Potent 2-[(pyrimidin-4-yl)amine}-1,3-thiazole-5-carbonitrile-based inhibitors of VEGFR-2 (KDR) kinase

Article abstract of DOI:10.1016/j.bmcl.2005.11.089

Pyrimidino-thiazolyl carbonitriles were prepared that are potent VEGFR-2 (KDR) kinase inhibitors. The modification of lead structures resulted in 3m which exhibited the best overall profile in KDR inhibitory activity, iv/po pharmacokinetics, and reduced hERG affinity.

Full text of DOI:10.1016/j.bmcl.2005.11.089

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1146–1150
Potent 2-[(pyrimidin-4-yl)amine}-1,3-thiazole-5-carbonitrile-based
inhibitors of VEGFR-2 (KDR) kinase
John T. Sisko,a,* Thomas J. Tucker,a Mark T. Bilodeau,a Carolyn A. Buser,b
Patrice A. Ciecko,a Kathleen E. Coll,b Christine Fernandes,b Jackson B. Gibbs,b
Timothy J. Koester,a Nancy Kohl,b Joseph J. Lynch,c Xianzhi Mao,b Debra McLoughlin,c
Cynthia M. Miller-Stein,d Leonard D. Rodman,a Keith W. Rickert,b
Laura Sepp-Lorenzino,b Jennifer M. Shipman,b Kenneth A. Thomas,b
Bradley K. Wongd and George D. Hartmana
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
bDepartment of Cancer Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
cDepartment of Pharmacology, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
dDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 13 October 2005; revised 23 November 2005; accepted 28 November 2005
Available online 20 December 2005
Abstract—Pyrimidino-thiazolyl carbonitriles were prepared that are potent VEGFR-2 (KDR) kinase inhibitors. The modification of
lead structures resulted in 3m which exhibited the best overall profile in KDR inhibitory activity, iv/po pharmacokinetics, and
reduced hERG affinity.
Ó 2005 Elsevier Ltd. All rights reserved.
Vascular endothelial growth factor (VEGF) activation is
a critical rate-limiting step in physiological and patho-
logical angiogenesis.1 The biological effects of VEGF
N
N
N
CN
R1
R2
CN
are mediated by two receptor tyrosine kinases, VEG-
FR-12 kinase and VEGFR-2 kinase [also known as
kinase domain region (KDR kinase)]3, and it has been
demonstrated that the disruption of VEGF signaling
can retard angiogenesis and inhibit tumor growth.4 A
number of VEGF inhibitors are currently undergoing
advanced-stage clinical trials for the treatment of cancer,
including Bevacizumab, a recently marketed antibody
against VEGF4 and a soluble VEGF decoy receptor.5
In addition, small molecule inhibitors of KDR kinase6
are being investigated that have been shown to be effica-
cious in in vivo tumor xenograft models.
S
S
HN
HN
S
HN
N
N
N
N
N
N
R
1
2a, R = H
2b, R = CH2N(R1R2)
3
Figure 1. Lead structures.
the analogous pyridine thiazole series revealed that the
phenyl moiety could be replaced with a cyano group
with no loss in potency (compound 2a, KDR
IC50 = 36 nM). Further SAR revealed that the pyridine
ring could also be substituted with a basic amine, as in
compound 2b, to improve the physical properties and
the cell potency of the analogs.8 Unfortunately, some
analogs of compound 2b were shown to be potent bind-
ers to the potassium channel encoded by the human
ether-a-go-go-related gene (hERG). This delayed rectifi-
er potassium channel is involved in ventricular repolar-
ization and underlies the rapid component of the
Previous work from our laboratories7 has shown com-
pound 1 (Fig. 1) to have good potency against KDR
kinase (KDR IC50 = 36 nM). Exploring the SAR in
Keywords: KDR; VEGFR-2.
*
Corresponding author. Tel.: +1 215 652 3150; fax: +1 215 652 3971;
e-mail: jack_sisko@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.089

