Discovery of a novel and potent series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases

Article abstract of DOI:10.1016/j.bmcl.2008.04.009

A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds demonstrated potency with IC₅₀ values in the low nanomolar range in vitro while the lead compound also showed in vivo activity against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.

Full text of DOI:10.1016/j.bmcl.2008.04.009

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2793–2798
Discovery of a novel and potent series of thieno[3,2-b]pyridine-based
inhibitors of c-Met and VEGFR2 tyrosine kinases
Stephen Claridge,a,* Franck Raeppel,a Marie-Claude Granger,a Naomy Bernstein,a
Oscar Saavedra,a Lijie Zhan,a David Llewellyn,a Amal Wahhab,a Robert Deziel,a
Jubrail Rahil,c Normand Beaulieu,b Hannah Nguyen,b Isabelle Dupont,b Annie Barsalou,b
Carole Beaulieu,b Ian Chute,b Serge Gravel,b Marie-France Robert,b Sylvain Lefebvre,b
Marja Dubay,b Roussen Pascal,d Jeff Gillespie,d Zhiyun Jin,d James Wang,d
Jeffrey M. Besterman,b A. Robert MacLeodb,ꢀ and Arkadii Vaisburga
aDepartment of Medicinal Chemistry, MethylGene Inc., 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
bDepartment of Cell Biology and Pharmacology, MethylGene Inc., 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
cDepartment of Lead Discovery, MethylGene Inc., 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
dDepartment of PK/Analytical Chemistry, MethylGene Inc., 7220 Frederick-Banting, Montre´al, Que., Canada H4S 2A1
Received 19 February 2008; revised 2 April 2008; accepted 2 April 2008
Available online 9 April 2008
Abstract—A series of thieno[3,2-b]pyridine-based inhibitors of c-Met and VEGFR2 tyrosine kinases is described. The compounds
demonstrated potency with IC50 values in the low nanomolar range in vitro while the lead compound also showed in vivo activity
against various human tumor xenograft models in mice. Further exploration of this class of compounds is underway.
ꢀ 2008 Elsevier Ltd. All rights reserved.
c-Met (the receptor for hepatocyte growth factor/scatter
factor, (HGF/SF)) and members of the vascular endo-
thelial growth factor receptor (VEGFR) family1 are
among the attractive receptor tyrosine kinases (RTKs)
pursued actively as potential targets for the development
of cancer therapeutics. c-Met is primarily expressed on
cells of epithelial and mesenchymal origin. Upon bind-
ing to its ligand, HGF, c-Met is activated, resulting in
the phosphorylation of tyrosine residues within its ki-
nase domain followed by phosphorylation of key resi-
dues in its unique multi-substrate-binding site. The
phosphorylated residues provide sites for the recruit-
ment of cellular adaptors and docking proteins leading
to the activation of signaling pathways culminating in
numerous biological responses, including cell migration
and invasion, proliferation and survival, as well as mor-
phogenesis and angiogenesis.2
While c-Met is involved in normal processes during
development and wound healing, its deregulation is
associated with tumorigenesis. The overexpression of
c-Met or mutations within and outside of its kinase do-
main leading to its constitutive activation have been de-
tected in cancer patients.3 In addition, autocrine or
paracrine activation of c-Met by its ligand, HGF, has
also been described.4 Together these mechanisms of c-
Met activation have been associated with poor progno-
sis. In addition to its role in tumor cell survival and
metastasis, c-Met is also implicated in angiogenesis
and has been shown to cooperate synergistically with
the vascular endothelial growth factor receptors (VEG-
FRs) including VEGFR1 (Flt1) and VEGFR2 (KDR).
These receptors have also been implicated in the devel-
opment and progression of various human cancers
and, therefore, have been central in the development
of anticancer therapies.5 Molecules that potently inhibit
both c-Met and VEGFR may have advantages over
VEGFR-selective or c-Met-selective molecules since
they can exploit several mechanisms of antitumor activ-
ity including anti-angiogenic activity from c-Met and
VEGFR inhibition, potential synergistic anti-angiogenic
activity from c-Met/VEGFR inhibition, and antitumor
Keywords: c-Met; VEGFR2; Kinase; Thieno[3,2-b]pyridine; Cancer.
