Identification of a novel series of potent RON receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.03.073

A novel series of N-(3-fluoro-4-(2-substituted-thieno[3,2-b]pyridin-7-yloxy)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamides targeting RON receptor tyrosine kinase was designed and synthesized. SAR study of the series allowed us to identify compounds possessing either inhibitory activity of RON kinase enzyme in the low nanomolar range with low residual activity against the closely related c-Met or potent dual inhibitory activity against RON and c-Met, - with no significant activity against VEGFR2 in both cases.

Full text of DOI:10.1016/j.bmcl.2010.03.073

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2745–2749
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of a novel series of potent RON receptor tyrosine kinase inhibitors
a,
Stéphane Raeppel a, , Frédéric Gaudette , Michael Mannion a, Stephen Claridge a, Oscar Saavedra a,
*
Ljubomir Isakovic a, Robert Déziel a, Normand Beaulieu b, Carole Beaulieu b, Isabelle Dupont b,
Hannah Nguyen b,à, James Wang c, A. Robert Macleod b,§, Christiane Maroun b, Jeffrey M. Besterman b,
Arkadii Vaisburg a
a Department of Medicinal Chemistry, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
b Department Cell Biology and Pharmacology, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
c Department PK/Analytical Chemistry, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of N-(3-fluoro-4-(2-substituted-thieno[3,2-b]pyridin-7-yloxy)phenyl)-1-phenyl-5-(trifluo-
romethyl)-1H-pyrazole-4-carboxamides targeting RON receptor tyrosine kinase was designed and syn-
thesized. SAR study of the series allowed us to identify compounds possessing either inhibitory
activity of RON kinase enzyme in the low nanomolar range with low residual activity against the closely
related c-Met or potent dual inhibitory activity against RON and c-Met, À with no significant activity
against VEGFR2 in both cases.
Received 19 January 2010
Revised 17 March 2010
Accepted 17 March 2010
Available online 19 March 2010
Keywords:
RON
Ó 2010 Elsevier Ltd. All rights reserved.
c-Met
RTK inhibitors
Thieno[3,2-b]pyridine
Oncology
Receptor tyrosine kinases are implicated in tumor development
and progression and are validated targets for the development of
therapeutics in oncology.1 Deregulation of RON (recepteur d’orig-
ine nantais) has been described in numerous types of cancers
including colorectal, breast, lung, pancreas, prostate and bladder
and occurs mainly through wild type receptor overexpression or
expression of variants harboring different deletions within the
extracellular domain, leading to constitutive receptor activation.2
Importantly, increased RON activation in tumor tissues correlated
with histological grading and tumor staging, and was a predictor
of poorer patient prognosis.3 RON is mainly expressed on epithelial
cells but also on macrophages, and is activated following ligation
by macrophage stimulating protein (MSP). The molecular mecha-
nisms through which RON contributes to tumorigenesis are begin-
ning to unfold and involve multiple biological functions including
proliferation, migration, invasion and the induction of angio-
genic/chemotactic factors in endothelial cells.
Recently, ImClone Systems (now a division of Eli Lilly & Co.)
developed IMC-41A10, a human IgG1 monoclonal antibody that
binds with high affinity to human RON RTK and blocks MSP ligand
binding.4 IMC-41A10 inhibited RON phosphorylation and signaling
following activation of cancer cell lines with MSP, and suppressed
biological functions such as MSP-dependent cell migration. Impor-
tantly, IMC-41A10 inhibited tumor growth by 50–60% in several
human xenograft tumor models including colon, lung and pancre-
atic carcinoma models, thus further validating RON as a valuable
oncology target. Small molecule inhibitors of RON have been de-
scribed as well. These chemical entities inhibit both Ron and the
closely related c-Met kinase.5 c-Met is found to be activated in a
large number of different cancers and small molecule inhibitors
targeting Met/Ron are presently under clinical evaluation in
patients with solid tumors.6 For example, a potent small-molecule
dual inhibitor of c-Met/RON was disclosed by Amgen.7 This
quinoline based compound having the 1-(2-hydroxy-2-methylpro-
pyl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-car-
boxamide head group (Fig. 1) inhibits both Met and RON enzymes
in the low nanomolar range and demonstrates anti-tumor activity
in a colorectal xenograft model in mice when dosed po at 100 mg/
kg once daily. Furthermore, Bristol-Myers Squib described BMS-
777607 as a new pyridine based selective and orally efficacious
inhibitor of the Met/RON kinase superfamily having a new cyclic
head group that has advanced into phase I clinical trials.8 To the
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: raeppels@methylgene.com (S. Raeppel).
