Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin- 6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor

Article abstract of DOI:10.1016/j.bmc.2013.01.074

Vascular endothelial growth factor (VEGF) plays important roles in tumor angiogenesis, and the inhibition of its signaling pathway is considered an effective therapeutic option for the treatment of cancer. In this study, we describe the design, synthesis, and biological evaluation of 2-acylamino-6-phenoxy-imidazo[1,2-b]pyridazine derivatives. Hybridization of two distinct imidazo[1,2-b]pyridazines 1 and 2, followed by optimization led to the discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl} oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (23a, TAK-593) as a highly potent VEGF receptor 2 kinase inhibitor with an IC₅₀ value of 0.95 nM. The compound 23a strongly suppressed proliferation of VEGF-stimulated human umbilical vein endothelial cells with an IC₅₀ of 0.30 nM. Kinase selectivity profiling revealed that 23a inhibited platelet-derived growth factor receptor kinases as well as VEGF receptor kinases. Oral administration of 23a at 1 mg/kg bid potently inhibited tumor growth in a mouse xenograft model using human lung adenocarcinoma A549 cells (T/C = 8%).

Full text of DOI:10.1016/j.bmc.2013.01.074

Bioorganic & Medicinal Chemistry 21 (2013) 2333–2345
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-
5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor
Naoki Miyamoto, Nozomu Sakai, Takaharu Hirayama, Kazuhiro Miwa, Yuya Oguro, Hideyuki Oki,
Kengo Okada, Terufumi Takagi, Hidehisa Iwata, Yoshiko Awazu, Seiji Yamasaki, Toshiyuki Takeuchi,
⇑
Hiroshi Miki, Akira Hori, Shinichi Imamura
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 2-26-1 Muraokahigashi, 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:
Vascular endothelial growth factor (VEGF) plays important roles in tumor angiogenesis, and the inhibi-
tion of its signaling pathway is considered an effective therapeutic option for the treatment of cancer.
In this study, we describe the design, synthesis, and biological evaluation of 2-acylamino-6-phenoxy-
imidazo[1,2-b]pyridazine derivatives. Hybridization of two distinct imidazo[1,2-b]pyridazines 1 and 2,
followed by optimization led to the discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo
[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (23a, TAK-593)
as a highly potent VEGF receptor 2 kinase inhibitor with an IC50 value of 0.95 nM. The compound 23a
strongly suppressed proliferation of VEGF-stimulated human umbilical vein endothelial cells with an
IC50 of 0.30 nM. Kinase selectivity profiling revealed that 23a inhibited platelet-derived growth factor
receptor kinases as well as VEGF receptor kinases. Oral administration of 23a at 1 mg/kg bid potently
inhibited tumor growth in a mouse xenograft model using human lung adenocarcinoma A549 cells (T/
C = 8%).
Received 27 December 2012
Revised 30 January 2013
Accepted 31 January 2013
Available online 13 February 2013
Keywords:
VEGF
VEGFR2
Kinase inhibitor
Imidazo[1,2-b]pyridazine
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
increased stability of immature tumor vessels. Platelet-derived
growth factor (PDGF)-B/PDGF receptor b (PDGFRb) signaling is nec-
Angiogenesis is the formation of new capillary blood vessels
from existing vasculature. Tumor growth is dependent on the
nutrients and oxygen delivered by the vessels formed during angi-
ogenesis.1 Vascular endothelial growth factor (VEGF) signaling
through VEGF receptor 2 (VEGFR2, also known as KDR) has been
shown to play a major role in the regulation of tumor angiogene-
sis.2,3 Expression of VEGF is enhanced in several types of human tu-
mors, and its expression levels correlate with poor prognosis and
clinical stage in patients with solid tumors.4–11 Therefore, VEGF/
VEGFR2 signaling is an attractive therapeutic target in the treat-
ment of cancer. A humanized anti-VEGF monoclonal antibody
(bevacizumab)12 and several small molecule VEGFR2 kinase inhib-
itors (sorafenib,13 sunitinib,14 pazopanib,15 and axitinib16) have
been approved as antiangiogenic drugs, and they have shown clin-
ical benefits in the treatment of some tumors, with manageable
side effects. Many other new angiogenesis inhibitors are being
evaluated in clinical trials.17 Following the formation of new tumor
vessels, pericyte recruitment is an essential step leading to
essary for pericyte recruitment during angiogenesis.18 It has also
been suggested that inhibition of PDGF/PDGFR signaling is an
effective way to block further growth of end-stage tumors.19 In
addition to stabilizing tumor vessels, expression of PDGF has been
known to correlate with autocrine stimulation of both tumor
growth and the recruitment of tumor stroma fibroblasts.20 Conse-
quently, it is conceivable that simultaneous inhibition of VEGFR2
and PDGFR kinases may lead to synergistic antiangiogenic effects
in tumor therapy.
We previously reported that the 6-phenoxy-imidazo[1,2-b]pyr-
idazine derivative 1, possessing a benzamide unit, functions as a
VEGFR2 kinase inhibitor (Fig. 1).21 Compound 1 showed potent VEG-
FR2 kinase inhibitory activity with an IC50 value of 7.1 nM. However,
it only modestly suppressed VEGF-stimulated proliferation of hu-
man umbilical vein endothelial cells (HUVEC) with an IC50 of
650 nM. Meanwhile, searching our compound library of inhibitors
targeting other kinases, we found that the 2-acylamino-imi-
dazo[1,2-b]pyridazine derivative 2 moderately inhibited VEGFR2 ki-
nase (IC50 = 1300 nM). To determine its mode of binding to VEGFR2
kinase, the X-ray crystal structure of VEGFR2 complexed with 2 was
solved using a soaking method (Fig. 2a). The N1-nitrogen of the imi-
⇑
Corresponding author. Tel.: +81 466 32 1166; fax: +81 466 29 4448.
E-mail address: shin-ichi.imamura@takeda.com (S. Imamura).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.074

2334
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
H
N
CF3
X
O
H
N
H
N
R2
CF3
N
N
O
I
OH
O
N
N
O
O
O
HN
O
N
N
O
R1
N
O
N
O
1
N
N
HN
O
N
N
N
O
N
N
4
A
HN
N
3
2
Figure 1. Design of hybrid compound A.
Figure 2. (a) Binding mode of 2 to VEGFR2. (b) Binding mode of 3 to VEGFR2. (c) Superposition of 2 (pink) and 3 (blue). Dashed lines represent hydrogen bonds.
dazo[1,2-b]pyridazine forms a hydrogen bonding interaction with
the NH group of Cys919 in the VEGFR2 hinge region, and the iodine
atom at the 6-position is located near the entrance of the back pock-
et. The CH proton at the 8-position interacts with the carbonyl group
of Glu917. The acylamino group at the 2-position is situated in the
solvent accessible region, where the NH group interacts with the
carbonyl group of Cys919. In addition, the 6-phenoxy-imi-
dazo[1,2-b]pyridazine 3 was identified by fragment library screen-
ing as a weak binding fragment of VEGFR2 kinase.22 Its binding
mode was determined by X-ray crystallography (Fig. 2b), and the
bindingmodes ofthe imidazo[1,2-b]pyridazine cores in 2 and 3 were
found to be virtually identical (Fig. 2c). Based on the structural
resemblance of 1 to 3, it was speculated that the imidazo[1,2-b]pyr-
idazine group of 1 would be located at the same site. In that case, the
benzamide moiety of 1 would occupy the back pocket, and the sol-
vent accessible region would remain vacant. We hypothesized that
the introduction of an NH group at the 2-position of the imi-
dazo[1,2-b]pyridazine in 1 would provide an additional hydrogen
bonding site with the carbonyl oxygen of Cys919, which might en-
hance the interaction between the inhibitor and VEGFR2. As we pre-
dicted, the hybrid compound 4 potently inhibited VEGFR2 kinase
activity (IC50 = 6.9 nM). Furthermore, 4 was found to strongly sup-
press the growth of HUVEC (IC50 = 9.1 nM). Encouraged by these re-
sults, we designed compounds of type A in order to discover VEGFR2
kinase inhibitors with potent cellular activity, as well as physico-
chemical properties suitable for oral administration. In this study,
we report the synthesis, structure–activity relationships (SAR),
and characterization of these new inhibitors.
2. Chemistry
Target compounds were prepared according to the synthetic
routes shown in Schemes 1–3. Scheme 1 shows the synthesis of
the 2-acylamino-6-phenoxyimidazo[1,2-b]pyridazine derivatives
10a–g. Ring formation of 3-amino-6-iodopyridazine (5) with ethyl
(chloroacetyl)carbamate in the presence of Na2HPO4 afforded the
6-iodoimidazo[1,2-b]pyridazine 6. Removal of the ethoxycarbonyl
group of 6 using aqueous Ba(OH)2 gave the amine 7, which was re-
NH2
O
O
R1
R1
N
I
N
I
N
I
N
O
a
c
d
N
N
N
N
RHN
HN
HN
N
N
N
H2N
5
6
8a–g
9a–g
: R = CO2Et
b
7: R = H
S
H
N
R1
=
O
F
F3C
Me
c
e
O
a
b
d
O
R1
N
O
N
HN
F3C
Me
N
10a–g
e
f
g
Scheme 1. Reagents: (a) ethyl (chloroacetyl)carbamate, Na2HPO4, DMA; (b) Ba(OH)2, NMP/H2O; (c) R1COCl, DMA; (d) 3-aminophenol, K2CO3, DMF; (e) 3-fluorobenzoyl
chloride, DMA.

