N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.01.068

A series of N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The compounds were potent against these two enzymes with IC₅₀

Full text of DOI:10.1016/j.bmcl.2009.01.068

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1323–1328
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimi-
dazolidine-1-carboxamides: A novel series of dual c-Met/VEGFR2 receptor
tyrosine kinase inhibitors
a
a
a
a
Stéphane Raeppel a, , Stephen Claridge , Oscar Saavedra , Frédéric Gaudette , Lijie Zhan ,
Michael Mannion a, Nancy Zhou a, Franck Raeppel a, Marie-Claude Granger a, Ljubomir Isakovic a,
Robert Déziel a, Hannah Nguyen b, Normand Beaulieu b, Carole Beaulieu b, Isabelle Dupont b,
Marie-France Robert b, Sylvain Lefebvre b, Marja Dubay c, Jubrail Rahil c, James Wang d, Hélène Ste-Croix b,
A. Robert Macleod b, , Jeffrey Besterman b, Arkadii Vaisburg a
*
a Department of Medicinal Chemistry, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
b Department of Cell Biology and Pharmacology, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
c Department of Lead Discovery, MethylGene Inc., 7220 rue Frederick-Banting, Montréal, QC, Canada H4S 2A1
d 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 series of N-(3-fluoro-4-(2-arylthieno[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-
carboxamides targeting c-Met and VEGFR2 tyrosine kinases was designed and synthesized. The com-
pounds were potent against these two enzymes with IC50 values in the low nanomolar range in vitro, pos-
sessed favorable pharmacokinetic profiles and showed high efficacy in vivo in several human tumor
xenograft models in mice.
Received 10 December 2008
Revised 20 January 2009
Accepted 21 January 2009
Available online 27 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
c-Met
VEGFR2
RTK inhibitors
Thieno[3,2-b]pyridine scaffold
Oncology
Receptor tyrosine kinases (RTKs) play crucial roles in numerous
signal transduction pathways and cellular processes. Many are
implicated in cancer,1 including the Hepatocyte Growth Factor
Receptor c-Met,2 which is over-expressed and/or mutated in vari-
ous human tumor types, and the Vascular Endothelial Growth Fac-
tor Receptors (VEGFR), which play key roles in tumor
angiogenesis.3 Dysregulation of these RTKs affects cell prolifera-
tion, survival and motility, leading to tumor growth, angiogenesis,
and metastasis. The combined inhibition of several RTKs is a prom-
ising approach for cancer therapy, as it targets multiple pathways
involved in angiogenesis and tumor survival.4
tent inhibitor of VEGFR2 but does not inhibit c-Met. Conversely,
PHA-665752 (2)8 is a potent inhibitor of c-Met but a weak inhibitor
of VEGFR2. In contrast to both molecules, representative quinoline
compounds of Kirin (3)9 and Exelixis (4)10 are effective against
both enzymes.
Recently, we reported on a series of 2-substituted thieno[3,2-
b]pyridine-based c-Met/VEGFR2 inhibitors exemplified by com-
pounds 5–10 (Table 1) bearing acyclic structural fragments, such
as arylacetyl thioureas, arylacetyl ureas or various arylmalona-
mides.11 Of particular interest was compound 8, which showed
the same potency against both c-Met and VEGFR2 as its des-
methyl analogue 7. We hypothesized that connecting the N-methyl
with the central methylene of the malonamide head group of com-
pound 8 via a CH2-linker, as illustrated in the N-phenylpyrroli-
done-type compounds 11 and 12, could preserve the same kinase
inhibitory properties. It turned out that compound 11 was indeed
similar to compound 8 (Table 1).12,13 Moving the carbonyl-group
(compound 13) or creating a quarternary center (compound 14)
resulted in a significant loss of activity against VEGFR2 for both
compounds compared to compound 11. The activities of both
Figure 1 highlights some of the known inhibitors of c-Met5 and/
or VEGFR6 enzymes from the patent and scientific literature.
Sutent (1),7 a marketed anticancer drug for patients with ad-
vanced renal cancer and gastrointestinal stromal tumors, is a po-
* Corresponding author. Tel.: +1 514 337 3333; fax: +1 514 337 0550.
E-mail address: raeppels@methylgene.com (S. Raeppel).
