Discovery and biological evaluation of novel dual EGFR/c-Met inhibitors

Article abstract of DOI:10.1021/ml4003309

Activating mutations in the epidermal growth factor receptor (EGFR) have been identified in a subset of non-small cell lung cancer (NSCLC), which is one of the leading cancer types worldwide. Application of EGFR tyrosine kinase inhibitors leads to acquire

Full text of DOI:10.1021/ml4003309

Letter
pubs.acs.org/acsmedchemlett
Discovery and Biological Evaluation of Novel Dual EGFR/c-Met
Inhibitors
Balint Szokol,†,○ Pal Gyulavari,‡,○ Ibolya Kurko,† Ferenc Baska,†,§ Csaba Szantai-Kis,† Zoltan Greff,†
́
́
́
́
́
́
†,#
§,∥
∥
∥
∥
Zoltan Orfi, Istvan Petak,‡,⊥ Kinga Penzes, Robert Torka, Axel Ullrich, Laszlo Orfi,
̋
̋
́
́
́
́
́
́
Tibor Vantus,*,‡,△ and Gyorgy Keri*,†,‡,§,△
†Vichem Chemie Research Ltd., 1022 Budapest, Hungary
́
́
̈
‡MTA-SE Pathobiochemistry Research Group, Department of Medical Chemistry, Semmelweis University, 1085 Budapest, Hungary
§Rational Drug-Design Laboratory Cooperation Research Centre, Semmelweis University, 1085 Budapest, Hungary
∥Max Planck Institute of Biochemistry, Munich 82152, Germany
⊥KPS Medical Biotechnology and Healthcare Services Ltd., 1022 Budapest, Hungary
#Department of Pharmaceutical Chemistry, Semmelweis University, 1085 Budapest, Hungary
S
* Supporting Information
ABSTRACT: Activating mutations in the epidermal growth factor
receptor (EGFR) have been identified in a subset of non-small cell
lung cancer (NSCLC), which is one of the leading cancer types
worldwide. Application of EGFR tyrosine kinase inhibitors leads to
acquired resistance by secondary EGFR mutations or by
amplification of the hepatocyte growth factor receptor (c-Met)
gene. Although several EGFR and c-Met inhibitors have been
reported, potent dual EGFR/c-Met inhibitors, which can overcome
this latter resistance mechanism, have hitherto not been published
and have not reached clinical trials. In the present study we have
identified dual EGFR/c-Met inhibitors and designed novel N-[4-
(quinolin-4-yloxy)-phenyl]-biarylsulfonamide derivatives, which in-
hibit the c-Met receptor and both the wild-type and the activating
mutant EGFR kinases in nanomolar range. We have demonstrated by Western blot analysis that compound 10 inhibits EGFR
and c-Met phosphorylation at cellular level and effectively inhibits viability of the NSCLC cell lines.
KEYWORDS: NSCLC, EGFR, c-Met, kinase inhibitor, acquired resistance
such as proliferation, survival, migration, invasion, and wound
dentification of oncogene-driven signaling pathways har-
Inessed by tumor cells and their inhibition by small- healing.11 Amplification of the c-Met gene causes secondary
resistance against EGFR TKI therapy in 18−20% of the cases,
and it can occur parallel with T790M EGFR mutation.12,13
Pharmacological inhibition of c-Met kinase restores sensitivity
of both T790M and c-Met gene amplification-derived resistant
lung cancer cells to EGFR TKIs.14 On the basis of these results,
the simultaneous inhibition of EGFR and c-Met kinases seems
desirable; however, to date only the quinazoline-based MJ-56
has been reported to decrease both c-Met and EGFR
phosphorylation in HT-29 colorectal cancer cells at relatively
high concentration (15 μmol).15 Potent EGFR/c-Met dual
inhibitors have not been developed until now and no such drug
candidates are in clinical trials, but several EGFR and c-Met
selective inhibitors have been reported as drug candidates or are
in clinical development.16,17 In the present study, we report
molecules is a key process for drug development resulting in
new medicines such as EGFR inhibitor gefitinib, erlotinib, or
lapatinib.1 EGFR-related signaling pathways play an important
role in several malignant processes including NSCLC and have
been proven to be potential therapeutic targets.2 Examination
of mutations in the EGFR domain is a useful tool for predicting
efficacy of EGFR TKIs.3,4 Those tumor cells, which harbor
activating mutations in the EGFR kinase domain (e.g., point
mutation L858R or in-frame deletions), respond impressively
to current EGFR TKIs;5 nevertheless, the emergence of
secondary mutations in the EGFR gene eliminates the efficacy
of these drugs.6 Nearly half of all acquired resistance is due to
the secondary T790M point mutation in exon 207−9 on the
EGFR gene.