Present address: Tranzyme Pharma Inc., 3001, 12ème Avenue nord, Sherbrooke,
QC, Canada J1H 5N4.
à
Present address: Eli Lilly & Co., Oncology Drug Discovery Research, 8A Biomedical
Grove #02-05 Immunos, 138648 Singapore, Singapore.
§
Present address: Isis Pharmaceuticals, 1896 Rutherford Road, Carlsbad, CA 92008,
USA.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.073

2746
S. Raeppel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2745–2749
Me
OH
EtO
Me
O
Me
H
N
N
F
N
N
H
F
N
O
O
O
F
O
O
Cl
H2N
N
Compound I
from Amgen
BMS-777607
from BMS
MeO
N
H
N
F
HG
N
N
(1), HG =
(2), HG =
Multi-targeted RTK
inhibitors
O
O
O
from MethylGene
N
S
H
H
N
N
N
N
Me
CF3
O
Figure 1. c-Met and/or VEGFR-2 inhibitors 1 and 2 as well as the Amgen and BMS c-Met/Ron inhibitors.
best of our knowledge, small molecules that specifically target RON
(but not c-Met) have not been described yet. The high sequence
similarity between the kinase catalytic domains of Met and RON
makes the design of such inhibitors challenging.6i
was identified as a weak inhibitor of both c-Met and VEGFR2,12
surprisingly showed activity against TPR-RON comparable to that
of 1. Thus, we hypothesized that combining the structural features
of the head groups of compounds 1 and 3, that is, rigidifying the
4,4,4-trifluoro-3-(phenylamino)butanamide moiety into the
We have previously reported on the two novel thieno[3,2-
b]pyridine based series of either dual c-Met/VEGFR2 inhibitors
with a five-membered cyclic urea head group9 or selective VEGFR
family inhibitors with an acyclic amide isostere head group,10
exemplified by structures 1 and 2, respectively (Fig. 1). These com-
pounds were evaluated for their inhibitory activity against the RON
kinase in a cell-based assay we developed, where TPR-RON was
stably expressed in a cell line and is constitutively active.11 Com-
pound 1 showed a submicromolar activity against TPR-RON while
compound 2 was a micromolar inhibitor of this enzyme (Table 1).
However compound 3—the regioisomer of compound 2—which
1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide
frag-
ment as illustrated by compound 4, may affect the kinase selectiv-
ity profile of the resultant chemical entities, and perhaps yield a
molecule with an even greater selectivity towards RON. Indeed,
when compound 4 was synthesized and evaluated it showed a dif-
ferent kinase selectivity profile compared to compounds 1 and 3: it
demonstrated a potent RON kinase inhibition in a Ron-specific cell-
based assay, reduced activity against c-Met and lack of activity
against VEGFR2 in in vitro kinase assays. As a consequence, we ini-
tiated the SAR study around compound 4 as a prototype of selec-
tive RON inhibitor.
First, we exercised a limited scaffold hopping—thieno[3,2-
b]pyridine, quinoline or pyridine—by keeping unchanged the new
cyclic head group and the central aromatic ring (Table 2). These
scaffolds are thought to bind to the ATP catalytic site in the hinge
region of the target kinase enzymes. Not surprisingly, the quino-
Table 1
Thieno[3,2-b]pyridine based kinase inhibitors 1–4 with different head groups
HG
F
O
line-based
1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carbox-
N
S
amide 5 was equipotent to compound 4 and showed a similar
kinase profile. However, the N-methylpicolinamide-based ana-
logue 6 was completely inactive against both TPR-RON and c-Met
enzymes. With the ultimate goal to improve both the enzymatic
and cellular activities of the newly discovered 1-aryl-5-(trifluoro-
methyl)-1H-pyrazole-4-carboxamides against RON we could ex-
plore either analogues of 4 or congeners of 5. We chose to focus
on the thieno[3,2-b]pyridine series of compounds—analogues of 4
since we had in place chemistry leading to the substituted thieno-
pyridine derivatives.9 A typical synthesis of the target molecules is
illustrated in Scheme 1 in which the aniline derivatives A9,10 react
with 1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxylic acid
(commercially available from Maybridge) in the presence of HATU
reagent and DIPEA to afford compounds B.13
Next, we investigated the substitution pattern of the pyrazole
ring, which is thought to occupy the hydrophobic back pocket of
the kinase catalytic domain of the target enzymes (Table 3).