J. T. Sisko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1146–1150
1147
delayed rectifier K+ current (IKr). Blockade of this
potassium channel has been shown to lead to QT pro-
longation and to potentially fatal ventricular arrhyth-
mias.9 The hERG binding was addressed in the
pyridine thiazole series (analogs of compound 2b) by
modifying the physical properties and basicity of the
molecules. In a similar effort to design novel and potent
KDR kinase inhibitors with reduced hERG channel
binding in the pyrimidine series, we merged some of
the features of compounds 1 and 2, and focused on
the synthesis of compounds of generic structure 3. We
targeted a series of pyrimidines in which the side chain
was directly attached to the pyrimidine ring (structure
3) and was engaged in a program of modifying the
basicity of this side chain in an effort to address the
hERG binding issues. In this paper, we describe our
efforts to further refine these leads and report the
discovery of 2-[(pyrimidin-4-yl)amine}-1,3-thiazole-5-
carbonitriles as a new structural class of potent KDR
kinase inhibitors.
azine in n-butanol at 100 °C, followed by hydrogenolysis
of each product to yield 3d and 3e, respectively. Finally,
compounds 3f–o were prepared (Method D) by treating
13 with ammonium hydroxide in a sealed tube at 100 °C
to give 4-amino-6-chloropyrimidine 14 as the only prod-
uct. None of the bis-amino displacement products was
observed. Alkylation of 14 with sodium hydride and
2-chloro-5-cyanothiazole 5 in THF provided compound
15. Exposure of 15 to various amines in n-butanol at
100 °C afforded the desired compounds 3f–o as filterable
solids.
Table 1 summarizes the in vitro data for compounds
3a–o. Target compounds were evaluated for their ability
to inhibit the KDR kinase enzyme in a standard enzyme
assay as previously described.10 Compounds were also
evaluated in a cell-based KDR autophosphorylation
inhibition assay (KDR Phos.)11 in HEK293 cells stably
transfected to express high levels of full length human
KDR. The affinity of compounds for the hERG channel
was also evaluated in radioligand competition experi-
ments analogous to [3H]-dofetilide binding assays.12 Pre-
vious work in our laboratories8 had shown that some
lipophilic, basic amine-containing analogs of compound
2b had high levels of potency in our hERG binding as-
say, and it was critical to track this activity as we synthe-
sized new compounds. Initially, we synthesized the
unsubstituted pyrimidino-5-cyanothiazole 3a (Table 1)
and found it to be 30-fold less potent in the KDR kinase
assay than was 1 and the pyridino-5-cyanothiazole 2a.7
Interestingly, this simple, unsubstituted compound (3a)
had some hERG binding activity, indicating that hERG
activity might be an issue with this series of compounds.
Scheme 1 outlines the synthesis of target compounds
3a–o (Table 1). Alkylation of 4-aminopyrimidine 4 in
THF with sodium hydride and 2-chloro-5-cyanothiazole
5 (Method A) provided compound 3a. In a similar man-
ner, compound 7 was obtained by treating 4-amino-2,
6-dichloropyrimidine 6 with NaH and chloride 5. Com-
pound 7 was treated with ammonium hydroxide in n-bu-
tanol at 100 °C in a sealed tube to provide regioisomers
8 and 9. Compounds 8 and 9 were individually hydro-
genolyzed to yield compounds 3b and 3c, respectively
(Method B). Compounds 11 and 12 were prepared
(Method C) by exposing compound 7 to 1-acetylpiper-
Method A
NaH
THF
N
NH2
R
N
HN
R =
CN
S
N
N
Cl
N
CN
HN
S
N
3a
5
4
Method B
R
R
R
HN
HN
NH2
NaH
THF
NH3
n-BuOH
100oC
Cl
1) chromatography
2) Pd/C/H2
NH2
+
3b, 3c
N
N
N
N
5
H2N
N
Cl Cl
N
Cl
N
Cl
Cl
N
8
9
6
7
ratio 8:9= 1:1
Method C
R
O
R
HN
HN
N
NH
= R'
1) chromatography
2) Pd/ C/ H2
H3C
+
N
N
3d, 3e
7
N
Cl
R'
Cl
N
R'
n-BuOH
100OC
11
12
ratio 11:12 = 1:1
Method D
N
CN
N
Cl
CN
NH2
S
NaH
THF
HN
S
NH4OH
100O
HN
N
N
HNR1R2 n-
BuOH / 100oC
N
C
R1
N
5
N
Cl
N
Cl
N
N
R2
N
Cl
13
14
3f-o
15
Scheme 1.

