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4385
cMET in the presence of mouse plasma.21 The biochemical potency
against two cMET Y1230 mutants was reduced by 3–4ꢁ compared
to wild-type cMET.22 To establish the biochemical selectivity pro-
file, 6 was tested against a panel of 96 kinases.23 Besides cMET,
Aurora, Arg, and Abl were the only kinases that showed >50% inhi-
bition at 1 lM of 6; RON was not present in this screening panel.
The potent biochemical Aurora B activity in that assay did not
translate to the cellular setting with a mechanistic IC50 in the pres-
Table 6
Rodent PK of 6 (OSI-296)a
6 (OSI-296)
Mouse
Rat
iv Dosing
Dose (mg/kg)
Cl (mL/min/kg)
Vss (L/kg)
2
51
3.8
2.9
2
39
6.8
2.0
t
(h)
½
po Dosing
Dose (mg/kg)
Salt form
ence of mouse plasma of >10
impact on polyploidy in a FACS assay up to 10
l
M; in addition, 6 had no significant
M.24 It showed
20
20
l
Mesylate saltb
Free baseb
2.5
minimal activity against KDR in biochemical and cellular assays,
thus achieving differentiation from cMET/KDR inhibitors such as
XL880 and XL184.8 Compound 6 was less potent against ALK than
against cMET and RON and showed no significant activity in the
ALK-driven Karpas-299 xenograft model at doses of up to
200 mg/kg qd.25 While 6 inhibited proliferation of cMET-driven cell
lines such as MKN45 at concentrations near the respective cell
mechanistic IC50, no antiproliferative activity was observed in
non-driven lines, for example, HCT-116 and MDA-MB-231 (IC50
Cmax
(
lM)
4.8
5425
83
AUC0–last (ng h/mL)
%F
6204
72
a
PK parameters calculated by non-compartmental modeling using the median
concentration at each timepoint (three animals per timepoint).
b
Both formulated as solution in 40% HPCD in 0.01 M HCl.
Table 7
Selectivity data for 6 (OSI-296)a
>2 lM). This indicates that the antiproliferative effects of 6 are
Kinase
IC50
(
lM)
Kinase
IC50 (lM)
based on inhibition of the intended targets and not general cyto-
toxicity. Thus, OSI-296 (6) is best described as a selective cMET/
RON inhibitor.
The favorable pharmacokinetic profile displayed by 6 in the
mouse, with good exposure and oral bioavailability, led to its eval-
uation in pharmacodynamic and efficacy studies.
Pharmacodynamic (PD) studies were performed in the MKN45
xenograft model, evaluating effects on phosphorylation of the
cMET receptor in relation to plasma drug levels.
Following a 100 mg/kg single oral dose of 6, a >80% sustained
inhibition of MET auto-phosphorylation (pY1234/1235) was ob-
cMET biochem.
cMET Y1230C
cMET Y1230H
cMET cell mech.
cMET mech. + mpb
RON biochem.
sfRON cell mech.
0.026
0.072
0.11
0.042
0.60
ALK biochem.
0.040
0.50
1.3
6.1
0.011
1.0
ALK cell mech.
KDR biochem.
KDR cell mech.
AurB biochem.
AurB cell mech.
AurB mech. + mpb
0.025
0.20
>10
a
Cell mechanistic assays monitored the autophosphorylation sites of cMET
(MKN45), RON (HeLa-sfRON), ALK (Karpas-299), and KDR (HUVEC) or p-Ser10 of
histone H3 for Aurora B (HT-29). Biochemical IC50 values were determined using
the Invitrogen Omnia format at an ATP concentration equal to Km
.
b
Cell mechanistic assay in the presence of 50% mouse plasma.
served up to 8 h, corresponding to plasma levels of >3 lM. This cor-
related with the plasma concentration being above the cMET
mech. + mouse plasma IC50 value for the entire 8 h time period.
Drug levels dropped to 0.52 lM by 16 h, with slow recovery of
pMET content (45% inhibition at 24 h) (Fig. 3). Similar effects were
observed for pY1349 cMET (site essential for creating docking site
for downstream signaling; data not shown).
The in vivo efficacy of 6 dosed orally on a once-daily schedule
for 14 days was initially evaluated in the MKN45 xenograft model
in female nu/nu CD-1 mice (Fig. 4). The compound exhibited a clear
dose response, showing an ED50 of 50 mg/kg and reaching 100% tu-
mor growth inhibition (TGI) with regressions (27%) at 100 mg/kg
qd. Analysis of cMET pY1234/1235 phosphorylation status in tu-
mor samples collected at the end of dosing indicated that >90%
inhibition of pMET for 24 h correlated with complete inhibition
from the amide NH with the hinge backbone carbonyl of Met1160.
However, sub-micromolar cellular potencies could not be achieved
with these compounds.
From among these proximal rings, the tetrahydropyridine was
selected for further optimization (compounds 5x–5ab, 6). Gratify-
ingly, cellular potency was retained upon acetylation (5x), and the
PAMPA assay indicated good permeability (Pe at pH
7.4 = 199 ꢁ 10ꢂ6 cm/s). While the carbamate 5v and dimethylurea
5z had reduced potencies, the methyl urea 5aa and primary urea 6
showed comparable or better cell potencies than (R)-5a. Based on
the structural information, this may be explained by a hydrogen
bond between the urea NH and the backbone carbonyl of
Lys1161 at the tip of the hinge region. The sulfamide analog 5ab,
in contrast, was less potent.
Compound 6 (OSI-296) met the in vitro criteria of cellular cMET
potencies equal to or better than (R)-5a, significant permeability in
the PAMPA assay (Pe at pH 7.4 = 359 ꢁ 10ꢂ6 cm/s), and metabolic
stability as measured by extraction ratios (ERs) of 0.7 in both
mouse and human liver microsomes and was progressed to sin-
gle-dose mouse PK. It showed a significantly improved PK profile
Plasma Drug Conc
% pMET Content
10
8
120
100
80
60
40
20
0
6
4
compared to (R)-5a, achieving a Cmax of 2.4 lM with moderate
2
clearance and good oral exposure when dosed as an HCl salt in
PEG400/water. With an optimized formulation suitable for repeat
dosing in efficacy and toxicology studies [methanesulfonate (mes-
0
0
6
12
Time (h )
18
24
ylate) salt of 6 in 40% w/v HPCD (TrappsolÒ) in 0.01 M HCl], Cmax
,
oral exposure, and bioavailability were further improved (Table 6).
Good PK was also observed in female Sprague–Dawley rats. The
Figure 3. Correlation of inhibition of cMET phosphorylation and plasma drug
exposure in MKN45 tumor xenografts following oral dosing with 6 (OSI-296). The
blue line corresponds to median plasma concentrations of 6 at indicated time
following a 100 mg/kg qd dose. The orange line represents median pMET (pY1234/
1235) content normalized with total cMET in MKN45 tumors and expressed as a
percentage of control pMET content from vehicle-treated animals. The dotted blue
line indicates the cMET cell mechanistic IC50 in the presence of mouse plasma
high solubiliy of 6 at low pH (pH 3, >100
lM; pH 4.5, 11 lM) de-
creased to approx. 1 M around physiological pH.
l
Biochemical and cellular IC50 values are summarized in Table 7.
Compound 6 showed potent inhibition of cMET and RON in bio-
chemical and cellular assays with submicromolar activity against
(0.60 lM).