Replacement of the bulky and hydrophobic trifluoromethyl group
by a small alkyl substituent (compounds 7 and 8) decreased the
activity against TPR-RON and slightly increased the activity against
c-Met and VEGFR2. The ‘unsubstituted’ compound 9 demonstrated
a further decrease of activity against TPR-RON showing the same
N
N
Compd Head groups (HG)
TPR-RON
c-Metb
IC50
(lM)
VEGFR2b
IC50 M)
ELISAa IC50
(l
(
lM)
H
N
N
N
1
0.17
0.02
0.01
O
O
H
N
H
N
2
3
1.03
0.41
0.12
0.70
0.01
0.21
CF3
O
O
H
N
H
N
CF3
N
H
N
N
4
0.05
0.15
>10
CF3
O
a
Cell-based assay.
Enzymatic assays.
b

S. Raeppel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2745–2749
2747
Table 2
Table 3
1-Phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamide kinase inhibitors based on
Head group variations (compounds 4 and 7–12)
different scaffolds
HG
F
N
N
H
F
N
O
N
S
CF3
O
O
N
N
Ar
c-Metb
VEGFR2b
M) IC50 M)
Compd Head groups (HG)
TPR-RON
c-Metb
IC50
M)
VEGFR2b
Compd Ar
TPR-RON
ELISAa IC50
IC50
(lM)
ELISAa IC50
IC50
(l
(l
(lM)
(
l
(lM)
N
H
N
N
S
N
4
7
0.05
0.15
0.21
0.59
>1
0.15
0.07
0.07
0.08
0.18
0.06
0.07
>10
4
0.05
0.08
0.15
>10
N
CF3
O
O
O
O
O
O
O
N
N
N
H
N
MeO
MeO
2.0
1.9
1.4
0.8
0.7
3.0
5
6
0.20
>15
>2
CH3
N
N
N
H
N
8
H
N
>10
>20
n-Pr
N
N
N
O
H
N
9
a
Cell-based assay.
Enzymatic assays.
b
N
N
H
N
10
11
12
activity against c-Met and VEGFR2. Replacement of the trifluoro-
methyl group by an amino group, a potential hydrogen bond donor
(compound 10) abolished the activity against TPR-RON, slightly
decreased the activity against c-Met and maintained measurable
activity against VEGFR2. The replacement of the terminal phenyl
ring by a methyl group (compound 11) reduced the inhibition of
TPR-RON, slightly improving the activity against c-Met, and to a
great extent restored the activity against VEGFR2. Finally, intro-
duction of the bulky tert-butyl group instead of the methyl re-
stored the activity against RON and maintained the inactivity
against the VEGFR2 enzyme (compound 12).
Thus, a bulky and an electronegative trifluoromethyl group at
the 5-position of the pyrazole system as well as an aromatic phenyl
ring at the position 1 of the same ring seem to be essential for the
potent inhibition of RON enzyme and the desired decreased inhibi-
tion against both c-Met and VEGFR2. The presence of these two
substituents in compound 4 may allow the head group to adopt
a specific conformation and interact favorably with the hydro-
phobic back pocket of the RON kinase active site, possibly via a
hydrogen bond network and/or hydrophobic contacts of the tri-
NH2
N
N
H
N
Me
>1
CF3
N
N
H
N
0.20
CF3
a
Cell-based assay.
Enzymatic assays.
b
to the kinase active site of VEGFR2 and—although to a lesser
extent—to that of c-Met as well.