Present address: Isis Pharmaceuticals, 1896 Rutherford Road, Carlsbad, CA 92008,
USA.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.068

1324
S. Raeppel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1323–1328
H
N
H
N
Cl
O
O
H
H
S
F
N
N
O
O
Cl
H
N
N
O
O
Et2 N
Pfizer/Sugen, Sutent (1)
Pfizer, PHA-665752 (2)
c-Met (38 nM)
VEGFR-2 (863 nM)
N
µ
c-Met (> 5 M)
VEGFR-2 (185 nM)
H
N
H
N
F
c-Met (80 nM)
VEGFR-2 (32 nM)
R
Kirin (3), R =
S
O
O
MeO
MeO
H
N
c-Met (21 nM)
VEGFR-2 (7 nM)
F
Exelixis (4), R =
N
O
O
Figure 1. c-Met and/or VEGFR inhibitors with their IC50 values (generated in house).
Table 1
Enzymatic c-Met and VEGFR2 and cellular TPR-Met phosphorylation assay results for compounds 5–15 of general structure
H
F
N
2
R
O
N
S
N
1
N
R
Compound
R1
R2
c-Met IC50
0.037
(
lM)
VEGFR2 IC50
0.026
(lM)
Phospho-TPR-Met IC50
0.05
(lM)
H
N
5
Me
S
O
O
O
H
N
6
7
Me
Me
0.025
0.028
0.037
0.015
n.a.
O
O
H
N
0.31
Me
N
8
Me
0.043
0.010
0.16
O
O
H
N
9
Me
Me
0.028
1
0.009
0.057
0.06
1.44
O
O
O
O
Me
N
10
N
11
12
Me
0.035
0.039
0.039
0.016
0.37
0.21
O
O
O
N
i-Pr
O
O
N
13
14
15
i-Pr
Me
Me
0.3
0.8
n.a.
O
Me
N
0.067
0.023
0.23
0.008
1.00
0.12
O
O
O
O
N
N

S. Raeppel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1323–1328
1325
F
NO2
Cl
N
Cl
O
N
S
S
a
b
c
N
S
N
N
Me
N
B
A
N
Me
d)
N
F
NH2
HO
Cl
O
O
O
O
N
S
or
C
N
e)
N
Me
N
N
O
H
N
F
N
X
O
O
O
N
S
11
X = CH,
X = N, 15
N
N
Me
Scheme 1. Synthesis of 11 and 15. Reagents and conditions: (a) i—n-BuLi, THF, À78 °C; ii—ZnCl2, THF, À78 °C to rt; iii—Pd(PPh3)4, 4-iodo-1-methyl-1H-imidazole, THF, reflux.
(b) 2-Fluoro-4-nitrophenol, K2CO3, Ph2O, 180 °C. (c) NiCl2, NaBH4, MeOH. (d) HATU reagent, DIPEA, DMF. (e) DIPEA, DCM.
compounds against c-Met changed as well—only slightly for com-
pound 14 but more significantly for compound 13. We then re-
placed the stereogenic center in the pyrrolidone fragment of
compound 11 with a nitrogen atom to form the corresponding cyc-
lic urea analogue 15. This compound turned out to be equipotent
to compound 8 at both the enzymatic14 and cellular15 level. Thus,
rigidification of the arylmalonamide fragment into the arylimidaz-
olidine motif is well tolerated, giving rise to a novel class of c-Met/
VEGFR2 kinase inhibitors, the N-(3-fluoro-4-(2-arylthieno[3,2-b]-
pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carbox-
amides (e.g. compound 15).12,13
Synthesis of compounds 11 and 15 is shown in Scheme 1. 7-
Chlorothieno[3,2-b]pyridine and 4-iodo-1-methyl-1H-imidazole
underwent a Negishi coupling reaction to form a methylimidazolyl
substituted thienopyridine A.16 This material was then reacted
with 2-fluoro-4-nitrophenol under basic conditions at high tem-
perature to form the nitro compound B. The nitro group of B was
reduced using a mixture of sodium borohydride and nickel chlo-
ride in methanol to form the aniline C. The intermediate C was
treated either with 2-oxo-1-phenylpyrrolidine-3-carboxylic acid
in the presence of HATU reagent and diisopropylethylamine to
form compound 11, or with 2-oxo-3-phenylimidazolidine-1-car-
bonyl chloride17 and diisopropylethylamine to give the cyclic urea
15.
