Design and Discovery of an Orally Efficacious Spiroindolinone-Based Tankyrase Inhibitor for the Treatment of Colon Cancer

Article abstract of DOI:10.1021/acs.jmedchem.0c00045

Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase family. Inhibition of their enzymatic activities attenuates the Wnt/β-catenin signaling, which plays an important role in cancer pathogenesis. We previously reported the discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582, which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM mouse xenograft model when orally administered. In addition to the dose-dependent elevation and attenuation of the levels of biomarkers AXIN2 and β-catenin, respectively, results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.

Full text of DOI:10.1021/acs.jmedchem.0c00045

Subscriber access provided by UNIV OF WESTERN ONTARIO
Article
Design and Discovery of an Orally Efficacious Spiroindolinone-
based Tankyrase Inhibitor for the Treatment of Colon Cancer
Fumiyuki Shirai, Anna Mizutani, Yoko Yashiroda, Takeshi Tsumura, Yuko Kano, Yukiko
Muramatsu, Tsubasa Chikada, Hitomi Yuki, Hideaki Niwa, Shin Sato, Kenichi Washizuka,
Yasuko Koda, Yui Mazaki, Myung-Kyu Jang, Haruka Yoshida, Akiko Nagamori, Masayuki
Okue, Takashi Watanabe, Kouichi Kitamura, Eiki Shitara, Teruki Honma, Takashi Umehara,
Mikako Shirouzu, Takehiro Fukami, Hiroyuki Seimiya, Minoru Yoshida, and Hiroo Koyama
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00045 • Publication Date (Web): 23 Mar 2020
Downloaded from pubs.acs.org on March 26, 2020
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Division, Pharmaceutical Research Center
Honma, Teruki; RIKEN Yokohama Branch , Center for Life Science
Technologies
Umehara, Takashi; Institute of Physical and Chemical Research, Center
for Biosystems Dynamic Research
Shirouzu, Mikako; RIKEN Center for Life Science Technologies ,
Fukami, Takehiro; RIKEN Program for Drug Discovery and Medical
Technology Platforms
Seimiya, Hiroyuki; Public Interest Incorporated Foundation Japanese
Foundation for Cancer Research, Divisoin of Molecular Biotherapy,
Cancer Chemotherapy Center
Yoshida, Minoru; RIKEN Center for Sustainable Resource Science,
Synthetic Organic Chemistry Laboratory
Koyama, Hiroo; RIKEN Center for Sustainable Resource Science, Drug
Discovery Chemistry Platform Unit
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Design and Discovery of an Orally Efficacious
Spiroindolinone-based Tankyrase Inhibitor for
the Treatment of Colon Cancer
,
†
‡‡
‡
#
#
Fumiyuki Shirai,* Anna Mizutani, Yoko Yashiroda, Takeshi Tsumura, Yuko Kano,
‡
‡
¶ꢀ
∥
⊥
⊥ꢀ
Yukiko Muramatsu, Tsubasa Chikada, Hitomi Yuki, Hideaki Niwa, Shin Sato,
Kenichi Washizuka, Yasuko Koda, Yui Mazaki, Myung-Kyu Jang,‡‡,§§ꢀHaruka
†
†
‡ꢀ
‡
‡
‡‡ꢀ
††
#
Yoshida, Akiko Nagamori, Masayuki Okue, Takashi Watanabe, Kouichi Kitamura,
∇
○ꢀ
∥
⊥
⊥ꢀ
Eiki Shitara, Teruki Honma, Takashi Umehara, Mikako Shirouzu, Takehiro
§
‡‡,§§
Minoru Yoshida,‡,§§§ and Hiroo Koyama*,†
Fukami, Hiroyuki Seimiya,
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†
‡
Drug Discovery Chemistry Platform 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
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∥
⊥
Drug Discovery Computational Chemistry Platform Unit; Drug Discovery Structural
Biology Platform Unit, RIKEN Center for Biosystems Dynamics Research, 1-7-22
Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
#
¶
∇
Medicinal Chemistry Laboratory; Pharmacokinetics and Analysis Laboratory;
○
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 Computational Biology and Medical Sciences, Graduate School of
Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8850,
Japan
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§§§Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
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KEYWORDS: tankyrase, Wnt/β-catenin signaling, AXIN, β-catenin, spiroindoline,
spiroindolinone, pharmacokinetics, P-glycoprotein efflux, mouse xenograft model
ABSTRACT: Tankyrases (TNKS/TNKS2) belong to the poly(ADP-ribose) polymerase
family. Inhibition of their enzymatic activities attenuates the Wnt/β-catenin signaling,
which plays an important role in cancer pathogenesis. We previously reported the
discovery of RK-287107, a spiroindoline-based, highly selective, potent tankyrase
inhibitor. Herein we describe the optimization process of RK-287107 leading to RK-582,
which exhibits a markedly improved robust tumor growth inhibition in a COLO-320DM
mouse xenograft model when orally administered. In addition to the dose-dependent
elevation and attenuation of the levels of biomarkers AXIN2 and -catenin, respectively,
results of the TCF reporter and cell proliferation studies on COLO-320DM are discussed.
