Structure-based design, synthesis, and evaluation of imidazo[1,2-b] pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.10.028

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a] pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.

Full text of DOI:10.1016/j.bmc.2013.10.028

Bioorganic & Medicinal Chemistry 21 (2013) 7686–7698
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design, synthesis, and evaluation
of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives
as novel dual c-Met and VEGFR2 kinase inhibitors
⇑
Shigemitsu Matsumoto , Naoki Miyamoto, Takaharu Hirayama, Hideyuki Oki, Kengo Okada,
Michiko Tawada, Hidehisa Iwata, Kazuhide Nakamura, Seiji Yamasaki, 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:
To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthe-
size compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular
endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using
co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to
the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2
kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form
intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One
compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone
ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC50 = 1.9, 2.2 nM), as well as
proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells
(IC50 = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45
(treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-
daily) mouse xenograft models.
Received 31 August 2013
Revised 17 October 2013
Accepted 19 October 2013
Available online 30 October 2013
Keywords:
c-Met
VEGFR2
Imidazo[1,2-b]pyridazine
Imidazo[1,2-a]pyridine
Pyridone
Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Vascular endothelial growth factor receptor 2 (VEGFR2) is a
tyrosine kinase receptor expressed in endothelial cells. Binding of
c-Mesenchymal epithelial transition factor (c-Met), a tyrosine
kinase receptor for hepatocyte growth factor (HGF), is widely
over-expressed in human tumors.1,2 Binding of HGF to c-Met in-
duces phosphorylation of tyrosine residues on c-Met and activates
its downstream signaling pathway. The c-Met signaling pathway
has been implicated in cell proliferation, invasion, metastasis,
and angiogenesis of various tumors.1,2 Recently, it was reported
that dysregulation of c-Met correlated with a poor prognosis in
clinical studies.3 Thus, c-Met is an attractive therapeutic target
for cancer therapy.3 Anti-HGF antibodies, anti-c-Met antibodies,3
and c-Met tyrosine kinase inhibitors (TKIs) are well-known agents
for targeting the c-Met pathway.3 Since c-Met TKIs are thought to
be effective against both ligand-dependent and -independent acti-
vation of c-Met, they are the most attractive means of targeting the
c-Met pathway. A number of c-Met TKIs have been developed and
evaluated in clinical trials for the treatment of cancer patients.3
VEGF to VEGFR induces a conformational change in VEGFR, fol-
lowed by receptor dimerization and phosphorylation of tyrosine
residues. The VEGF/VEGFR2 signaling pathway plays an important
role in tumor angiogenesis, which stimulates tumor growth by
supplying oxygen and nutrients.4,5 Overexpression of VEGF corre-
lates with poor prognoses and clinical outcomes for patients with
solid tumors.6–13 Therefore, the VEGF/VEGFR2 pathway is regarded
as an attractive target for cancer therapy. Indeed, a monoclonal
anti-VEGF antibody, bevacizumab,14 and kinase inhibitors target-
ing VEGFRs such as sunitinib,15 sorafenib,16 pazopanib,17 and axiti-
nib18 have proved efficacious in clinical trials.
On the basis of this background information, we anticipated
that dual c-Met and VEGFR2 TKIs might show synergistic inhibi-
tory effects on both tumor growth and angiogenesis across a broad
spectrum of tumor types. We therefore started synthetic studies of
dual c-Met and VEGFR2 TKIs with potent antitumor efficacy.
Several kinase inhibitors, including cabozantinib19 and foretinib,20
were previously reported to suppress c-Met and VEGFR2 kinases
simultaneously.
⇑
Corresponding author. Tel.: +81 466 32 1170; fax: +81 466 29 4458.
E-mail address: shigemitsu.matsumoto@takeda.com (S. Matsumoto).
0968-0896/$ - see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.028

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7687
Me
X = N, CH
R
O
HN
O
H
N
X
O
N
N
O
N
O
N
N
HN
Me
N
O
N
H
N
R
Het.
1
2
A
R = Me
R = H
(TAK-593)
Figure 1. Design of compound A for dual c-Met and VEGFR2 inhibitors.
We recently reported the discovery of the imidazo[1,2-b]pyrid-
azine derivatives 1 (TAK-593) and 2 as potent VEGFR2 kinase
inhibitors (Fig. 1).21,22 In the VEGFR2 kinase inhibitor program, a
variety of 5,6-fused heterocyclic scaffolds including imidazo
[1,2-b]pyridazine and imidazo[1,2-a]pyridine were identified as
novel hinge-binding templates for type-II kinase inhibitors.21,22
We expected that these scaffolds could be utilized as hinge binders
of dual c-Met and VEGFR2 TKIs. In order to confirm the structure of
c-Met protein with a type-II inhibitor, we performed an X-ray crys-
tal structure analysis of c-Met in complex with the c-Met inhibitor
N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N-(2-phenylacetyl)
thiourea, which was reported by scientists from Kirin Brewery.23
The crystal structure revealed that the c-Met protein possesses a
lipophilic back pocket region and adopts an inactive DFG-out con-
formation, which is typically observed in type-II inhibitor binding
mode.24 This information prompted us to design type-II inhibitors
of both c-Met and VEGFR2 kinases, by using the hinge binders from
the VEGFR2 inhibitor program.
nol or 4-amino-2-fluorophenol under basic conditions to obtain
aniline derivatives 5a and 5b in 67% and 71% yield, respectively.
The target compounds 3a and 3b were obtained by a condensation
reaction of the aniline 5a with acid chloride 6 or carboxylic acid 725
in 27% and 65% yield, respectively. Compounds 3c–g were synthe-
sized by utilizing O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetrameth-
yluronium hexafluorophosphate (HATU) with the anilines 5a,b
and carboxylic acids 826, 9, 10, and 11.27
The carboxylic acid 10 was prepared as shown in Scheme 2.
Diethyl isopropylidenemalonate (12) and aniline were heated at
200 °C to produce the anilide 13 in 62% yield. We then developed
the cyclization reaction of compound 13 with 1,1-dimethoxy-
N,N-dimethylmethanamine.28 The obtained pyridone derivative
14 was hydrolyzed in aqueous NaOH to provide the carboxylic acid
10 in 79% yield.
Imidazo[1,2-a]pyridine derivatives 25a and 26 were synthe-
sized according to the route shown in Scheme 3. The hydroxy
group of commercially available 2-chloro-5-hydroxypyridine (15)
was protected with a p-methoxybenzyl (PMB) group (64% yield).
The obtained compound 16 was treated under Buchwald’s cou-
pling conditions [lithium hexamethyldisilazide (LHMDS), Pd2
(dba)3, (2-biphenyl)dicyclohexylphosphine]29 followed by hydro-
lysis with hydrochloric acid to provide the desired amino pyridine
17 in 82% yield. The amino group of 17 was protected by a p-tolu-
enesulfonyl (Ts) group (89% yield), and the resulting amide 18 was
alkylated with 2-iodoacetamide to afford 19 in 80% yield. The sub-
sequent cyclization of 19 with trifluoroacetic anhydride produced
In this paper, we describe the design, structure–activity
relationships (SAR), and characterization of novel dual c-Met and
VEGFR2 inhibitors bearing imidazo[1,2-b]pyridazine and imi-
dazo[1,2-a]pyridine scaffolds. (Fig. 1 A)
2. Chemistry
The synthesis of imidazo[1,2-b]pyridazine derivatives 3a–g is
shown in Scheme 1. Compound 421 was coupled with 4-aminophe-
c (for 3a)
R1
R1
a
b
5a
O
HN
(for
(for
)
d (for 3b)
O
O
5b
N
O
)
e (for 3c-g)
N
O
N
I
N
O
N
N
HN
HN
N
N
H
R2
N
N
NH2
1 = H 67%
1 = F 71%
5a
5b
R
R
4
3a g
-
R1 = H
R2
R1 = H
R2
R1 = H
R
1 = F
R
1 = F
R1 = F
R1 = F
R2
Me
Me
N
=
=
Me R2
=
Me R2
=
R2
=
R2
=
=
N
N
Me
N
N
N
Ph
N
N
O
N
Ph
O
N
Me
O
O
N
Ph
O
Me
Ph
Ph
Ph
3a
3b
3c
3d
3e
3f
3g
27%
65%
64%
62%
31%
79%
76%
O
O
O
Me
O
O
O
O
O
Me
N
HO
HO
HO
HO
Cl
HO
Me
N
N
Ph
Me
N
N
N
Ph
N
Ph
O
N
Ph
N
Ph
Me
O
Me
6
7
8
9
10
11
Scheme 1. Reagents and conditions: (a) 4-aminophenol, KOtBu, K2CO3, DMF, 140 °C; (b) 4-amino-2-fluorophenol, Cs2CO3, DMSO, 100 °C; (c) 5a, 6, Et3N, THF, rt; (d) (1) 7,
i
(COCl)2, DMF, THF, rt (2) 5a, DMA, rt; (e) 5a or 5b, 8–11, HATU, Pr2NEt, DMF, rt.