To study the SAR of compound 15, we first explored the substit-
uents at position 2 of the thieno[3,2-b]pyridine template (Table 2).
Elongation of the chain (compound 16) and/or incorporation of a
basic center (compounds 17–19) were well tolerated and provided
potent molecular entities. Changing the orientation of the imidaz-
olyl ring system—proceeding from compound 15 and 16 to com-
pounds 20 and 21—did not affect potency against c-Met but were
slightly less active against VEGFR2. Replacement of the imidazole
ring by a para-substituted phenyl ring (compounds 22–25) was
well tolerated. However, having three basic nitrogens, as in com-
pound 25 resulted in a dramatic decrease in cell-based activity.
The m-acetophenyl group in compound 26 proved to be detrimen-
tal and resulted in reduced enzyme inhibitory activity. A last mod-
ification which was explored was the replacement of the
heteroaryl or aryl appendages by either an amide (compound 27)
or a tetrahydropyridine (compound 28). Interestingly, compound
28 was significantly less active against VEGFR2.
We next investigated the presence of a small substituent on the
phenyl ring of the phenylimidazolidine fragment of the ethyl
substituted analogues of compound 16. (Table 3). The ortho- and
para-substituted fluoride atom allowed us to slightly improve
VEGFR2 inhibitory activity (compounds 29 and 30, compared to
compound 16). On the other hand, the presence of an ortho-fluoro
group (compound 30) had a slightly negative impact on cell-based
activity. Finally, although compound 31 with an ortho-methoxy
group was equipotent to compound 16, it showed reduced activity
in our c-Met-driven cellular assays (data not shown).
We also examined the replacement of the oxygen atoms by sul-
fur atoms in the dicarbonyl framework of compound 15 (Table 4).
Compound 32 (an analogue of compound 5) was as potent as com-
pound 15, while compound 33 was inactive against c-Met and
VEGFR2.
Rat pharmacokinetic analyses were performed on a small set of
compounds (Table 5). Compounds with N-methylimidazole frag-
ments (e.g. compounds 11 and 15) showed a short to medium
half-life and poor bioavailability. Results were encouraging, partic-
ularly for compound 29 as it displayed a long half-life and decent
bioavailability, an appreciable improvement when compared to
compound 5. Compounds that had basic substituents generally
had a shorter half-life and lower bioavailability (data not shown).
Compound 29 was further evaluated in other cell-based assays.
First, it was tested for its ability to inhibit two c-Met-specific func-
tional endpoints (Table 6). Compound 29 dramatically impaired
HGF-induced epithelial cell migration and scattering, showing that
it is able to inhibit c-Met-dependent cell motility events. Second,
compound 29 was tested in a variety of VEGF-dependent cellular
assays (Table 6). It potently inhibited the VEGF-induced phosphor-
ylation of ERK, a downstream effector of the VEGF pathway, and
also impeded the VEGF-dependent proliferation of human umbili-
cal vein endothelial cells (HUVEC). In an in vitro angiogenesis assay,
which measures the formation of tubules generated by a co-culture
of endothelial and fibroblast cells (AngiokitTM, TCS Cellworks),
compound 29 potently inhibited tubule growth and was also able
to almost completely inhibit tubule formation at the 100 nM dose

1326
S. Raeppel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1323–1328
Table 2
Enzymatic c-Met and VEGFR2 and cellular TPR-Met phosphorylation assay results for compounds 16–28 of general structure
H
1'
N
F
N
N
1
3
4'
O
O
O
7"
S
R
2"
N
Compound
R
c-Met IC50
0.025
(lM)
VEGFR2 IC50
0.012
(lM)
Phospho-TPR-Met IC50
0.04
(lM)
N
16
N
Et
N
N
17
18
19
20
21
0.034
0.035
0.021
0.026
0.024
0.014
0.02
0.06
0.32
0.30
0.12
0.14
N
Me N
2
N
N
N
0.008
0.031
0.026
N
N
N
N
Me
N
N
Et
N
22
23
24
0.022
0.029
0.016
0.011
0.012
0.008
0.42
0.25
0.08
HN
Me
N
N
N
N
O
MeHN
N
N
25
26
27
28
0.028
0.2
0.003
0.88
0.83
0.27
0.23
0.30
N
O
O
0.042
0.033
0.023
0.17
N
Me N
2
Me
N

S. Raeppel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1323–1328
1327
Table 3
Table 7
Enzymatic c-Met and VEGFR2 and cellular TPR-Met phosphorylation assay results for
Effect of compound 29 on human cancer cell proliferation
compounds 29–31 of general structure
Compound MKN-45 IC50
M)
MNNG-HOS
IC50 M)
HCT 116 IC50
M)
MDA MB-231
R
(l
(
l
(
l
IC50
(lM)
H
N
F
N
N
29
0.01
0.033
13
20
O
O
O
N
S
Third, compound 29 was evaluated for its ability to inhibit the
proliferation of a variety of human cancer cell lines, using a 72 h
MTT assay. It displayed nanomolar IC50s in tumor lines whose
growth is known to be sensitive to c-Met inhibition, namely the
MKN-45 gastric carcinoma and the TPR-Met-expressing MNNG-
HOS cell lines. In contrast, significantly higher concentrations of
compound 29 were required to inhibit the growth of c-Met-inde-
pendent HCT116 colon carcinoma and MDA-MB-231 breast adeno-
carcinoma cells (Table 7). These results show that compound 29
specifically targets c-Met-driven cancer cell proliferation in
culture.
