Discovery of a new series of imidazo[1,2-a]pyridine compounds as selective c-Met inhibitors
Acta Pharmacologica Sinica
Discovery of a new series of imidazo[1,2-a]pyridine compounds as selective c-Met inhibitors
Tong-chao LIU 0 1
Xia PENG 2
Yu-chi MA 1
Yin-chun JI 2
Dan-qi CHEN 1
Ming-yue ZHENG 1
Dong-mei ZHAO 0
Mao-sheng CHENG 0
Mei-yu GENG 2
Jing-kang SHEN 1
Jing AI 2
Bing XIONG 1
0 Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University , Shenyang 110016 , China
1 Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203 , China
2 Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203 , China
Aim: Aberrant c-Met activation plays a critical role in cancer formation, progression and dissemination, as well as in development of resistance to anticancer drugs. Therefore, c-Met has emerged as an attractive target for cancer therapy. The aim of this study was to develop new c-Met inhibitors and elaborate the structure-activity relationships of identified inhibitors. Methods: Based on the predicted binding modes of Compounds 5 and 14 in docking studies, a new series of c-Met inhibitor-harboring 3-((1H-pyrrolo[3,2-c]pyridin-1-yl)sulfonyl)imidazo[1,2-a]pyridine scaffolds was discovered. Potent inhibitors were identified through extensive optimizations combined with enzymatic and cellular assays. A promising compound was further investigated in regard to its selectivity, its effects on c-Met signaling, cell proliferation and cell scattering in vitro. Results: The most potent Compound 31 inhibited c-Met kinase activity with an IC50 value of 12.8 nmol/L, which was >78-fold higher than those of a panel of 16 different tyrosine kinases. Compound 31 (8, 40, 200 nmol/L) dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and ERK signaling cascades in c-Met aberrant human EBC-1 cancer cells. In 12 human cancer cell lines harboring different background levels of c-Met expression/activation, Compound 31 potently inhibited c-Metdriven cell proliferation. Furthermore, Compound 31 dose-dependently impaired c-Met-mediated cell scattering of MDCK cells. Conclusion: This series of c-Met inhibitors is a promising lead for development of novel anticancer drugs.
c-Met inhibitors; hepatocyte growth factor receptor; imidazo[1; 2-a]pyridine; anticancer agents; drug discovery
Introduction c-Met has been linked to many types of cancers that occur as
c-Met, also known as hepatocyte growth factor receptor a consequence of gene amplification or rearrangement,
tran(HGFR), was discovered in 1984 as an oncogenic fusion pro- scriptional regulation, as well as autocrine or paracrine ligand
]. Since then, extensive investigations on the structure stimulation[
]. Importantly, both c-Met and HGF elevation
and functions of c-Met have shown that it belongs to a unique have been associated with poor clinical outcomes[
subfamily of receptor tyrosine kinases (RTKs)[
] and forms a aberrant c-Met activation plays a critical role in cancer
formaheterodimer by connecting a short extracellular ? chain and a tion, progression, and dissemination and in the development
membrane spanning ? chain through a disulfide bond. After of resistance against approved therapies. Therefore, c-Met has
binding to its natural ligand, hepatocyte growth factor (HGF), emerged as an attractive target for cancer therapy [
c-Met initiates its kinase phosphorylation activity and triggers Currently, the most promising approach for disrupting
a series of downstream signaling pathways, including PI3K- c-Met signaling is to use small molecular inhibitors to target
AKT-mTOR and Ras-MEK-ERK[
]. Abnormal activation of the intracellular kinase domain. Through the analysis of
binding modes, small molecule inhibitors of c-Met can be roughly
classified into three types. Type I c-Met inhibitors bind to
E*-Tmoawilhboxmioncgo@rrseismpmon.adce.nccne(BshinoguXldIObNeGa)d;dressed. ATP binding pockets in a ?U? shape, which usually interacts
(Jing AI) with residue Met1211 at the hinge part to anchor the
inhibiReceived 2015-11-10 Accepted 2016-01-05 tor and forms a typical ?-? stacking interaction with residue
]. As implied by the unique U-shaped binding mode,
type I inhibitors (such as crizotinib 1) all show good selectivity
for c-Met and are expected to cause fewer side effects in cancer
]. Type II c-Met inhibitors (such as cabozantinib 2)
are usually multi-kinase inhibitors and adopt extended
conformations, starting from the solvent-accessible part to hinge and
further stretching to the deep hydrophobic Ile1145 subpocket
near the C-helix region[
]. Except for the above-mentioned
well-classified inhibitors, there are other atypical c-Met
inhibitors, such as ARQ197 (3), that are all classified as type III c-Met
Previously, we elaborated on the synthesis of a series of
pyrazol[4,3-b]pyridine compounds and their potent and
selective activities as c-Met inhibitors (lead compound 4)[
the optimization process, we synthesized an interesting
compound (5) containing two possible hinge binders, an imidazole
ring and an imidazo[1,2-a]pyridine ring, which could form an
essential hydrogen bonding interaction with the backbone of
Met1160. Considering its interesting binding mode and good
enzymatic activity, we initialized a medicinal chemistry
modification with the aim of finding a novel series of c-Met inhibitors
for the further development of anti-cancer drugs.
