
group induced a twisted conformation between the piperadine and the phenyl rings because of
steric reasons, which was observed in the co-crystal structure (Fig. 3).
Table 2. Modification of tetrahydroquinazoline ring of 1a
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
6
X
NH
7
N
N
2'
8
OMe
N
3'
4'
TCF
Reporter
IC50
TNKS
TNKS2
PARP1
COLO-
320DM
Aq.Sol.
(pH7.4)
(g/mL)
IC50
IC50
[a]
(nM)
IC50
[b]
(nM)
Cmpd
X
Position
[c]
[
a]
GI (M)
(nM)
[a]
5
0
(nM)
[
d]
H
135*
20.5
39.4
35.0
2.5
58.9*
19.4
39.7
45.5
2.3
-
1,536
233
-
4.9
10.0
5.2
1
[d]
2’-MeO
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
-
3.2
1
a
CH2
[e]
3
’-MeO
1,066
1,294
23.2
14.2
>20
3.8
m-1a
p-1a
[e]
4’-MeO
2’-MeO
3’-MeO
4’-MeO
2’-MeO
4’-MeO
2’-MeO
4’-MeO
4.6
1
b
0.5
m-1b
p-1b
S
6.2
4.8
148
7.4
1.7
3.4
2.6
114
>50*
17.1
>20*
0.9
1
c
67.6*
220*
19.4
79.6
1,350*
112*
148*
65.5
36.4
3,476*
1,009
2,246
403
12.3
6.0
O
p-1c
1
d
15.5*
0.8
CF2
p-1d
1,971
7,282
>10,000
>10,000
>10,000
>10,000
>20*
1.1
CHCH3
1
e
-
1.8
C(CH3)2
1
f
>20,000* >20,000*
-
-
2.4
CH CH
1
g
5,919*
7,779*
9,336*
6,984*
-
-
6.9
2
2
1h
NH
-
-
-
66.4
69.7
8.8
2
’-MeO
NCH3
SO
1
i
>20,000* >20,000*
6,168* 5,345*
-
1ｊ
>20,000 >10,000
>20*
[
a] Mean values are shown from two or three independent experiments carried out in
duplicate or triplicate, except that data indicated by asterisks are from a single
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experiment carried out in duplicate or triplicate. [b] A single experiment was performed
in duplicate. [c] Three or more independent experiments were performed in triplicate.
The asterisk indicates that a single experiment was performed in triplicate. COLO-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
320DM growth inhibition was evaluated by CellTiter-Glo cell viability assay method. [d]
Compounds 1 and 1a were commercially available. [e] Tankyrase activities of
compounds m-1a, p-1a were disclosed in a PCT patent application by Merck GMBH
(
2013) after evaluation of the compounds. 33
Interaction of 1a and TNKS2 in a co-crystal structure. In the crystal structure of the 1a -
TNKS2 complex (Fig. 3; Green), the THQ moiety of 1a binds to the nicotinamide pocket,
2
2
placing the methoxyphenyl group in the “nook” region. Residues that form the nook region
including His1048, Tyr1050, Ile1051, and Gly1053, are TNKS specific and not shared with other
PARPs. Thus, close interaction between a ligand and the nook region may contribute to
increased PARP selectivity. The carbonyl oxygen of the THQ forms two hydrogen bonds with
the hydroxyl group of Ser1068 and the NH group of Gly1032. The nitrogen of the THQ also
interacts with the carbonyl group of Gly1032. In addition, the THQ has a - interaction with
Tyr1071. At the other side of 1a, the methoxyphenyl group fills the hydrophobic space and
forms T-shaped - and CH- interactions with Phe1035 and Ile1075, respectively. The
perpendicular orientation of the THQ and terminal benzene rings was thought to be important to
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
keep the aforementioned close interactions with both the nicotinamide pocket and the nook
region. It appeared as though the piperazine moiety connecting the THQ and the terminal phenyl
group was simply holding the terminal phenyl ring in a right angle and space without interacting
with the enzyme per se. (PDB ID: 5ZQO; Supporting Information Table S10)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
SAR of the right-hand substructure. Structural modification of the connecting piece between
the THQ and the phenyl ring was conducted with the intention of placing the phenyl group
perpendicular to the connecting ring structure (Table 3).
