Discovery of pyrrolopyridine-pyridone based inhibitors of met kinase: Synthesis, X-ray crystallographic analysis, and biological activities

Article abstract of DOI:10.1021/jm800476q

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC₅₀ value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC₅₀ values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

Full text of DOI:10.1021/jm800476q

5330
J. Med. Chem. 2008, 51, 5330–5341
Discovery of Pyrrolopyridine-Pyridone Based Inhibitors of Met Kinase: Synthesis, X-ray
Crystallographic Analysis, and Biological Activities
Kyoung Soon Kim,*,† Liping Zhang,† Robert Schmidt,† Zhen-Wei Cai,† Donna Wei,† David K. Williams,† Louis J. Lombardo,†
George L. Trainor,† Dianlin Xie, Yaquan Zhang, Yongmi An, John S. Sack, John S. Tokarski, Celia Darienzo,§
Amrita Kamath,§ Punit Marathe,§ Yueping Zhang,‡ Jonathan Lippy,∆ Robert Jeyaseelan, Sr.,‡ Barri Wautlet,‡ Benjamin Henley,‡
Johnni Gullo-Brown,‡ Veeraswamy Manne,‡ John T. Hunt,‡ Joseph Fargnoli,‡ and Robert M. Borzilleri†
Departments of Oncology Chemistry, Oncology Drug DiscoVery, Structural Biology and Modeling, and Metabolism and Pharmacokinetics,
Bristol-Myers Squibb Pharmaceutical Research & DeVelopment, P.O. Box 4000, Princeton, New Jersey 08543-4000
ReceiVed April 24, 2008
Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of
1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine
N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP
binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR
of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities
against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2
kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in
mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft
model.
Introduction
The Met receptor tyrosine kinase is a transmembrane growth
factor receptor that plays a critical role in both normal
development and tumorigenesis.1 The disulfide-linked het-
erodimeric Met protein consists of an R- and ꢀ-subunit with
the receptor tyrosine kinase domain residing in the ꢀ-chain. Met
is expressed predominantly on epithelial and endothelial cells.
It mediates a variety of epithelial functions, and deregulation
of the HGF/Met pathway has been implicated in a wide variety
of malignancies.2 Upon binding to its ligand, hepatcyte growth
Figure 1. Pyrrolopyridine N-acylurea 1.
factor (HGF), also known as scatter factor (SF), Met autophos-
phorylates tyrosines 1234 and 1235 within the activation loop
(A-loop).
inhibitory activity (Figure 1).9 Replacement of the acylurea
moiety by a conformationally constrained 2-pyridone as in
analogue 2 was envisioned as a new Met kinase inhibitor
chemotype (Figure 2). Structure-activity relationship (SAR)
studies on the 2-pyridone analogues led to the identification of
4-pyridone 3 and pyridine N-oxide 4 (Figure 2). The synthesis,
SAR, structural biology, and in vivo biological activities of these
compounds are described.
Subsequently, phosphorylation of Y1349 and Y1356 in the
multifunctional docking site at the C terminus occurs, which
mediates the docking of multiple downstream signal transducers
such as PI3K, SRC, and SHC.1,3,4 The biological consequences
of HGF/Met signal transduction include tumor growth, induction
of cell motility, invasiveness, metastases, and angiogenesis.1
Overexpression of Met or HGF and/or amplification of the Met
gene is frequently observed in a variety of human tumors
including liver, breast, pancreas, lung, kidney, bladder, ovary,
brain, prostate, and myeloma, among others.5,6 The HGF/Met
signaling pathway has been a popular target for diagnosis and
therapeutic intervention of human cancer. The growing list of
experimental reagents with therapeutic potential that target the
HGF/Met signaling pathway includes small molecules as well
as various biologicals that include neutralizing antibodies against
human HGF/SF and Met.7,8
Chemistry
Aminophenoxypyrrolopyridine 8 was prepared by coupling
4-chloropyrrolopyridine 510 with 2-fluoro-4-nitrophenol 6, fol-
lowed by reduction of the nitro group of intermediate 7 using
zinc metal (Scheme 1). Michael addition of 4-fluoroaniline 10
to pyrone ester 9 followed by intramolecular cyclization of the
Michael adduct provided p-fluorophenyl-2-pyridone ester 11,
which was hydrolyzed to afford p-fluorophenyl-2-pyridone acid
12. Coupling of amine 8 with 2-pyridone acid 12 produced
pyrrolopyridine-2-pyridone analogue 2. 2-Pyridone analogues
13-20 (Table 1) were synthesized using similar procedures
illustrated in Scheme 1.
The syntheses of various 4-pyridone substituted analogues
are depicted in Schemes 2-4. Fluorophenyl-ꢀ-keoester 24,
which was prepared from Meldrum’s acid adduct 23 according
to the literature procedure,11 was reacted with triazine 25 to
obtain ester 26.12 Ester 26 was hydrolyzed to yield 3-fluorophe-
The pyrrolopyridine derivative 1, containing an acylurea
moiety, was previously reported to have potent Met kinase
* To whom correspondence should be addressed. Phone: (609) 252-5181.
Fax: (609) 252-6601. E-mail: kyoung.kim@bms.com.
†
Department of Oncology Chemistry.
Department of Structural Biology and Modeling.
Department of Metabolism and Pharmacokinetics.
Department of Discovery Biology.
Department of Lead Evaluation.
§
‡
∆
10.1021/jm800476q CCC: $40.75
2008 American Chemical Society
Published on Web 08/09/2008

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5331
Figure 2. 2-Pyridone, 4-pyridone, and pyridine N-oxide inhibitors.
Scheme 1a
a Reagents and conditions: (a) i-Pr2NEt, microwave, 200 °C, NMP, 43%; (b) Zn, NH4Cl, MeOH/THF, room temp, overnight, 77%; (c) DMF, 0 °C, 7 h;
EDCI, DMAP, room temp, overnight, 64%; (d) 2 N NaOH, MeOH, 65 °C, 1.5 h, 99%; (e) TBTU, DMF/CH3CN, 0 °C to room temp, 3 h, 81%.
nylpyridine-4-pyridone acid 27. Coupling of acid 27 with
pyrrolopyridine amine 8 provided 4-pyridone analogue 3
(Scheme 2). N-Methyl or N-hydroxyethyl substituted 4-pyridone
analogues 34 and 37 were obtained from intermediate 3-bromo-
4-hydroxypyrine-5-carboxylic acid 29, which was obtained from
3,5-dibromo-4-hydroxypyridine 2813 via metalation and car-
boxylation (Scheme 3). Alkylation of acid 29 with methyl iodide
or the methyl ester of 29 with 2-hydroxybromoethane yielded
N-substituted 4-pyridone esters 30 and 35, respectively.
