Discovery of 3H-Imidazo[4,5-b]pyridines as Potent c-Met Kinase Inhibitors: Design, Synthesis, and Biological Evaluation

Article abstract of DOI:10.1002/cmdc.201200120

To identify novel c-Met inhibitors, sequences and crystal structures of the human kinome were analyzed to find interesting hinge binders that have been underexplored within the tyrosine kinase subfamily. Through this study, the imidazolopyridine ring was selected as a novel c-Met hinge-binding inhibitor scaffold. A series of derivatives was prepared, and the structure-activity relationships were studied. Among these, one compound in particular showed excellent activities in enzymatic and cellular assays, good invitro metabolic stability, and favorable pharmacokinetic parameters. When administered orally, the compound inhibited tumor growth in an NIH-3T3/TPR-Met xenograft model and did not show adverse effects on body weight. The present work not only conceptually demonstrates a new route for designing novel kinase inhibitors by using known structural information of ligand-hinge interactions but also provides a series of imidazolopyridine derivatives as potent c-Met inhibitors.

Full text of DOI:10.1002/cmdc.201200120

MED
DOI: 10.1002/cmdc.201200120
Discovery of 3H-Imidazo[4,5-b]pyridines as Potent c-Met
Kinase Inhibitors: Design, Synthesis, and Biological
Evaluation
Danqi Chen,[a] Ying Wang,[b] Yuchi Ma,[a] Bing Xiong,[a] Jing Ai,[b] Yi Chen,[b] Meiyu Geng,*[b]
and Jingkang Shen*[a]
To identify novel c-Met inhibitors, sequences and crystal struc-
tures of the human kinome were analyzed to find interesting
hinge binders that have been underexplored within the tyro-
sine kinase subfamily. Through this study, the imidazolopyri-
dine ring was selected as a novel c-Met hinge-binding inhibitor
scaffold. A series of derivatives was prepared, and the struc-
ture–activity relationships were studied. Among these, one
compound in particular showed excellent activities in enzymat-
ic and cellular assays, good in vitro metabolic stability, and fa-
vorable pharmacokinetic parameters. When administered
orally, the compound inhibited tumor growth in an NIH-3T3/
TPR-Met xenograft model and did not show adverse effects on
body weight. The present work not only conceptually demon-
strates a new route for designing novel kinase inhibitors by
using known structural information of ligand–hinge interac-
tions but also provides a series of imidazolopyridine derivatives
as potent c-Met inhibitors.
Introduction
c-Met is a unique member of the receptor tyrosine kinase
(RTK) family that is expressed in both normal and malignant
cells. It is a cell surface receptor for hepatocyte growth factor
(HGF), a pleiotropic cytokine that conveys a unique combina-
tion of pro-migratory, anti-apoptotic, and mitogenic signals.[1,2]
Aberrant HGF/c-Met signaling has been identified in a wide
range of human malignancies, including brain, colorectal, gas-
tric, lung, stomach, breast, head, and neck cancers. Moreover,
both c-Met overexpression and MET amplification have been
associated with poor clinical outcomes of cancers. Of particular
note, amplification of the MET oncogene is observed in EGFR-
mutant NSCLCs after TKI failure.[3,4] Therefore, c-Met has
become an attractive target in cancer therapy, and a number
of c-Met inhibitors have been submitted to clinical trials, such
as cabozantinib (Exelixis), crizotinib (Pfizer), and tivantinib
(ArQule).
The vast majority of known kinase inhibitors bind to the
ATP-binding site of the kinase catalytic domain, which is highly
conserved between all protein kinases. Thus, inhibitor chemo-
types for different protein kinases may overlap with each other
as they target similar sites on the protein. As for small mole-
cule c-Met inhibitors, various hinge binders have been report-
ed, including aminopyridine, pyrrolopyridine, quinoline,
pyrrolo[2,1][1,2,4]triazine, and thieno[3,2-b]pyridine.[5–9] Though
these compounds are diverse in overall structure, most contain
[a] Dr. D. Chen,+ Y. Ma, Dr. B. Xiong, Prof. Dr. J. Shen
Department of Medicinal Chemistry, State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China)
E-mail: jkshen@mail.shcnc.ac.cn
[b] Y. Wang,+ Dr. J. Ai, Prof. Y. Chen, Prof. Dr. M. Geng
Division of Antitumor Pharmacology
State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
555 Zuchongzhi Road, Zhangjiang Hi-tech Park
Shanghai, 201203 (P.R. China)
E-mail: mygeng@mail.shcnc.ac.cn
[+] These authors contributed equally to this work.
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a pyridine or polycyclic system containing a pyridine structure
in which the nitrogen atom can form a hydrogen bond with
the hinge region, a loop structure connecting the N-terminal
and C-terminal lobes of the catalytic kinase domain. Some of
these have also been reported to have inhibition activity
toward other kinases such as IGF-1R, JAK2, and checkpoint
kinase 2.[10–12] To explore other c-Met inhibitor scaffolds,
a hinge binding analysis of the crystal structures of kinase–in-
hibitor complexes in the RCSB Protein Data Bank (PDB) was
conducted. From this analysis, imidazolopyridine was selected
for investigation in present study. After proof-of-concept evalu-
ation, a series of imidazo[4,5-b]pyridines was studied further in
order to explore their ability to inhibit c-Met. Herein we de-
scribe the syntheses, structure–activity relationships (SAR), and
in vitro and in vivo biological evaluation of these compounds.
Strategy and Discovery of Novel Chemotypes
Hinge binding analysis
To obtain novel hinge binders for designing c-Met inhibitors,
sequences of human kinases were first downloaded from the
Human Kinome website (http://kinase.com/human/kinome/).
These kinase sequences were used as queries to search the
entire PDB sequence database with a locally installed blast pro-
gram. In total, 1350 kinase structures that were highly homo-
logical to human kinase (at least 80% identity in a sequence
similarity calculation) were selected from the PDB. The ligands
in these crystal structures were extracted, and the ring struc-
tures located in the vicinity of the hinge region were analyzed
with in-house software based on the OpenBabel software
package, which can detect the smallest set of smallest rings
(SSSR), find fused two-ring structures, and calculate the occur-
rence probability in a kinase subfamily. From these results, the
ring structures that did not appear in tyrosine kinase (TK) or ty-
rosine kinase-like (TKL) subfamily complex crystals were select-
ed so as to avoid conflicts with known patents for kinase inhib-
itors. Due to the importance of the hydrogen bond between
the ligand and Met1160 in the hinge region of c-Met, only
those structures containing a pyridine ring were investigated
in detail. As shown in Figure 1a, a total of eight ring fragments
were identified. The imidazolopyridine ring was chosen, based
on synthetic accessibility and the deduced interaction mode in
which two possible hydrogen bonds could form with the
Met1160 residue. Literature review regarding the imidazolopyr-
idine ring scaffold revealed that it was originally identified in
inhibitors of Aurora A kinase (PDB IDs: 2X6D and 2X6E). Com-
parison of Aurora A with c-Met showed that these kinases only
share 24% sequence identity, although the binding sites over-
lap well, particularly within the hinge region (Figure 1b). Peer-
reviewed and patent literature was searched to confirm the
novelty of imidazolopyridine in designing c-Met inhibitors. To
the best of our knowledge, imidazolopyridine has not been
used as a hinge binder among c-Met inhibitors, although this
ring structure is common in medicinal chemistry.
Figure 1. a) Bicyclic scaffolds containing a pyridine ring identified from
hinge binding analysis of kinase crystal structures in the PDB. (PDB ID: 3L8V;
number of the structure appearing in certain subfamilies of kinases given in
brackets). b) Superimposition of the binding sites of an Aurora A kinase crys-
tal structure 2X6D (yellow) with a c-Met crystal structure 3L8V (green). The
hinge binders fit well, as conserved hydrogen bonds are essentially identical
in both structures.
ioureas, benzylacetyl, cyclized pyridine, and cyclopropyl malo-
namide, were adapted to attach to the selected hinge binder
describe above. These attached R groups (Figure 2) were as-
sumed to extend toward the hydrophobic DFG pocket and
make considerable interactions with the binding pocket of c-
Met, which further locks the c-Met into a unique inactive con-
formation. Meanwhile, an oxazolopyridine compound was also
prepared for comparison to test the importance of the 7-nitro-
gen atom in imidazolopyridine, which should act as hydrogen
donor to the backbone of the Met1160.
Chemistry
Commercially available 2-amino-3-hydoxypyridine, 4-fluoroben-
zoic acid, and polyphosphoric acid were heated at 2508C for
4 h to provide oxazolopyridine 7, which was oxidized with
mCPBA and chlorinated with phosphorus oxychloride to form
9. The 4-chloro moiety of 9 was replaced by 4-nitrophenol
after 2 h microwave irradiation at 2008C. The nitro group of
For assembling fragments into c-Met inhibitors, four
common moieties in c-Met inhibitors, including arylacetylth-
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treatment under common coupling conditions, then acetic
acid under microwave irradiation, to yield 27.[18] After depro-
tection, target compound 28 was obtained.
Compound 18a (R=phenyl) was combined with phenylace-
tyl chloride to give 29. Compound 33 was synthesized using
the method described by Kim et al.[6] Desired products 34–42
were achieved by coupling 18a–i with 33 (Scheme 4).
As shown in Scheme 5, compounds 43–46 were prepared
by coupling 24 with corresponding compounds 18a, e, f, or g
using the same conditions as in Scheme 3.
Results and Discussion
Structure–activity relationships and structural studies
Figure 2. Suggested interactive mode of the imidazolopyridine or oxazolo-
pyridine ring with the hinge region Met1160 residue of c-Met.
Upon hinge binding analysis, imidazolopyridine was believed
to have potential activity against c-Met kinase. This chemotype
was tested and compared with the oxazolopyridine scaffold. It
compound 10 was reduced to
an amino group by zinc powder.
Final product 12 was obtained
by reaction of 11 with phenyla-
cetyl chloride and sodium thio-
cyanate to afford an acylthiourea
moiety.[13,14] (Scheme 1)
2-Amino-4-chloropyridine (13)
was treated with fuming nitric
acid and concentrated sulfuric
acid to give nitration product
14.[15] The chloride atom of com-
pound 14 was replaced by the
hydroxy group of 4-aminophe-
nol to yield 15,[16] the phenyl
ring amino group of which was
protected by Fmoc to give 16.
After reduction of the nitro
Scheme 1. Reagents and conditions: a) polyphosphoric acid, 2508C; b) mCPBA, CH2Cl2; c) POCl3, reflux; d) 4-nitro-
group to an amino by zinc phenol, DIEA, NMP, 2008C; e) Zn, NH4Cl, MeOH, THF, Ar, RT; f) phenylacetyl chloride, NaSCN, acetone, reflux.
powder, the resulting 17 was
heated with different aryl acids
and polyphosphoric acid at
2508C to give the corresponding
imidazolopyridines 18. Com-
pounds 19–22, containing the
acylthiourea moieties, were pre-
pared using the conditions de-
scribed above (Scheme 2).