Materials and methods
Reagents and conditions: (a) ClSO 3H, CHCl3, Reflux, 24 h,
98%; (b) (4-fluorophenyl)boronic acid, Pd(dppf)Cl2, K2CO3,
90 ?C, 3 h, 70.9%; (c) phosphorus oxychloride, reflux, 24 h,
51%; (d) NaH, DMF, room temperature, 4 h.
Compounds 5?19 were prepared according to the procedure
shown in Scheme 1. Commercially available 32 was
sulfonylated to afford 33. Conventional Suzuki coupling of 33 with
(4-fluorophenyl)boronic acid afforded compound 34.
Treatment of compound 34 with phosphorus oxychloride afforded
compound 35. Compounds 5-19 were prepared by
subjecting compound 35 to condensation with the appropriate
Reagents and conditions: (a) phosphorus oxychloride, reflux,
24 h, 51%; (b) 1H-pyrrolo[3,2-c]pyridine, NaH, DMF, room
temperature, 4 h, 60%; (c) R-boronic acid or R-boronic acid
pinacol ester, PdCl2(dppf)-CH2Cl2, K2CO3, 90 ?C, 30 min, 31%?
Compounds 20?31 were synthesized according to the
procedures outlined in Scheme 2. Compound 33 was treated with
phosphorus oxychloride to afford compound 36. Compound
37 was prepared by deprotonating 5-azaindole, which was
followed by the addition of compound 36. A variety of aryl
groups were introduced at the 6-position of compound 37 via
Suzuki coupling reactions to provide compounds 20?31.
1H NMR (400 MHz) spectra were recorded using a Varian
Mercury-400 High Performance Digital FT-NMR spectrometer
using tetramethylsilane (TMS) as an internal standard.
Abbreviations for peak patterns in the NMR spectra are as follows:
br=broad, s=singlet, d=doublet, and m=multiplet.