7688
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
Me
O
O
O
Me
O
O
Me
Me
OEt
Me
Me
O
O
a
b
c
EtO
HO
EtO
EtO
62%
33%
79%
N
Ph
O
N
Ph
NH
Ph
12
13
14
10
Scheme 2. Reagents and conditions: (a) aniline, imidazole, 200 °C; (b) 1,1-dimethoxy-N,N-dimethylmethanamine, 100 °C; (c) 1 N NaOH, 100 °C.
OPMB
OH
OPMB
a
OPMB
b
c
d
N
N
N
N
HN
Ts
Cl
H2N
Cl
64%
82%
89%
80%
15
16
17
18
H2N
O
O
e
f
g
OPMB
OPMB
OPMB
N
N
N
H2N
HN
68%
83%
88%
N
N
N
Ts
21
20
19
F
O
F
O
h
i
O
O
OH
O
N
N
N
N
HN
F
HN
HN
NH2
N
N
95%
NO2
86%
24
23
22
F
O
O
j for 25a
k for 25b
l
O
O
N
O
O
N
O
HN
HN
N
N
H
N
N
H
79%
HCl
N
Ph
R
O
N
Me
26
Ph
25a R = H 71%
25b R = Me 66%
Scheme 3. Reagents and conditions: (a) PMB-Cl, K2CO3, DMF, 80 °C; (b) LHMDS, Pd2(dba)3, (2-biphenyl)dicyclohexylphosphine, THF, 80 °C then 2 N HCl, rt; (c) TsCl, pyridine,
rt; (d) 2-iodoacetamide, iPr2NEt, DMF, rt; (e) TFAA, THF, 0 °C, then 8 N NaOH, MeOH, rt; (f) cyclopropanecarbonyl chloride, DMA, 0 °C; (g) TFA, anisole, trifluoromethyl
benzene, rt; (h) 1,2-difluoro-4-nitrobenzene, Cs2CO3, DMSO, rt; (i) FeCl3, activated carbon, NH2NH2ꢀH2O, MeOH/THF, reflux; (j) 9, HATU, iPr2NEt, DMA, rt.; (k) 11, EDC, HOBt,
TEA, DMA, rt; (l) HCl, MeOH, rt.
the imidazo[1,2-a]pyridine scaffold,30 followed by removal of the
trifluoroacetyl group with 8 N NaOH to give the 2-aminoimi-
dazo[1,2-a]pyridine derivative 20 in 68% yield. The amino group
of 20 was reacted with cyclopropanecarbonyl chloride to give the
amide 21, and the PMB group was removed by trifluoroacetic acid
with anisole to provide compound 22 in 88% yield. Compound 22
was coupled with 1,2-difluoro-4-nitrobenzene under basic condi-
tions, followed by reduction using hydrazine monohydrate and
iron chloride to afford the aniline 24 in 86% yield. Condensation
of 24 with carboxylic acids 9 and 11 proceeded in the presence
of HATU or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) to give the target compounds 25a and 25b.
The hydrochloride salt of 25b was formed by 6 N HCl in methanol
to lead to 26 in 79% yield.
At first we attempted to dock compound 221 to the c-Met crystal
structure derived from the complex of c-Met and Kirin’s patent
compound.23 However, compound 2 could not be stabilized in
the c-Met protein because of the steric hindrance of the meta-
substituted central phenyl ring with gatekeeper Leu1157 (Fig. 2A,
pink). To avoid this clash, we designed a para-substituted com-
pound 3a that could adopt a suitable configuration to fit the
c-Met protein (Fig. 2A, white). Furthermore, this model suggested
that introduction of a hydrophobic group on the pyrazole of 3a
could enhance c-Met inhibitory activity by additional hydrophobic
interactions with residues Phe1134 and Phe1200 (Fig. 2A).
Regarding VEGFR2, compound 3a was not predicted to cause
steric repulsion of the gatekeeper residue (Val916), which was sup-
ported by a docking study of 3a with VEGFR2 protein (PDB 3VO3)
as shown in Figure 2B. In addition, VEGFR2 possesses a space
around the pyrazole ring that can accommodate the hydrophobic
group (Fig. 2B). On the basis of both the c-Met and VEGFR2 model
studies, we also designed compound 3b, with a phenyl group on
the pyrazole ring.
3. Results and discussion
The inhibitory activities of the compounds prepared against
c-Met and VEGFR2 kinases were determined by the AlphaScreenÒ
assay using anti-phosphotyrosine antibodies. The effects of the com-
pounds on cell proliferation were evaluated using MKN45 cells,
in which the c-Met protein is constitutively activated, and in
VEGF-stimulated human umbilical vein endothelial cells (HUVEC).
As shown in Table 1, the meta-substituted derivative 221 was inac-
tive against c-Met kinase (IC50 >10,000 nM), while showing potent
VEGFR2 inhibitory activity (IC50 = 1.4 nM). On the other hand, the
para-substituted derivative 3a was found to display both c-Met

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7689
Me
O
O
HN
H
N
N
N
O
N
O
N
N
N
O
Me
O
HN
Me
O
N
N
N
N
H
N
H
N
Me
N
N
Ph
Me
2
3a
3b
A
B
Figure 2. (A) Docking model of compounds 2 (pink) and 3a (white) with c-Met (3VW8). (B) Docking model of compound 3a (white) with VEGFR2 (3VO3).
(IC50 = 940 nM) and VEGFR2 (IC50 = 12 nM) inhibitory activities, as
suggested by the docking model studies. Furthermore, compound
3b showed enhanced c-Met inhibitory activity (IC50 = 89 nM), sug-
gesting that the additional phenyl group on the pyrazole ring inter-
acted with lipophilic residues in the back pocket region, such as
Phe1134 and Phe1200. The VEGFR2 inhibitory activity of 3b slightly
decreased, but it was still potent with an IC50 of 89 nM (Table 1).
Next, we focused on the modification of the pyrazole moiety to
further enhance c-Met/VEGFR2 kinase inhibitory activity. Docking
studies of 3b with c-Met and VEGFR2 kinases indicated that the
pyrazole and amide groups were almost in the same plane
(Fig. 3). We speculated that the introduction of an additional car-
bonyl group on the pyrazole should assist molecules to adopt the
preferred conformation by formation of an intramolecular hydro-
gen bond (Fig. 3, 3c,d). We also designed 2-pyridone derivatives
3e–g (Fig. 3), which could adopt a conformation similar to that of
the pyrazolones 3c and 3d.
VEGFR2 (IC50 = 7.1 nM) kinases, as shown in Table 1. Compound
3d, with a fluorine atom at the 3-position of the central phenyl
ring, also potently inhibited the kinases (c-Met IC50 = 2.3 nM, VEG-
FR2 IC50 = 5.6 nM). Interestingly, the fluorine compound 3d
(IC50 = 880 nM) exhibited a more potent inhibitory effect on the
growth of MKN45 cells than that of the parent 3c (IC50 = 4000 nM),
as showed in Table 1. As for the VEGF-stimulated HUVEC, com-
pound 3d (IC50 = 170 nM) inhibited cell proliferation as strongly
as 3c (IC50 = 210 nM). When we investigated other substituents
(fluoro group at 2-position, chloro or methyl group at 2- or 3-posi-
tion) on the central phenyl ring in a related derivative, a 3-fluoro
compound showed the best inhibitory effect on the proliferation
of both MKN45 and HUVEC cells.
Replacement of the pyrazolone ring in 3d with a pyridone (3e)
produced a slight increase in kinase inhibitory activity (c-Met
IC50 = 1.7 nM, VEGFR2 IC50 = 2.5 nM). Surprisingly, in the cell
growth assay, compound 3e showed 10-fold more inhibition of
The pyrazolone compound 3c was found to exhibit single digit
nanomolar IC50 values against both c-Met (IC50 = 3.2 nM) and
MKN45 (IC50 = 92 nM), and
HUVEC (IC50 = 7.0 nM), compared to that shown by 3d (Table 1).
a 24-fold increased inhibition of

7690
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
Table 1
SAR of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivativesa,b
R1
O
X
O
R2
R3
N
HN
N
Compd.
X
R1
R2
R3
c-Met
VEGFR2
MKN45
IC50 (nM)
HUVEC
Me
N
H
N
2
N
H
H
>10,000
1.4 (1.3–1.5)
NT
1.7 (0.66–3.8)
N
Me
O
O
O
O
Me
N
N
H
N
3a
3b
3c
3d
N
N
N
N
H
H
H
F
H
H
H
H
940 (590–1500)
89 (52–150)
3.2 (2.8–3.6)
2.3 (2.1–2.6)
12 (9.1–16)
89 (61–130)
7.1 (3.8–13)
5.6 (5.0–6.3)
NT
140 (140–150)
Me
N
H
Me
Me
Me
>10,000
>1000
N
N
Ph
N
H
N
N
Ph
4000 (2100–7800)
880 (410–1900)
210 (170–260)
170 (120–240)
O
O
N
H
N
N
Ph
O
O
N
H
3e
N
F
H
1.7 (1.4–2.2)
2.5 (2.2–2.8)
92 (35–240)
7.0 (4.0–10)
N
Ph
Me
4
O
O
N
H
3f
N
N
F
F
H
H
5.2 (4.1–6.5)
1.9 (1.6–2.3)
2.8 (2.5–3.0)
3.4 (3.1–3.7)
>10,000
210 (120–360)
40 (27–58)
N
Ph
O
O
N
H
3g
62 (28–140)
6
N
Ph
Me
O
O
N
H
25a
26c
CH
CH
F
F
H
H
1.7 (1.3–2.2)
1.9 (1.7–2.0)
2.1 (1.7–2.6)
2.2 (2.0–2.3)
48 (23–100)
5.0 (2.0–10)
5.4 (3.0–9.8)
1.8 (1.0–3.0)
N
Ph
O
O
N
H
6
N
Me
O
Ph
NT, not tested.
a
Numbers in parentheses represent 95% confidence intervals.
Prior to the initiation of the kinase reaction with ATP (2
HCl salt.
b
c
lM for c-Met, 10 lM for VEGFR2), compound and enzyme were incubated for 5 min.
Next, we synthesized 4- and 6-substituted pyridone derivatives
and found that the c-Met inhibitory activity of the C4-methyl
derivative 3f was weaker than that of 3e. A possible reason for this
might be a difficulty in forming an intramolecular hydrogen bond
between the pyridone-carbonyl and amide-NH groups, because of
steric hindrance between the pyridone-C4-methyl and