Another important oncogene in NSCLC is the hepatocyte
growth factor (HGF) receptor also known as scatter factor
(SF) or c-Met and its natural ligand, HGF.10 The receptor
tyrosine kinase c-Met is implicated in many cellular processes
Received: August 25, 2013
Accepted: January 30, 2014
Published: January 30, 2014
© 2014 American Chemical Society
298
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303

ACS Medicinal Chemistry Letters
Letter
novel small-molecule kinase inhibitors that inhibit both EGFR
and c-Met activity at nanomolar range in enzymatic assays and
induce apoptosis in a clinically relevant HCC827 NSCLC cell
line. As the initial step, our in-house compound collection of
Nested Chemical Library was screened by recombinant wt
EGFR and c-Met kinase assays, and we have identified 1 and 2
as very effective c-Met inhibitors. These compounds were
ineffective on EGFR while strongly inhibiting InsR, which is
considered undesirable. Potent AXL inhibitory effect has been
reported for (4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-car-
boxamide scaffold of BMS-777607 (IC50: 1.1 nM) and
quinoline-based sulphonamides (IC50: 16 nM).18 Taking the
structural similarity between c-Met and AXL kinases into
account, we assumed that the antipyrine-carboxamide motif is
also bioisostere with the biaryl-sulfonamide scaffold in regard to
c-Met (Figure 1). To verify this, we performed docking
both in enzymatic and cellular assays. The quality of the side
chain is also pivotal for the efficacy of these compounds:
especially, the 3-(4-methylpiperazin-1-yl)-propoxy side-chain
enhanced the inhibitory effect dramatically (e.g., wt EGFR, 6
3860 nM vs 9 168 nM; wt EGFR, 7 1370 nM vs 8 84 nM),
which prompted us to focus on it in our synthetic efforts. Three
compounds were selected for further characterization as
potential dual EGFR/c-Met inhibitors, and compound 10 was
found the most effective and therefore selected for testing in
various biochemical and cellular assays. To investigate the
selectivity of this series, compound 10 was screened at 1 μM
concentration on a recombinant kinase selectivity panel of 34
clinically relevant kinases, and it showed more than 70%
inhibition against only six other receptor tyrosine kinases:
DDR1 (111%), AXL (109%), c-KIT (91%), ErbB2 (81%),
RET (78%), and FLT3 (76%) (see Supporting Information).
On the basis of the preliminary data, it was hypothesized that
all the compounds were ATP-analogue, reversible kinase
inhibitors, and to assess whether the molecules bind the
ATP-binding cleft in the kinases, we analyzed the enzyme
activity data on double reciprocal plots. The trendlines intersect
in one point suggesting the ATP-competitive nature of
inhibition of compound 10 on both EGFR and c-Met (Figure
2).