We also explored the nature of the substituent R—presumed to
extend into the solvent exposed area and potentially capable
of modulating physicochemical and pharmacokinetic properties
of the inhibitors—in N-(3-fluoro-4-(thieno[3,2-b]pyridin-7-yloxy)
phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamides
(compounds 13–21, Table 4). Compound 13—an isomer of 4 (both
bearing a rather small N-methylimidazole substituent)—maintained
relatively weak inhibitory activity against c-Met (150 nM), a micro-
molar activity against VEGFR2 enzyme but importantly, showed
fluoromethyl group and a p–p stacking and/or hydrophobic inter-
actions of the phenyl group (e.g., compound 4 vs compounds 9 and
11). At the same time, the presence of these two substituents on
the head group may preclude compound 4 from binding properly
N
N
HO
N
N
CF3
O
F
NH2
H
F
N
HATU reagent
DIPEA
CF3
O
O
O
S
DMF
rt
S
R
A
B
R
N
N
Scheme 1. General synthesis of N-(3-fluoro-4-(2-substituted-thieno[3,2-b]pyridin-7-yloxy)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamides.

2748
S. Raeppel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2745–2749
Table 4
EphB4, FAK, GSK3b, Haspin, IKKb, JAK2, JAK3, LIMK1, MEK1,
PDK1, PI3-b, Pim-1, PKB , Ret and Tie2.
Compound 4 was evaluated for its ability to inhibit c-Met and
RON in multiple cell-based assays (Table 5). Consistent with its
sub-micromolar inhibitory activity against the c-Met enzyme
Variation of substituents in N-(3-fluoro-4-(2-substituted-thieno[3,2-b]pyridin-7-
yloxy)phenyl)-1-phenyl-5-(trifluoromethyl)-1H-pyrazole-4-carboxamides series
a
N
H
N
F
N
(0.15 lM), compound 4 displayed weak inhibition of c-Met phos-
CF3
O
phorylation in the MKN-45 gastric carcinoma cell line which
over-expresses c-Met, as well as c-Met-dependent cell migration
and scattering mediated by HGF, in two other cancer cell lines, a
non-small-lung carcinoma cell line A549 and a prostate carcinoma
cell line DU145. As predicted, these results demonstrate that com-
pound 4 is a weak inhibitor of c-Met when compared to compound
1. However, when compound 4 was evaluated for it ability to inhi-
bit RON phosphorylation, it demonstrated potent inhibition of RON
phosphorylation in several models. RON has recently been de-
scribed to play an important role in prostate tumorigenesis: knock-
O
S
R
N
Compd
R
TPR-RON
c-Metb VEGFR2b
IC50
M)
ELISAa IC50 IC50
(l
M)
(l
M)
(l
N
4
0.05
0.06
0.15
0.15
>10
N
down of RON in
a prostate carcinoma cell line, PC3, that
N
overexpresses RON, resulted in reduced tumor growth.14 There-
fore, we determined in this model whether compound 4 inhibited
the phosphorylation of endogenously expressed full length RON.
Compound 4 potently inhibited RON phosphorylation in these can-
cer cells with an IC50 of 30–40 nM (Fig. 2).15 This is consistent with
the low-nanomolar IC50 values for compound 4 obtained in our
RON kinase cell-based assay (50 nM). Furthermore, the inhibition
of RON phosphorylation was also analyzed in another cancer cell
model, the HCT116 colon carcinoma, where RON is expressed
and phosphorylated in the absence of stimulation with exoge-
nously added RON ligand (MSP).16 In these cells, compound 4 sig-
nificantly inhibited RON phosphorylation in a dose-dependent
manner (Fig. 3).15 Interestingly, although the proliferation of these
cells may not be driven by RON exclusively, compound 4 potently
inhibited cellular proliferation in this model, as detected in a MTT
assay (Table 5).
13
1.4
N
O
N
MeO
14
0.06
0.03
2.5
MeO2S
NH
15
16
0.08
0.03
0.06
0.05
1.1
1.4
O
Me2N
O
17
0.09
0.03
0.7
N
Me2N
Compound 4 was further evaluated for its pharmacokinetic prop-
erties in the rat (Table 6). Compound 4 showed a 2.7 h half-life, a rea-
sonably low rate of clearance and good oral bioavailability.
O
18
19
0.10
0.03
0.04
0.04
1.0
2.5
N
Me2N
O
Table 5
Effect of compounds 1 and 4 on c-Met- mediated cellular endpoints and on human
cancer cell proliferation
Me2N
N
O
Compd
c-Met-driven cell-based assay
Tumor
MTT
assay
HCT116
IC50
20
21
0.04
0.09
0.06
0.05
5.3
1.4
NH
N
MeO
O
N
c-Met in
A549 Wound
Healing inh. IC50
DU145
Scattering inh.