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Table 3. Optimization of the connecting structure to improve potency and solubility
O
NH
N
Connecting piece
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TCF
COLO-
TNKS
IC50
TNKS2 PARP1
Hu/Mo
Mic.Stab.
(%)
Aq.Sol.
(pH7.4)
Reporter 320DM
IC50
[a]
Connecting
piece - Ar
IC50
[a]
(nM)
IC50
[b]
(nM)
Cmpd
Y
GI50
[a]
(M)
[a]
(
g/mL)
10.0
69.6
-
(nM)
@30min
(nM)
N
O
N
1a
7
-
-
-
-
20.5
19.4
>20,000
233
3.2
84.1/0.0
O
N
6,008*
4,386* >20,000
9,144
5,401
1,161
41.9*
19.6*
6.3
-
N
O
O
O
N
8
14,300* 6,417*
-
-
N
N
9
35.0
65.4
49.7
67.6
>20,000
14.2
1.0
99.3/91.7
62.1/0.0
[c]
-
>20,000
>20,000
531
608
4.6
1
0
1
1
N
CH2
43.4
30.2
31.5
33.7
28.5
27.6
118*
514*
14.6
18.3
35.7
21.0
20.7
25.1
29.3*
258*
10.8
>50
0.67
1.17
1.07
31.6
1.6
64.3/0.1
28.1/0.1
76.3/0.0
34.3/0.3
77.9/34.7
84.3/0.0
-
12
Y
C=O
CH2
>20,000 >10,000
O
1
3
N
>20,000
>20,000
>20,000
>20,000
>20,000
342
1,347
352
5.8
14
O
C=O
CH2
26.9
13.1
11.2
22.4*
97.7*
Y
N
N
1
5
Y
16
N
H
C=O
CH2
591
64.2
67.0
70.1
1
7
1,605
18
HN
C=O
>20,000 >10,000
-
Y
[a] Mean values are shown from two or more independent experiments performed in
duplicate or triplicate. The asterisk indicates that a single experiment was performed in
duplicate or triplicate. [b] A single experiment was performed in duplicate. [c] Tankyrase
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
activities of compound 10 were disclosed in a PCT patent application by Merck GMBH
2013) after evaluation of the compound had been completed.33
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparison of the activities of 7 and 8 with 1a suggested that the enzyme activities are
sensitive to the placement of the terminal phenyl ring. Compound 9, which replaces the
piperazine moiety of 1a with a 1,2,3,6-tetrahydropyridine, showed comparable enzyme and
33
cellular functional activity with 1a. Compound 10 , which replaces the piperazine moiety of 1a
with a piperidine moiety, also showed enzyme and cellular functional activities comparable to
1
a. These observations prompted us to investigate a new series of compounds that would hold
the terminal phenyl ring perpendicular to the connecting ring structure, namely compounds with
bicyclic spiro structures (11-18).(Fig. 2)
Analogues comparable in enzymatic potencies to 1a include benzofuran derivatives (11 and
12), whose structures could be viewed as the products of connecting the methoxy group of the
terminal phenyl group and the piperidine C-4 carbon of 10 (Fig. 2), isobenzofurane (13 and 14)
and indoline derivatives (15 and 16) (TNKS/TNKS2 IC = 15 - 45 nM) although cellular
5
0
activities were somewhat diminished. While none of the newly synthesized compounds showed
superior in vitro profiles both in liver microsomal stability and aqueous solubility as compared
with 1a, spiroindoline compound 15 and spiroindolinone compound 16 showed superior
microsomal stability, and significantly higher aqueous solubility, respectively. Based on these
findings, we decided to continue with the SAR development of the spiroindoline and
spiroindolinone series.