All of the compounds showed time-dependent kinetics for
inhibition of c-Met, which was proved by applying prolonged
time of preincubation resulting in lower IC50 values similarly to
XL-880 (foretinib), quinoline-based c-Met inhibitor.25 The IC50
values of all compounds were determined on clinically relevant
NSCLC cell lines and on the NIH3T3 cell line, as
nontumorous control. The NSCLC cell lines harbor various
abnormalities in the EGFR signaling axis; therefore, the
following different reference inhibitors were used: BMS-
77760726 or crizotinib27 for H1993, erlotinib28 for HCC827,
and afatinib29 for H1975 and A549. In accordance with their
enzyme inhibitory profiles, 1 and 2 impressively reduced the
viability of c-Met amplified H1993 cell line but showed a weaker
effect against A549, HCC827, and H1975 cell lines containing
EGFR or KRAS mutations. Furthermore, compounds 1 and 2
also diminished the viability of NIH3T3 cell line. Compounds
8−10 had weaker but more uniform effect on the four cell lines,
without considerably inhibiting NIH3T3. Compound 10 was
the most efficient among them on H1993 and on HCC827 cell
lines, probably due to its more potent kinase inhibition profile.
Cell viability inhibition of the H1975 cell line is probably also
due to the c-Met inhibitory ability of 10.14 However, the effect
on A549 cell line was weak, presumably because of its KRAS
mutation beside the wt EGFR.30,31 To assess whether these
compounds downregulate EGFR and c-Met kinase activation in
living cells, we investigated downstream ERK-mediated MAPK
and PI3K/AKT/mTOR signaling pathways and performed
Western blot analysis on the tested cell lines. Compounds 1, 2,
8, and 9 inhibited c-Met phosphorylation but had no such effect
on EGFR (data not shown). Compound 10 downregulated
both c-Met and EGFR phosphorylation at 1 μM but abrogated
downstream phosphorylation only on the c-Met inhibitor-
sensitive H1993 cell line and not on the EGFR inhibitor-
sensitive HCC827 cell line, probably due to its stronger effect
on c-Met kinase activity than on EGFR (Figure 3).
Figure 1. Structures of the identified inhibitor and its analogues. (A)
Structure of c-Met inhibitor BMS-777607. (B) Structure of the original
hits, compounds 1 and 2. (C) General structure of the EGFR/c-Met
inhibitors, compounds 6−15.
simulations that led to effective biaryl-sulfonamide derivatives
against c-Met (Supporting Information). In vitro assays of the
biaryl-sulfonamide analogues demonstrated increased EGFR
inhibition and decreased c-Met and InsR inhibition compared
to 1 and 2.
To examine the structure−activity relationship (SAR), we
prepared further derivatives (6−15) regarding to the previously
reported synthetic routes of c-Met inhibitors (Table 1).19−21 In
our synthetic strategy we applied a similar method22,23 to built
up side-chain fitted quinoline derivatives affording the 3a−b
nitro compounds. It was reduced by catalytic hydrogenation
affording the amine intermediates 4a−b, which were reacted
with aromatic sulfonyl-chlorides containing bromine providing
5a−c. These were cross-coupled with five-membered hetero-
aromatic boronic acids under palladium-catalyzed Suzuki-
Myaura conditions24 using microwave irradiation at 140 °C
in 1,2-dimethoxyethane to give biaryl derivatives in moderate
yield (Scheme 1).
By comparing the analogues, we found 8 (wt EGFR: 84 nM,
c-Met: 564 nM), 9 (wt EGFR, 168 nM; c-Met, 1048 nM), and
10 (wt EGFR, 94 nM; c-Met, 398 nM) derivatives to be the
most effective against EGFR and c-Met kinases, while the
corresponding 4-substitued analogues showed less potency
The HCC827 and H1993 cell lines were exposed to control
compounds and compound 10, which induced apoptosis on the
HCC827 cell line almost as efficiently as erlotinib and afatinib,
299
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303

ACS Medicinal Chemistry Letters
Letter
Table 1. Stucture−Activity Relationship, Enzymatic Data, and Cell Viability Inhibition of N-[4-(Quinolin-4-yloxy)-phenyl]-
a
biarylsulfonamides
a
Enzyme activity and cell viability inhibition. IC50 values (μM) of the compounds on four recombinant enzymes, on various NSCLC, and on the
NIH 3T3 cell lines. Cell viability data were gathered with MTT assay. All values are an average of at least three independent experments. N.D., not
determined.