As shown in Scheme 3, com-
pound 24 was synthesized ac-
cording
to
the
reported
method.[17] Compound 25 was
obtained by coupling of 24 to
the amino group on the phenyl
ring of 15. The nitro group on
the pyridyl ring was reduced to
an amino group to produce 26,
which was further coupled with
Scheme 2. Reagents and conditions: a) fuming HNO3, conc. H2SO4, 0–48C, 5 days; b) 4-aminophenol, tBuOK, dry
DMF, Ar, RT!708C, 20 h; c) Fmoc-Cl, NaHCO3, dioxane, H2O, 08C!RT, overnight; d) Zn, CaCl2, 95% EtOH, reflux;
N-carbobenzyloxyglycine
by e) substituted aromatic carboxylic acids, polyphosphoric acid, 2508C; f) phenylacetyl chloride, NaSCN, TEA, DMF.
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several reported representative
structures to investigate struc-
ture–activity relationships. Ben-
zylacetyl,[19] arylacetylthioureas,
cyclized pyridine,[6] and cyclo-
propyl malonamide[9] were se-
lected from the patent literature
for this purpose (Table 2). The re-
sults showed that the shorter
benzylacetyl decreased the activ-
ity drastically, while cyclized pyri-
done gave excellent activity (up
to 11.0 nm). Arylacetylthioureas
and cyclopropyl malonamide
also had potent activity against
c-Met at sub-micromolar levels.
Thus cyclized pyridine, arylace-
tylthioureas, and cyclopropyl
malonamide were selected for
further SAR studies.
Scheme 3. Reagents and conditions: a) THF, TEA, SOCl2, 08C; b) 24, DIEA, HATU, DMF, RT, overnight; c) Zn, NH4Cl,
THF/MeOH (1:2); d) N-carbobenzyloxyglycine, EDC·HCl, HOAt, DMF, NMM, RT, overnight; e) AcOH, microwave
1508C, 30 min; f) 10% Pd/C, H2, AcOH/MeOH (1:1).
The R2 moiety, selected from
different substituted phenyl
groups and heterocyclic groups,
was found that 2-aryl-oxazolo[4,5-b]pyridine (12) showed low
inhibition of c-Met, while 2-aryl-imidazo[4,5-b]pyridine (19) dis-
played enzymatic inhibition with an IC50 value of 182.5 nm. To
assess the effect of the benzene moiety attached to imidazo-
[4,5-b]pyridine, 2-alkyl-imidazo[4,5-b]pyridine (28) was evaluat-
ed and showed an approximate tenfold decrease in enzyme
activity, which suggested that an aryl group may be necessary
in this region. Based on these results, 2-aryl-imidazo[4,5-b]pyri-
dine was selected as a basic scaffold for further investigation
(Table 1).
was combined with arylacetylthioureas, pyridine, and cyclo-
propyl malonamide through parallel synthesis to yield more
SAR information (Table 3). For the arylacetyl thiourea moiety,
different aryl R2 groups had different impacts on activity. Elec-
tron-donating substituents such as a methyl group at the 4-po-
sition showed a threefold decrease in activity, while heterocy-
clic groups such as pyridyl or thiophenyl groups exhibited
a threefold improvement in inhibitory activity to ~50 nm. The
cyclopropyl malonamide moiety showed improved activities
over the arylacetyl thiourea moiety. A chloride substituent at
position 2 of the benzyl ring had no effect on the IC50 values
relative to the benzyl group. An electron-donating dimethyla-
As reported, the R group extended into the hydrophobic
DFG pocket.[6] The imidazolopyridine scaffold was attached to
Scheme 4. Reagents and conditions: a) phenylacetyl chloride, TEA, DMF, RT; b) EDCI·HCl, DMAP, DMF, 08C!RT, overnight; c) 2n NaOH, 658C, 3 h; d) TBTU,
DIEA, DMF/CH3CN (1:1), overnight.
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Imidazolopyridines as c-Met Kinase Inhibitors
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mino substituent at position 4
resulted in a large increase in ac-
tivity (IC50 values <20 nm),
which was different from the ac-
tivity observed for arylacetylth-
ioureas. The heterocyclic thio-
phenyl group also showed in-
creased activity was similar to
that observed with the arylacetyl
Scheme 5. Reagents and conditions: a) DIEA, HATU, DMF, RT.
Table 1. Preliminary evaluation of oxazolopyridine and imidazolopyridine
Table 3. c-Met inhibitory potency of imidazolopyridine derivatives.
scaffolds with c-Met kinase.
Compd
Structure
IC50 [nm]
12
ꢀ10000.0
Compd
R1
R2
IC50 [nm]
19
182.5ꢁ1.7
19
28
182.5ꢁ1.7
20
21
433.8ꢁ67.2
58.8ꢁ1.6
1247.8ꢁ13.5
22
34
53.2ꢁ12.8
11.0ꢁ0.0018
Table 2. c-Met inhibitory potency of imidazolopyridine derivatives.
35
36
37
38
39
40
20.9ꢁ1.3
43.4ꢁ7.8
315.6ꢁ105.4
15.0ꢁ4.9
12.4ꢁ2.5
14.2ꢁ4.4
Compd
R
IC50 [nm]
19
182.5ꢁ1.7
29
34
ꢀ10000.0
11.0ꢁ0.0018
100.1ꢁ25.6
43
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Table 3. (Continued)
Table 4. Effect of pyridine series compounds on cell proliferation.
Cell line
IC50 [mm]
Compd 34
Compd 41
BaF3/TPR-Met
NIH-3T3/TPR-Met
NIH-3T3
1.8ꢁ0.57
3.9ꢁ1.3
ꢀ50
0.59ꢁ0.14
1.6ꢁ0.3
17.0ꢁ4.6
Compd
R1
R2
IC50 [nm]
strongly inhibited the proliferation of BaF3/TPR-Met cells with
an IC50 value of 0.59ꢁ0.14 mm, twofold greater potency than
that of 34. Similar results were obtained with the NIH-3T3/TPR-
Met cells upon treatment with these compounds. In addition,
both compounds exhibited much lower potency in NIH-3T3
cells in which TPR-Met was not expressed, suggesting that c-
Met is the main cellular target of growth inhibition for these
compounds. Given the clear advantage in cellular potency ob-
served in this assay, compound 41 was selected as the lead
compound for subsequent in vitro and in vivo evaluation.
41
10.7ꢁ0.43
42
107.5ꢁ6.4
43
44
45
46
100.1ꢁ25.6
103.6ꢁ1.8
16.3ꢁ3.1
38.4ꢁ0.8
Kinase selectivity of compound 41
The selectivity of compound 41 toward tyrosine kinases was
assessed by screening against 11 tyrosine kinases including c-
Src, PDGFRa, EPH-B2, EPH-A2, ALK, Flt-1, c-Kit, RET, EGFR,
ErbB2, IGF1R, and FGFR1. As shown in Table 5, compound 41
potently inhibited the kinase activity of c-Met with an IC50
value of 10.7ꢁ0.43 nm. Although 41 showed inhibitory effects
against c-Src, PDGFRa, and EPH-B2 as well, the potency was
two- to sixfold lower than against c-Met. Moreover, compound
41 only slightly inhibited kinase activity against the other eight
tyrosine kinases tested (IC50 >2 mm). These results suggest that
compound 41 is a relatively selective inhibitor of c-Met.
thiourea moiety. The 2-pyridone moiety generally exhibited
the highest inhibition activity among the three structures,
most of which had IC50 values below 50 nm. No drastic differ-
ence was observed among electron-donating substituents
such as methyl or 4-dimethylamino groups, or electron-with-
drawing substituents such as a fluoro group or heterocyclic
groups such as pyridyl or thiophenyl groups, which was quite
different from the activity observed for the arylacetyl thiourea
and cyclopropyl malonamide moieties. However, upon com-
parison of compounds 19 and 20 and comparison of 34 and
35, a small decrease in activity (2.5- and twofold, respectively)
was observed upon attachment of a methyl group to the
phenyl ring. When the electron density of R2 was further de-
creased, as is apparent for 3,4-difluorophenyl and pyridazin-4-
yl, lower IC50 values were observed. Of all compounds tested,
34 and 41 exhibited the best IC50 values (11.0ꢁ0.0018 and
10.7ꢁ0.43 nm, respectively). These two compounds were sub-
mitted for further evaluation.
Table 5. Inhibition of tyrosine kinases by compound 41.
Kinases
IC50 [nm]
c-Met
c-Src
PDGFRa
EPH-A2
Flt-1
c-Kit
RET
EGFR
ErbB2
IGF1R
FGFR1
10.7ꢁ0.43
21.0ꢁ11.0
21.0ꢁ13.0
2774.0ꢁ387.8
ꢀ10000
ꢀ10000
ꢀ10000
ꢀ10000
ꢀ10000
ꢀ10000
ꢀ10000
Compound 41 inhibits c-Met phosphorylation and its down-
stream signaling pathways
We next investigated the in vitro and in vivo c-Met-targeting
activity of compound 41. Both natural (MKN-45 and SUN-5
human gastric carcinoma cells which express elevated levels of
constitutively active c-Met) and genetic (NIH-3T3/TPR-Met and
BaF3/TPRMet cells) c-Met-expressing cell lines were selected.
We found that compound 41 inhibited c-Met autophosphory-
Effects on cell proliferation
As activated c-Met signaling drives cell proliferation, we next
evaluated the effect of compounds 34 and 41 on cell prolifera-
tion in c-Met-constitutively activated NIH-3T3/TPR-Met and
BaF3/TPR-Met cells.[20,21] As shown in Table 4, compound 41
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nificantly inhibited proliferation of BaF3-TPR-Met and
NIH-3T3-TPR-Met cells, clearly demonstrated that
compound 41 inhibited Met-mediated cell prolifera-
tion.
Compound 41 inhibits HGF-stimulated c-Met--
mediated cell motility
Activation of the c-Met pathway is known to increase
cell motility, which contributes to the metastatic
characteristics of malignant cells.[2,23] To examine the
effect of compound 41 on cell motility, MDCK cells
were treated with HGF in the presence of compound
41, and cell migration was determined using an in vi-
tro wound healing assay. As shown in Figure 4a,
compound 41 strongly suppressed MDCK cell motili-
ty in a dose-dependent manner. Notably, compound
41 was sufficient to block the movement of most
cells at a dose of 1 mm. These results indicated that
compound 41 strongly inhibited c-Met activation-
mediated cell motility.