Low-resolution mass spectra were obtained with a Finnigan LCQ Deca
XP mass spectrometer using a CAPCELL PAK C18 (50 mm?2.0
mm, 5 ZM) or an Agilent ZORBAX Eclipse XDB C18 (50
mm?2.1 m, 5 ZM) column in positive or negative electrospray
mode. The purities of all the compounds were determined by
analytical Gilson high-performance liquid chromatography
(HPLC) using an YMC ODS3 column (50 mm?4.6 mm, 5 ZM)
using the following conditions: CH3CN/H2O eluent at 2.5
mL/min flow [containing 0.1% trifluoroacetic acid (TFA)] at
35 ?C, 8 min, gradient 5% CH3CN to 95% CH3CN, monitored
by UV absorption at 214 nm and 254 nm. TLC analyses were
carried out using glass precoated silica gel GF254 plates. The
TLC spots were visualized under UV light. Flash column dazo[1,2-a]pyridine (5). NaH (10.3 mg, 0.258 mmol) was
chromatography was performed using a Teledyne ISCO Com- first dissolved in 1 mL of anhydrous DMF. Imidazole (10.5
biFlash Rf system. All the solvents and reagents were used mg, 0.155 mmol) dissolved in 1 mL of anhydrous DMF was
as received unless otherwise noted. Anhydrous dimethylfor- slowly added dropwise, and the mixture was stirred for 30
mamide was purchased from Acros and was used without min. Compound 35 (40 mg, 0.129 mmol) dissolved in 1 mL of
further drying. All air- and moisture-sensitive reactions were anhydrous DMF was slowly added dropwise, and the mixture
carried out under an atmosphere of dry argon with heat-dried was stirred for 3 h at room temperature. The reaction
soluglassware using standard syringe techniques. tion was poured into 0.1 mol/L hydrochloric acid, which was
then turned basic using an aqueous sodium bicarbonate
soluGeneral procedure for the syntheses of 5?19 tion and extracted with ethyl acetate. The organic layer was
6-Bromoimidazo[1,2-a]pyridine-3-sulfonic acid (33). Chol- collected, and distilled under reduced pressure. The residue
rosulfonic acid (1.01 mL, 15.3 mmol) was dissolved in chlo- was purified by flash chromatography to afford compound 5
roform (10 mL), and this solution was added dropwise to (26 mg, 60%). MS m/z (ESI) found 343 (M+H)+; 1H NMR (400
6-bromoimidazo[1,2-a]pyridine (1.00 g, 5.1 mmol) in chloro- MHz, chloroform-d) ? 8.67 (s, 1H), 8.43 (s, 1H), 8.13 (s, 1H),
form (15 mL) over 20 min. The reaction mixture was refluxed 7.88 (d, J=9.0 Hz, 1H), 7.74 (d, J=9.6 Hz, 1H), 7.56?7.39 (m, 2H),
for 24 h, allowed to cool to room temperature and concen- 7.36 (s, 1H), 7.27?7.19 (m, 2H), 7.12 (s, 1H). Retention time 3.02
trated to dryness under vacuum. The crude oily product was min, 100% pure.
treated with diethyl ether (20 mL) and ethanol (10 mL), which The details of compounds 6-19 are provided in the
Supportresulted in the collection of a white precipitate. The solid was ing Material.
collected by filtration, washed with EtOH and dried to afford
6-bromoimidazo[1,2-a]pyridine-3-sulfonic acid (33) (1.38 g, General procedure for the syntheses of 20?31
98% yield). MS m/z (ESI) found 275, 277 (M-H) +; 1H NMR (400 6-Bromoimidazo[1,2-a]pyridine-3-sulfonyl chloride ( 36).
MHz, DMSO-d6) ? 8.93 (dd, J=1.8, 0.8 Hz, 1H), 8.30 (s, 1H), Compound 36 was prepared according to the procedure for
8.13 (dd, J=9.5, 1.8 Hz, 1H), 7.95 (dd, J=9.5, 0.8 Hz, 1H); the 35. 34% yield; MS m/z (EI) found 296(M)+; 1H-NMR (400 MHz,
OH on sulfonic acid is missing. CDCl3) ? 8.97 (m, 1H), 8.47 (s, 1H), 7.89 (d, J = 9.6 Hz, 1H), 7.83
6-(4-fluorophenyl)imidazo[1,2-a]pyridine-3-sulfonic acid (dd, J = 9.6, 1.7 Hz, 1H).
