Design, synthesis, and evaluation of novel VEGFR2 kinase inhibitors: Discovery of [1,2,4]triazolo[1,5-a]pyridine derivatives with slow dissociation kinetics

Article abstract of DOI:10.1016/j.bmc.2013.04.042

For the purpose of discovering novel type-II inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2) kinase, we designed and synthesized 5,6-fused heterocyclic compounds bearing a anilide group. A co-crystal structure analysis of imidazo[1,2-b]pyridazine derivative 2 with VEGFR2 revealed that the N1-nitrogen of imidazo[1,2-b]pyridazine core interacts with the backbone NH group of Cys919. To retain this essential interaction, we designed a series of imidazo[1,2-a]pyridine, [1,2,4]triazolo[1,5-a]pyridine, thiazolo[5,4-b]pyridine, and 1,3-benzothiazole derivatives maintaining a ring nitrogen as hydrogen bond acceptor (HBA) at the corresponding position. All compounds thus designed displayed strong inhibitory activity against VEGFR2 kinase, and the [1,2,4]triazolo[1,5-a]pyridine 13d displayed favorable physicochemical properties. Furthermore, 13d inhibited VEGFR2 kinase with slow dissociation kinetics and also inhibited platelet-derived growth factor receptor (PDGFR) kinases. Oral administration of 13d showed potent anti-tumor efficacy in DU145 and A549 xenograft models in nude mice.

Full text of DOI:10.1016/j.bmc.2013.04.042

Bioorganic & Medicinal Chemistry 21 (2013) 4714–4729
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and evaluation of novel VEGFR2 kinase inhibitors:
Discovery of [1,2,4]triazolo[1,5-a]pyridine derivatives with slow
dissociation kinetics
⇑
Yuya Oguro , Douglas R. Cary, Naoki Miyamoto, Michiko Tawada, Hidehisa Iwata, Hiroshi Miki,
Akira Hori, Shinichi Imamura
Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd, 2-26-1 Muraoka-higashi, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
For the purpose of discovering novel type-II inhibitors of vascular endothelial growth factor receptor 2
(VEGFR2) kinase, we designed and synthesized 5,6-fused heterocyclic compounds bearing a anilide
group. A co-crystal structure analysis of imidazo[1,2-b]pyridazine derivative 2 with VEGFR2 revealed
that the N1-nitrogen of imidazo[1,2-b]pyridazine core interacts with the backbone NH group of
Received 19 February 2013
Revised 12 April 2013
Accepted 13 April 2013
Available online 23 April 2013
Cys919. To retain this essential interaction, we designed
a series of imidazo[1,2-a]pyridine,
[1,2,4]triazolo[1,5-a]pyridine, thiazolo[5,4-b]pyridine, and 1,3-benzothiazole derivatives maintaining
a ring nitrogen as hydrogen bond acceptor (HBA) at the corresponding position. All compounds thus
designed displayed strong inhibitory activity against VEGFR2 kinase, and the [1,2,4]triazolo[1,5-a]pyr-
idine 13d displayed favorable physicochemical properties. Furthermore, 13d inhibited VEGFR2 kinase
with slow dissociation kinetics and also inhibited platelet-derived growth factor receptor (PDGFR)
kinases. Oral administration of 13d showed potent anti-tumor efficacy in DU145 and A549 xenograft
models in nude mice.
Keywords:
Type-II VEGFR2 kinase inhibitor
PDGFR
[1,2,4]Triazolo[1,5-a]pyridine
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
Therefore, abrogation of angiogenesis by VEGFR inhibition is re-
garded as a promising approach for the treatment of cancer. Indeed,
Angiogenesis, the formation of new blood vessels, plays impor-
tant roles in the growth and metastasis of solid tumors.1 It is gen-
erally thought that angiogenesis is required for the expansion of
tumor mass beyond a certain size to supply oxygen and nutrients.
Inhibition of tumor angiogenesis, therefore, has thus been targeted
as a rational approach for anticancer therapy.2
Tumor angiogenesis is regulated by various kinds of angiogenesis
factors. Vascular endothelial growth factors (VEGF) play a major role
in tumor angiogenesis.3 VEGF induction in tumor cells can result
from cancer-related changes including proto-oncogene activation,4
loss of tumor suppressor function,5,6 growth factor stimuli,7 and
hypoxic status.8,9 The high-affinity VEGF receptors (VEGFR) consist
of FLT1 (Fms-like tyrosine kinase 1; VEGFR1), KDR (VEGFR2), and
FLT4 (VEGFR3).10–12 Binding of VEGF with VEGFR specifically exerts
mitogenic and chemotactic activity on endothelial or lymphendo-
thelial cells via receptor dimerization, leading to autophosphoryla-
tion of tyrosine residues in the intracellular kinase domain.13
Overactivation of VEGFR signaling positively correlates with poor
prognosis and metastasis in the majority of solid tumor patients.14,15
several VEGFR kinase inhibitors (sunitinib,16 sorafenib,17 pazopa-
nib,18 axitinib,19 and regorafenib20) have been approved for various
types of cancer, such as renal cell carcinomas.
Except for allosteric inhibitors, the majority of known kinase
inhibitors are classified as type-I or type-II kinase inhibitors by
their mode of binding with the target kinase.21 Although both type
of inhibitors bind in and around the region occupied by the ade-
nine ring of ATP, type-II kinase inhibitors induce the inactive con-
formation of kinases, enabling them to interact with a deeper
region called the back pocket. Type-II kinase inhibitors have sev-
Me
F
H
N
H
N
H
N
N
N
O
O
N
Me
O
Me
N
O
N
CF3
N
N
O
N
N
H
1
2
⇑
Corresponding author. Tel.: +81 466 32 1180; fax: +81 466 29 4458.
E-mail address: yuuya.ooguro@takeda.com (Y. Oguro).
Figure 1. Pyrrolo[3,2-d]pyrimidine derivative
derivative 2.
1 and imidazo[1,2-b]pyridazine
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.042

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4715
Figure 2. X-ray crystal structure of 2 bound to VEGFR2 (PDB ID 3VO3).
eral advantages over type-I kinase inhibitors such as slow off-rate
profiles.22,23 We previously reported the urea-possessing pyrrol-
o[3,2-d]pyrimidine derivatives as type-II VEGFR2 kinase inhibi-
tors.24 The representative compound 1 (Fig. 1) inhibited VEGFR2
with slow dissociation kinetics and demonstrated potent inhibition
of tumor growth in a DU145 human prostate cancer cell xenograft
model in nude mice without serious toxicity. In our recent efforts
to identify novel VEGFR2 kinase inhibitors, we also discovered
the imidazo[1,2-b]pyridazine 2 (Fig. 1) as another type-II inhibitor
of VEGFR2 kinase (VEGFR2 IC50 = 1.4 nM).25 The crystal structure of
the complex between 2 and VEGFR2 is shown in Figure 2. The
kinase adopted an inactive conformation (DFG-out) so that the
pyrazole-5-yl amide moiety occupies a hydrophobic back pocket.
When compound 2 binds with VEGFR2, the N1-nitrogen of the imi-
dazo[1,2-b]pyridazine core and the NH group of the cyclopropyla-
mide at the 2-position interact with the backbone NH group and
carbonyl of Cys919, respectively. CHÁ Á ÁO interaction is also ob-
served between the 8-position of the imidazo[1,2-b]pyridazine
and the backbone carbonyl group of Glu917. The amide group be-
tween central benzene ring and pyrazole ring interacts with the
protein through two hydrogen bonds; the amide proton forms a
hydrogen bond with the side chain of Glu885 and the carbonyl
interacts with the main chain of Asp1046. The external pyrazole
moiety occupies the hydrophobic pocket created by the rearrange-
ment of protein.
Based on this information, we expanded our investigation into
different 5,6-fused heterocyclic ring systems to discover novel
hinge-binding templates for type-II inhibitors of VEGFR2 kinase.
As part of our strategy, the imidazo[1,2-b]pyridazine scaffold was
replaced with other 5,6-fused heterocycles possessing a ring
nitrogen as hydrogen bond acceptor (HBA) at the position
corresponding to the N1-nitrogen of 2 so that the compounds
could retain the essential hydrogen-bonding interaction with the
main chain of Cys919 (Fig. 3(a)). Among the possible structures
that meet this requirement, we reasoned that compounds possess-
ing [1,2,4]triazolo[1,5-a]pyridine (A), imidazo[1,2-a]pyridine (B),
(a)
Glu885
Hydrophobic
O
O
O
Pocket
Me
N
H
R
N
R'
H
N
N
O
O
N
Me
O
O
N
ring
N
O
N
H
N
N
H
CH2COO-
Asp1046
H
N
ring =
O
H
N
O
N
N
S
N
S
N
N
N
N
Cys919
N
(A)
(B)
(C)
(D)
Me
N
(b)
H
N
N
Me
O
O
N
O
ring
N
H
13a
17a
22a
33
Compd
Ring
2
N
N
N
S
S
N
N
N
N
N
N
N
N
(a)
(a)
(a)
(a)
(a)
Atomic charge
-0.66
-0.61
-0.58
-0.58
-0.60
on the N(a)a
Figure 3. (a) Inhibitor Design. (b) Atomic charges of the representative compounds. aAtomic charges were calculated at the Hartree–Fock 6-31G⁄ level using the Gaussian03.

4716
Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
thiazolo[5,4-b]pyridine (C), and 1,3-benzothiazole (D) scaffolds
might have good potential based on consideration of the atomic
charge on nitrogen N(a) (Fig. 3(b)). Calculation of atomic charges
revealed that the nitrogen atom (N(a)) of 13a, 17a, 22a, and 33
had similar or greater negative charge compared to that of the cor-
responding imidazo[1,2-b]pyridazine 2. These results suggested
that scaffolds A–D might be promising hinge-binding templates
because they could form tight hydrogen-bonding with the main
chain of Cys919. Thus, to develop novel orally active type-II
VEGFR2 kinase inhibitors, we designed and synthesized a series
of imidazo[1,2-a]pyridine, [1,2,4]triazolo[1,5-a]pyridine, thiazolo-
[5,4-b]pyridine, and 1,3-benzothiazole derivatives.
stituent on the phenyl ring was generally unsuccessful. To obtain
6b–d, therefore, the aminophenols 7a–c were converted to the
corresponding amides 9a–c using 8, followed by coupling with
5-bromo-2-nitropyridine and hydrogenation of 10a–c with palla-
dium–carbon (Pd/C) to afford desired compounds 6b–d. Com-
pounds 6a–d were then converted to the corresponding
thioureas 11a–d using ethoxycarbonyl isothiocyanate, followed
by treatment with hydroxylamine hydrochloride and N,N-diiso-
propylethylamine (DIEA) to give the 2-amino-[1,2,4]triazolo[1,5-
a]pyridines 12a–d in 63–75% yield. According to the literature,26
we postulate that the 2-amino-[1,2,4]triazolo[1,5-a]pyridines
12a–d were formed by oxadiazole formation of 11a–d and
subsequent rearrangement. Acylation of 12a,b with cyclopropan-
ecarbonyl chloride in the presence of triethylamine provided not
only the desired amide derivatives 13a,b but also the diacylated
byproducts which had two cyclopropane carbonyl groups. Thus,
the diacylated byproducts were converted to the monoacylated
compounds 13a,b by hydrolysis using aqueous sodium carbonate.
On the other hand, acylation of 12c,d with cyclopropanecarbonyl
chloride in DMA without triethylamine provided only the
desired monoacylated derivatives 13c,d in 53% and 71% yield,
respectively.
2. Chemistry
The general synthetic route for the preparation of [1,2,4]triaz-
olo[1,5-a]pyridine derivatives is shown in Scheme 1. The coupling
reaction of commercially available 3-nitrophenol (3) with 5-bro-
mo-2-nitropyridine in the presence of cesium carbonate gave 4,
the nitro groups of which were converted to amino groups by
hydrogenation to afford the diamine 5. The selective acylation
of 5 using acid chloride 8 furnished 6a in 61% yield. Unfortu-
nately, this route was limited to the synthesis of 6a because the
selective acylation of diamine analogues of 5 possessing an R sub-
The preparation of imidazo[1,2-a]pyridine derivatives is shown
in Scheme 2. Treatment of 6a,b with p-toluenesulfonyl chloride
Me
R
H
N
NO2
NH2
N
N
HO
NO2
Me
O
b
c
6a
)
a
(for
O
O
O
N
N
N
H2N
O2N
H2N
3
4
5
6a
: R = H
6b: R = 2-Me
6c
: R = 3-Me
6d: R = 2-F
Me
N
Me
N
H
R
R
3
2
Me
N
f
6b-d)
(for
N
NH2
N
R
H
N
e
d
Me
N
O
4
N
ClOC
Me
O
O
OH
Me
N
OH
O2N
7a: R = 2-Me
7b: R = 3-Me
7c: R = 2-F
8
9a: R = 2-Me
9b: R = 3-Me
9c: R = 2-F
10a: R = 2-Me
10b: R = 3-Me
10c: R = 2-F
Me
N
Me
N
R
H
N
H
N
R
N
N
h
g
Me
O
6a-d
Me
O
O
O
S
N
N
N
H2N
EtOOC
N
H
N
N
11a:
12a
: R = H
R = H
H
11b: R = 2-Me
12b: R = 2-Me
12c
11c:
R = 3-Me
: R = 3-Me
12d: R = 2-F
11d: R = 2-F
Me
H
R
N
i
13a b
, )
(for
N
N
O
j (for 13c,d)
Me
O
O
N
N
HN
N
13a
: R = H
13b: R = 2-Me
13c
: R = 3-Me
13d: R = 2-F
Scheme 1. Reagents and yields: (a) 5-Bromo-2-nitropyridine, Cs2CO3, DMF, 33%; (b) H2, Pd/C, MeOH, 96%; (c) 8, pyridine, THF, 61%; (d) Et3N, THF, 63–99%; (e) 5-Bromo-
2-nitropyridine, Cs2CO3, DMF, 44–59%; (f) H2, Pd/C, MeOH, 88–95%; (g) EtOOCNCS, DMSO; (h) NH2OHÁHCl, DIEA, MeOH, EtOH, 63–75% for two steps; (i) (1)
cyclopropanecarbonyl chloride, Et3N, THF, (2) Na2CO3, MeOH, H2O, 60% (13a), 60% (13b); (j) cyclopropanecarbonyl chloride, DMA, 53% (13c), 71% (13d).