MKN-45 IC50
(
lM)
(l
M)
IC50
(l
M)
(lM)
1
4
0.07
0.5
0.08
0.4
0.08
2
n.a.
0.08
a
Cell-based assay.
Enzymatic assays.
b
nanomolar activity in the TPR-RON assay. However to our surprise,
compounds 14–21, bearing bulkier substituents R of different shape
and/or electronic properties, while maintaining good activities
against RON (30–100 nM), were equally active against c-Met in the
range of 30–90 nM and generally much less active against VEGFR2.
Thus, compounds 4, 13–21 showed good selectivity for the RON
and c-Met enzymes with compounds 4 and 13 being the most
RON-selective.
Compound 4 was profiled and tested at 0.1 lM against 23 ki-
nases using Millipore’s Kinaseprofiler™ assay services. In addition
to targeting RON receptor tyrosine kinase (>95% of inhibition), it
was also active against Flt3 (96% of inhibition) and moderately ac-
tive against TrkA (81% of inhibition) but importantly, did not show
significant inhibitory activity against a representative panel of hu-
man kinases (<40% of inhibition): ALK, Bmx, CHK1, cKit, c-Raf,
Figure 2. Effect of compound 4 against endogenous RON enzyme in the PC-3
prostate cancer cells.

S. Raeppel et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2745–2749
2749
7. (a) Zhang, Y.; Kaplan-Lefko, P. J.; Rex, K.; Yang, Y.; Moriguchi, J.; Osgood, T.;
Mattson, B.; Coxon, A.; Reese, M.; Kim, T.-S.; Lin, J.; Chen, A.; Burgess, T. L.;
Dussault, I. Cancer Res. 2008, 68, 6680; (b) Liu, L.; Siegmund, A.; Xi, N.; Kaplan-
Lefko, P.; Rex, K.; Chen, A.; Lin, J.; Moriguchi, J.; Berry, L.; Huang, L.; Teffera, Y.;
Yang, Y.; Zhang, Y.; Bellon, S. F.; Lee, M.; Shimanovich, R.; Bak, A.; Dominguez,
C.; Norman, M. H.; Harmange, J.-C.; Dussault, I.; Kim, T.-S. J. Med. Chem. 2008,
51, 3688.
8. Schroeder, G. M.; An, Y.; Cai, Z.-W.; Chen, X.-T.; Clark, C.; Cornelius, L. A. M.; Dai,
J.; Gullo-Brown, J.; Gupta, A.; Henley, B.; Hunt, J. T.; Jeyaseelan, R.; Kamath, A.;
Kim, K.; Lippy, J.; Lombardo, L. J.; Manne, V.; Oppenheimer, S.; Sack, J.; 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. J. Med. Chem.
2009, 52, 1251.
9. Raeppel, S.; Claridge, S.; Saavedra, O.; Gaudette, F.; Zhan, L.; Mannion, M.; Zhou,
N.; Raeppel, F.; Granger, M.-C.; Isakovic, L.; Déziel, R.; Nguyen, H.; Beaulieu, N.;
Beaulieu, C.; Dupont, I.; Robert, M.-F.; Lefebvre, S.; Dubay, M.; Rahil, J.; Wang, J.;
Ste-Croix, H.; Macleod, A. R.; Besterman, J.; Vaisburg, A. Bioorg. Med. Chem. Lett.
2009, 19, 1323.
Figure 3. Effect of compound 4 against endogenous RON enzyme in the HCT116
10. Gaudette, F.; Raeppel, S.; Nguyen, H.; Beaulieu, N.; Beaulieu, C.; Dupont, I.;
Macleod, A. R.; Besterman, J.; Vaisburg, A. Bioorg. Med. Chem. Lett. 2010, 20, 848.
11. The catalytic domain of RON was cloned downstream of a dimerizing domain
from the TPR gene in a homologous fashion to the documented activated TPR-
Met gene (Park, M.; Dean, M.; Cooper, C. S.; Schmidt, M.; O’Brien, S. J.; Blair, D.