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SAR of substituted spiro indoline/indolinone derivatives to improve in vitro activities. Then
we shifted our focus to the substituents at the 4- and/or 6-position of the phenyl ring for
improved enzyme activity. (Table 4)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Introduction of halogen atoms, such as F and Cl to the 4- and 4, 6-positions of 15 and 16
resulted in improved enzyme, cellular TCF reporter and cell growth inhibitory activities (15a, c,
and d; 16a, c, and d), while other substitution patterns on the phenyl ring (e.g. ethers, amines,
and amides) were not fruitful (data not shown). Low aqueous solubility of these derivatives
discouraged us from further investigation.
In an attempt to explain the improved activities of the halogen-substituted indoline series, we
applied the fragment molecular orbital (FMO) method34, 35, 36 for compounds 15 and, 15a-d. Pair
interaction energy decomposition analysis based on FMO calculations showed high correlation
between TNKS2 enzyme IC values and the interaction energies of two fragments, Tyr1050 and
5
0
Pro1034. A hydrogen bond was observed between the hydrogen attached to the nitrogen of
indoline and the main-chain carbonyl oxygen of Ala1049 (assigned to the Tyr1050 fragment, see
computational methods section for details) in the X-ray structure of TNKS2 and 15d (PDB ID:
6
A84; Supporting Information Fig. S9; Table S10). Introduction of halogen(s) causes a small
increase of the positive charge of the indoline hydrogen, which enhances the hydrogen-bonding
interaction. In addition, introduction of halogen(s) to the indoline, especially at the 4-position,
causes a charge transfer interaction between the phenyl ring and Pro1034. These two interactions
with the main-chain carbonyl oxygen of Ala1049 and Pro1034 are thought to be responsible for
the improved enzyme activities (Supporting Information Fig. S2; Table S5 and S6).
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 4. Optimization of the substituents of the spiroindoline/indolinone derivatives for
enzyme and cellular activities
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
NH
4
R
N
N
6
Y
N
H
TNKS
TNKS2
TCF
Reporter
COLO-
320DM
Aq.Sol.
(pH7.4)
(μg/mL)
IC50
a]
IC50
[a]
(nM)
Compound
Y
R
[a]
[a]
[
IC (nM)
GI (μM)
(nM)
50
50
1
5
H
4-F
28.5
4.4
20.7
2.8
352
13.1
3.3
1.6
4.2
1
5a
5b
5c
15d
5e
15f
5g
15h
72.4
657
110
247
818
609
454
799
591
31.1
369
43.9
21.5
623
533
91.2
496
1
6-F
16.7
5.4
7.6
10.3
5.2
3.2
1
4,6-diF
4-Cl
3.3
6.3
CH2
6.4
5.3
4.9
2.6
1
6-Cl
8.4
5.8
6.9
9.2
4,6-diCl
4-Br
7.5
10.4
15.6
18.5*
25.1
4.7
7.5
11.0
8.9
1
16.0
9.9*
27.6
5.8
10.4
23.6*
11.2
2.3
4-CN
H
69.2
64.2
3.1
1
6
1
6a
6b
6c
6d
6e
16f
6g
16h
4-F
1
6-F
11.0
6.4
12.0
7.5
15.0
4.2
62.9
0.8
1
4,6-diF
4-Cl
1
C=O
10.2
11.1
5.3
4.9
5.0
2.8
1
6-Cl
14.4
7.1
19.9
21.4
8.8
1.8
4,6-diCl
4-Br
15.7
1.9
1
5.8
13.4
26.8*
4-CN
16.8*
20.4*
5.3
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[
a] Mean values are shown from two or more independent experiments performed in
duplicate or triplicate. The asterisk indicates that a single experiment was performed in
duplicate or triplicate.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 5.