300
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303

ACS Medicinal Chemistry Letters
Letter
Scheme 1. Synthesis Route of N-[4-(Quinolin-4-yloxy)-
a
phenyl]-biarylsulfonamides
Figure 4. Flow cytometry analysis of HCC827 and H1993 cell lines
treated with erlotinib, crizotinib, afatinib, and compound 10. Data are
means SD from at least 3 independent experiments. The significance
of differences in the percentage of apoptosis was evaluated with
unpaired 2-tailed Student’s t test. The * marks p < 0.05 versus the
corresponding value for cells treated with DMSO.
a
Reagents and conditions: (a) methanol/10% Pd/C, rt; (b) anhydrous
pyridine, 3- or 4-Br-PhSO2Cl, 50 °C, 24 h; (c) Pd(0)PPh4, Na2CO3,
Ar−B(OH)2, 140 °C, μW, (20−40%).
HGF and treated by compound 10 to demonstrate the
inhibitory effect of HGF-induced cell scattering (Supporting
Information). In the case of DU145 cells, compound 10
showed to be less effective (IC50: 1.1 μM) than the reference
compound BMS-777607 (IC50: 200 nM), which was used as a
positive control due to its stronger c-Met inhibition. On
MDCK cells, compound 10 induced cell death at 1 μM
concentration, while at lower concentrations a low level of
inhibition of cell scattering was observed. To assist further
chemical modifications and to understand the mode of action,
the EGFR/c-Met inhibitors were docked into the ATP binding
site of both enzyme in silico using Protein Data Bank
cocrystallization data (c-Met, 3LQ8; EGFR, 1XKK). By
studying the predicted binding mode of the compounds on c-
Met kinase, the following conclusions can be made: the
quinoline N forms a hydrogen bond to Met1160 at the hinge
region. The sulfonamide group interacts with the DFG motif at
Asp1222 and with Lys1110 near the αC helix. The substituted
phenyl, thiophene, or pyrazole groups insert into the small
hydrophobic pocket near the DFG motif. The side chain 1-
methyl-piperazine and the morpholine rings form other
hydrogen bonds with the binding site improving the binding
affinity of the compounds. In the case of the EGFR kinase, the
quinoline nitrogen forms a hydrogen bond to Met793 at the
Figure 2. Assessing the binding mode of compound 10 to
recombinant (A) wt EGFR and (B) c-Met enzyme. Enzyme activity
(Vmax) measurements were performed at various ATP and kinase
inhibitor concentrations. The measured signal shifts (ΔS) were
depicted on a Lineweaver−Burk double reciprocal plot.
hinge region and the sulfonamide group interacts with Asn842
,
which can be found near the DFG motif of the kinase (Figure
5).
In conclusion, we have developed a novel N-[3-fluoro-4-
[[6,7-disubstitued]-4-quinolinyl]oxy]phenyl]-biaryl-benzene-
sulfonamide-based multikinase inhibitor cotargeting EGFR and
c-Met and characterized it in various biochemical assays. We
found that compound 10 inhibited both EGFR and c-Met
activity in nanomolar range in enzymatic assays and acts as a
reversible, ATP-competitive inhibitor without considerably
inhibiting InsR. Furthermore, compound 10 effectively
inhibited cell viability on NSCLC cell lines A549, H1975,
and especially HCC827 and H1993. We verified that this
compound blocks phosphorylation of both enzymes at cellular
level and induces apoptosis on HCC827 cell line comparable to
reference compounds. On the cell motility model, inhibition of
HGF-induced cell scattering was observed on the DU145 cell
line at about 1 μM IC50. Compound 10, as a novel dual
inhibitor of EGFR and c-Met, can be considered as a good
Figure 3. Western blot analysis of NSCLC cell lysates. (A) HCC827
cell line was treated with erlotinib and compound 10. (B) H1993 cell
line was treated with BMS-777607 and compound 10.
while none of the compounds had significant effect on H1993
cell line (Figure 4).