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2335
H
N
CF3
a
O
9a
O
N
O
O
N
HN
N
4
Me
N
N
Me
Me
Me
N
N
N
R2
=
H
N
O
O
N
Me
R2
b
O
11
12
13
14
15
Me
16
9f
O
N
O
N
Me
N
Me
N
CF3
HN
Me
N
N
N
N
O
Me
11–21
N
N
N
N
Me
Me
Ph
Me
17
18
19
20
21
Scheme 2. Reagents: (a) 3-(trifluoromethyl)benzoyl chloride, DMA; (b) R2COCl, DMA.
Table 1
X
Effect of the amide substituent R1 on VEGFR2 kinase inhibitory activitya
2
NH2
O
a
N
I
4
6
H
N
F
O
N
O
HN
N
O
N
N
HN
O
HN
N
R1
N
O
N
8f
22a–f
N
R1
VEGFR2
Me
Compound
IC50 (nM)
X
H
N
2
N
X
4b
6.9
(6.4–7.4)
N
b
a
b
c
d
e
f
2-Me
4-Me
6-Me
2-Et
2-F
Me
O
4
6
O
2.7
(2.6–2.9)
11
O
N
O
10a
10b
N
HN
N
2-Cl
(10–12)
23a–f
S
19
(16–21)
10c
Scheme 3. Reagents: (a) aminophenols, K2CO3, DMF for 22a,c–f; 5-amino-2-
methylphenol, NaH, DMF for 22b; (b) 1,3-dimethyl-1H-pyrazole-5-carbonyl chlo-
ride, DMA for 23a,e,f; 1,3-dimethyl-1H-pyrazole-5-carbonyl chloride, Et3N, THF for
23b,c; 1,3-dimethyl-1H-pyrazole-5-carbonyl chloride, NMP for 23d.
Me
160
(120–200)
10d
10e
F3C
F3C
120
(110–140)
1.0
(0.96–1.1)
1.1
(1.0–1.2)
acted with a variety of acid chlorides in N,N-dimethylacetamide
(DMA) to provide the corresponding amides 8a–g. Substitution of
the iodine atom in 8a–g by 3-aminophenol produced the anilines
9a–g, which were acylated with 3-fluorobenzoyl chloride to give
the amides 10a–g.
The aniline 9a was also converted into the 3-(trifluoromethyl)benz-
amide derivative 4 by treatment with 3-(trifluoromethyl)benzoyl chlo-
ride in DMA (Scheme 2). In addition, compounds possessing various acyl
groups (11–21) were synthesized by reaction of 9f with the correspond-
ing acid chlorides.
Compounds having a substituent (X) on the central phenyl ring
(23a–f) were synthesized as shown in Scheme 3. The 6-iodoimi-
dazo[1,2-b]pyridazine 8f was coupled with appropriate aminophe-
nols to give the corresponding anilines 22a–f. Treatment of 22a–f
with 1,3-dimethyl-1H-pyrazole-5-carbonyl chloride afforded the
corresponding amides 23a–f.
10f
10g
Me
a
Numbers in parentheses represent 95% confidence interval.
See Figure 1 for the structure of 4.
b
proliferation assay was performed to determine the ability of VEG-
FR2 inhibitors to suppress VEGF-stimulated growth of HUVEC.
A preliminary study revealed that the 3-fluorobenzamide deriva-
tive 10a (IC50 = 2.7 nM) showed more potent VEGFR2 kinase inhibi-
tory activity than that of 4 (IC50 = 6.9 nM). Thus, optimization of the
amide substituent R1 at the 2-position of the imidazo[1,2-b]pyrida-
zine core was performed starting from 10a (Table 1). Replacement
of the 4-tetrahydropyranyl group in 10a with various sizes of aro-
matic or aliphatic groups revealed that small aliphatic substituents
were optimal for VEGFR2 kinase inhibition. The cyclohexyl derivative
10b (IC50 = 11 nM) was about four-fold less active than 10a, while the
thienyl (10c, IC50 = 19 nM) and (trifluoromethyl)phenyl (10d,
IC50 = 160 nM; 10e, IC50 = 120 nM) compounds exhibited even low
3. Results and discussion
The imidazo[1,2-b]pyridazine derivatives depicted in Tables 1–3
were evaluated for their inhibitory activity against VEGFR2 by a
non-RI assay using the AlphaScreen system.23 Subsequently, a cell

2336
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
Table 2
Effect of the amide substituent R2 on VEGFR2 kinase inhibitory activitya
H
N
R2
O
O
N
O
N
HN
N
Compound
R2
VEGFR2
Compound
R2
VEGFR2
IC50 (nM)
IC50 (nM)
N
N
1.0
(0.96–1.1)
1.3
(1.2–1.5)
10f
11
16
17
N
Me
Me
F
12
(11–14)
0.96
(0.81–1.1)
N
Me
N
Me
Me
Me
Me
N
O
650
(550–750)
25
(19–33)
12
13
18
19
Me
Me
N
1.3
(1.2–1.5)
1.4
(1.3–1.5)
N
N
Me
Me
N
O
O
1.3
(1.2–1.3)
1.2
(1.2–1.3)
N
14
15
20
21
N
N
Ph
CF3
Me
N
1.4
(1.2–1.8)
3.7
(3.3–4.2)
N
Me
a
Numbers in parentheses represent 95% confidence interval.
Table 3
cyclopropyl group (11) resulted in a 12-fold reduction in inhibitory
activity against VEGFR2 kinase (IC50 = 12 nM), and the compound
with a tert-butyl group (12) had a 650-fold loss of potency
(IC50 = 650 nM). These results suggested that large planar substitu-
ents might have a better fit with the VEGFR2 protein. Indeed, except
for 18, the aromatic amide derivatives 10f, 13–17, and 19–21 more
potently inhibited VEGFR2 kinase than the aliphatic amide deriva-
tives 11 and 12. No clear difference in activity was observed among
the aromatic compounds 10f, 13–17, and 19–21. For example, the
fluorophenyl (10f), pyridyl (13), and isoxazolyl (14) derivatives had
similarly strongactivity against VEGFR2 kinase, although the ring size
and number of heteroatoms differed between them. The methylisox-
azolyl (15) and methyltriazolyl (16) compounds also showed similar
inhibition of enzymatic activity with IC50 values around 1 nM, indi-
cating that mono-methyl substitution on the heteroaryl did not affect
VEGFR2 kinase inhibition. Conversely, the isoxazole 18 possessing
two methyl groups at the ortho position of the carbonyl group was
found to be about 20-fold less active (IC50 = 25 nM) than the dimeth-
ylpyrazole derivatives 17 and 19 (IC50 = 0.96, 1.4 nM) which had dif-
ferent substitution patterns. Regarding the size of substituents on the
heteroaryl group, even replacement of the 1-methyl group of the pyr-
azole in 19 with a much larger phenyl group (20) was acceptable
(IC50 = 1.2 nM). Replacement of the 3-methyl group of the pyrazole
in 19 with a trifluoromethyl group (21) resulted in a slightly de-
creased potency (IC50 = 3.7 nM). Further evaluation revealed that
the heteroaryl derivatives tended to show lower potency against B-
raf kinase than the fluorophenyl compound 10f. For instance,
although 10f moderately inhibited B-raf kinase with an IC50 of
650 nM, the pyrazole derivative 19 did not significantly inhibit B-
raf kinase(IC50 >10,000 nM). Thus, interms ofkinaseselectivity, com-
pound 19 was judged to be preferable to compound 10f. In addition,
19 strongly suppressed the growth of HUVEC with an IC50 of 1.7 nM,
Effect of the central phenyl substituent X on VEGFR2 kinase activity and HUVEC
growtha
Me
X
H
N
2
N
N
Me
O
4
6
O
O
N
N
HN
N
Compound
X
VEGFR2
IC50 (nM)
HUVECb
IC50 (nM)
19
H
1.4 (1.3–1.5)
0.95 (0.82–1.1)
1.8 (1.5–2.1)
4.7 (4.2–5.3)
5.1 (4.2–6.2)
1.1 (1.0–1.3)
1.3 (1.1–1.5)
1.7 (0.66–3.8)
0.30 (0.28–0.32)
11 (6.8–17)
97 (73–130)
93 (66–130)
0.56 (0.14–1.5)
3.0 (1.4–5.8)
23a (TAK-593)
2-Me
4-Me
6-Me
2-Et
2-F
23b
23c
23d
23e
23f
2-Cl
a
Numbers in parentheses represent 95% confidence interval.
Inhibition of VEGF-stimulated HUVEC proliferation.
b
potency. These results implied that the binding pocket of VEGFR2 for
R1 is rather small. This was supported by the fact that compounds
having
IC50 = 1.1 nM) group were more potent than 10b. Considering these
results, we selected the cyclopropyl group as the R1 substituent for
further study.
Next, we examined theeffect of amidesubstituents atthe meta po-
sition of the 6-phenoxy moiety (R2, Table 2). In contrast to the results
of R1, the small aliphatic substituents seemed to be less preferable as
the R2 group. Replacement of the fluorophenyl group in 10f with a
a
cyclopropyl (10f, IC50 = 1.0 nM) or methyl (10g,

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2337
Table 4
thereby emerging as a new lead compound for the further develop-
ment of VEGFR2 inhibitors.
Inhibition of VEGFR2 kinase in various conditionsa
The co-crystal structure of 19 in complex with VEGFR2 was
solved, and the result is shown in Figure 3. Compound 19 binds
to the inactive (DFG-out) conformation of VEGFR2 in the same
manner as the so-called type-II kinase inhibitors.24 When 19 is
bound to VEGFR2, the N1-nitrogen of the imidazo[1,2-b]pyridazine
core interacts with the NH proton of Cys919. The CH proton at the
8-position of the core forms a CHÁ Á ÁO hydrogen bond with the car-
bonyl group of Glu917. The 6-phenoxy moiety extends toward the
back pocket, accompanied by interaction of the amide proton with
Glu885, the amide carbonyl group with Asp1046, and the terminal
pyrazole ring with the hydrophobic pocket. As for the amide group
at the 2-position of the imidazo[1,2-b]pyridazine, the amide proton
is involved in hydrogen bonding with the carbonyl group of
Cys919, and the cyclopropane moiety is suitably located in the sol-
vent accessible region. In addition, a small hydrophobic pocket was
observed around the central phenyl ring, which indicated the pos-
sibility of introducing a substituent on the phenyl moiety.
Thus, the effects of substituents on the central phenyl group
were investigated as shown in Table 3. Methyl substitution at the
2-position (23a) was found to produce potent HUVEC growth inhi-
bition. While the 2-methyl (23a, IC50 = 0.95 nM), 4-methyl (23b,
IC50 = 1.8 nM), and 6-methyl (23c, IC50 = 4.7 nM) derivatives
showed similar inhibitory activity against VEGFR2 kinase, com-
pound 23a inhibited growth of HUVEC (IC50 = 0.30 nM) much
stronger than 23b or 23c (IC50 = 11, 97 nM). Conversely, replace-
ment of the methyl group in 23a with an ethyl group (23d) de-
creased both the inhibition of VEGFR2 kinase (IC50 = 5.1 nM) and
the inhibition of HUVEC growth (IC50 = 93 nM). This SAR is consis-
tent with the crystal structure analysis of 19 with VEGFR2, in
which the space for introducing substituents around the central
phenyl ring is limited. Further investigation showed that substitu-
tion with the electron-withdrawing group at this position was tol-
erated. In fact, the 2-fluoro (23e) and 2-chloro (23f) derivatives
exhibited IC50 values below 10 nM in both the enzyme activity
(VEGFR2 IC50 = 1.1, 1.3 nM) and cell proliferation (HUVEC
IC50 = 0.56, 3.0 nM) assays.
Compound
VEGFR2 IC50 (nM)
ATP 1000 l
Mb
HUVECd
IC50 (nM)
ATP 10
5 minc
l
Mb
5 minc
60 minc
1
4
19
23a
7.1
6.9
1.4
0.95
35 (30–39)
10 (9.9–11)
49 (42–57)
1.1 (1.0–1.1)
650
9.1
1.7
1.8 (1.5–2.1) 0.19 (0.17–0.22)
1.1 (1.0–1.1) 0.093 (0.087–0.098)
0.30
a
b
c
Numbers in parentheses represent 95% confidence interval.
Concentration of ATP.
Preincubation time.
d
Inhibition of VEGF-stimulated HUVEC proliferation.
ATP concentration from 10 to 1000 lM resulted in a fivefold increase
of the IC50 value (7.1 ? 35 nM). On the other hand, the IC50 values for
4 were approximately the same (6.9 ? 10 nM). More interestingly,
increasing the preincubation time from 5 to 60 min led to an approx-
imate 10-fold decrease in IC50 for 4 (10 ? 1.1 nM). This time depen-
dency indicated that compound 4 is a slow-binding inhibitor of
VEGFR2. In contrast, compound 1 did not exhibit an increase in activ-
ity after preincubation for 60 min, suggesting that it is a fast-binding
inhibitor. The enzyme inhibition produced with the longer preincu-
bation time was found to be better correlated with the inhibition of
cell proliferation. The VEGFR2 IC50 value obtained with 60-min prein-
cubation for compound 4 was about 50-fold lower than that for 1 (1.1
vs 49 nM), which roughly corresponds to the ratio of IC50 values ob-
served in the HUVEC proliferation assay (9.1 vs 650 nM).
We also evaluated the time dependency of compounds 19 and
23a, which were again found to be slow-binding inhibitors. After
preincubation for 60 min, compounds 19 and 23a inhibited VEG-
FR2 kinase with IC50 values of 0.19 and 0.093 nM, respectively. A
typical inhibition curve, exemplified by 23a, is shown in Figure 4.
Due to their strong inhibition of the kinase, compounds 19 and
23a also exhibited significant inhibitory activity in the cell growth
assay. Recently, we showed that 23a has a long residence time on
VEGFR2.25 The slow dissociation kinetics of 23a might contribute
to its strong inhibitory potency on HUVEC growth.
During the course of our study, we observed a discrepancy
betweentheenzymeand cell growth inhibition assays. In fact, the ini-
tial compound 1 inhibited VEGFR2 enzyme phosphorylation and
VEGF-stimulated growth of HUVEC with IC50 values of 7.1 and
650 nM, while the IC50 values for compound 4 were 6.9 and 9.1 nM,
respectively. To better understand the reason for this difference, we
performed the kinase assay with varying ATP concentrations and pre-
incubation times (Table 4). In the case of compound 1, an increase of
The pharmacokinetic properties of selected compounds that
showed strong inhibition of HUVEC growth (19 and 23a,e) were
evaluated in mice (Table 5). It was found that the presence of a
substituent at the 2-position is important for increasing the area
under the plasma concentration–time curve (AUC) values. After
oral administration of compound 19 (10 mg/kg) to mice, an
AUC0–8h value of 0.73
l
gÁh/mL was observed. In contrast, introduc-
Figure 3. Crystal structure of 19 in complex with VEGFR2 (PDB 3VO3). Dashed lines represent hydrogen bonds.