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4717
Me
N
Me
Me
N
R
O
R
R
O
H
N
H
N
N
H
N
N
N
N
a
b
Me
O
Me
Me
O
H2N
O
N
O
O
N
N
H2N
TsHN
Me
TsN
6a
14a:
15a:
: R = H
R = H
R = H
6b: R = Me
14b: R = Me
15b: R = Me
Me
R
R
H
H
N
N
N
Me
N
N
N
c
d
Me
O
O
O
O
O
N
N
H2N
HN
N
N
16a
17a
: R = H
: R = H
16b: R = Me
17b: R = Me
Scheme 2. Reagents and yields: (a) TsCl, pyridine, 99% (14a) and 77% (14b); (b) iodoacetamide, DIEA, DMF, 71% (15a) and 72% (15b); (c) (1) TFAA, CH2Cl2 (2) NaOH, H2O,
EtOH; (d) cyclopropanecarbonyl chloride, Et3N, THF, 21% (17a) and 41% (17b), for two steps.
gave 14a,b, which were converted to 15a,b using iodoacetamide.
Formation of the imidazo[1,2-a]pyridine ring was achieved by
the reaction of 15a,b with trifluoroacetic anhydride (TFAA),27 and
subsequent hydrolysis to afford 16a,b. Acylation of 16a,b with
cyclopropanecarbonyl chloride in the presence of triethylamine
provided only the desired amide derivatives 17a,b, respectively.
Thiazolo[5,4-b]pyridine derivatives were synthesized as illus-
trated in Scheme 3. Amide 18, prepared from the reaction of 3-
aminophenol with 8, and compound 9a were coupled with 2-
chloro-5-nitropyridine in the presence of potassium carbonate
to give 19a,b. After hydrogenation of 19a,b using Pd/C, the 3-
aminopyridines 20a,b were cyclized to the corresponding thiazol-
o[5,4-b]pyridines 21a,b by treatment with bromine and potas-
sium isothiocyanate in 51% and 47% yield, respectively.28
Similar to the synthesis of 13a,b, compound 22a was synthesized
by acylation of 21a with cyclopropanecarbonyl chloride in the
presence of triethylamine followed by hydrolysis of the
diacylated byproduct with sodium carbonate. The selective
monoacylation of 21b was also accomplished by addition of
cyclopropanecarbonyl chloride in DMA without any other base
to give 22b in 66% yield.
[1,2,4]Triazolo[1,5-a]pyridine analogues were also synthesized
as shown in Scheme 4. In a similar manner to the synthesis of
6b–d, compounds 25a,b were prepared by the coupling of 23a,b
with 5-bromo-2-nitropyridine and subsequent hydrogenation of
the resulting nitro derivatives 24a,b. Compounds 25a,b were then
cyclized to give the [1,2,4]triazolo[1,5-a]pyridines 26a and 26b in
87% and 49% yield, respectively. The 2-amino groups of 26a,b were
acylated followed by removal of the Boc protecting groups in acidic
condition to give anilines 27a,b. Acylation of 27a,b with the corre-
sponding acid chlorides furnished 28 and 29a–f in 45–87% yield.
Scheme 5 depicts the synthesis of the 1,3-benzothiazole deriv-
ative 33. Commercially available 30 was acylated with cyclopro-
panecarbonyl chloride to provide 31, which was coupled with 1-
fluoro-3-nitrobenzene to give nitro derivative 32. Reduction of
the nitro group of 32 and the subsequent acylation with acid chlo-
ride 8 afforded the desired product 33 in 5% yield.
3. Results and discussion
The compounds in Tables 1 and 3 were evaluated for their
inhibitory activity against human VEGFR2 kinase in a non-RI assay
Me
N
Me
N
Me
N
R
O
R
O
H
N
H
N
R
N
N
H
N
a
b
Me
Me
N
O
O
Me
O
N
N
OH
O2N
H2N
18
: R = H
19a
: R = H
20a
: R = H
9a: R = Me
19b: R = Me
20b: R = Me
Me
Me
R
R
O
H
H
N
N
N
N
Me
d (for 22a)
N
N
c
e
22b
(for )
Me
O
O
O
N
O
N
S
N
S
N
H2N
HN
21a
: R = H
22a
: R = H
21b: R = Me
22b: R = Me
Scheme 3. Reagents and yields: (a) 2-Chloro-5-nitropyridine, K2CO3, DMF, 94% (19a) and 94% (19b); (b) H2, Pd/C, MeOH, 86% (20a) and quant. (20b); (c) Br2, KNCS, AcOH, 51%
(21a) and 47% (21b); (d) (1) cyclopropanecarbonyl chloride, Et3N, THF, (2) Na2CO3, MeOH, H2O, 56%; (e) cyclopropanecarbonyl chloride, DMA, 66%.

4718
Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
Me
Me
NHBoc
NHBoc
Me
3
2
NHBoc
a
b
4
O
O
N
N
OH
O2N
H2N
23a: 4-yl
23b: 2-yl
24a: 4-yl
24b: 2-yl
25a: 4-yl
25b: 2-yl
Me
Me
NHBoc
NH2
c
d
O
O
O
N
N
N
N
N
H2N
HN
N
26a: 4-yl
26b: 2-yl
27a: 4-yl
27b: 2-yl
Me
Me
O
H
N
H
N
R
N
N
e
f
O
27a
27b
Me
O
O
Me
O
N
N
N
O
N
HN
HN
N
N
28
29a-f
Scheme 4. Reagents and yields: (a) 5-Bromo-2-nitropyridine, Cs2CO3, DMF, 44% (24a) and 53% (24b); (b) H2, Pd/C, MeOH, 99% (25a) and 98% (25b); (c) (1) EtOOCNCS, DMSO,
(2) NH2OHÁHCl , DIEA, MeOH, EtOH, 87% (26a) and 49% (26b); (d) (1) cyclopropanecarbonyl chloride, DMA, (2) TFA, 91% (27a), 86% (27b); (e) 8, DMA, 91%; (f) RCOCl, DMA, 45–
87%.
NO2
O
OH
S
N
a
b
OH
S
N
O
H2N
HN
O
S
N
HN
30
31
32
Me
N
H
N
N
c
Me
O
O
O
S
HN
N
33
Scheme 5. Reagents and yields: (a) cyclopropanecarbonyl chloride, Et3N, THF, 97%; (b) 1-fluoro-3-nitrobenzene, K2CO3, DMF, 37%; (c) (1) H2, Pd/C, MeOH, (2) Fe, CaCl2, EtOH,
H2O, (3) 8, Et3N, THF, 5%, for three steps.
using the AlphascreenÒ system.29 Compounds that showed potent
VEGFR2 kinase inhibition were evaluated for their cellular activity
in human umbilical vein endothelial cells (HUVEC).
tency observed for 22a and 33 revealed that the bridgehead nitro-
gen of 2 was not important for potency and the larger sulfur atom
of 22a and 33 did not perturb the hinge interactions. Compounds
13a, 17a, 22a, and 33 also displayed similar cellular potency to 2
in the VEGF-driven HUVEC proliferation assay, revealing that
[1,2,4]triazolo[1,5-a]pyridine, imidazo[1,2-a]pyridine, thiazol-
o[5,4-b]pyridine, and 1,3-benzothiazole are promising hinge-bind-
ing cores for VEGFR2 kinase inhibitors.
Table 2 shows the effect of the hinge-binding core on inhibitory
activity against other kinases. Similar to the imidazo[1,2-b]pyrida-
zine derivative 2, compounds 13a, 17a, 22a and 33 strongly inhib-
ited VEGFR1 and PDGFRb kinase with IC50 values of less than
100 nM. It has been reported that some type-II kinase inhibitors
show inhibitory activity against not only the original target kinase
but also other kinases which can adopt the DFG-out conforma-
Table 1 summarizes the results of modifications to the hinge-
binding core scaffold. Replacement of imidazo[1,2-b]pyridazine
(2) with other 5,6-fused heterocycles possessing a nitrogen at the
1-position (13a, 17a, 22a, and 33) retained strong VEGFR2 inhibi-
tory activity. This result suggested that these compounds adopt
similar binding modes to 2 with VEGFR2, with the N1-nitrogen
forming a tight hydrogen bond with the NH group of Cys919. Mov-
ing the nitrogen of 2 from the 5-position to the 3-position (13a) as
well as replacement of the N5-nitrogen of 2 with carbon (17a) did
not affect VEGFR2 inhibition. These results suggest that there are
no amino acid residues that could potentially hydrogen bond with
these compounds in this region. In addition, the retention of po-

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4719
Table 1
Effect of modifications to the hinge-binding corea
Compd
Ring
R
VEGFR2 IC50 (nM)
1.4 (1.3–1.5)
HUVECb IC50 (nM)
1.7 (0.66–3.8)
Solubility JP2c
1.7
(
lg/mL)
ClogPd
2
H
2.3
13a
H
1.1 (1.0–1.3)
3.9 (2.0–7.5)
1.2
2.4
13b
13c
28
2-Me
3-Me
4-Me
2-F
1.9 (1.6–2.2)
2.1 (1.8–2.4)
3.7 (3.3–4.3)
2.0 (1.8–2.3)
1.3 (0.59–2.6)
47 (17–136)
67 (48–92)
53
2.2
2.9
2.9
1.9
3.2
3.7
65
13d
5.0 (3.0–8.0)
17a
17b
H
1.1 (1.0–1.2)
1.2 (1.1–1.3)
1.1 (0.34–2.6)
0.80 (0.38–1.5)
0.73
2.9
3.3
3.1
2-Me
22a
22b
H
1.3 (1.1–1.4)
1.2 (1.0–1.4)
4.4 (1.9–9.9)
3.6 (1.4–8.6)
<0.04
4.8
3.3
3.2
2-Me
33
H
3.6 (3.0–4.3)
0.93 (0.12–3.7)
0.22
4.2
a
b
c
Numbers in parentheses represent 95% conidence interval.
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
Kinetic solubility in the 2nd fluid of Disintegration Test of the Japanese Pharmacopoeia (pH 6.8).
d
ClogP value was calculated by Daylight Software. ClogP, version 4.95, Daylight Software, Daylight chemical information systems, Inc., Aliso Viejo, CA; http://
www.daylight.com.
Table 2
Kinase selectivity of compounds 2, 13a, 17a, 22a, and 33a
Compd
Ring
IC50 (nM)b
VEGFR1
VEGFR2
PDGFRb
c-kit
Tie-2
Aurora A
B-raf
p38a
2
7.4 (6.4–8.4)
1.4 (1.3–1.5)
5.5 (3.8–7.9)
23 (19–27)
>1000
360 (240–530)
>1000
>1000
13a
17a
22a
33 (25–43)
6.4 (5.4–7.7)
56 (36–87)
1.1 (1.0–1.3)
1.1 (1.0–1.2)
1.3 (1.1–1.4)
19 (14–25)
5.9 (4.6–7.8)
8.3 (4.6–15)
130 (110–170)
21 (16–26)
>1000
>1000
>1000
120 (44–330)
170 (110–260)
>1000
290 (42–2000)
>1000
>1000
>1000
>1000
170 (110–260)
>1000
33
69 (62–77)
3.6 (3.0–4.3)
21 (14–33)
240 (190–290)
>1000
680 (150–3200)
>1000
>1000
a
Abbreviations of kinases are described in Section 5.
Numbers in parentheses represent 95% confidence interval.
b
tion.21 For instance, a pyrrolo[3,2-d]pyrimidine-based VEGFR2 ki-
nase inhibitor we previously reported has strong Tie-2 kinase
inhibitory activity.24 Thus, we investigated kinase selectivity
against c-kit,30 Aurora A,31 B-raf,32 Tie-2,33 and p38 mitogen-acti-
vated protein kinase a (p38a)
22 whose crystal structures with their
type-II inhibitors have been reported. Interestingly, inhibitory
activity against c-kit, Aurora A, and B-raf was different among
these chemotypes, while Tie-2 and p38a were not strongly inhib-