1148
J. T. Sisko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1146–1150
Table 1. Properties of selected compounds
N
CN
S
Y
HN
N
X
N
Compound
X
Y
KDR
IC50 (nM)
KDR Phos
IC50 (nM)
HERG binding
IP (nM)
Method
3a
3b
3c
H
H
990
557
41
ND
ND
ND
2932
>10000
>10000
A
B
B
H2N
H
H
NH2
O
3d
H
153
ND
44
34
23
59
21
15
32
22
ND
C
N
N
O
3e
H
13
16
10
10
16
9
4
3291
21,500
923
C
N
N
N
3f
H
H
H
H
H
H
H
H
H
H
D
D
D
D
D
D
D
D
D
D
NH
3g
2
3
N
N CH3
NH2
3h
6643
3891
1012
2951
4029
5000
4003
383
N
N
3i (rac)
3j (rac)
3k
NH2
NH2
N
27
9
8
3
5
2
4
NH
N
N
N
3l
O CH3
N
N
3m
3n
12
10
9
19
23
11
4
N
O
N
N
N
N
N
O
3o
N
For determinations where n > 2, the standard deviation is given.
In analogy to the earlier pyridine-based series of com-
pounds of generic structure 2, we wanted to examine
the meta substitution pattern in an attempt to enhance
the potency of these inhibitors. In an effort to simplify
the synthesis of our target compounds as well as to re-
move the potentially metabolically labile methylene
group of compound 2b, we chose to pursue pyrimidines
that were substituted directly with basic side chains as
indicated in the generic structure 3. Entries 3b–e in
Table 1 summarize our efforts to discern the optimal
substitution pattern on the pyrimidine ring to maximize
activity against KDR. Comparing the results from com-
pounds 3b and 3d versus those for 3c and 3e clearly
indicates that the 4,6-pyrimidine substitution pattern re-
sults in more potent analogs than the 2,4-pyrimidine
series in the enzymatic assay. Further elaboration of
the amine-containing side chains led to a series of com-
pounds with a 2- to 5-fold enhancement of their inhib-
itory potency. Simple cyclic side chains such as those in
compounds 3e–k all provided potent inhibitory activity
in both the enzymatic and cell-based assays. Activity in
the hERG assay varied, with the 4-alkyl piperazine 3g