2338
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
The methyl derivative 23a, which showed the largest AUC va-
lue, was selected for further evaluation. Compound 23a was still
detected in plasma even when measured 8 h after administration
(C8h = 0.60
microsomes (CL = 31
in human hepatic microsomes (CL = 2
l
g/mL) reflecting its high stability in mouse hepatic
L/min/mg). Since 23a showed high stability
L/min/mg), it would be ex-
l
l
pected to have sufficient oral bioavailability in humans.
Pharmacokinetic parameters of 23a were measured in rats and
cynomolgus monkeys (Table 6). After intravenous administration
(0.25 mg/kg), 23a exhibited low plasma clearance (191–438 mL/
h/kg) and low to moderate volume of distribution (471–1028 mL/
kg) in these species. Oral administration (0.5 mg/kg) to monkeys
showed a peak plasma concentration of 0.129
the plasma concentration decreased slowly with a mean residence
time of 11.0 h. The AUC0–48h value was 1.511
lg/mL at 2.8 h, and
l
gÁh/mL, and the oral
Figure 4. VEGFR2 inhibition curves for 23a in various conditions. Data represent
the mean SD (n = 4).
bioavailability was 57.6%. These data also predicted desirable phar-
macokinetic profiles in humans.
Compound 23a was examined for its selectivity against a panel
of over 260 kinases.25 It showed potent inhibitory activity against
VEGFR (VEGFR1–3: IC50 = 3.2, 0.95, 1.1 nM) and PDGFR (PDGFRa,b:
tion of methyl (23a) and fluoro (23e) groups dramatically in-
creased AUC values (15 and 7.6
lgÁh/mL). We supposed that these
IC50 = 4.3, 13 nM) families. Against other kinases, the IC50 values of
23a were above 100 nM, except for Fms (IC50 = 10 nM) and Ret
(IC50 = 18 nM) kinases. Thus, 23a proved to be a potent VEGFR
and PDGFR kinase inhibitor with high selectivity within the human
kinome.
We evaluated the antitumor efficacy of 23a in a nude mouse
xenograft model using A549 human lung adenocarcinoma epithe-
lial cells (Fig. 5).28 Tumor growth inhibition was calculated as
treatment over control (T/C) values, measured as the increase in
treated tumor volumes divided by the increase in control tumor
volumes. Consistent with its potent in vitro activity and good oral
exposure levels, 23a showed significant antitumor activity. Upon
oral administration of 23a twice daily at doses of 0.25, 1, and
4 mg/kg for 2 weeks, the T/C values were 34, 8, and À8%, respec-
tively. No obvious weight loss or morbidity was observed over
the entire treatment period.
higher AUC values were attributed to the improved solubility of
23a and 23e compared to 19 in Japanese Pharmacopoeia disinte-
gration test solution 2 (pH 6.8) containing a bile acid (Table 5).
To clarify the effect of the 2-substituents on solubility, logD val-
ues and melting points of 19 and 23a,e were measured, since gen-
eral solubility equations include both lipophilicity and melting
point.26 The results revealed that there is a relationship between
their solubilities and melting points (Table 5). Compound 23a,
which showed the lowest melting point (223 °C), was the most sol-
uble (38
lg/mL), while 19 showed the highest melting point
(266 °C) and the lowest solubility (3.7
lg/mL). The logD values at
pH 7.4 of 19 and 23a,e were nearly identical. There are similar
examples of this relationship in the literature,27 and the authors
of this previous report concluded that disruption of molecular pla-
narity by introduction of a substituent resulted in a decreased crys-
tal packing energy and a lower melting point, leading to improved
solubility. Thus, the methyl group of 23a and the fluoro group of
23e may contribute to distortion of the planar conformation of
the test compounds.
We then studied the pharmacokinetic and pharmacodynamic
properties of 23a, to gain insight into its potent antitumor activity
(Fig. 6).28 Oral administration of 23a at 1 mg/kg to athymic nude
mice showed that the concentration of 23a in plasma (0.451 lg/
Table 5
Pharmacokinetic and physicochemical profiles of 19 and 23a,e
Compound
Mouse PKa
Metabolic stabilityd
Mouse
L/min/mg)
Solubilitye
JP2 + BA
Mpf
(°C)
logD7.4
b
c
AUC0–8h
gÁh/mL)
C8h
g/mL)
Human
L/min/mg)
(
l
(
l
(
l
(
l
(lg/mL)
19
23a
23e
0.73
15
7.6
0.053
0.60
0.30
6
2
9
43
31
54
3.7
38
14
266
223
234
2.28
2.24
2.21
a
b
c
d
e
f
Compounds (10 mg/kg) were orally administered by cassette dosing.
Area under plasma concentration–time curve from 0 to 8 h.
Plasma concentration at 8 h after administration.
Metabolic stability in hepatic microsomes.
Thermodynamic solubility in Japanese Pharmacopoeia disintegration test solution 2 (pH 6.8) containing a bile acid.
Determined by differential scanning calorimetry.
Table 6
Pharmacokinetic parameters of 23a in rats and cynomolgus monkeysa
Species
iv (0.25 mg/kg)
po (0.5 mg/kg)
AUCb
gÁh/mL)
BA
(%)
AUCb
gÁh/mL)
Vdss
(mL/kg)
CLtot
(mL/h/kg)
Cmax
g/mL)
Tmax
(h)
MRT
(h)
(
l
(
l
(l
Rat
Monkey
0.571 0.009
1.313 0.064
471 85
1028 109
438
191
7
9
0.056 0.012
0.129 0.011
1.3 0.6
2.8 1.5
0.226 0.092
1.511 0.100
3.3 0.8
11.0 1.4
19.8 8.1
57.6 2.1
a
Pharmacokinetic parameters were obtained by discrete dosing. Data represent mean values SD (rat, n = 3; monkey, n = 4).
b
Area under plasma concentration–time curve: rat, AUC0–24h; monkey, AUC0–48h
.