 INTRODUCTION
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Globally, colorectal cancer (CRC) is the third most common cause of cancer deaths
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in men and the second in women. Over 90% of CRC cases have been reported to
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accompany with a hyper-activated WNT signaling pathway. Moreover, loss-of-function
mutations of the tumor suppressor adenomatous polyposis coli (APC) 2,3 are found in
approximately 80% of the cases. Tankyrases (tankyrase-1/TNKS/TNKS1/PARP5a and
tankyrase-2/TNKS2/PARP5b) are members of the poly(ADP-ribose) polymerases
(
PARPs) and possess PARP catalytic domains. Tankyrase-1 and tankyrase-2 are
reported to be functionally redundant.4
Tankyrases add multiple ADP-ribose moieties (PARsylate) to various target proteins,
including AXIN, which is a negative regulator of the WNT signaling pathway.
PARsylated AXIN undergoes ubiquitin-dependent degradation in the proteasomes,
leading to the translocation of β-catenin to the nucleus and activation of the WNT
signaling pathway.
In 2009, Huang et al. reported that inhibition of the tankyrase enzymatic activity by
XAV939 attenuates the WNT signaling via AXIN stabilization. In addition, XAV939
5
inhibits colony formation of APC-mutant CRC cells (DLD-1 cells). Recent reports
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indicate that tankyrase inhibitors suppress CRC with truncated APC in in vivo and ex
vivo mouse models as well as patient-derived cell lines.6,7 These findings provided
important insights to the therapeutic utility of tankyrase inhibitors to treat cancer
harboring aberrant Wnt/β-catenin signaling.
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Since tankyrases possess highly divergent functions, including regulation of the
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WNT/-catenin pathway, telomere elongation, Hippo pathway, mitotic checkpoint,
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and vesicle trafficking, they have attracted significant interest as molecular targets for
cancer treatment.12-14 To date, X-ray crystallographic studies have identified three
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different binding motifs: 1) an adenosine pocket binder such as IWR-1 (2), IWR-2 (3)
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18,19
and G007-LK (4), 2) a nicotinamide pocket binder such as XAV-939 (5)
and RK-
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87107 (1),20,21 and 3) a dual pocket binder such as NVP-TNKS656 (6) (Figure 1).
An important issue in cancer drug development is the balance between efficacy and
toxicity. In a 2013 report, the antitumor activity and the intestinal toxicity of G007-LK, an
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adenosine pocket binder, were carefully studied in a xenograft model. Although G007-
LK showed potent tumor growth inhibition in APC-mutant CRC cell lines (COLO-320DM
and SW403) mouse xenograft models, dose-limiting epithelial degeneration and a
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reduction of crypt proliferation in the small intestine were observed among the mice
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treated for 21 days. In addition, a recent report demonstrated that tankyrase inhibitor
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G-631 (7), a nicotinamide pocket binder, caused dose-dependent intestinal toxicity in
mice while showing modest antitumor effects in a SW403 xenograft model at 100 mg/kg
daily oral dosing. Based on these findings, they concluded that the clinical utility of orally
administered tankyrase inhibitors would be limited due to the “on-target” intestinal
toxicity.24
We recently discovered a tetrahydroquinazoline-based potent tankyrase inhibitor RK-
287107 (1), which shows >200-fold selectivity against other PARP enzymes and modest
20
antitumor effects in a mouse xenograft model without notable weight loss. Given that
multiple binding motifs of tankyrase inhibitors have been reported and that previously
reported tankyrase inhibitors showed variable levels of anticancer effects and intestinal
toxicity, we decided to find tankyrase inhibitors with a reasonable therapeutic window.