G.; Vande Woude, G. F. Cell 1986, 45, 895). A cellular clone of 293T kidney
epithelial cells stably expressing this activated form of RON under a CMV
promoter was derived. Cells were treated with compounds dilutions for
150 min and lysate samples from treatment wells were transferred to high
binding white polysterene 96 wells plates (Corning). TPR-RON
autophosphorylated levels were detected by ELISA using the primary
antibodies anti-phospho-Tyrosine (Millipore, 4G10) and a reporter antibody
(HRP-cross linked anti-mouse from Sigma), and plates were washed on a plate
washer (SkanWasher, Molecular Devises) and subsequently incubated with
chemiluminescent substrate solution (ECL, Roche). Luminescence signal was
captured on a Polar Star Optima apparatus (BMG LabTech). Average values of
lung cancer cells.
Table 6
Rat pharmacokinetic profile of compound 4
PK propertiesa
Compd 4
T1/2 (h), iv
CL (L/h/kg)
Vss (L/kg)
Tmax (h), po
2.7
0.63
2.4
6.0
Cmax
(
lM/(mg/kg)), po
0.25
0.84
>30
AUC0_6 (lM h/(mg/kg)), po
% F
triplicate treatment points were used to prepare IC50 curves using
a 4-
a
parameter fit model. These curves were calculated using GraFit 5.0 software.
TPR-RON was first described by Santoro, M. M.; Collesi, C.; Grisendi, S.;
Gaudino, G.; Comoglio, P. M. Mol. Cell Bio. 1996, 16, 7072.
iv dose: 2.5 mg/kg, po dose: 5.0 mg/kg.
12. In vitro Kinase Assays (c-Met and VEGFR-2/KDR): Preparation of GST fusion
proteins: recombinant baculovirus containing the catalytic domain of c-Met
and of the VEGFR-2/KDR receptor fused to glutathione S-transferase (GST)
fusion genes were 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
In conclusion, a novel series of N-(3-fluoro-4-(2-substituted-thi-
eno[3,2-b]pyridin-7-yloxy)phenyl)-1-phenyl-5-(trifluoromethyl)-
1H-pyrazole-4-carboxamides was designed and synthesized. The
most attractive compounds of the series—4 and 13—are selective
for RON kinase enzyme with only residual activity against c-Met
and no significant inhibitory activity against VEGFR2. These novel
chemical entities represent a valuable starting point in design and
synthesis of RON selective inhibitors both as a tool to further vali-
date RON as a biological target and for the development of potential
anti-cancer therapeutics.
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 DELFIA™ assay (Perkin Elmer). The substrate poly(Glu4,Tyr) was
immobilized onto black high-binding polystyrene 96-well plates (Nunc
Maxisorp). The c-Met kinase reaction was conducted 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
References and notes
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lM for c-Met (5Â the Km) and 0.6 lM for
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VEGFR-2/KDR (2Â the Km). Enzyme concentration was 25 nM (c-Met) or 5 nM
(VEGFR-2/KDR).The recombinant enzymes were pre-incubated with inhibitor
and Mg-ATP on ice in polypropylene 96-well plates for 4 min, and then
transferred to the substrate coated plates. The subsequent kinase reaction took
place at 30 °C 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
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detected by time-resolved fluorescence in
a Gemini SpectraMax reader
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P.; Covino, N.; Bassi, R.; Prewett, M.; Gottfredsen, K. J.; Thobe, M. N.; Cheng, Y.;
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(Molecular Devices). Inhibitors were tested at 7 different concentrations each
in triplicate. IC50s were calculated in a 4 parameters equation curve plotting
inhibition (%).
13. The details for the synthesis and the characterization of all the new compounds
are described in WO 2007/107005 A1, US 2008/0004273 A1 and WO 2008/
046216.
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15. To confirm the cell based efficacy of the lead compound on endogenous RON,
PC-3 cancer cells or HCT116 cancer cells were treated for 3 h with the indicated
doses of compounds. Endogenously expressed RON proteins were
immunoprecipitated from cell lysates with anti-RON-beta total (c-20) (Santa
cruz biotechnologies) and subjected to immunoblotting analysis using anti-
phospho-tyrosine (Millipore, 4G10) or anti-RON-beta total (c-20) (Santa cruz
biotechnologies) as indicated. Protein detection was completed using anti-
mouse or anti-rabbit horseradish peroxidase (trueblot from e-Bioscience), and
subsequently incubated with chemiluminescent substrate solution (ECL plus,
Roche). Fluorescent signal was captured on a Storm apparatus (Amersham
Biosciences).
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