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7691
R1
O
O
HN
N
N
O
O
N
O
N
O
O
O
HN
N
N
N
H
N
H
N
H
Me
Me
R2
N
N
N
N
N
Ph
O
Ph
Ph
3b
3c,d
3e-g
(A)
(B)
Figure 3. (A) Docking model of 3b with c-Met (3VW8). (B) Docking model of 3b with VEGFR2 (3VO3).
amide-carbonyl groups. Conversely, the C6-methyl derivative 3g
maintained c-Met (IC50 = 1.9 nM) and VEGFR2 (IC50 = 3.4 nM)
inhibitory activities, as well as good cellular activity against
MKN45 (IC50 = 62 nM). The inhibition activity of 3g in the HUVEC
(IC50 = 40 nM) proliferation assay was however slightly lower than
that of 3e.
(IC50 = 4000 nM). In contrast, pyridone 26 significantly suppressed
the growth of MKN45 cells (IC50 = 5.0 nM), although it had almost
the same kinase inhibition activity as 3c (IC50 = 1.9 nM). To inves-
tigate these discrepancies between kinase and cell growth inhibi-
tion, we modified the assay conditions. In the case of 3c,
increasing the ATP concentration from 2 to 1000 lM resulted in
Pyridone-based c-Met inhibitors have been recently reported,
and in their crystal structures in complex with c-Met, the carbonyl
group of the pyridone interacts with the backbone NH of
Asp1222.31–33 Similar to these reports, the pyridone-carbonyl of
3e or 3g might not only contribute to the formation of an intramo-
lecular hydrogen bond, but also interact with the Asp1222 of c-Met
protein.
an over 1000-fold increase of its IC50 for c-Met kinase
(3.2 ? 4400 nM). Increasing the preincubation time from 5 to
60 min decreased this IC50 value but it still remained high
(4400 ? 320 nM). For compound 26, the IC50 value was reduced
to 0.61 nM under the modified conditions (ATP 1000
bation 60 min). Using the c-Met kinase inhibition activities at this
higher ATP concentration (1000 M) and longer preincubation
lM, preincu-
l
To obtain more potent compounds, we replaced the hinge bind-
ing imidazo[1,2-b]pyridazine moiety in 3e and 3g by an imi-
dazo[1,2-a]pyridine scaffold,22 which led to the identification of
25a and 26. These compounds strongly inhibited c-Met (25a,
IC50 = 1.7 nM; 26, IC50 = 1.9 nM) and VEGFR2 (25a, IC50 = 2.1 nM;
26, IC50 = 2.2 nM) kinases at the same level as the parent com-
pounds 3e and 3g. Of these, compound 26 exhibited the most po-
tent inhibitory activities against MKN45 and HUVEC proliferation,
with IC50 values of 5.0 and 1.8 nM, respectively.
We previously demonstrated that the VEGFR2 kinase inhibition
activity of a compound, determined in the presence of a higher ATP
concentration with a longer preincubation time, correlated more
closely to its growth inhibition activity against VEGF-stimulated
HUVEC.21,22 To investigate the enzymatic properties of the present
inhibitors in detail, we carried out the c-Met and VEGFR2 kinase
assays with varying ATP concentrations and preincubation times.
Typical inhibition curves for compound 26 are shown in Figure 4,
and the kinase inhibitory activities of each compound under vari-
ous conditions are presented in Table 2 (c-Met) and Table 3
(VEGFR2).
time (60 min), we observed a better correlation between the en-
zyme and cell growth inhibition for the compounds tested, as
shown in Table 2. Similarly, VEGFR2 inhibition under the modified
conditions (ATP 1000 lM, preincubation 60 min) also correlated
more closely to growth inhibition activities against HUVEC
(Table 3). Most of the pyridone derivatives 3e,g, 25a, and 26
showed time-dependent inhibition of c-Met and VEGFR2 kinases,
where increasing preincubation time decreased the IC50 values of
the compounds. These results indicated that 3e,g, 25a, and 26 are
slow-binding inhibitors for both c-Met and VEGFR2.34
The imidazo[1,2-a]pyridines 25a and 26 displayed a tendency
to be more potent c-Met and VEGFR2 inhibitors under the modified
assay conditions (ATP 1000 lM, preincubation 60 min) than the
imidazo[1,2-b]pyridazines 3e and 3g. This phenomenon might be
caused by a difference in basicity of their hinge-binding nitrogen
atoms. The N1 nitrogen of imidazo[1,2-a]pyridine actually has
higher basicity (pKa = 6.9) than that of imidazo[1,2-b]pyridazine
(pKa = 4.6).35,36 Therefore, we assumed that higher basicity at the
N1 position of imidazo[1,2-a]pyridines 25a and 26 might contrib-
ute to the formation of tight hydrogen-bonding with the main
chain NH of the hinge region (Met1160 in c-Met, Cys919 in
VEGFR2), resulting in strong inhibitory effects against the kinases
and cell proliferation.
As shown in Table 2, with a low ATP concentration (2 lM) and a
short preincubation (5 min), the compounds 3c–g, 25a, and 26
suppressed c-Met kinase activity with IC50 values ranging from
1.7 to 5.2 nM, while IC50 values for inhibiting MKN45 cell growth
ranged widely from 5.0 to over 10,000 nM. For example, pyrazo-
lone 3c inhibited c-Met kinase with a single-digit nanomolar IC50
value (3.2 nM), while showing weak MKN45 growth inhibition
We next evaluated the pharmacokinetic and solubility profiles
of selected compounds (Table 4). The solubility of pyridones 3e
(3.2
lg/mL) and 25a (4.7 lg/mL) was relatively low in the Japanese
Pharmacopoeia disintegration test solution (pH 6.8) containing a

7692
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
Figure 4. c-Met and VEGFR2 inhibition curves for 26 under the conditions indicated.
Table 2
c-Met inhibitory activities under a range of conditionsa
Compd.
c-Met IC50 (nM)
MKN45 IC50 (nM)
ATP 2
l
Mb
ATP 1000 l
Mb
5 minc
5 minc
60 minc
3c
3d
3e
3f
3g
3.2
2.3
1.7
5.2
1.9
1.7
1.9
4400 (1900–10,000)
170 (130–210)
26 (23–31)
>10,000
10 (9.2–11)
330 (210–520)
80 (59–110)
4.0 (3.5–4.6)
>10,000
0.77 (0.7–0.86)
0.68 (0.58–0.80)
0.61 (0.55–0.67)
4000
880
92
>10,000
62
25a
9.4 (7.9–11)
11 (8.5–15)
48
5.0
26d
a
b
c
Numbers in parentheses represent 95% confidence intervals.
Concentration of ATP.
Preincubation time.
HCl salt.
d
Table 3
Table 4
VEGFR2 inhibitory activities under a range of conditionsa
Pharmacokinetic and solubility profiles of 3e,g, 25a,b, and 26
Compd.
VEGFR2 IC50 (nM)
ATP 1000
HUVEC IC50 (nM)
Compd.
Mouse PKa
Metabolic stabilityd
Human Mouse
L/min/mg) L/min/mg)
Solubilitye
JP2 + BA
ATP 10
5 minc
l
Mb
l
Mb
AUC0–8h
C8h
g/mL)
b
c
(
l
g h/mL)
(l
(l
(l
(lg/mL)
5 minc
60 minc
3e
3g
25a
25b
26f
9.3
38
6.3
17
37
1.5
5.1
0.66
2.1
4.3
3
11
5
—
6
11
8
15
—
3.2
>140
4.7
92
>110
3c
3d
3e
3f
3g
7.1
5.6
2.5
2.8
3.4
2.1
2.2
26 (20–33)
28 (23–33)
5.2 (4.2–6.5) 0.42 (0.38–0.47) 7.0
9.7 (5.2–18)
7.5 (6.6–8.7) 1.4 (1.2–1.7)
20 (12–32)
26 (22–31)
210
170
9.0 (4.1–19)
210
40
10
a
b
c
25a
1.3 (1.2–1.5) 0.14 (0.12–0.16) 5.4
9.6 (9.0–10) 0.54 (0.51–0.58) 1.8
Compound (10 mg/kg) was orally administrated 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.
Kinetic solubility in the 2nd fluid of disintegration test of the Japanese Phar-
26d
a
b
c
Numbers in parentheses represent 95% confidence interval.
d
e
Concentration of ATP.
Preincubation time.
HCl salt.
macopoeia (pH 6.8) containing a bile acid.
d
f
HCl salt.
bile acid. Introduction of a methyl group was found to improve sol-
ubility, as exemplified by 6-methylpyridones 3g (>140 g/mL) and
25b (92 g/mL). This improvement of solubility might be because
of the methyl group (introduced at the 6-position) twisting the
adjacent phenyl ring, leading to reduced crystal packing energy.
The hydrochloride salt 26 showed better solubility than the free
base 25b. After oral administration of 26 (10 mg/kg, cassette dos-
tumor xenograft models.37 Compound 26 potently suppressed
MKN45 tumor growth with treated/control ratio (T/C) values of
57%, 30%, and 4% at doses of 0.5, 1.5, and 5 mg/kg, respectively
(once-daily, 14 days) in mouse xenografts, without significant
body weight loss.37 Furthermore, 26 showed tumor regression at
l
l
a
dose of 15 mg/kg. The level of c-Met phosphorylation in
MKN45 tumors was measured after a single oral administration
of 26, and dose-dependent inhibition of phosphorylation was
observed.37
Compound 26 also showed antitumor efficacy in the COLO205
tumor xenograft model in mice with T/C of 30% and 13% at doses
of 5 and 15 mg/kg, respectively, while COLO205 cells showed no
sign of constitutively active c-Met and were not sensitive to 26
in vitro.37 In this case, the antitumor efficacy of 26 was considered
to be primarily mediated by an antiangiogenic mechanism.
ing) to mice, an AUC0–8h value of 37
the compound was detected at a high level (4.3
even 8 h after administration. Because 26 displayed favorable sol-
ubility and stability in human hepatic microsomes (CL = 6 L/min/
l
gꢀh/mL was observed, and
l
g/mL) in plasma
l
mg), it would be expected to have good oral bioavailability in
humans.
Based on its excellent in vitro potency and pharmacokinetic
properties, 26 was selected for in vivo efficacy studies in several