SAR of hydroxyethyl substituted spiroindoline/indolinone derivatives to
improve aqueous solubility
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
N
NH
4
R
6
N
Y
N
OH
COLO-
320DM
(MTT)
GI50
TCF
Reporter 320DM
IC50 GI50
COLO-
RKO
(MTT)
GI50
AXIN2
up-
regulation
TNKS TNKS2
PARP-1
IC50
Aq.Sol.
(pH7.4,
PAMPA
IC50
IC50
-6
Cmpd
Y
R
(x10
cm/s)
[e]
[a]
[a]
[b]
[a]
[c]
[c]
g/mL)
(nM)
(nM)
(nM)
@
0.1M
(nM)
(M)
3.9
1.4
1.5
1.6
2.9
12.7
1.8
1.0
2.4
2.6
[c]
(M)
0.22
0.09
(M)
4
0
H
4-F
49.3
9.2
26.9
11.8
17.0
10.6
16.4
26.0
8.2
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
>20,000
362
83.9
52.9
77.6
119
918
36.5
13.9
79.4
19.8
1.77
0.26
0.30
0.98
3.63
4.86
4.42
4.03
0.98
4.10
5.75
3.93
8.03
>76.1
74.6
2.5*
17.4
7.6
-
40a
0d
0c
0f
[d]
4
CH2
4-Cl
16.6
14.3
18.2
19.0
6.8
>10
[f]
[d, f]
4
4,6-diF
4,6-diCl
H
0.45
>10
72.1
5.2*
2.0
-
4
4.65
2.67
0.19
0.25
0.47
0.45
0.04
4
3
3.40
2.03
78.7
80.1
84.5
76.9
55.9
2.9
3.6
1.1
7.9
43.0
43a
3d
3c
3f
4-F
[
d]
4
C=O
4-Cl
4.4
6.8
>10
>10
[d]
4
4,6-diF
4,6-diCl
10.0
9.0
15.1
9.5
4
2.61
[a] Mean values are shown from two or more independent experiments performed in
duplicate or triplicate. [b] A single experiment was performed in duplicate. [c] Mean
values are shown from three or more independent experiments performed in triplicate.
[d] While the cell number was marginally reduced at 0.3 µM of the compound to the
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maximal extent, the relative cell numbers were maintained higher than 50% of untreated
cells at any doses examined (see Figure S4). [e] In general, single experiment has been
performed. The asterisk indicates two experiments have been performed. [f] Data were
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
disclosed in ref. 41.
Introduction of the hydroxyethyl substituent to improve drug-like properties. For improved
aqueous solubility, we decided to introduce a hydroxyethyl group to the 1-NH position of the
indoline/indolinone series (Table 5). Introduction of a hydroxyethyl moiety to halogen
substituted derivatives was tolerated in enzyme (IC = 4 - 18 nM) and TCF reporter (14 - 120
5
0
nM) inhibitory activities, and cell proliferation inhibition (GI = 1.0 - 2.9 μM) while contributing
5
0
to improved aqueous solubility (40a, 40c, 43a, 43c, 43d, and 43f). Meanwhile, compounds (40a,
40f, 43a and 43f) exhibited moderate to potent growth inhibition of both COLO-320DM and
RKO cells, presumably due to unspecified off-target effects. Compounds 40c, 40d, 43c, and 43d
were selected for further evaluations (Supporting Information Fig. S4).
Crystal structure of spiro compounds with TNKS2. According to the crystal structure of
compound 12 (Fig. 4; magenta), the THQ moiety forms the same hydrogen bonds and

 interactions as 1a (Fig. 4; green) in the nicotinamide pocket. The spirobenzofuranone of 12
also fits in the hydrophobic pocket and makes -stacking interactions with Phe1035 and interacts
with Ile1075, just like the methoxyphenyl group of 1a (PDB ID: 5ZQP). As compared with the
rotating methoxyphenyl group of 1a, the structurally rigid spirobenzofuranone of 12 may
contribute to binding affinity more effectively. Since 1a and 12 share the same spiropiperidine
ring, placement of the spirobenzofuranone group is nearly superimposable, but the phenyl rings
were in opposite orientations. The inversion of the benzofuranone ring seems to be set by the -
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
interaction with Phe1035, CH- interaction with Ile1075, and long-range weak interaction
between the oxygen in the furan moiety and the nitrogen of Ile1075 via a water molecule. In
contrast, X-ray crystallography of 52, an N-methyl derivative of 16, indicated that the indole
phenyl ring was overlapping with the phenyl ring of 1a as a result of the  interaction with
both Phe1035 and Ile1075 (PDB ID: 5ZQQ; Supporting Information Fig. S10, Table S10).