Figure 3. Compound 41 inhibits phosphorylation of c-Met and its downstream signaling
pathways: a) MKN-45, b) SNU-5, c) BaF3/TPR-Met, and d) NIH-3T3/TPR-Met cells were in-
cubated with the indicated compounds for 2 h at 378C. Protein extracts were analyzed
by immunoblotting using the specific antibodies as described.
Compound 41 inhibits tumor growth in vivo
To evaluate the ability of compound 41 to inhibit c-
Met activity in vivo, antitumor activity was investigat-
ed in the NIH-3T3/TPR-Met xenograft model. In this
lation at Tyr1234/1235 (autophosphorylation site) and Tyr1349
(growth factor receptor binding protein 2 binding site) in both
MKN-45 and SNU-5 cells (Figure 3a and b). In addition, Erk1/2
and AKT, the key downstream molecules of c-Met which play
important roles in c-Met functioning,[2,22] were also significantly
inhibited upon treatment with compound 41 (Figure 3a and
b). Similar results were obtained using NIH-3T3/TPR-Met cells
and BaF3/TPR-Met cells (Figure 3c and d). These data further
support that compound 41 inhibits c-Met activity and thus,
subsequent c-Met downstream signaling, at a cellular level.
model, tumor growth is specifically driven by constitutive c-
Met activation. We found that oral administration of com-
pound 41 at the indicated doses for 11 days inhibited tumor
growth (tumor growth inhibition for 100 or 50 mgkgꢂ1 doses
Table 6. Effect of compound 41 on cell proliferation.
Cell line
IC50 [mm]
MKN-45
SNU-5
4.3ꢁ0.25
5.3ꢁ0.39
Compound 41 inhibits the prolif-
eration of c-Met-addicted cell
lines
Inhibitory activity of compound
41 on signaling was next con-
firmed by determining its effect
on cell growth in MKN-45 and
SNU-5 human cancer cell lines,
while c-Met was constitutively
activated due to MET genomic
amplification. As shown in
Table 6, compound 41 strongly
inhibited the proliferation of
MKN-45 and SNU-5 cells with
IC50 values of 4.3ꢁ0.25 and
5.3ꢁ0.39 mm, respectively. These
Figure 4. Compound 41 suppressed HGF-induced cell migration of MDCK cells. MDCK cells were treated with the
indicated compounds and HGF (30 ngmLꢂ1). Time-lapse microscopy of cell migration was performed at 0 and
findings, together with the ob-
servation that compound 41 sig- 10 h after wounding.
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Table 8. Pharmacokinetic profiles of compound 41.
Parameter
Route
p.o.
i.v.
Dose [mgkgꢂ1
]
10
2166
1
2.07
2.71
6277
10
C
max [ngmLꢂ1
]
7397
0.083
5.71
4.46
23197
tmax [h]
1/2 [h]
MRT [h]
t
AUC0–24 h [nghꢂ1 mLꢂ1
]
F [%]
28.3
availability of 28.3% from a solution formulation of PEG 400/
water/methanesulfonic acid (10:9:0.1), which suggested that
compound 41 could be administered orally.
Figure 5. Antitumor efficacy of compound 41 in the NIH-3T3/TPR-Met xeno-
graft model. Tumor-bearing nude mice were randomly divided into groups
after tumor volume (Vtum) reached 100–200 mm3 and were given compound
41 p.o. at the indicated dosage levels or vehicle alone over the designated
treatment schedule. Data are presented as means ꢁSEM; n=6 mice per
group; **p<0.01, ***p<0.001, versus control group, determined by Stu-
dent’s t-test.
Conclusions
Most kinase inhibitors bind to the ATP-binding site, which
makes it possible that a “core structure” in one kinase may
also be effective on other kinases containing a similar binding
site. The imidazolopyridine ring was selected on the basis of
analysis of kinase crystal structures in the PDB, and analogues
were prepared for SAR studies by attaching four reported moi-
eties to the scaffold. A number of these novel molecules
showed good inhibition activities with IC50 values below
100 nm, which proved that 2-aryl-imidazolopyridine was
a potent c-Met inhibitor. Among the prepared compounds,
compound 41 showed the most potent enzyme activity with
an IC50 value of 10 nm and was selected for further evaluation.
It strongly inhibited the proliferation of BaF3/TPR-Met, NIH-
3T3/TPR-Met, MKN-45 and SNU-5 cells with IC50 values of 0.59,
1.6, 4.3, and 5.3 mm respectively. It also inhibited c-Met auto-
phosphorylation and subsequent c-Met downstream signaling
at a cellular level and strongly suppressed MDCK cell motility
in a dose-dependent manner. To evaluate its drugability, com-
pound 41 was submitted to in vitro metabolic stability testing.
The results showed good stability in rat and human liver mi-
crosomes with no clear inhibition of CYPs. Favorable pharma-
cokinetic parameters were also obtained for 41. In the NIH-
3T3/TPR-Met xenograft model, 41 inhibited tumor growth
(30.9% inhibition after oral administration of 100 mgkgꢂ1) with
no significant loss of body weight. Based on all of the results,
41 was proven to be a potent c-Met inhibitor and is now
under further investigation.
was 30.9 and 28.0%, respectively; Figure 5). In addition, com-
pound 41 was well tolerated, as evidenced by no significant
loss of body weight in mice after treatment (data not shown).
Compound 41 shows good in vitro metabolic stability
To evaluate the in vitro metabolic stability of compound 41, it
was tested on rat and human liver microsome (RLM and HLM,
respectively) tissues. If the numbers of RLM and HLM are
below 100, a compound is believed to be stable. Compound
41 showed good stability, with numbers of 46 and 10, respec-
tively. It also showed no clear inhibitory activity in direct inhibi-
tion or time-dependent inhibition tests on CYPs. These results
indicate that compound 41 has good in vitro metabolic stabili-
ty (Table 7).
Pharmacokinetic (PK) profiles of compound 41
Pharmacokinetic parameters of compound 41 in mice after i.v.
and p.o. dosing (both 10 mgkgꢂ1) are summarized in Table 8.
Compound 41 had a favorable half-life of 2.2 h and oral bio-
Table 7. Metabolic stability and inhibition of rat or human liver micro-
somes by compound 41.
Experimental Section
Parameter[a]
Value
Chemistry
RLM stability mean: CLint [mLminꢂ1 (mg protein)ꢂ1
]
]
46
10
HLM stability mean: CLint [mLminꢂ1 (mg protein)ꢂ1
General: Reagents (chemicals) were purchased from Alfa-Aesar
(Karlsruhe, Germany), Acros (Geel, Belgium), Aldrich (St. Louis, MO,
USA) and Shanghai Chemical Reagent Company (Shanghai, China)
and were used without further purification. Analytical thin-layer
chromatography was performed on HSGF 254 (150–200 mm thick-
ness; Yantai Huiyou Company, Yantai, Shandong, China). 1H NMR
(300 MHz or 400 MHz) spectra were recorded on a Varian Mercury-
300 or 400 High Performance Digital FT-NMR with TMS as an inter-
Direct inhibition mean [%]
3A4
2D6
1A2
3
22
29
NI
TDI [10–4minꢂ1
]
[a] RLM: rat liver microsome; HLM: human liver microsome; TDI: time-de-
pendent inhibition of CYPs (NI: no inhibition).
1064
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1057 – 1070

Imidazolopyridines as c-Met Kinase Inhibitors
MED
nal standard. HPLC analysis was performed using a Gilson HPLC
system with UV detection at 214 and 254 nm. LC–MS spectra were
obtained on an LCQ Deca XP ion trap mass spectrometer (Thermo-
Finnigan, San Jose, CA, USA). Accurate mass measurements were
carried out on a Q-TOF ultima Globe mass spectrometer (Micro-
mass, Manchester, UK).
(9 mg, 0.11 mmol) was dissolved in acetone (2 mL), followed by the
dropwise addition of phenylacetyl chloride (15 mL, 0.11 mmol)
under Ar. The mixture was stirred at room temperature for 5 min,
then a suspension of 11 (32 mg, 0.1 mmol) in acetone (2 mL) was
added, and the mixture was stirred at reflux for 2 h. The mixture
was cooled, diluted with water, and extracted with EtOAc. The or-
ganic layer was washed with brine, dried with Na2SO4, and concen-
trated. The crude product was purified by silica gel chromatogra-
phy (CH2Cl2/MeOH, 15:1) to yield 12 as a yellow solid (20 mg,
2-(4-Fluorophenyl)oxazolo[4,5-b]pyridine (7): 2-Amino-3-hydroxy-
pyridine (1.1 g, 10 mmol) and 4-fluorobenzoic acid (1.4 g, 10 mmol)
were heated in polyphosphoric acid (80 g, 0.24 mol) at 2508C for
4 h. The reaction mixture was cooled to room temperature and
poured into a mixture of ice water and NaOH. The resulting solid
was filtered, washed with 1n NaOH and water, and dried to yield
1
40%): H NMR (300 MHz, [D6]DMSO): d=8.42 (d, 1H, J=5.5 Hz), 8.2
(m, 2H), 7.74 (d, 2H, J=8.4 Hz), 7.4 (m, 8H), 6.94 (d, 1H, J=5.5 Hz),
5.74 (s, 1H), 3.82 ppm (s, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
164.0, 158.6, 149.4, 148.6, 130.9, 129.9, 128.5, 127.0, 126.9, 125.0,
121.3, 121.0, 117.3, 117.1, 42.7 ppm; LC–MS (ESI): 499 [M+H]+;
HRMS: m/z [M+Na]+ calcd for C27H19N4O3FNaS: 521.1061, found:
521.1083.
1
product 7 (1.55 g, 72%): H NMR (300 MHz, [D6]DMSO): d=8.57 (d,
1H, J=3.4 Hz), 8.3 (m, 3H), 7.52 ppm (m, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=165.9, 163.9, 163.4, 155.4, 146.5, 142.8, 130.4, 122.5,
120.7, 119.0, 116.8, 116.5 ppm; LC–MS (ESI): 215 [M+H]+.
4-Chloro-3-nitropyridin-2-amine (14): 2-Amino-4-chloropyridine
(15 g, 0.12 mol) was carefully dissolved in concentrated H2SO4
(100 mL). The reaction mixture was cooled to 08C, then fuming
HNO3 (95%, 5 mL) was added dropwise while stirring. The mixture
was stirred for 5 days at 48C, or until a drop mixed with water
gave no precipitate (the nitramine is insoluble in dilute acid). The
reaction mixture was then poured onto crushed ice and brought
to pH 5–6 by adding ammonia water (35%). The resulting yellow
precipitate was filtered, washed with ice water, and dried. The
crude product was repeatedly extracted with EtOAc (~750 mL)
until a light yellow product remained. The extracts were combined
and evaporated to dryness, and the residue was crystallized from
EtOH to yield 14 as bright yellow needles (8.5 g, 42%): 1H NMR
(300 MHz, CDCl3): d=8.1 (1H, d, J=5.5 Hz), 6.8 ppm (1H, d, J=
5.3 Hz); 13C NMR (100 MHz, [D6]DMSO): d=153.0, 151.7, 135.9,
129.2, 112.6 ppm; LC–MS (ESI): 174 [M+H]+.