Herein we describe the design and discovery of an orally bioavailable, potent and
novel tankyrases RK-582. RK-582 exhibits robust tumor growth inhibitory activity in a
mouse COLO-320DM xenograft model either with oral or intraperitoneal administration.
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RK-582 possesses an excellent pharmacokinetic profile and exhibits a comparable level
of efficacy with RK-287107, at a much lower dose. Concomitant measurements of the
biomarker levels revealed dose-dependent attenuation of active -catenin and elevation
of AXIN2, clearly indicating the inhibition of WNT signaling.
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
RESULTS AND DISCUSSION
Chemistry. Scheme 1 depicts synthesis of tetrahydroquinazolinone analogues. 8-
Methylquinazoline compounds 8 and 9 were synthesized in one step with good yields
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via a substitution reaction between 2-chloro-8-methylquinazolin-4(3H)-one (17) and 1-
hydroxyethylspiroindoline or 1-hydroxyethylspiroindolinone intermediates (20 or 21), 21
respectively. 8-Methyltetrahydropyridopyrimidinone compounds 10 and 11 were
synthesized in three steps in good yields via a substitution reaction between 2-
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chloropyrido[2,3-d] pyrimidin-4(3H)-one (18) and 20 or 21, respectively, methylation of
the pyridine moiety, and hydrogenation with platinum oxide. Pyrrolopyrimidinone
compounds 12 and 13 were prepared via a substitution reaction between 2-chloro-7-
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methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (19) and 20 or 21, respectively.
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Dihydropyrrolopyrimidinone compounds 14 and 15 were prepared by hydrogenation of
12 or 13, respectively.
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Scheme 2 outlines the synthesis of 1-substituted spiroindolinone derivatives.
Following the same or a slightly modified procedure of Scheme 1, 1-methyl substituted
spiroindolinone derivative 11a was synthesized in three steps in a moderate yield from
2-chloropyrido[2,3-d] pyrimidin-4(3H)-one (18) and 1-methyl spiro-intermediate 21a,
21
which was prepared by the published procedure. Compounds (11b–h) were
synthesized efficiently in one step via a substitution reaction between 2-chloro-8-methyl-
5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-one (28), which was prepared from 18 in
four steps, and spiro-intermediates (21b–h) prepared from 24 by the published
procedure.21
Scheme 3 outlines the synthesis of 6-oxysubstituted spiroindolinone derivatives.
Following the same procedure as described above, compounds (37a–j) were
synthesized from the intermediate 28 and spiro-intermediates (36a–j) in one step. To
synthesize the spiro-intermediates (36a–j), regioselective 6-substituted spiro-3-
piperidinylindolinone formation was the key step. tert-Butyl 1-methyl-2-oxospiro[indoline-
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,4'-piperidine]-1'-carboxylate (33) was obtained by 1) gem-bis alkylation of 2-(2,6-
difluoro-4-methoxyphenyl)acetonitrile, which was prepared from palladium coupling
between 2-bromo-1,3-difluoro-5-methoxybenzene and tert-butyl 2-cyanoacetate, and
decarboxylation of tert-butylcarboxylate, piperidine ring cyclization using Boc-
bis(chloroethyl)amine to provide a piperidine-4-carbonitrile (31), 2) subsequent
hydrolysis of the nitrile group, 3) nucleophilic aromatic substitution to form a indolinone
and methylation of the 1-NH position of the indolinone to furnish 33. Spiroindolinone
precursors (36b–j) were obtained by demethylation of 6-methoxy group of 33 using
boron tribromide, restoration of the Boc group (34), and introduction of R groups via an
alkylation (36b and 36j) or a Mitsunobu reaction (36c–i) followed by the deprotection of
the Boc group.
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Syntheses of the 6-aminosubstituted spiroindolinone analogs with various saturated
heterocyclic substituents were accomplished with the procedure outlined in Scheme 4.
Compounds (41a–g) were synthesized from the intermediate 28 and spiro-intermediates
(40a–g) by the same procedure as described above. Spiro-intermediates (40b–g) were
prepared by palladium coupling between cyclic amines and an intermediate 38, which
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was prepared from 34 and triflic anhydride, and deprotection of the Boc group. The
spiro-intermediate 40a was prepared by palladium coupling between dimethylamine
hydrochloride and an intermediate 42 followed by deprotection of the Cbz group.