(34). A solution of 33 (4 g, 14.4 mmol), (4-fluorophenyl)
3-((1H-pyrrolo[3,2-c]pyridin-1-yl)sulfonyl)-6-bromoimiboronic acid (2.4 g, 17.3 mmol), PdCl2(dppf)-CH2Cl2 (590 mg, dazo[1,2-a]pyridine (37). NaH (0.88 g, 22 mmol) was
sus0.72 mmol) and K2CO3 (7.97 g, 57.8 mmol) in 1,4-dioxane:water pended in 10 mL of anhydrous DMF and cooled to 0 ?C in an
(40 mL, 2:1, v/v) in a microwave tube was flushed with N2 for ice bath. 1H-pyrrolo[3,2-c]pyridine (1.3 g, 11 mmol) dissolved
5 min and then sealed. The tube was placed in the microwave in 10 mL of anhydrous DMF was slowly added dropwise,
cavity and heated at 90 ?C for 1 h. Then, the reaction mixture and the mixture was stirred for 30 min at 0 ?C. Compound 36
was evaporated to dryness. The residue was diluted with (3.9 g, 13.2 mmol) dissolved in 15 mL of anhydrous DMF was
water (60 mL), filtered and washed with water (20 mL). The added in dropwise, and the reaction mixture was stirred for 4
pH of the filtrate was adjusted to 1?2 with 1 mol/L aqueous h at room temperature. The reaction was monitored by TLC.
HCl, and white precipitate appeared. The precipitate was The reaction solution was poured into 0.1 mol/L hydrochloric
filtered and dried under vacuum to give 34 (2.98 g, 70.9% acid, which was then turned basic using an aqueous sodium
yield). MS m/z (ESI) found 291 (M-H) +,293(M+H) +; 1H NMR bicarbonate solution and extracted with ethyl acetate. The
(400 MHz, DMSO-d6) ? 8.97 (s, 1H), 8.33 (d, J=2.3 Hz, 1H), organic layer was collected and distilled under reduced
pres8.30 (d, J=9.3 Hz, 1H), 8.05 (d, J=9.4 Hz, 1H), 8.04 (s, 0H), 7.81 sure. The remaining substance was purified by column
chro? 7.67 (m, 2H), 7.44 (t, J = 8.3 Hz, 2H); the OH on sulfonic matography to give purified compound 37 (2.5 g, 60%). MS
acid is missing. m/z (ESI) found 377 (M+H)+; 1H NMR (400 MHz,
chloroform6-(4-fluorophenyl)imidazo[1,2-a]pyridine-3-sulfonyl chlo- d) ? 8.91 (d, J=1.0 Hz, 1H), 8.77 (dd, J=1.8, 0.9 Hz, 1H), 8.53 (d,
ride (35). Compound 34 (2.98 g, 10.2 mmol) was treated with J=5.8 Hz, 1H), 8.35 (s, 1H), 7.84 (d, J=5.8 Hz, 1H), 7.67 (d, J=3.7
phosphorus oxychloride (60 mL) and refluxed for 24 h. The Hz, 1H), 7.63 (dd, J=9.5, 0.9 Hz, 1H), 7.53 (dd, J=9.5, 1.8 Hz,
reaction mixture was cooled to room temperature and treated 1H), 6.80 (dd, J=3.7, 0.9 Hz, 1H).
with DCM (100 mL), poured over ice-cold water (100 mL), and
3-((1H-pyrrolo[3,2-c]pyridin-1-yl)sulfonyl)-6-phenylthen extracted with DCM (4?50 mL). The organic layers were imidazo[1,2-a]pyridine (20). A solution of 37 (50 mg,
combined, dried (Na2SO4), filtered, and concentrated to dry- 0.133 mmol), phenylboronic acid (24.2 mg, 0.199 mmol),
ness under vacuum to give crude 35. The crude product was PdCl2(dppf)-CH2Cl2 adduct (5.4 mg, 0.007 mmol) and K2CO3
purified by flash chromatography to give purified compound (55 mg, 0.398 mmol) in 1,4-dioxane:water (4 mL, 2:1, v/v) in a
35 (1.6 g, 51%). MS m/z (ESI) found 311 (M+H)+; 1H NMR (400 microwave tube was flushed with N2 for 5 min then sealed.
