3-Cyano-6-(5-methyl-3-pyrazoloamino)pyridines: Selective Aurora A kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.119

A new class of Aurora A kinase inhibitor was created by transforming 4-(5-methyl-3-pyrazoloamino)pyrimidine moiety of VX-680 to 3-cyano-6-(5-methyl- 3pyrazoloamino)pyridine. Compound 6 exhibited a potent Aurora A kinase inhibitory activity, excellent selectivity to Aurora B kinase and other 60 kinases, good cell permeability and good PK profile. Therefore compound 6 was effective in antitumor mice model at a dose of 30 mg/kg po qd without decrease of body weight.

Full text of DOI:10.1016/j.bmcl.2010.04.119

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4709–4711
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
3-Cyano-6-(5-methyl-3-pyrazoloamino)pyridines: Selective Aurora A
kinase inhibitors
Ryoichi Ando a, Hiroshi Ikegami a, Makoto Sakiyama a, Shinsuke Ooike b, Masayuki Hayashi b,
Yasuhiro Fujino b, Daisuke Abe a, Hideo Nakamura b, Tadashi Mishina a, Harutoshi Kato c, Yumiko Iwase d,
Hideo Tomozane a, Masahiko Morioka a,
*
a Medicinal Chemistry Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
b Pharmacology Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
c DMPK Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda, Saitama 335-8505, Japan
d Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1-1-1, Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of Aurora A kinase inhibitor was created by transforming 4-(5-methyl-3-pyrazoloami-
no)pyrimidine moiety of VX-680 to 3-cyano-6-(5-methyl-3pyrazoloamino)pyridine. Compound 6 exhib-
ited a potent Aurora A kinase inhibitory activity, excellent selectivity to Aurora B kinase and other 60
kinases, good cell permeability and good PK profile. Therefore compound 6 was effective in antitumor
mice model at a dose of 30 mg/kg po qd without decrease of body weight.
Received 23 February 2010
Revised 23 April 2010
Accepted 27 April 2010
Available online 25 May 2010
Ó 2010 Published by Elsevier Ltd.
Keywords:
Aurora kinase
Cyanopyridine
Aminopyrazole
The Aurora kinase is a family of highly conserved serine/threo-
nine protein kinases1 found to be involved in multiple mitotic
events.2 There are three homologs in human. Aurora A and Aurora
B are ubiquitously present in various cells, whereas Aurora C is
highly expressed only in testis.3
N
N
N
N
H
N
NH
N
N
NH
N
N
Aurora A or Aurora B kinase is one of attractive targets for can-
cer therapy. Some Aurora kinase inhibitors have proven effective
on a wide range of tumor types in clinical trials.4 These clinical data
are very encouraging and promising for development of structur-
ally different Aurora kinase inhibitors.
O
N
N
H
S
N
N
H
ENMD-2076
VX-680
Figure 1. Structures of VX-680 and ENMD-2076.
The most widely studied Aurora kinase inhibitor would be
VX-680 (MK-0457) (Fig. 1) which inhibits Aurora A, B, and C
kinases with Ki values of 0.6, 18, and 4.6 nM, respectively.5 It also
inhibits FLT-3 (fms-related tyrosine kinase 3), which is also attrac-
tive as antitumor target, with a Ki of 30 nM, but it shows good
selectivity against other protein kinases.5,6 One drawback of VX-
680 is poor bioavailability. So it was administrated by iv infusion.7
VX-689 (MK-5108) is a successor of VX-680 by Vertex and
Merck group (structure is unknown). It is administrated as po
formulation. ENMD-2076 (Fig. 1) is developed as an orally active
analogue of VX-680. IC50 values against Aurora A and Aurora B
kinase inhibition are 14 and 290 nM, respectively, but this is a
non-selective kinase inhibitor.8
We report herein design and synthesis of a new class of orally
active Aurora A kinase inhibitor by transformation of core struc-
ture of VX-680.
We focused on a bioisoster structure of 4-(5-methyl-3-pyrazolo-
amino)pyrimidine moiety of VX-680. In our hypothesis, 5-methyl-3-
pyrazoloamino moiety and near by one nitrogen atom of pyrimidine
ring was essential to form multiple hydrogen-bonds with main-
N
N
NH
NH
N
N
N
H
N
N
H
N
* Corresponding author. Tel.: +81 45 963 3369; fax: +81 45 963 4293.