SAR of analogs beyond the tetrahydroquinazolinone structure. Recently discovered
RK-287107 (1), which is a nicotinamide pocket binder, exhibits potent TNKS/TNKS2
enzyme inhibitory activities with an excellent selectivity against other PARP family
enzymes (PARP1, PARP2, and PARP10) as well as TCF reporter activity in HEK293
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4
5
6
7
8
9
0
21
and DLD-1cells, and cell proliferation of COLO-320DM cells. However, the relatively
high doses of 1 required during the xenograft study prompted us to search for
compounds with significantly improved cell growth inhibition, a 50% growth inhibition
value (GI ) of <0.3 µM, and efficacy against tumor proliferation in a mouse xenograft
50
model.20
We prepared various nicotinamide pocket binders, including the derivatives of 8-
methylquinazolin-4(3H)-one (8 and 9), 8-methyl-5,6,7,8-tetrahydropyrido[2,3-
d]pyrimidin-4(3H)-one (MTPP) (10 and 11), 7-methyl-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (12 and 13) and 7-methyl-3,5,6,7-tetrahydro-4H-pyrrolo[2,3-
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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
2
0
d]pyrimidin-4-one (14 and 15). Compared to 1, the enzyme inhibitory activities of these
newly synthesized compounds are maintained or improved (Table 1). In addition,
compound 10 and 11, derivatives with the MTPP moiety, possess improved activities in
both the cellular TCF reporter and cell proliferation assays. These compounds,
exhibiting the similar TNKSs inhibitory and improved cell growth inhibitory activities with
a GI value of 0.25 M when compared to 1, suggested that the introduction of the
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
50
MTPP moiety may improve the ability of cellular penetration. Table 2 summarizes the
results of physicochemical studies and liver microsomal stability for 10 and 11, and
indicated that PAMPA values of 10 and 11 were almost same as that of 1. These
findings seem to suggest that an increase in the cellular permeability of those
compounds with MTPP moiety does not come from increased lipid membrane
permeability, but other factor(s) influencing an ability of cellular penetration.
Although the human and mouse liver microsomal stabilities of 10 and 11 need to be
improved, the slightly superior in vitro activities, aqueous solubility, free fraction ratio in
blood plasma and microsomal stability of spiroindolinone 11 caught our attention.
Hence, we focused on the indolinone derivatives with the MTPP moiety for further
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optimization.
Table 1. SAR study of analogs of the tetrahydroquinazolinone moiety.
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
O
NH
F
Ring
N
F
N
Y
N
OH
O
Enzyme
TCF reporter
COLO-
20DM
[a]
[b]
NH
IC50 (nM)
IC50 (nM)
3
Cmpd
Y
Ring
[c]
GI50 (M)
N
TNKS
14.3
14.9
5.5
TNKS2
10.6
20.6
8.5
HEK293
DLD-1
77.6
71.2
13.7
4.8
O
1
7
8
CH
2
12.1
2.1
3.1
0.2
1.63
2.27
0.72
2.49
NH
N
C=O
O
CH
2
NH
N
O
C=O
5.3
3.4
9
CH3
10
11
12
13
14
CH
2
13.6
7.5
21.4
13.5
2.7
1.1
7.0
3.9
0.25
0.25
NH
N
N
C=O
CH3
O
CH
2
3.8
4.5
7.0
1.1
1.5
7.1
6.3
3.56
NH
N
N
O
C=O
10.2
>20.0
H3C
CH
2
53.5
14.1
73.6
27.6
36.5
4.4
140.6
32.9
5.01
2.37
NH
N
N
C=O
15
H3C
[
a] Mean values are shown from two independent experiments conducted in duplicate.
[b] Mean values are shown from two independent experiments conducted in triplicate.
[
c] COLO-320DM growth inhibition was evaluated by the CellTiter-Glo cell viability
assay method. Three independent experiments were conducted in triplicate.
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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
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
1
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Table 2. ADME properties of 8-methyl-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-4(3H)-
O
N
O
N
NH
NH
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
F
F
N
N
F
N
N
F
O
N
NH
F
N
OH
N
OH
O
N
F
Compound
1
N
10
11
OH
Aqueous solubility
(pH7.4 PBS; g/mL)
7
2.1
40.2
3.3
82.9
3.0
PAMPA
2
.0
-6
(
4 h; x 10 cm/s)
Microsomal stability: Mouse
18.3 / 3.2
12.8 / 3.2
5.9 / 2.2
0.3 / 0.2
0.4 / 0.2
7.4 / 1.4
21.2 / 8.5
2.0 / 0.5
21.0 / 6.3
3
0min / 60min ꢀ Rat
(Remaining %) Humanꢀꢀ
Protein binding[a]
Human, Mouse; % free)
Hu: 1.5%, Mo:0.4%
Hu: 0.5%, Mo: 0.2%
Hu: 2.2%, Mo: 8.3%
(
one derivatives (10 and 11).