Miyaura-Suzuki coupling reaction14 of the bromo-4-pyridone
esters 30 and 35 with p-fluorophenylboronic acid, followed by
hydrolysis to the corresponding acids, and coupling of the acids
33 and 36 with pyrrolopyridine amine 8 afforded N-substituted
4-pyridone analogues 34 and 37, respectively. Alternatively,
N-substituted 4-pyridone analogues 41-43 were prepared
directly from 4-oxopyran 40 using the corresponding amine or
aniline (Scheme 4). Pyrone aldehyde 38,15 which was obtained
by Vilsmeier-Haack reaction of 4-fluorophenylacetone, was
oxidized and converted to the acyl chloride 39. Coupling
between acyl chloride 39 and pyrrolopyridine amine 8 afforded
4-pyrone 40. Reaction of pyrone 40 with various amines
provided N-substituted 4-pyridone analogues 41-43.
phenylboronic acid followed by oxidation. Coupling of 6-phe-
nylpicoloinic acid 45 or 6-phenylpicoloinic acid N-oxide 46 with
pyrrolopyridine amine 8 produced pyridine analogue 47 and
pyridine N-oxide 4.
Structure-Activity Relationships and Structural Studies
2-Pyridone analogues with hydrophobic phenyl substituents
2 and 13 (Table 1) displayed potent Met kinase inhibitory
activities with IC50 values in the single digit nanomolar range.
An X-ray crystal structure of 2 bound to the Met kinase domain
revealed that it binds to the ATP-binding pocket of the Met
protein in an inactive conformation; i.e., the activation loop folds
back toward the ATP-binding pocket (Figure 3).16 The DFG
motif, which is conserved among kinases and marks the
beginning of the activation loop, is observed in the structure,
but most of the remaining loop is undefined. The N1 nitrogen
of the pyrrolopyridine ring engages in hydrogen bonding with
the backbone NH of Met1160 of the hinge region, and the NH
of the pyrrolopyridine ring hydrogen-bonds with the Met1160
carbonyl oxygen. The central aminophenyl ring is flanked on
one side by the gatekeeper residue Leu1157 and on the opposite
face by the side chain of Phe1223 (of the DFG motif) in a face-
to-face aromatic interaction. The 2-pyridone carbonyl oxygen
hydrogen-bonds with the backbone NH of Asp1222. The
4-fluorophenyl ring lies in a hydrophobic pocket comprising
Phe1134, Phe1200, Met1131, and Leu1195. Replacement of the
The synthesis of pyridine and pyridine N-oxide analogues
47 and 4 are described in Scheme 5. Phenylpicolinic acid 45
and 6-phenylpicoloinic acid N-oxide 46 were obtained by
Miyaura-Suzuki coupling of 2-bromopicolinic acid 44 with

5332 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kim et al.
Scheme 2a
a Reagents and conditions: (a) pyridine, CH2Cl2, 0 °C, 2.5 h; (b) EtOH, reflux, 16 h, 48% overall; (c) NaOEt, EtOH, room temp, 2 h, 78%; (d) 2 N NaOH,
MeOH, 97%; (e) 8, HATU, i-Pr2NEt, DMF, 0 °C to room temp, 40%.
Table 1. SAR of 2-Pyridone Analogues
a polar substituent as seen with the poor activity of phenylcar-
boxamide 20. However, the poor activity of 20 may alternatively
be attributed to the fact that the pocket appears limited in size
in the para-substituent direction.
The antiproliferative cellular activity of the Met inhibitors
was evaluated in vitro using the GTL-16 gastric carcinoma cell
line. GTL-1617 is a subclone derived from MKN-45,18 a poorly
differentiated gastric carcinoma cell line carrying an amplifica-
tion of the Met gene. The Met protein in the GTL-16 cell line
is constitutively activated as observed by HGF ligand indepen-
dent phosphorylation.19,20 The GTL-16 cell line was previously
shown to be highly sensitive to small molecule inhibitors of
Met20,21 and was adopted to be the primary in vitro cell line
for screening Met inhibitors. In addition to these cell lines,
inhibitors were routinely evaluated in the N87 gastric carcinoma
cell line lacking Met activation.22 Compounds 2 and 13 possess
potent antiproliferative cellular activities against the GTL-16
IC50 (nM)
Met
(biochemical)
GTL-16
(cellular)
N87
(cellular)
compd
R
2
p-F-phenyl
C6H5
tert-butyl
cyclopropyl
CH2CH2CH(CH3)2
p-F-benzyl
2-pyridyl
1.8 ( 0.7
1.9 ( 0.1
1000 ( 0.0
1000 ( 0.0
30 ( 6.4
8.2 ( 1.4
10 ( 0.7
60
140
NT
NT
3700
2100
970
4000
>5000
>5000
>5000
NT
13
14
15
16
17
18
19
20
gastric tumor cell line, while they lack cellular activity (IC50
>
NT
>5000
>5000
>5000
>5000
NT
5 µM) against the N87 cell line (Table 1). We believe the
antiproliferative cellular activity difference in these two cell lines
suggests that the Met receptor is the target of cellular growth
inhibition.
3-pyridyl
C6H4-p-CONH2
70 ( 0.0
1000 ( 0.0
As an extension of the 2-pyridone chemotype, 4-pyridone
analogues (3, 34, 37, 41-43, Table 2) were also prepared. The
SAR of the 4-pyridones is consistent with predictions from
the X-ray crystal structure of Met kinase bound to 2-pyridone
analogue 2, which reveals that the unsubstituted positions of
the pyridinone ring are viable vectors for derivatization. The
environment around the N-1 region of the 4-pyridinone ring is
directed toward helix RC, which displayed mobility in its
orientation in the X-ray structures (data not shown). The SAR
revealed little difference in in vitro Met kinase inhibitory activity
of analogues with N-1 substituents of different size and
hydrophobicity (IC50 ) 2.3-5.9 nM), which confirmed the
flexibility of this region of the protein. All of these 4-pyridone
analogues exhibit potent antiproliferative cellular activities
terminal phenyl ring by tert-butyl or cyclopropyl (14, 15)
significantly reduced the kinase inhibitory activitiy, perhaps
because both the tert-butyl and cyclopropyl groups are not
sufficiently large enough to fill the hydrophobic pocket or lack
the aromaticity to engage in π-electron interactions with the
aromatic residues comprising the pocket. Interestingly, the larger
alkyl isopentyl substituted analogue 16 restores most of the
inhibitory activity. The weaker activity of the fluorobenzyl 17
compared to the fluorophenyl analogue suggests that the phenyl
ring without a methylene linker is optimal for fitting into the
hydrophobic pocket. Pyridyl substituted compounds (18, 19)
showed good activities, albeit weaker than the phenyl substituted
analogues. The hydrophobic pocket does not appear to tolerate

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5333
Scheme 3a
a Reagents and conditions: (a) MeLi, n-BuLi, CO2, -78 °C, 85%; (b) CH3I, Cs2CO3, DMF, room temp, 4 h, 86%; (c) Pd(PPh3)4, K2CO3, dioxane, 90 °C,
23 h, 21%; (d) 2 N NaOH, MeOH, 60 °C, 2 h, 86%; (e) 8, HATU, i-Pr2NEt, DMF, room temp, 1.5 h, 66%; (f) SOCl2, MeOH, 0 to 70 °C, 93%; (g)
BrCH2CH2OH, K2CO3, DMF, 50 °C, 87%; (h) 31, Pd(PPh3)4, K2CO3, dioxane, 90 °C, 20 h, 22%; (i) 6 N NaOH, MeOH, room temp, 2 h, 89%; (j) 8, HATU,
i-Pr2NEt, DMF, room temp, 1.5 h, 67%.
against the GTL-16 gastric tumor cell line with good selectivity
over the N87 cell line.