E-mail address: Morioka.Masahiko@ma.mt-pharma.co.jp (M. Morioka).
Scheme 1. Transformation of core structure of VX-680.
0960-894X/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2010.04.119

4710
R. Ando et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4709–4711
H
N
N
H
N
N
NH
N
NH
N
O
O
S
N
N
H
S
N
N
H
2
1
Figure 2. Structures of lead compounds.
chain amide residues of E211 and A213 at the hinge region of the
kinase.9,10 So we designed 3-cyano-6-(5-methyl-3-pyrazoloami-
no)pyridine as a new core structure in the point of electronically
similar property of six-membered aromatic ring (Scheme 1).
As first specific compound, we designed compound 1 (Fig. 2);
the substituent at 4-position of pyridine ring was phenyl group,
not 4-methyl-1-piperazinyl group as seen in VX-680 or ENMD-
2076, because of easiness of synthesis.11
A Ki value of compound 1 against Aurora A kinase inhibition
was 310 nM.12 It was about 150 times less potent than VX-680
whose Ki value was 2.0 nM by our assay system, but we thought
that 310 nM was potent enough as a new lead compound. One of
problems of compound 1 was a high C log P value (6.03).
Further transformation of compound 1 encouraged us. Namely,
replacement of 4-phenyl moiety of pyridine ring to methyl group
improved inhibitory activity. For example, a Ki value of compound
2 (Fig. 2) was 5.3 nM. This is great advancement because the
inhibitory activity was markedly improved, the starting compound
3-cyano-2,6-dichloro-4-methylpyridine 3 was commercially avail-
able, synthesis of derivatives is very easy, and C log P value became
3.94 which was much preferable as a lead compound to 6.03 of
compound 1.
Table 1
Inhibitory activity against Aurora A kinase
Compound
R–X–
Kia (nM)
5a
5b
5c
5d
5e
5f
CH3(CH2)5–S–
HO(CH2)6–S–
H2N(CH2)6–S–
HO(CH2)2–NH–
HO(CH2)6–NH–
HO(CH3)2C(CH2)5–S–
HO(CH3)2C(CH2)5–NH–
3.8
8.5
28
20
4.5
9.7
2.3
5g
a
Values are means of three experiments.
HCl
N
NH
N
N
H
HO
N
H
N
6
Figure 3. Structure of a representative compound.
tion of glucuronic acid or oxidation to carboxylic acid followed
by b-oxidation. Indeed, their PK profiles were markedly improved.
For example, Cmax and AUC values of compound 5f were 4000 times
and 5700 times as high as those of compound 5b at 30 mg/kg po in
rats.
Thus, we synthesized various derivatives of compound 2.
Firstly, reaction of compound 3 with 3-amino-5-methylpyrazole
in the presence of diisopropylethylamine in DMSO afforded an
intermediate 4 in 60% yield by substitution of the chlorine atom
at 6-position of pyridine ring. Secondly, substitution reaction of
the chlorine atom of compound 4 with nucleophiles such as alkyl-
amines or alkylthiols in DMSO gave designed compounds 5a–5g
(Scheme 2).
We introduced various types of substituted alkyl moiety as
R–X–, and typical results are summarized in Table 1. As a result
of transformation of first step (5a–5e), hydroxyalkyl groups, espe-
cially 6-hydroxyhexyllthio (5b) or 6-hydroxyhexylamino (5e)
group gave good results in the points of inhibitory activity against
Aurora A kinase, cell permeability, and physiochemical properties.
However, pharmacokinetic (PK) profile of these two compounds
was very poor after po administration in rats.
An optimized compound in this alkyl series was compound 613
(Fig. 3) which inhibited Aurora A kinase with a Ki value of 1.2 nM,14
whereas a Ki value against Aurora B kinase inhibition was 101 nM.
This compound exhibited good kinase selectivity. We examined
inhibitory activity against typical 68 kinases, and IC50 values
against 60 kinases were over 1000 nM.15 Compound 6 also exhib-
ited good cell permeability. It inhibited proliferation of HCT116
cells with an IC50 value of 115 nM.