*
Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337
0550; e-mail: claridges@methylgene.com
ꢀ
Present address: Takeda San Diego, 10410 Science Center Drive,
San Diego, CA 92121, USA.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.009

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S. Claridge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2793–2798
activity and anti-metastatic/anti-invasive activity from
c-Met inhibition.6 Thus, the combined inhibition of both
c-Met and VEGFR signaling represents a promising ap-
proach to cancer treatment by directly targeting multiple
pathways involved in tumor cell survival, as well as angi-
ogenesis and metastasis.7
moiety and this report will disclose their synthesis and
the SAR of the 2-position substitution of these thieno-
pyridine-based inhibitors, Scheme 1.
Thus, chlorination of 1 with POCl3 afforded 210 which
upon selective deprotonation11 with n-BuLi followed
by quenching of the intermediate carbanion with vari-
ous electrophiles afforded compounds 3a–e. Heating
3a–e in diphenyl ether at 180 ꢂC with 2-fluoro-4-nitro-
phenol and potassium carbonate gave the desired nitro-
phenyl ethers 4a–e, treatment of which with a mixture of
NaBH4 and NiCl2 Á6H2O12 gave anilines 5a–e. Reaction
of the aforementioned anilines with phenylacetyl isothi-
ocyanate13 afforded 6a–e, Scheme 1. A detailed descrip-
tion of the syntheses and characterization of 6a–e and
subsequent compounds mentioned in this letter is
provided.14
Figure 1 shows two small molecule inhibitors of VEG-
FR: Sunitinib (Sutentꢁ) from Pfizer is approved for
the treatment of both renal cell carcinomas (RCC) and
imatinib mesylate-resistant gastrointestinal stromal tu-
ꢁ
mor (GIST). Sorafenib (Nexavar ) from Bayer/Onyx
is approved for the treatment of advanced RCC and
hepatocellular carcinoma (HCC).
The report of the c-Met/VEGFR2 inhibitor (III) discov-
ered by Kirin Brewery in 20038 and shown in Figure 1
led to the genesis of the project with the initial goal to
identify an alternative to the heavily utilized quinoline
scaffold of III. Our preliminary investigations identified
thieno[3,2-b]pyridine9 as a good mimic of the quinoline
From Table 1, it may be seen that compounds 6d and 6e
were the most potent against both enzymes when
assayed in vitro. Although c-Met tolerated a range of
‘simple’ substituents in the 2-position of the thieno[3,2-
b]pyridine ring system, VEGFR2 preferred a carbonyl.
Thus, an additional set of molecules was made in efforts
to obtain more potent inhibitors of both enzymes.
H
H
N
N
NEt2
O
O
N
O
N
Cl
H
Table 1. In vitro15 profile for compounds III and 6a–e
CF3
N
H
F
H
N
H
H
N
F
N
O
N
H
O
S
O
O
I
Sorafenib (Nexavar®)
S
II
X
®
Bayer/Onyx
Sunitinib
(Sutent )
N
Pfizer
H
H
F
N
N
Compound
X
c-Met
IC50 (nM)
VEGFR2
IC50 (nM)
S
O
O
III
6a
6b
6c
6d
6e
—
H
121
116
132
130
130
113
32
1134
561
346
127
90
MeO
CH(OH)Me
SMe
III
MeO
N
Kirin Brewery
–CO-(2-Furyl)
CO2Me
Figure 1. Examples of some known VEGFR and/or c-Met inhibitors.
OH
N
Cl
N
Cl
c
S
S
a
b
S
X
N
1
3a-e
2
H
N
H
F
NO2
F
NH2
F
N
S
O
O
O
N
O
N
d
e
S
S
S
X
X
X
N
5a-e
6a-e
4a-e
Scheme 1. Reagents and conditions: (a) POCl3, reflux; (b) n-BuLi, À78 ꢂC, THF then electrophile X; (c) 2-fluoro-4-nitrophenol, Ph2O, K2CO3,
180 ꢂC; (d) NaBH4, NiCl2 Á6H2O, 0 ꢂC, MeOH, water; (e) BnCONCS, THF, rt or EtOH/toluene, rt.