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7693
and the mixture was stirred at room temperature for 30 min. Com-
pound (4 g, 12.2 mmol) and potassium carbonate (0.84 g,
4. Conclusion
4
6.07 mmol) were added to the reaction mixture, and the mixture
was stirred at 140 °C for 4 h. After cooling the reaction mixture,
water was added to the mixture, and the mixture was extracted
with EtOAc/THF. The organic layer was washed with brine, dried
over anhydrous magnesium sulfate, and filtrated. The solvent
was evaporated under reduced pressure, and the residue was puri-
fied by silica gel column chromatography to give the 5a (2.53 g,
67%) as a dark brown powder. 1H NMR (DMSO-d6) d 0.76–0.81
(4H, m), 1.88–1.95 (1H, m), 5.09 (2H, s), 6.57–6.61 (2H, m), 6.88–6.93
(3H, m), 7.88 (1H, s), 7.94 (1H, d, J = 9.9 Hz), 11.03 (1H, s).
To develop dual c-Met and VEGFR2 TKIs, a series of imidazo[1,2-b]
pyridazine and imidazo[1,2-a]pyridine derivatives were designed
and synthesized, utilizing data from docking studies of c-Met and
VEGFR2 proteins. Compounds incorporating a para-substituted
central phenyl ring (e.g., 3a) possessed both c-Met and VEGFR2
inhibitory activities. The introduction of carbonyl and phenyl
groups on the pyrazole ring in 3a enhanced c-Met inhibitory activ-
ity (3c,d). Moreover, pyridone derivatives (3e,g) strongly sup-
pressed c-Met and VEGFR2 kinase activities, even in the presence
of a higher ATP concentration and longer preincubation time, and
showed potent inhibitory activity against cell proliferation
(MKN45, VEGF-stimulated HUVEC). Eventually, replacement of
the imidazo[1,2-b]pyridazine (3g) scaffold with an imidazo[1,2-a]
pyridine led to compound 26, exhibiting the most potent inhibitory
effect on cell growth. Compound 26 significantly inhibited c-Met-
dependent (MKN45) and -independent (COLO205) tumor growth
in mouse xenograft models in vivo. Our findings indicated that
compound 26 has promising therapeutic potential for the treat-
ment of human cancers.
5.2. N-[6-(4-Amino-2-fluorophenoxy)imidazo[1,2-b]pyridazin-2-
yl] cyclopropanecarboxamide (5b)
To a solution of 4 (4.80 g, 14.6 mmol) and 4-amino-2-fluorophe-
nol (2.48 g, 19.5 mmol) in DMSO (25 mL) was added cesium car-
bonate (9.77 g, 30.0 mmol), and the mixture was stirred at 100 °C
for 5 h. H2O (100 mL) and THF (100 mL) was added to the mixture.
The precipitate was filtered through Celite pad, and the filtrate was
extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous magnesium sulfate, and filtrated. The solvent
was evaporated under reduced pressure, and the residue was puri-
fied by silica gel column chromatography (NH, EtOAc/hexane) to
give 5b (3.50 g, 71%) as white solid. 1H NMR (DMSO-d6) d 0.49–
0.96 (4H, m), 1.77–2.02 (1H, m), 5.41 (2H, s), 6.37–6.43 (1H, m),
6.49 (1H, dd, J = 13.2, 2.5 Hz), 6.99–7.09 (2H, m), 7.88 (1H, s),
7.99 (1H, d, J = 9.6 Hz), 11.04 (1H, s).
5. Experimental
Melting points were determined on a SRS OptiMelt melting point
apparatus, and were not corrected. Proton nuclear magnetic reso-
nance (1H NMR) spectra were recorded on Varian Mercury 300
(300 MHz) or Bruker AVANCE II (300 MHz) or Bruker DPX300
(300 MHz) instruments. Chemical shifts are reported as d values
(ppm) downfield from internal tetramethylsilane of the indicated
organic solution. Peak multiplicities are expressed as follows: s,
singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet;
ddd, doublet of doublet of doublets; br s, broad singlet; m, multi-
plet. Coupling constants (J values) are given in hertz (Hz). Mass
spectra (MS) were acquired using an Agilent LC/MS system
(Agilent1200SL/Agilent6130MS), Shimadzu LC/MS system (LC-
10ADvp high pressure gradient system/LCMS-2010A) or Shimadzu
UFLC/MS (Prominence UFLC high pressure gradient system/LCMS-
2020) operating in electron spray ionization mode (ESI+) and were
used to confirm the purity of each compound. The column used
5.3. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1,3-dimethyl-1H-pyrazole-5-
carboxamide (3a)
To a solution of 5a (0.22 g, 0.71 mmol) in THF (6.60 mL) was
added triethyl amine (0.30 mL, 2.13 mmol) and
6 (0.23 g,
1.42 mmol), and the mixture was stirred at room temperature for
6 h. Water was added to the mixture, and the mixture was ex-
tracted with EtOAc/THF. The organic layer was washed with brine,
dried over anhydrous magnesium sulfate, and filtrated. The solvent
was evaporated under reduced pressure, and the residue was puri-
fied by silica gel column chromatography (hexane/EtOAc/EtOH) to
give white solid. The solid was recrystallized from EtOAc/hexane to
get 3a (84 mg, 27%) as white crystals. Mp 255–259 °C; 1H NMR
(DMSO-d6) d 0.78–0.82 (4H, m), 1.87–1.96 (1H, m), 2.21 (3H, s),
4.00 (3H, s), 6.82 (1H, s), 7.03 (1H, d, J = 9.6 Hz), 7.25 (2H, d,
J = 9.0 Hz), 7.78 (2H, d, J = 9.0 Hz), 7.92 (1H, s), 8.02 (1H, d,
J = 9.6 Hz), 10.22 (1H, s), 11.07 (1H, s); LC–MS m/z 432 [M+H];
Anal. Calcd for C22H21N7O3: C, 61.24; H, 4.91; N, 22.73. Found: C,
61.03; H, 4.97; N, 22.47.
was an L-column 2 ODS (3.0 ꢁ 50 mm ID, 3
lm, CERI, Japan) with
a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min. Mobile
phase A was 0.05% TFA in ultrapure water. Mobile phase B was 0.05%
TFA in acetonitrile which was increased linearly from 5% to 90% over
2 min, 90% over the next 1.5 min, after which the column was equil-
ibrated to 5% for 0.5 min. Elemental analyses were carried out by
Takeda Analytical Laboratories. Reaction progress was determined
by thin layer chromatography (TLC) analysis on silica gel 60 F254
plate (Merck) or NH TLC plates (Fuji Silysia Chemical Ltd). Chro-
matographic purification was carried on silica gel columns 60
(0.063–0.200 or 0.040–0.063 mm, Merck), basic silica gel (Chromat-
orex NH, 100–200 mesh, Fuji Silysia Chemical Ltd) or Purif-Pack (Si
or NH, MORITEX Corporation). Commercial reagents and solvents
were used without additional purification. Abbreviations are used
as follows: DMSO-d6, dimethyl sulfoxide-d6; EtOAc, ethyl acetate;
DMF, N,N-dimethylformamide; MeOH, methanol; THF, tetrahydro-
furan; DMSO, dimethyl sulfoxide; DMA, N,N-dimethylacetamide;
iPr2NEt, diisopropylethyl amine; HATU, O-(7-azabenzotriazol-1-
yl)-N,N,N0,N0-tetramethyluronium hexafluorophosphate.
5.4. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-5-methyl-1-phenyl-1H-pyrazole-3-
carboxamide (3b)
To a solution of 7 (196 mg, 0.97 mmol) in THF (2.00 mL) was
added DMF (30.0 lL, 0.39 mmol), oxalyl chloride (85.0 lL,
0.97 mmol) at 0 °C, and the mixture was stirred at room tempera-
ture for 30 min. The mixture was added to a solution of 5a (200 mg,
0.65 mmol) in DMA (4.00 mL), and the mixture was stirred at room
temperature for 2 h. Saturated NaHCO3 aqueous solution was
added to the mixture, and the mixture was extracted with
EtOAc/THF. The organic layer was washed with brine, dried over
anhydrous magnesium sulfate, and filtrated. The solvent was evap-
orated under reduced pressure, and the residue was washed with
EtOAc to give 3b (209 mg, 65%) as white crystals. Mp 248 °C; 1H
5.1. N-[6-(4-Aminophenoxy)imidazo[1,2-b]pyridazin-2-yl]
cyclopropanecarboxamide (5a)
To a solution of 4-aminophenol (1.60 g, 14.6 mmol) in DMF
(40 mL) was added potassium tert-butoxide (1.71 g, 15.2 mmol),