[a] Human (Hu) /Mouse (Mo) plasma protein binding, % value of the free fraction of the
compound.
SAR of the right side spiroindolinone substructure. Analysis of the effects of the
substitution pattern on the spiroindoline moiety in our previous study indicated that 4-
2
halo substituents, especially 4-fluoro substituent (R = F), provide the most potent
21
enzyme, cellular TCF reporter, and cell growth inhibitory activities. The addition of a 5-
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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
3
substituent (R ) attenuates the inhibitory activity, presumably due to the destabilizing
effect by steric repulsion between the 5-substituent and Pro1034 and Gly1074 residues
of tankyrase-2 (data not shown). In contrast, our previous study also suggested the
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
1
4
5
addition of 1, 6, and 7-substituents (R , R , and R , respectively) would have little
adverse effect on the enzyme inhibitory activity, because the direction of these
substituents is toward cytosolic space of the enzyme. However, such an addition should
exert various effects on the drug-like properties (e.g. aqueous solubility and microsomal
stability etc.). Thus, we investigated the structure-activity relationship of the MTPP
1
4
derivatives with substituents at the 1- and 6-position (R and R , respectively) on the
spiroindolinone moiety (Figure 2).
SAR of the 1-substituted spiroindolinone series. Initially, we focused on the effects of
1
the substituent at the 1-position (R ) of the indolinone (Table 3). All the 1-substituted
spiroindolinone derivatives except 11g predictably show the comparable enzymatic and
cellular activities as 11. The aqueous solubility increases for substituents containing
hydroxyl (11 and 11h), nitrile (11d), and carboxymethyl (11g) groups. Only compounds
11g and 11h show improved aqueous solubility and microsomal stability. However, the
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P-glycoprotein (P-gp) efflux ratio of the compounds containing hydroxyl (11 and 11h),
nitrile (11d), sulfone (11e) or oxetane (11f) increases to more than eight as measured
by a cell system method, which involves MDR1 overexpressing MDCKII cells (MDCKII-
MDR1). In particular, the efflux ratio (ER) of 11h is extremely high (ER = 43.7).
Although 11g shows superior in vitro profiles, including aqueous solubility and
microsomal stability, and P-gp efflux activity, its TCF reporter (HEK293 and DLD-1 IC :
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
50
2
21 and 1185 nM, respectively) and cell growth inhibitory (GI : 12.6 M) activities are
50
substantially lower, presumably due to its low cellular permeability. Based on these
findings, further optimizations were performed with derivatives with the 1-methyl moiety.
We comprehensively preferred this moiety on account of the balance of biological
activities and drug-like properties such as solubility and P-gp efflux activity. Next we
shifted our focus to the substituent at the 6-position of the spiroindolinone derivatives.
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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 3. SAR development of 1-substituted spiroindolinone series.
O
NH
F
4
N
N
N
F
0
1
2
3
4
5
6
7
8
9
0
6
N
1
O
R1
[
d
Enzyme
TCF reporter
COLO- Aq.Sol.
P-gp ER[
f]
Hu/Mo
[
a]
[b]
]
IC50 (nM)
IC50 (nM)
320DM
R1
[e]
Cmpd
MDCK-MDR1
+ / - (NER)
Mic.Stab.
GI50
(
pH7.4,
[
c]
(% @ 60 min)
TNKS
TNKS2
HEK293
DLD-1
(M)

g/mL)
1 11
2
CH
CH
OH
7.5
7.9
13.5
10.7
11.7
32.4
6.1
1.1
3.9
0.3
0.2
0.2
0.1
0.3
221
0.7
3.9
14.4
3.0
0.25
0.22
0.05
0.07
0.44
0.53
0.20
12.6
0.71
82.9
16.0
6.3
6.3 / 2.0
0.3 / 0.0
1.8 / 0.1
8.0 / 1.0
6.1 / 1.2
4.1 / 0.1
0.0 / 0.3
100 /100
23.9 / 42.7
24.6 / 6.4 (3.8)
3.1 / 1.1 (2.8)
4.8 / 1.3 (3.7)
2.0 / 0.7 (2.9)
11.7 / 2.3 (5.1)
12.0 / 3.3 (3.6)
8.4 / 1.2 (7.0)
0.5 / 0.4 (1.3)
43.7 /11.2 (3.9)
2
2
11a
11b
11c
11d
11e
CH
CH
CH CF
CH CN
CH SO Me
3
3
4
5
6
7
8
9
0
1
2
CH
2
2
OMe
10.3
16.2
3.7
2
3
2.4
1.3
2
1.6
50.5
3.0
2
2
4.3
5.5
5.7
O
11f
7.8
12.2
29.8
12.5
7.2
9.3
3 11g
4
CH
2
COOH
28.5
8.9
1185
14.5
92.1
88.2
11h CH
2
CHOHCH OH
2
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
[a] Mean values are shown from two independent experiments conducted in duplicate.