4720
Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
Table 3
Modification of the acyl groupa
Compd
R
VEGFR2 IC50 (nM)
1.9 (1.6–2.2)
HUVECb IC50 (nM)
1.3 (0.59–2.6)
Solubility JP2c
53
(lg/mL)
ClogPd
13b
2.2
29a
29b
29c
29d
1.5 (1.3–1.7)
0.48 (0.43–0.55)
1.9 (1.7–2.2)
1.6 (1.4–1.7)
4.3 (1.5–12)
1.6 (0.9–26)
3.8 (1.5–9.3)
9.9 (4.3–23)
0.16
1.2
3.5
3.7
1.9
1.9
>93
>80
29e
29f
2.0 (1.7–2.2)
3.0 (2.6–3.5)
56 (33–96)
12
3.4
2.8
88 (59–133)
>78
a
b
c
Numbers in parentheses represent 95% confidence interval.
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
Kinetic solubility in the 2nd fluid of Disintegration Test of the Japanese Pharmacopoeia (pH 6.8).
ClogP value was calculated by Daylight Software. ClogP, version 4.95, Daylight Software, Daylight chemical information systems, Inc., Aliso Viejo, CA; http://
d
www.daylight.com.
ited by any of the evaluated compounds even at a concentration of
1000 nM. Imidazo[1,2-b]pyridazine 2 and imidazo[1,2-a]pyridine
17a inhibited c-kit more potently than the other three compounds,
suggesting that the carbon atom at the 3-position of their bicyclic
cores is favorable for c-kit inhibition. On the other hand, the fact
that the [1,2,4]triazolo[1,5-a]pyridine 13a showed the strongest
inhibitory activity against B-raf kinase may suggest the importance
of the nitrogen atom at the 3-position for B-raf inhibition. Unlike c-
kit and B-raf, inhibition data for Aurora A showed less dependence
on the hinge-binding core. Except for the thiazolo[5,4-b]pyridine
22a, most of these compounds displayed moderate inhibition
against Aurora A. These results revealed that modification of the
hinge-binding core can be utilized to adjust kinase selectivity.
We next evaluated kinetic solubility for compounds in the neu-
tral aqueous solution (JP2: the 2nd fluid of Disintegration Test of
the Japanese Pharmacopoeia, pH 6.8). The measured solubility for
2 in JP2 was 1.7 lg/mL. Compared to 2, compounds 13a and 17a
showed similar solubility, while 22a and 33 were found to be less
soluble. The poor solubility of 22a and 33 may be attributable to
their higher lipophilicity.34 Another hypothesis is that the intramo-
lecular interaction between the S3-sulfur and the carbonyl oxygen
at the 2-position might stabilize the planar conformations of 22a
and 33, thus enabling tight crystal packing. Indeed, SÁ Á ÁO interac-
tions have been observed in a large number of organosulfur com-
pounds controlling the conformation of small and large
molecules.35 On the other hand, introduction of a methyl group
into the central benzene ring (13b, 17b, 22b) resulted in improve-
ment of solubility, especially for the triazolo[1,5-a]pyridine 13b
(JP2 solubility: 53 lg/mL). The improved solubility brought by
the methyl substituent might be due to distortion from molecular
planarity and/or symmetry. Recently, Ishikawa et al. reported that
disruption of molecular planarity leads to improvement of aqueous
Table 4
Pharmacokinetic properties of 2, 13d, and 29c
Compd
Mouse PKa
Metabolic stabilityd
L/min/mg) Mouse (lL/min/mg)
Solubility JP2e
(lg/mL)
Cmax
(lg/mL)
AUC0–8h
(lgÁh/mL)
C8h
(l
g/mL)c
Human (
l
b
2
13d
29c
0.15
3.11
3.21
0.730
7.39
2.31
0.053
0.247
0.011
6.0
10
6.0
43
45
78
1.7
65
>93
a
b
c
Compounds were administered at an oral dose of 10 mg/kg as a cassette dosing.
Area under the plasma concentration–time curve.
Plasma concentration at 8 h after administration.
Metabolic stability in hepatic microsomes.
Kinetic solubility in the 2nd fluid of Disintegration Test of the Japanese Pharmacopoeia (pH 6.8).
d
e

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4721
Table 5
solubility and lower melting points by decreased crystal packing
Preincubation time-dependent inhibition of 13d and 29fa
energy.36 In fact, the melting point (217–220 °C) of 13b was lower
than that of 13a (244–245 °C).
Compd
VEGFR2 IC50 (nM) pre-incubationb
HUVECc IC50 (nM)
Since 13b exhibited not only strong inhibitory activity against
VEGFR2 kinase but also improved solubility, we investigated more
substitutions on the central benzene ring. Moving the methyl
group of 13b from the 2-position to the 3- or 4-position (13c, 28)
retained inhibitory activity against VEGFR2 kinase. On the other
hand, 13c and 28 were less potent than 13b in HUVEC proliferation
assay despite their retained enzyme activity, revealing that substi-
tution at the 2-position was optimal. Further investigation re-
vealed that substitution of the electron-withdrawing group at
this position was also tolerated. In fact, the 2-fluoro derivative
(13d) exhibited single-digit nanomolar IC50 values in both of en-
zyme and cellular assays. As a result of kinetic solubility screening,
5 min
60 min
13d
29f
2.2 (2.0–2.5)
3.9 (3.4–4.4)
0.26 (0.25–0.28)
3.1 (2.4–4.0)
5.0 (3.0–8.0)
88 (59–133)
a
Numbers in parentheses represent 95% conidence interval.
The assay was performed with preincubation of the compounds and VEGFR2
before addition of 1 mM of ATP.
b
c
Growth inhibition of human umbilical vein endothelial cells (HUVEC).
peak plasma concentrations (Cmax) as well as area under the plas-
ma concentration-time curve (AUC0–8h) values than that of 2, indi-
cating that their improved solubilities might contribute to their
improved pharmacokinetic properties. In particular, 13d exhibited
not only a favorable AUC value but also high plasma concentration
at 8 h after administration (C8h) reflecting its high stability in
mouse hepatic microsomes. Since 13d also showed high stability
in human hepatic microsomes, it would be expected to have suffi-
cient oral bioavailability in humans.
13d (JP2 solubility: 65 lg/mL) showed improved solubility compa-
rable to the 2-methyl derivative 13b. One possible explanation for
the improved solubility observed for 13d could be due to its lower
lipophilicity compared to 13a.
The crystal structure of the complex between 2 and VEGFR2
(Fig. 2) indicated that modification of the terminal 1,3-dimethyl-
pyrazole moiety would be possible because no pyrazole-specific
interaction with the surrounding amino acid residues was ob-
served. Thus, we examined the effect of the terminal acyl group
(Table 3). As the co-crystal structure analysis suggested, a variety
of acyl groups were tolerated with respect to enzymatic activity.
Replacement of a pyrazole group (13b) with not only other hetero-
aromatic rings (29a,b) but also aliphatic groups (29e,f) retained
strong VEGFR2 inhibitory activity. In contrast, cell activity and sol-
ubility showed a more marked dependence on the acyl group. As
for HUVEC inhibition, the five-membered heterocycle derivatives
(29a–d) were more potent than the aliphatic derivatives (29e,f).
The fact that the compounds showing similar ClogP value (e.g.
29a and 29e) have different HUVEC inhibitory activity revealed
that lipophilicity of compounds has little relation to their cellular
activity. The planar structure of heteroaryl groups may rather con-
tribute to enhance cellular activity. On the other hand, lipophilicity
of compounds seemed to influence their solubility. For example,
the monomethyl pyrazole derivative 29d displayed improved sol-
ubility compared to the corresponding dimethyl pyrazole deriva-
tive 13b. Similar tendency was observed in the dimethyl-
substituted heteroaryl derivatives (13b and 29a,c). Compared to
13b, more polar 29c showed improved solubility while less polar
29a was much less soluble.
Recently, we demonstrated that pyrrolo[3,2-d]pyrimidine and
imidazo[1,2-b]pyridazine derivatives inhibited VEGFR2 with very
slow, reversible (pseudo-irreversible) kinetics.24,37,38 These favor-
able properties may contribute to their enhanced cell activity.
Thus, we evaluated the VEGFR2 inhibition kinetics of the triazol-
o[1,5-a]pyridine derivatives presented in this work. Table 5 shows
the pre-incubation time dependency of compounds in VEGFR2
inhibition. Pre-incubation of 13d and VEGFR2 for 60 min before
addition of ATP to start the kinase reaction resulted in 10-fold
enhancement of enzymatic activity (IC50 = 0.26 nM) compared to
that for 5 min of pre-incubation time (IC50 = 2.2 nM). This time
dependency indicated that 13d possesses slow-binding kinetics
against VEGFR2. On the other hand, 29f did not show pre-incuba-
tion time dependency, suggesting that it is a fast-binding inhibitor.
Inhibitory activity of the compounds with the longer preincubation
was found to be better correlated with their HUVEC inhibitory
activities. The VEGFR2 IC50 value obtained with 60-min preincuba-
tion for compound 13d was about 10-fold lower than that for 29f
(0.26 nM vs 3.1 nM), which supports the ratio of IC50 values ob-
served in the HUVEC proliferation assay (5.0 nM for 13d vs
88 nM for 29f). To better understand inhibition kinetics against
VEGFR2 kinase, dilution assay of VEGFR2–13d complex was per-
formed. When the pre-formed complex of 13d and VEGFR2 was di-
luted to one tenth of IC50 to dissociate the complex and the kinase
reaction was initiated, the recovery of kinase activity was much
slower than in the absence of compound (Fig. 4). The above results
revealed that 13d also possesses slow-dissociation kinetics. Advan-
tages of compounds showing slow-dissociation kinetics include
improvement of off-target selectivity and in vivo efficacy.39 Thus,
Compounds that displayed strong HUVEC inhibition and mod-
erate solubility were selected for further evaluation (Table 4).
Pharmacokinetic parameters for 2, 13d, and 29c were measured
in mice (cassette dosing). Compounds 13d and 29c showed higher
200000
control
13d
Table 6
150000
100000
50000
0
Kinase selectivity of 13da,b
Kinase
IC50 (nM)
Kinase
IC50 (nM)
VEGFR2
VEGFR1
2.0 (1.8–2.3)
3.6 (3.3–3.8)
12 (10–14)
14 (11–17)
2700 (1900–3900)
>10,000
>10,000
>10,000
>10,000
IGF1-R
c-kit
Src
FAK
B-raf
ERK1
PKCh
GSK3b
Aurora A
>10,000
150 (120–200)
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2900 (1800–4500)
PDGFR
a
PDGFRb
FGFR1
Tie-2
HER2
EGFR
IR
0
1000 2000 3000 4000 5000 6000
time,sec
Figure 4. Dilution assay of VEGFR2–13d complex. Phosphorylation of peptide
substrate as a function of time is shown. The reaction was initiated by diluting a
preformed enzyme–inhibitor complex to one tenth of IC50 with reaction buffer
p38
a
>10,000
a
Abbreviations of kinases are described in Section 5.
Numbers in parentheses represent 95% confidence interval.
containing 1 mM ATP and 0.1
lg/mL biotinylated poly-Glu-Tyr (4:1). The recovery
b
of activity was measured using the AlphaScreenÒ system.