7694
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
NMR (DMSO-d6) d 0.77–0.83 (4H, m), 1.86–1.96 (1H, m), 2.36
(3H, s), 6.80 (1H, s), 7.03 (1H, d, J = 9.5 Hz), 7.21–7.25 (2H, m),
7.48–7.67 (5H, m), 7.86–7.91 (2H, m), 7.92 (1H, s), 8.01 (1H, d,
J = 9.5 Hz), 10.18 (1H, s), 11.05 (1H, s); LC–MS m/z 494 [M+H];
Anal. Calcd for C27H23N7O3ꢀ0.5H2O: C, 64.53; H, 4.81; N, 19.51.
Found: C, 64.83; H, 4.80; N, 19.60.
5.9. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-3-fluorophenyl]-6-methyl-2-oxo-1-phenyl-
1,2-dihydropyridine-3-carboxamide (3g)
The compound 3g was prepared from 11 and 5b in a manner
similar to that described for 3c to yield a white crystals (76%).
Mp 287 °C; 1H NMR (DMSO-d6) d 0.73–0.84 (4H, m), 1.83–1.96
(1H, m), 2.07 (3H, s), 6.71 (1H, d, J = 7.9 Hz), 7.14 (1H, d,
J = 9.7 Hz), 7.36–7.67 (7H, m), 7.89 (1H, s), 7.98 (1H, dd, J = 13.1,
2.0 Hz), 8.05 (1H, d, J = 9.7 Hz), 8.50 (1H, d, J = 7.9 Hz), 11.07 (1H,
s), 12.09 (1H, s); LC–MS m/z 539 [M+H]; Anal. Calcd for C29H23FN6-
O4ꢀ0.5H2O: C, 63.61; H, 4.42; N, 15.35. Found: C, 63.35; H, 4.47; N,
15.15.
5.5. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)phenyl]-1-methyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazole-4-carboxamide (3c)
To a solution of 8 (212 mg, 0.97 mmol), iPr2NEt (251 mg,
1.94 mmol) and HATU (369 mg, 0.97 mmol) in DMF (4.00 mL)
was added 5a (200 mg, 0.65 mmol), and the mixture was stirred
at room temperature for 3 h. The solvent was evaporated under re-
duced pressure, the residue was diluted with THF, H2O and EtOH.
The whole was extracted with EtOAc/THF. The organic layer was
washed with brine, dried over anhydrous MgSO4, and concen-
trated. The residue was purified by silica gel column chromatogra-
phy (NH, EtOAc/hexane) to get 3c (212 mg, 64%) as white crystals.
Mp 282–287 °C; 1H NMR (DMSO-d6) d 0.75–0.84 (4H, m), 1.87–
1.96 (1H, m), 3.48 (3H, s), 7.03 (1H, d, J = 9.6 Hz), 7.20–7.27 (2H,
m), 7.46–7.64 (5H, m), 7.65–7.71 (2H, m), 7.93 (1H, s), 8.01 (1H,
d, J = 9.6 Hz), 8.62 (1H, s), 10.39 (1H, s), 11.06 (1H, s); LC–MS m/z
510 [M+H]; Anal. Calcd for C27H23N7O4: C, 63.65; H, 4.55; N,
19.24. Found: C, 63.47; H, 4.65; N, 19.10.
5.10. Ethyl 2-(anilinocarbonyl)-3-methylbut-2-enoate (13)
A
mixture of 12 (15.0 g, 74.9 mmol), aniline (1.71 mL,
18.7 mmol) and imidazole (1.27 g, 18.7 mmol) were heated at
200 °C for 3 h. The mixture was concentrated in vacuo, the residue
was purified by silica gel column chromatography (EtOAc/hexane)
to get 13 (2.86 g, 62%) as yellow solid. 1H NMR (DMSO-d6) d 1.16
(3H, t, J = 7.0 Hz), 1.89 (3H, s), 2.16 (3H, s), 4.13 (2H, q,
J = 7.0 Hz), 7.00–7.13 (1H, m), 7.27–7.45 (2H, m), 7.50–7.88 (2H,
m), 10.20 (1H, s).
5.11. Ethyl 4-methyl-2-oxo-1-phenyl-1,2-dihydropyridine-3-
carboxylate (14)
5.6. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-3-fluorophenyl]-1-methyl-3-oxo-2-phenyl-
2,3-dihydro-1H-pyrazole-4-carboxamide (3d)
A mixture of 13 (2.86 g, 11.6 mmol) and 1,1-dimethoxy-N,N-
dimethylmethanamine (6.00 mL, 45.0 mmol) was heated at
100 °C for 2 h. The reaction mixture was evaporated in vacuo, the
residue was purified by silica gel column chromatography (NH,
EtOAc/hexane) to get 14 (1.00 g, 33%) as yellow oil. 1H NMR
(DMSO-d6) d 1.25 (3H, t, J = 7.0 Hz), 2.17 (3H, s), 4.25 (2H, q,
J = 7.0 Hz), 6.28 (1H, d, J = 6.9 Hz), 7.34–7.58 (5H, m), 7.66 (1H, d,
J = 6.9 Hz).
The compound 3d was prepared from 5b and 8 in a manner
similar to that described for 3c to yield a white crystals (62%).
Mp 292–297 °C; 1H NMR (DMSO-d6) d 0.74–0.84 (4H, m), 1.86–
1.96 (1H, m), 3.49 (3H, s), 7.14 (1H, d, J = 9.6 Hz), 7.30–7.35 (1H,
m), 7.41 (1H, t, J = 8.8 Hz), 7.47–7.64 (5H, m), 7.88–7.97 (2H, m),
8.05 (1H, d, J = 9.6 Hz), 8.65 (1H, s), 10.52 (1H, s), 11.06 (1H, s);
LC–MS m/z 528 [M+H]; Anal. Calcd for C27H22FN7O4ꢀ0.1H2O: C,
61.27; H, 4.23; F, 3.59; N, 18.52; O, 12.39. Found: C, 61.20; H,
4.29; N, 18.22.
5.12. 4-Methyl-2-oxo-1-phenyl-1,2-dihydropyridine-3-
carboxylic acid (10)
A mixture of 14 (1.00 g, 3.88 mmol) in 1 N NaOH (20 mL) was
heated at 100 °C for 3 h. The mixture was washed with EtOAc.
The aqueous solution was treated with 1 N HCl (30 mL), the precip-
itate was collected and washed with water to get 10 (700 mg, 79%)
as light yellow solid. 1H NMR (DMSO-d6) d 2.54 (3H, s), 6.57 (1H, d,
J = 7.0 Hz), 7.37–7.64 (5H, m), 7.92 (1H, d, J = 7.0 Hz), 14.55 (1H, br
s).
5.7. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-3-fluorophenyl]-2-oxo-1-phenyl-1,2-
dihydropyridine-3-carboxamide (3e)
The compound 3e was prepared from 9 and 5b in a manner sim-
ilar to that describe for 3c to yield a white crystals (31%). Mp
182 °C; 1H NMR (DMSO-d6) d 0.70–0.89 (4H, m), 1.83–2.00 (1H,
m), 6.69–6.78 (1H, m), 7.15 (1H, d, J = 9.4 Hz), 7.36–7.66 (7H, m),
7.90 (1H, s), 7.96–8.08 (2H, m), 8.14 (1H, dd, J = 6.6, 2.1 Hz), 8.60
(1H, dd, J = 7.3, 2.2 Hz), 11.08 (1H, s), 12.13 (1H, s); LC–MS m/z
525 [M+H]; Anal. Calcd for C28H21FN6O4ꢀ0.3H2O: C, 63.46; H,
4.11; N, 15.86. Found: C, 63.17; H, 4.03; N, 15.88.
5.13. 2-Chloro-5-[(4-methoxybenzyl)oxy]pyridine (16)
To a solution of 15 (50.0 g, 386 mmol) and potassium carbonate
(80.0 g, 580 mmol) in DMF (300 mL) was added p-methoxybenzyl
chloride (66.5 g, 425 mmol), and the mixture was stirred at 80 °C
for 4 h. EtOAc and water were added to the reaction mixture, and
the mixture was extracted with EtOAc. The organic layer was
washed with water and brine, dried over anhydrous magnesium
sulfate and filtered. The solvent was evaporated under reduced
pressure, and the residue was washed with methanol to give 16
(61.3 g, 64%) as a white solid. 1H NMR (DMSO-d6) d 3.76 (3H, s),
5.10 (2H, s), 6.82–7.06 (2H, m), 7.34–7.46 (3H, m), 7.50–7.59 (1H,
m), 8.17 (1H, d, J = 2.6 Hz).
5.8. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-b]
pyridazin-6-yl}oxy)-3-fluorophenyl]-4-methyl-2-oxo-1-phenyl-
1,2-dihydropyridine-3-carboxamide (3f)
The compound 3f was prepared from 10 and 5b in a manner
similar to that described for 3c to yield a white crystals (79%).
Mp 280 °C; 1H NMR (DMSO-d6) d 0.65–0.88 (4H, m), 1.86–1.96
(1H, m), 2.31 (3H, s), 6.37 (1H, d, J = 7.2 Hz), 7.14 (1H, d,
J = 9.8 Hz), 7.35–7.61 (7H, m), 7.73–7.91 (1H, m), 7.84–7.94 (2H,
m), 8.05 (1H, d, J = 10.2 Hz), 10.88 (1H, s), 11.06 (1H, s); LC–MS
m/z 539 [M+H]; Anal. Calcd for C29H23FN6O4ꢀ0.5H2O: C, 63.61; H,
4.42; N, 15.35. Found: C, 63.53; H, 4.54; N, 15.50.
5.14. 5-[(4-Methoxybenzyl)oxy]pyridine-2-amine (17)
To a solution of 16 (45.0 g, 182 mmol), tris(dibenzylideneace-
tone)dipalladium (5.00 g, 5.46 mmol) and (2–biphenyl)dicyclohex-