b] Mean values are shown from two independent experiments conducted in triplicate.
c] COLO-320DM growth inhibition was evaluated by the CellTiter-Glo cell viability
[
[
assay method. Three independent experiments were conducted in triplicate. [d] Aq.Sol.:
Aqueous solubility in pH 7.4 phosphate buffer. [e] Hu/Mo Mics.Stab.: Liver microsomal
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stability was determined by the remaining % of compound after 60 min of incubation
with human (Hu) or mouse (Mo) liver microsome according to the protocol described in
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
21
our previous report. [f] P-gp ER MDCK-MDR1+ / - : Efflux ratio (ER:
PappBtoA/PappAtoB) of MDR1 overexpressing MDCKII (+) or wild-type MDCKII (-) cells
was determined. Net efflux ratio (NER) was calculated as the ratio of the value of
MDCKII-MDR1 to MDCKII.
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5
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
Effects of 6-alkoxy substituents of the spiroindolinone series. Table 4 summarizes the
in vitro activities and ADME profiles of compounds with various substituents at the 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
4
position of the indolinone ring. Replacement of the 6-fluoro group (R ) of 11a with
substituted methoxy groups improves or maintains the enzyme, cellular TCF reporter
and cell growth inhibitory activities (37a–i). In contrast, the TCF reporter inhibitory
activity of 37j, which possesses a carboxymethoxy moiety, is reduced, presumably by
the poor cellular permeability. Surprisingly 37j maintains the cell growth inhibitory
activity with a GI value of 0.3 M. To confirm whether this phenomenon is caused by
50
tankyrase inhibition or not, we investigated the protein expression levels of AXIN2 and
-catenin, which are biomarkers of Wnt/-catenin signaling. Given that the biomarker
levels and cell growth inhibitory activity are not well correlated with each other, other
mechanisms may be involved (Supporting Information Figure S1).
From the viewpoint of ADME properties, 37b, which has a 2-hydroxyethoxy group,
exhibits an improved aqueous solubility, but unfortunately indicates the poor microsomal
stability and elevated P-gp efflux activity. Compounds (37d–i), which are substituted
with a basic amino group, show excellent aqueous solubility and microsomal stability,
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while the P-gp efflux liability deteriorates.
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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5
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
Table 4. Effects of 6-alkoxy substituents of the spiroindolinone series.
O
NH
F
4
N
N
N
R4
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
6
N
1
O
Enzyme
TCF reporter
[d
COLO-
320D
Hu/Mo
Aq.Sol.
P-gp ER[
f]
[a]
[b]
[
e]
IC50 (nM)
IC50 (nM)
]
Mic.Stab.
R4
cmpd
MDCK-MDR1
/ - (NER)
M GI50
(pH7.4,
(% @ 60
min)
TNKS
+
TNKS
HEK293
DLD-1
[c]
(M)
g/mL)
2
3
7a
7b
-OCH
3
4.4
3.5
6.2
4.7
0.2
0.8
0.1
0.3
0.4
2.5
4.5
2.3
1.3
6.1
6.6
5.0
0.13
0.16
0.05
0.81
1.08
1.09
4.8
75.1
11.4
95.0
100.1
100.7
1.9 / 0.0
19.8 / 0.7
1.8 / 13.7
54.4 / 83.7
61.5 / 100
18.5 / 56.0
2.7 / 1.1 (2.5)
14.9 / 6.2 (2.4)
7.5 / 2.1 (3.6)
22.7 / 9.2 (2.5)
21.9 / 7.1 (3.1)
6.1 / 3.2 (1.9)
3
-OCH
2
CH
2
OH
37c -OCH
2
CH
2
OCH
3
4.8
6.4
O
N
N
3
7d
7e
4.9
11.1
12.4
33.3
O
O
O
3
11.1
12.0
N
37f
N
N
3
7g
7h
9.2
17.1
0.3
2.8
0.33
101.1
1.5 / 41.4
29.1 / 5.1 (5.7)
O
N
O
O
3
7.9
6.1
4.2
18.7
21.2
9.9
1.2
0.1
6.3
2.7
0.74
0.66
0.30
188.0
102.5
98.4
56.5 / 83.5
22.4 / 60.3
97.6 / 93.3
86.4 / 13.1 (6.6)
11.7 / 4.6 (2.5)
0.3 / 0.3 (1.0)
37i
7j
N
3
-OCH
2
CO
2
H
507
1877
[
[
[
a] Mean values are shown from two independent experiments conducted in duplicate.