S. Claridge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2793–2798
2795
Hence, a series of amides was synthesized using the
chemistry shown in Scheme 2 and their in vitro activities
are shown in Table 2.
search for an additional class of molecules in which
the amide moiety was replaced with various heterocycles
as amide isosteres. The chemistry used to achieve this is
shown in Schemes 3 and 4.
These data demonstrate that the amides are potent
nanomolar inhibitors of both enzymes and that the
amide functionality is well tolerated.
Table 3 shows the different 2-heteroaryl derivatives syn-
thesized. The choice of the chemical method used was
dependent on the availability of the respective halides
or boronic acids. The chemistry in Scheme 4 was prefer-
able over that shown in Scheme 3 due to the avoidance
of the use of tin reagents.
Unsubstituted (8a) or monosubstituted (8b) amides were
reasonably potent against c-Met and slightly less so
against VEGFR2. While dimethylamide 8c showed a
slight increase in potency against c-Met and a more pro-
nounced increase in activity against VEGFR2, bulkier
amides such as 8d, 8e, and 8g, on the contrary, were less
potent against both enzymes. Interestingly, compound
8f, which was synthesized as a constrained analogue of
compound 8d, showed a 40-fold increase in activity
against VEGFR2 suggesting that the active site of the
VEGFR2 enzyme has a tolerance for only rigid and
‘small’ amides, such as 8c and 8f. The weak in vivo effi-
cacy of the amides (data not shown), probably resulting
from their poor pharmacokinetics, necessitated the
Although all the compounds were active inhibitors, the
imidazole substituted compounds were among the most
potent of these analogues, when tested in in vitro kinase
assays against the c-Met and VEGFR2 enzymes. Inter-
estingly, in the TPR-Met assay, a cell-based assay which
detected the phosphorylation of Y1230-34-35 in a fusion
Cl
N
Cl
S
a
S
R
N
2
9a-k
Cl
N
Cl
N
H
N
H
S
a
S
F
N
CONR1R2
S
O
O
N
2
7a-g
S
b,c,d
R
H
H
F
N
N
S
O
10a-k
O
Scheme 3. Reagents and conditions: (a) i—n-BuLi, THF, À78 ꢂC, then
Bu3SnCl; ii—heteroaryl bromide or iodide, (PPh3)4Pd, toluene, reflux,
N2; (b) 2-fluoro-4-nitrophenol, K2CO3, 180 ꢂC, Ph2O; (c) NaBH4,
NiCl2 Á6H2O, 0 ꢂC, MeOH, water; (d) BnCONCS, THF, rt or EtOH/
toluene, rt.
S
b,c,d
CONR1R2
N
8a-g
Scheme 2. Reagents and conditions: (a) i— n-BuLi, À78 ꢂC, THF then
CO2 (g); ii—(COCl)2, DCM, reflux; iii—R1R2NH, DCM, rt; (b) 2-
fluoro-4-nitrophenol, K2CO3, 180 ꢂC, Ph2O; (c) NaBH4, NiCl2 Á6H2O,
0 ꢂC, MeOH, water; (d) BnCONCS, THF, rt or EtOH/toluene, rt.
F
NO2
Cl
N
O
Table 2. In vitro15 profile for compounds 8a–g
S
a
S
I
H
H
N
F
N
N
2
11
S
O
O
H
N
H
F
N
S
CONR1R2
S
O
N
O
N
S
b,c,d
Compound R1
R2 c-Met
IC50 (nM) IC50 (nM)
VEGFR2
R
8a
8b
8c
8d
8e
8f
H
H
H
65
80
129
133
19
10l-o
Me
Me
Et
Me
Et
48
Scheme 4. Reagents and conditions: (a) i—n-BuLi, THF, À78 ꢂC,
iodine,11
ii—2-fluoro-4-nitrophenol, K2CO3, 180 ꢂC, Ph2O; (b)
heteroaryl boronic acid, (PPh3)4Pd, DME, NaHCO3, CsF, H2O,
reflux, N2; (c) NaBH4, NiCl2Æ6H2O, 0 ꢂC, MeOH, water; (d)
BnCONCS, THF, rt or EtOH/toluene, rt.