It has been reported that inhibition of c-Met leads to
blocking of c-Met-triggered cell scattering in MDCK cells21,32
and DU145 cells,33 which is an excellent model for invasive
growth and cell motility of tumor cells. Cell scattering of
DU145 prostate cancer cells and MDCK cells were induced by
301
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303

ACS Medicinal Chemistry Letters
Letter
insulin receptor; μW, microwave irradiation; MAPK, micro-
tubule-associated protein kinase; PI3K, phosphatidylinositol-3-
kinase; AKT (PKB), protein kinase B; mTOR, mammalian
target of rapamycin
REFERENCES
■
(1) Lynch, T. J.; Adjei, A. A.; Bunn, P. A., Jr.; Eisen, T. G.; Engelman,
J.; Goss, G. D.; Haber, D. A.; Heymach, J. V.; Janne, P. A.; Johnson, B.
̈
E.; Johnson, D. H.; Lilenbaum, R. C.; Meyerson, M.; Sandler, A. B.;
Sequist, L. V.; Settleman, J.; Wong, K. K.; Hart, C. S. Summary
statement: novel agents in the treatment of lung cancer: advances in
epidermal growth factor receptor-targeted agents. Clin. Cancer Res.
2006, 12, 4365−4371.
(2) Mendelsohn, J.; Baselga, J. The EGF receptor family as targets for
cancer therapy. Oncogene 2000, 19, 6550−6565.
(3) Paez, J. G.; Janne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.;
̈
Gabriel, S.; Herman, P.; Kaye, F. J.; Lindeman, N.; Boggon, T. J.;
Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.; Sellers, W. R.; Johnson, B.
E.; Meyerson, M. EGFR mutations in lung cancer: correlation with
clinical response to gefitinib therapy. Science 2004, 304, 1497−1500.
(4) Sordella, R.; Bell, D. W.; Haber, D. A.; Settleman, J. Gefitinib-
sensitizing EGFR mutations in lung cancer activate anti-apoptotic
pathways. Science 2004, 305, 1163−1167.
(5) Lynch, T. J.; Bell, D. W.; Sordella, R.; Gurubhagavatula, S.;
Okimoto, R. A.; Brannigan, B. W.; Harris, P. L.; Haserlat, S. M.; Supko,
J. G.; Haluska, F. G.; Louis, D. N.; Christiani, D. C.; Settleman, J.;
Haber, D. A. Activating mutations in the epidermal growth factor
receptor underlying responsiveness of non-small-cell lung cancer to
gefitinib. N. Engl. J. Med. 2004, 350, 2129−2139.
(6) Pao, W.; Miller, V. A.; Politi, K. A.; Riely, G. J.; Somwar, R.;
Zakowski, M. F.; Kris, M. G.; Varmus, H. Acquired resistance of lung
adenocarcinomas to gefitinib or erlotinib is associated with a second
mutation in the EGFR kinase domain. PLoS Med. 2005, 2, e73.
Figure 5. Predicted binding mode of compound 10 to (A) c-Met and
(B) EGFR kinases. The formed hydrogen bonds are marked with
yellow.
starting point to seek more effective EGFR/c-Met inhibitors as
drug candidates.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and biological measuring methods for
1−15. This material is available free of charge via the Internet
at http://pubs.acs.org.
■
S
(7) Kobayashi, S.; Boggon, T. J.; Dayaram, T.; Janne, P. A.; Kocher,
̈
O.; Meyerson, M.; Johnson, B. E.; Eck, M. J.; Tenen, D. G.; Halmos, B.
EGFR mutation and resistance of non-small-cell lung cancer to
gefitinib. N. Engl. J. Med. 2005, 352, 786−792.
(8) Kosaka, T.; Yatabe, Y.; Endoh, H.; Yoshida, K.; Hida, T.; Tsuboi,
M.; Tada, H.; Kuwano, H.; Mitsudomi, T. Analysis of epidermal
growth factor receptor gene mutation in patients with non-small cell
lung cancer and acquired resistance to gefitinib. Clin. Cancer Res. 2006,
12, 5764−5769.