J. T. Sisko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1146–1150
Table 2. Dog iv PK data for selected compounds
1149
and the amino azepine 3j having moderately potent
activity. The 4-substituted piperazine derivatives 3l–n
all possessed a good balance of KDR kinase inhibitor
activity, cell potency, and decreased affinity for the
hERG channel. Compound 3o was highly potent in
both the enzymatic and cell-based assays, however this
compound had very high affinity for the hERG channel.
Attempts at directly correlating basicity and/or lipophil-
icity to hERG activity were unsuccessful. It appears
that the overall properties of the molecules are critical,
and the combination of lipophilicity and basicity is a
warning sign but not necessarily predictive for potential
hERG binding activity. While it was not always appar-
ent how to design a compound that lacked hERG chan-
nel activity, by preparing a series of compounds of
varying side chain basicities one could modulate the
hERG activity and find compounds of varying affinities.
Employing this strategy provided a variety of potent
inhibitors of KDR kinase, a number of which showed
greatly reduced levels of hERG activity.
Compound
AUC
(lM h)
Vd
Cl
(mL/min/kg)
T1/2
(h)
(L/kg)
3h
3l
1.91
4.81
3.65
1.31
30.20
10.50
4.11
1.34
2.00
10.50
7.97
3m
3n
3o
10.20
3.08
6.07
2.40
14.80
14.05
13.10
7.26
23.00
(Beagles, 0.25 mg/kg iv, DMSO) Average of two dogs, individual
determinations are within 25% of the mean unless noted otherwise.
Table 3. Pharmacokinetic data for 3ma in three species
Dogb
Ratc
Rhesusd
T1/2
11.26
4.11
2.72
51
1.58
38.67
3.32
3.23
22.70
10.12
54
Cl (mL/min/kg)
Vdss (L/kg)
F (%)
>99
a Dosed iv as a DMSO solution and po as a solution in 0.05 M citric
acid.
b Average of two dogs dosed at 1 mg/kg iv and 1 mg/kg po. The
individual determinations are within 25% of the mean.
c Average of three rats dosed at 2 mg/kg iv and 10 mg/kg po. Individual
determinations are within 25% of the mean.
Based on their biological profiles, several compounds
were chosen for pharmacokinetic evaluation in dogs fol-
lowing iv administration (Table 2). Compound 3h,
which bears a primary amine substituent on the side
chain, showed low AUC, high clearance, and relatively
short half-life. Analogs containing 4-substituted pipera-
zine side chains (3l–o) consistently exhibited better phar-
macokinetic profiles, with higher exposures and longer
half-lives. In the case of compound 3m, low clearance
was likely a major contributor to the long iv half-life ob-
served for this compound. However, in the case of com-
pounds 3n–o where the observed clearances were higher,
large increases in volume of distribution were likely
responsible for the long iv half-lives observed for these
analogs. This may be attributable to the more highly ba-
sic amines present in the side chain of compounds 3n–o,
as well as the slightly higher lipophilicity of these two
analogs.
d Average of two rhesus dosed at 1 mg/kg iv and 1 mg/kg po. Indi-
vidual determinations are within 25% of the mean.
Table 4. Kinase selectivity of compound 3m
Kinase
3m
Flk-1
Flt-1
Flt-3
5
6.5
4
2
Flt-4
PDGFRb
c-kit
15
35
13
52
14
26
c-fms
FGFR1
FGFR2
Src
Based on its overall excellent in vitro and iv pharmaco-
kinetic performance, compound 3m was selected for
further pharmacokinetic evaluation. The pharmacoki-
netic profile of 3m in rat, dog, and rhesus monkey after
iv/po dosing is detailed in Table 3. The compound
exhibited moderate to long half-life, low to moderate
clearance, and excellent oral bioavailability in all three
species.
pyrimidine ring provided compounds of varying affinities
for the hERG channel. Representative compounds from
this series possess good pharmacokinetic profiles after iv
and po dosing. Compound 3m possessed the best overall
profile of KDR inhibitory activity, reduced hERG affinity,
and iv/po pharmacokinetics, and became a second-genera-
tion lead structure for further work in this series. These
compounds represent a novel class of KDR kinase inhibi-
tors and are the target of further efforts in our laboratories
to obtain potent inhibitors that have good pharmacokinet-
ic profiles and greatly reduced affinity for the hERG chan-
nel. The results of these efforts will be reported in due
course.
Compound 3m was also evaluated against a panel of
related tyrosine kinases (Table 4). Data are expressed
as a ratio of the given kinase IC50 to the KDR kinase
IC50. In general, the compound showed low levels of
selectivity versus the closely related Fit and Flk families
of kinases, but more moderate levels of selectivity versus
other tyrosine kinases.
In summary, we have developed a new class of KDR
kinase inhibitors based on the 2-[(pyrimidin-4-yl)-
amine}-1,3-thiazole-5-carbonitrile template. Compounds
within this series are potent in enzyme and cell-based
assessments of KDR kinase inhibitory activity. Modulat-
ing the basicity of the side chain in the 4-position of the
Acknowledgments
We thank Dr. Art Coddington, Dr. Chuck Ross, and
Dr. Harri Ramjit for mass spectral analyses.

1150
J. T. Sisko et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1146–1150
8. Bilodeau, M. T.; Balitza, A. E.; Koester, T. J.; Manley, P.
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