b] Mean values are shown from two independent experiments conducted in triplicate.
c] COLO-320DM growth inhibition was evaluated by the CellTiter-Glo cell viability
assay method. Three independent experiments were conducted in triplicate. [d] Aq.Sol.:
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Aqueous solubility in a pH 7.4 phosphate buffer. [e] Hu/Mo Mics.Stab.: Human/mouse
liver microsomal stability. [f] P-gp ER MDCK-MDR1+ / - : Efflux ratio (ER:
PappBtoA/PappAtoB) of MDR1 overexpressing MDCKII (+) or wild-type MDCKII (-) cells
was determined. Net efflux ratio (NER) was calculated as the ratio of the value of
MDCKII-MDR1 to MDCKII.
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
Effects of 6-amino substituents of the spiroindolinone series. Table 5 summarizes
the results of the SAR studies of compounds with various saturated heterocycle
substituents at the 6-position of the indolinone moiety.
The enzyme (IC = 7–40 nM) and TCF reporter (0.1–30 nM) inhibitory activities can
50
tolerate with the introduction of a saturated heterocyclic moiety at the 6-position,
displaying the improved cell proliferation inhibitory activity (GI : <1.2 μM) and the
50
acceptable aqueous solubility (41c–g) when compared to 1. Compounds 41a and 41b
show the good enzyme and TCF reporter inhibitory activities, and the acceptably low P-
gp efflux ratios, but disappointingly exhibited the poor aqueous solubility and
microsomal stability. Compounds (41c–e and 41g) exhibited the moderate stability in
human and mouse liver microsomes. But compounds (41c, 41d, and 41e) with basic
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2
3
4
5
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
amino groups (pKa: >7.5) and 41f with a 4-morpholinyl moiety show the moderate to
high P-gp efflux ratio.
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
Compound 41g with a 2,6-dimethylmorpholine moiety at the 6-position, shows the
excellent P-gp efflux activity, improved microsomal stability and aqueous solubility.
Compared to unsubstituted morpholine compound 41f, the P-gp efflux ratio (MDCK-
MDR1) of 41g is dramatically decreased from 7.8 to 1.0 due to the introduction of two
methyl groups on the morpholine moiety.
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Table 5. Effects of 6-amino substituents of the spiroindolinone series.
O
NH
F 4
N
N
N
R4
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
6
N
O
1
Enzyme
TCF reporter
[d
COLO-
320DM
Hu/Mo
Aq.Sol.
P-gp ER[f]
[
a]
[b]
[
e]
IC50 (nM)
IC50 (nM)
]
Mic.Stab.
R4
cmpd
MDCK-MDR1
GI50
(
pH7.4,
(% @ 60
min)
TNKS
2
+
/ - (NER)
TNKS
HEK293
DLD-1
[c]
(
M)
g/mL)
4
1a
41b
1c
41d
1e
1f
1g
6.3
21.3
12.8
7.7
13.0
49.1
23.4
12.3
20.2
10.5
39.2
4.6
0.7
3.2
0.9
0.1
0.1
0.3
20.7
6.4
1.17
0.30
1.12
0.19
0.85
0.10
0.23
7.4
1.8 / 1.8
1.5 / 10.5
28.6 / 63.7
30.5 / 54.5
33.0 / 82.7
6.3 / 46.5
28.7 / 55.9
2.7 / 1.1 (2.5)
2.3 / 1.0 (2.3)
9.1 / 6.8 (1.3)
13.2 / 3.1 (4.3)
23.1 / 6.2 (3.7)
7.8 / 1.9 (4.1)
1.0 / 0.9 (1.1)
N
N
5.0
NH
N
N
N
4
29.1
8.4
97.3
94.5
102.1
72.2
34.1
NH
O
N
N
4
11.1
14.9
36.1
4.3
4
2.0
O
N
4
3.1
[
[
[
a] Mean values are shown from two independent experiments conducted in duplicate.