In the co-crystal structure of 40c with TNKS2 (Fig.5; blue), 40c fits in the nicotinamide
binding pocket with the indole phenyl ring overlapping the phenyl ring of 1a and 52. The
hydroxyethyl group on the nitrogen of the spiroindoline 40c forms a hydrogen bond with the OH
group of Ser1033 via a water molecule (PDB ID: 5ZQR; Supporting Information Table S10).
Pharmacokinetic profiles. To assess the potential for in vivo efficacy, we studied serum protein
binding, liver microsomal stability, and P-glycoprotein (P-gp/MDR1) mediated efflux ratio using
a method of cell systems which involve MDR1 overexpressing MDCKII cells.37
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In the serum protein binding studies, free fractions of the indolinones (43c and 43d) were
higher than that of indolines (40c and 40d). By the introduction of the hydroxyethyl group,
mouse liver microsomal stability of the indolinones improved as compared with the indolines.
As compared with the indolines, the indolinones exhibited significantly higher aqueous
solubility, lower human serum protein binding, and better liver microsomal stability, whereas the
efflux ratios of both MDR1-overexpressed and normal MDCK cells were higher with the
indolinones than the indolines. Increased efflux ratios of 1-hydroxyethylindolinones may be
attributed to the additional substituent change of the indolinone with a lone pair of the carbonyl
moiety (Table 6).
P-glycoprotein is a clinically important transmembrane transporter in chemotherapy due to its
wide-ranging effects on the absorption and excretion of drugs, and it therefore confers a multi-
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38
drug resistance phenotype to cancer cells. Based on a report of a drug resistant tumor xenograft
model using a P-gp inhibitor, we assumed that the P-gp efflux ratio is an important factor when
selecting compounds for in vivo efficacy studies.39
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 6. Biochemical properties and pharmacokinetic profiles of selected compounds
F
F
Cl
THQ
Cl
THQ
N
THQ
THQ
O
N
N
N
F
N
F
NH
N
O
N
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
THQ=
OH
OH
OH
OH
Compound
40c
40d
43c
43d
[
a]
0.4 / 1.5
.9 / 18.3
0.9 / 1.0
6.1 / 1.1
1.7 / 7.7
1.8 / 4.2
Hu/Mo PB (% free)
Hu/Mo Mics.Stab. (%)
remaining@30min
MDCK
5
39.5 / 39.1
3.3
28.9 / 19.1
3.7
1
.8
.3
1.7
5.1
efflux ratio
MDCK-MDR1
efflux ratio
5
16.1
18.8
[b]
Mouse IP
max (mol/L)
199.2
75.6
48.1
53.5
C
t1/2 (h)
2.8
4.8
3.6
4.1
AUCinf (μmol/L*h)
555
667
106
157
[a] Hu/Mo PB: Human/Mouse serum protein binding, % value of free fraction. [b] Mouse
intraperitoneal (IP) injection, Dose 50mg/kg, ICR mouse.
Pharmacokinetic studies (mouse intraperitoneal injection) indicated that the maximum
concentration (Cmax) of 40c was four times higher than that of 43c and 43d, and that the exposure
levels (compared with the area under the curve [AUC ]) of 40c and 40d were about four times
inf
higher than that of 43c and 43d. Lower Cmax of 40d was thought to be due to low aqueous
solubility. These results may be derived from the intestinal and renal efflux, rather than
differences in lipophilicity, given that the differences in membrane permeability were relatively
small and the value of 40c was rather lower when 40c and 43c were compared (2.0 and 7.9 x10-6
cm/s, respectively; Table 5).
