Identification of potent 5-pyrimidinyl-2-aminothiazole CDK4, 6 inhibitors with significant selectivity over CDK1, 2, 5, 7, and 9

Article abstract of DOI:10.1016/j.bmcl.2006.04.048

5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin-dependent kinases (CDKs) by a screening of the Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the gate

Full text of DOI:10.1016/j.bmcl.2006.04.048

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3751–3754
Identification of potent 5-pyrimidinyl-2-aminothiazole CDK4, 6
inhibitors with significant selectivity over CDK1, 2, 5, 7, and 9
Tadashi Shimamura, Jun Shibata, Hideki Kurihara, Takashi Mita, Sachie Otsuki,
Takeshi Sagara, Hiroshi Hirai and Yoshikazu Iwasawa*
Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan
Received 28 February 2006; revised 31 March 2006; accepted 19 April 2006
Available online 6 May 2006
Abstract—5-Pyrimidinyl-2-aminothiazole 1 was identified as an inhibitor of cyclin-dependent kinases (CDKs) by a screening of the
Merck sample repository. The introduction of a methyl group at the C-5 or C-6 position on the pyrimidine ring, directed toward the
gate keeper residue of CDK4 (Phe93), led to significant enhancement of selectivity for CDK4 over other CDKs. Compound 3 exhib-
ited more than 300-fold selectivity for CDK4 over CDK1, 2, 5, 7, and 9. Subsequent improvements in aqueous solubility afforded
compound 4, which is available for further in vivo studies and this compound inhibited pRb phosphorylation and BrdU incorpo-
ration in tumor models.
Ó 2006 Elsevier Ltd. All rights reserved.
CDKs are a prominent family of protein kinases which
plays key roles in regulation of the cell cycle in eukary-
otic cells.1 Orderly cell cycle progression requires activa-
tion of CDKs, which is mainly controlled by expression
of their activator subunit, cyclin. CDK4 and CDK6
complex with cyclin D, and CDK2 complex with cyclin
E or A sequentially phosphorylate retinoblastoma pro-
tein (pRb) to facilitate the G1/S progression. CDK4
and CDK6 are closely related proteins with basically
indistinguishable biochemical properties. pRb is a nega-
tive regulator of transcription factor E2F, with which it
forms a complex. When hyperphosphorylated by CDKs,
pRb loses its binding activity to E2F and the released
E2F activates transcription of genes whose products
are critical for the cell cycle progression. CDK activity
is also regulated by CDK inhibitory proteins (CDKIs).
Among them, INK4 family protein represented by
p16INK4a selectively inhibits cyclin D/CDK4 and
CDK6 complexes, and induces G1 cell cycle arrest when
overexpressed in pRb-positive mammalian cells. Dereg-
ulation of the cell cycle is a hallmark of cancer.2 Indeed,
genetic or epigenetic mutations in p16INK4a/cyclin
D/CDK4 and CDK6/pRb pathway are commonly
observed in a variety of many types of human cancers,
suggesting CDK4, 6 may be an attractive target for
development of anti-cancer drugs.
To date, 13 types of CDKs have been identified in the
human genome, and there are some with functions unre-
lated to cell cycle regulations.3 CDK7 and CDK9 are
components of transcription regulation factor TFIIH
and P-TEFb, respectively,4 and CDK5 plays numerous
functions in the nervous system.5 Thus, for development
of anti-cancer agents, a small molecule inhibitor of
CDK4, 6 with selectivity over non-cell cycle CDKs
would be important to further understanding of biology
of CDKs and development for anti-cancer agents. Here,
we report the development of novel CDK4, 6 selective
inhibitors with a 5-pyrimidinyl-2-aminothiazole scaffold.
We previously reported that the diarylurea class of
compounds was identified to be a novel class of CDK4
selective inhibitors.6 Further characterization of this
class of compounds showed limited CDK4 selectivity
over some other CDKs, except for CDK1 and 2. We
attempted to find new classes and strategies for generat-
ing inhibitors with selectivity for CDK4 over other
CDKs including CDK5, 7, and 9 as well as 1 and 2.
In the first place, we screened the Merck sample reposi-
tory, and a series of compounds with a 5-pyrimidinyl-2-
aminothiazole scaffold was identified to show potent
inhibitory activities against all CDKs tested. Struc-
ture–activity relationships (SARs) obtained during
screening indicated that the cyclohexyloxy group at the
Keywords: Cancer; Anti-cancer agent; CDK; CDK inhibitor; 5-Pyri-
midinyl-2-aminothiazole.
*
Corresponding author. Tel.: +81 29 877 2197; fax: +81 29 877
2199; e-mail: yoshikazu_iwasawa@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.048