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2339
substituents at the 2-position showed that small aliphatic groups
were suitable to maintain inhibitory activity. The heteroaryl groups
were suitable for the amide substituent at the meta position of the 6-
phenoxy moiety. Furthermore, the introduction of a methyl group
on the central phenyl ring resulted in the discovery of 23a, the com-
pound possessing the strongest potency in the cell-based assay and
favorable pharmacokinetic properties based on its improved solu-
bility. Compound 23a showed potent activity against PDGFR kinases
as well as VEGFR kinases with high kinase selectivity. It exhibited
potent antitumor efficacy in an A549 xenograft mouse model. Ulti-
mately, 23a (TAK-593) was selected as a clinical candidate for the
treatment of human cancer.
700
600
500
400
300
200
100
0
Vehicle
0.25 mg/kg, bid
1 mg/kg, bid
4 mg/kg, bid
T/C
34%*
8%*
–8%*
5. Experimental section
5.1. Chemistry
13
18
23
28
Days after inoculation
Figure 5. Antitumor efficacy of 23a in an A549 xenograft mouse model. A549
tumor-bearing nude mice were orally administered 23a (0.25, 1, and 4 mg/kg) twice
daily for 14 days (day 14–27). Data represent mean SD (n = 5). ⁄p 60.025 versus
vehicle control as determined by a one-tailed Williams’ test. Antitumor effects are
expressed as T/C values (increase in treated tumor volume/increase in control
tumor volume) Â 100.
Commercial reagents and solvents were used without addi-
tional purification. Microwave assisted reactions were carried out
in a Biotage Initiator microwave reactor. Reaction progress was
determined by thin layer chromatography (TLC) on Silica Gel 60
F254 plates (Merck) or NH TLC plates (Fuji Silysia). Column chro-
matography was carried out using a Purif Purification System
(Moritex) with silica gel (Purif-Pack SI-60, Fuji Silysia) or basic sil-
ica gel (Purif-Pack NH-60, Fuji Silysia) cartridges. Melting points
were determined on a Yanagimoto micro melting point, BÜCHI
Melting Point B-545, or OptiMelt melting point apparatus and were
not corrected. Proton nuclear magnetic resonance (1H NMR) spec-
tra were recorded on a Bruker DPX 300 (300 MHz), Varian Mercury
300 (300 MHz), or Bruker AV 400 (400 MHz) instrument. Chemical
shifts (d) are reported in ppm relative to internal tetramethylsilane.
Peak multiplicities are expressed as follows: s, singlet; d, doublet;
t, triplet; q, quartet; dd, doublet of doublet; dt, doublet of triplet;
td, triplet of doublet; ddd, doublet of doublet of doublet; m, multi-
plet; br s, broad singlet. Coupling constants (J) are given in hertz
(Hz). Elemental analyses and high resolution mass spectrometry
(HRMS) were performed at the Takeda Analytical Research Labora-
tories, Ltd. Abbreviations are used as follows: DMA, N,N-dimethyl-
acetamide; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; EDCÁHCl, 1-ethyl-3-[3-(dimethylamino)propyl]carbodi-
imide hydrochloride; HOBt, 1-hydroxybenzotriazole; NMP, N-
methylpyrrolidone; THF, tetrahydrofuran.
0.6
0.5
0.4
0.3
0.2
0.1
0.0
120
100
80
60
40
20
0
Plasma
Lung
pVEGFR2
-20
0
4
8
12
16
20
24
Time after administration (h)
Figure 6. PK/PD analysis of 23a. Nude mice (n = 3) were orally administered 23a
(1 mg/kg), and the concentration of 23a in plasma and lung tissue was determined
by LC/MS/MS. For PD analysis, nude mice (n = 4) were treated with an oral dose of
23a (1 mg/kg), and the lung tissue was collected 5 min after VEGF (20
lg/mouse)
injection. VEGFR2 phosphorylation in the lung tissue was assessed by Western blot.
5.1.1. Ethyl (6-iodoimidazo[1,2-b]pyridazin-2-yl)carbamate (6)
To a stirred solution of 3-amino-6-iodopyridazine (5)30 (27.0 g,
122 mmol) in DMA (270 mL) were added ethyl (chloroacetyl)car-
bamate (32.4 g, 196 mmol) and Na2HPO4 (43.4 g, 306 mmol), and
the mixture was stirred at 110 °C for 3 h. After cooling to room
temperature, the mixture was diluted with water (810 mL), and
the precipitate was collected by filtration and washed with CH3CN
followed by Et2O to give 6 (33.0 g, 81%) as a dark brown solid. 1H
NMR (300 MHz, DMSO-d6) d 1.26 (3H, t, J = 7.1 Hz), 4.17 (2H, q,
J = 7.1 Hz), 7.47 (1H, d, J = 9.2 Hz), 7.70 (1H, d, J = 9.2 Hz), 8.06
(1H, s), 10.51 (1H, br s).
mL) and lung tissue (0.242 lg/g) at 1 h after dosinghad achieved lev-
els sufficient to inhibit VEGFR2 kinase. At 8 h after dosing, the con-
centration of 23a was below or near the detectable limit in either
plasma (<0.003 lg/mL) or lung tissue (<0.015 lg/g). On the other
hand, phospho-VEGFR2 in lung tissue was almost completely sup-
pressed at not only 1 h, but also 8 h after oral administration of
23a, and restored to baseline after 16 h. Compound 23a has a long-
lasting inhibitory effect on VEGFR2 kinase,25 which might contrib-
ute to in vivo efficacy.29 Consequently, we speculated that the potent
in vivo efficacy of 23a was achieved by a combination of its favorable
pharmacokinetic profile and its long residence time on VEGFR2.
5.1.2. 6-Iodoimidazo[1,2-b]pyridazin-2-amine (7)
A suspension of Ba(OH)2Á8H2O (14.5 g, 46.0 mmol) in water
(240 mL) was stirred at 80 °C for 15 min. To the mixture was added
a solution of 6 (10.2 g, 30.7 mmol) in NMP (80 mL), and the mix-
ture was stirred at 120 °C for 8 h. After cooling to room tempera-
ture, the mixture was diluted with water (480 mL) and stirred for
1 h. The mixture was extracted with EtOAc/THF, and the organic
layer was washed with brine, dried over MgSO4, and filtered. The
filtrate was concentrated in vacuo, and the residue was triturated
with i-Pr2O to give 7 (6.10 g, 76%) as a brown solid. 1H NMR
4. Conclusion
To discover potent VEGFR2 kinase inhibitors with strong in vivo
antitumor efficacy, we designed and synthesized 2-acylamino-6-
phenoxyimidazo[1,2-b]pyridazine derivatives, which were a hybrid
of 6-phenoxyimidazo[1,2-b]pyridazine
dazo[1,2-b]pyridazine 2. These efforts led to a significant enhance-
1 and 2-acylaminoimi-
ment of cell growth inhibition. Optimization of the amide

2340
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
(300 MHz, DMSO-d6) d 5.61 (2H, s), 7.22 (1H, d, J = 8.7 Hz), 7.36
(1H, s), 7.39 (1H, d, J = 8.7 Hz).
give 9f (468 mg, 62%) as a brown solid. 1H NMR (300 MHz,
DMSO-d6) d 0.75–0.85 (4H, m), 1.87–1.97 (1H, m), 5.31 (2H, s),
6.27–6.32 (1H, m), 6.35 (1H, t, J = 2.2 Hz), 6.40–6.46 (1H, m), 6.95
(1H, d, J = 9.5 Hz), 7.05 (1H, t, J = 8.0 Hz), 7.98 (1H, s), 8.00 (1H, d,
J = 9.5 Hz), 11.08 (1H, s).
5.1.3. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)
cyclopropanecarboxamide (8f)
To a stirred solution of 7 (1.00 g, 3.85 mmol) in DMA (10 mL)
was added cyclopropanecarbonyl chloride (384 lL, 4.23 mmol),
Compounds 9a–e,g were prepared from the corresponding io-
dides 8a–e,g in a manner similar to that described for 9f.
and the mixture was stirred at room temperature for 4 h. The mix-
ture was diluted with water and extracted with EtOAc/THF (2:1).
The organic layer was washed with brine, dried over MgSO4, and
filtered. The filtrate was concentrated in vacuo, and the residue
was triturated with n-hexane/EtOAc (4:1) to give 8f (1.01 g, 80%)
as a dark brown solid. 1H NMR (300 MHz, DMSO-d6) d 0.82–0.86
(4H, m), 1.90–2.00 (1H, m), 7.49 (1H, d, J = 9.3 Hz), 7.73 (1H, d,
J = 9.3 Hz), 8.23 (1H, s), 11.20 (1H, s).
5.1.11. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]
tetrahydro-2H-pyran-4-carboxamide (9a)
Yield 42%, white solid. 1H NMR (300 MHz, DMSO-d6) d 1.57–
1.73 (4H, m), 2.64–2.77 (1H, m), 3.26–3.39 (2H, m), 3.84–3.94
(2H, m), 5.30 (2H, s), 6.27–6.45 (3H, m), 6.95 (1H, d, J = 9.6 Hz),
7.05 (1H, t, J = 8.1 Hz), 7.97–8.03 (2H, m), 10.78 (1H, s).
Compounds 8a–e,g were prepared from 7 and the appropriate
acyl chloride in a manner similar to that described for 8f.
5.1.12. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-
yl]cyclohexanecarboxamide (9b)
Yield 51%, colorless oil. 1H NMR (400 MHz, CDCl3) d 1.21–1.37
(2H, m), 1.45–1.59 (2H, m), 1.65–2.02 (6H, m), 2.21–2.34 (1H,
m), 3.77 (2H, br s), 6.50 (1H, t, J = 2.1 Hz), 6.52–6.57 (2H, m),
6.81 (1H, d, J = 9.6 Hz), 7.17 (1H, t, J = 8.0 Hz), 7.70 (1H, d,
J = 9.6 Hz), 8.01–8.14 (1H, m), 8.22 (1H, s).
5.1.4. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)tetrahydro-2H-
pyran-4-carboxamide (8a)
Yield 73%, light brown solid. 1H NMR (300 MHz, DMSO-d6) d
1.54–1.76 (4H, m), 2.66–2.80 (1H, m), 3.27–3.40 (2H, m), 3.84–
3.96 (2H, m), 7.49 (1H, d, J = 9.3 Hz), 7.74 (1H, d, J = 9.3 Hz), 8.28
(1H, s), 10.91 (1H, s).
5.1.13. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]-3-
methylthiophene-2-carboxamide (9c)
5.1.5. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)
cyclohexanecarboxamide (8b)
Yield 58%, pale yellow solid. 1H NMR (400 MHz, CDCl3) d 2.62
(3H, s), 3.78 (2H, br s), 6.52 (1H, t, J = 2.1 Hz), 6.56 (2H, dd,
J = 8.1, 2.0 Hz), 6.83 (1H, d, J = 9.5 Hz), 6.95 (1H, d, J = 4.9 Hz),
7.18 (1H, t, J = 8.1 Hz), 7.37 (1H, d, J = 5.1 Hz), 7.67 (1H, dd,
J = 9.5, 1.7 Hz), 8.32 (1H, s), 8.36 (1H, br s).
Yield 50%, white solid. 1H NMR (300 MHz, DMSO-d6) d 1.12–
1.49 (6H, m), 1.59–1.85 (5H, m), 7.48 (1H, d, J = 9.3 Hz), 7.73 (1H,
d, J = 9.3 Hz), 8.22–8.31 (1H, m), 10.80 (1H, s).
5.1.6. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)-3-
methylthiophene-2-carboxamide (8c)
5.1.14. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]-3-
(trifluoromethyl)benzamide (9d)
Yield 61%, pale yellow solid. 1H NMR (400 MHz, DMSO-d6) d
2.47–2.50 (3H, m), 7.03 (1H, d, J = 4.9 Hz), 7.53 (1H, d, J = 9.4 Hz),
7.69 (1H, d, J = 4.9 Hz), 7.77 (1H, d, J = 9.2 Hz), 8.40 (1H, s), 10.96
(1H, s).
Yield 54%, white solid. 1H NMR (400 MHz, CDCl3) d 3.80 (2H, br
s), 6.53 (1H, t, J = 2.1 Hz), 6.55–6.60 (2H, m), 6.84 (1H, d, J = 9.5 Hz),
7.20 (1H, t, J = 8.0 Hz), 7.58 (1H, d, J = 9.5 Hz), 7.64 (1H, t,
J = 7.8 Hz), 7.82 (1H, d, J = 7.8 Hz), 8.11 (1H, d, J = 7.8 Hz), 8.21
(1H, s), 8.39 (1H, s), 9.16 (1H, br s).
5.1.7. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)-3-
(trifluoromethyl)benzamide (8d)
5.1.15. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]-4-
(trifluoromethyl)benzamide (9e)
Yield 63%, white solid. 1H NMR (300 MHz, DMSO-d6) d 7.55 (1H,
d, J = 9.3 Hz), 7.74–7.85 (2H, m), 7.98 (1H, d, J = 7.8 Hz), 8.37 (1H, d,
J = 7.7 Hz), 8.46 (1H, s), 8.51 (1H, s), 11.77 (1H, s).
Yield 44%, white solid. 1H NMR (400 MHz, CDCl3) d 3.80 (2H, br
s), 6.53 (1H, t, J = 2.2 Hz), 6.55–6.60 (2H, m), 6.83 (1H, d, J = 9.6 Hz),
7.20 (1H, t, J = 8.0 Hz), 7.54 (1H, d, J = 9.6 Hz), 7.76 (2H, d,
J = 8.5 Hz), 8.04 (2H, d, J = 8.3 Hz), 8.39 (1H, s), 9.13 (1H, s).
5.1.8. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)-4-
(trifluoromethyl)benzamide (8e)
Yield 49%, white solid. 1H NMR (300 MHz, DMSO-d6) d 7.55 (1H,
d, J = 9.3 Hz), 7.81 (1H, d, J = 9.3 Hz), 7.91 (2H, d, J = 8.3 Hz), 8.26
(2H, d, J = 8.1 Hz), 8.51 (1H, s), 11.72 (1H, s).
5.1.16. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]
acetamide (9g)
Yield 72%, white solid. 1H NMR (400 MHz, DMSO-d6) d 2.07 (3H,
s), 5.31 (2H, br s), 6.30 (1H, dd, J = 7.9, 1.6 Hz), 6.36 (1H, t,
J = 2.2 Hz), 6.43 (1H, dd, J = 8.1, 1.2 Hz), 6.96 (1H, d, J = 9.8 Hz),
7.05 (1H, t, J = 8.1 Hz), 7.97–8.01 (2H, m), 10.79 (1H, s).
5.1.9. N-(6-Iodoimidazo[1,2-b]pyridazin-2-yl)acetamide (8g)
Yield 43%, white solid. 1H NMR (300 MHz, DMSO-d6) d 2.09 (3H,
s), 7.49 (1H, d, J = 9.3 Hz), 7.74 (1H, d, J = 9.3 Hz), 8.25 (1H, s), 10.91
(1H, s).
5.1.17. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-3-fluorobenzamide (10f)
5.1.10. N-[6-(3-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]
cyclopropanecarboxamide (9f)
To a stirred solution of 9f (100 mg, 0.32 mmol) in DMA (3 mL)
was added 3-fluorobenzoyl chloride (43 lL, 0.36 mmol), and the
A mixture of 8f (805 mg, 2.45 mmol), 3-aminophenol (430 mg,
3.94 mmol), and K2CO3 (847 mg, 6.13 mmol) in DMF (6 mL) was
stirred at 180 °C for 30 min using a microwave synthesizer. The
mixture was concentrated in vacuo. The residue was diluted with
water and extracted with EtOAc/THF (1:1). The organic layer was
washed with brine, dried over Na2SO4, and filtered. The filtrate
was concentrated in vacuo, and the residue was purified by silica
gel column chromatography (n-hexane/EtOAc 70:30 to 0:100) to
mixture was stirred at room temperature overnight. The mixture
was diluted with water and extracted with EtOAc. The organic layer
was washed with water and brine, dried over MgSO4, and filtered.
The filtrate was concentrated in vacuo, and the residue was purified
by basic silica gel column chromatography (n-hexane/EtOAc 100:0
to 0:100) to give 10f (56 mg, 40%) as a white solid. Mp 263–264 °C.
1H NMR (300 MHz, DMSO-d6) d 0.76–0.85 (4H, m), 1.87–1.98 (1H,
m), 7.02 (1H, dd, J = 8.1, 2.2 Hz), 7.07 (1H, d, J = 9.5 Hz), 7.41–7.49