7-Chloro-2-(4-fluorophenyl)oxazolo[4,5-b]pyridine
(9):
Com-
pound 7 (1.2 g, 5.6 mmol) was dissolved in CH2Cl2 (15 mL), cooled
to 08C, and mCPBA (3 g, 17.4 mmol) in CH2Cl2 (25 mL) was added.
The mixture was stirred for 4 days or until there was no starting
material. After filtration, the filtrate was washed with saturated
Na2CO3 and brine, dried with Na2SO4, and concentrated to yield 8
as a yellow solid (700 mg, 55%). Compound 8 (700 mg, 3 mmol)
was stirred at reflux with POCl3 (10 mL, 0.1 mmol) overnight. The
mixture was poured into ice water, neutralized with NaHCO3, and
extracted with EtOAc. The organic layer was washed with brine,
dried with Na2SO4, and concentrated. The crude product was puri-
fied by silica gel chromatography (petroleum/EtOAc, 8:1) to yield 9
1
as a white solid (200 mg, 46%): H NMR (300 MHz, [D6]DMSO): d=
8.5 (d, 1H, J=5.6 Hz), 8.31 (m, 2H), 7.65 (d, 1H, J=5.6 Hz), 7.5 ppm
(t, 2H, J=9.1 Hz); 13C NMR (100 MHz, [D6]DMSO): d=166.9, 165.0,
164.3, 157.0, 147.0, 130.8, 130.7, 125.6, 122.0, 120.9, 116.6,
116.4 ppm; LC–MS (ESI): 249 [M+H]+.
4-(4-Aminophenoxy)-3-nitropyridin-2-amine (15): 4-Hydroxyani-
line (5.6 g, 0.05 mol) was dissolved in dry DMF (80 mL), and the so-
lution was degassed with bubbling argon for 10 min. Potassium
tert-butoxide (6 g, 0.053 mol) was added, and stirring and argon
bubbling continued for 1 h. Compound 14 (8 g, 0.046 mol) was dis-
solved in 40 mL dry DMF and added to the mixture. The reaction
mixture was heated and stirred at 708C for 20 h under argon. The
solvent was evaporated, and the residue was extracted with EtOAc
and aqueous Na2CO3 containing 5% KOH. The organic layer was
dried with Na2SO4 and concentrated. The crude product was puri-
fied by silica gel chromatography (petroleum/EtOAc, 1:1) to yield
2-(4-Fluorophenyl)-7-(4-nitrophenoxy)oxazolo[4,5-b]pyridine
(10): Compound 9 (248 mg, 1 mmol) and 4-nitrophenol (350 mg,
2.5 mmol) were dissolved in NMP (3 mL). After adding DIEA (1 mL),
the mixture was heated under microwave at 2008C for 2 h. The re-
action mixture was diluted with EtOAc, washed with saturated
KH2PO4, and 1m Na2CO3. The organic layer was dried with Na2SO4
and concentrated. The crude product was purified by silica gel
chromatography (petroleum/EtOAc, 1:1) to yield 10 as a yellow
1
solid (130 mg, 37%): H NMR (300 MHz, [D6]DMSO): d=8.5 (d, 1H,
J=5.7 Hz), 8.35 (d, 2H, J=8.7 Hz), 8.19 (m, 2H), 7.56 (d, 2H, J=
9.3 Hz), 7.46 (t, 2H, J=8.4 Hz), 7.19 ppm (d, 1H, J=5.9 Hz);
13C NMR (100 MHz, [D6]DMSO): d=174.3, 165.3 (J=251 Hz), 164.5,
159.7, 159.1, 148.8, 146.4, 144.8, 140.0, 133.6, 131.1, 126.7, 122.7,
120.4, 117.4, 117.2, 116.3, 111.2 ppm; LC–MS (ESI): 352 [M+H]+.
1
15 as an orange solid (8.8 g, 80%): H NMR (300 MHz, [D6]DMSO):
d=7.94 (d, 1H, J=5.9 Hz), 7.06 (brs, 2H), 6.84 (d, 2H, J=9.3 Hz),
6.61 (d, 2H, J=8.6 Hz), 5.89 (d, 1H, J=6.2 Hz), 5.17 ppm (brs, 2H);
13C NMR (100 MHz, [D6]DMSO) d=164.99, 158.45, 157.64, 151.87,
147.40, 126.20, 119.45, 104.50 ppm; LC–MS (ESI): 246 [M+1].
4-((2-(4-Fluorophenyl)oxazolo[4,5-b]pyridin-7-yl)oxy)aniline (11):
Compound 10 (120 mg, 0.34 mmol) was dissolved in MeOH (4 mL)
and THF (2 mL), then zinc powder (135 mg, 2 mmol) and ammoni-
um chloride (115 mg, 2.1 mmol) were added. The mixture was
stirred overnight under argon. The reaction mixture was filtered
and the filtrate was washed with EtOAc. The combined organic
layers were concentrated to yield 11 as a yellow solid (108 mg,
(9H-Fluoren-9-yl)methyl(4-((2-amino-3-nitropyridin-4-yl)oxy)phe-
nyl)carbamate (16): NaHCO3 (1.36 g, 0.13 mol) was dissolved in
water (8 mL), followed by the addition of dioxane (8 mL) and com-
pound 15 (1 g, 4 mmol). The mixture was cooled to 08C, and 9-flu-
orenylmethyl chloroformate (1.15 g, 4.4 mmol) in dioxane (4 mL)
was added. The reaction mixture was stirred overnight at room
temperature, then concentrated to half volume and filtered. The
solid was washed with saturated NaHCO3 and water and dried to
obtain 16 as a yellow solid (1.5 g, 79%): 1H NMR (300 MHz,
[D6]DMSO): d=7.97 (d, 1H, J=5.7 Hz), 7.91 (d, 2H, J=7.2 Hz), 7.74
(d, 2H, J=7.8 Hz), 7.38 (m, 6H), 7.11 (m, 2H), 5.9 (d, 1H, J=6.2 Hz),
4.51 (d, 2H, J=6.7 Hz), 4.31 ppm (t, 1H, J=6.6 Hz); 13C NMR
(100 MHz, [D6]DMSO) d=160.3, 153.7, 152.9, 147.07, 142.7, 142.6,
1
98%): H NMR (300 MHz, [D6]DMSO): d=8.24 (d, 1H, J=6 Hz), 8.14
(m, 2H), 7.37 (t, 2H, J=8.4 Hz), 6.96 (d, 2H, J=8.5 Hz), 6.66 ppm
(m, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.7, 165.9, 163.4,
157.9, 157.8, 150.2, 149.8, 148.0, 147.0, 143.1, 132.1, 122.4, 116.8,
116.6, 116.2, 115.5, 114.7, 107.3 ppm; LC–MS (ESI): 322 [M+H]+.
N-((4-((2-(4-Fluorophenyl)oxazolo[4,5-b]pyridin-7-yl)oxy)phenyl)-
carbamothioyl)-2-phenylacetamide (12): Sodium thiocyanate
ChemMedChem 2012, 7, 1057 – 1070
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1065

M. Geng, J. Shen, et al.
MED
139.5, 137.48, 129.0, 127.4, 121.5, 120.1, 114.8, 109.9, 99.8,
66.4 ppm; LC–MS (ESI): 469 [M+H]+.
[D6]DMSO): d=150.6, 146.9, 146.5, 146.4, 144.0, 143.2, 136.8, 121.6,
121.4, 120.4, 114.9, 103.3, 102.8 ppm; LC–MS (ESI): 304 [M+H]+.
(9H-Fluoren-9-yl)methyl(4-((2,3-diaminopyridin-4-yl)oxy)phenyl)-
carbamate (17): Compound 16 (450 mg, 0.96 mmol), zinc powder
(625 mg, 9.6 mmol), and anhydrous CaCl2 (780 mg, 7 mmol) were
suspended in 95% EtOH (20 mL), and the mixture was stirred at
reflux for 1 h. After cooling, the reaction mixture was filtered, and
the filtrate was evaporated to dryness. The residue was purified by
silica gel chromatography (CH2Cl2/MeOH, 10:1) to yield 17 as
4-((2-(Thiophen-3-yl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18h): Brown solid (42%): H NMR (300 MHz, [D6]DMSO): d=13.45
1
(s, 1H), 8.4 (s, 1H), 8.08 (d, 1H, J=5.5 Hz), 7.84 (d, 1H, J=4.9 Hz),
7.76 (m, 1H), 6.93 (m, 2H), 6.67 (d, 2H, J=8.3 Hz), 6.34 ppm (d, 1H,
J=5.7 Hz); 13C NMR (100 MHz, [D6]DMSO): d=146.4, 145.1, 143.5,
132.1, 127.8, 126.4, 121.6, 115.0, 103.1 ppm; LC–MS (ESI): 309 [M+
H]+.
1
a pink solid (200 mg, 48%): H NMR (300 MHz, [D6]DMSO): d=9.68
4-((2-(Pyridazin-4-yl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(s, 1H), 7.91 (d, 2H, J=7.3 Hz), 7.75 (d, 2H, J=7.1 Hz), 7.46–7.32
(m, 5H), 7.23 (d, 1H, J=5.4 Hz), 6.94 (d, 2H, J=8.6 Hz), 6.65 (dd,
1H, J=51.5, 9.3 Hz), 5.95 (d, 1H, J=6 Hz), 5.54 (brs, 2H), 4.49 (d,
2H, J=6.8 Hz), 4.31 ppm (t, 1H, J=6.2 Hz); LC–MS (ESI): 439 [M+
1].
1
(18i): Brown solid (21%): H NMR (300 MHz, [D6]DMSO): d=9.97 (s,
1H), 9.45 (d, 1H, J=4.7 Hz), 8.36 (s, 1H), 8.23 (d, 1H, J=5.4 Hz),
6.96 (d, 2H, J=8.5 Hz), 6.68 (d, 2H, J=9 Hz), 6.43 ppm (d, 1H, J=
5.2 Hz); LC–MS (ESI): 305 [M+H]+.
The general synthetic method for compounds 19–22 followed the
same procedure as used for compound 12.