4722
Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
Table 7
(300 MHz) or Bruker DPX300 (300 MHz) instruments. Chemical
shifts are reported as d values (ppm) downfield from internal tetra-
methylsilane of the indicated organic solution. Peak multi-plicities
are expressed as follows: s, singlet; d, doublet; t, triplet; q, quartet;
dd, doublet of doublet; ddd, doublet of doublet of doublets; dt, dou-
blet of triplet; br s, broad singlet; m, multiplet. Coupling constants
(J values) are given in hertz (Hz). Elemental analyses were carried
out by Takeda Analytical Laboratories. Reaction progress was deter-
mined by thin layer chromatography (TLC) analysis on silica gel 60
F254 plate (Merck) or NH TLC plates (Fuji Silysia chemical Ltd.).
Chromatographic purification was carried on silica gel columns
60 (0.063–0.200 mm or 0.040–0.063 mm, Merck), basic silica gel
(ChromatorexNH, 100–200 mesh, Fuji silysia chemical Ltd) or Pur-
if-Pack (SI 60 lM or NH 60 lM, Fuji Silysia, Ltd). Commercial reagents
and solvents were used without additional purification. Abbrevia-
tions are used as follows: CDCl3, deuterated chloroform; DMSO-
d6, dimethyl sulfoxide-d6; AcOEt, ethyl acetate; DMF, N,N-dimeth-
ylformamide; MeOH, methanol; THF, tetrahydrofuran; EtOH, etha-
nol; DMSO, dimethyl sulfoxide; DMA, N,N-dimethylactamide.
Antitumor efficacy of 13d in nude mice bearing human tumor xenografts
Tumor model
Dosea (mg/kg)
T/Cb (%)
DU145 (prostate)
0.5
1.5
5.0
0.5
1.5
5.0
60
37
27
57
34
14
A549 (NSCLC)
a
Compound was dosed twice daily for14 days.
Treatment per control (T/C %), an index of antitumor efficacy, was calculated by
b
comparison of the mean change in tumor volume over the treatment period for the
control and treated groups.
13d is expected to show prolonged anti-tumor activity with fewer
off-target effects in an in vivo setting.
We evaluated the kinase selectivity of compound 13d, and the
results are shown in Table 6. Compound 13d was found to potently
inhibit platelet-derived growth factor receptor (PDGFR) family ki-
nases in addition to the VEGFR kinases. Platelet-derived growth
factor (PDGF) and the PDGFR signaling pathway are necessary for
the pericyte recruitment during angiogenesis.40 It was also sug-
gested that inhibition of PDGF/PDGFR signaling is an effective
way for blocking further tumor growth in end-stage tumors.41
Thus, simultaneous inhibition of VEGFR and PDGFR kinases may
lead to synergistic effects in anti-angiogenic tumor therapy. The
inhibitory potency of 13d against several other protein kinases,
such as human epidermal growth factor receptor 2 (HER2), epider-
mal growth factor receptor (EGFR), insulin receptor (IR), and pro-
tein kinase C h (PKCh) were all over 1000 nM, with c-kit showing
the most potent off-target inhibition (IC50 = 150 nM). These results
revealed that 13d is a highly selective and potent inhibitor of VEG-
FR/PDGFR kinases.
5.1. 2-Nitro-5-(3-nitrophenoxy)pyridine (4)
A mixture of 3 (7.76 g, 55.8 mmol), 5-bromo-2-nitropyridine
(10.3 g, 50.7 mmol), cesium carbonate (24.8 g, 76.1 mmol), and
DMF (150 mL) was stirred at 50 °C for 15 h. The mixture was di-
luted with water and extracted with AcOEt. The extract was
washed with water and brine, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue
was purified by basic silica gel column chromatography (AcOEt/
hexane) to give 4 (4.37 g, 33%) as a yellow solid: 1H NMR
(DMSO-d6) d 7.73–7.85 (3H, m), 8.10 (1H, t, J = 2.1 Hz), 8.15–8.19
(1H, m), 8.38 (1H, dd, J = 9.0, 0.6 Hz), 8.52–8.54 (1H, m).
Finally, we evaluated the antitumor efficacy of 13d in DU145
(human prostate cancer) and A549 (non-small cell lung cancer)
xenograft models in nude mice (Table 7). Consistent with its potent
cellular activity and good oral exposure profiles, 13d showed sig-
nificant anti-tumor efficacy in the both models tested when dosed
orally at 10 mg/kg daily (5.0 mg/kg, bid). Under these conditions,
neither body weight loss nor obvious signs of toxicity were ob-
served (data not shown).
5.2. 5-(3-Aminophenoxy)pyridin-2-amine (5)
A mixture of 4 (1.33 g, 5.07 mmol), 10% palladium on carbon
(water ꢀ50%, 100 mg), and MeOH (10 mL) was stirred under a
hydrogen atmosphere at room temperature for 12 h. The catalyst
was filtered off, and the filtrate was concentrated in vacuo to give
5 (980 mg, 96%) as a yellow oil: 1H NMR (DMSO-d6) d 5.15 (2H, br
s), 5.82 (2H, br s), 6.00–6.04 (2H, m), 6.18–6.22 (1H, m), 6.47 (1H, d,
J = 8.9 Hz), 6.90 (1H, t, J = 7.7 Hz), 7.14 (1H, dd, J = 8.9, 3.0 Hz), 7.69
(1H, d, J = 3.0 Hz).
4. Conclusion
By replacement of the imidazo[1,2-b]pyridazine core of 2 with
[1,2,4]triazolo[1,5-a]pyridine (13a), the imidazo[1,2-a]pyridine
(17a), the thiazolo[5,4-b]pyridine (22a), and 1,3-benzothiazole
(33), we developed a novel series of type-II VEGFR2 kinase inhibi-
tors. Further optimization revealed that introduction of a substitu-
ent into the central benzene ring of the [1,2,4]triazolo[1,5-
a]pyridine 13a and subsequent modification of the terminal pyra-
zole moiety (13d and 29c) led to improvement of solubility, which
resulted in improved pharmacokinetic properties. Results of fur-
ther studies revealed that representative compound 13d inhibited
VEGFR2 kinase with slow dissociation kinetics and showed strong
inhibitory activity against PDGFR kinases. Consistent with its
favorable profile described above, 13d showed potent antitumor
efficacy without obvious signs of toxicity in DU145 and A549
mouse xenograft models. Thus, based on its slow dissociation
kinetics and potent VEGFR/PDGFR kinase inhibitory activities,
compound 13d shows great promise as an anticancer agent.
5.3. N-{3-[(6-Aminopyridin-3-yl)oxy]phenyl}-1,3-dimethyl-1H-
pyrazole-5-carboxamide (6a)
To a solution of 5 (975 mg, 4.85 mmol), pyridine (410 lL,
5.09 mmol) in THF (10 mL) was added a solution of 8 (807 mg,
5.09 mmol) in THF (10 mL) dropwise at 0 °C, and the mixture
was stirred at room temperature for 2 h. The mixture was diluted
with water and extracted with AcOEt. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography (AcOEt/hexane) followed by recrystalliza-
tion from AcOEt–hexane to give 6a (964 mg, 61%) as a white
solid: 1H NMR (DMSO-d6) d 2.18 (3H, s), 3.96 (3H, s), 5.91 (2H, br
s), 6.50 (1H, d, J = 8.9 Hz), 6.65–6.68 (1H, m), 6.78 (1H, s), 7.11
(1H, dd, J = 8.9, 3.0 Hz), 7.24–7.30 (2H, m), 7.43–7.47 (1H, m),
7.75 (1H, d, J = 3.0 Hz), 10.10 (1H, br s).
5.4. N-{5-[(6-Aminopyridin-3-yl)oxy]-2-methylphenyl}-1,3-
dimethyl-1H-pyrazole-5-carboxamide (6b)
5. Experimental
Melting points were determined on a BÜCHI Melting Point B-
545, and were not corrected. Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on Varian Mercury 300
A mixture of 10a (3.38 g, 9.20 mmol), 10% palladium on carbon
(water ꢀ50%, 300 mg), and MeOH (20 mL) was stirred under a

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4723
hydrogen atmosphere at room temperature for 4 h. The catalyst
was filtered off, and the filtrate was concentrated in vacuo to give
6b (2.94 g, 95%) as a white solid: 1H NMR (DMSO-d6) d 2.15 (3H, s),
2.18 (3H, s), 3.97 (3H, s), 5.88 (2H, br s), 6.49 (1H, dd, J = 9.0,
0.6 Hz), 6.73–6.78 (2H, m), 6.88 (1H, d, J = 2.4 Hz), 7.17–7.22 (2H,
m), 7.73–7.75 (1H, m), 9.71 (1H, br s).
due was purified by silica gel column chromatography (AcOEt/
hexane) and basic silica gel column chromatography (AcOEt/hex-
ane) to give 10a (3.38 g, 44%) as a white solid: 1H NMR (DMSO-
d6) d 2.20 (3H, s), 2.27 (3H, s), 3.98 (3H, s), 6.82 (1H, s), 7.09 (1H,
dd, J = 8.3, 2.5 Hz), 7.31 (1H, d, J = 2.5 Hz), 7.41 (1H, d, J = 8.3 Hz),
7.62 (1H, dd, J = 8.8, 2.8 Hz), 8.36 (1H, d, J = 8.8 Hz), 8.42 (1H, d,
J = 2.8 Hz), 9.82 (1H, s).
5.5. N-{3-[(6-Aminopyridin-3-yl)oxy]-5-methylphenyl}-1,3-
dimethyl-1H-pyrazole-5-carboxamide (6c)
5.11. 1,3-Dimethyl-N-{3-methyl-5-[(6-nitropyridin-3-
yl)oxy]phenyl}-1H-pyrazole-5-carboxamide (10b)
The compound 6c was prepared from 10b in a manner similar
to that described for 6b to yield a pale yellow oil (88%): 1H NMR
(CDCl3) d 2.28 (3H, s), 2.31 (3H, s), 4.12 (3H, s), 4.40 (2H, s), 6.40
(1H, s), 6.50–6.57 (2H, m), 6.92 (1H, t, J = 2.0 Hz), 7.17 (1H, s),
7.21 (1H, dd, J = 8.6, 2.6 Hz), 7.65 (1H, s), 7.89–7.92 (1H, m).
The compound 10b was prepared from 9b in a manner similar to
that described for 10a to yield a pale yellow oil (54%): 1H NMR
(CDCl3) d 2.29 (3H, s), 2.39 (3H, s), 4.12 (3H, s), 6.73 (1H, s), 7.19
(1H, s), 7.43 (1H, t, J = 2.0 Hz), 7.46 (1H, dd, J = 8.8, 3.0 Hz), 7.74
(1H, s), 8.01 (1H, s), 8.25 (1H, d, J = 8.8 Hz), 8.34 (1H, d, J = 3.0 Hz).
5.6. N-{5-[(6-Aminopyridin-3-yl)oxy]-2-fluorophenyl}-1,3-
dimethyl-1H-pyrazole-5-carboxamide (6d)
5.12. N-{2-Fluoro-5-[(6-nitropyridin-3-yl)oxy]phenyl}-1,3-
dimethyl-1H-pyrazole-5-carboxamide (10c)
The compound 6d was prepared from 10c in a manner similar
to that described for 6b to yield a white solid (88%): 1H NMR
(DMSO-d6) d 2.18 (3H, s), 3.96 (3H, s), 5.90 (2H, s), 6.50 (1H, d,
J = 9.0 Hz), 6.76–6.85 (2H, m), 7.10 (1H, dd, J = 6.2, 3.2 Hz), 7.16–
7.28 (2H, m), 7.76 (1H, d, J = 3.0 Hz), 9.97 (1H, s).
The compound 10c was prepared from 9c in a manner similar to
that described for 10a to yield a white solid (59%): 1H NMR (DMSO-
d6) d 2.20 (3H, s), 3.99 (3H, s), 6.87 (1H, s), 7.16–7.22 (1H, m), 7.46
(1H, dd, J = 10.5, 8.9 Hz), 7.59 (1H, dd, J = 6.3, 3.0 Hz), 7.66 (1H, dd,
J = 9.5, 2.9 Hz), 8.36 (1H, d, J = 9.3 Hz), 8.44 (1H, d, J = 2.7 Hz), 1H
not detected.
5.7. N-(5-Hydroxy-2-methylphenyl)-1,3-dimethyl-1H-pyrazole-
5-carboxamide (9a)
5.13. N-{3-[(2-Amino[1,2,4]triazolo[1,5-a]pyridin-6-
To a solution of 7a (3.71 g, 30.1 mmol), triethylamine (4.38 mL,
31.6 mmol) in THF (30 mL) was added a solution of 8 (5.02 g,
31.6 mmol) in THF (10 mL) dropwise at 0 °C, and the mixture
was stirred at room temperature for 15 h. The mixture was diluted
with water and extracted with AcOEt. The extract was washed with
brine, dried over anhydrous magnesium sulfate, and concentrated
under reduced pressure. The residue was collected by filtration and
washed with AcOEt–hexane to give 9a (4.67 g, 63%) as a white so-
lid: 1H NMR (DMSO-d6) d 2.09 (3H, s), 2.19 (3H, s), 3.98 (3H, s), 6.57
(1H, dd, J = 8.5, 2.4 Hz), 6.76–6.79 (2H, m), 7.02 (1H, d, J = 8.5 Hz),
9.26 (1H, br s), 9.59 (1H, s).
yl)oxy]phenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (12a)
To a solution of 6a (184 mg, 0.569 mmol) in DMSO (4 mL) was
added ethoxycarbonyl isothiocyanate (89.6 mg, 0.682 mmol), and
the mixture was stirred at room temperature for 15 h. The mixture
was diluted with water and extracted with AcOEt. The extract was
washed with brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure to give 11a (268 mg, 99%)
as a colorless oil. A mixture of 11a (238 mg, 0.524 mmol), hydrox-
ylammonium chloride (364 mg, 5.24 mmol), N,N-diisopropylethyl-
amine (548 lL, 3.14 mmol), EtOH (3 mL), and MeOH (3 mL) was
stirred at 60 °C for 5 h and 80 °C for 2 h. The mixture was diluted
with water and extracted with AcOEt. The extract was washed with
water and brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (AcOEt/hexane and AcOEt/
MeOH) and recrystallized from AcOEt–hexane to give 12a
(131 mg, 69%) as a white solid: 1H NMR (DMSO-d6) d 2.18 (3H,
s), 3.96 (3H, s), 6.03 (2H, s), 6.78–6.82 (2H, m), 7.30–7.43 (4H,
m), 7.52–7.57 (1H, m), 8.65 (1H, d, J = 2.4 Hz), 10.13 (1H, s).
5.8. N-(3-Hydroxy-5-methylphenyl)-1,3-dimethyl-1H-pyrazole-
5-carboxamide (9b)
The compound 9b was prepared from 7b and 8 in a manner
similar to that described for 9a to yield a pale yellow oil (96%):
1H NMR (CDCl3) d 2.29 (3H, s), 2.30 (3H, s), 4.13 (3H, s), 5.58 (1H,
s), 6.39 (1H, s), 6.49 (1H, s), 6.80 (1H, s), 7.13 (1H, t, J = 2.0 Hz),
7.55 (1H, s).
5.9. N-(2-Fluoro-5-hydroxyphenyl)-1,3-dimethyl-1H-pyrazole-
5-carboxamide (9c)
5.14. N-{5-[(2-Amino[1,2,4]triazolo[1,5-a]pyridin-6-yl)oxy]-2-
methylphenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (12b)
The compound 9c was prepared from 7c and 8 in a manner sim-
ilar to that described for 9a to yield a white solid (99%): 1H NMR
(DMSO-d6) d 2.19 (3H, s), 3.98 (3H, s), 6.58–6.64 (1H, m), 6.83
(1H, s), 6.97–7.09 (2H, m), 9.87 (1H, s), 1H not detected.
The compound 12b was prepared from 6b in a manner similar
to that described for 12a to yield a white solid (75%): 1H NMR
(DMSO-d6) d 2.18 (6H, s), 3.96 (3H, s), 5.99 (2H, s), 6.77 (1H, s),
6.87 (1H, dd, J = 8.6, 2.6 Hz), 7.02 (1H, d, J = 2.6 Hz), 7.23–7.29
(2H, m), 7.38 (1H, d, J = 9.3 Hz), 8.55 (1H, d, J = 1.8 Hz), 9.73 (1H, s).
5.10. 1,3-Dimethyl-N-{2-methyl-5-[(6-nitropyridin-3-
yl)oxy]phenyl}-1H-pyrazole-5-carboxamide (10a)
5.15. N-{3-[(2-Amino[1,2,4]triazolo[1,5-a]pyridin-6-yl)oxy]-5-
methylphenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (12c)
A mixture of 9a (4.67 g, 19.0 mmol), 5-bromo-2-nitropyridine
(3.68 g, 18.1 mmol), cesium carbonate (9.29 g, 28.5 mmol), and
DMF (20 mL) was stirred at room temperature for 15 h. The mix-
ture was diluted with water and extracted with AcOEt. The extract
was washed with water and brine, dried over anhydrous magne-
sium sulfate, and concentrated under reduced pressure. The resi-
The compound 12c was prepared from 6c in a manner similar to
that described for 12a to yield a white solid (63%): 1H NMR (DMSO-
d6) d 2.17 (3H, s), 2.28 (3H, s), 3.96 (3H, s), 6.01 (2H, s), 6.63 (1H, s),
6.77 (1H, s), 7.17 (1H, t, J = 1.9 Hz), 7.25–7.34 (1H, m), 7.36–7.45
(2H, m), 8.61 (1H, d, J = 1.9 Hz), 10.03 (1H, s).