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2341
(2H, m), 7.54–7.83 (5H, m), 7.98 (1H, s), 8.06 (1H, d, J = 9.5 Hz),
10.43 (1H, s), 11.08 (1H, s). Anal. Calcd for C23H18FN5O3: C, 64.03;
H, 4.21; N, 16.23. Found: C, 63.76; H, 4.12; N, 16.10.
5.1.24. N-{6-[3-({[3-(Trifluoromethyl)phenyl]carbonyl}amino)
phenoxy]imidazo[1,2-b]pyridazin-2-yl}tetrahydro-2H-pyran-4-
carboxamide (4)
To an ice-cooled stirred solution of 9a (200 mg, 0.57 mmol) in
DMA (2 mL) was added 3-(trifluoromethyl)benzoyl chloride
(100 lL, 0.68 mmol), and the mixture was stirred at room temper-
Compounds 10a–e,g were prepared from the corresponding
anilines 9a–e,g in a manner similar to that described for 10f.
ature for 2 h. The mixture was diluted with aqueous NaHCO3 and
extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4 and filtered. The filtrate was concentrated in va-
cuo, and the residue was purified by silica gel column chromatog-
raphy (EtOAc/MeOH 100:0 to 95:5) followed by recrystallization
from n-hexane/EtOAc to give 4 (221 mg, 74%) as a white solid.
Mp 205–206 °C. 1H NMR (300 MHz, DMSO-d6) d 1.56–1.73 (4H,
m), 2.62–2.78 (1H, m), 3.26–3.38 (2H, m), 3.83–3.93 (2H, m),
7.00–7.06 (1H, m), 7.08 (1H, d, J = 9.6 Hz), 7.46 (1H, t, J = 8.1 Hz),
7.64–7.83 (3H, m), 7.95–7.99 (1H, m), 8.03 (1H, s), 8.06 (1H, d,
J = 9.6 Hz), 8.23–8.29 (2H, m), 10.60 (1H, s), 10.80 (1H, s). Anal.
Calcd for C26H22F3N5O4Á0.3H2O: C, 58.82; H, 4.29; N, 13.19. Found:
C, 58.80; H, 4.36; N, 13.06.
5.1.18. N-[6-(3-{[(3-Fluorophenyl)carbonyl]amino}phenoxy)
imidazo[1,2-b]pyridazin-2-yl]tetrahydro-2H-pyran-4-
carboxamide (10a)
Yield 34%, white solid. Mp 230–231 °C. 1H NMR (400 MHz,
DMSO-d6) d 1.58–1.74 (4H, m), 2.65–2.76 (1H, m), 3.28–3.37 (2H,
m), 3.86–3.92 (2H, m), 7.02 (1H, dd, J = 8.1, 1.5 Hz), 7.08 (1H, d,
J = 9.5 Hz), 7.42–7.49 (2H, m), 7.56–7.62 (1H, m), 7.64–7.68 (1H,
m), 7.72–7.78 (2H, m), 7.80 (1H, d, J = 7.8 Hz), 8.03 (1H, s), 8.06
(1H, d, J = 9.5 Hz), 10.45 (1H, s), 10.81 (1H, s). Anal. Calcd for
C
25H22FN5O4Á0.25H2O: C, 62.56; H, 4.72; N, 14.59. Found: C,
62.64; H, 4.63; N, 14.82.
5.1.19. N-[3-({2-[(Cyclohexylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-3-fluorobenzamide (10b)
Yield 54%, white solid. Mp 216–217 °C. 1H NMR (300 MHz,
DMSO-d6) d 1.09–1.49 (5H, m), 1.57–1.84 (5H, m), 2.39–2.48 (1H,
m), 6.98–7.10 (2H, m), 7.40–7.50 (2H, m), 7.54–7.69 (2H, m),
7.71–7.83 (3H, m), 8.00–8.08 (2H, m), 10.44 (1H, s), 10.69 (1H, s).
Anal. Calcd for C26H24FN5O3Á0.2H2O: C, 65.45; H, 5.15; N, 14.68.
Found: C, 65.49; H, 5.21; N, 14.63.
Compounds 11–21 were prepared from 9f and the appropriate
acyl chloride in a manner similar to that described for 10f.
5.1.25. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]cyclopropanecarboxamide (11)
Yield 70%, white solid. Mp 273–274 °C (n-hexane/EtOAc). 1H
NMR (300 MHz, DMSO-d6) d 0.77–0.82 (8H, m), 1.70–1.78 (1H, m),
1.87–1.97 (1H, m), 6.90 (1H, dt, J = 6.9, 2.2 Hz), 7.05 (1H, s), 7.30–
7.42 (2H, m), 7.57 (1H, s), 7.96 (1H, s), 8.03 (1H, d, J = 9.5 Hz),
10.34 (1H, s), 11.08 (1H, s). Anal. Calcd for C20H19N5O3Á0.3H2O: C,
62.75; H, 5.16; N, 18.30. Found: C, 62.74; H, 5.18; N, 18.00.
5.1.20. N-[6-(3-{[(3-Fluorophenyl)carbonyl]amino}phenoxy)
imidazo[1,2-b]pyridazin-2-yl]-3-methylthiophene-2-
carboxamide (10c)
Yield 33%, white solid. Mp 203–204 °C. 1H NMR (300 MHz,
DMSO-d6)
d
2.47 (3H, s), 6.99–7.07 (2H, m), 7.12 (1H, d,
5.1.26. N-(6-{3-[(2,2-Dimethylpropanoyl)amino]phenoxy}
imidazo[1,2-b]pyridazin-2-yl)cyclopropanecarboxamide (12)
Yield 60%, white solid. Mp 234–235 °C (n-hexane/EtOAc). 1H
NMR (400 MHz, CDCl3) d 0.86–0.93 (2H, m), 1.08–1.14 (2H, m),
1.31 (9H, s), 1.55–1.59 (1H, m), 6.83 (1H, d, J = 9.5 Hz), 6.94 (1H,
ddd, J = 8.0, 2.3, 1.0 Hz), 7.26–7.40 (3H, m), 7.60 (1H, t, J = 2.2 Hz),
7.72 (1H, d, J = 9.5 Hz), 8.14 (1H, s), 8.46 (1H, s). HRMS–ESI (m/z):
[M+H]+ calcd for C21H23N5O3, 394.1874; found, 394.1857.
J = 9.5 Hz), 7.41–7.50 (2H, m), 7.55–7.70 (3H, m), 7.73–7.85 (3H,
m), 8.01–8.18 (2H, m), 10.45 (1H, s), 10.86 (1H, s). HRMS–ESI (m/
z): [M+H]+ calcd for C25H18FN5O3S, 488.1187; found, 488.1166.
5.1.21. 3-Fluoro-N-(3-{[2-({[3-(trifluoromethyl)phenyl]
carbonyl}amino)imidazo[1,2-b]pyridazin-6-yl]oxy}phenyl)
benzamide (10d)
Yield 52%, amorphous solid. 1H NMR (300 MHz, DMSO-d6) d
7.06 (1H, ddd, J = 8.0, 2.4, 0.9 Hz), 7.15 (1H, d, J = 9.8 Hz), 7.41–
7.51 (2H, m), 7.60 (1H, td, J = 8.0, 6.1 Hz), 7.66–7.72 (1H, m),
7.74–7.85 (4H, m), 7.97 (1H, d, J = 7.6 Hz), 8.10–8.16 (1H, m),
8.25 (1H, s), 8.36 (1H, d, J = 8.0 Hz), 8.45 (1H, s), 10.47 (1H, s),
11.68 (1H, s). HRMS–ESI (m/z): [M+H]+ calcd for C27H17F4N5O3,
536.1340; found, 536.1327.
5.1.27. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]pyridine-4-carboxamide (13)
Yield 78%, white solid. Mp 257–259 °C (EtOH). 1H NMR
(300 MHz, DMSO-d6) d 0.75–0.85 (4H, m), 1.84–2.00 (1H, m),
7.01–7.07 (1H, m), 7.08 (1H, d, J = 9.6 Hz), 7.46 (1H, t, J = 8.1 Hz),
7.63–7.69 (1H, m), 7.73 (1H, t, J = 2.1 Hz), 7.82–7.87 (2H, m), 7.98
(1H, s), 8.06 (1H, d, J = 9.6 Hz), 8.76–8.81 (2H, m), 10.62 (1H, s),
11.09 (1H, s). Anal. Calcd for C22H18N6O3Á0.2H2O: C, 63.21; H,
4.44; N, 20.10. Found: C, 63.28; H, 4.55; N, 19.97.
5.1.22. 3-Fluoro-N-(3-{[2-({[4-(trifluoromethyl)phenyl]
carbonyl}amino)imidazo[1,2-b]pyridazin-6-yl]oxy}phenyl)
benzamide (10e)
Yield 55%, white solid. Mp 211–212 °C. 1H NMR (300 MHz,
DMSO-d6) d 7.06 (1H, dd, J = 8.0, 2.2 Hz), 7.11–7.18 (1H, m),
7.40–7.51 (2H, m), 7.54–7.64 (1H, m), 7.65–7.71 (1H, m), 7.73–
7.84 (3H, m), 7.89 (2H, d, J = 8.4 Hz), 8.13 (1H, d, J = 9.5 Hz),
8.21–8.29 (3H, m), 10.46 (1H, s), 11.61 (1H, s). Anal. Calcd for
5.1.28. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]isoxazole-5-carboxamide (14)
Yield 57%, white solid. Mp 237–239 °C. 1H NMR (300 MHz,
DMSO-d6) d 0.75–0.85 (4H, m), 1.85–1.99 (1H, m), 7.02–7.10 (2H,
m), 7.25 (1H, d, J = 1.7 Hz), 7.46 (1H, t, J = 8.2 Hz), 7.63–7.72 (2H,
m), 7.98 (1H, s), 8.06 (1H, d, J = 9.4 Hz), 8.81 (1H, d, J = 1.7 Hz),
10.87 (1H, s), 11.08 (1H, s). Anal. Calcd for C20H16N6O4: C, 59.40;
H, 3.99; N, 20.78. Found: C, 59.09; H, 3.97; N, 20.73.
C
27H17F4N5O3Á0.5H2O: C, 59.56; H, 3.33; N, 12.86. Found: C,
59.75; H, 3.46; N, 12.80.
5.1.23. N-(3-{[2-(Acetylamino)imidazo[1,2-b]pyridazin-6-yl]
oxy}phenyl)-3-fluorobenzamide (10g)
5.1.29. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-3-methylisoxazole-5-carboxamide
(15)
Yield 76%, white solid. Mp 304–305 °C. 1H NMR (300 MHz,
DMSO-d6) d 2.07 (3H, s), 6.99–7.11 (2H, m), 7.39–7.51 (2H, m),
7.53–7.70 (2H, m), 7.71–7.84 (3H, m), 7.97–8.09 (2H, m), 10.44
(1H, s), 10.80 (1H, s). Anal. Calcd for C21H16FN5O3Á0.2H2O: C,
61.67; H, 4.04; N, 17.12. Found: C, 61.94; H, 4.09; N, 16.86.
Yield 22%, white solid. Mp 241–243 °C (n-hexane/EtOAc). 1H
NMR (300 MHz, DMSO-d6) d 0.73–0.87 (4H, m), 1.86–1.98 (1H,
m), 2.32 (3H, s), 7.01–7.14 (3H, m), 7.45 (1H, t, J = 8.2 Hz), 7.62–