(9) Balak, M. N.; Gong, Y.; Riely, G. J.; Somwar, R.; Li, A. R.;
Zakowski, M. F.; Chiang, A.; Yang, G.; Ouerfelli, O.; Kris, M. G.;
Ladanyi, M.; Miller, V. A.; Pao, W. Novel D761Y and common
secondary T790M mutations in epidermal growth factor receptor-
mutant lung adenocarcinomas with acquired resistance to kinase
inhibitors. Clin. Cancer Res. 2006, 12, 6494−6501.
AUTHOR INFORMATION
Corresponding Authors
*(G.K.) E-mail: gykeri@vichem.hu.
*(T.V.) E-mail: vantus.tibor@med.semmelweis.univ.hu.
■
Author Contributions
○B.S. and P.G. contributed equally to this work. The
manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
(10) Looyenga, B. D.; Cherni, I.; Mackeigan, J. P.; Weiss, G. J.
Tailoring tyrosine kinase inhibitors to fit the lung cancer genome.
Transl. Oncol. 2011, 4, 59−70.
Funding
This work is supported by NKFP_07_A2-NANODRUG grant.
Notes
(11) Stella, M. C.; Comoglio, P. M. HGF: a multifunctional growth
factor controlling cell scattering. Int. J. Biochem. Cell Biol. 1999, 31,
1357−1362.
The authors declare no competing financial interest.
△T.V. and G.K. are shared last authors.
(12) Engelman, J. A.; Zejnullahu, K.; Mitsudomi, T.; Song, Y.;
Hyland, C.; Park, J. O.; Lindeman, N.; Gale, C. M.; Zhao, X.;
Christensen, J.; Kosaka, T.; Holmes, A. J.; Rogers, A. M.; Cappuzzo,
ACKNOWLEDGMENTS
We are grateful to Dr. Lars Neumann for the technical
assistance and to Dr. Andras
■
F.; Mok, T.; Lee, C.; Johnson, B. E.; Cantley, L. C.; Janne, P. A. MET
̈
́
Dancso
́
(EGIS Pharmaceuticals
amplification leads to gefitinib resistance in lung cancer by activating
ERBB3 signaling. Science 2007, 316, 1039−43.
PLC, Budapest, Hungary) for providing help in molecular
modeling studies.
(13) Bean, J.; Brennan, C.; Shih, J. Y.; Riely, G.; Viale, A.; Wang, L.;
Chitale, D.; Motoi, N.; Szoke, J.; Broderick, S.; Balak, M.; Chang, W.
C.; Yu, C. J.; Gazdar, A.; Pass, H.; Rusch, V.; Gerald, W.; Huang, S. F.;
Yang, P. C.; Miller, V.; Ladanyi, M.; Yang, C. H.; Pao, W. MET
amplification occurs with or without T790M mutations in EGFR
mutant lung tumors with acquired resistance to gefitinib or erlotinib.
Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20932−20937.
ABBREVIATIONS
■
EGFR, epidermal growth factor receptor; wt, wild-type; c-Met,
mesenchymal-epithelial transition factor; NSCLC, non-small
cell lung cancer; HGF, hepatocyte growth factor; RTKs,
receptor tyrosine kinases; TKI, tyrosine kinase inhibitor; InsR,
302
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303

ACS Medicinal Chemistry Letters
Letter
(14) Tang, Z.; Du, R.; Jiang, S.; Wu, C.; Barkauskas, D. S.; Richey, J.;
Molter, J.; Lam, M.; Flask, C.; Gerson, S.; Dowlati, A.; Liu, L.; Lee, Z.;
Halmos, B.; Wang, Y.; Kern, J. A.; Ma, P. C. Dual MET-EGFR
combinatorial inhibition against T790M-EGFR-mediated erlotinib-
resistant lung cancer. Br. J. Cancer 2008, 99, 911−922.