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Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
5.16. N-{5-[(2-Amino[1,2,4]triazolo[1,5-a]pyridin-6-yl)oxy]-2-
fluorophenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (12d)
8.91 (1H, d, J = 2.3 Hz), 10.05 (1H, s), 11.04 (1H, s); Anal. Calcd
for C23H23N7O3: C, 62.01; H, 5.20; N, 22.01. Found: C, 62.00; H,
5.22; N, 22.10.
The compound 12d was prepared from 6d in a manner similar
to that described for 12a to yield a yellow solid (69%): 1H NMR
(DMSO-d6) d 2.18 (3H, s), 3.96 (3H, s), 6.02 (2H, s), 6.82 (1H, s),
6.92–6.98 (1H, m), 7.23–7.33 (3H, m), 7.40 (1H, dd, J = 9.3,
0.6 Hz), 8.61 (1H, dd, J = 2.1, 0.6 Hz), 10.01 (1H, s).
5.20. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-fluorophenyl]-1,3-dimethyl-1H-pyrazole-
5-carboxamide (13d)
The compound 13d was prepared from 12d in a manner similar
to that described for 13c to yield white crystals (71%); mp 209 °C;
1H NMR (DMSO-d6) d 0.81–0.84 (4H, m), 1.99–2.08 (1H, m), 2.18
(3H, s), 3.96 (3H, s), 6.81 (1H, s), 6.98–7.03 (1H, m), 7.29–7.35
(2H, m), 7.52 (1H, dd, J = 9.6, 2.4 Hz), 7.73 (1H, d, J = 9.6 Hz), 8.87
(1H, d, J = 2.4 Hz), 10.02 (1H, s), 11.02 (1H, s); Anal. Calcd for
5.17. N-[3-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (13a)
To a solution of 12a (117 mg, 0.322 mmol), triethylamine
(134
bonyl chloride (38.0
was stirred at room temperature for 12 h. Cyclopropanecarbonyl
chloride (29.3 L, 0.322 mmol) was added to the mixture, and
l
L, 0.966 mmol) in THF (5 mL) was added cyclopropanecar-
C22H20FN7O3: C, 58.79; H, 4.49; N, 21.82. Found: C, 58.61; H,
4.36; N, 21.65.
lL, 0.419 mmol) at 0 °C, and the mixture
l
5.21. 1,3-Dimethyl-N-{3-[(6-{[(4-
the resulting mixture was stirred at room temperature for 3 h.
The mixture was diluted with water and extracted with AcOEt.
The extract was washed with water and brine, dried over anhy-
drous magnesium sulfate, and concentrated under reduced pres-
sure. To the residue thus obtained was added Na2CO3 (100 mg),
methylphenyl)sulfonyl]amino}pyridin-3-yl)oxy]phenyl}-1H-
pyrazole-5-carboxamide (14a)
A mixture of 6a (750 mg, 2.32 mmol), para-toluenesulfonyl
chloride (486 mg, 2.55 mmol), and pyridine (6 mL) was stirred at
80 °C for 15 h. The mixture was diluted with water and extracted
with AcOEt. The extract was washed with brine, dried over anhy-
drous magnesium sulfate, and concentrated under reduced pres-
sure to give 14a (1.13 g, 99%) as a colorless oil: 1H NMR (DMSO-
d6) d 2.18 (3H, s), 2.35 (3H, s), 3.33 (3H, s), 6.71–6.77 (2H, s),
7.13 (1H, d, J = 8.7 Hz), 7.29–7.53 (6H, m), 7.78 (2H, d, J = 8.4 Hz),
8.01 (1H, d, J = 3.0 Hz), 10.14 (1H, s), 11.06 (1H, br s).
MeOH (2 mL), and water (100 lL). The mixture was stirred at
50 °C for 30 min. The mixture was diluted with water and ex-
tracted with AcOEt. The extract was washed with water and brine,
dried over anhydrous magnesium sulfate, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (AcOEt) followed by recrystallization from AcOEt
to give 13a (112 mg, 60%) as white crystals: mp 244–245 °C; 1H
NMR (DMSO-d6) d 0.80–0.85 (4H, m), 2.00–2.01 (1H, m), 2.17
(3H, s), 3.96 (3H, s), 6.77–6.85 (2H, m), 7.35 (1H, t, J = 8.3 Hz),
7.40 (1H, t, J = 2.1 Hz), 7.51–7.58 (2H, m), 7.74 (1H, d, J = 9.3 Hz),
8.94 (1H, d, J = 2.4 Hz), 10.14 (1H, s), 11.05 (1H, s); Anal. Calcd
for C22H21N7O3Á0.25AcOEt: C, 60.92; H, 5.11; N, 21.62. Found: C,
61.10; H, 5.21; N, 21.38.
5.22. 1,3-Dimethyl-N-{2-methyl-5-[(6-{[(4-
methylphenyl)sulfonyl]amino}pyridin-3-yl)oxy]phenyl}-1H-
pyrazole-5-carboxamide (14b)
The compound 14b was prepared from 6b in a manner similar
to that described for 14a to yield a white solid (77%): 1H NMR
(DMSO-d6) d 2.19 (6H, s), 2.34 (3H, s), 3.97 (3H, s), 6.78–6.84
(2H, m), 6.98 (1H, d, J = 2.7 Hz), 7.13 (1H, d, J = 8.9 Hz), 7.25 (1H,
d, J = 8.7 Hz), 7.36 (2H, d, J = 8.1 Hz), 7.44 (1H, dd, J = 8.9, 3.0 Hz),
7.77 (2H, d, J = 8.1 Hz), 7.97 (1H, d, J = 3.0 Hz), 9.73 (1H, s), 11.02
(1H, br s).
5.18. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (13b)
The compound 13b was prepared from 12b in a manner similar
to that described for 13a to yield white crystals (60%): mp 217–
220 °C; 1H NMR (DMSO-d6) d 0.81–0.83 (4H, m), 1.98–2.10 (1H,
m), 2.18 (3H, s), 2.19 (3H, s), 3.96 (3H, s), 6.78 (1H, s), 6.92 (1H,
dd, J = 8.4, 2.6 Hz), 7.07 (1H, d, J = 2.6 Hz), 7.27 (1H, d, J = 8.4 Hz),
7.49 (1H, dd, J = 9.5, 2.3 Hz), 7.71 (1H, d, J = 9.5 Hz), 8.53 (1H, d,
J = 2.3 Hz), 9.76 (1H, s), 11.04 (1H, s); Anal. Calcd for
5.23. N-(3-{[1-(2-Amino-2-oxoethyl)-6-{[(4-
methylphenyl)sulfonyl]imino}-1,6-dihydropyridin-3-
yl]oxy}phenyl)-1,3-dimethyl-1H-pyrazole-5-carboxamide (15a)
A mixture of 14a (1.11 g, 2.32 mmol), N,N-diisopropylethyl-
C
23H23N7O3Á0.25EtOAc: C, 61.66; H, 5.39; N, 20.97. Found: C,
amine (424 lL, 2.44 mmol), and DMF (7 mL) was stirred at room
61.28; H, 5.36; N, 20.95.
temperature for 2 h. Iodoacetamide (451 mg, 2.44 mmol) was
added to the mixture, and the resulting mixture was stirred at
room temperature for 15 h. The mixture was diluted with water
and extracted with AcOEt. The extract was washed with brine,
dried over anhydrous magnesium sulfate, and concentrated under
reduced pressure. The residue was collected by filtration and
washed with AcOEt–hexane to give 15a (883 mg, 71%) as a white
solid: 1H NMR (DMSO-d6) d 2.19 (3H, s), 2.34 (3H, s), 3.97 (3H, s),
4.83 (2H, s), 6.72–6.79 (2H, m), 7.26–7.43 (6H, m), 7.52–7.56
(1H, m), 7.66–7.77 (4H, m), 8.12 (1H, d, J = 2.7 Hz), 10.18 (1H, s).
5.19. N-[3-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-5-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (13c)
To a solution of 12c (303 mg, 0.803 mmol) in DMA (10 mL) was
added cyclopropanecarbonyl chloride (74.0 lL, 0.815 mmol) drop-
wise at 0 °C, and the mixture was stirred at room temperature for
15 h. The mixture was diluted with water and extracted with
AcOEt. The extract was washed with brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure.
The residual solid was crystallized from AcOEt–hexane to give
13c (188 mg, 53%) as white crystals: mp 233 °C; 1H NMR (DMSO-
d6) d 0.77–0.87 (4H, m), 1.96–2.10 (1H, m), 2.17 (3H, s), 2.29 (3H,
s), 3.96 (3H, s), 6.67 (1H, s), 6.78 (1H, s), 7.21 (1H, t, J = 1.9 Hz),
7.43 (1H, s), 7.52 (1H, dd, J = 9.6, 2.3 Hz), 7.74 (1H, d, J = 9.6 Hz),
5.24. N-(5-{[1-(2-Amino-2-oxoethyl)-6-{[(4-
methylphenyl)sulfonyl]imino}-1,6-dihydropyridin-3-yl]oxy}-2-
methylphenyl)-1,3-dimethyl-1H-pyrazole-5-carboxamide (15b)
The compound 15b was prepared from 14b in a manner sim-
ilar to that described for 15a to yield a white solid (72%): 1H