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7695
ylphosphine (5.10 g, 14.6 mmol) in THF (500 mL) was added drop-
5.18. N-{6-[(4-Methoxybenzyl)oxy]imidazo[1,2-a]pyridin-2-yl}
wise lithium hexamethyldisilazide (1.6 mol/L THF solution,
170 mL, 272 mmol) at room temperature, and the mixture was
stirred at 80 °C for 4 h under a nitrogen atmosphere. 2 N Hydro-
chloric acid (300 mL, 600 mmol) was added to the reaction mix-
ture. EtOAc and saturated aqueous sodium hydrogen carbonate
solution were added to the reaction mixture, and the mixture
was extracted with EtOAc. The organic layer was washed with
water and brine, dried over anhydrous magnesium sulfate and fil-
tered. The solvent was evaporated under reduced pressure, and the
residue was washed with EtOAc to give 17 (34.4 g, 82%) as a yellow
solid. 1H NMR (DMSO-d6) d 3.75 (3H, s), 4.90 (2H, s), 5.45 (2H, s),
6.40 (1H, d, J = 8.9 Hz), 6.86–6.98 (2H, m), 7.14 (1H, dd, J = 8.9,
3.0 Hz), 7.28–7.38 (2H, m), 7.67 (1H, d, J = 2.6 Hz).
cyclopropanecarboxamide (21)
To a solution of 20 (8.5 g, 31.6 mmol) in DMA (80.0 mL) was
added
a solution of cyclopropanecarbonyl chloride (3.43 mL,
37.9 mmol) in DMA (20.0 mL) at 0 °C, and the mixture was stirred
at 0 °C for 3 h. The reaction mixture was diluted with water, and
the precipitate was collected by filtration and washed with water
and EtOAc to give 21 (8.90 g, 83%) as a gray solid. 1H NMR
(DMSO-d6) d 0.62–0.89 (4H, m), 1.87–1.96 (1H, m), 3.76 (3H, s),
4.96 (2H, s), 6.89–6.99 (2H, m), 7.02 (1H, dd, J = 9.6, 2.3 Hz), 7.32
(1H, d, J = 9.6 Hz), 7.37–7.45 (2H, m), 7.96 (1H, s), 8.36 (1H, d,
J = 1.7 Hz), 10.88 (1H, s).
5.19. N-(6-Hydroxyimidazo[1,2-a]pyridin-2-yl)
cyclopropanecarboxamide (22)
5.15. N-{5-[(4-Methoxybenzyl)oxy]pyridin-2-yl}-4-
methylbenzenesulfonamide (18)
To a suspension of 21 (10.1 g, 29.9 mmol) and anisole (26.0 mL,
239 mmol) in trifluoromethyl benzene (200 mL) was added triflu-
oroacetic acid (40.4 mL), and the mixture was stirred at room tem-
perature for 3 h. Saturated sodium hydrogen carbonate aqueous
solution was added to the reaction mixture, and the mixture was
extracted with EtOAc. The organic layer was washed with brine,
dried over anhydrous magnesium sulfate and filtered. The solvent
was evaporated under reduced pressure. The obtained residue was
washed with EtOAc and collected by filtration to give 22 (5.70 g,
88%) as a pale brown solid. 1H NMR (DMSO-d6) d 0.67–0.86 (4H,
m), 1.83–1.97 (1H, m), 6.89 (1H, dd, J = 9.6, 2.3 Hz), 7.25 (1H, d,
J = 9.4 Hz), 7.91 (1H, s), 7.96–8.00 (1H, m), 9.43 (1H, br s), 10.81
(1H, s).
To a solution of 17 (34 g, 148 mmol) in pyridine (300 mL) was
added 4-methylbenzenesulfonyl chloride (29.6 g, 155 mmol) at
0 °C, and the mixture was stirred at room temperature for 4 h.
The reaction mixture was diluted with water and EtOAc. The pre-
cipitate was filtrated and washed with water and EtOAc to give
18 (40 g, 70%) as a pale red solid. The filtrate was extracted with
EtOAc. The organic layer was washed with brine, dried over anhy-
drous magnesium sulfate and filtered. The solvent was evaporated
under reduced pressure, and the residue was washed with EtOAc
to give 18 (11 g, 19%) as a yellow solid. 1H NMR (DMSO-d6) d
2.34 (3H, s), 3.75 (3H, s), 4.98 (2H, s), 6.82–7.00 (2H, m), 7.07
(1H, d, J = 8.9 Hz), 7.26–7.46 (5H, m), 7.66–7.77 (2H, m), 7.91
(1H, d, J = 2.6 Hz), 10.74 (1H, br s).
5.16. 2-[5-[(4-Methoxybenzyl)oxy]-2-{[(4-methylphenyl)
sulfonyl]imino}pyridin-1(2H)-yl]acetamide (19)
5.20. N-[6-(2-Fluoro-4-nitrophenoxy)imidazo[1,2-a]pyridin-2-yl]
cyclopropanecarboxamide (23)
To a suspension of 18 (35 g, 91 mmol) in DMF (300 mL) were
added iPr2NEt (23.8 mL, 136.5 mmol) and 2-iodoacetamide
(20.2 g, 109 mmol), and the mixture was stirred at rt for 2 days.
EtOAc and water were added to the mixture, and the mixture
was extracted with EtOAc. The organic layer was washed with
water and brine. The organic layer was holded for 30 min. The pre-
cipitate was collected by filtration and washed with EtOAc to give
19 (25 g) as white solid. The filtrate was concentrated in vacuo. The
residue was washed with EtOAc to get 19 (7 g) as white solid. The
total yield of 19 was 80%. 1H NMR (DMSO-d6) d 2.33 (3H, s), 3.75
(3H, s), 4.82 (2H, s), 4.89 (2H, s), 6.81–7.03 (2H, m), 7.20–7.41
(6H, m), 7.60–7.69 (3H, m), 7.76 (1H, s), 7.92 (1H, d, J = 3.0 Hz).
To a solution of 22 (30 g, 138 mmol) and 1,2-difluoro-4-nitro-
benzene (24.2 g, 152 mmol) in DMSO (180 mL) was added cesium
carbonate (58.5 g, 180 mmol), and the mixture was stirred at room
temperature for 5 h under argon atmosphere. Water (1.08 L) was
added to the reaction mixture, and the mixture was stirred for
20 min. The precipitate was collected by filtration and washed
with water and EtOAc. After drying at room temperature for one
day, the solid was dried under reduced pressure at 100 °C for 5 h
to give 23 (46.5 g, 95%) as a yellow solid. 1H NMR (DMSO-d6) d
0.76–0.84 (4H, m), 1.88–1.99 (1H, m), 7.22 (1H, d, J = 9.5 Hz),
7.21–7.27 (1H, m), 7.54 (1H, d, J = 9.5 Hz), 8.01–8.08 (1H, m),
8.08 (1H, s), 8.36 (1H, dd, J = 11.1, 2.9 Hz), 8.74 (1H, d, J = 1.8 Hz),
11.02 (1H, s).
5.17. 6-[(4-Methoxybenzyl)oxy]imidazo[1,2-a]pyridine-2-amine
(20)
5.21. N-[6-(4-Amino-2-fluorophenoxy)imidazo[1,2-a]pyridin-2-
yl]cyclopropanecarboxamide (24)
To a suspension of 19 (20.8 g, 47.1 mmol) in THF (200 mL) was
added trifluoroacetic anhydride (49.5 g, 236 mmol) at 0 °C, and the
mixture was stirred for 1 h at 0 °C. Methanol (100 mL) was added
to the reaction mixture, and 8 N sodium hydroxide aqueous solu-
tion was added until pH reached 12–13. The mixture was stirred
at room temperature for 3 h. Water and EtOAc were added to the
reaction mixture, and the mixture was extracted with EtOAc. The
organic layer was washed with brine, dried over anhydrous mag-
nesium sulfate and filtered. The solvent was evaporated under re-
duced pressure. The obtained residue was washed with EtOAc and
collected by filtration to give 20 (8.60 g, 68%) as a brown solid. 1H
NMR (DMSO-d6) d 3.76 (3H, s), 4.90 (2H, s), 4.93 (2H, s), 6.81 (1H,
dd, J = 9.5, 2.4 Hz), 6.88–7.00 (3H, m), 7.08 (1H, d, J = 9.6 Hz), 7.32–
7.44 (2H, m), 8.12 (1H, d, J = 1.7 Hz).
To a suspension of 23 (4.50 g, 12.6 mmol) in THF (20 mL)/meth-
anol (20 mL) were added iron chloride hexahydrate (170 mg),
hydrazine monohydrate (3.67 mL, 75.6 mmol) and activated car-
bon (450 mg), and the mixture was refluxed for 16 h. After cooling
to 40 °C, the mixture was filtered through celite pad. The filtrate
was evaporated under reduced pressure, and the residue was
washed with water and EtOAc and collected by filtration to give
23 (3.53 g, 86%) as a white solid. 1H NMR (DMSO-d6) d 0.52–0.98
(4H, m), 1.72–1.98 (1H, m), 5.34 (2H, s), 6.38 (1H, ddd, J = 8.7,
2.5, 1.0 Hz), 6.49 (1H, dd, J = 13.4, 2.6 Hz), 6.88–7.07 (2H, m),
7.38 (1H, d, J = 9.6 Hz), 8.00 (1H, s), 8.21 (1H, d, J = 2.3 Hz), 10.88
(1H, s).