MHz, chloroform-d) ? 8.82 (s, 1H), 8.40 (s, 1H), 7.94 (d, J=9.3 The tube was placed in a microwave cavity and heated at
Hz, 1H), 7.84 (d, J=9.3 Hz, 1H), 7.60 (dd, J=8.7, 5.2 Hz, 2H), 90 ?C for 60 min. Then, the reaction mixture was evaporated
7.30?7.17 (m, 2H). to dryness. The residue was purified by flash chromatography
3-((1H-imidazol-1-yl)sulfonyl)-6-(4-fluorophenyl)imi- to give 20 (39.7 mg, 80%). MS m/z (ESI) found 375 (M+H)+;
1H NMR (400 MHz, chloroform-d) ? 8.91 (s, 1H), 8.71?8.69
(m, 1H), 8.53 (d, J=5.8 Hz, 1H), 8.43 (s, 1H), 7.91 (d, J=6.2 Hz,
1H), 7.79 (dd, J=9.4, 0.9 Hz, 1H), 7.75 ?7.63 (m, 2H), 7.59?7.37
(m, 5H), 6.79 (dd, J=3.7, 0.7 Hz, 1H). Retention time 2.82 min,
The details of compounds 21?31 are provided in the
The X-ray complex structure of an azaindole compound bound
to c-Met (PDB entry: 2WD1[
]) was downloaded from the PDB
database. The Schr?dinger software package was used for the
modeling studies. First, the structure was subjected to Protein
Preparation Wizard to add the hydrogen atoms and refine the
structure to eliminate the improper interactions. Then, the
Glide program was used to generate the grid file. The receptor
grid was defined as an enclosed box centered at the ligand in
the ATP binding site. Docking was performed using the Glide
software in standard precision (SP) mode with the default
]. Finally, the binding interactions were analyzed
and illustrated with the Pymol program.
ELISA kinase assay
The effects of the compounds on the activities of various
tyrosine kinases were determined using enzyme-linked
immunosorbent assays (ELISAs) with purified recombinant proteins.
Briefly, 20 ?g/mL poly(Glu, Tyr) 4:1 (Sigma, St Louis, MO,
USA) was pre-coated in 96-well plates as a substrate. A 50-?L
aliquot of 10 ?mol/L ATP solution diluted in kinase reaction
buffer (50 mmol/L HEPES [pH 7.4], 50 mmol/L MgCl 2, 0.5
mmol/L MnCl2, 0.2 mmol/L Na3VO4, and 1 mmol/L DTT)
was added to each well. Then, 1 ?L of various concentrations
of compounds diluted in 1% DMSO ( v/v) (Sigma, St Louis,
MO, USA) were then added to each reaction well. DMSO (1%,
v/v) was used as a negative control. The kinase reaction was
initiated adding purified tyrosine kinase proteins diluted in
49 ?L of kinase reaction buffer. After incubation for 60 min
at 37 ?C, the plate was washed three times with
phosphatebuffered saline (PBS) containing 0.1% Tween 20 (T-PBS).
Antiphosphotyrosine (PY99) antibody (100 ?L; 1:500, diluted in 5
mg/mL BSA T-PBS) was then added. After a 30-min
incubation at 37 ?C, the plate was washed three times, and 100 ?L
of horseradish peroxidase-conjugated goat anti-mouse IgG
(1:2000, diluted in 5 mg/mL BSA T-PBS) was added. The
plate was then incubated at 37?C for an additional 30 min and
washed 3 times. A 100-?L aliquot of a solution containing
0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 mol/L
citrate buffer (pH 5.5) was added. The reaction was
terminated by adding 50 ?L of 2 mol/L H2SO4; as the color changed,
the plate was analyzed using a multi-well spectrophotometer
(SpectraMAX 190, from Molecular Devices, Palo Alto, CA,
USA) at 490 nm. The inhibition rate (%) was calculated using
the following equation: [1-(A490/A490 control)]?100. The IC50
values were calculated from the inhibition curves in two separate
EBC-1, MKN-45, and MKN-1 cells were purchased from
Japanese Research Resources Bank (Tokyo, Japan).
NCIH661, A549, KATOIII and DU145 cells were purchased from
the American Type Culture Collection (Manassas, VA, USA).