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4725
NMR (DMSO-d6) d 2.17 (3H, s), 2.19 (3H, s), 2.34 (3H, s), 3.98
5.28. N-(3-Hydroxyphenyl)-1,3-dimethyl-1H-pyrazole-5-
carboxamide (18)
(3H, s), 4.82 (2H, s), 6.79 (1H, s), 6.84 (1H, dd, J = 8.3, 2.6 Hz),
7.04 (1H, d, J = 2.6 Hz), 7.24–7.29 (3H, m), 7.37–7.42 (2H, m),
7.65–7.71 (3H, m), 7.76 (1H, br s), 8.10 (1H, d, J = 3.0 Hz), 9.77
(1H, s).
The compound 18 was prepared from 3-aminophenol and 8 in a
manner similar to that described for 9a to yield a white solid (90%):
1H NMR (DMSO-d6) d 2.19 (3H, s), 3.98 (3H, s), 6.47–6.53 (1H, m),
6.79 (1H, s), 7.08–7.13 (2H, m), 7.28 (1H, s), 9.41 (1H, s), 9.97 (1H, s).
5.25. N-{3-[(2-Aminoimidazo[1,2-a]pyridin-6-yl)oxy]phenyl}-
1,3-dimethyl-1H-pyrazole-5-carboxamide (16a)
5.29. 1,3-Dimethyl-N-{3-[(5-nitropyridin-2-yl)oxy]phenyl}-1H-
A mixture of 15a (882 mg, 1.65 mmol), trifluoroacetic anhy-
dride (6 mL), and dichloromethane (8 mL) was stirred at room
temperature for 2.5 h. The mixture was concentrated under re-
duced pressure and the residue was partitioned with AcOEt and
saturated aqueous sodium hydrogen carbonate. The organic layer
was separated and washed with brine, dried over anhydrous mag-
nesium sulfate, and concentrated under reduced pressure. The res-
idue was purified by silica gel column chromatography (AcOEt/
pyrazole-5-carboxamide (19a)
A mixture of 18 (1.58 g, 6.83 mmol), 2-chloro-5-nitropyridine
(1.03 g, 6.51 mmol), potassium carbonate (1.42 g, 10.2 mmol),
and DMF (10 mL) was stirred at 60 °C for 15 h. The mixture was di-
luted with water and extracted with AcOEt. The extract was
washed with water and brine, dried over anhydrous magnesium
sulfate, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (AcOEt/hexane)
to give 19a (2.26 g, 94%) as a white solid: 1H NMR (DMSO-d6) d 2.20
(3H, s), 3.98 (3H, s), 6.83 (1H, s), 6.96–7.00 (1H, m), 7.28 (1H, d,
J = 9.3 Hz), 7.44 (1H, t, J = 8.3 Hz), 7.58–7.62 (1H, m), 7.69 (1H, t,
J = 2.1 Hz), 8.61–8.65 (1H, m), 9.04 (1H, d, J = 3.3 Hz), 10.27 (1H, s).
hexane)
to
give
1,3-dimethyl-N-[3-({2-[(trifluoroace-
tyl)amino]imidazo[1,2-a]pyridin-6-yl}oxy)phenyl]-1H-pyrazole-
5-carboxamide (338 mg, 45%) as a white solid. The solid thus ob-
tained (328 mg, 0.715 mmol) was dissolved in 1 N NaOH (3 mL),
EtOH (3 mL), and the solution was stirred at 40 °C for 2 h. The mix-
ture was diluted with water and extracted with AcOEt. The extract
was washed with brine, dried over anhydrous magnesium sulfate,
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (AcOEt/MeOH) to give
16a (205 mg, 79%) as a white solid: 1H NMR (DMSO-d6) d 2.17
(3H, s), 3.96 (3H, s), 5.09 (2H, s), 6.75–6.78 (2H, m), 6.86 (1H, dd,
J = 9.4, 2.0 Hz), 7.01 (1H, s), 7.22 (1H, d, J = 9.4 Hz), 7.32 (1H, d,
J = 8.3 Hz), 7.37 (1H, t, J = 2.3 Hz), 7.50–7.60 (1H, m), 8.33 (1H, d,
J = 2.0 Hz), 10.12 (1H, s).
5.30. 1,3-Dimethyl-N-{2-methyl-5-[(5-nitropyridin-2-
yl)oxy]phenyl}-1H-pyrazole-5-carboxamide (19b)
The compound 19b was prepared from 3-aminophenol and 9a
in a manner similar to that described for 19a to yield a white solid
(94%): 1H NMR (DMSO-d6) d 2.19 (3H, s), 2.25 (3H, s), 3.97 (3H, s),
6.81 (1H, s), 7.04 (1H, dd, J = 8.7, 2.9 Hz), 7.24–7.27 (2H, m), 7.35
(1H, d, J = 8.7 Hz), 8.62 (1H, dd, J = 8.9, 2.9 Hz), 9.01 (1H, dd,
J = 2.7, 0.6 Hz), 9.81 (1H, s).
5.26. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-
a]pyridin-6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (17a)
5.31. N-{3-[(5-Aminopyridin-2-yl)oxy]phenyl}-1,3-dimethyl-
1H-pyrazole-5-carboxamide (20a)
To a solution of 16a (270 mg, 0.745 mmol), triethylamine
A mixture of 19a (2.26 g, 6.40 mmol), 10% palladium on carbon
(water ꢀ50%, 200 mg), and MeOH (20 mL) was stirred under a
hydrogen atmosphere at room temperature for 5 h. The catalyst
was filtered off, and the filtrate was concentrated in vacuo. The res-
idue was collected by filtration and washed with AcOEt–hexane to
give 20a (1.77 g, 86%) as a white solid: 1H NMR (DMSO-d6) d 2.19
(3H, s), 3.97 (3H, s), 5.14 (2H, s), 6.67–6.71 (1H, m), 6.76–7.00 (2H,
m), 7.08 (1H, dd, J = 8.4, 3.0 Hz), 7.28 (1H, t, J = 8.1 Hz), 7.36 (1H, t,
J = 2.1 Hz), 7.43–7.47 (1H, m), 7.56 (1H, d, J = 3.0 Hz), 11.10 (1H, s).
(310
lL, 2.24 mmol) in THF (5 mL) was added a solution of cyclo-
propanecarbonyl chloride (88.0
l
L, 0.969 mmol) at 0 °C, and the
mixture was stirred at room temperature for 15 h. The mixture
was diluted with water and extracted with AcOEt. The extract
was washed with water and brine, dried over anhydrous magne-
sium sulfate, and concentrated under reduced pressure. The resi-
due was purified by silica gel column chromatography (AcOEt/
hexane) followed by recrystallization from AcOEt–hexane to give
17a (191 mg, 60%) as white crystals: mp 226–230 °C; 1H NMR
(DMSO-d6) d 0.79–0.81 (4H, m), 1.89–1.97 (1H, m), 2.17 (3H, s),
3.95 (3H, s), 6.77–6.81 (2H, m), 7.08 (1H, dd, J = 9.8, 2.4 Hz), 7.33
(1H, t, J = 8.3 Hz), 7.39 (1H, t, J = 2.1 Hz), 7.46–7.56 (2H, m), 8.06
(1H, s), 8.58 (1H, d, J = 2.4 Hz), 10.12 (1H, s), 10.97 (1H, s); Anal.
Calcd for C23H22N6O3: C, 64.17; H, 5.15; N, 19.52. Found: C,
63.97; H, 5.19; N, 19.48.
5.32. N-{5-[(5-Aminopyridin-2-yl)oxy]-2-methylphenyl}-1,3-
dimethyl-1H-pyrazole-5-carboxamide (20b)
The compound 20b was prepared from 19b in a manner similar
to that described for 20a to yield a white solid (quant): 1H NMR
(DMSO-d6) d 2.17 (3H, s), 2.18 (3H, s), 3.97 (3H, s), 5.10 (2H, s),
6.74–6.80 (3H, m), 6.93 (1H, d, J = 2.7 Hz), 7.07 (1H, dd, J = 8.7,
3.0 Hz), 7.20 (1H, d, J = 9.0 Hz), 7.53–7.54 (1H, m), 9.72 (1H, s).
5.27. N-[5-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-
a]pyridin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (17b)
5.33. N-{3-[(2-Amino[1,3]thiazolo[5,4-b]pyridin-5-
yl)oxy]phenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (21a)
The compound 17b was prepared from 15b in a manner sim-
ilar to that described for 16a and 17a to yield white crystals
(41%): mp 265–268 °C; 1H NMR (DMSO-d6) d 0.78–0.81 (4H, m),
1.91–1.94 (1H, m), 2.18 (3H, s), 2.19 (3H, s), 3.96 (3H, s), 6.78
(1H, s), 6.88 (1H, dd, J = 8.5, 2.9 Hz), 7.02–7.08 (2H, m), 7.26
(1H, d, J = 8.5 Hz), 7.46 (1H, d, J = 9.4 Hz), 8.06 (1H, s), 8.53–8.55
(1H, m), 9.74 (1H, s), 10.97 (1H, s); Anal. Calcd for
To a suspension of 20a (1.75 g, 5.41 mmol), potassium thiocya-
nate (4.21 g, 43.3 mmol) in AcOH (10 mL) was added a solution of
bromine (1 mL) in AcOH (4 mL) at 0 °C, and the mixture was stirred
at 0 °C for 2 h and at room temperature for 15 h. Water (10 mL)
was added to the mixture, and the resulting mixture was warmed
to 80 °C. The resulting solid was filtrated off, and the filtrate was
neutralized with 8 N NaOH. The resulting solid was collected and
washed with water to give 21a (1.05 g, 51%) as a white solid: 1H
C24H24N6O3Á0.25H2O: C, 64.20; H, 5.50; N, 18.72. Found: C,
64.47; H, 5.63; N, 18.40.