4. In addition to potent Met kinase inhibitory activity, compound
2 also exhibits potent inhibitory activities notably against Flt-3
(IC50 ) 4 nM) and VEGFR-2 (IC50 ) 27 nM) kinases with
weaker activities against LCK and c-kit kinases.
Interestingly, pyrone 40 shows only weak activity despite its
overall structural similarity. It is speculated that the vinylogous
amide carbonyl in the 4-pyridone analogues makes it more basic
and hence a better hydrogen bond acceptor than the carbonyl
in pyrone 40.
In Vitro Metabolic Stability and Pharmacokinetics of
Compound 2. Compound 2 showed good metabolic stability
in both human and mouse liver microsomes with metabolic rates
of <0.01 and 0.055 (nmol/min)/mg of protein, respectively, at
a concentration of 3 µM. Pharmacokinetic parameters of
compound 2 in mice after iv (5 mg/kg) and po (10 mg/kg)
dosing are summarized in Table 5. The systemic serum clearance
(Cl) of compound 2 was about 10% of hepatic blood flow, and
the large steady state volume of distribution (Vss ) 1.6 L/kg) is
indicative of extensive extravascular distribution. Compound 2
demonstrated a favorable half-life (t1/2 ) 2.2 h) and mean
residence time (MRT ) 4.5 h) after po dosing. The measured
oral bioavailability of compound 2 was 100% from a solution
formulation of 70:30 PEG 400/water. The serum protein binding
of compound 2 was high (99.4 ( 0.3% and 99.9 ( 0% in mouse
and human serum, respectively) using the equilibrium dialysis
method at a concentration of 10 µM.
With the concept that the carbonyl oxygen of the pyridone
analogues plays an important role in hydrogen-bonding with
the backbone NH of Asp1222, the pyridine and pyridine N-oxide
analogues were prepared (Table 3). Pyridine N-oxides 4 and49
displayed potent Met kinase inhibitory activities with IC50 values
of 1.3 and 1.8 nM, respectively, while pyridine analogue 47
showed much weaker inhibitory activity with an IC50 of 470
nM. Compounds 4 and 49 also possess potent antiproliferative
cellular activities against GTL-16 with IC50 of 40 nM with good
selectivity over the N87 cell line. Despite the presence of a basic
pyridine nitrogen in 47, it would likely be positioned at a longer
distance than the N-oxide oxygen from the hydrogen-bonding
partner backbone NH of Asp1222, which results in a weaker
activity than the N-oxide compounds.
Compound 2 was screened broadly against many other
kinases, and some of its kinase activitites are shown in Table
In Vivo Antitumor Activity. To determine the in vivo
antitumor effects due to Met kinase inhibition, compound 2 was

5334 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kim et al.
Scheme 4a
a Reagents and conditions: (a) POCl3, DMF, 59%; (b) oxone, THF/H2O, room temp, 2 h, 78%; (c) SOCl2, CH2Cl2, room temp, 1 h, 100%; (d) 8, 2,6-
lutidine, CH2Cl2, 0 °C, 1 h, 52%; (e) RNH2, THF, room temp, 14 h, 43-61%.
Scheme 5a
a Reagents and conditions: (a) C6H5B(OH)2, PdCl2[(t-Bu)2P(OH)]2, Cs2CO3, DMF/H2O, 110 °C, 24 h, 18%; (b) 8, EDCI, HOBt, DMF, room temp,
overnight, 19%; (c) m-CPBA, K2HPO4, CH2Cl2, 60 °C, overnight, 68%; (d) 8, EDCI, HOBt, DMF, room temp, overnight, 54%.
evaluated against GTL-16 human gastric carcinoma cell line
xenografts in nude mice. Good in vivo efficacy was observed
at multiple dose levels, as shown in Figure 4. Treatment of
compound 2 (po, qd) was initiated 14 days after tumor
implantation, at which time tumors were staged to a volume of
approximately 125 mm3. Antitumor activity of 2 was dose
proportional. Tumor regressions were observed at 50 and 100
mg/kg, and 100 mg/kg was determined to be the maximum
tolerated dose.23 At 25 mg/kg, this compound induced complete
tumor stasis, and the minimum efficacious dose was identified
at 12.5 mg/kg, based on greater than or equal to 50% tumor
growth inhibition for more than one tumor volume doubling
time (7 days). Compound 2, however, was ineffective at the
6.25 mg/kg dose level. Although we cannot quantify the level
of contribution from VEGFR-2 inhibition to the antitumor
efficacy of compound 2, it is likely VEGFR-2 inhibition
contributed to the in vivo antitumor activity because of its
important role in angiogenesis.24 In order to confirm the in vivo
antitumor activity of compound 2 in the GTL-16 model is due
to inhibition of Met kinase phosphorylation levels of the Met
protein in the GTL-16, tumor extracts from the tumor bearing
animals were examined (Figure 5). Both subefficacious (6.25
mg/kg) and maximally efficacious (25 mg/kg) dose levels were
evaluated. As indicated in Figure 5, Met receptor phosphory-
lation remained relatively unchanged at the subefficacious dose
(6.25 mg/kg). At the maximally efficacious dose (25 mg/kg),
complete and sustained inhibition of Met receptor phosphory-
lation was observed up to 8 h, and at 24 h, phosphorylated Met
levels began returning to control levels. The Met receptor
phosphorylation levels are consistent with the in vivo antitumor
efficacy results demonstrated by the compound.
Conclusions
Replacement of the N-acylurea moiety in pyrrolopyridine
substituted analogue 1 with the conformationally constrained
2-pyridone, 4-pyridone, and pyridine N-oxide in analogues 2-4

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5335
Table 2. SAR of 4-Pyridone Analogues
Table 3. SAR of Pyridine and Pyridine N-Oxide Analogues
Figure 3. Surface representation and depiction of the binding of
compound 2 to the Met kinase domain in X-ray crystal structure.
resulted in a series of potent Met kinase inhibitors. Many of
these analogues also demonstrated potent antiproliferative
cellular activity in the Met-dependent gastric carcinoma GTL-
16 cell line while lacking cellular activity against the Met-
independent N87 cell line. Compound 2 also possesses potent
inhibitory activity against Flt-3 and VEGFR-2 kinases. Com-
pound 2 showed favorable metabolic and pharmacokinetic
properties, and it demonstrated good in vivo efficacy in a GTL-
16 human gastric carcinoma xenograft model upon oral admin-
istration in nude mice. Tumor regressions were observed in the
GTL-16 tumor model at 50 and 100 mg/kg, and the minimum
efficacious dose was 12.5 mg/kg. Phosphorylation of the Met
receptor in GTL-16 tumor extracts decreased in a time and dose
dependent manner, consistent with the in vivo antitumor efficacy
of the Met-driven GTL-16 tumor model.