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Moreover, as for the mouse pharmacokinetics of 40c, the exposure level (AUC ) when orally
inf
administered at the 50mg/kg dose was about a half of that of the intraperitoneal administration
(Supporting Information Fig. S7), and the bioavailability (F value) for rat pharmacokinetics was
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
9.8% (Table 7, Supporting Information Fig. S8). Compound 40c was well absorbed from the
gastrointestinal tract resulting in high plasma drug levels. Overall, 40c appeared to have the most
desirable mouse pharmacokinetic properties for in vivo experiments, albeit with slightly lower
liver microsomal stability.
Profile of tankyrase inhibitor 40c. To assess the biological target specificity of 40c, inhibition
of human PARP family member enzymes PARP1, PARP2 (poly ADP-ribosylation) and PARP10
4
0
(mono ADP-ribosylation) was tested. None of the tested PARP enzymes were strongly
inhibited by 40c, comparing inhibitory activity of PARP1 (>100,000 nM), PARP2 (2,717 nM)
and PARP10 (19,807 nM) with the activity of TNKS and TNKS2 (14.3 nM and 10.6 nM,
respectively) (Table 7; Supporting Information Fig. S5). Furthermore, 40c was evaluated on the
screen panel of forty-four kinds of protein kinases, phosphatases, ion channels, GPCRs and
transporters at 10 M concentration (SafetyScreen44, Eurofins), and no hit protein with over
20% inhibition was observed. (Supporting Information Table S7)
Quantitative RT-PCR assay of 40c with the benchmark compound G007-LK to specify the
target genes of β-catenin and hippo pathway was evaluated. After treatment of COLO-320DM
cells with the inhibitors for 48 h, relative expression levels of AXIN2, TCF7, AMOTL2, CTGF
41
and CYR61 transcripts were determined by normalization with those of ACTB expression. It
revealed that down-regulation of AXIN2 and TCF7 transcripts by β-catenin pathway were
inevitably observed, but obvious changes of AMOTL2, CTGF and CYR61 transcripts by hippo
pathway was not observed in COLO-320DM cells. (Supporting Information Fig. S6)
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 40c has been reported to demonstrate efficacy in mouse COLO-320DM xenograft
4
2
models with intraperitoneal and oral administration. Upon 150 mg/kg twice daily (BID)
intraperitoneal administration to mice (n = 9) bearing human COLO-320DM tumors for 11 days,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
0c exhibited statistically significant tumor growth inhibition (TGI = 47.2%, p < 0.01) without
notable weight loss or toxicity. In contrast, G007-LK was reported to inhibit tumor growth (48%
TGI) at 40 mg/kg daily intraperitoneal administration together with detectable weight loss.21
Meanwhile, 40c exhibited statically significant tumor growth inhibition (TGI = 51.9%, p < 0.05)
with slightly lower body weight when administered orally at 300 mg/kg, BID to the model
mouse (n = 9). Recently, Anumala et al. also reported no animal discomforts or body weight
differences in xenograft model with their tankyrase inhibitor.43

CONCLUSION
Multifactorial optimization of a HTS hit compound 1a, led to the identification of a
spiroindoline derivative 40c (RK-287107). RK-287107 is a potent TNKS/TNKS2 inhibitor with
high selectivity against other PARP family members, PARP1 (IC = >100,000 nM: >7,000-fold),
5
0
PARP2 (IC = 2,717 nM: ~200-fold) and PARP10 (IC = 19,807 nM: >1,000-fold). At the