General synthetic method for 18a–h: Compound 17 (1 mmol)
and the appropriate aryl acids (1 mmol) were heated in polyphos-
phoric acid (24 g) at 2508C for 4 h. The mixture was cooled to
room temperature, poured over crushed ice, and brought to
pH 10–12 by the addition of NaOH. The resulting solid was filtered,
washed with water, and dried with Na2SO4. The residue was puri-
fied by silica gel chromatography (CH2Cl2/MeOH, 15:1) to yield
18a–i.
2-Phenyl-N-((4-((2-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phe-
1
nyl)carbamothioyl)acetamide (19): Off-white solid (17%): H NMR
(300 MHz, [D6]DMSO): d=12.42 (s, 1H), 8.23 (m, 3H), 7.74 (d, 2H,
J=7.9 Hz), 7.56 (m, 3H), 7.37 (m, 3H), 7.28 (m, 4H), 6.58 (d, 1H, J=
5.6 Hz), 4.11(s, 1H), 3.85 ppm (s, 2H); 13C NMR (100 MHz, [D6]DMSO)
d=179.0, 173.1, 164.1, 154.9, 148.8, 145.4, 134.6, 134.2, 130.4,
129.5, 129.3, 128.9, 127.0, 126.7, 126.3, 120.2, 105.0, 42.4 ppm; LC–
MS (ESI): 480 [M+H]+; HRMS: m/z [M+Na]+ calcd for
C27H21N5O2Na: 502.1314, found: 502.1318.
4-((2-Phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18a):
1
Brown solid (23%): H NMR (300 MHz, CDCl3): d=8.32 (d, 2H, J=
7.4 Hz), 8.21 (d, 1H, J=6 Hz), 7.54 (m, 3H), 7.07 (d, 2H, J=8.6 Hz),
6.76 (d, 2H, J=9 Hz), 6.54 ppm (d, 1H, J=5.8 Hz); LC–MS (ESI): 304
[M+H]+.
2-Phenyl-N-((4-((2-(p-tolyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-
phenyl)carbamothioyl)acetamide (20): Off-white solid (13%):
1H NMR (300 MHz, [D6]DMSO): d=8.12 (m, 2H), 7.74 (m, 2H), 7.3
(m, 10H), 6.48 (dd, 1H, J=34.9, 4.7 Hz), 3.83 (s, 1H), 3.7 (s, 1H),
3.55 (s, 1H), 2.37 ppm (s, 3H); 13C NMR (100 MHz, [D6]DMSO) d=
169.2, 154.5, 152.8, 148.9, 140.9, 136.9, 136.0, 129.7, 129.6, 129.4,
129.2, 128.4, 128.1, 127.0, 126.6, 126.4, 121.1, 121.0, 104.1, 43.4,
21.1 ppm; LC–MS (ESI): 494 [M+H]+; HRMS: m/z [M+Na]+ calcd
for C28H23N5O2NaS: 516.1470, found: 516.1442.
4-((2-(p-Tolyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18b):
Off-white solid (56%); 1H NMR (300 MHz, [D6]DMSO): d=8.12 (m,
3H), 7.38 (d, 2H, J=8.2 Hz), 6.94 (d, 2H, J=8.3 Hz), 6.68 (d, 2H, J=
8.3 Hz), 6.35 (d, 1H, J=5.7 Hz), 2.4 ppm (s, 3H); 13C NMR (100 MHz,
[D6]DMSO): d=146.5, 145.2, 13.4, 140.2, 129.6, 127.0, 126.6, 121.6,
114.9, 103.1, 21.1 ppm; LC–MS (ESI): 317 [M+H]+.
4-((2-(4-Fluorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18c): White solid (49%): 1H NMR (300 MHz, [D6]DMSO): d=13.56
(brs, 1H), 8.27 (m, 2H), 8.08 (d, 1H, J=5.1 Hz), 7.41 (t, 2H, J=
8.9 Hz), 6.95 (m, 2H), 6.65 (d, 2H, J=9.1 Hz), 6.35 (d, 1H, J=
5.4 Hz), 5.12 ppm (brs, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
164.6, 162.1, 146.4, 145.4, 143.4, 129.0, 126.3, 121.6, 116.2, 116.0,
115.0, 103.2 ppm; LC–MS (ESI): 215 [M+H]+.
2-Phenyl-N-((4-((2-(pyridin-4-yl)-3H-imidazo[4,5-b]pyridin-7-yl)-
oxy)phenyl)carbamothioyl)acetamide (21): Brown solid (39%):
1H NMR (300 MHz, [D6]DMSO): d=8.76 (d, 2H, J=4.2 Hz), 8.2 (m,
3H), 7.73 (d, 2H, J=8.8 Hz), 7.32 (m, 8H), 6.59 (d, 1H, J=5.6 Hz),
3.82 (s, 2H); 13C NMR (100 MHz, [D6]DMSO) d=169.2, 150.2, 149.0,
137.1, 136.9, 136.0, 129.2, 128.4, 126.6, 121.2, 120.8, 120.7, 104.2,
43.4 ppm; LC–MS (ESI): 481 [M+H]+; HRMS: m/z [M+Na]+ calcd
for C26H20N6O3NaS: 503.1266, found: 503.1251.
4-((2-(3,4-Difluorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)ani-
line (18d): Brown solid (35%): 1H NMR (300 MHz, [D6]DMSO): d=
8.15 (m, 3H), 7.65 (m, 1H), 6.95 (m, 2H), 6.66 (d, 2H, J=8.3 Hz),
6.36 ppm (d, 1H, J=5.7 Hz); LC–MS (ESI): 339 [M+H]+.
2-Phenyl-N-((4-((2-(thiophen-3-yl)-3H-imidazo[4,5-b]pyridin-7-
yl)oxy)phenyl)carbamothioyl)acetamide (22): Yellow solid (18%):
1
mp: 217–2208C; H NMR (300 MHz, CDCl3): d=12.39 (s, 1H), 11.75
4-((2-(2-Chlorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18e): Brown solid (28%): 1H NMR (300 MHz, [D6]DMSO): d=8.12
(d, 1H, J=5.6 Hz), 7.8 (d, 1H, J=6.9 Hz), 7.65 (d, 1H, J=7.4 Hz),
7.54(m, 2H), 6.92 (d, 2H, J=8.5 Hz), 6.64(d, 2H, J=8.2 Hz),
6.36 ppm (d, 1H, J=5.5 Hz); LC–MS (ESI): 337 [M+H]+.
(s, 1H), 8.36 (s, 1H), 8.16 (d, 1H, J=4.5 Hz), 7.75 (m, 4H), 7.3 (m,
8H), 6.54 (d, 1H, J=6 Hz), 3.82 ppm (s, 2H); 13C NMR (100 MHz,
[D6]DMSO) d=169.2, 148.9, 136.8, 136.0, 135.8, 131.7, 131.5, 129.6,
129.1, 128.4, 128.1, 126.6, 126.4, 121.1, 120.9, 120.4, 43.4 ppm; LC–
MS (ESI): 486 [M+H]+; HRMS: m/z [M+Na]+ calcd for
C25H19N5O2NaS2: 508.0878, found: 508.0893.
4-(7-(4-Aminophenoxy)-3H-imidazo[4,5-b]pyridin-2-yl)-N,N-di-
methylaniline (18 f): Brown solid (30%): 1H NMR (300 MHz,
[D6]DMSO): d=8.02 (m, 3H), 6.9 (d, 2H, J=7.4 Hz), 6.81 (d, 2H, J=
8.6 Hz), 6.64(d, 2H, J=9 Hz), 6.28 (d, 2H, J=5.7 Hz), 2.99 ppm (s,
6H); LC–MS (ESI): 346 [M+H]+.
1-((4-Fluorophenyl)carbamoyl)cyclopropanecarboxylic acid (24):
TEA (5.52 mL, 39.7 mmol) was added dropwise to a mixture of cy-
clopropane-1,1-dicarboxylic acid (4.24 g, 32.6 mmol) in anhydrous
THF (40 mL) under nitrogen while stirring for 30 min at 08C, fol-
lowed by the addition of thionyl chloride (2.36 mL, 32.6 mmol)
while stirring for an additional 30 min at 08C. A solution of 4-fluo-
roaniline (3.44 mL, 36.2 mmol) in anhydrous THF (20 mL) was
added dropwise to the resulting mixture under nitrogen while stir-
4-((2-(Pyridin-4-yl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)aniline
(18g): Brown solid (51%): 1H NMR (300 MHz, [D6]DMSO): d=8.77
(d, 2H, J=5.8 Hz), 8.17 (m, 3H), 6.96 (d, 2H, J=8.7 Hz), 6.68 (d, 2H,
J=8.3 Hz), 6.4 ppm (d, 1H, J=6 Hz); 13C NMR (100 MHz,
1066
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Imidazolopyridines as c-Met Kinase Inhibitors
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ring for 1.5 h at 08C. The reaction mixture was diluted with EtOAc
(40 mL) and washed with 1n NaOH. The layers were separated,
and the EtOAc layer was concentrated in vacuo to give a light
brown solid. The solid was washed with small amount of cold
EtOAc, filtered, and dried under vacuum to yield 24 as a white
1H NMR (300 MHz, [D6]DMSO): d=13.63 (s, 1H), 8.23 (m, 4H), 7.73
(d, 2H, J=8.0 Hz), 7.54 (m, 3H), 7.36 (m, H), 7.23 (m, 3H), 6.46 (d,
1H, J=5.3 Hz), 3.67 (s, 2H); 13C NMR (100 MHz, [D6]DMSO) d=
169.1, 145.4, 136.5, 136.0, 130.3, 129.6, 129.1, 128.9, 128.3, 126.5,
120.9, 120.8, 104.1, 43.3 ppm; LC–MS (EI): 420 [M]+; HRMS: m/z
[M]+ calcd for C26H20O2N4: 420.1586, found: 420.1593.
1
solid (2.92 g, 41%): H NMR (300 MHz, [D6]DMSO): d=13.11 (s, 1H),
10.58 (s, 1H), 7.62 (m, 2H), 7.16 (m, 2H), 1.41 ppm (m, 4H); LC–MS
Methyl 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxyl-
ate (32): 4-Fluoroaniline (0.62 mL, 6.5 mmol) was added to a solu-
tion of methyl 2-oxo-2H-pyran-3-carboxylate (1 g, 6.5 mmol) in
DMF (9 mL) at 08C, and the reaction mixture was stirred for 7 h.