IC50 (nM)
compd
n
X
Met (biochemical) GTL-16 (cellular) N87 (cellular)
47
4
49
0
1
1
H
H
F
470 ( 77.8
1.3 ( 0.1
1.8 ( 0.2
NT
40
40
NT
>5000
4900
Table 4. In Vitro Kinase Inhibition of 2
protein kinase
IC50 (nM)
Met
Flt-3
VEGFR-2
LCK
1.9
4
4 27
290
c-kit
610
Experimental Section
CDK2/cyclin E
PKCR
PKA
IGF-1R
InsR
>1000
>30000
>30000
>5000
>5000
>10000
All new compounds were homogeneous by thin layer chroma-
tography and reversed-phase HPLC (>99%). Flash chromatography
was carried out on E. Merck Kieselgel 60 silica gel (230-400
mesh). All the reaction solvents used such as tetrahydrofuran (THF),
N,N-dimethylformamide (DMF), and dichloromethane (CH2Cl2)
were anhydrous solvents that were purchased from Aldrich Chemi-
cal Co. EDCI, HOBT, and TFA refer to ethyl dimethylaminopro-
pylcarbodiimide hydrochloride salt, 1-hydroxybenzotriazole, and
trifluoroacetic acid, respectively. Preparative HPLC was run on
YMC OD S-10 50 mm × 500 mm column eluting with a mixture
of solvents A and B (starting from 10% of solvent B to 100%
MK-2
solvent B over 30 min gradient time; solvent A, 10% MeOH-90%
H2O-0.1% TFA; solvent B, 90% MeOH-10% H2O-0.1% TFA;
flow rate 84 mL/min; UV 220 nm). 1H and 13C NMR spectra were
obtained on a JEOL 500 MHz Eclipse NMR spectrometer operating
at 500.16 and 125.77 MHz, respectively. Chemical shifts are

5336 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Table 5. Pharmacokinetic Properties of Compound 2 in Micea
Kim et al.
Table 6. Summary of the Crystallographic Data Collection, Reduction,
and Refinement
iv dose
5
po dose
parameter
2 bound to Met kinase protein
dose (mg/kg)
Cmax (µM)
10
11.5
0.5
50.2
2.2
4.5
space group
P21 21 21
43.040
49.137
Tmax (h)
a (Å)
b (Å)
c (Å)
AUC0-24h (µM·h)
T1/2 (h)
MRT (h)
clearance ((mL/min)/kg)
Vss (L/kg)
19.8
2.5
3.0
9.2
1.6
159.324
max observed resolution (Å)
no. of observations
no. of unique reflections
Rsymm (%)
2.40 (2.40-2.49)
41954 (2165)
10949 (897)
14.4 (46.7)
10. 0 (1.1)
81.3 (69.9)
BUSTER-TNT
20.90 (22.42)
27.24 (25.03)
71.58
F (%)
100
mean I/σI
a Composite serum concentration-time profiles were constructed for the
PK analysis. Vehicle: PEG 400/water (70:30).
completeness (%)
refinement program
R-factor (%)
Rfree (%)
av B-factor (protein) (Å2)
av B-factor (ligand) (Å2)
no. of residues in final model
no. of atoms
49.02
293
2419
no. of protein atoms
no. of ligand atoms
no. of solvent atoms
rms (bond) (Å)
2323
34
62
0.006
rms (angle) (deg)
rms (torsion) (deg)
0.866
24.454
Hz, 1H), 6.25 (d, J ) 5.33 Hz, 1H); 13C NMR (DMSO-d6) δ 152.4
(d, JCF ) 217.2 Hz), 151.3, 148.2, 144.3, 143.7 (d, JCF ) 7.7 Hz),
144.3, 126.0, 121.4, 121.3, 113.5 (d, JCF ) 23 Hz), 110.3, 103.6,
96.6. HRMS for C13H8N3O3F (M + H)+ calcd 274.0622, found
274.0623.
Figure 4. Antitumor activity of compound 2 in the GTL-16 human
gastric carcinoma model (% TGI on day 29).
4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluoroaniline (8). To
a suspension of nitro compound 7 (300 mg, 1.1 mmol) in THF (5
mL) and MeOH (10 mL) were added zinc powder (350 mg, 5.5
mmol) and ammonium chloride (294 mg, 5.5 mmol). The mixture
was stirred at room temperature overnight. The reaction mixture
was filtered through a pad of Celite and rinsed with methanol, and
the filtrate was concentrated in vacuo. The product was purified
by flash column chromatography (silica gel, eluting with 1-5%
MeOH in CH2Cl2) to afford the 8 (205 mg, 77%) as an off-white
solid, mp 185-188 °C. HPLC/UV purity 95%; 1H NMR (DMSO-
d6) δ 11.67 (br s, 1H), 8.02 (m, 1H), 7.32 (m, 1H), 7.01 (t, J )
8.8, 1H), 6.51 (dd, J ) 13.2, 2.8 Hz, 1H), 6.43 (dd, J ) 8.8, 2.8
Hz, 1H), 6.28 (d, J ) 5.0 Hz, 1H), 6.24 (m, 1H), 5.42 (s, 2H); 13
C
NMR (DMSO-d6) δ 158.3, 154.7 (d, JCF ) 241.6 Hz), 151.0, 148.2
(d, JCF ) 10.7 Hz), 144.2, 130.1, 124.4, 124.2, 110.0, 109.3, 101.4
(d, JCF ) 23 Hz), 100.1, 96.9; HRMS for C13H10N3O3F (M + H)+
calcd 244.0881, found 244.0881.
Figure 5. Phospho Met protein levels in GTL-16 tumor extracts. Met
and pMet proteins were simultaneously detected in GTL-16 tumor
extracts using two IR fluorophores. Proteins were immunoprecipitated
using an anti-Met antibody and resolved by SDS-PAGE. Immunoblots
were incubated with rabbit anti-Met and mouse anti-PY-20 primary
antibodies, followed by Alexa Fluor 680 goat antimouse (red) and
IRDye 800 goat antirabbit (green). Both colors were imaged in a single
scan.