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Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
NMR (DMSO-d6) d 2.19 (3H, s), 3.98 (3H, s), 6.81–6.86 (2H, m), 6.92
(1H, d, J = 8.1 Hz), 7.35 (1H, t, J = 8.1 Hz), 7.50 (1H, t, J = 2.1 Hz),
7.57 (1H, d, J = 8.1 Hz), 7.68–7.74 (3H, m), 10.31 (1H, s).
5.40. tert-Butyl {5-[(6-aminopyridin-3-yl)oxy]-2-
methylphenyl}carbamate (25b)
The compound 25b was prepared from 24b in a manner sim-
ilar to that described for 5 to yield a pale yellow oil (98%): 1H
NMR (DMSO-d6) d 1.50 (9H, s), 2.20 (3H, s), 6.30 (1H, s), 6.55
(1H, dd, J = 8.3, 2.7 Hz), 6.60 (1H, d, J = 8.7 Hz), 7.05 (1H, d,
J = 8.3 Hz), 7.25–7.31 (1H, m), 7.59 (1H, s), 7.75 (1H, d,
J = 2.7 Hz), 8.02 (2H, s).
5.34. N-{5-[(2-Amino[1,3]thiazolo[5,4-b]pyridin-5-yl)oxy]-2-
methylphenyl}-1,3-dimethyl-1H-pyrazole-5-carboxamide (21b)
The compound 21b was prepared from 20b in a manner similar
to that described for 21a to yield a white solid (47%): 1H NMR
(DMSO-d6) d 2.19 (3H, s), 2.21 (3H, s), 3.98 (3H, s), 6.80 (1H, s),
6.88–6.94 (2H, m), 7.09 (1H, d, J = 2.4 Hz), 7.28 (1H, d, J = 8.7 Hz),
7.62 (2H, s), 7.71 (1H, dd, J = 8.4, 0.9 Hz), 9.77 (1H, s).
5.41. tert-Butyl {3-[(2-amino[1,2,4]triazolo[1,5-a]pyridin-6-
yl)oxy]-4-methylphenyl}carbamate (26a)
5.35. N-[3-({2-[(Cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-
b]pyridin-5-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (22a)
The compound 26a was prepared from 25a in a manner similar
to that described for 12a to yield a white solid (87%): 1H NMR
(DMSO-d6) d 1.39 (9H, s), 2.19 (3H, s), 6.00 (2H, s), 6.98 (1H, s),
7.12–7.18 (2H, m), 7.24 (1H, dd, J = 9.5, 2.3 Hz), 7.39 (1H, dd,
J = 9.5, 0.3 Hz), 8.43 (1H, d, J = 1.8 Hz), 9.23 (1H, s).
The compound 22a was prepared from 21a in a manner similar
to that described for 13a to yield white crystals (56%): mp 225–
228 °C; 1H NMR (DMSO-d6) d 0.94–0.98 (4H, m), 1.96–2.05 (1H,
m), 2.19 (3H, s), 3.97 (3H, s), 6.81 (1H, s), 6.89–6.94 (1H, m), 7.14
(1H, d, J = 8.9 Hz), 7.40 (1H, t, J = 8.4 Hz), 7.57–7.61 (2H, m), 8.17
(1H, d, J = 8.9 Hz), 10.20 (1H, s), 12.70 (1H, s); Anal. Calcd for
5.42. tert-Butyl {5-[(2-amino[1,2,4]triazolo[1,5-a]pyridin-6-
yl)oxy]-2-methylphenyl}carbamate (26b)
The compound 26b was prepared from 25b in a manner similar
to that described for 12a to yield a white solid (49%): 1H NMR
(CDCl3) d 1.50 (9H, s), 2.22 (3H, s), 4.42 (2H, s), 6.32 (1H, s), 6.61
(1H, dd, J = 8.3, 2.7 Hz), 7.09 (1H, d, J = 8.3 Hz), 7.23 (1H, d,
J = 2.3 Hz), 7.32–7.38 (1H, m), 7.70 (1H, d, J = 1.9 Hz), 8.07 (1H, d,
J = 1.9 Hz).
C
22H20N6O3SÁ2H2O: C, 54.53; H, 4.99; N, 17.34. Found: C, 54.65;
H, 4.85; N, 17.31.
5.36. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,3]thiazolo[5,4-
b]pyridin-5-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (22b)
5.43. N-[6-(5-Amino-2-methylphenoxy)[1,2,4]triazolo[1,5-
a]pyridin-2-yl]cyclopropanecarboxamide (27a)
The compound 22b was prepared from 21b in a manner similar
to that described for 13c to yield white crystals (66%): mp 228–
231 °C; 1H NMR (DMSO-d6) d 0.94–0.97 (4H, m), 1.95–2.02 (1H,
m), 2.19 (3H, s), 2.24 (3H, s), 3.97 (3H, s), 6.80 (1H, s), 7.00 (1H,
dd, J = 8.3, 2.6 Hz), 7.10 (1H, d, J = 8.4 Hz), 7.17 (1H, d, J = 2.4 Hz),
7.32 (1H, d, J = 8.7 Hz), 8.16 (1H, d, J = 8.4 Hz), 9.78 (1H, s), 12.68
(1H, s); Anal. Calcd for C23H22N6O3SÁ0.25H2O: C, 59.15; H, 4.86;
N, 17.99. Found: C, 58.96; H, 4.77; N, 17.90.
To a solution of 26a (1.50 g, 4.22 mmol) in DMA (5 mL) was
added cyclopropanecarbonyl chloride (1.15 mL, 12.7 mmol) at
0 °C, and the mixture was stirred at room temperature for 2 h.
The mixture was diluted with water and extracted with AcOEt.
The extract was washed with water and brine, dried over anhy-
drous magnesium sulfate, and concentrated under reduced pres-
sure. The residual solid was collected and washed with AcOEt–
hexane to give tert-butyl [3-({2-[(cyclopropylcarbonyl)amino]
[1,2,4]triazolo[1,5-a]pyridin-6-yl}oxy)-4-methylphenyl]carbamate
(1.59 g, 89%) as a white solid. The solid thus obtained (1.50 g,
3.54 mmol) was dissolved in trifluoroacetic acid (5 mL), and the
solution was stirred at 0 °C for 30 min. The mixture was evapo-
rated and the residue was dissolved in water. The solution was
neutralized with saturated aqueous sodium hydrogen carbonate
and extracted with AcOEt. The organic layer was separated and
washed with water, brine, dried over anhydrous magnesium sul-
fate, and concentrated under reduced pressure. The residual solid
was collected and washed with AcOEt–hexane to give 27a
(1.04 g, 91%) as a white solid: 1H NMR (DMSO-d6) d 0.81–0.84
(4H, m), 1.99–2.08 (1H, m), 2.09 (3H, s), 5.09 (2H, br s), 6.08 (1H,
d, J = 2.1 Hz), 6.30 (1H, dd, J = 8.2, 2.1 Hz), 6.93 (1H, d, J = 8.2 Hz),
7.42 (1H, dd, J = 9.6, 2.3 Hz), 7.69 (1H, d, J = 9.6 Hz), 8.61 (1H, d,
J = 2.3 Hz), 10.98 (1H, s).
5.37. tert-Butyl {4-methyl-3-[(6-nitropyridin-3-
yl)oxy]phenyl}carbamate (24a)
The compound 24a was prepared from 23a in a manner similar
to that described for 4 to yield a colorless oil (44%): 1H NMR
(DMSO-d6) d 1.45 (9H, s), 2.06 (3H, s), 7.24–7.34 (3H, m), 7.45
(1H, dd, J = 9.2, 2.9 Hz), 8.32 (1H, d, J = 9.2 Hz), 8.38 (1H, d,
J = 2.9 Hz), 9.50 (1H, s).
5.38. tert-Butyl {2-methyl-5-[(6-nitropyridin-3-
yl)oxy]phenyl}carbamate (24b)
The compound 24b was prepared from 23b in a manner similar
to that described for 4 to yield a pale yellow oil (53%): 1H NMR
(DMSO-d6) d 1.51 (9H, s), 2.28 (3H, s), 6.40 (1H, s), 6.72 (1H, dd,
J = 8.3, 2.6 Hz), 7.20 (1H, d, J = 8.3 Hz), 7.40 (1H, dd, J = 9.0,
2.6 Hz), 7.84 (1H, d, J = 1.9 Hz), 8.22 (1H, d, J = 9.0 Hz), 8.31 (1H,
d, J = 2.6 Hz).
5.44. N-[6-(3-Amino-4-methylphenoxy)[1,2,4]triazolo[1,5-
a]pyridin-2-yl]cyclopropanecarboxamide (27b)
5.39. tert-Butyl {3-[(6-aminopyridin-3-yl)oxy]-4-
methylphenyl}carbamate (25a)
The compound 27b was prepared from 26b in a manner similar
to that described for 27a to yield a white solid (86%): 1H NMR
(CDCl3) d 0.87–0.97 (2H, m), 1.15–1.23 (2H, m), 1.65 (1H, s), 2.14
(3H, s), 3.70 (2H, s), 6.29–6.39 (2H, m), 7.01 (1H, d, J = 7.5 Hz),
7.33–7.41 (1H, m), 7.55 (1H, dd, J = 9.8, 0.8 Hz), 8.27 (1H, d,
J = 1.9 Hz), 9.56 (1H, s).
The compound 25a was prepared from 24a in a manner similar
to that described for 5 to yield a colorless oil (99%): 1H NMR
(DMSO-d6) d 1.40 (9H, s), 2.16 (3H, s), 5.80 (2H, s), 6.46 (1H, d,
J = 9.0 Hz), 6.89 (1H, s), 7.01–7.13 (3H, m), 7.66 (1H, d, J = 3.0 Hz),
9.50 (1H, s).

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
4727
5.45. N-[3-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-4-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (28)
(3H, s), 3.79 (3H, s), 6.86 (1H, dd, J = 8.5, 2.8 Hz), 7.17 (1H, d,
J = 2.6 Hz), 7.25 (1H, d, J = 8.7 Hz), 7.49 (1H, dd, J = 9.6, 2.5 Hz),
7.72 (1H, dd, J = 9.4, 0.8 Hz), 8.23 (1H, s), 8.81 (1H, dd, J = 2.3,
0.8 Hz), 9.16 (1H, s), 11.03 (1H, s); Anal. Calcd for
To a solution of 27a (265 mg, 0.820 mmol) in DMA (5 mL) was
added a solution of 1,3-dimethyl-1H-pyrazole-5-carbonyl chloride
(260 mg, 1.64 mmol) dropwise at 0 °C, and the mixture was stirred
at room temperature for 15 h. The mixture was diluted with water
and extracted with AcOEt. The extract was washed with brine,
dried over anhydrous magnesium sulfate, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (AcOEt/hexane) followed by recrystallization
from AcOEt to give 28 (333 mg, 91%) as white crystals: mp 222–
225 °C; 1H NMR (DMSO-d6) d 0.81–0.84 (4H, m), 1.99–2.08 (1H,
m), 2.15 (3H, s), 2.27 (3H, s), 3.93 (3H, s), 6.72 (1H, s), 7.21–7.30
(2H, m), 7.48–7.56 (2H, m), 7.74 (1H, dd, J = 9.5, 0.7 Hz), 8.78–
8.80 (1H, m), 10.02 (1H, s), 11.04 (1H, s); Anal. Calcd for
C
23H23N7O3Á0.5H2O: C, 60.78; H, 5.31; N, 21.57. Found: C, 61.20;
H, 5.76; N, 19.77.
5.49. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-1-methyl-1H-pyrazole-5-
carboxamide (29d)
Yield 47%, white crystals: mp 131–135 °C; 1H NMR (DMSO-d6) d
0.78–0.87 (4H, m), 2.04 (1H, br s), 2.20 (3H, s), 4.05 (3H, s), 6.94
(1H, dd, J = 8.3, 2.7 Hz), 7.02 (1H, d, J = 1.9 Hz), 7.09 (1H, d,
J = 2.7 Hz), 7.29 (1H, d, J = 8.7 Hz), 7.46–7.53 (2H, m), 7.72 (1H,
dd, J = 9.8, 0.8 Hz), 8.82–8.85 (1H, m), 9.86 (1H, s), 11.02 (1H, s);
Anal. Calcd for C22H21N7O3: C, 61.24; H, 4.91; N, 22.73. Found: C,
61.24; H, 4.98; N, 22.58.
C23H23N7O3Á0.5H2O: C, 60.78; H, 5.32; N, 21.57. Found: C, 61.04;
H, 5.29; N, 21.55.
5.50. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-2-
methylpropanecarboxamide (29e)
5.46. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-2,5-dimethyl-1,3-
thiazole-4-carboxamide (29a)
Yield 72%, white crystals: mp 217–219 °C; 1H NMR (DMSO-d6) d
0.79–0.85 (4H, m), 0.93 (6H, d, J = 6.4 Hz), 1.96–2.11 (2H, m) 2.16–
2.23 (5H, m) 6.79 (1H, dd, J = 8.3, 2.7 Hz) 7.17–7.25 (2H, m) 7.47
(1H, dd, J = 9.7, 2.5 Hz) 7.71 (1H, dd, J = 9.5, 0.8 Hz) 8.79 (1H, dd,
J = 2.3, 0.8 Hz) 9.21 (1H, s) 11.01 (1H, s); Anal. Calcd for C22H25N5O3:
C, 64.85; H, 6.18; N, 17.19. Found: C, 64.71; H, 6.23; N, 17.24.
To a solution of 2,5-dimethyl-1,3-thiazole-4-carboxylic acid
(107 mg, 0.681 mmol) in THF (5 mL) was added thionyl chloride
(118 lL, 1.36 mmol) and two drops of DMF. The mixture was stir-
red at room temperature for 2 h and concentrated under reduced
pressure. The residue was dissolved in DMA (6 mL), and 27b
(200 mg, 0.619 mmol) was added to the solution at 0 °C. The mix-
ture was stirred at room temperature for 2 h. The mixture was di-
luted with saturated aqueous sodium hydrogen carbonate and
extracted with AcOEt. The extract was washed with saturated
aqueous sodium hydrogen carbonate, brine, dried over anhydrous
magnesium sulfate, and concentrated under reduced pressure.
The residual solid was recrystallized from EtOH to give 29a
(248 mg, 87%) as white crystals: mp 239–240 °C; 1H NMR
(DMSO-d6) d 0.78–0.86 (4H, m), 2.04 (1H, br s), 2.23–2.28 (3H,
m), 2.65 (3H, s), 2.70 (3H, s), 6.82 (1H, dd, J = 8.3, 2.7 Hz), 7.27
(1H, d, J = 8.7 Hz), 7.50 (1H, dd, J = 9.5, 2.3 Hz), 7.66 (1H, d,
J = 2.7 Hz), 7.69–7.75 (1H, m), 8.82 (1H, dd, J = 2.3, 0.8 Hz), 9.66
(1H, s), 11.02 (1H, s); Anal. Calcd for C23H22N6O3S: C, 59.73; H,
4.79; N, 18.17. Found: C, 59.48; H, 4.80; N, 18.00.
5.51. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]cyclobutanecarboxamide
(29f)
Yield 45%, white crystals: mp 229–230 °C; 1H NMR (DMSO-d6) d
0.78–0.87 (4H, m), 1.72–1.85 (1H, m), 1.85–1.98 (1H, m), 1.98–2.14
(3H, m), 2.15 (3H, s), 2.17–2.27 (2H, m), 3.21–3.35 (1H, m), 6.80
(1H, dd, J = 8.3, 2.7 Hz), 7.20 (1H, d, J = 8.7 Hz), 7.25 (1H, d,
J = 1.9 Hz), 7.47 (1H, dd, J = 9.5, 2.3 Hz), 7.66–7.74 (1H, m), 8.76–
8.80 (1H, m), 9.05 (1H, s), 11.01 (1H, s); Anal. Calcd for
C22H23N5O3: C, 65.17; H, 5.72; N, 17.27. Found: C, 65.24; H, 5.70;
N, 17.34.
5.52. N-(6-Hydroxy-1,3-benzothiazol-2-
The following compounds 29b–f were prepared from 27b using
a procedure similar to that described for 29a.
yl)cyclopropanecarboxamide (31)
To a solution of 30 (9.03 g, 54.3 mmol), triethylamine (8.27 mL,
59.7 mmol) in THF (100 mL) was added a solution of cyclopropan-
ecarbonyl chloride (5.19 mL, 57.0 mmol) in THF (25 mL) at 0 °C,
and the mixture was stirred at room temperature for 2 h. The mix-
ture was diluted with water and extracted with AcOEt. The extract
was washed with water and brine, dried over anhydrous magne-
sium sulfate, and concentrated under reduced pressure. The resid-
ual solid was collected and washed with AcOEt–hexane to give 31
(12.3 g, 97%) as a white solid: 1H NMR (DMSO-d6) d 0.95–1.05 (4H,
m), 1.84–1.99 (1H, m), 6.93 (1H, dd, J = 8.8, 2.6 Hz), 7.29 (1H, d,
J = 8.8 Hz), 7.45–7.50 (3H, m).
5.47. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-3-chloro-4-
methylthiophene-2-carboxamide (29b)
Yield 78%, pale yellow crystals: mp 218–219 °C; 1H NMR
(DMSO-d6) d 0.79–0.86 (4H, m), 2.04 (1H, br s), 2.20 (3H, d,
J = 1.1 Hz), 2.27 (3H, s), 6.89 (1H, dd, J = 8.3, 2.6 Hz), 7.28 (1H, d,
J = 8.7 Hz), 7.42 (1H, d, J = 2.6 Hz), 7.50 (1H, dd, J = 9.8, 2.7 Hz),
7.66 (1H, d, J = 1.1 Hz), 7.69–7.76 (1H, m), 8.84 (1H, dd, J = 2.3,
0.8 Hz), 9.54 (1H, s), 10.96–11.09 (1H, m); Anal. Calcd for
C23H20ClN5O3S: C, 57.32; H, 4.18; N, 14.53. Found: C, 57.28; H,
4.04; N, 14.51.
5.53. N-[6-(3-Nitrophenoxy)-1,3-benzothiazol-2-
yl]cyclopropanecarboxamide (32)
5.48. N-[5-({2-[(Cyclopropylcarbonyl)amino][1,2,4]triazolo[1,5-
a]pyridin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-4-carboxamide (29c)
A mixture of 31 (3.54 g, 15.1 mmol), 1-fluoro-3-nitrobenzene
(2.24 g, 15.9 mmol), potassium carbonate (6.26 g, 45.3 mmol),
and DMF (30 mL) was stirred at 150 °C for 15 h. The mixture was
diluted with water and extracted with AcOEt. The extract was
washed with water and brine, dried over anhydrous magnesium
Yield 58%, pale brown crystals: mp 192–194 °C; 1H NMR
(DMSO-d6) d 0.79–0.86 (4H, m), 2.04 (1H, br s), 2.20 (3H, s), 2.32