NCI-H358, BGC-823, MGC-803 and NCI-H460 were obtained
from the Typical Culture Preservation Commission Cell Bank
at the Chinese Academy of Sciences. NCI-H3122 was obtained
from the National Cancer Institute. MDCK cells were a kind
gift from Dr H Eric XU at the Shanghai Institute of Materia
Medica. The cells were routinely maintained according to the
recommendations of their suppliers[
Cell proliferation assay
Cells were seeded in 96-well tissue culture plates. On the next
day, the cells were exposed to various concentrations of
compounds and further cultured for 72 h. Cell proliferation was
then determined using sulforhodamine B (SRB, from
SigmaAldrich, St Louis, MO, USA) or a Cell Counting Kit (CCK-8)
assay. The IC50 values were calculated by fitting
concentration-response curves using a SoftMax pro-based
Western blot analysis
EBC-1 cells were treated with the indicated dose of compound
31 for 2 h at 37 ?C and then lysed in 1?SDS sample buffer. The
cell lysates were subsequently resolved by 10% SDS-PAGE
and transferred to nitrocellulose membranes. The membranes
were probed with the appropriate primary antibodies (ie,
[c-Met (Santa Cruz, CA, USA), phospho-c-Met, phospho-ERK,
ERK, phospho-AKT, AKT (all from Cell Signaling Technology,
Beverly, MA, USA), and GAPDH (KangChen Biotech,
Shanghai, China)) and then with horseradish peroxidase-conjugated
anti-rabbit or anti-mouse IgG[
]. The immunoreactive proteins
were detected using an enhanced chemiluminescence
detection reagent (Thermo Fisher Scientific, Rockford, IL, USA).
MDCK cells (1.5?103 cells per well) were plated in 96-well
plates and grown overnight. Increasing concentrations
of Compound 31 and HGF (50 ng/mL) were added to the
appropriate wells, and the plates were incubated at 37 ?C and
5% CO2 for 24 h. The cells were fixed with 4%
paraformaldehyde for 15 min at room temperature and then stained with
0.2% crystal violet. The assay was performed in triplicate.
Images were obtained using an Olympus IX51 microscope.
Results and Discussion
To identify the binding mode of compound 5, we utilized
the Glide program to perform a docking study on 5 in the
ATP binding site of c-Met. The crystal structure of 2WD1
was selected as a template, and the protein structure was first
refined for glide grid generation. Then, the minimized
compound 5 was docked into the binding site. As shown in Figure
2A, the binding conformation of 5 indicated that the hinge
binder was an imidazole group and that the 4-fluoro-benzene fied bicycle-aromatic rings containing the H-bond acceptor
group was situated below the side chain of residue Tyr1230 to N atom were introduced, and the synthesized compounds
form a ?-? stacking interaction. To verify whether the imid- were tested in enzymatic assays (Table 2). Compounds 13, 14
azole unit was the hinge binder, we synthesized 7 compounds and 15 had similar enzyme inhibitory activities against c-Met.
by replacing imidazole with different substituted aromatic When the pyridine ring was replaced by a phenyl ring ( 16),
5-member rings. As shown in Table 1, when the N atom at the the compounds lost their activities, reinforcing the finding that
3-position was removed or changed to the 2-position (6 and pyridine plays an important role in protein-ligand interactions.
7), the activities dramatically decreased, which informed us When a halogen atom was introduced on the carbon adjacent
that it may be advantageous to have an H-bond receptor at the to the N atom (17, 18 and 19), the activities also decreased
dra3-position to interact with the essential hinge part of the bind- matically, suggesting that the halogen atoms could interfere
ing site. To test our hypothesis, some H-bond receptors such with nearby residues in the hinge part of the binding site.
as a nitryl group or an aldehyde group were installed. The The nearly identical activities of 13 and 14 puzzled us
results demonstrated that compounds with substitutions at the regarding their binding conformations because the position
3-position exhibited significantly better activities than those of the important nitrogen atoms were different in the
pyrwith substitutions at the 2-position (comparing 8 to 9 and 10 to rolopyridine rings, which were thought to be essential in
11, respectively). However, compound 12, which had an etha- H-bond interactions. From the interaction pattern found in
none structure at the 3-position, did not show good inhibitory the crystal structure 2WD1, compound 13 would bind to the
activity, indicating that the methyl group caused steric hin- ATP site by forming a hydrogen bond with the backbone of
drance when the compound approached the carbonyl group residue Met1160. However, compound 14 had a shifted
nitroof the binding site (comparing compounds 10 and 12). gen atom, which could not fulfill the requirement of
From Table 1, we found H-bond receptors at the 3-position ing with the hinge part of the protein. Thus, we performed a
of the imidazole ring improved activities. Therefore, diversi- docking study with the aim of predicting the interaction mode
The inhibition values are estimated values from two separate experiments. The IC50 values were calculated by the Logit method from the results of at
least two independent tests with six concentrations each and expressed as mean?SD.