Methyl 1-(4-Fluorophenyl)-2-oxo-1,2-dihydropyridine-3-car-
boxylate (11). To a solution of pyran 9 (30.0 g, 195 mmol, Aldrich)
in DMF (175 mL) at 0 °C was added 4-fluoroaniline 10 (18.6 mL,
196 mmol), and the reaction mixture was stirred for 7 h. To the
reaction mixture was added EDCI ·HCl (48.6 g, 254 mmol) and
DMAP (6.0 g, 49 mmol) at room temperature, and it was stirred
overnight. Most of the solvent was removed in vacuo, and the
residue was diluted with water and ethyl acetate. The EtOAc layer
was separated, washed with brine, dried over MgSO4, and concen-
trated in vacuo to obtain a semisolid material. It was triturated with
i-PrOH to obtain the first crop of 11 (24.1 g). The filtrate solution
was concentrated in vacuo to a small volume which was mixed
with diethyl ether, and the mixture was stirred for 30 min. The
solid was filtered to give light-yellow solid of a second crop of 11
(6.5 g) (total 30.6 g, 64%), mp 101-104 °C. HPLC/UV purity
100%; 1H NMR (DMSO-d6) δ 8.10 (dd, J ) 7.2, 2.2 Hz, 1H),
7.93 (dd, J ) 7.2, 2.2 Hz, 1H), 7.46-7.47 (m, 2H), 7.32-7.37 (m,
2H), 6.39 (dd, J ) 7.2, 7.2 Hz, 1H) 3.76 (s, 3H); 13C NMR (DMSO-
d6) δ 164.7, 160.3 (d, JCF ) 245.9 Hz), 157.8, 144.5, 143.8, 136.4,
128.9 (d, JCF ) 7.6 Hz), 120.4, 115.6 (d, JCF ) 22.9 Hz), 104.2,
51.5. Anal. (C13H10NO3F) C, H, N, F.
reported as ppm downfield from an internal tetramethylsilane
1
standard. The abbreviations of br s, d, t, and m in H NMR refer
to broad singlet, doublet, triplet, and multiplet, respectively. Melting
points are uncorrected.
4-(2-Fluoro-4-nitrophenoxy)-1H-pyrrolo[2,3-b]pyridine (7). A
mixture of chloropyrrolopyridine 5 (457 mg, 3.0 mmol), 2-fluoro-
4-nitrophenol 6 (703 mg, 4.2 mmol), and N,N-diisopropylethylamine
(583 mg, 4.2 mmol) in 1-methyl-2-pyrrolidinone (NMP) (3 mL)
was heated at 200 °C under microwave irradiation for 1 h. The
reaction mixture was diluted with EtOAc (150 mL), washed with
saturated aqueous KH2PO4 solution and Na2CO3 (aqueous 1 M),
and dried over Na2SO4. The product was purified by flash column
chromatography (silica gel, eluting with CH2Cl2 to 30% EtOAc/
CH2Cl2) to afford 7 as a brown solid (350 mg, 43%), mp 221-223
1
°C. HPLC/UV purity 95%; H NMR (DMSO-d6) δ 11.99 (br s,
1H), 8.43 (dd, J ) 13.4, 2.7 Hz, 1H), 8.20 (d, J ) 5.42 Hz, 1H),
8.15 (dd, J ) 13.0, 2.7 Hz, 1H), 7.45 (m, 2H), 6.73 (d, J ) 5.33

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5337
1-(4-Fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxylic Acid
(12). A mixture of methyl ester 11 (30.0 g, 121 mmol) and 2 N
aqueous NaOH (160 mL) in methanol (30 mL) was heated at 65
°C for 1.5 h. To the reaction mixture was added 2 N HCl solution
with stirring at room temperature (pH 1.0). The precipitate that
formed was filtered, washed with a small amount water, and dried
to obtain the desired acid 12 (28.3 g, 99%) as a white solid, mp
213-215 °C. HPLC purity 99%; 1H NMR (DMSO-d6) δ 14.22
(br s, 1H), 8.47 (dd, J ) 7.1, 2.2 Hz, 1H), 8.19 (dd, J ) 6.6, 2.2
Hz, 1H), 7.59-7.63 (m, 2H), 7.39-7.44 (m, 2H), 6.78 (dd, J )
135.65 (d, J ) 12.72 Hz), 129.26 (2 carbons, d, J ) 7.63 Hz),
127.15, 123.95, 120.03, 116.65, 116.06 (2 carbons, d, J ) 7.63
Hz), 111.86, 108.64 (d, J ) 22.89 Hz), 107.01, 101.45, 98.58; MS
(ESI) 459 (M + H)+. Anal. (C25H16N4O3F2 ·1.0HCl) C, H, N, F,
Cl. Karl Fischer/water analysis KF water ) 0.17 wt %.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-2-
oxo-1-phenyl-1,2-dihydropyridine-3-carboxamide (13). HPLC
purity 97%; 1H NMR (acetone-d6) δ 13.40 (br s, 1H), 12.32 (br s,
1H), 8.67 (dd, J ) 7.1, 2.2 Hz, 1H), 8.43 (d, J ) 6.6 Hz, 1H), 8.18
(dd, J ) 13.2, 2.2 Hz, 1H), 8.03 (dd, J ) 6.6, 2.2 Hz, 1H),
7.72-7.48 (m, 8H), 6.89 (d, J ) 6.6 Hz, 1H), 6.75 (t, J ) 6.3 Hz,
1H), 6.58 (d, J ) 3.3 Hz, 1H); MS (ESI) 441 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-
tert-butyl-2-oxo-1,2-dihydropyridine-3-carboxamide (14). HPLC
purity 95%; 1H NMR (CD3OD) δ 8.54 (dd, J ) 7.2, 2.2 Hz, 1H),
8.28 (d, J ) 7.1 Hz, 1H), 8.17 (dd, J ) 7.1, 1.7 Hz, 1H), 8.07 (dd,
J ) 12.7, 2.2 Hz, 1H), 7.53 (d, J ) 3.8 Hz, 1H), 7.52 (s, 1H), 7.50
(d, J ) 1.1 Hz, 1H), 7.44 (t, J ) 8.8 Hz, 1H), 6.85 (d, J ) 6.6 Hz,
1H), 6.65 (d, J ) 3.8 Hz, 1H), 6.55 (t, J ) 7.1 Hz, 1H), 1.78 (s,
9H); MS (ESI) 421 (M + H)+.