EDC·HCl (1.62 g, 8.45 mmol) and DMAP (0.2 g, 1.62 mmol) were
added to the mixture at room temperature, and the mixture was
stirred overnight. A majority of the solvent was removed in vacuo,
and the residue was diluted with water and EtOAc. The organic
layer was separated, washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel chro-
matography (petroleum/EtOAc, 1:2) to yield 31 as a light yellow
solid (900 mg, 57%): 1H NMR (300 MHz, [D6]DMSO): d=8.11 (dd,
1H, J=7.1, 2.8 Hz), 7.94 (dd, 1H, J=7.1, 2.2 Hz), 7.48 (m, 2H), 7.36
(m, 2H), 6.4 (t, 1H, J=7.1 Hz), 3.74 ppm (s, 3H); LC–MS (ESI): 248
[M+H]+.
(ESI): 224 [M+H]+.
N-(4-((2-Amino-3-nitropyridin-4-yl)oxy)phenyl)-N-(4-fluorophe-
nyl)cyclopropane-1,1-dicarboxamide (25): Compounds 15
(741 mg, 1.9 mmol) and 24 (932 mg, 4.2 mmol) were dissolved in
dry DMF (50 mL). DIEA (2.6 mL, 15 mmol) and HATU (2.6 g,
6.8 mmol) were added to the solution, and the mixture was stirred
overnight at room temperature. The mixture was then diluted with
EtOAc and washed with 1n NaOH and brine. The organic layer
was dried with Na2SO4 and concentrated. The product was purified
by silica gel chromatography (CH2Cl2/MeOH, 10:1) to yield 25 as
1
a yellow solid (973 mg, 72%): H NMR (300 MHz, CD3OD): d=8.59
(d,1H, J=5.8 Hz), 8.33 (d, 2H, J=8.8 Hz), 8.22 (m, 2H), 7.76 (m,
4H), 6.70 (d, 1H, J=5.6 Hz), 2.28 ppm (s, 4H); LC–MS (ESI): 452
[M+H]+.
N-(4-((2-(Aminomethyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phen-
1-(4-Fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic acid
(33): A mixture of compound 32 (300 mg, 1.21 mmol) and 2n
aqueous NaOH (2 mL, 4 mmol) were heated at 658C for 3 h. A 2n
solution of HCl was added to the reaction mixture while stirring at
room temperature (pH 1.0). The resulting precipitate was filtered,
washed with water, and dried to obtained 33 as a white solid
(270 mg, 96%): H NMR (300 MHz, [D6]DMSO): d=8.48 (dd, 1H, J=
7.3, 2.3 Hz), 8.2 (dd, 1H, J=6.1, 2.2 Hz), 7.61 (m, 2H), 7.42 (t, 2H,
J=9 Hz), 6.78 ppm (t, 1H, J=6.4 Hz); LC–MS (ESI): 234 [M+H]+.
yl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide
(28):
Compound 25 (223 mg, 0.5 mmol), zinc powder (150 mg,
2.3 mmol), and ammonium chloride (150 mg, 2.8 mmol) were sus-
pended in MeOH (4 mL) and THF (2 mL). After stirring overnight at
room temperature, the mixture was filtered, and the filtrate was
concentrated to give 26 as a brown solid (180 mg). The product
was used in the next step without purification. Compound 26
1
(270 mg,
0.64 mmol),
N-carbobenzyloxyglycine
(135 mg,
0.64 mmol), EDC·HCl (149 mg), and HOAt (88 mg, 0.64 mmol) were
dissolved in DMF (2 mL), followed by the addition of N-methylmor-
pholine (300 mL, 2.72 mmol). The mixture was stirred overnight at
room temperature, then diluted with EtOAc and washed with satu-
rated NaHCO3 and brine. The organic layer was dried and concen-
trated to obtain a brown oil, to which AcOH (5 mL) was added.
The reaction was heated under microwave at 1608C for 45 min.
After cooling, MeOH (5 mL) and 10% Pd/C (300 mg) was added
and the mixture was stirred under H2 at room temperature for
about two days. The mixture was filtered, and the filtrate was con-
centrated to less than half of the original volume. The residue was
neutralized with saturated Na2CO3 and extracted with EtOAc. The
organic layer was washed with brine, dried with Na2SO4, and con-
centrated to dryness in vacuo. The residue was purified by silica
gel chromatography (CH2Cl2/MeOH, 5:1) to yield 28 as an off-white
solid (70 mg, total yield 31%): 1H NMR (300 MHz, [D6]DMSO): d=
10.15 (s, 1H), 10.06 (s, 1H), 8.10 (d, 1H, J=5.3 Hz), 7.71 (d, 2H, J=
8 Hz), 7.64 (m, 2H), 7.15 (m, 4H), 6.45 (d, 1H, J=5.4 Hz), 3.95 (s,
2H), 1.46 ppm (s, 4H); 13C NMR (100 MHz, [D6]DMSO) d=168.1,
168.0, 158.2 (d, J=239 Hz), 149.9, 144.6, 135.9, 135.2, 122.4, 122.3,
122.1, 120.0, 115.1, 114.9, 104.0, 31.5, 15.4 ppm; LC–MS (ESI): 461
[M+H]+; HRMS: m/z [M+H]+ calcd for C24H22N6O3F: 461.1737,
found: 461.1749.
General synthetic method for compounds 34–42: Compounds
18a–i (0.1 mmol), compound 33 (0.1 mmol), and TBTU (0.13 mmol)
were dissolved in DMF/CH3CN (2 mL, 1:1). The mixture was cooled
to 08C and DIEA (0.36 mmol) was added. The reaction mixture was
stirred at room temperature overnight, then quenched with aque-
ous 1n NaOH, and the resulting solution was stirred for 30 min at
room temperature. The mixture was concentrated in vacuo, and
the resulting slurry was diluted with water. The solid precipitate
was filtered, washed with aqueous 10% NaOH and water, and
dried. The crude product was purified by silica gel chromatography
(CH2Cl2/MeOH, 15:1) to yield 34–42.
1-(4-Fluorophenyl)-2-oxo-N-(4-((2-phenyl-3H-imidazo[4,5-b]pyri-
din-7-yl)oxy)phenyl)-1,2-dihydropyridine-3-carboxamide
(34):
1
White solid (44%): H NMR (300 MHz, [D6]DMSO): d=11.99 (s, 1H),
8.58 (dd, 1H, J=7.2, 2.4 Hz), 8.2 (m, 3H), 8.1 (dd, 1H, J=6, 1.7 Hz),
7.82 (d, 2H, J=9 Hz), 7.57 (m, 5H), 7.42 (t, 2H, J=9.1), 7.26 (d, 2H,
J=8.3 Hz), 6.72 (t, 1H, J=7.1 Hz), 6.51 ppm (d, 1H, J=5.5 Hz);
13C NMR (100 MHz, [D6]DMSO): d=170.5, 161.9 (d, J=244.8 Hz),
161.8, 161.1, 158.0, 152.3, 151.6, 144.7, 143.9, 141.9, 136.4, 134.6,
134.3, 129.4, 129.3, 128.3, 128.1, 126.7, 121.2, 120.6, 120.1, 116.3,
116.0, 107.0, 102.8 ppm; LC–MS (ESI): 518 [M+H]+; HRMS: m/z
[M+Na]+ calcd for C30H20N5O3FNa: 540.1448, found: 540.1457.
2-Phenyl-N-(4-((2-phenyl-3H-imidazo[4,5-b]pyridin-7-yl)oxy)phe-
nyl)acetamide (29): Compound 18a (31 mg, 0.1 mmol) was dis-
solved in DMF (1 mL), followed by the addition of TEA (15.7 mL,
0.11 mmol). Phenylacetyl chloride (13.5 mL, 0.1 mmol) was added
dropwise to the mixture in an ice bath. The mixture was stirred at
room temperature overnight, then diluted with water and extract-
ed with EtOAc. The organic layer was dried with Na2SO4 and con-
centrated. The product was purified by silica gel chromatography
(CH2Cl2/MeOH, 15:1) to yield 29 as a yellow solid (36 mg, 85%):
1-(4-Fluorophenyl)-2-oxo-N-(4-((2-(p-tolyl)-3H-imidazo[4,5-b]pyri-
din-7-yl)oxy)phenyl)-1,2-dihydropyridine-3-carboxamide
(35):
White solid (40%): 1H NMR (300 MHz, [D6]DMSO): d=13.52 (brs,
1H), 11.97 (brs, 1H), 8.59 (dd, 1H, J=7.3, 2.2 Hz), 8.12 (m, 3H), 7.8
(d, 2H, J=8.9 Hz), 7.61 (m, 2H), 7.4 (m, 4H), 7.23 (d, 2H, J=8.8 Hz),
6.72 (t, 1H, J=7 Hz), 6.51 (d, 1H, J=5.5 Hz), 2.38 ppm (s, 3H); LC–
MS (ESI): 532 [M+H]+; HRMS: m/z [M+H]+ calcd for C31H23N5O3F:
532.1785, found: 532.1791.
ChemMedChem 2012, 7, 1057 – 1070
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1067

M. Geng, J. Shen, et al.
MED
1-(4-Fluorophenyl)-N-(4-((2-(4-fluorophenyl)-3H-imidazo[4,5-
b]pyridin-7-yl)oxy)phenyl)-2-oxo-1,2-dihydropyridine-3-carboxa-
mide (36): Light yellow solid (30%): H NMR (300 MHz, [D6]DMSO):
mide (42): Brown solid (59%): 1H NMR (300 MHz, [D6]DMSO): d=
11.93 (s, 1H), 9.90 (s, 1H), 9.19 (d, 1H, J=5.2 Hz), 8.59 (dd, 1H, J=
7.6, 2.1 Hz), 8.24 (dd, 1H, J=5.2, 1.8 Hz), 8.10 (dd, 1H, J=6.2,
1.8 Hz), 8.01 (d, 1H, J=5.3 Hz), 7.75 (d, 2H, J=9.1 Hz), 7.61 (m,
2H), 7.42 (t, 2H, J=8.8 Hz), 7.15 (d, 2H, J=9.0 Hz), 6.72 (t, 1H, J=
7.2 Hz), 6.28 ppm (d, 1H, J=5.2 Hz); 13C NMR (100 MHz, [D6]DMSO):
d=161.86 (d, J=245 Hz), 161.8, 161.1, 154.2, 153.4, 151.7, 151.3,
149.2, 144.7, 143.8, 142.8, 136.3, 134.5, 133.3, 129.4, 139.3, 121.9,
121.3, 120.5, 120.3, 116.2, 116.0, 107.0, 102.3 ppm; LC–MS (ESI): 520
[M+H]+; HRMS: m/z [M+Na]+ calcd for C28H18N7O3FNa: 542.1353,
found: 542.1356.