7696
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
5.22. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-a]
pyridin-6-yl}oxy)-3-fluorophenyl]-2-oxo-1-phenyl-1,2-
dihydropyridine-3-carboxamide (25a)
TCEP, and 5 mM DTT. Fractions containing the monomeric protein
were pooled, concentrated to 16 mg/ml and stored in ꢂ80 °C free-
zer for further crystallization experiments. N-{4-[(6,7-Dimethoxy-
4-quinolyl)oxy]phenyl}-N-(2-phenylacetyl)thiourea23 in DMSO
solution was added to a solution of approximately 16 mg/mL pro-
tein with gentle stirring to give a final concentration of 1 mM, and
the enzyme compound complex was then incubated for 3 h on ice.
Crystallization experiments were performed at 277 K by mixing
100 nL of enzyme/N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-
N-(2-phenylacetyl)thiourea23 solution with 100 nL of precipitant
solution containing 0.1 M HEPES, pH 7.5%, 25% PEG2000 mono-
methyl ether, and 8% isopropanol. Crystals were harvested by mix-
ing precipitant solution with the cryoprotectant ethyleneglycol to
a final concentration of 20% and flash-frozen by direct immersion
in liquid nitrogen. X-ray diffraction data of the crystals were col-
lected at 100 K on the beam line 5.0.3 at Advanced Light Source
(ALS) of Lawrence Berkeley National Laboratory in Berkeley, Cali-
fornia. The crystal belongs to the orthorhombic space group
P212121 with approximate cell dimensions a = 42.6 Å, b = 78.7 Å,
and c = 89.9 Å, diffracting to 2.0 Å resolution. The structure was
solved by molecular replacement with MOLREP (CCP4 program
suite) using the apo structure of c-Met kinase domain and refined
with REFMAC5 (CCP4 program suite). The final refined crystallo-
graphic statistics for the co-crystal structure with N-{4-[(6,7-dime-
The compound 25a was prepared from 9 and 24 in a manner
similar to that described for 3c to 71% yield a white crystal. Mp
192 °C; 1H NMR (DMSO-d6) d 0.75–0.82 (4H, m), 1.85–1.96 (1H,
m), 6.70–6.75 (1H, m), 7.08–7.20 (2H, m), 7.34–7.61 (7H, m),
7.94–8.00 (1H, m), 8.02 (1H, s), 8.10–8.15 (1H, m), 8.46 (1H, d,
J = 1.8 Hz), 8.56–8.60 (1H, m), 10.93 (1H, s), 12.06 (1H, s). LC–MS
m/z 524 (M+H); Anal. Calcd for C29H22FN5O4ꢀ0.7H2O: C, 64.97; H,
4.40; N, 13.06; Found: C, 64.83; H, 4.70; N, 13.11.
5.23. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-a]
pyridin-6-yl}oxy)-3-fluorophenyl]-6-methyl-2-oxo-1-phenyl-
1,2-dihydropyridine-3-carboxamide (25b)
To a solution of 24 (1.00 g, 3.06 mmol) and 11 (843 mg,
3.68 mmol) in DMA (10 mL) were added 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (822 mg, 4.29 mmol),
1-hydroxybenzotriazole (580 mg, 4.29 mmol) and triethylamine
(854 lL, 6.13 mmol). The mixture was stirred at room temperature
for 16 h. Water (60 mL) was added to the reaction mixture, and the
mixture was stirred for 20 min. The precipitate was collected by fil-
tration and washed with water and EtOAc. The obtained powder
was recrystallized from methyl ethyl ketone/water to give 25b
(1.08 g, 66%) as white crystals. Mp 247 °C; 1H NMR (DMSO-d6) d
0.70–0.85 (4H, m), 1.86–1.96 (1H, m), 2.06 (3H, s), 6.71 (1H, d,
J = 8.3 Hz), 7.07–7.17 (2H, m), 7.32–7.65 (7H, m), 7.96 (1H, dd,
J = 13.4, 2.5 Hz), 8.02 (1H, s), 8.42–8.52 (2H, m), 10.93 (1H, s),
12.02 (1H, s); LC–MS m/z 538 (M+H).
thoxy-4-quinolyl)oxy]phenyl}-N-(2-phenylacetyl)thiourea23
are
R = 22.2% (Rfree = 28.3%) with root-mean-square deviations (RMSD)
in bond lengths and angles of 0.010 and 1.22 Å, respectively. The
refined co-ordinates do not contain nine amino acid residues be-
tween Ile1043 and Thr1051 because of the weak electron densities.
The relatively high free-R factor may be due to highly anisotropic
diffraction data caused by the extremely thin plate morphology.
5.24. N-[4-({2-[(Cyclopropylcarbonyl)amino]imidazo[1,2-a]
pyridin-6-yl}oxy)-3-fluorophenyl]-6-methyl-2-oxo-1-phenyl-
1,2-dihydropyridine-3-carboxamide hydrochloride (26)
5.26. Docking study in c-Met and VEGFR2 protein
The X-ray crystal structures of c-Met complexed with
N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]phenyl}-N-(2-phenylacetyl)
thiourea23 and VEGFR2 complexed with compound 221 were uti-
lized in the docking calculations. The compounds were docked into
the protein using Gold 5.1.38 After the automatic docking, the con-
formations were energy-minimized at the MMFF94s force field
using MOE 2010.10.39
A solution (5–10%) of hydrogen chloride/MeOH (5 ml) was
heated to 50 °C, and 25b (500 mg, 0.93 mmol) was added. The mix-
ture was stirred for 5 min at 50 °C and at room temperature for
30 min. The precipitate was collected by filtration and washed
with MeOH to give 26 (420 mg, 79%) as white crystals. Mp
239 °C; 1H NMR (DMSO-d6) d 0.35–1.61 (4H, m), 1.77–2.04 (1H,
m), 2.07 (3H, s), 6.72 (1H, d, J = 8.3 Hz), 7.04–7.36 (1H, m),
7.36–7.49 (4H, m), 7.49–7.80 (4H, m), 7.93–8.26 (2H, m), 8.49
(1H, d, J = 7.6 Hz), 8.56 (1H, d, J = 1.9 Hz), 11.49 (1H, br s), 12.06
(1H, s); LC–MS m/z 538 (M+HꢂHCl); Anal. Calcd for C30H24FN5O4-
ꢀHCl: C, 62.77; H, 4.39; N, 12.20; Cl, 6.18. Found: C, 62.77; H,
4.34; N, 12.07; Cl, 6.08.
5.27. In vitro c-Met and VEGFR2 enzyme assay
VEGFR2 and c-Met kinase activity were determined by use of an
anti-phosphotyrosine antibody with quantitation performed
through the AlphaScreenÒ system (PerkinElmer, USA). The N-ter-
minal FLAG-tagged cytoplasmic domain of VEGFR2 (residues
790–1356) was prepared with the baculovirus expression system.
For VEGFR2, 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
5.25. Protein preparation, crystallization, and structure
determination
The gene for human c-Met (residue 1023–1360) was cloned into
a pFastBacHT (Invitrogen, USA) baculovirus expression vector with
an N-terminal 6-histidine tag containing a recombinant Tobacco
Etch Virus (rTEV) protease cleavage site. The protein was expressed
in Sf9 insect cells (Invitrogen) and purified by immobilized metal-
chelate affinity chromatography (IMAC). Protein bound to the
IMAC column was eluted with 30 ml buffer containing 50 mM
Tris–HCl pH 8.5, 0.2 M NaCl, 10% glycerol, 0.25 mM TCEP,
200 mM imidazole, and then diluted into 120 ml of the same buffer
but without imidazole. The above eluted protein was dephospho-
rylated by PTP1b and meanwhile treated by rTEV protease to re-
move the N-terminal His tag, which followed by Nickel reverse
purification. The c-Met protein was further purified through a
Superdex 200 size exclusion column in the buffer containing
25 mM Tris–HCl pH 8.5, 150 mM NaCl, 10% glycerol, 0.25 mM
DTT, containing 10 lM ATP, 0.1 lg/ml biotinylated poly-GluTyr
(4:1) and 0.3 nM of VEGFR2. Prior to the catalytic initiation with
ATP, compound and enzyme were incubated for 5 min at RT (pre-
incubation). After 10 min, the reactions were quenched by the
addition of 25
l
l of 100 mM EDTA, 10
lg/ml AlphaScreen strepta-
vidine donor beads and 10
l
g/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 EnVision 2102 Multilabel
Reader (PerkinElmer).
For c-Met, kinase assay was performed as described above with
0.02 nM of c-Met (Millipore, UK) and 2 lM ATP with 20 min of
reaction. Wells containing the substrate and the enzyme without
the compound were used as total reaction control. Wells contain-
ing the biotinylated poly-GluTyr (4:1) and the enzyme without
ATP were used as basal control. The concentration of inhibitor