(15) Chen, H. J.; Jiang, Y. L.; Lin, C. M.; Tsai, S. C.; Peng, S. F;
Fushiya, S.; Hour, M. J.; Yang, J. S. Dual inhibition of EGFR and c-Met
kinase activation by MJ-56 reduces metastasis of HT29 human
colorectal cancer cells. Int. J. Oncol. 2013, 43, 141−50.
(16) Liu, X.; Newton, R. C.; Scherle, A. P. Development of c-MET
pathway inhibitors. Expert Opin. Investig. Drugs 2011, 20, 1225−1241.
(17) Kao, H. F.; Lin, C. C.; Yang, J. C. EGFR inhibitors as the first-
line systemic treatment for advanced non-small-cell lung cancer. Future
Oncol. 2013, 9, 991−1003.
(18) Mollard, A.; Warner, S. L.; Call, L. T.; Wade, M. L.; Bearss, J. J.;
Verma, A.; Sharma, S.; Vankayalapati, H.; Bearss, D. J. Design,
synthesis, and biological evaluation of a series of novel AXL kinase
inhibitors. ACS Med. Chem. Lett. 2011, 2 (12), 907−912.
(19) D’Angelo, N. D.; Bellon, S. F.; Booker, S. K.; Cheng, Y.; Coxon,
A.; Dominguez, C.; Fellows, I.; Hoffman, D.; Hungate, R.; Kaplan-
Lefko, P.; Lee, M. R.; Li, C.; Liu, L.; Rainbeau, E.; Reider, P. J.; Rex,
K.; Siegmund, A.; Sun, Y.; Tasker, A. S.; Xi, N.; Xu, S.; Yang, Y.;
Zhang, Y.; Burgess, T. L.; Dussault, I.; Kim, T. S. Design, synthesis,
and biological evaluation of potent c-Met inhibitors. J. Med. Chem.
2008, 51, 5766−79.
(20) Li, S.; Zhao, Y.; Wang, K.; Gao, Y.; Han, J.; Cui, B.; Gong, P.
Discovery of novel 4-(2-fluorophenoxy)quinoline derivatives bearing
4-oxo-1,4-dihydrocinnoline-3-carboxamide moiety as c-Met kinase
inhibitors. Bioorg. Med. Chem. 2013, 21, 2843−2855.
kinase oncogenic mutants and kinetic studies with aminopyridine and
triazolopyrazine inhibitors. Biochemistry 2009, 48, 5339−5349.
(28) Moyer, J. D.; Barbacci, E. G.; Iwata, K. K; Arnold, L.; Boman, B.;
Cunningham, A.; DiOrio, C.; Doty, J.; Morin, M. J.; Moyer, M. P.;
Neveu, M.; Pollack, V. A.; Pustilnik, L. R.; Reynolds, M. M.; Sloan, D.;
Theleman, A.; Miller, P. Induction of apoptosis and cell cycle arrest by
CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine
kinase. Cancer Res. 1997, 57, 4838−4848.
(29) Reid, A.; Vidal, L.; Shaw, H.; de Bono, J. Dual inhibition of
ErbB1 (EGFR/HER1) and ErbB2 (HER2/neu). Eur. J. Cancer 2007,
43, 481−489.
(30) Krypuy, M.; Newnham, G. M.; Thomas, D. M.; Conron, M.;
Dobrovic, A. High resolution melting analysis for the rapid and
sensitive detection of mutations in clinical samples: KRAS codon 12
and 13 mutations in non-small cell lung cancer. BMC Cancer 2006, 6,
295.
(31) Suda, K.; Tomizawa, K.; Mitsudomi, T. Biological and clinical
significance of KRAS mutations in lung cancer: an oncogenic driver
that contrasts with EGFR mutation. Cancer Metastasis Rev. 2010, 29,
49−60.
́
(32) Patane, S.; Pietrancosta, N.; Hassani, H.; Leroux, V.; Maigret, B.;
Kraus, J. L.; Dono, R.; Maina, F. A new Met inhibitory-scaffold
identified by a focused forward chemical biological screen. Biochem.
Biophys. Res. Commun. 2008, 375, 184−189.
