Diaminopyridine-based potent and selective Mps1 kinase inhibitors binding to an unusual flipped-peptide conformation

Article abstract of DOI:10.1021/ml3000879

Monopolar spindle 1 (Mps1) is an attractive cancer drug target due to the important role that it plays in centrosome duplication, the spindle assembly checkpoint, and the maintenance of chromosomal stability. A design based on JNK inhibitors with an aminopyridine scaffold and subsequent modifications identified diaminopyridine 9 with an IC₅₀ of 37 nM. The X-ray structure of 9 revealed that the Cys604 carbonyl group of the hinge region flips to form a hydrogen bond with the aniline NH group in 9. Further optimization of 9 led to 12 with improved cellular activity, suitable pharmacokinetic profiles, and good in vivo efficacy in the mouse A549 xenograft model. Moreover, 12 displayed excellent selectivity over 95 kinases, indicating the contribution of its unusual flipped-peptide conformation to its selectivity.

Full text of DOI:10.1021/ml3000879

Letter
pubs.acs.org/acsmedchemlett
Diaminopyridine-Based Potent and Selective Mps1 Kinase Inhibitors
Binding to an Unusual Flipped-Peptide Conformation
Ken-ichi Kusakabe,*,† Nobuyuki Ide,† Yataro Daigo,‡,§ Takeshi Itoh,† Kenichi Higashino,†
Yousuke Okano,† Genta Tadano,† Yuki Tachibana,† Yuji Sato,† Makiko Inoue,† Tooru Wada,†
Motofumi Iguchi,† Takayuki Kanazawa,† Yukichi Ishioka,† Keiji Dohi,† Sachie Tagashira,† Yasuto Kido,†
Shingo Sakamoto,† Kazuya Yasuo,† Masahiro Maeda,† Takahiko Yamamoto,† Masayo Higaki,†
Takeshi Endoh,† Kazuo Ueda,† Takeshi Shiota,† Hitoshi Murai,† and Yusuke Nakamura‡
†Medicinal Research Laboratories, Drug Developmental Research Laboratories, and Innovative Drug Discovery Research
Laboratories, Shionogi Pharmaceutical Research Center, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka 561-0825, Japan
‡Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1
Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
§Department of Medical Oncology, Shiga University of Medical Science, Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
S
* Supporting Information
ABSTRACT: Monopolar spindle 1 (Mps1) is an attractive cancer drug
target due to the important role that it plays in centrosome duplication, the
spindle assembly checkpoint, and the maintenance of chromosomal
stability. A design based on JNK inhibitors with an aminopyridine scaffold
and subsequent modifications identified diaminopyridine 9 with an IC50 of
37 nM. The X-ray structure of 9 revealed that the Cys604 carbonyl group
of the hinge region flips to form a hydrogen bond with the aniline NH
group in 9. Further optimization of 9 led to 12 with improved cellular
activity, suitable pharmacokinetic profiles, and good in vivo efficacy in the
mouse A549 xenograft model. Moreover, 12 displayed excellent selectivity
over 95 kinases, indicating the contribution of its unusual flipped-peptide
conformation to its selectivity.
KEYWORDS: monopolar spindle 1, Mps 1, TTK, inhibitor, diaminopyridine, peptide flip, cancer
onopolar spindle 1 (Mps1), also known as TTK, is a
dual-specificity protein kinase that is important for
M
centrosome duplication, the spindle assembly checkpoint, and
the maintenance of chromosomal stability.1−3 Mps1 is activated
during mitosis and highly expressed in cancer cells.4−7 A recent
report has shown that siRNA depletion of Mps1 resulted in
aberrant mitoses and induction of apoptosis in breast cancer
cells, which ultimately decreased the chance of survival. In
contrast, there has been no significant increase in apoptosis in
Mps1-depleted nonmalignant cells. Furthermore, Mps1 deple-
tion by shRNA leads to apoptosis and reduced tumor volume in
breast cancer xenograft models.7 Such evidence points to Mps1
as a promising target in the development of anticancer
therapeutics.