1
d=11.99 (s, 1H), 8.58 (dd, 1H, J=7.4, 1.5 Hz), 8.08 (dd, 1H, J=6.6,
2 Hz), 7.8 (d, 2H, J=8.6 Hz), 7.59 (m, 2H), 7.4 (m, 9H), 6.72 (t, 1H,
J=7 Hz), 6.51 ppm (d, 1H, J=5.3 Hz); 13C NMR (100 MHz,
[D6]DMSO): d=164.2, 161.9, 161.3, 158.3 (d, J=239 Hz), 155.6,
152.1, 135.9, 135.3, 134.8, 129.4 (d, J=9 Hz), 129.0 (d, J=9 Hz),
128.8, 121.5 (d, J=8 Hz), 120.9, 120.4, 119.8, 115.8 (d, J=7 Hz),
115.6 (d, J=8 Hz), 106.6, 104.7 ppm; LC–MS (ESI): 536 [M+H]+;
HRMS: m/z [M+Na]+ calcd for C30H19N5O3F2Na: 558.1354, found:
558.1365.
General synthetic method for compounds 43–46: Compound 18
(0.075 mmol) was dissolved in DMF (1 mL), followed by the addi-
tion of compound 24 (0.075 mmol), HATU (0.1 mmol), and DIEA
(0.2 mmol). The mixture was stirred at room temperature over-
night. The reaction mixture was quenched with aqueous 1n
NaOH, and the resulting solution was stirred for 30 min at room
temperature. The mixture was then concentrated in vacuo, and the
resulting slurry was diluted with water. The solid precipitate was fil-
tered, washed with aqueous 10% NaOH and water, and dried. The
residue was purified by silica gel chromatography (CH2Cl2/MeOH,
10:1) to yield 43–46.
N-(4-((2-(3,4-Difluorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-
phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxa-
1
mide (37): White solid (46%): H NMR (300 MHz, [D6]DMSO): d=8.6
(dd, 1H, J=7.4, 1.6 Hz), 8.25 (m, 2H), 8.12 (dd, 2H, J=6.3, 1.6 Hz),
7.83 (d, 2H, J=8.9 Hz), 7.64 (m, 3H), 7.43 (t, 2H, J=8.9 Hz), 7.27 (d,
2H, J=8.7 Hz), 6.73 (t, 1H, J=7 Hz), 6.53 ppm (d, 1H, J=5.3 Hz);
LC–MS (ESI): 554 [M+H]+; HRMS: m/z [M+Na]+ calcd for
C30H18N5O3F3Na: 576.1259, found: 576.1255.
N-(4-((2-(2-Chlorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-
phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxa-
mide (38): Off-white solid (54%): 1H NMR (300 MHz, [D6]DMSO):
d=12.0 (s, 1H), 8.61 (dd, 1H, J=7.2, 2.1 Hz), 8.23 (d, 1H, J=
5.3 Hz), 8.13 (dd, 1H, J=6.3, 1.7 Hz), 7.84 (m, 3H), 7.6 (m, 5H), 7.44
(t, 2H, J=9 Hz), 7.28 (d, 2H, J=9 Hz), 6.74 (t, 1H, J=6.9 Hz), 6.57
(d, 1H, J=5.7 Hz); 13C NMR (100 MHz, [D6]DMSO): d=161.9 (d, J=
245 Hz), 161.8, 161.2, 149.9, 145.6, 144.8, 143.9, 136.3, 135.5, 132.1,
131.5, 130.3, 129.9, 129.4, 129.3, 127.4, 121.4, 121.0, 120.5, 116.2,
116.0, 107.0, 104.3 ppm; LC–MS (ESI): 552 [M+H]+; HRMS: m/z
[M+H]+ calcd for C30H20N5O3FCl: 552.1239, found: 552.1255.
N-(4-Fluorophenyl)-N-(4-((2-phenyl-3H-imidazo[4,5-b]pyridin-7-
yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide (43): White solid
(73%): 1H NMR (300 MHz, [D6]DMSO): d=10.17 (s, 1H), 10.08 (s,
1H), 8.23 (m, 2H), 8.16 (d, 1H, J=5.4 Hz), 7.74 (d, 2H, J=8.6 Hz),
7.65 (m, 2H), 7.56 (m, 3H), 7.19 (m, 3H), 6.47 (d, 1H, J=6.1 Hz),
1.47 ppm (s, 4H); 13C NMR (100 MHz, [D6]DMSO): d=168.1, 168.0,
159.2, 156.2 (d, J=224 Hz), 151.8, 149.7, 145.4, 136.1, 135.2, 130.3,
129.6, 129.0, 126.6, 122.4, 122.3, 122.2, 120.6, 115.1, 114.9, 31.5,
15.4 ppm; LC–MS (ESI): 508 [M+H]+; HRMS: m/z [M+H]+ calcd for
C29H22N5O3FNa: 530.1604, found: 530.1635.
N-(4-((2-(4-(Dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-
yl)oxy)phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-
carboxamide (39): Off-white solid (74%): 1H NMR (300 MHz,
[D6]DMSO): d=11.99 (s, 1H), 8.6 (m, 1H), 8.10(m, 4H), 7.82 (d, 2H,
J=8.3 Hz), 7.62 (m, 2H), 7.44 (t, 2H, J=8.8 Hz), 7.25 (d, 2H, J=
8.6 Hz), 6.84 (d, 2H, J=8.4 Hz), 6.73 (m, 1H), 6.49 (s, 1H), 3.02 ppm
(s, 6H); LC–MS (ESI): 561 [M+H]+; HRMS: m/z [M+H]+ calcd for
C32H25N6O3F: 561.2050, found: 561.2033.
N-(4-((2-(2-Chlorophenyl)-3H-imidazo[4,5-b]pyridin-7-yl)oxy)-
phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (44):
Off-white solid (80%): 1H NMR (300 MHz, [D6]DMSO): d=10.11 (d,
2H, J=5.8 Hz), 8.21 (d, 1H, J=5.3 Hz), 7.83 (dd, 1H, J=7.5,
1.69 Hz), 7.74 (d, 2H, J=8.7 Hz), 7.6 (m, 5H), 7.23 (d, 2H, J=
8.6 Hz), 7.15 (t, 2H, J=8.6 Hz), 6.5 (d, 1H, J=5.6 Hz), 1.46 ppm (s,
4H); 13C NMR (100 MHz, [D6]DMSO): d=168.1, 158.3 (d, J=238 Hz),
149.6, 145.7, 136.2, 135.2, 132.1, 131.6, 130.3, 139.8, 127.4, 122.4,
122.2, 120.6, 115.1, 114.9, 104.0, 31.6, 15.4 ppm; LC–MS (ESI): 542
[M+H]+; HRMS: m/z [M+H]+ calcd for C29H22N5O3FCl: 542.1395,
found: 542.1412.
1-(4-Fluorophenyl)-2-oxo-N-(4-((2-(pyridin-4-yl)-3H-imidazo[4,5-
b]pyridin-7-yl)oxy)phenyl)-1,2-dihydropyridine-3-carboxamide
1
(40): Brown solid (48%): H NMR (300 MHz, [D6]DMSO): d=12.0 (s,
1H), 8.77 (d, 2H, J=5 Hz), 8.59 (dd, 1H, J=7.3, 2.1 Hz), 8.16 (m,
4H), 7.83 (d, 2H, J=8.3 Hz), 7.61 (m, 2H), 7.42 (t, 2H, J=9.1 Hz),
7.28 (m, 2H), 6.72 (t, 1H, J=6.9 Hz), 6.54 ppm (d, 1H, J=5.6 Hz);
LC–MS (ESI): 519 [M+H]+; HRMS: m/z [M+H]+ calcd for
C29H20N6O3F: 519.1581, found: 519.1564.
N-(4-((2-(4-(Dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-
yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxa-
mide (45): Off-white solid (86%): 1H NMR (300 MHz, [D6]DMSO):
d=8.04 (m, 3H), 7.70 (d, 2H, J=8.8 Hz), 7.62 (m, 2H), 7.15 (m, 4H),
6.81 (dm 2H, J=9.1 Hz), 6.39 (d, 1H, J=5.5 Hz), 3.0 (s, 6H),
1.45 ppm (s, 4H); 13C NMR (100 MHz, [D6]DMSO): d=168.2, 158.2
(d, J=239 Hz), 151.5, 149.9, 144.0, 136.0, 135.3, 127.9, 122.4, 122.2,
120.4, 116.8, 115.1, 114.9, 111.7, 104.0, 39.8, 31.5, 15.4 ppm; LC–MS
(ESI): 551 [M+H]+; HRMS: m/z [M+H]+ calcd for C31H28N6O3F:
551.2207, found: 551.2229.
1-(4-Fluorophenyl)-2-oxo-N-(4-((2-(thiophen-3-yl)-3H-imidazo-
[4,5-b]pyridin-7-yl)oxy)phenyl)-1,2-dihydropyridine-3-carboxa-
mide (41): White solid (73%): 1H NMR (300 MHz, [D6]DMSO): d=
8.58 (dd, 1H, J=6.6, 1.8 Hz), 8.31 (d, 1H, J=1.5 Hz), 8.1 (m, 2H),
7.8 (m, 3H), 7.7 (m, 1H), 7.6 (m, 2H), 7.41 (t, 2H, J=8.3 Hz), 7.22 (d,
2H, J=8.5 Hz), 6.71 (t, 1H, J=6.7 Hz), 6.45 ppm (d, 1H, J=5.7 Hz);
13C NMR (100 MHz, [D6]DMSO): d=166.5, 161.9 (d, J=244.8 Hz),
161.8, 161.2, 153.6, 150.2, 149.1, 144.9, 143.9, 136.4, 135.4, 135.3,
132.6, 129.4, 129.3, 127.6, 126.5, 125.9, 121.4, 121.0, 120.5, 116.3,
116.0, 107.0, 104.2 ppm; LC–MS (ESI): 524 [M+H]+; HRMS: m/z
[M+Na]+ calcd for C28H18N5O3FSNa: 546.1012, found: 546.1029.
N-(4-Fluorophenyl)-N-(4-((2-(thiophen-3-yl)-3H-imidazo[4,5-b]pyr-
idin-7-yl)oxy)phenyl)cyclopropane-1,1-dicarboxamide
(46):
1
Brown solid (78%): H NMR (300 MHz, [D6]DMSO): d=10.16 (s, 1H),
10.06 (s, 1H), 8.35 (s, 1H), 8.14 (s, 1H), 7.7 (m, 5H), 7.18 (m, 3H),
6.45 (d, 1H, J=5 Hz), 1.47 ppm (s, 4H); 13C NMR (100 MHz,
[D6]DMSO): d=168.6, 168.5, 159.7, 157.8, 145.6, 135.7, 132.4, 128.3,
126.8, 126.5, 122.9, 122.8, 122.7, 121.1, 115.6, 115.4, 32.0, 15.9 ppm;
1-(4-Fluorophenyl)-2-oxo-N-(4-((2-(pyridazin-4-yl)-3H-imidazo-
[4,5-b]pyridin-7-yl)oxy)phenyl)-1,2-dihydropyridine-3-carboxa-
1068
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1057 – 1070

Imidazolopyridines as c-Met Kinase Inhibitors
MED
LC–MS (ESI): 514 [M+H]+; HRMS: m/z [M+H]+ calcd for
C27H21N5O3SF: 514.1349, found: 514.1347.