6.6, 7.1 Hz, 1H); 13C NMR (DMSO-d6) δ 164.3, 163.4 (d, JCF
)
245.9 Hz), 160.8, 146.0, 144.9, 135.1, 128.8 (d, JCF ) 7.6 Hz),
117.1, 115.9 (d, JCF ) 22.9 Hz), 108.0. Anal. (C12H8NO3F) C, H,
N, F.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (2). To
a heterogeneous mixture of pyrrolopyridine 8 (11.5 g, 47.2 mmol),
acid 12 (11.0 g, 47.2 mmol), and 2-(1H-benzotriazole-1-yl)-1,1,3-
tetramethyluronium tetrafluoroborate (TBTU) (16.7 g, 51.9 mmol)
in DMF/CH3CN (1:1, 200 mL) at 0 °C was added diisopropyl-
ethylamine (24.6 mL, 141 mmol) with stirring. Upon addition of
diisopropylethylamine, the mixture became homogeneous. The
reaction mixture was allowed to warm to room temperature and
was stirred for 2 h. Additional TBTU (3.0 g, 9.40 mmol), 12 (1.10
g, 4.72 mmol), and diisopropylethylamine (4.90 mL, 28.3 mmol)
were added to the reaction mixture, and stirring was continued for
3 h at room temperature. The reaction mixture was quenched with
aqueous 1 N NaOH, and the resultant solution was stirred for 30
min at room temperature. The reaction mixture was concentrated
in vacuo, and the resulting slurry was diluted with water. The
precipitated solid was filtered, and it was washed with aqueous 10%
NaOH followed by water. The solid was dried by azetroping with
3/1 MeOH/toluene. This procedure was repeated 3 times. Purifica-
tion was accomplished by preabsorbing the crude product on Celite
and loading the preabsorbed material on a silica gel flash chroma-
tography column. The product was eluted using a 0-1.5% MeOH
in CHCl3 gradient. The appropriate fractions were collected and
concentrated in vacuo and dried to obtain 2 (16.6 g, 81% yield) as
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-cy-
clopropyl-2-oxo-1,2-dihydropyridine-3-carboxamide (15). HPLC
1
purity 95%; H NMR (DMF-d7) δ 12.56 (br s, 1H), 12.00 (br s,
1H), 8.55 (dd, J ) 7.2, 2.2 Hz, 1H), 8.22 (d, J ) 5.5 Hz, 1H), 8.17
(dd, J ) 13.2, 2.2 Hz, 1H), 8.13 (dd, J ) 6.6, 2.2 Hz, 1H),
7.58-7.50 (m, 3H), 6.65 (t, J ) 7.2 Hz, 1H), 6.57 (d, J ) 5.5 Hz,
1H), 6.43 (d, J ) 2.2 Hz, 1H), 3.60-3.62 (m, 1H), 1.19-1.20 (m,
2H), 1.10-1.11 (m, 2H); MS (ESI) 405 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-
isopentyl-2-oxo-1,2-dihydropyridine-3-carboxamide (16). Mp
183-184 °C; HPLC purity 98%; 1H NMR (CDCl3) δ 12.26 (br s,
1H), 9.05 (br s, 1H), 8.62 (d, 1H, J ) 5.04 Hz), 8.14 (d, 1H, J )
5.56 Hz), 7.95 (d, J ) 12.32, 2.52 Hz, 1H), 7.58 (dd, 1H, J )
6.56, 2.28 Hz, 2H), 7.40-7.45 (m, 1H), 7.16-7.24 (m, 2H), 6.51
(t, J ) 7.08 Hz, 1H), 6.42-6.47 (m, 2H), 4.09 (t, J ) 7.52 Hz,
2H), 1.65-1.80 (m, 3H), 1.02 (d, J ) 6.28 Hz, 6H); MS (ESI) 435
(M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(4-
fluorobenzyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (17).
1
a white solid, mp 212-214 °C. H NMR (DMSO-d6) δ 12.10 (br
1
s, 1H), 11.78 (br s, 1H), 8.59 (dd, J ) 7.3, 2.1 Hz, 1H), 8.14 (d, J
) 2.1 Hz, 1H), 8.12 (d, J ) 2.1 Hz, 1H), 8.05 (dd, J ) 20.5, 5.4
Hz, 1H), 7.62 (m, 2H), 7.42 (m, 5H), 6.73 (t, J ) 7.1 Hz, 1H),
6.39 (d, J ) 5.5 Hz, 1H), 6.26 (m, 1H); 13C NMR (DMSO-d6) δ
161.78 (d, J ) 246.68 Hz), 161.55, 160.64, 157.05, 154.82, 153.21
(d, J ) 244.14 Hz), 151.07, 144.93, 144.20, 144.15,136.24, 129.33,
129.24, 124.86, 123.84, 120.14, 116.41, 116.18, 115.95, 109.36,
108.67, 108.44, 106.99, 100.77, 96.72; MS (ESI) 459 (M + H)+.
Anal. (C25H16N4O3F2) C, H, N, F. Karl Fischer/water analysis KF
water ) 0.20 wt %.
Mp 140-142 °C (dec); H NMR (DMSO-d6) δ 12.43 (br s, 1H),
12.27 (br s, 1H), 8.51 (dd, J ) 7.2, 1.9 Hz, 1H), 8.36 (dd, J ) 6.5
Hz, 2.0 Hz, 1H), 8.26 (d, J ) 6.1 Hz, 1H), 8.07 (dd, J ) 12.90,
2.1 Hz, 1H), 7.55-7.49 (m, 5H), 7.48-7.24 (m, 2H), 6.71 (t, J )
6.89 Hz, 1H), 6.66-6.64 (m, 1H), 6.43 (br s, 1H), 5.32 (s, 2H);
MS (ESI) 473 (M + H)+. Anal. (C26H18N4O3F2 ·1.12HCl·1.44H2O)
C, H, N, Cl.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-2-
oxo-1-(pyridin-2-yl)-1,2-dihydropyridine-3-carboxamide (18).
1
HPLC purity 97%; H NMR (DMF-d7) δ 12.16 (br s, 1H), 11.99
(br s, 1H), 8.71-8.72 (m, 2H), 8.37 (d, J ) 3.9 Hz, 1H), 8.21-7.47
(m, 8H), 6.87 (t, J ) 7.2 Hz, 1H), 6.56 (t, J ) 5.5 Hz, 1H), 6.42
(d, J ) 3.3 Hz, 1H); MS (ESI) 442 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(4-
fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide Hy-
drochloride Salt (2 ·HCl). Compound 2 (40.0 g, 87.3 mmol, 1.0
eq) was suspended in 7:2 THF/MeOH (900 mL), and the hetero-
geneous solution was cooled to 0 °C. Anhydrous 4 M HCl in
dioxane (40.0 mL, 160 mmol, 1.8 equiv) was added in portions, at
which point the reaction mixture became homogeneous. After the
mixture was stirred for 10 min at 0 °C, a thick white precipitate
formed and the reaction mixture continued to be stirred for 1 h at
0 °C. The reaction mixture was concentrated in vacuo, and the
resultant solid was triturated with diethyl ether. The solid was
filtered, washed with additional diethyl ether, and dried under high
vacuum for 12 h. The resultant white solid afforded the product 2
(43 g, 99%) as a mono HCl salt, mp 274-277 °C (dec). HPLC
purity 99%; 1H NMR (DMSO-d6) δ 12.79 (br s, 1H), 12.15 (br s,
1H), 8.59 (dd, J ) 7.4, 2.2 Hz, 1H), 8.31 (d, J ) 6.3 Hz, 1H), 8.14
(dd, J ) 6.6, 1.9 Hz, 1H), 8.08 (dd, J ) 12.92, 2.20 Hz, 1H), 7.61
(m, 2H), 7.58 (m, 1H), 7.55 (m, 1H), 7.50 (t, J ) 8.8 Hz, 1H),
7.43 (t, J ) 8.8 Hz, 2H), 6.74 (m, 1H), 6.73 (d, J ) 3.3 Hz, 1H),
6.50 (d, J ) 3.0 Hz, 1H); 13C NMR (DMSO-d6) δ 161.79 (d, J )
246.68 Hz), 161.75, 161.69, 160.59, 153.32 (d, J ) 244.14 Hz),
145.00, 144.24, 143.91, 139.06, 137.65 (d, J ) 7.63 Hz), 136.17,
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-2-
oxo-1-(pyridin-3-yl)-1,2-dihydropyridine-3-carboxamide (19).