Figure 1. Structure of NMS-P715 (1).
et al. screened 577 compounds against 203 kinases, successfully
analyzing them and creating a kinase structure−activity
relationship (SAR) similarity tree (which is analogous to the
kinome phylogenetic tree), using similarity matrices derived
from their SAR data. According to the SAR similarity tree,
Only a few classes of Mps1 inhibitors have been
reported.8−10 For example, Nerviano Medical Sciences
disclosed pyrimidine-based inhibitor 1 (NMS-P715), which
shows antiproliferation effects over a large cancer cell line panel
and inhibits tumor growth in A2780 ovary carcinoma and A375
melanoma xenograft models (Figure 1).8
Received: April 7, 2012
Accepted: June 6, 2012
Published: June 6, 2012
c-Jun N-terminal kinase (JNK) inhibitors seem to be good
templates for the design of novel Mps1 inhibitors. Bamborough
© 2012 American Chemical Society
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Mps1 and JNK1 are grouped together, while they are in
different sub-branches in the kinome phylogenetic tree.11
Interestingly, despite sharing only 27% kinase domain sequence
identity between Mps1 and JNK1, they possess SAR similarity.
One possible explanation for this is that the gatekeeper residues
between these kinases are conserved (Met). However, Met is a
frequent gatekeeper residue12 and thus using other specific
ATP site-residues may make it possible to gain this similarity in
addition to the gatekeeper residues. Indeed, JNK inhibitor
SP600125 is a highly potent inhibitor of Mps1.13,14 Although
SP600125 is also known as a pan-kinase inhibitor, it has
induced mitotic phenotypes in human cells consistent with
Mps1 inhibition.3,15 Taken together, it is likely that analogue
synthesis and isosteric replacements of JNK inhibitors provide
an effective method for identifying novel Mps1 inhibitors.
Abbott Laboratories has reported the development of
aminopyridine-based JNK inhibitors (Figure 2).16,17 Amino-
Scheme 1. Synthesis of 6
a
Scheme 2. Synthesis of 9
a
Reagents and conditions: (a) Et3N, DMSO, 180 °C (microwave). (b)
Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux. (c) (i) Aqueous NaOH,
DMSO, rt; (ii) HATU, Et3N, aqueous NH3, DMF, rt.
Figure 2. Abbott JNK inhibitors.
pyridine is an attractive scaffold for the lead compound because
these analogues show a unique binding mode and excellent
selectivity over 77 kinases. As a first step, we synthesized
aminopyridine-based JNK inhibitor 2 and evaluated its
inhibitory activity. As expected, 2 showed good potency for
Mps1 (IC50 = 568 nM), which encouraged us to initiate our
medicinal chemistry efforts by utilizing the aminopyridine
scaffold to discover a selective Mps1 inhibitor.
a
Scheme 3. Synthesis of 12
In this paper, we report on the design and synthesis of highly
selective Mps1 inhibitors, which were found to reduce cancer
cell proliferation and inhibit tumor growth in vivo. The X-ray
structure of diaminopyridine 9 revealed that the Cys604
carbonyl group of the hinge region flips to form a hydrogen
bond with the aniline NH group in 9, which is likely contribute
to the high selectivity of the optimized compound 12 for other
kinases.
The synthesis of aminopyridine analogues is described in
Scheme 1−3. Compound 6 was synthesized by heating
compound 5 with phenethylamine (Scheme 1).17 The synthesis
of compounds 7 and 9 is illustrated in Scheme 2. Compound 5
was coupled with aniline and methyl 4-aminobenzoate using
palladium-mediated amination to give compounds 7 and 8,
respectively. The hydrolysis of ester 8 followed by a HATU-
mediated amide coupling gave compound 9. Scheme 3 depicts
the synthesis of compound 12. Bromopyridine 1016 was reacted
with t-butylamine in N-methylpyrrolidone (NMP) at 250 °C
using a microwave reactor to give triaminopyridine 11, which
was then coupled with methyl 4-iodobenzoate followed by
hydrolysis and amide coupling to give the final compound 12.
We initially evaluated the SAR for compounds 2−4 as
reported by Abbott (Table 1). Under our assay conditions,
a
Reagents and conditions: (a) t-Butylamine, NMP, 250 °C (micro-
wave). (b) (i) Methyl 4-iodobenzoate, Pd(OAc)2, xantphos, Cs2CO3,
dioxane, reflux; (ii) aqueous NaOH, DMSO, rt; (iii) HATU, Et3N,
aqueous NH3, DMF, rt.