S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
7697
producing 50% inhibition of the kinase activities of VEGFR2 and
c-Met (IC50 values) and 95% confidential interval (95% CI) were
analysed using GraphPad Prism version 5.01, GraphPad Software
(CA, USA). Sigmoidal dose–response (variable slope) curves were
fitted using nonlinear regression analysis, with the top and bottom
of the curve constrained at 100 and 0, respectively.
For time dependent IC50 determinations, each assay was per-
formed as described above with 1 mM of ATP after 5 or 60 min of
preincubation at RT and 10 min reaction for both of kinases.
Japan CLEA. For subcutaneous tumor models, cancer cells in Hanks’
balanced salt solution (HBSS; Gibco, Invitrogen Corp.) were subcu-
taneously (sc) inoculated into the hind flank of 6- to 7-week-old
mice (day 0). For the study with MKN45, cells were suspended in
a 1:1 mixture of matrigel and HBSS and then inoculated. After
the tumors were established, mice bearing tumors of appropriate
size were randomized. Once-daily oral administration of 26 or
vehicle was initiated on the next day after randomization. Tumor
volume was measured twice weekly with Vernier calipers and
calculated as (length ꢁ width2) ꢁ 0.5. Percentage treated/control
ratio (T/C%) was calculated by dividing the change in volume of
the tumors in the treated mice by the change in volume in the mice
treated by vehicle, and statistical analysis was performed as de-
scribed previously.40
5.28. Cell proliferation assay
MKN45 and HUVECs were seeded in 96-well plates in
RPMI1640 medium with 10% fetal bovine serum (FBS) and human
endothelial serum free medium (Invitrogen), respectively. The fol-
lowing day, serial dilutions of compounds or 0.1% DMSO were
added to each well. MKN45 were then incubated for further 72 h.
Proliferation of HUVECs was stimulated with 60 ng/mL recombi-
nant human VEGF (R&D Systems, Minneapolis, MN) for 120 h. After
incubation, cell proliferation was determined using Cell Counting
Kit-8 (DOJINDO Laboratories, Kumamoto, Japan). IC50 values were
calculated by nonlinear regression analysis using GraphPad Prism
(GraphPad Software, Inc.).
Acknowledgments
We thank Akio Mizutani, Yuichi Kakoi and Yoshiko Awazu for
in vitro and in vivo assays. We thank Bi-Ching Sang, Hua Zou and
Ryan Bertsch for co-crystal X-ray analysis of c-Met. We thank
Tomoyuki Kitazaki, Tomoyasu Ishikawa and Keiji Kamiyama for
helpful discussion.
Supplementary data
5.29. Pharmacokinetic studies in mice using cassette dosing
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.028.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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-
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.
References and notes
1. Birchmeier, C.; Birchmeier, W.; Gherardi, E.; Vande Woude, G. F. Nat. Rev. Mol.
Cell Biol. 2003, 4, 915. and references cited therein.
2. Christensen, J. G.; Burrows, J.; Salgia, R. Cancer Lett. 2005, 225, 1.
3. Liu, X.; Newton, R. C.; Scherle, P. S. Expert Opin. Investig. Drugs 2011, 20, 1225.
4. Folkman, J. New Engl. J. Med. 1971, 285, 1182.
5. Shibuya, M.; Claesson-Welsh, L. Exp. Cell Res. 2006, 312, 549.
6. Yoshiji, H.; Kuriyama, S.; Hicklin, D. J.; Huber, J.; Yoshii, J.; Miyamoto, Y.;
Kawata, M.; Ikenaka, Y.; Nakatani, T.; Tsujinoue, H.; Fukui, H. Hepatology 1999,
30, 1179.
7. Crew, J. P.; O’Brien, T.; Bradburn, M.; Fuggle, S.; Bicknell, R.; Cranston, D.;
Harris, A. L. Cancer Res. 1997, 57, 5281.
8. Balbay, M. D.; Pettaway, C. A.; Kuniyasu, H.; Inoue, K.; Ramirez, E.; Li, E.; Fidler,
I. J.; Dinney, C. P. N. Clin. Cancer Res. 1999, 5, 783.
9. Samoto, K.; Ikezaki, K.; Ono, M.; Shono, T.; Kohno, K.; Kuwano, M.; Fukui, M.
Cancer Res. 1995, 55, 1189.
10. Takahashi, A.; Sasaki, H.; Kim, S. J.; Tobisu, K.; Kakizoe, T.; Tsukamoto, T.;
Kumamoto, Y.; Sugimura, T.; Terada, M. Cancer Res. 1994, 54, 4233.
11. Guetz, G. D.; Uzzan, B.; Nicolas, P.; Cucherat, M.; Morere, J. F.; Benamouzig, R.;
Breau, J. L.; Perret, G. Y. Br. J. Cancer 2006, 94, 1823.
12. Yuan, A.; Yu, C. J.; Chen, W. J.; Lin, F. Y.; Kuo, S. H.; Luh, K. T.; Yang, P. C. Int. J.
Cancer 2000, 89, 475.
13. Jacobsen, J.; Grankvist, K.; Rasmuson, T.; Bergh, A.; Landberg, G.; Ljungberg, B.
Br. J. Urol. Int. 2004, 93, 297.
14. Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W. Nat. Rev. Drug Disc. 2004, 3,
391.
15. Motzer, R. J.; Michaelson, M. D.; Redman, B. G.; Hudes, G. R.; Wilding, G.;
Filglin, R. A.; Ginsberg, M. S.; Kim, S. T.; Baum, C. M.; DePrimo, S. E.; Li, J. Z.;
Bello, C. L.; Theuer, C. P.; George, D. J.; Rini, B. I. J. Clin. Oncol. 2006, 24, 16.
16. Strumberg, D. Drugs Today 2005, 41, 773.
17. Harris, P. A.; Boloor, A.; Cheung, M.; Kumar, R.; Crosby, R. M.; Davis-Ward, R.
G.; Epperly, A. H.; Hinkle, K. W.; Hunter, R. N. I. I. I.; Johnson, J. H.; Knick, V. B.;
Laudeman, C. P.; Luttrell, D. K.; Mook, R. A.; Nolte, R. T.; Rudolph, S. K.;
Szewczyk, J. R.; Truesdale, A. T.; Veal, J. M.; Wang, L.; Stafford, J. A. J. Med. Chem.
2008, 51, 4632.
5.30. 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
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.
5.31. Solubility measurement
Small volumes of the compound DMSO solutions were added to
the aqueous buffer. After incubation, precipitates were separated
from by filtration through a filter plate. The filtrates were analyzed
for compound in solution by HPLC analysis.
18. Rini, B. I.; Escudier, B.; Tomczak, P.; Kaprin, A.; Szczylik, C.; Hutson, T. E.;
Michaelson, M. D.; Gorbunova, V. A.; Gore, M. E.; Rusakov, I. G.; Negrier, S.; Ou,
Y. C.; Castellano, D.; Lim, H. Y.; Uemura, H.; Tarazi, J.; Cella, D.; Chen, C.;
Rosbrook, B.; Kim, S.; Motzer, R. J. Lancet 2011, 378, 1931.
19. Yakes, F. M.; Chen, J.; Tan, J.; Yamaguchi, K.; Shi, Y.; Yu, P.; Qian, F.; Chu, F.;
Bentzien, F.; Cancilla, B.; Orf, J.; You, A.; Laird, A. D.; Engst, S.; Lee, L.; Lesch, J.;
Chou, Y. C.; Joly, A. H. Mol. Cancer Ther. 2011, 10, 2298.
20. Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K. A.; Mock, L.; Lou, T.; Tan, J.; Li,
C.; Tam, D.; Lougheed, J.; Yakes, F. M.; Bentzien, F.; Xu, W.; Zaks, T.; Wooster,
R.; Greshock, J.; Joly, A. H. Cancer Res. 2009, 69, 8009.
5.32. Tumor xenograft models
Animal experiments were conducted according to the guide-
lines of the Takeda Experimental Animal Care and Use Committee.
Athymic nude mice (BALB/cA Jcl-nu/nu) were purchased from

7698
S. Matsumoto et al. / Bioorg. Med. Chem. 21 (2013) 7686–7698
21. Miyamoto, N.; Sakai, N.; Hirayama, T.; Miwa, K.; Oguro, Y.; Oki, H.; Okada, K.;
Takagi, T.; Iwata, H.; Awazu, Y.; Yamasaki, S.; Takeuchi, T.; Miki, H.; Hori, A.;
Imamura, S. Bioorg. Med. Chem. 2013, 21, 2333.
22. Oguro, Y.; Cary, D. R.; Miyamoto, N.; Tawada, M.; Iwata, H.; Miki, H.; Hori, A.;
Imamura, S. Bioorg. Med. Chem. 2013, 21, 4714.
23. Fujiwara, Y.; Senga, Y.; Nishitoba, T.; Osawa, T.; Miwa, A.; Nakamura, K. PCT Int.
Appl. WO2003/000660. N-{4-[(6,7-Dimethoxy-4-quinolyl)oxy]phenyl}-N-(2-
phenylacetyl)thiourea is compound of example 60 in this patent.
24. The atomic coordinates of c-Met kinase complexed with N-{4-[(6,7-
dimethoxy-4-quinolyl)oxy]phenyl}-N-(2-phenylacetyl)thiourea have been
deposited in RCSB Protein Data Bank (PDB code 3VW8).
Kim, K.; Lippy, J.; Lombardo, L. J.; Manne, V.; Oppenheimer, S.; Sack, J. S.;
Schmidt, R. J.; Shen, G.; Stefanski, K.; Tokarski, J. S.; Trainor, G. L.; Wautlet, B. S.;
Wei, D.; Williams, D. K.; Zhang, Y.; Zhang, Y.; Fargnoli, J.; Borzilleri, R. M. J. Med.
Chem. 2009, 52, 1251.
32. Kim, K. S.; Zhang, L.; Schmidt, R.; Cai, Z. W.; Wei, D.; Williams, C. K.; Lombardo,
L. J.; Trainor, G. L.; Xie, D.; Zhang, Y.; An, Y.; Sack, J. S.; Tokarski, J. S.; Darienzo,
C.; Kamath, A.; Marathe, P.; Zhang, Y.; Lippy, J.; Jeyaseelan, R., Sr.; Wautlet, B.;
Henley, H.; Gullo-Brown, J.; Manne, V.; Hunt, J. T.; Fargnoli, J.; Borzilleri, R. M. J.
Med. Chem. 2008, 51, 5330.
33. Zhao, A.; Gao, X.; Wang, Y.; Ai, J.; Wang, Y.; Chen, Y.; Geng, M.; Zhang, A. Bioorg.
Med. Chem. 2011, 19, 3906.
25. Kashima, C.; Shibata, S.; Yokoyama, H.; Nishio, T. J. Heterocycl. Chem. 2003, 40,
773.
34. Iwata, H.; Imamura, S.; Hori, A.; Hixon, M. S.; Kimura, H.; Miki, H. Biochemistry
2011, 50, 738.
26. Kim, T. S.; Bauer, D.; Bellon, S.; Boezio, A.; Booker, S.; Choquette, D.; D’amico, D.
C.; D’angelo, N.; Dominguez, C.; Fellows, I. M.; Germain, J.; Graceffa, R.;
Harmange, J. C.; Hirai, S.; LA, D.; Lee, M.; Liu, L.; Norman, M. H.; Potashman, M.;
Roveto, P.; Siegmund, A. C.; Xi, N.; Yang, K. PCT Int. Appl. WO2006/116713.
27. Bladh, H.; Klingstedt, T.; Larsson, J.; Lawitz, K.; Lepisto, M.; Lohn, H.; Nikitidis,
G. PCT Int. Appl. WO 2004/043924.
35. Humphries, A. C.; Gancia, E.; Gilligan, M. T.; Goodacre, S.; Hallett, D.; Merchant,
K. J.; Thomas, S. R. Bioorg. Med. Chem. Lett. 2006, 16, 1518.
36. Armarego, W. L. F. J. Chem. Soc. 1965, 2778.
37. Awazu, Y.; Nakamura, K.; Mizutani, A.; Kakoi, Y.; Iwata, H.; Yamasaki, S.;
Miyamoto, N.; Imamura, S.; Miki, H.; Hori, A. Mol. Cancer Ther. 2013, 12, 913.
38. GOLD (version 5.1); The Cambridge Crystallographic Data Centre (CCDC): 12
Union Road, Cambridge CB2 1EZ2, UK, 2011.
28. Bayer, H. O.; McNulty, P. J. US3711488, 1973.
29. Huang, X.; Buchwald, S. L. Org. Lett. 2001, 3, 3417.
30. Bochis, R. J.; Olen, L. E.; Fisher, M. H.; Reamer, R. A.; Wilks, G.; Taylor, J. E.;
Olson, G. J. Med. Chem. 1981, 24, 1483.
31. Schroeder, G. M.; An, Y.; Cai, Z. W.; Chen, X. T.; Clark, C.; Cornelius, L. A. M.; Dai,
J.; Gullo-Brown, J.; Gupta, A.; Henley, B.; Hunt, J. T.; Jeyaseelan, R.; Kamath, A.;
39. MOE (version 2010.10); Chemical Computing Group Inc. (CCG): 1010
Sherbrooke St. W, Suite 910 Montreal, Quebec, Canada, 2010.
40. Awazu, Y.; Mizutani, A.; Nagase, Y.; Tsuchiya, S.; Nakamura, K.; Kakoi, Y.;
Kitahara, O.; Takeuchi, T.; Yamasaki, S.; Miyamoto, N.; Iwata, H.; Miki, H.;
Imamura, S.; Hori, A. Cancer Sci. 2013, 104, 486.