N
N
Et
Compound
R
c-Met IC50
M)
VEGFR2 IC50
M)
Phospho- TPR-Met IC50
(lM)
(
l
(l
16
29
30
31
H
0.025
0.027
0.021
0.026
0.012
0.007
0.003
0.014
0.04
0.05
0.10
0.06
p-F
o-F
o-
OMe
Lastly, compound 29 was also profiled against a small set of ki-
nases [using Millipore’s KinaseprofilerTM assay services] (Table 8).
It strongly inhibited VEGFR1, VEGFR3, Ron and Tie-2, moderately
inhibited Flt-3 and was not active against Aurora A.
Table 4
Enzymatic c-Met and VEGFR2 and cellular TPR-Met phosphorylation assay results for
compounds 32 and 33 of general structure
Finally, compound 29 was tested in a mouse xenograft model
bearing tumors derived from the c-Met-driven MKN-45 cells
(Fig. 2). Interestingly, oral administration of a 40 mg/kg dose once
daily for 12 days caused significant regression of MKN-45 tumor
growth.
H
N
F
N
N
Y
X
O
N
S
N
In conclusion, a series of 2-oxo-3-aryl-N-(4-(thieno[3,2-b]pyri-
din-7-yloxy)phenyl)imidazolidine-1-carboxamides was designed
and synthesized. The compounds show low nanomolar inhibitory
activity of both c-Met and VEGFR2 enzymes.
N
Me
Compound
X
Y
c-Met IC50
M)
VEGFR2 IC50
M)
Phospho-TPR-Met IC50
M)
(
l
(
l
(l
15
32
33
O
S
O
O
O
S
0.023
0.024
2
0.008
0.012
1.8
0.12
0.08
>10
Table 8
% enzyme inhibition at 100 nM of 29
VEGFR-1
99
VEGFR-3
99
Ron
100
Tie-2
99
FLT-3
80
Aurora A
9
Table 5
Rat PK analysis: iv half-life and bioavailability (F%) for select compounds with general
structure
1200.0
1000.0
800.0
600.0
400.0
200.0
0.0
H
N
2
F
N
X
R
O
O
no treatment
O
Compound 29 p.o. at
40mg/kg
N
S
N
1
N
R
Compound
R1
X
R2
t1/2 iv (h)a
F (%)b
5
Me
Me
Me
Et
1.2
1.1
2.3
6.3
12
14
13
37
11
15
29
CH
N
N
H
H
F
a
iv doses 2.17–2.86 mg/kg.
po doses 4.28–4.98 mg/kg.
b
-8 -6 -4 -2
0
2
4
6
8
10 12 14
Day
where cells were purposely supplemented with VEGF (Table 6).
These results show that compound 29 can potently inhibit VEGF-
dependent functional activity in cells.
Figure 2. Oral anti-tumor activity of compound 29 in the MKN-45 tumor xenograft
model. Compound 29 was dosed po, once daily, at 40 mg/kg (6 mice per group)
starting on Day 0.