The inhibition values are estimated values from two separate experiments. The IC50?s were calculated by the Logit method from the results of at least
two independent tests with six concentrations each and expressed as mean?SD.
of compound 14. As shown in Figure 2B, the binding mode
of compound 14 was dramatically different from that of
compound 13 because the structure was reversed and a hydrogen
bond between the imidazo[1,2-a]pyridine ring and residue
Met1160 was observed. This surprising binding
conformation triggered us to pursue further optimizations based on this
As demonstrated by the prediction of the binding
tion of compound 14, the 4-fluoro-benzene group coupled to many of them did not show good cellular activities in EBC-1
imidazo[1,2-a]pyridine pointed to the solvent-accessible part cancer cells. The enzyme IC50 values of compounds 24 and
of the c-Met. Therefore, different heterocycles or substituted 29 were 6.6?1.9 and 224.1?74.8 nmol/L, respectively, while
phenyl groups were evaluated for their occupancy of the sol- their IC50 values in EBC-1 cells were similar (ie, their IC50
valvent accessible subpocket (Table 3). Most of the compounds ues were approximately 500 nmol/L). On the whole, R2 with
showed excellent c-Met inhibition in an enzymatic assay, but substituted phenyl groups showed better activities than those
The inhibition values are estimated values from two separate experiments. The IC50 values were calculated by the Logit method from the results of at
least two independent tests with six concentrations each and expressed as mean?SD.
with heterocycles; in particular, compound 31 exhibited strong
inhibition on both molecular and cellular levels.
Compound 31 is a potent and selective inhibitor of c-Met
In an enzymatic screen designed to identify c-Met
inhibitors, compound 31 was distinguished for its potency against
recombinant human c-Met kinase and exhibited an average
IC50 value of 12.8 nmol/L (Table 3). Accordingly, we were
prompted to investigate whether this potency was
specifically against c-Met. Thus, the activity of Compound 31 was
evaluated against a panel of kinases (Table 4). In contrast to
its high potency against c-Met, Compound 31 barely inhibited
the kinase activity of other tested tyrosine kinases, including
c-Met family member Ron and highly homologous kinases
Axl, Tyro3, c-Mer (IC50>1 ?mol/L), indicating that compound
31 is a selective c-Met inhibitor.
The ability of Compound 31 to inhibit the enzymatic activities of a
panel of recombinant tyrosine kinases was evaluated by ELISA assays,
representing IC50s as mean?SD or estimated values.
Compound 31 inhibits c-Met phosphorylation and its downstream
To further assess the cellular activity of Compound 31 against
c-Met kinase, we measured its effects on the phosphorylation
of c-Met and downstream signaling molecules in EBC-1 cells
that harbor an amplified MET gene. As shown in Figure 3,
Compound 31 significantly inhibited the phosphorylation of
c-Met with a complete abolishment at 40 nmol/L in EBC-1
cells, including the phosphorylation of Akt and ERK, which
are key downstream molecules of c-Met[
]. These results
suggested that Compound 31 exhibits effective inhibition of c-Met
activation and its signaling.
Compound 31 significantly inhibits c-Met-addicted proliferation
Activated c-Met is known to trigger cancer cell proliferation[
Therefore, we next assessed the effect of Compound 31 on cell
proliferation in human cancer cells and genetically engineered
cells that harbor different backgrounds of c-Met expression
and activation. Compound 31 significantly inhibited the
proliferation of the c-Met-constitutively activated EBC-1 and
MKN45 cells, with IC50 values of 19.8 and 9.9 nmol/L,
respectively (Table 5). In contrast, compound 31 showed over
500fold less potency in cells with low c-Met expression or
activation (Table 5). These data indicate that Compound 31
specifically inhibits c-Met-dependent cancer cell growth.