2342
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
7.74 (2H, m), 7.98 (1H, s), 8.06 (1H, d, J = 9.8 Hz), 10.82 (1H, s),
11.10 (1H, s). HRMS–ESI (m/z): [M+H]+ calcd for C21H18N6O4,
419.1462; found, 419.1429.
C22H18F3N7O3: C, 54.43; H, 3.74; N, 20.20. Found: C, 54.48; H,
3.75; N, 19.97.
5.1.36. N-[6-(3-Amino-4-methylphenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22a)
5.1.30. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1-methyl-1H-1,2,3-triazole-5-
carboxamide (16)
A mixture of 8f (743 mg, 2.3 mmol), 3-amino-4-methylphenol
(446 mg, 3.6 mmol), and K2CO3 (782 mg, 5.7 mmol) in DMF
(6 mL) was stirred at 180 °C for 30 min using a microwave synthe-
sizer. The mixture was concentrated in vacuo. The residue was di-
luted with water and extracted with EtOAc/THF (1:1). The organic
layer was washed with brine, dried over Na2SO4, and filtered. The
filtrate was concentrated in vacuo, and the residue was purified
by silica gel column chromatography (n-hexane/EtOAc 80:20 to
25:75) to give 22a (424 mg, 58%) as a brown solid. 1H NMR
(300 MHz, DMSO-d6) d 0.72–0.86 (4H, m), 1.84–1.97 (1H, m),
2.04 (3H, s), 5.06 (2H, s), 6.28 (1H, dd, J = 8.1, 2.4 Hz), 6.42 (1H, d,
J = 2.4 Hz), 6.93 (1H, d, J = 9.3 Hz), 6.95 (1H, d, J = 8.1 Hz), 7.96
(1H, s), 7.98 (1H, d, J = 9.3 Hz), 11.07 (1H, s).
Yield 67%, white solid. Mp 282–284 °C (EtOAc). 1H NMR
(300 MHz, DMSO-d6) d 0.79–0.82 (4H, m), 1.87–1.96 (1H, m),
4.23 (3H, s), 7.02–7.09 (2H, m), 7.46 (1H, t, J = 8.1 Hz), 7.56–7.65
(2H, m), 7.96 (1H, s), 8.05 (1H, d, J = 9.6 Hz), 8.37 (1H, s), 10.61
(1H, s), 11.09 (1H, s). Anal. Calcd for C20H18N8O3Á0.1EtOAc: C,
57.35; H, 4.44; N, 26.23. Found: C, 57.24; H, 4.58; N, 26.00.
5.1.31. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1,5-dimethyl-1H-pyrazole-3-
carboxamide (17)
Yield 42%, white solid. Mp 226–227 °C (n-hexane/EtOAc). 1H
NMR (300 MHz, DMSO-d6) d 0.78–0.83 (4H, m), 1.88–1.96 (1H,
m), 2.30 (3H, s), 3.82 (3H, s), 6.54 (1H, s), 6.91–6.96 (1H, m), 7.05
(1H, d, J = 9.6 Hz), 7.37 (1H, t, J = 8.1 Hz), 7.69–7.73 (1H, m),
7.76–7.78 (1H, m), 7.97 (1H, s), 8.04 (1H, d, J = 9.6 Hz), 10.10 (1H,
s), 11.09 (1H, s). Anal. Calcd for C22H21N7O3Á1.2H2O: C, 58.32; H,
5.21; N, 21.64. Found: C, 58.41; H, 5.32; N, 21.36.
5.1.37. N-[6-(5-Amino-2-methylphenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22b)
To a suspension of NaH (60% in oil, 56 mg, 1.4 mmol) in DMF
(2 mL) was added
a
solution of 5-amino-2-methylphenol
(173 mg, 1.4 mmol) in DMF (1 mL), and the mixture was stirred
at 0 °C for 30 min. To the mixture was added a solution of 8f
(306 mg, 0.93 mmol) in DMF (1.5 mL), and the mixture was stirred
at 110 °C for 24 h. The mixture was concentrated in vacuo. The res-
idue was diluted with water and extracted with EtOAc. The organic
layer was washed with brine, dried over Na2SO4, and filtered. The
filtrate was concentrated in vacuo, and the residue was purified
by silica gel column chromatography (n-hexane/EtOAc 83:17 to
10:90) to give 22b (94 mg, 31%) as a brown solid. 1H NMR
(300 MHz, DMSO-d6) d 0.73–0.89 (4H, m), 1.84–2.01 (1H, m),
1.97 (3H, s), 5.08 (2H, s), 6.30 (1H, d, J = 2.3 Hz), 6.40 (1H, dd,
J = 8.0, 2.3 Hz), 6.94 (1H, d, J = 8.0 Hz), 6.95 (1H, d, J = 9.5 Hz),
7.93 (1H, s), 7.99 (1H, d, J = 9.5 Hz), 11.06 (1H, s).
5.1.32. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-3,5-dimethylisoxazole-4-
carboxamide (18)
Yield 73%, white solid. Mp 227–228 °C (n-hexane/EtOAc). 1H
NMR (300 MHz, DMSO-d6) d 0.79–0.82 (4H, m), 1.88–1.95 (1H,
m), 2.32 (3H, s), 2.54 (3H, s), 6.98–7.02 (1H, m), 7.06 (1H, d,
J = 9.6 Hz), 7.42 (1H, t, J = 8.1 Hz), 7.48–7.51 (1H, m), 7.61–7.64
(1H, m), 7.96 (1H, s), 8.05 (1H, d, J = 9.6 Hz), 10.19 (1H, s), 11.09
(1H, s). Anal. Calcd for C22H20N6O4: C, 61.10; H, 4.66; N, 19.43.
Found: C, 60.83; H, 4.74; N, 19.31.
5.1.33. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (19)
5.1.38. N-[6-(3-Amino-2-methylphenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22c)
Yield 54%, white solid. Mp 266 °C (EtOH/THF). 1H NMR
(300 MHz, DMSO-d6) d 0.79–0.83 (4H, m), 1.87–1.97 (1H, m),
2.19 (3H, s), 3.98 (3H, s), 6.82 (1H, s), 6.99–7.03 (1H, m), 7.07
(1H, d, J = 9.6 Hz), 7.43 (1H, t, J = 8.1 Hz), 7.60–7.64 (1H, m),
7.65–7.68 (1H, m), 7.97 (1H, s), 8.06 (1H, d, J = 9.6 Hz), 10.24 (1H,
s), 11.10 (1H, s). Anal. Calcd for C22H21N7O3Á0.2EtOH: C, 61.05; H,
5.08; N, 22.25. Found: C, 61.03; H, 5.04; N, 22.24.
Compound 22c was prepared form 8f and 3-amino-2-methyl-
phenol in a manner similar to that described for 22a. Yield 58%,
brown solid. 1H NMR (300 MHz, DMSO-d6) d 0.71–0.87 (4H, m),
1.83–1.98 (1H, m), 1.89 (3H, s), 5.13 (2H, s), 6.32 (1H, d,
J = 8.0 Hz), 6.55 (1H, d, J = 6.8 Hz), 6.93 (1H, dd, J = 8.0, 6.8 Hz),
6.94 (1H, d, J = 9.5 Hz), 7.91 (1H, s), 7.98 (1H, d, J = 9.5 Hz), 11.05
(1H, s).
5.1.34. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-3-methyl-1-phenyl-1H-pyrazole-5-
carboxamide (20)
5.1.39. N-[6-(3-Amino-4-ethylphenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22d)
A mixture of 8f (984 mg, 3.0 mmol), 3-amino-4-ethylphenol
(617 mg, 4.5 mmol), and K2CO3 (829 mg, 6.0 mmol) in DMF
(9 mL) was stirred at 150 °C for 24 h. The mixture was diluted with
water and extracted with EtOAc/THF (3:1). The organic layer was
concentrated in vacuo, and the residue was purified by silica gel
column chromatography (n-hexane/EtOAc 50:50 to 0:100) fol-
lowed by trituration with EtOAc/i-Pr2O to give 22d (706 mg, 70%)
as a khaki solid. 1H NMR (300 MHz, DMSO-d6) d 0.73–0.88 (4H,
m), 1.14 (3H, t, J = 7.4 Hz), 1.85–2.00 (1H, m), 2.43 (2H, q,
J = 7.4 Hz), 5.07 (2H, s), 6.31 (1H, dd, J = 8.1, 2.7 Hz), 6.42 (1H, d,
J = 2.7 Hz), 6.93 (1H, d, J = 9.6 Hz), 6.95 (1H, d, J = 7.8 Hz), 7.97
(1H, s), 7.98 (1H, d, J = 9.0 Hz), 11.05 (1H, s).
Yield 66%, white solid. Mp 255–256 °C (EtOAc). 1H NMR
(300 MHz, DMSO-d6) d 0.76–0.84 (4H, m), 1.84–1.96 (1H, m),
2.29 (3H, s), 6.85 (1H, s), 6.96–7.01 (1H, m), 7.04 (1H, d,
J = 9.6 Hz), 7.30–7.58 (8H, m), 7.95 (1H, s), 8.03 (1H, d, J = 9.6 Hz),
10.64 (1H, s), 11.08 (1H, s). Anal. Calcd for C27H23N7O3Á0.8H2O: C,
63.85; H, 4.88; N, 19.30. Found: C, 63.91; H, 4.97; N, 19.31.
5.1.35. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1-methyl-3-(trifluoromethyl)-1H-
pyrazole-5-carboxamide (21)
Yield 78%, white solid. Mp 249–251 °C (n-hexane/EtOAc). 1H
NMR (300 MHz, DMSO-d6) d 0.73–0.85 (4H, m), 1.84–1.97 (1H,
m), 4.15 (3H, s), 7.02–7.10 (2H, m), 7.46 (1H, t, J = 8.2 Hz), 7.50
(1H, s), 7.59–7.64 (1H, m), 7.66 (1H, t, J = 2.1 Hz), 7.98 (1H, s),
8.06 (1H, d, J = 9.6 Hz), 10.53 (1H, s), 11.10 (1H, s). Anal. Calcd for
5.1.40. N-[6-(3-Amino-4-fluorophenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22e)
Compound 22e was prepared form 8f and 3-amino-4-fluoro-
phenol in a manner similar to that described for 22d. Yield 73%,