(33) Fram, S. T.; Wells, C. M.; Jones, G. E. HGF-induced DU145 cell
scatter assay. Methods Mol. Biol. 2011, 769, 31−40.
(21) Furlan, A.; Colombo, F.; Kover, A.; Issaly, N.; Tintori, C.;
Angeli, L.; Leroux, V.; Letard, S.; Amat, M.; Asses, Y.; Maigret, B.;
Dubreuil, P.; Botta, M.; Dono, R.; Bosch, J.; Piccolo, O.; Passarella, D.;
Maina, F. Identification of new aminoacid amides containing the
imidazo[2,1-b]benzothiazol-2-ylphenyl moiety as inhibitors of tumori-
genesis by oncogenic Met signaling. Eur. J. Med. Chem. 2012, 47, 239−
54.
(22) Cai, Z. W.; Wei, D.; Schroeder, G. M.; Cornelius, L. A.; Kim, K.;
Chen, X. T.; Schmidt, R. J.; Williams, D. K.; Tokarski, J. S.; An, Y.;
Sack, J. S.; Manne, V.; Kamath, A.; Zhang, Y.; Marathe, P.; Hunt, J. T.;
Lombardo, L. J.; Fargnoli, J.; Borzilleri, R. M. Discovery of orally active
pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.
Bioorg. Med. Chem. Lett. 2008, 18, 3224−3229.
(23) Li, S.; Huang, Q.; Liu, Y.; Zhang, X.; Liu, S.; He, C.; Gong, P.
Design, synthesis and antitumour activity of bisquinoline derivatives
connected by 4-oxy-3-fluoroaniline moiety. Eur. J. Med. Chem. 2013,
64, 62−73.
(24) Li, J.; Wu, N.; Tian, Y.; Zhang, J.; Wu, S. Aminopyridyl/
pyrazinyl spiro[indoline-3,4′-piperidine]-2-ones as highly selective and
efficacious c-Met/ALK inhibitors. ACS Med. Chem. Lett. 2013, 4, 806−
810.
(25) Qian, F.; Engst, S.; Yamaguchi, K.; Yu, P.; Won, K. A.; Mock, L.;
Lou, T.; Tan, J.; Li, C.; Tam, D.; Lougheed, J.; Yakes, F. M.; Bentzien,
F.; Xu, W.; Zaks, T.; Wooster, R.; Greshock, J.; Joly, A. H. Inhibition
of tumor cell growth, invasion, and metastasis by EXEL-2880 (XL880,
GSK1363089), a novel inhibitor of HGF and VEGF receptor tyrosine
kinases. Cancer Res. 2009, 69, 8009−8016.
(26) Schroeder, G. M.; An, Y.; Cai, Z. W.; Chen, X. T.; Clark, C.;
Cornelius, L. A.; Dai, J.; Gullo-Brown, J.; Gupta, A.; Henley, B.; Hunt,
J. T.; Jeyaseelan, R.; Kamath, A.; Kim, K.; Lippy, J.; Lombardo, L. J.;
Manne, V.; Oppenheimer, S.; Sack, J. S.; Schmidt, R. J.; Shen, G.;
Stefanski, K.; Tokarski, J. S.; Trainor, G. L.; Wautlet, B. S.; Wei, D.;
Williams, D. K.; Zhang, Y.; Zhang, Y.; Fargnoli, J.; Borzilleri, R. M.
Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-
4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide
(BMS-777607), a selective and orally efficacious inhibitor of the Met
kinase superfamily. J. Med. Chem. 2009, 52, 1251−1254.
(27) Timofeevski, S. L.; McTigue, M. A.; Ryan, K.; Cui, J.; Zou, H.
Y.; Zhu, J. X.; Chau, F.; Alton, G.; Karlicek, S.; Christensen, J. G.;
Murray, B. W. Enzymatic characterization of c-Met receptor tyrosine
303
dx.doi.org/10.1021/ml4003309 | ACS Med. Chem. Lett. 2014, 5, 298−303