Table 6
Effect of compound 29 on c-Met and VEGF-mediated cellular endpoints
Compound A549 wound healing
DU145 scattering
VEGF-dept phospho-
VEGF- dept HUVEC
Angiokit, Tubule
Angiokit, Tubule length
with VEGF (% inh.)
inh. IC50
(lM)
inh. IC50
(l
M)
ERK IC50
(lM)
proliferation IC50
(l
M)
length IC50
(lM)
29
0.24
0.08
0.001
0.006
0.006
98

1328
S. Raeppel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1323–1328
Armand, J.-P.; Scigalla, P.; Raymond, E. J. Clin. Oncol. 2006, 24, 25; (d) Faivre, S.;
Compound 29, the lead molecule, shows an excellent in vitro
Demetri, G.; Sargent, W.; Raymond, E. Nat. Rev. Drug Discov. 2007, 6, 734.
8. (a) Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti, P.; Chan, E.; Le, P.;
Chen, J.; Wang, X.; Ruslim, L.; Blake, R.; Lipson, K. E.; Ramphal, J.; Do, S.; Cui, J.
J.; Cherrington, J. M.; Mendel, D. B. Cancer Res. 2003, 63, 7345; (b) Ma, P. C.;
Schaefer, E.; Christensen, J. G.; Salgia, R. Clin. Cancer Res. 2005, 11, 2312; (c)
Smolen, G. A.; Sordella, R.; Muir, B.; Mohapatra, G.; Barmettler, A.; Archibald,
H.; Kim, W. J.; Okimoto, R. A.; Bell, D. W.; Sgroi, D. C.; Christensen, J. G.;
Settleman, J.; Haber, D. A. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 2316.
9. WO 2003/000660 A1.
profile and favorable pharmacokinetic characteristics and exhibits
significant in vivo efficacy in the MKN-45 human xenograft mouse
model. Efforts to further optimize pharmacokinetic characteristics
and physicochemical properties of N-(3-fluoro-4-(2-arylthie-
no[3,2-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-
1-carboxamides as well as attempts to expand this series of com-
pounds towards chemical entities with a different kinase inhibi-
tory profile is in progress.
10. WO 2005/030140 A2.
11. (a) WO 2006/010264 A1.; (b) US 2006/0287343 A1.; (c) US 2007/0004675 A1.;
(d) Claridge, S.; Raeppel, F.; Granger, M.-C.; Bernstein, N.; Saavedra, O.; Zhan, L.;
Llewellyn, D.; Wahhab, A.; Deziel, R.; Rahil, J.; Beaulieu, N.; Nguyen, H.;
Dupont, I.; Barsalou, A.; Beaulieu, C.; Chute, I.; Gravel, S.; Robert, M.-F.;
Lefebvre, S.; Dubay, M.; Pascal, R.; Gillespie, J.; Jin, Z.; Wang, J.; Besterman, J.;
Macleod, A. R.; Vaisburg, A. Bioorg. Med. Chem. Lett. 2008, 18, 2793.
12. The details for the synthesis and the characterization of all the new compounds
are described in US 2007/0004675 A1 and WO 2007/107005 A1.
13. The present work was first presented (poster session) at the International
Conference on Medicinal Chemistry. Drug Discovery and Selection. 43èmes
Rencontres internationales de Chimie Thérapeutique. July 4–6, 2007, Faculté
de Pharmacie, Lille, France.
14. 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
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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 VEGFR-2/KDR was
measured in a DELFIATM 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
MnCl2, 1.2 mM b-Mercaptoethanol, 0.1 mg/mL BSA and 3 uM vanadate. ATP
concentrations in the assay were 10
lM for c-Met (5Â the Km) and 0.6 lM for
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. (VEGFR-2/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 (%).
15. A cellular clone of 293T cells stably expressing TPR-Met (Park, M.; Dean, M.;
Cooper, C. S.; Schmidt, M.; O’Brien, S. J.; Blair, D. G.; Vande Woude, G. F. Cell
1986, 45, 895), the activated mutated form of the receptor Met, 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-well
plates
(Corning).
TPR-Met
autophosphorylated levels were detected by ELISA using the primary
antibodies anti-phospho-Tyrosine (Millipore, 4G10) and a reporter antibody,
anti-mouse -horseradish peroxidase (Sigma). 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
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triplicate treatment points were used to prepare IC50 curves using
a 4-
parameter fit model. These curves were calculated using GraFit 5.0 software.
16. 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
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