Compound 6 showed good PK profile; Cmax value was 4930 nM
(Tmax = 1.2 h) and serum concentration after 24 h was 52 nM
(T1/2 = 3.3 h) at 30 mg/kg po in rats. So we tested antitumor activity
in HCT-116 subcutaneous Xenograft model in mice. Compound 6
was administrated at 30 mg/kg po qd. Growth of tumor was inhib-
ited by 59% after 14 days;16 body weight was comparable to that of
control mice at this dose during all period administrated.
In summary, we have created a new class of Aurora A kinase
inhibitor by transforming the 4-(5-methyl-3-pyrazoloamino)-
pyrimidine moiety of VX-680 to 3-cyano-6-(5-methyl-3pyrazoloa-
mino)pyridine. Various substituted alkylthio or alkylamino group
were introduced in its 2-position of pyridine ring. The optimized
compound in this alkyl series (compound 6) exhibited a potent
Aurora A kinase inhibitory activity, excellent selectivity to Aurora
B kinase and other 60 kinases, good cell permeability and good PK
profile. Therefore compound 6 was effective in antitumor mice mod-
el at a dose of 30 mg/kg po qd without decrease of body weight.
We presumed that one of its reasons was rapid metabolism of
the primary alcohol moiety. So we transformed the primary
alcohol moiety to tertiary alcohol (5f and 5g) to prevent conjuga-
N
N
NH
N
a
b
Cl
X
N
Cl
Cl
N
N
H
3
4
N
R
NH
N
N
N
H
5a-5g
X = NH or S
References and notes
Scheme 2. Reagents and conditions: (a) 3-amino-5-methylpyrazole, diisopropyl-
ethylamine, DMSO, 100 °C, 60%; (b) R-XH, NaHCO3, DMSO, 100 °C.
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J = 6.5 Hz, 3H), 1.30–1.50 (m, 5H), 1.64 (m, 1H), 2.24 (s, 3H), 2.28 (s, 3H), 4.13
(m, 1H), 6.20 (s, 1H), 6.28 (s, 1H), 6.76 (s, 1H), 10.54 (s, 1H); 13C NMR (400 MHz,
DMSO-d6) d 10.736, 20.203, 20.731, 29.107, 29.235, 36.377, 43.369, 47.119,
68.610, 79.933, 94.817, 98.711, 117.227, 141,635, 145.995, 153.232, 153.845,
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14. Racemate 6 was separated using chiral preparative HPLC (CHIRALPAK AD-H,
Daicel Chemical Industries). Ki values of eutomer and distomer against aurora
A kinase inhibition were 0.59 and 66 nM, respectively. The eutomer inhibited
proliferation of HCT116 cells with an IC50 value of 52 nM. Intrinsic clearance
(CLint) in human hepatic microsomes of the racemate, the eutomer, and the
distomer were 0.07, 0.05, and 0.11 mL/min/mg, respectively. Absolute
configuration of the eutomer was (S)-form which was determined by
chemical synthesis via an alternative route starting from (R)-6-methyl-5-
hepten-2-ol.
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NC
9. There was no report regarding X-ray co-complex structure of VX-680 bound to
Aurora A kinase when our project started.
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11. All new compounds were fully characterized.
12. Recombinant Aurora A kinase was prepared using gene from Hela cells (ATCC
CCL-2). Inhibition of phosphotransferases activity was measured by using
NH
N
OH
N
H
HO
N
H
N
eutomer
15. The less selective kinases were SRC-family; Ki values against yes, cSRC, and Fyn
inhibition were 24, 26, and 28 nM, respectively.
16. Growth inhibitions by VX-680 (30 and 100 mg/kg ip qd) after 14 days were 60%
and 68%, respectively.
peptide substrate kemptide and
measurement of kinase activity inhibition. A Km value of kemptide was 28
13. Mp 177–179 °C; IR 3246, 2966, 2930, 2199, 1637, 1582, 1538, 1451, 1361,
1204, 763, 730 cmÀ1 1H NMR (400 MHz, DMSO-d6) d 1.04 (s, 6H), 1.20 (d,
[
c
-32P]ATP in
a
usual manner for
l
M.
;