1
HPLC purity 97%; H NMR (DMF-d7) δ 12.26 (br s, 1H), 12.18
(br s, 1H), 8.90 (d, J ) 2.2 Hz, 1H), 8.78 (d, J ) 3.9 Hz, 1H), 8.70
(dd, J ) 7.1, 2.2 Hz, 1H), 8.27 (dd, J ) 6.6, 2.2 Hz, 2H), 8.17-8.19
(m, 2H), 8.03 (s, 1 H), 7.72-7.50 (m, 3H), 6.86 (t, J ) 7.2 Hz,
1H), 6.63 (d, J ) 5.5 Hz, 1H), 6.47 (s, 1H); MS (ESI) 442 (M +
H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(4-
carbamoylphenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (20).
Mp 311-313 °C; HPLC purity 96%; 1H NMR (DMF-d7) δ 12.26
(br s, 1H), 12.21 (br s, 1H), 8.72-8.70 (m, 1H), 8.30-8.15 (m,
6H), 7.75-7.60 (m, 2H), 7.57-7.48 (m, 4H), 6.85-6.83 (m, 1H),
6.63-6.62 (m, 1H), 6.45-6.44 (m, 1H); MS (ESI) 484 (M + H)+.
Ethyl 4-(4-Fluorophenyl)-3-oxobutanoate (24). To a solution
of Meldrum’s acid 21 (20.0 g, 139 mmol) in anhydrous CH2Cl2
(160 mL) was added anhydrous pyridine (28 mL), followed by
addition of 4-fluorophenylacetyl chloride 22 (20 mL, 142 mmol)
under nitrogen at ice bath temperature. The reaction mixture was

5338 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kim et al.
stirred at ice bath temperature for 2.5 h. The reaction mixture was
extracted with a mixture of CH2Cl2 and aqueous HCl solution (1
N). The organic layer was washed with brine, dried over MgSO4,
and concentrated. To the residue was added absolute ethanol (200
mL), and the reaction mixture was heated at 85 °C for 16 h. The
solvent was removed in vacuo, and the resulting residue was purified
by silica gel chromatography eluting with 0-10% of ethyl acetate
in hexane. The desired product 24 was obtained as a colorless oil
(15.0 g, 48%). 1H NMR (CDCl3) δ 7.25-7.15 (m, 2H), 7.05-6.98
(m, 2H), 4.18 (q, J ) 7.2 Hz, 2H), 3.81 (s, 2H), 3.46 (s, 2H), 1.26
(t, J ) 7.2 Hz, 3H); MS (ESI) 225.3 (M + H)+.
Ethyl 5-(4-Fluorophenyl)-4-oxo-1,4-dihydropyridine-3-car-
boxylate (26). To a solution of 24 (20.1 g, 89.7 mmol) in absolute
ethanol (200 mL) was added triazine 25 (7.63 g, 94.2 mmol)
followed by addition of 21% NaOEt solution in EtOH (35.2 mL,
94.2 mmol) under nitrogen at room temperature. The resulting
mixture was heated at 85 °C for 2 h. The reaction mixture was
concentrated to about 20 mL. To the residue was added 1 N aqueous
HCl solution with stirring. The precipitate was filtered, washed with
distilled water, ethyl acetate, and dried to give 26 as a yellow solid
(18.2 g, 78%). 1H NMR (CDCl3) δ 8.97 (s, 1H), 8.55 (s, 1H),
7.58-7.52 (m, 2H), 7.17 (t, J ) 8.8 Hz, 2H), 4.50 (q, J ) 7.2 Hz,
2H), 1.46 (t, J ) 7.2 Hz, 3H); MS (ESI) 262 (M + H)+.
5-(4-Fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxylic Acid
(27). A mixture of ester 26 (18.2 g, 69.7 mmol) in MeOH (30 mL)
and 2 N aqueous NaOH solution (120 mL) was heated for 2 h at
60 °C. After cooling to room temperature, the mixture was poured
into 2 N aqueous HCl. The solid that formed was filtered, washed
with distilled water and a small amount of ethyl acetate, and dried
to give 27 as a light-yellow solid (15.8 g, 97%). 1H NMR (DMSO-
d6) δ 8.60 (s, 1H), 8.24 (s, 1H), 7.72-7.68 (m, 2H), 7.27 (t, J )
8.6 Hz, 2H); MS (ESI) 234.3 (M + H)+. Anal. (C12H8FNO3) C,
H, N, F.
with CH2Cl2/EtOAc/MeOH (1:1:0.05) to obtain 30 as a yellow solid
(1.94 g, 86%). H NMR (CD3OD) δ 8.42 (s, 1H), 8.24 (s, 1H),
1
3.82 (s, 3H), 3.80 (s, 3H); 13C NMR (CD3OD) δ 171.0, 165.8,
147.8, 143.9, 119.2, 117.9, 52.3, 44.6; MS (ESI) 246 (M + H)+.
Methyl 5-(4-Fluorophenyl)-1-methyl-4-oxo-1,4-dihydropyri-
dine-3-carboxylate (32). A mixture of 30 (1.80 g, 7.32 mmol),
4-fluorophenylboronic acid 31 (1.43 g, 10 mmol), and K2CO3 (1.52
g, 10.2 mmol) in 1,4-dioxane (20 mL) was purged with argon, and
Pd(PPh3)4 (658 mg, 0.57 mmol) was added to the mixture. The
reaction mixture was heated for 23 h at 90 °C under argon. The
reaction mixture was filtered through a Celite pad and washed with
ethyl acetate and the filtrate solution was concentrated and purified
by silica gel chromatography, eluting with CH2Cl2/EtOAc/MeOH
1
(1:1:0.1) to give 32 as a yellow solid (400 mg, 21%). H NMR
(CD3OD) δ 8.40 (d, J ) 2.2 Hz, 1H), 7.84 (d, J ) 2.2 Hz, 1H),
7.59 (dd, J ) 5.5 Hz, 2H), 7.11 (t, J ) 8.8 Hz, 2H), 3.81 (s, 6H);
MS (ESI) 262 (M + H)+.