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2343
pale yellow solid. 1H NMR (300 MHz, DMSO-d6) d 0.74–0.86 (4H,
5.1.45. N-[5-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
m), 1.86–1.98 (1H, m), 5.36 (2H, s), 6.30–6.38 (1H, m), 6.58 (1H,
dd, J = 7.7, 2.9 Hz), 6.97 (1H, d, J = 9.6 Hz), 7.03 (1H, dd, J = 11.4,
8.7 Hz), 7.96 (1H, s), 8.00 (1H, dd, J = 9.6, 0.6 Hz), 11.06 (1H, s).
pyridazin-6-yl}oxy)-2-ethylphenyl]-1,3-dimethyl-1H-pyrazole-
5-carboxamide (23d)
To a stirred solution of 22d (202 mg, 0.60 mmol) in NMP (3 mL)
was added 1,3-dimethyl-1H-pyrazole-5-carbonyl chloride (143 mg,
0.90 mmol), and the mixture was stirred at room temperature for
8 h. The mixture was diluted with 1 N aqueous NaOH and extracted
with EtOAc. The organic layer was concentrated in vacuo, and the
residue was purified by silica gel column chromatography (EtOAc/
MeOH 100:0 to 80:20) followed by recrystallization from EtOAc/i-
Pr2O to give 23d (167 mg, 61%) as a white solid. Mp 199 °C. 1H
NMR (300 MHz, DMSO-d6) d 0.76–0.84 (4H, m), 1.15 (3H, t,
J = 7.6 Hz), 1.84–2.00 (1H, m), 2.19 (3H, s), 2.63 (2H, q, J = 7.6 Hz),
3.97 (3H, s), 6.80 (1H, s), 7.04 (1H, d, J = 9.6 Hz), 7.15 (1H, dd,
J = 8.6, 2.6 Hz), 7.23 (1H, d, J = 2.6 Hz), 7.37 (1H, d, J = 8.6 Hz), 7.94
(1H, s), 8.03 (1H, dd, J = 9.6, 0.6 Hz), 9.82 (1H, s), 11.07 (1H, s). Anal.
Calcd for C24H25N7O3Á0.3H2O: C, 62.00; H, 5.55; N, 21.09. Found: C,
61.95; H, 5.51; N, 21.09.
5.1.41. N-[6-(3-Amino-4-chlorophenoxy)imidazo[1,2-b]
pyridazin-2-yl]cyclopropanecarboxamide (22f)
Compound 22f was prepared from 8f and 3-amino-4-chloro-
phenol in a manner similar to that described for 22a. Yield 73%,
brown solid. 1H NMR (300 MHz, DMSO-d6) d 0.72–0.87 (4H, m),
1.84–1.98 (1H, m), 5.56 (2H, s), 6.40 (1H, dd, J = 8.5, 2.8 Hz), 6.61
(1H, d, J = 2.8 Hz), 7.00 (1H, d, J = 9.8 Hz), 7.23 (1H, d, J = 8.5 Hz),
7.98 (1H, s), 8.02 (1H, d, J = 9.8 Hz), 11.09 (1H, s).
5.1.42. N-[5-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (23a)
To an ice-cooled stirred suspension of 22a (485 mg,
1.50 mmol) in DMA (3 mL) was added 1,3-dimethyl-1H-pyra-
zole-5-carbonyl chloride (262 mg, 1.65 mmol), and the mixture
was stirred at room temperature for 15 h. The mixture was di-
luted with 0.25 N aqueous NaOH and extracted with EtOAc/THF
(2:1). The organic layer was washed with water and concentrated
in vacuo. The residue was purified by silica gel column chroma-
tography (EtOAc/MeOH 100:0 to 80:20) followed by recrystalliza-
tion from EtOH to give 23a (517 mg, 77%) as a white solid. Mp
5.1.46. N-[5-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-2-fluorophenyl]-1,3-dimethyl-1H-pyrazole-
5-carboxamide (23e)
To an ice-cooled stirred solution of 22e (1.64 g, 5.0 mmol) in
DMA (30 mL) was added a solution of 1,3-dimethyl-1H-pyrazole-
5-carbonyl chloride (0.83 g, 5.2 mmol) in DMA (5 mL), and the
mixture was stirred at room temperature for 1.5 h. The mixture
was diluted with aqueous NaHCO3 under ice cooling and extracted
with EtOAc. The organic layer was washed with brine, dried over
MgSO4, and filtered. The filtrate was concentrated in vacuo, and
the residue was purified by silica gel column chromatography
(EtOAc/MeOH 100:0 to 95:5) followed by recrystallization from
EtOAc/MeOH to give a pale yellow solid (1.82 g). The solid was
recrystallized from EtOH/THF to give 23e (1.48 g, 66%) as a white
solid. Mp 234 °C. 1H NMR (300 MHz, DMSO-d6) d 0.72–0.88 (4H,
m), 1.84–2.00 (1H, m), 2.19 (3H, s), 3.97 (3H, s), 6.84 (1H, s), 7.07
(1H, d, J = 9.6 Hz), 7.14–7.25 (1H, m), 7.39 (1H, t, J = 9.6 Hz),
7.48–7.58 (1H, m), 7.93 (1H, s), 8.03 (1H, d, J = 9.6 Hz), 10.09 (1H,
s), 11.06 (1H, s). Anal. Calcd for C22H20FN7O3: C, 58.79; H, 4.49;
N, 21.82. Found: C, 58.61; H, 4.44; N, 21.74.
223 °C. 1H NMR (300 MHz, DMSO-d6)
d 0.76–0.85 (4H, m),
1.86–1.98 (1H, m), 2.19 (3H, s), 2.25 (3H, s), 3.97 (3H, s), 6.81
(1H, s), 7.04 (1H, d, J = 9.5 Hz), 7.10 (1H, dd, J = 8.4, 2.5 Hz),
7.27 (1H, d, J = 2.5 Hz), 7.35 (1H, d, J = 8.4 Hz), 7.93 (1H, s), 8.03
(1H, d, J = 9.5 Hz), 9.80 (1H, s), 11.07 (1H, s). Anal. Calcd for
C23H23N7O3: C, 62.01; H, 5.20; N, 22.01. Found: C, 61.98; H,
5.18; N, 22.08.
5.1.43. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-4-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (23b)
To an ice-cooled stirred solution of 22b (73 mg, 0.23 mmol) in
THF (5 mL) were added Et3N (68 mg, 0.67 mmol) and 1,3-di-
methyl-1H-pyrazole-5-carbonyl chloride (46 mg, 0.29 mmol), and
the mixture was stirred at room temperature for 1 h. The mixture
was diluted with saturated aqueous NaHCO3 and extracted with
EtOAc. The organic layer was washed with brine, dried over
Na2SO4, and filtered. The filtrate was concentrated in vacuo to a
volume of approximately 1 mL, and the precipitate was collected
by filtration and washed with n-hexane/EtOAc to give 23b
(64 mg, 64%) as a white solid. Mp 137 °C. 1H NMR (300 MHz,
DMSO-d6) d 0.70–0.88 (4H, m), 1.85–1.97 (1H, m), 2.14 (3H, s),
2.18 (3H, s), 3.97 (3H, s), 6.81 (1H, s), 7.08 (1H, d, J = 9.5 Hz), 7.32
(1H, d, J = 8.3 Hz), 7.55 (1H, dd, J = 8.3, 1.9 Hz), 7.60 (1H, d,
J = 1.9 Hz), 7.92 (1H, s), 8.05 (1H, d, J = 9.5 Hz), 10.18 (1H, s),
11.08 (1H, s). Anal. Calcd for C23H23N7O3Á1.5H2O: C, 58.47; H,
5.55; N, 20.75. Found: C, 58.40; H, 5.56; N, 20.69.
5.1.47. N-[2-Chloro-5-({2-[(cyclopropylcarbonyl)amino]imidazo
[1,2-b]pyridazin-6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (23f)
Compound 23f was prepared from 22f in a manner similar to
that described for 23e to yield 66% as a white solid. Mp 255 °C
(EtOH/THF). 1H NMR (300 MHz, DMSO-d6) d 0.72–0.86 (4H, m),
1.85–1.98 (1H, m), 2.20 (3H, s), 3.98 (3H, s), 6.84 (1H, s), 7.10
(1H, d, J = 9.6 Hz), 7.26 (1H, dd, J = 8.9, 2.8 Hz), 7.54 (1H, d,
J = 2.8 Hz), 7.64 (1H, d, J = 8.9 Hz), 7.96 (1H, s), 8.06 (1H, d,
J = 9.6 Hz), 9.99 (1H, s), 11.09 (1H, s). Anal. Calcd for C22H20ClN7O3:
C, 56.72; H, 4.33; Cl, 7.61; N, 21.04. Found: C, 56.65; H, 4.30; Cl,
7.51; N, 21.03.
5.2. X-ray crystal structure determination
5.1.44. N-[3-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-
pyrazole-5-carboxamide (23c)
Purified VEGFR2 kinase domain was prepared as described pre-
viously.31 Prior to crystallization with 19, 10 mg/mL of the enzyme
was incubated with 1 mM of 19 on ice for 1 hour, followed by cen-
trifugation to remove precipitation. The given enzyme/inhibitor
complex was crystallized by sitting drop vapor diffusion method
under the condition of a 1:1 mixture of the protein solution with
the reservoir solution of 0.1 M HEPES pH 8.5, 1.2 M tri-sodium cit-
rate. Crystals were harvested by mixing with the cryoprotectant
ethylene glycerol to a final concentration of 25% in mother liquor
and flash-frozen by direct immersion in liquid nitrogen. X-ray dif-
Compound 23c was prepared from 22c in a manner similar to
that described for 23b. Yield 82%, white solid. Mp 262 °C (n-hex-
ane/EtOAc). 1H NMR (300 MHz, DMSO-d6) d 0.72–0.87 (4H, m),
1.86–1.98 (1H, m), 2.06 (3H, s), 2.20 (3H, s), 4.01 (3H, s), 6.84
(1H, s), 7.09 (1H, d, J = 9.5 Hz), 7.15 (1H, dd, J = 6.8, 2.3 Hz), 7.25–
7.36 (2H, m), 7.88 (1H, s), 8.05 (1H, d, J = 9.5 Hz), 9.95 (1H, s),
11.07 (1H, s). Anal. Calcd for C23H23N7O3Á0.3H2O: C, 61.27; H,
5.28; N, 21.75. Found: C, 61.31; H, 5.03; N, 21.51.