The IC50 values are shown as the mean?SD (nmol/L) or estimated values
from two separate experiments.
Compound 31 inhibits c-Met-dependent cell scattering
Activated HGF/c-Met signaling is also known to promote
cell scattering that stimulates cells to abandon their original
environment, a hallmark of cancer invasiveness and
]. It has been well documented that MDCK cells, which
normally grow in clusters, are disruptive and scatter cell
nies upon HGF stimulation. We thus determined the effect
of compound 31 on this cell scattering behavior using MDCK
cells stimulated by HGF. As shown in Figure 4, treatment
with compound 31 reduced the HGF-induced cell scattering of
MDCK cells in a dose-dependent manner, completely blocking
the spreading of cells at a dose of 500 nmol/L.
Based on the previously identified lead compound 4, we
synthesized an interesting compound 5 during the development
of c-Met inhibitors. According to the docking prediction, we
proposed that the imidazole of compound 5 would form a
hydrogen bonding interaction with the hinge part of the ATP
binding site of c-Met. The structure-activity relationships
of synthesized compounds 6?12 were consistent with this
hypothesis. Further optimization resulted in a novel
compound, 14, which contained a pyrrolo[3,2-c]pyridine scaffold.
A docking study of this compound suggested that it could
interact with c-Met in a reversed conformation by using the
imidazo[1,2-a]pyridine as a hinge binder. Following this
finding, further optimization resulted in the synthesis of
compound 31, the most potent compound, which exhibited potent
enzymatic inhibition activity with an IC50 of 12.8 nmol/L.
Compound 31 effectively inhibited overactivated c-Met
signaling in EBC-1 cancer cells. In turn, compound 31 suppressed
c-Met-dependent cell proliferation and cell scattering. This
discovery will benefit other researchers and enable the
development of a novel series of c-Met inhibitors as anti-cancer
An interesting feature of Compound 31 was its selectivity
against c-Met. Compound 31 presented IC50 values for c-Met
in the nanomolar range in a kinase assay and showed more
than a 78-fold selectivity over a panel of 16 human kinases,
including c-Met family member Ron and highly homologous
kinases, such as Axl, Tyro3 and Mer. Consistently, the
antiproliferative activity of compound 31 was more than 500-fold
potent for c-Met-addicted cells in contrast to a panel of tumor
cell lines with low c-Met expression and activation levels. In
fact, most c-Met inhibitors currently undergoing clinical trials
are multi-target inhibitors, which may result in unwanted
]. Specific c-Met inhibitors could largely avoid
toxicity arising from the targeting of extra molecules and thus
provide a better option for the sub-population of c-Met-driven
cancers in the new era of precision medicine. The high
specificity and potency of compound 31 give it the potential to act
as a tool inhibitor in preclinical use and allows it to be a
promising novel drug candidate for further development.
We are grateful for financial support from the Foundation
of China Postdoctoral Science, the National Natural Science
Foundation of China (No 81202391, 91229205, 81473243,
81321092 and 81330076) and the National Science &
Technology Major Project "Key New Drug Creation and
Manufacturing Program" of China (No 2014ZX09507002,
2012ZX09301001007 and 2013ZX09507001). The SA-SIBS Scholarship Program
is also gratefully acknowledged.
Bing XIONG, Jing AI and Dong-mei ZHAO designed the
research; Tong-chao LIU, Xia PENG, Yu-chi MA and Yin-chun
JI conducted the research; Dan-qi CHEN, Ming-yue ZHENG,
Mao-sheng CHENG, Mei-yu GENG and Jing-kang SHEN
analyzed the data; Bing XIONG, Jing AI, and Dong-mei
ZHAO wrote the paper.
Chemical experimental procedures and analytical data for the
mentioned compounds are available in the supplementary
Information at the website of Acta Pharmacologica Sinica.
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