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T. Shimamura et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3751–3754
C-2 position on the pyrimidine ring is an important
component of the compounds’ potent inhibitory activity
against CDK4.
(Fig. 1); therefore, a methyl group was introduced at
either the C-5 or the C-6 position on the pyrimidine ring.
Surprisingly, these gate site-directed modifications were
found to greatly reduce the inhibitory activity against
other CDKs than CDK4, while CDK4 inhibitory activ-
ity was retained. In particular, the C6-methyl derivative
3 showed more than 300-fold selectivity for CDK4 over
other CDKs (Table 1).
We investigated the docking study of compound 1 with
the ATP-binding pocket of a CDK4 model (Fig. 1). As a
result of this study, it seemed that the gate keeper7 and
the solvent accessible site6 were accessible from com-
pound 1. The gate keeper residue is generally accepted
as the target site for influencing selectivity in protein
kinases. Also as reported previously, non-conserved
residue, Thr102 in CDK4, was found to be the key
residue for selectivity of CDK4 over other CDKs,
especially CDK1 and 2. Therefore, we decided to intro-
duce substituents at the C-5 and C-6 positions on the
pyrimidine ring, directed toward the gate keeper and
the C-5 position on the pyridine ring, directed toward
the solvent accessible site.
Selective inhibitors of CDK4 have been reported in the
literatures8; however, there are few reports of showing
the inhibitory activity against non-cell cycle-related
CDKs, such as CDK5, 7, and 9 among current selective
CDK4 inhibitors. To our knowledge, this is the first
identification of an inhibitor that displays high selectiv-
ity for CDK4 over CDK1, 2, 5, 7, and 9. In the course of
medicinal chemistry efforts to generate compounds with
adequate solubility, compound 3 led us to identify com-
pound 49 (Table 2) which has an amino alkyl group on
the pyrazine ring. The compound 4 also exhibited signif-
icant selectivity for CDK4, 6 over other CDKs and
showed sufficient aqueous solubility for intravenous
administration. Compound 4 potently inhibited more
than 80% of pRb phosphorylation at Ser780 residue,
which is specifically phosphorylated by CDK4, 6, and
consequently roughly 90% of cells were arrested at G1
in Eol-1 human eosinophilic leukemia cells at 300 nM
The results of the gate keeper-directed modification are
shown in Table 1. It was concluded from the docking
study that the space available between the pyrimidine
ring and Phe93 gate keeper residue was limited
Gate Keeper Residue
Phe93
N
N
N
N
Table 2. CDK4/6 selective inhibition by compound 4
O
N
H
O
S
N
H
O
N
N
Hinge Region
S
N
N
N
NMe
N
N
Solvent Accessible Site
Region
H
4
OH
Thr
Lys
Val
Gly
CDK4, 6
CDK1, 2, 5
CDK7
Inhibitory activity (IC50, nM)
CDK4 CDK6 CDK1 CDK2 CDK5 CDK7 CDK9
CDK9
Figure 1. Schematic representation of predicted binding mode of
compound 1 in CDK4 model.
9.2
7.8
600
1700
3000
530
2500
Table 1. Structures and enzyme inhibitory activities of 5-pyrimidinyl-2-aminothiazole-based compounds
O
O
O
N
N
N
N
N
N
S
N
S
N
S
N
N
N
N
N
N
N
H
H
H
1
2
3
Compound
Inhibitory activity (IC50, nM)
CDK2 CDK5
14 34
CDK4
CDK1
24
>1000
CDK7
CDK9
1
2
3
4.2
13
20
190
2.5
270
>10000
>10000
>10000
26
>10000
34
>10000
>10000