Wound healing assay: MDCK cells (2ꢁ105 cells per well) were
added to the wells of 12-well plates and cultured for 24–48 h until
a uniform monolayer formed. All wound healing assays were per-
formed in serum-free medium by first pre-incubating the cells in
the serum-free medium for 30 min before the start of the experi-
ments. A micropipette was used to create a wound in the mono-
layer by scraping. After washing to remove the detached cells,
compounds were added at the indicated concentrations and the
plates were incubated for 60 min at 378C. Subsequently, the cells
were incubated with and without 30 ngmLꢂ1 recombinant human
HGF and placed in a cell incubator (378C). Images were captured
at 0 h and 10 h after incubation using a phase-contrast micro-
scope.[26]
Biology
ELISA kinase assay: Tyrosine kinase activities were evaluated ac-
cording to the reported protocol.[24] Briefly, in an enzyme-linked im-
munosorbent assay (ELISA), 20 mgmLꢂ1 Poly (Glu/Tyr, 4:1; Sigma)
was pre-coated as a substrate in 96-well plates. ATP (50 mL of
a 10 mm solution) diluted in kinase reaction buffer (50 mm HEPES
pH 7.4, 50 mm MgCl2, 0.5 mm MnCl2, 0.2 mm Na3VO4, 1 mm DTT)
was added to each well. Various concentrations of compounds di-
luted in 10 mL of 1% DMSO (v/v) were added to each reaction well,
with 1% DMSO (v/v) used as the negative control. The kinase reac-
tion was initiated by the addition of purified tyrosine kinase pro-
teins diluted in kinase reaction buffer solution (40 mL). After incuba-
tion for 60 min at 378C, the plate was washed three times with
phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T-PBS).
Next, 100 mL of anti-phosphotyrosine (PY99) antibody (1:500 dilut-
ed in 5 mgmLꢂ1 BSA T-PBS) was added. After 30 min incubation at
378C, the plate was washed three times. A solution of 100 mL
horseradish peroxidase-conjugated goat anti-mouse IgG (1:2000 di-
luted in 5 mgmLꢂ1 BSA T-PBS) was added. The plate was re-incu-
bated at 378C for 30 min, and washed as before. Finally, 100 mL of
a solution containing 0.03% H2O2 and 2 mgmLꢂ1 o-phenylenedia-
mine in 0.1 mm citrate buffer (pH 5.5) was added, and samples
were incubated at room temperature until color emerged. The re-
action was terminated by the addition of 2m H2SO4 (50 mL), and
the plate was read using a multiwell spectrophotometer (VERSA-
max, Molecular Devices, Sunnyvale, CA, USA) at 490 nm. The inhibi-
tion rate (%) was calculated using the following equation:
[1ꢂ(A490/A490 control)]ꢁ100%. IC50 values were calculated from the in-
hibition curves.
In vitro metabolic stability: Microsomes (0.5 mgmLꢂ1; human micro-
some: Xenotech, Lot No. H0610; rat microsome: Xenotech, Lot No.
R1000) were pre-incubated with 1 mm test compound for 5 min at
378C in 0.1m phosphate buffer (pH 7.4) with 1 mm EDTA and
5 mm MgCl2. The reactions were initiated by adding pre-warmed
cofactor (1 mm NADPH). After 0, 5, 10, and 30 min incubations at
378C, reactions were quenched by adding an equal volume of cold
CH3CN. The samples were vortexed for 10 min, then centrifuged at
10000 g for 10 min. The supernatants were analyzed with LC–MS/
MS to determine the amount of parent compound remaining. The
corresponding loss of parent compound was determined by LC–
MS/MS. in vitro intrinsic clearance: CLint =[0.693/(in vitro t1/2)/(incu-
bation volume)/(mg microsomal protein)]. The criteria are
100 mLminꢂ1 mgꢂ1 protein.
Direct and time-dependent inhibition of microsomes: CYP enzymatic
activities were characterized based on their probe reactions:
CYP3A4 (midazolam 1-hydroxylation), CYP2D6 (dextromethorphan
O-demethylation), and CYP1A2 (phenacetin O-deethylation). Incu-
bation mixtures were prepared in a total volume of 100 mL as fol-
lows: 0.2 mgmLꢂ1 human microsome, 1 mm NADPH, 100 mm
phosphate buffer (pH 7.4), probe substrates cocktail (5 mm midazo-
lam, 5 mm dextromethorphan, 100 mm phenacetin), and 10 mm test
compound or positive control cocktail (10 mm ketoconazole, 10 mm
quinidine, 10 mm a-naphthoflavone) or negative control (PBS). The
final concentration of organic reagents in incubation mixtures was
less than 1% v/v. There was a 5 min pre-incubation period at 378C
before the reaction was initiated by adding the NADPH-generating
system. The reactions were conducted for 20 min for CYP3A4,
CYP2D6, and CYP1A2. For each probe drug, the percentage of me-
tabolite conversion was less than 20% of substrate added. Percent
inhibition was calculated as: [(formation of the metabolite of
probe substrates with 10 mm tested compound)/(formation of the
metabolite of probe substrates with PBS)]ꢁ100; criteria are 50%.
Western blot analysis: Cells were cultured under regular growth
conditions to exponential growth phase. The cells were then treat-
ed with the indicated concentrations of corresponding compounds
for 2 h at 378C and lysed in 1ꢁSDS sample buffer. The cell lysates
were subsequently resolved on 10% SDS-PAGE and were trans-
ferred to nitrocellulose membranes. Membranes were probed with
phospho-c-Met and c-Met, phospho-ERK1/2 and ERK1/2, phospho-
AKT and AKT (all from Cell Signaling Technology, Beverly, MA) and
GAPDH (KangChen Bio-tech) antibodies, then subsequently with
anti-rabbit or anti-mouse IgG horseradish peroxidase. Immunoreac-
tive proteins were detected using an enhanced chemilumines-
cence detection reagent.
Cell proliferation assay: Cells were seeded in 96-well tissue culture
plates. The following day, cells were exposed to various concentra-
tions of compounds and further cultured for 72 h. Finally, cell pro-
liferation was determined using a sulforhodamine B (SRB; Sigma)
or thiazolyl blue tetrazolium bromide (MTT; Sigma) assay.
Samples for the time-dependent inactivation screening assay were
pre-incubated for 0, 5, 10, or 20 min at 378C with 0.2 mgmLꢂ1
human microsome and 10 mm test compound or positive control
(10 mm troleandomycin, 10 mm paroxetine, 10 mm furafylline) with
or without 1 mm NADPH. The percentage of remaining activity of
CYP3A4, CYP2D6, and CYP1A2 was measured by the formation of
the metabolite of their marker substrates: midazolam, dextrome-
thorphan and phenacetin, at single concentrations approximating
their Km values (5 mm midazolam, 5 mm dextromethorphan, 100 mm
phenacetin). The percentage of remaining activity for microsomes
pre-incubated with NADPH was compared with that of microsomes
without pre-incubation. Kobs =slope of the decrease in enzymatic
Transwell assay: Cell migration was examined using the membrane
invasion culture system (transwell polycarbonate membranes,
6.5 mm diameter, 8 mm pore size). Briefly, 1.5ꢁ104 NCI-H441 cells
were seeded onto the upper chamber of the transwells. The lower
chamber of the transwells was filled with serum-free medium, the
indicated cytokines, and compound at the indicated concentra-
tions. After 24 h, noninvasive cells on the upper surface of the
membrane were removed with a cotton swab. Cells that passed
through the 8 mm pores of the transwell and attached to the lower
surface of membrane were fixed with 90% EtOH, stained with crys-
tal violet, and photographed.[25]
activity versus time. The criteria are 200ꢁ10ꢂ4 minꢂ1
.
In vivo antitumor activity assay: Animal experiments were per-
formed according to the institutional ethical guidelines for animal
ChemMedChem 2012, 7, 1057 – 1070
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1069

M. Geng, J. Shen, et al.
MED
care. Cells (density of 5–10ꢁ106 in 200 mL) were first implanted s.c.
into the right flank of each nude mice and then allowed to grow
to 700–800 mm3, defined as a well-developed tumor. After that,
the well-developed tumors were cut into 1 mm3 fragments and
transplanted s.c. into the right flank of nude mice using a trocar.
When the tumor volume reached 100 to 200 mm3, the mice were
randomly assigned into control and treatment groups. Control
groups were given vehicle alone, and treatment groups received
compound 32 at the indicated doses via p.o. administration 7 days
per week for 11 days. The sizes of the tumors were measured twice
per week using microcalipers. The tumor volume (Vtum) was calcu-
lated as: Vtum =(lengthꢁwidth2)/2. Tumor volume was shown on
indicated days as the median tumor volume ꢁSE indicated for
groups of mice. Percent (%) inhibition values were measured on
the final day of study for drug-treated mice as compared with ve-
hicle-treated mice and were calculated as 100ꢁ{1ꢂ[(treatedfinal
dayꢂtreatedday 1)/(controlfinal dayꢂcontrolday 1)]}. Significant differences
between the treated versus the control groups (pꢃ0.001) were de-
termined using Student’s t-test.
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Abbreviations
NSCLC: non-small cell lung cancer; TKI: tyrosine kinase inhibitor;
HGF: hepatocyte growth factor; DIEA: N,N-diisopropylethylamine;
NMP: N-methyl-2-pyrrolidone; Fmoc: fluorenylmethoxycarbonyl;
DMF: N,N-dimethylformamide; mCPBA: meta-chloroperoxybenzoic
acid; TEA: triethylamine; EDC·HCl: N-(3-dimethylaminopropyl)-N’-
ethylcarbodiimide hydrochloride; HOAt: 1-hydroxy-7-azabenzotria-
zole; NMM: N-methylmorpholine; HATU: O-(7-azabenzotriazol-1-yl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate; TBTU: O-ben-
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zotriazol-1-yl-N,N,N’,N’-tetramethyluronium
tetrafluoroborate;
HOAt: 1-hydroxy-7-azabenzotriazole; THF: tetrahydrofuran.
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This research was supported by grants from the Natural Science
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Keywords: c-Met · kinase inhibitors · imidazolopyridines ·
structure–activity relationships · hinge binders
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Received: March 5, 2012
Revised: March 28, 2012
Published online on May 13, 2012
1070
www.chemmedchem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2012, 7, 1057 – 1070