Table 1. SAR of Aminopyridines
IC50 (nM)
compd
Mps1
JNK1
4.7
2
3
568
529
4200
3970
228
37
350
260
4
6
>100000
1035
7
9
190
12
6.4
231
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ACS Medicinal Chemistry Letters
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JNK1 IC50 values of these compounds were comparable to the
published values.16,17 Introduction of a methylsulfonyl group
was important for the JNK1 inhibitory activity, while such
modification did not affect the Mps1 inhibitory activity as
shown by compounds 2 and 3. Reversal of the amide
connectivity resulted in compound 4 with an 8-fold loss in
Mps1 activity, although 4 retained JNK1 activity. The X-ray
crystal structures of Abbott's aminopyridine derivatives with
JNK1 confirmed that the carbonyl oxygen at the 6-position of
the pyridine forms a hydrogen bond with the Met111 NH
group.16,17 We therefore explored carbonyl surrogates for this
6-position. However, introduction of sulfonamides, ureas, and
heterocycles (oxazoles and oxadiazoles) led to reduced Mps1
activity (data not shown). Interestingly, removal of the amide
carbonyl group of compound 4 led to the phenethylamine
analogue 6, which retained Mps1 activity, while JNK1 activity
was completely abolished. Encouraged by this result, we
investigated a number of amine analogues and discovered that
aniline analogue 7 had a higher Mps1 potency than compounds
2 and 3. Finally, introduction of the amide group at the 4-
position of the phenyl group in 7 gave rise to compound 9 with
a 7-fold increase in Mps1 activity (IC50 = 37 nM).
binding site of Mps1 revealed that the Cys604 carbonyl group
of the hinge region flips to form a hydrogen bond with the
aniline NH group in 9 (2.90 Å). The amino group at the 4-
position of the pyridine in 9 also engages in a hydrogen bond
with the carbonyl of Glu603 (2.94 Å). The pyridine lies in a
hydrophobic pocket defined by Ile531, Val539, Leu654, and
Ile663, and the cyano group on the pyridine ring points to the
back pocket of the conserved Lys553. The phenyl ring also has
van der Waals contact with Ile531 and Leu654 and is pointed
toward the solvent. On the other hand, there are no clear
interactions between the amide group on the phenyl ring and
the protein in spite of the fact that this amide group contributes
to a 6-fold increase in activity, as found for compounds 7 and 9.
Similar peptide flips at the hinge region have been observed
in X-ray structures of quinazolinones,18,19 pyridol-pyrimi-
dines,18 phthalazines,20,21 pyridazinopyridinones,22 PH-
797804,23 and VX-74524 bound to p38 MAP kinase and a
quinazoline bound to Mps1.25 As illustrated in Figure 4a, the
peptide flip in the p38α complexed with pyridol-pyrimidine 13
occurred at the main chain nitrogen between Met109 and
Gly110. The G110A mutation of p38α resulted in a 14-fold
decrease in activity, indicating that Gly110 makes the peptide
flip more favorable. In our case, the peptide flip in Mps1
occurred at the main chain carbonyl of Cys604 (Figure 4b), and
Gly605 next to Cys604 is thought to contribute to the peptide
flip at Cys604.
Given the X-ray structure of aminopyridines with JNK1
reported by Abbott,16,17 we were surprised to discover that the
aniline analogues 7 and 9 exhibited Mps1 activity despite the
absence of the carbonyl group at the pyridine 6-position. To
elucidate the binding mode of aniline analogues, the X-ray
structure of 9 with Mps1 was obtained at 2.40 Å resolution
(Figure 3). To our surprise, the cocrystal structure in the ATP
Next, we turned our attention to the C2 side chain of the
pyridine ring to further improve enzymatic activity and
selectivity over JNK1. The crystal structure revealed that the
C2 side chain of the ethyl ether lies in the ribose pocket that
consists of hydrophobic residues Val539 and Ile663. Therefore,
we examined a small set of hydrophobic substituents and found
that substitution with a t-butylamino group (12) led to a 6-fold
improvement in potency with a 36-fold selectivity for Mps1
over JNK1.
Once the highly potent Mps1 inhibitors 9 and 12 were
identified, we examined the cellular effects of the compounds
(Table 2). To investigate the cellular inhibition of Mps1, we
Table 2. Cellular Activity and Mouse PK Profile of 12
cellular activity
pMps1 IC50 A549 IC
mouse PK po, 25 mg/kg
Cmax (ng/
Tmax
d
AUCpo (ng h
a
b50
c
e
(nM)
(nM)
mL)
(h)
/mL)
131
840
3542
0.25
6604
a
b
Figure 3. Crystal structure of 9 bound to Mps1 (PDB ID: 3VQU).