114
125
54
386
289
9
;
N-Morpholine
N-Pyrrolidine
N-(4-Methylpiperazine)
8g
193
344

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S. Claridge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2793–2798
Table 3. In vitro15 profile of compounds 10a–o
H
N
H
F
N
S
O
O
S
R
N
Compound
R
c-Met IC50 (nM)
VEGFR2 IC50 (nM)
Y1230-34-35 TPR-Met IC50 (nM)
10a
10b
10c
10d
10e
10f
10g
10h
10i
1-Methyl-1H-imidazol-4-yl
1-Ethyl-1H-imidazol-4-yl
1-Methyl-1H-imidazol-2-yl
1-Ethyl-1H-imidazol-2-yl
1-Methyl-1H-pyrazol-4-yl
1-Methyl-1H-1,2,4-triazol-5-yl
Thiazol-2-yl
29
52
10
11
12
22
51
10
25
2
35
108
24
25
28
ND
20
69
65
17
17
28
20
Pyridin-2-y
Pyridin-3-yl
62
126
181
60
113
94
52
10j
Pyrimidin-2-yl
1,3,4-Thiadiazol-2-yl
Thiophen-2-y
200
ND
300
108
ND
5000
10k
10l
145
32
70
49
10m
10n
10o
1-Methyl-1H-pyrrol-2-yl
Furan-3-yl
Pyrimidin-5-yl
31
74
63
593
175
Table 5. Pharmacokinetic profile for 10a in two species
protein between c-Met kinase domain sequences and
TPR, thepyrimidine-based compounds 10j and 10o were
among the least active, possibly due to poor cellular
penetration.
Parameter
Rata
Dogb
t1/2, iv (h)
1.2
0.33
0.25
3.5
5.8
1.1
CL (L/(kg h))
Vss (L/kg)
Tmax, po (h)
1.5
1.1
Compounds 10a and 10c were found to be potential lead
candidates. However, the low stability found for 10c in a
human liver microsome test (data not shown) coupled
with its poor solubility (1.7 lg/mL compared with
30 lg/mL for 10a) led us to select 10a for additional
testing.
Cmax, po (lM/(mg/kg))
AUC, po (lM h/(mg/kg))
% F
0.14
0.74
12
0.21
0.74
42
a Dose, iv 2.5 mg/kg (four animals used), po 5 mg/kg (nine animals
used) and 25 mg/kg (three animals used).
b Dose, iv 0.8 mg/kg (two animals used), po 5 mg/kg (four animals
used).
Table 4 shows a head to head comparison of the lead
compound 10a against Sutentꢁ in various HGF- and
VEGF-dependent cell-based assays. In HGF-driven cell
migration and scattering assays, 10a inhibited both re-
sponses efficiently, in contrast to Sutentꢁ, which did
not inhibit c-Met enzymatic activity. However, both
inhibitors were comparable in VEGF-driven assays.
0.1 N HCl in 40/60 PEG400/saline was used to dose rats
po. For the dog studies, 0.04 N HCl in 25% HpbCD was
used for iv dosing while a mixture of 5% DMSO, 1%
Tween 80 in water and 0.1 N HCl in 40/60 PEG400/sal-
ine was used for po dosing to the dogs. Plasma samples
were analyzed using an Agilent 1100 HPLC system cou-
pled with an MDS Sciex API2000 triple quadrupole
mass spectrometer. WinNonLin software was used to
calculate the PK parameters.
Given its good in vitro enzyme inhibition and cell-based
profiles, the pharmacokinetic properties of 10a were
evaluated in vivo in rat and dog, Table 5.