5
0
50
cellular level, RK-287107 inhibits the Wnt/-catenin signaling pathway as indicated by a TCF
reporter assay in DLD-1 and HEK293 cells (IC = 77.6 nM and 83.0 nM, respectively), and also
5
0
inhibits the proliferation of human colorectal cancer COLO-320DM cells to the same extent as
G007-LK (Table 7). Analysis of correlation between pIC value of TNKS/TNKS2 enzymes and
5
0
that of TCF reporter assay in DLD-1 cells revealed that the enzyme inhibitory activities are
significantly correlated with the functional inhibition of Wnt/-catenin signaling with Pearson
correlation coefficients (r = 0.767 / 0.747), respectively. RK-287107 modulates β-catenin
pathway by down-regulation of AXIN2 and TCF7 transcripts as revealed by quantitative RT-
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PCR assay in COLO-320DM cells. RK-287107 exhibited very weak or no activities with forty-
four kinds of important safety-related proteins. Furthermore, RK-287107 showed impressive
pharmacokinetic profiles with rat bioavailability of approximately 60%.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In a COLO-320DM xenograft model, RK-0287107 exhibited moderate tumor growth
inhibition at a relatively high dose, presumably because of insufficient cellular activity and drug
duration by low microsomal stability. It was noteworthy that weight loss and general toxicity
was not observed at doses where statistically significant efficacy was observed. These results
suggest that RK-287107 is an attractive lead compound for the development of new tankyrase
inhibitors and provide valuable insights into the potential utility of tankyrase inhibitors for the
treatment of various forms of cancer.
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. In vitro and rat pharmacokinetic properties of RK-287107 (40c) in comparison to 1a
and G007-LK
Compound
RK-287107 (40c)
1a
20.5
G007-LK
58.1
TNKS enz IC (nM)
1
4.3
0.6
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
TNKS2 enz IC (nM)
1
19.4
85.5
5
0
PARP1 enz IC (nM)
>
100,000
,717
9,807
>100,000
2,230
10,155
>100,000
>100,000
>100,000
5
0
PARP2 enz IC (nM)
2
50
PARP10 enz IC (nM)
1
5
0
TCF reporter
DLD-1 / HEK293 IC (nM)
77.6 / 12.1
.6
233 / 42.9
3.2
26.7 / 4.5
1.5
5
0
Colo-320DM (CellTiter-Glo)
1
GI (μM)
50
Colo-320DM (MTT)
[a]
[a]
[a]
0.45
1.82
0.43
GI (μM)
50
RKO (MTT) GI (μM)
>
10
-
8.90
5.84
5
0
[b]
AXIN2 up-regulation@0.1 μM
Aq.Sol. (pH7.4) (μg/mL)
4.42
3
.13
72.1
10.0
0.34
1.5
-6
2
.0
0.4 / 1.5
.9 / 18.3
11.4
PAMPA (x10 cm/s)
Hu/Mo PB (% free)
4.3 / 2.9
2.8 / 3.5
Hu/Mo Mics.Stab. (%)
remaining@30min.
5
84.1 / 0.0
1.0 / 1.0
100 / 95.8
1.0 / 3.5
MDCKII / MDCKII-MDR1
Flux ratio (AtoB vs BtoA)
1
.8 / 5.3
.84
.5
[c]
Rat IV
9
1.00
13.0
0.72
4.08
58.9
0.12
0.5
1.54
3.3
0
C (μmol/L)
T1/2 (hr)
3
AUCinf (μmol/L*hr)
CLtot (L/hr/kg)
Vdss (L/kg)
4.42
0.54
0.59
2.28
0.25
7.92
59.8
0.79
2.38
1.79
0.23
4.0
[d]
Rat PO Cmax (μmol/L)
Tmax (hr)
AUCinf (μmol/L*hr)
1.27
59.7
2.33
97.8
F (%)
27
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Page 29 of 81
Journal of Medicinal Chemistry
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Bold characters mean improved properties on 1a. [a] Data are from ref. 41. [b] AXIN2 up-
regulation at 0.33 µM. [c] Rat intravenous (IV) injection at 1 mg/kg. [d] Rat per oral (PO)
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injection at 3 mg/kg, Sprague-Dawley rat.
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