2344
N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
fraction data were collected at the Advanced Light Source (ALS)
beamline 5.0.3 (Berkeley, CA), and processed using the program
HKL2000.32 The crystal was diffracted to 1.52 Å, providing an
unambiguous electron density for 19. The structure was deter-
mined by molecular replacement using MOLREP,33 utilizing the
previously reported coordinate of VEGFR2 with the PDB accession
code 3VHE.31 Subsequently, structure refinement and model build-
ing were performed utilizing REFMAC.34 The solved structure was
modeled with WinCoot (version 0.3.3).35
ing an internal standard. After centrifugation, the supernatant was
diluted with a mixture of 0.01 mol/L ammonium formate solution
and acetonitrile (9:1, v/v) and centrifuged again. The compound
concentrations in the supernatant were measured by LC/MS/MS.
5.6. Metabolic stability assay
Human and mouse liver microsomes were purchased from
Xenotech, LLC (Lenexa, KS). An incubation mixture consisted of
microsomes in 50 mmol/L KH2PO4/K2HPO4 buffer (pH 7.4) and
5.3. VEGFR2 kinase inhibition assay
1 lmol/L test compound. The concentration of microsomes was
0.2 mg protein/mL. An NADPH-generating system containing
25 mmol/L MgCl2, 25 mmol/L glucose-6-phosphate, 2.5 mmol/L
beta-NADP+ and 7.5 unit/mL glucose-6-phosphate dehydrogenase
was added to the incubation mixture with a 20% volume of the
reaction mixture to initiate the enzyme reaction. After the addition
of the NADPH-generating system, the mixture was incubated at
37 °C. The reaction was terminated by the addition of acetonitrile
equivalent to the volume of the reaction mixture. Test compound
in the reaction mixture was measured by HPLC equipped with a
UV detector or LC/MS/MS. For metabolic stability determinations,
chromatograms were analyzed for parent compound disappear-
ance from the reaction mixtures.
The kinase activity of VEGFR2 was measured by use of an anti-
phosphotyrosine antibody with the AlphaScreenÒ system (Perkin-
Elmer, USA). Enzyme reactions were performed in 50 mM Tris-
HCl pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.01% Tween-20 and
2 mM DTT, containing 10 lM ATP, 0.1 lg/mL biotinylated poly-
GluTyr (4:1) and 0.1 nM of VEGFR2 (Millipore, UK). Prior to cata-
lytic initiation with ATP, compound and enzyme were incubated
for 5 min at room temperature (preincubation). The reactions were
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 pH 7.4, 250 mM NaCl, and 0.1% BSA.
Plates were incubated in the dark overnight and then read by EnVi-
sion 2102 Multilabel Reader (PerkinElmer, USA). Wells containing
the substrate and the enzyme without compound were used as to-
tal reaction control. Wells containing biotinylated poly-GluTyr
(4:1) and enzyme without ATP were used as basal control. The con-
centration of inhibitor producing 50% inhibition of the kinase activ-
ities (IC50 values) and 95% confidence intervals (95% CI) were
analyzed using GraphPad Prism version 5.01, GraphPad Software
(USA). Sigmoidal dose–response (variable slope) curves were fitted
using non-linear regression analysis, with the top and bottom of
the curve constrained at 100 and 0, respectively.
5.7. Solubility measurement
Solubility was measured by concentration quantification of the
filtrate of a suspension including drug substance and aqueous solu-
tion after shaking for 18 h at 37 °C. The quantification was per-
formed by HPLC equipped with a photo diode array detector.
5.8. Pharmacokinetic studies in rats and monkeys
The animals used in this study were male albino rats
(Crl:CD(SD) rat; weight, 278.2–313.4 g; Charles River Laboratories
Japan, Inc., Ibaraki, Japan) and male cynomolgus monkeys (weight,
2.53–3.27 kg; Keari Co., Ltd., Wakayama, Japan).
For time-dependent inhibition analyses, enzyme assays were
performed with the same method as above in four conditions;
10 lM ATP with 5 min of preincubation, 10 lM ATP with 60 min
of preincubation, 1 mM ATP with 5 min of preincubation and
1 mM ATP with 60 min of preincubation. Each assay was per-
formed in at least duplicate.
Compound 23a was suspended in 0.5% (weight/vol) methylcel-
lulose solution for oral (po) administration at the doses of 0.5 mg/
10 mL/kg to rats or 0.5 mg/2 mL/kg to monkeys. 23a was dissolved
in mixture of dimethylacetamide/saline (1:1, by vol) for intrave-
nous (iv) injection at the doses of 0.25 mg/mL/kg to rats or
0.25 mg/0.2 mL/kg to monkeys. The formulated test compound
was administered to nonfasted rats or fed monkeys. The iv admin-
istration in rats was conducted under anesthesia with diethyl
ether. At 5, 10 (only for iv dosing), 15, 30 min, and 1, 2, 3, 4, 6, 8,
12, 24, 32 (only for monkeys) and 48 h (only for monkeys) after
dosing, blood was taken from the tail vein in rats or from the fem-
oral vein in monkeys. Then, the blood was centrifuged to obtain the
plasma fraction. The plasma was kept frozen at À20 °C until
analysis.
5.4. Cell growth inhibition assay
HUVECs (Cambrex, USA) were seeded into a 96-well plate at
3000 cells/well in Human Endothelial-SFM Growth Medium (Invit-
rogen) containing 3% fetal bovine serum (FBS) (Hyclone, USA) and
were incubated overnight at 37 °C in a 5% CO2 incubator. Various
concentrations of the test compounds were added in the presence
of 60 ng/mL VEGF (R&D systems, USA), and the cells were cultured
for a further 5 days. Cellular proliferation was determined by the
WST-8 formazan assay using Cell Counting Kit-8 (DOJINDO Labora-
tories, Japan). Briefly, 10
l
L/well of Cell Counting Kit-8 was added
The concentration of 23a in plasma was determined by the
and the cells were cultured for several hours. Then, the absorbance
value at 450 nm was measured using a Benchmark Plus Microplate-
reader (Bio-Rad Labs., USA). The IC50 values and 95% confidence
intervals (95% CI) were calculated from a dose–response curve gen-
erated by least-squares linear regression of the response using NLIN
procedure of the SAS software (SAS Institute Japan, Inc., Japan).
high-performance liquid chromatography with a fluorescence
detector. The excitation and emission were 346 and 420 nm,
respectively.
Data were expressed as the mean values with standard devia-
tions (SD) for the results of three to four animals, unless otherwise
indicated. Values for maximum plasma concentration (Cmax) and
time to reach Cmax (Tmax) were noted directly from the data. The
area under the plasma concentration–time curve from 0 to 24 or
48 h after administration (AUC0–24h or AUC0–48h), plasma clearance
(CLtot), volume of distribution at steady state (Vdss) and mean res-
idence time (MRT) were calculated by the non-compartmental
model using WinNonlin Ver.4.1 (Pharsight Corporation). The AUC
was calculated by the trapezoidal rule. The bioavailability (BA)
was determined after dose normalization.
5.5. Pharmacokinetic studies in mice using cassette dosing
Test compounds were administered at a dose of 10 mg/kg as a
cassette dosing to nonfasted mice (BALB/cAJcl; female; CLEA Japan,
Inc.). After oral administration, blood samples were collected. The
blood samples were centrifuged to obtain the plasma fraction.
The plasma samples were deproteinized with acetonitrile contain-

N. Miyamoto et al. / Bioorg. Med. Chem. 21 (2013) 2333–2345
2345
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twice-weekly after inoculation and calculated as volume
=
length  width2  1/2, where length was taken to be the longest
diameter across the tumor and width the corresponding perpen-
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volume over the treatment period for the control and treated
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Acknowledgments
The authors would like to thank Ms. T. Yoshida for kinase assay;
Dr. A. Mizutani, Mr. Y. Nagase, and Dr. K. Nakamura for pharmaco-
logical in vitro and in vivo assays; Dr. K. Iwamoto and Ms. A. Hirok-
awa for protein production for X-ray crystallography; Ms. Y.
Watanabe for PK evaluation; Dr. K. Kamiyama and Dr. T. Kitazaki
for helpful discussion and supports; and all TAK-593 project team
members for their valuable contributions.
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21. Miyamoto, N.; Oguro, Y.; Takagi, T.; Iwata, H.; Miki, H.; Hori, A.; Imamura, S.
Bioorg. Med. Chem. 2012, 20, 7051.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.074.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
22. The fragment library screening was performed by an enzyme inhibition assay
using VEGFR2 kinase domain, which was used in X-ray crystallography.
Compound 3 was identified as one of the hit fragments with an IC50 value of
34 lM.
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