Compound 10a (>95% chromatographic purity) was
administered to female Sprague–Dawley rats and male
beagle dogs. DMSO was used in iv dosing to rats while
a mixture of 5% DMSO, 1% Tween 80 in water and
The results in Table 5 show that 10a has a reasonable
half-life, 1.2 h in rats and 5.8 h in dogs, and has an
acceptable clearance, 0.33 L/(kg h) in rats and 1.1
Table 4. Comparison of 10a with Sutentꢁ
HGF-dependent
VEGF-dependent
A549 cell migration
IC50 (lM)
DU145 cell scattering
IC50 (lM)
HUVEC ERK phosphorylation
IC50 (lM)
HUVEC proliferation
IC50 (lM)
Sutent
10a
2
0.4
10
0.03
0.03
0.025
0.006
0.08

S. Claridge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2793–2798
2797
Table 6. The effect of oral dosage of 10a on various human tumor
models in vivo at 20 mg/kg once daily
3. (a) Maulik, G.; Shrikhande, A.; Kijima, T.; Ma, P. C.;
Morrison, P. T.; Salgia, R. Cytokine Growth Factor Rev.
2002, 13, 41; (b) Peschard, P.; Park, M. Oncogene 2007, 26,
1276; (c) Di Renzo, M.-F.; Olivero, M.; Martone, T.;
Maffe, A.; Maggiora, P.; De Stefani, A.; Valente, G.;
Giordano, S.; Cortesina, G.; Comoglio, P. M. Oncogene
2000, 19, 1547; (d) Lee, J.-H.; Han, S.-U.; Cho, H.;
Jennings, B.; Gerrard, B.; Dean, M.; Schmidt, L.; Zbar,
B.; Vande Woude, G. F. Oncogene 2000, 19, 4947; (e) Ma,
P. C.; Kijima, T.; Maulik, G.; Fox, E. A.; Sattler, M.;
Griffin, J. D.; Johnson, B. E.; Salgia, R. Cancer Res. 2003,
63, 6272.
Tumor model
Experiment % Tumor growth
duration (days) inhibition
Colo205 (colorectal)
DU145 (prostate)
HCT116 (colorectal)
16
11
14
41
57
41
61
94
MNNGHOS (osteosarcoma) 26
MKN45 (gastric) 13
4. (a) Corso, S.; Comoglio, P. M.; Giordano, S. Trends Mol.
Med. 2005, 11, 284; (b) Comoglio, P. M.; Boccaccio, C.
Semin. Cancer Biol. 2001, 11, 153; (c) Furge, K. A.; Zhang,
Y. W.; Vande Woude, G. F. Oncogene 2000, 19, 5582; (d)
Siegfried, J. M.; Weissfeld, L. A.; Singh-Kaw, P.; Weyant,
R. J.; Testa, J. R.; Landreneau, R. J. Cancer Res. 1997, 57,
439; (e) Moriyama, T.; Kataoka, H.; Tsubouchi, H.;
Koono, M. Fed. Eur. Biochem. Soc. Lett. 1995, 372, 78.
5. (a) Ribatti, D.; Vacca, A. Curr. Cancer Drug Targets 2005,
5, 573; (b) Olsson, A.-K.; Dimberg, A.; Kreuger, J.;
Claesson-Welsh, L. Nat. Rev. Mol. Cell Biol. 2006, 7, 359;
(c) Kiselyov, A.; Balakin, K. V.; Tkachenko, S. E. Expert
Opin. Investig. Drugs 2007, 16, 83.
6. (a) Xin, X.; Yang, S.; Ingle, G.; Rangell, L.; Kowalski, J.;
Schwall, R.; Ferrara, N.; Gerritsen, M. E. Am. J. Pathol.
2001, 158, 1111; (b) Van Belle, E.; Witzenbichler, B.;
Chen, D.; Silver, M.; Scwall, R.; Isner, J. M. Circulation
1998, 97, 381; (c) Gerritsen, M. E.; Tomlinson, J. E.; Zlot,
C.; Ziman, M.; Hwang, S. Br. J. Pharmacol. 2003, 140,
595.
L/(kg h) in dogs. The steady state volume of distribution
was low in rats (0.25 L/kg) and reasonable in dogs
(1.5 L/kg), while the oral bio-availability was deter-
mined to be 12% and 42% in rats and dogs, respectively.