b] Mean values are shown from two independent experiments conducted in triplicate.
c] COLO-320DM growth inhibition was evaluated by the CellTiter-Glo cell viability
assay method. Three independent experiments were conducted in triplicate. [d] Aq.Sol.:
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2
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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
Aqueous solubility in a pH 7.4 phosphate buffer. [e] Hu/Mo Mics.Stab.: Human/mouse
liver microsomal stability. [f] P-gp ER MDCK-MDR1+ / - : Efflux ratio (ER:
PappBtoA/PappAtoB) of MDR1 overexpressing MDCKII (+) or wild-type MDCKII (-) cells
was determined. Net efflux ratio (NER) was calculated as the ratio of the value of
MDCKII-MDR1 to MDCKII.
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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Table 6 summarizes the detailed characterization of compounds 41d and 41g. Most
of the biological and physicochemical properties of 41d are superior or equivalent to 41g.
Compound 41d provided slightly more favorable aqueous solubility and a free fraction
ratio of plasma protein binding (aqueous solubility: 94.4 g/mL; protein binding: 0.4 and
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
1.9% in human and mouse plasma, respectively) than 41g (aqueous solubility: 34.1
g/mL; protein binding: 0.3 and 0.6% in human and mouse plasma, respectively). And
the microsomal stabilities of both 41d and 41g are comparable. Although 41g does not
suffer from P-gp mediated efflux [Net efflux ratio (NER) = 1.1], 41d has some issues
with the P-gp mediated efflux [NER = 4.3, and the efflux ratio of MDCKII-MDR1 is high
(ER = 13.2)]. In mouse pharmacokinetic studies (50 mg/kg, IP), Cmax and AUC values
inf
of 41g are two- and four-times higher than those of 41d, respectively. This may be due
to the difference in the P-gp mediated efflux activity (Table 6; Supporting Information
Figure S2). Surprisingly, 41d was ineffective for tumor growth inhibition in a preliminary
mouse xenograft study, despite its superior cellular activity and lower protein binding.
(Data not shown) We speculated that P-gp, which is a clinically important
transmembrane transporter that confers a multi-drug resistance phenotype to cancer
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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
cells, might contribute to pharmacokinetic profile and drug resistance to result in the
28
lack of in vivo efficacy of 41d. Hence, compound 41g was chosen as the best
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
candidate for further development.
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Table 6. Comparison of the physicochemical/biochemical properties and PK profiles of
O
N
O
N
41d and 41g.
NH
NH
F
F
N
O
N
N
N
N
N
N
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
N
N
O
O
Compound
Aqueous solubility (pH7.4 in PBS; g/ml))
41d
94.5
12.8
41g
34.1
12.4
-6
PAMPA (4 h; x 10 cm/s)
P-gp efflux: NER[a]
MDCKII-MDR1
MDCKII
4.3
13.2
3.1
1.1
1.0
0.9
Liver microsomal stability: Mouseꢀꢀꢀ
54.9 / 30.5
80.2 / 65.3
72.7 / 54.5
49.7 / 28.7
57.3 / 44.0
73.0 / 55.9
3
0min / 60min
Ratꢀꢀꢀꢀ
(Remaining %)
Humanꢀꢀꢀ
Human/Mouse protein binding: (% free)
Hu: 0.4%, Mo: 1.9%
.5
H: 0.3%, Mo: 0.6%
1
[b, d]
0
Mouse IP administration:
t
max (h)
max (μmol/L)
1/2 (h)
C
53.6
102
t
2.5
207
0.25
1.9
607
1
AUCinf (μmol h/L)
[
c, d]
Mouse PO administration:
t
max (h)
max (μmol/L)
1/2 (h)
C
21.2
4.0
77
121
1.8
t
AUCinf (μmol h/L)
594
[
a] Net efflux ratio (NER) was calculated as the ratio of the value of MDCKII-MDR1 to
MDCKII. [b] Intraperitoneal administration of 41d and 41g at a dose of 50 mg/kg to male
ICR mouse. [c] Oral administration of 41d and 41g at a dose of 50 mg/kg to male ICR
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