T. Shimamura et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3751–3754
3753
b-d
a
N
N
N
OH
e, f
N
N
N
HOOC
BocHN
BocHN
A-1
A-2
A-3
S
N
OTBDPS
N
OTBDPS
g, h
SMe
SMe
N
N
H2N
N
N
N
H2N
H
A-4
A-5
N
N
l
m,n
S
N
OH
S
N
OTBS
N
N
SMe
N
SMe
N
SMe
N
N
k
N
N
i, j
N
N
H
N
SEM
N
OEt
C-1
C-2
Br
Cl
OEt
OEt
B-3
B-1
B-2
O
O
N
O
N
N
N
N
N
o, p
q-s
t
S
N
N
S
N
OTBS
S
N
N
N
N
NMe
N
NMe
N
N
N
N
N
N
SEM
SEM
H
C-3
C-4
Compound 4
t
Scheme 1. Synthesis of compound 4. Reagents and conditions: (a) DPPA, Et3N, 3 h, 100 °C, dioxane, BuOH, 88%; (b) NBS, AIBN, 12 h, 100 °C,
CCl4; (c) KOAc, 18-crown-6-ether, 1 h, rt, MeCN; (d) 3 N NaOH aq, 12 h, rt, MeOH, 29% in 3 steps; (e) TBDPSCl, imidazole, 12 h, rt, DMF, 99% ;
(f) TFA, 5 h, rt, CHCl3, 92%; (g) BzNCS, rt, 2 h, THF, 96%; (h) K2CO3, 45 °C, 5.5 h, THF/MeOH/H2O, 99%; (i) ethyl ethynyl ether, BH3 Æ THF, rt,
4 h, THF; (j) 3 N NaOH aq, PPh3, Pd(OAc)2, rt, 6 h, THF, 88% in 2 steps; (k) NBS, rt, 0.5 h, EtOH, 80%; (l) p-TsOH Æ H2O, 90 °C, 12 h, EtOH/H2O;
(m) TBSCl, imidazole, 3 h, rt, DMF; (n) SEMCl, EtNiPr2, 1 h, 0 °C, DMF, 27% in 3 steps; (o) mCPBA, 1.5 h, 0 °C, CHCl3, 99%; (p) NaH, cyclohexyl
alcohol, rt, 0.5 h, DMF; (q) 1 M TBAF in THF, 1 h, rt, THF, 40% in 2 steps; (r) MsCl, EtNiPr2, 1 h, 0 °C, CHCl3; (s) N-methylpiperazine, K2CO3,
1 h, 70 °C, CHCl3; (t) 4 N HCl in dioxane, 12 h, rt, MeOH, 45% in 3 steps.
in vitro. In addition, this compound inhibited pRb phos-
phorylation and BrdU incorporation by 99% and 91%,
respectively, in Eol-1 xenograft tumor models in nude
rats when administered via constant intravenous
infusion for 24 h at a plasma concentration of 510 nM
(manuscript in preparation).
and for further elucidation of CDK biology. Further
SAR analysis of the analogues and molecular modeling
studies to clarify the mechanism of their CDK4, 6 selec-
tive inhibitions and biological profiles of compound 4
will be reported elsewhere in due course.
The synthetic route for preparation of compound 4 is
illustrated in Scheme 1. Reaction of the thiourea A-5,
which was prepared from 5-methylpyrazine-2-carboxylic
acid A-1 in 8 steps, with 2-bromo-diethyl acetal B-3 gave
5-pyrimidinyl-2-aminothiazole C-1. Next, the methyl-
thio group was oxidized with mCPBA, followed by
introduction of a cyclohexyloxy group to give C-3. N-
Methylpiperazine was then introduced via the mesyla-
tion reaction. Finally, deprotection of SEM group
provided compound 4 as a HCl salt.
Acknowledgments
We acknowledge the excellent contribution of the fol-
lowing scientists to this work: for chemistry, Nobuhiko
Kawanishi, Takashi Hashihayata, and Mikako Kawam-
ura; for biology, Ikuko Suzuki-Takahashi and Yoko
Nakatsuru.
References and notes
In conclusion, we used compound screening and medic-
inal chemistry techniques, supported by molecular mod-
eling, to identify potent CDK4 inhibitors 3 and 4 which
show high selectivity over other CDK family kinases.
These compounds are based on a novel 5-pyrimidinyl-
2-aminothiazole scaffold. Introduction of a methyl
group at the C-6 position of the pyrimidine ring in this
series was found to greatly enhance CDK4 selectivity
over other CDK family kinases. Compound 4, which
showed significant selectivity for CDK4, 6 over non-cell
cycle CDKs, showed mechanism-based inhibition of
CDK4 in cultured cells and in vivo. These results suggest
that compound 4 may be useful in drug development
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