Inhibition of autophosphorylation. Cell viability in A549 cells at 72
c
d
h. Maximum plasma concentration. Time of maximum plasma
concentration. Area under the curve.
e
Figure 4. Comparison of the ligand-induced peptide flip in p38 Map kinase and Mps1. (a) Schematic diagram of compound 13 bound to p38 at the
hinge region. (b) Compound 9 bound to Mps1 at the hinge region. The flipped residues are colored blue.
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developed an autophosphorylation assay using a cell line that
stably expresses FLAG-tagged Mps1 under the control of a
tetracycline (Tet)-suppressible promoter. Compound 12
showed an IC50 value of 131 nM, while 9 showed weak
inhibition (1.8 μM) in this autophosphorylation assay. These
antiproliferative activities were also examined in A549 lung
carcinoma cell lines. Compounds 9 and 12 inhibited the growth
of these cell lines with IC50 values of 9.5 and 0.84 μM,
respectively. The antiproliferative effects correlated with the
cellular inhibition of Mps1.
advanced inhibitors of Mps1. Diaminopyridine 12 also
exhibited good cellular activity and pharmacokinetic properties
and was efficacious in the A549 lung cancer xenograft model.
Additionally, 12 has good leadlike profiles: low MW (324),
CLog P (2.8), and high LE (0.47). The selective Mps1 inhibitor
12 with in vivo efficacy could be used as a valuable tool for
elucidating the biological functions in cancer therapy.
ASSOCIATED CONTENT
■
S
* Supporting Information
To evaluate kinase selectivity, compound 12 was screened
against a panel of 95 kinases. We found that 12 displayed an
excellent selectivity profile in this panel, except for the Flt3 and
Flt3 mutants (D835Y) (50 and 86% inhibition at 1 μM,
respectively; Supporting Information). This selectivity profile
can be explained by the crystal structure of the enzyme and the
inhibitor complex. Our compounds form hydrogen bonds with
the flipped carbonyl of Cys604 next to Gly605 and the carbonyl
of Glu603 at the hinge region in an unusual manner. Therefore,
our Mps1 inhibitors do not have the ability to bind to the
“usual” hinge regions of other kinases, which accounts for the
excellent kinase selectivity of 12.
The pharmacokinetic (PK) profile of 12 was studied using
mice (Table 2). Compound 12 showed good PK properties
with a Cmax of 3542 ng/mL and AUC of 6604 ng h/mL at an
oral dose of 25 mg/kg. Encouraged by these results, the in vivo
efficacy of 12 was examined with the A549 xenograft model.
Mice bearing xenografts were dosed orally once daily with an
escalating dose from 25 to 100 mg/kg for 19 days. The results
showed that 12 inhibited the growth of A549 cells in a dose-
dependent manner (Figure 5). At a dose of 100 mg/kg, 12
exhibited 47% tumor growth inhibition without body weight
loss.
Experimental details for the synthesis and characterization of
compounds 6, 7, 9, and 12; details of Mps1 enzyme inhibition,
cell proliferation, autophosphorylation, and in vivo antitumor
assays; crystallographic methods for the crystal structure of
compound 9; and kinase selectivity data for compound 12. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Accession Codes
PDB ID: 3VQU (Figure 3 crystal structure).
AUTHOR INFORMATION
Corresponding Author
■
*Tel: +81(6)6331 8081. Fax: +81(6)6332 6385. E-mail: ken-
ichi.kusakabe@shionogi.co.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Natsuki Ishizuka, Kazuya Okamoto, and Masahiko
Hujioka for their synthetic support and Rikio Ikenishi and
Yumiko Takagi for the elemental analysis. K.K. also acknowl-
edges Robert Hall for his assistance in preparing this
manuscript. We gratefully thank Kenji Abe, Yoshiyuki Matsuo,
Hirosato Kondo, and Kohji Hanasaki for their helpful advice
and suggestions during the course of this research.
ABBREVIATIONS
■
BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; DMF,
N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Et3N,
triethylamine; JNK, c-Jun N-terminal kinase; Mps1, monopolar
spindle 1; NMP, N-methylpyrrolidone; SAR, structure−activity
relationship; Tet, tetracycline, Pd(OAc)2, palladium(II) acetate;
Xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene
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