As shown in Table 6, compound 10a performed well in
vivo against a panel of different human tumor types,
particularly those that are driven by or overexpress c-
Met (MNNGHOS and MKN45). Tumor growth inhibi-
tion at a dose of 20 mg/kg po once daily ranged from
41% to 94%. Compound 10a was found to show spill-
over inhibition of a number of kinases in addition to
the intended c-Met/VEGFR2 activity.16 Although dis-
appointing as it was hoped that 10a would be more
selective, the inhibition of multiple kinases could help
with the development of cancer treatments in which tu-
mor growth due to up-regulation of alternative kinase
signaling pathways, occurs. Furthermore, toxicity stud-
ies, using the MDS AdverseReactionEnzymeTM and Hit-
ProfilingTM Assay packages (MDS Pharma) showed that
10a did not exhibit any binding to receptors, channels,
or enzymes that could lead to potential toxicity liabili-
ties. Compound 10a (at 10 lM) did not inhibit
CYP450 3A4, however, it showed some activity against
CYP2C19, and 2DC (data not shown).
7. Peruzzi, B.; Bottaro, D. P. Clin. Cancer Res. 2006, 12,
3657.
8. (a) Fujiwara, A.; Senga, T.; Nishitoba, T.; Osawa, T.;
Miwa, A.; Nakamura, K. PCT Int. Patent Appl. WO
2003/000660; (b) Fujiwara, Y.; Senga, T.; Nishitoba, T.;
Osawa, T.; Nakamura, K. U.S. Patent 2004/0242603; (c)
The additional use of acyl thioureas as inhibitors of c-Met
may be found in the following references: Kung, Pei-Pei
PCT Int. Patent Appl. WO2005/121125; (d) Borzilleri, R.
M.; Chen, Z.; Huynh, T. N.; Vaccaro, W.; Xhen, X. T.;
Kim, K. S.; Cai, Z. W. PCT Int. Patent Appl. WO 2006/
004636; (e) Bannen, L. C.; Chan, D. S.; Dalrymple, L. E.;
Jammalamadaka, V.; Khoury, R. G.; Leahy, J. W.; Mac,
M. B.; Mann, G.; Mann, L. W.; Nuss, J. M.; Parks, J. J.;
Wang, Y.; Xu, W. PCT Int. Patent Appl. WO 2006/
014325.
9. Several compounds using scaffolds other than thieno[3,2-
b]pyridine were synthesized for example thieno[3,2-
b]pyrimidine, pyrrolo[3,2-b]pyridine, purine and pyrrol-
o[3,2-d]pyrimidine but these compounds were far inferior
to the thieno[3,2-b]pyridine in terms of potency. For
details please see Ref. 14.
In conclusion, a series of novel c-Met/VEGFR2 tyrosine
kinase inhibitors based upon the thieno[3,2-b]pyridine
scaffold were designed and synthesized. These com-
pounds exhibited excellent in vitro profiles and in partic-
ular, the most promising compound, 10a, has significant
antitumor activity in vivo. Additional SAR studies have
been undertaken and these will be reported in due
course.
Acknowledgment
10. (a) Barker, J. M.; Huddleston, P. R.; Holmes, D.; Keenan,
G. J.; Wright, B. J. Chem. Res. Miniprint 1984, 771; (b)
Barker, J. M.; Huddleston, P. R.; Holmes, D.; Keenan, G.
J.; Wright, B. J. Chem. Res. Miniprint 1982, 1726.
11. Ragan, J. A.; Raggon, J. W.; Hill, P. D.; Jones, B. P.;
Mcdermott, R. E.; Munchhof, M. J.; Marx, M. A.;
Casavant, J. M.; Cooper, B. A.; Doty, J. L.; Lu, Y. Org.
Process Res. Dev. 2003, 7, 676.
12. Nose, A.; Kudo, T. Chem. Pharm. Bull. 1981, 29, 1159.
13. Smith, P. A. S.; Kan, R. O. J. Org. Chem. 1964, 29, 2261.
14. Vaisburg, A.; Claridge, S. W.; Raeppel, F.; Saavedra, O.
M.; Bernstein, N.; Granger, M.-C.; Zhan, L.; Wahhab, A.;
Llewellyn, D. U.S. Patent 2006/0074056 A1.