4728
Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
sulfate, and concentrated under reduced pressure. The residual so-
lid was collected and washed with AcOEt–hexane to give 32
(1.82 g, 37%) as a white solid: 1H NMR (DMSO-d6) d 0.95–0.99
(4H, m), 1.96–2.05 (1H, m), 7.24 (1H, dd, J = 8.7, 2.7 Hz), 7.47–
7.51 (1H, m), 7.63–7.70 (2H, m), 7.78–7.83 (2H, m), 7.94–7.99
(1H, m), 12.67 (1H, s).
sion analysis, with the top and bottom of the curve constrained at
100 and 0, respectively.
5.56. Dilution assay of VEGFR2–13d complex
To test the dissociation kinetics of compounds an enzyme–
inhibitor dilution assay was performed.29,42 The recovery of
enzyme activity from a preformed enzyme–inhibitor complex
was measured using the AlphaScreenÒ system as described above.
VEGFR2 at 100-fold higher concentration than the standard
reaction condition and 13d at 10-fold higher concentration than
IC50 value were incubated together in reaction buffer for 60 min
at ambient temperature to form enzyme–inhibitor complex. This
complex was diluted 1:100 into reaction buffer containing 1 mM
5.54. N-[3-({2-[(Cyclopropylcarbonyl)amino]-1,3-benzothiazol-
6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide
(33)
A mixture of 32 (1.80 g, 5.53 mmol), 10% palladium on carbon
(water ꢀ50%, 100 mg), and MeOH (20 mL) was stirred under a
hydrogen atmosphere at room temperature for 5 h. The catalyst
was filtered off, and the filtrate was concentrated in vacuo. To
the residue thus obtained was added iron (309 mg), calcium chlo-
ride (613 mg, 5.53 mmol), EtOH (10 mL), and water (2 mL). The
mixture was stirred for 2 h. The mixture was filtered, and the fil-
trate was diluted with water and extracted with AcOEt. The organic
layer was separated and washed with water and brine, dried over
anhydrous magnesium sulfate, and concentrated under reduced
pressure. The residue was dissolved in THF (10 mL), and triethyl-
ATP and 0.1 lg/mL biotinylated poly-Glu-Tyr (4:1), to initiate the
kinase reaction.
5.57. Kinase profiling by IC50 measurement
Kinase profiling was performed as described previously.43
Briefly, kinase activities of VEGFR1, PDGFRa, Tie-2, Insulin Recep-
tor (IR), insulin-like growth factor 1 receptor (IGF1-R), c-kit, Src
and focal adhesion kinase (FAK) were determined by use of the
AlphaScreenÒ system. For VEGFR1, kinase assay was performed
amine (192 lL, 1.39 mmol), 8 (220 mg, 1.39 mmol) were added
to the solution at 0 °C. The mixture was stirred at room tempera-
ture for 3 h. The mixture was diluted with water and extracted
with AcOEt. The extract was washed with water and brine, dried
over anhydrous magnesium sulfate, and concentrated under re-
duced pressure. The residue was purified by silica gel column chro-
matography (AcOEt/hexane) followed by recrystallization from
AcOEt–THF to give 33 (126 mg, 5.1%) as white crystals: mp 192–
194 °C; 1H NMR (DMSO-d6) d 0.94–0.97 (4H, m), 1.96–2.05 (1H,
m), 2.17 (3H, s), 3.95 (3H, s), 6.76–6.80 (2H, m), 7.15 (1H, dd,
J = 8.6, 2.6 Hz), 7.34 (1H, t, J = 8.1 Hz), 7.40 (1H, t, J = 2.3 Hz),
7.51–7.56 (1H, m), 7.70–7.77 (2H, m), 10.13 (1H, s), 12.62 (1H,
s); Anal. Calcd for C23H21N5O3S: C, 61.73; H, 4.73; N, 15.65. Found:
C, 61.49; H, 4.52; N, 15.67.
with 20 ng/mL VEGFR1 (Millipore) and 0.5
kinase assay was performed with 10 ng/mL IGF1R (BIOMOL,
USA) and 10 M ATP. For FAK, kinase assay was performed with
62 ng/mL FAK (N-terminal FLAG-tagged recombinant proteins
using the baculovirus expression system) and 2 M of ATP. In
lM ATP. For IGF1-R,
l
l
these assays, wells containing the substrate and the enzyme
without the compound were used as total reaction control. Wells
containing the biotinylated poly-GluTyr (4:1) and the enzyme
without ATP were used as basal control. Kinase activities of B-
raf, extracellular signal-regulated kinases (ERK1), protein kinase
C h (PKCh), glycogen synthase kinase-3b (GSK3b) and Aurora A
were determined by use of radio labeled [
care, USA). For GSK3b, kinase assay was performed with 2
GSK3b, 4 g/well of pGS peptide and 0.5 M ATP (0.1 Ci/50
33P] ATP). For Aurora A, kinase assay was performed
g/mL Aurora A, 30 mol/L Aurora substrate peptide
M ATP (0.2 Ci/50 L/well of [
33P] ATP). EGFR and
HER2 kinase activity was determined by use of radio labeled
32P] ATP (GE Healthcare). In these assays, wells containing
c
-
33P] ATP (GE Health-
g/mL
L/
l
5.55. Measurement of inhibitory activities against VEGFR2,
PDGFRb and FGFR1
l
l
l
l
well of [
c-
with 10
l
l
Kinase activities of VEGFR2, PDGFRb and fibroblast growth fac-
tor receptor 1 (FGFR1) were determined by use of an anti-phospho-
tyrosine antibody with quantitation performed through the
AlphaScreenÒ system (PerkinElmer, USA). For VEGFR2, enzyme
reactions were performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl2,
and 0.5
l
l
l
c-
[c-
the substrate and the enzyme without the compound were used
as total reaction control. Wells containing the substrate and the
radio labeled ATP without the enzyme was used as basal control.
The concentration of inhibitor producing 50% inhibition of the ki-
nase activities (IC50 values) and 95% confidence intervals (95% CI)
were analyzed as described above.
5 mM MgCl2, 0.01% Tween-20 and 2 mM DTT, containing 10
ATP, 0.1 g/mL biotinylated poly-GluTyr (4:1) and 0.1 nM of VEG-
FR2 (Millipore, UK). For PDGFRb, kinase assay was performed as
described above with 0.8 nM of PDGFRb (Millipore) and 20 M of
ATP. For FGFR1, kinase assay was performed as described above with
0.1 nM of FGFR1 (ProQinase GmbH, Germany) and 0.2 M of ATP.
lM
l
l
l
5.58. Cell proliferation assays
Prior to the catalytic initiation with ATP, compound and enzyme
were incubated for 5 min at room temperature. The reactions were
HUVECs (Cambrex, USA) were seeded into a 96-well plate at
3000 cells/well in Human Endothelial-SFM Growth Medium (Invit-
rogen, USA) containing 3% fetal bovine serum (FBS) (Hyclone, USA)
and were incubated overnight at 37 °C in a 5% CO2 incubator. Var-
ious concentrations of the test compounds were added in the pres-
ence of 60 ng/mL VEGF (R&D systems, USA), and the cells were
cultured for a further 5 days. The cellular proliferation was deter-
mined by the WST-8 formazan assay using Cell Counting Kit-8
quenched by the addition of 25
lL of 100 mM EDTA, 10
lg/mL
AlphaScreen streptavidine donor beads and 10
lg/mL acceptor
beads in 62.5 mM HEPES pH7.4, 250 mM NaCl, and 0.1% BSA. Plates
were incubated in the dark overnight and then read by EnVision
2102 Multilabel Reader (PerkinElmer). Wells containing the sub-
strate and the enzyme without the compound was used as total
reaction control. Wells containing the biotinylated poly-GluTyr
(4:1) and the enzyme without ATP were used as basal control.
The concentration of inhibitor producing 50% inhibition of the ki-
nase activities of VEGFR2, PDGFRb and FGFR1 (IC50 values) and
95% confidence intervals (95% CI) were analyzed using GraphPad
Prism version 5.01, GraphPad Software (USA). Sigmoidal dose–re-
sponse (variable slope) curves were fitted using non-linear regres-
(DOJINDO Laboratories, Japan). Briefly, 10 lL/well of Cell Counting
Kit-8 was added and the cells were cultured for several hours.
Then, the absorbance value at 450 nm was measured using a
Benchmark Plus Microplatereader (Bio-Rad Labs., USA). The IC50
values and 95% confidence intervals (95% CI) were calculated from
a dose-response curve generated by least-squares linear regression

Y. Oguro et al. / Bioorg. Med. Chem. 21 (2013) 4714–4729
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Acknowledgements
The authors thank Ms. T. Yoshida, Ms. Y. Awazu, Dr. A. Mizutani,
and Mr. Y. Nagase for in vitro and in vivo assays. The authors thank
Mr. S. Yamasaki and Ms. Y. Watanabe for PK evaluation and Dr. T.
Kitazaki for helpful discussion.
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