We thank Dr. Christiane Maroun for her help in the
preparation of this manuscript.
References and notes
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2005, 225, 1; (b) Cohen, P. Nat. Rev. Drug Disc. 2002, 1,
309; (c) Ramos, M. J.; Moreira, I. S.; Fernandes, P. A.
Anticancer Agents Med. Chem. 2007, 7, 223; (d) Abidoye,
O.; Murukurthy, N.; Salgia, R. Rev. Recent Clin. Trials
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15. In vitro kinase assays (c-Met and VEGFR-2/KDR):
preparation of GST fusion proteins: recombinant baculo-
2. Birchmeier, C.; Birchmeier, W.; Gherardi, E.; Vande
Woude, G. F. Nat. Rev. Mol. Cell Biol. 2003, 4, 915.

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S. Claridge et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2793–2798
virus containing the catalytic domain of c-Met and of the
(VEGFR-2/KDR).The recombinant enzymes were pre-
incubated with inhibitor and Mg-ATP on ice in polypro-
pylene 96-well plates for 4 min, and then transferred to the
substrate coated plates. The subsequent kinase reaction
took place at 30 ꢂC for 30 min. (c-Met) or 10 min.
(VEGFR2/KDR). After incubation, the kinase reactions
were quenched with EDTA and the plates were washed.
Phosphorylated product was detected by incubation with
Europium-labeled anti-phosphotyrosine MoAb. After
washing the plates, bound MoAb was detected by time-
resolved fluorescence in a Gemini SpectraMax reader
(Molecular Devices). Inhibitors were tested at seven
different concentrations each in triplicate. IC50s were
calculated in a four parameters equation curve plotting
inhibition (%).
VEGFR-2/KDR receptor fused to glutathione S-transfer-
ase (GST) fusion genes was used to infect high five (c-Met)
or Sf9 (VEGFR-2/KDR) cells at a multiplicity of infection
of 1 or 0.1, respectively. Cell lysates were prepared after
ꢀ72 h of infection in 1% Triton X-100, 2 lg of leupeptin/
mL, and 2 lg of aprotinin/mL after ꢀ72 h of infection in
phosphate-buffered saline, and the fusion proteins were
purified over glutathione agarose (Sigma) according to the
manufacturer’s instructions. Biochemical kinase assays for
IC50 determination and kinetic studies: Inhibition of c-Met
and VEGFR2/KDR was measured in a DELFIATM assay
(Perkin-Elmer). The substrate poly(Glu4, Tyr) was immo-
bilized onto black high-binding polystyrene 96-well plates
(Nunc Maxisorp). The c-Met kinase reaction was con-
ducted in 25 mM Hepes, pH 7.5, containing 20 mM NaCl,
10 mM MgCl2, 5 mM b-mercaptoethanol, 0.1 mg/mL
bovine serum albumin (BSA) and 20 lM vanadate, while
the VEGFR-2/KDR reaction was conducted in 60 mM
Hepes, pH 7.5, containing 3 mM MgCl2, 3 mM MnCl2,
1.2 mM b-mercaptoethanol, 0.1 mg/mL BSA and 3 lM
vanadate. ATP concentrations in the assay were 10 lM for
c-Met (5· the Km) and 0.6 lM for VEGFR-2/KDR (2· the
Km). Enzyme concentration was 25 nM (c-Met) or 5 nM
16. Compound 10a at 100 nM concentration inhibited the
following enzymes: VEGFR-1 (91%), VEGFR-3 (100%),
Ron (80%), Tie-2 (81%), Flt-3 (96%), c-Kit (92%), Abl
(91%), and TrkA (99%). Compound 10a had <5%
inhibitory activity against the following enzymes CHK1,
EGFR, GSK3b, IGF-1R, IKK-b, JAK2, and JNKa1.
Compound 10a was evaluated using the KinaseprofilerTM
Kinase Selectivity Screening Service (radiometric protein
kinase assays) by Millipore.