5-(4-Fluorophenyl)-1-methyl-4-oxo-1,4-dihydropyridine-3-
carboxylic Acid (33). A mixture of 32 (110 mg, 0.42 mmol) in
MeOH (5.0 mL) and 2 N aqueous NaOH (1.0 mL) solution was
heated at 60 °C for 2 h. After cooling to room temperature, the
mixture was poured onto 1 N aqueous HCl solution. The resulting
solid was filtered, washed with water, and dried to give 33 as a
1
white solid (90 mg, 86%). H NMR (CD3OD) δ 8.70 (d, J ) 2.0
Hz, 1H), 8.18 (d, J ) 2.0 Hz, 1H), 7.73-7.70 (m, 2H), 7.23-7.19
(m, 2H), 4.01 (s, 3H); MS (ESI) 248 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-5-(4-
fluorophenyl)-1-methyl-4-oxo-1,4-dihydropyridine-3-carboxam-
ide (34). To a mixture of 33 (87 mg, 0.35 mmol) in anhydrous
DMF (2.0 mL) were added HATU (174 mg, 0.358 mmol) and
i-Pr2NEt (1.0 mL) followed by 8 (92 mg, 0.38 mmol) under
nitrogen. The reaction mixture was stirred at room temperature for
1.5 h. The reaction mixture was purified by preparative HPLC to
give 34 as a white solid (88 mg, 66%), mp 196-198 °C. HPLC
purity 97%; 1H NMR (DMSO-d6) δ 13.23 (br s, 1H), 12.09 (br s,
1H), 8.69 (d, J ) 2.2 Hz, 1H), 8.17-8.16 (m, 2H), 8.03 (dd, J )
2.2, 13.2 Hz, 1H), 7.71 (dd, J ) 5.5, 8.2 Hz, 2H), 7.50-7.38 (m,
3H), 7.28 (t, J ) 8.8 Hz, 2H), 6.53-6.51 (m, 1H), 6.35 (br s, 1H),
3.92 (s, 3H); HRMS (ESI) calcd for C26H18F2N4O3 (M + H)+
473.1420, found 473.1419.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-5-(4-
fluorophenyl)-4-oxo-1,4-dihydropyridine-3-carboxamide (3). To
a suspension of acid 27 (8.0 g, 34.3 mmol) in anhydrous DMF (56
mL) were added HATU (15.65 g, 41.2 mmol) and i-Pr2NEt (18
mL, 104 mmol) at ice bath temperature under nitrogen. The mixture
turned into a clear solution after being stirred for 5 min. To the
solution was added aniline 8 (7.9 g, 32.5 mmol) slowly. The reaction
mixture was stirred for 3 h at room temperature and was poured
into aqueous 1 N HCl solution. The solid that formed was filtered,
washed with distilled water, and dried. The crude product was
purified by silica gel chromatography, eluting with 1-5% of MeOH
in CH2Cl2 to obtain 3 as a light-yellow solid (5.9 g, 40%), mp
Methyl 5-Bromo-1-(2-hydroxyethyl)-4-oxo-1,4-dihydropyri-
dine-3-carboxylate (35). (A) To a mixture of acid 29 in anhydrous
MeOH (60 mL) at 0 °C was slowly added thionyl chloride (5.0
mL) under nitrogen. The resulting mixture was stirred at 0 °C for
15 min. It was warmed to room temperature and heated at 70 °C
for 4 h. The solvent was removed in vacuo, and the residue was
triturated with saturated NaHCO3. The solid was filtered and dried
to give a light-brown solid of methyl 5-bromo-4-oxo-1,4-dihydro-
pyridine-3-carboxylate (4.93 g, 93%): 1H NMR (DMSO-d6) δ 12.20
(br s, 1H), 8.26 (s, 2H), 3.73 (s, 3H); MS (ESI) 232.2 (M + H)+.
(B) A mixture of 5-bromo-4-oxo-1,4-dihydropyridine-3-carboxy-
1
188-190 °C. HPLC purity 97%; H NMR (DMF-d7) δ 13.53 (br
s, 1H), 13.03 (br s, 1H), 11.97 (br s, 1H), 9.0 (d, J ) 1.2 Hz, 1H),
8.39 (d, J ) 1.2 Hz, 1H), 8.37-8.32 (m, 2H), 8.04-7.99 (m, 2H),
7.72-7.61 (m, 3H), 7.49 (m, 2H), 6.67 (d, J ) 5.6 Hz, 1H), 6.56
(t, J ) 2.4 Hz, 1H); MS (ESI) 459 (M + H)+.
5-Bromo-4-hydroxynicotinic Acid (29). Methyllithium solution
in diethyl ether (8.0 mL of 1.5 M solution, 12.0 mmol) was slowly
added to a mixture of 28 (3.0 g, 11.9 mmol) in anhydrous THF
(25 mL) at -78 °C under argon. After the mixture was stirred for
30 min, n-BuLi solution in hexane (15.6 mL of 1.6 M solution,
25.0 mmol) was added to the reaction mixture at -78 °C and it
was stirred for 1.5 h at -78 °C. Carbon dioxide gas was bubbled
through the mixture for 30 min at -78 °C. To the reaction mixture
was added aqueous 4 N HCl solution, and the resulting mixture
was concentrated. Water was added to the residue and the solid
that formed was filtered, washed with water, and dried to afford
29 as a brown solid (2.2 g, 85%). 1H NMR (DMF-d7) δ 13.72 (br
s, 1H), 8.95 (d, J ) 1.1 Hz, 1H), 8.89 (d, J ) 1.4 Hz, 1H); MS
(ESI) 218 (M + H)+.
Methyl 5-Bromo-1-methyl-4-oxo-1,4-dihydropyridine-3-car-
boxylate (30). A mixture of acid 29 (2.0 g, 9.17 mmol) and Cs2CO3
(18.0 g, 55 mmol) in anhydrous DMF (20 mL) was stirred at room
temperature for 20 min under nitrogen. To the mixture was added
MeI (3.4 mL, 55 mmol), and the reaction mixture was stirred at
room temperature for 4 h. The reaction mixture was concentrated
and the residue was purified by silica gel chromatography, eluting
late (4.0 g, 17.2 mmol) and K
2CO3 (5.0 g, 34 mmol) in anhydrous
DMF (30 mL) was stirred for 30 min at room temperature. To the
mixture 2-bromoethnol (3.1 mL, 43 mmol) was added, and the
reaction mixture was heated at 50 °C for 22 h under nitrogen. After
cooling to room temperature, the reaction mixture was filtered
through a Celite pad and washed with ethyl acetate and the filtrate
solution was concentrated. The residue was triturated with ether to
obtain 35 as a light-yellow solid (4.16 g, 87%). 1
H NMR (CD3OD)
δ 8.45 (s, 1H), 8.27 (s, 1H), 4.07 (s, 2H), 3.79 (s, 5H); MS (ESI)
276.18, 278.1 (M + H)+
.
5-(4-Fluorophenyl)-1-(2-hydroxyethyl)-4-oxo-1, 4-dihydropy-
ridine-3-carboxylic Acid (36). (A) A mixture of 35 (0.56 g, 2.41
mmol), 4-fluorophenylboronic acid 31 (0.51 g, 3.62 mmol), and
K2CO3 (0.53 g, 3.61 mmol) in 1,4-dioxane (8.0 mL) was purged
with argon. Then Pd(PPh3)4 (195 mg, 0.17 mmol) was added to
the reaction mixture. The reaction mixture was heated at 90 °C for
20 h under argon. The reaction mixture was filtered through a Celite
pad and washed with ethyl acetate. The filtrate solution was
concentrated and purified by silica gel chromatography, eluting with
CH2Cl2/EtOAc/MeOH (5:2:0.4) to give the ester of 36 (160 mg,

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5339
1
22%) as a light-yellow solid. H NMR (CD3OD) δ 8.45 (d, J )
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(3-
fluoro-4-methoxyphenyl)-5-(4-fluorophenyl)-4-oxo-1,4-dihydro-
pyridine-3-carboxamide (42). Mp 238-240 °C (dec); HPLC purity
98%; 1H NMR (CD3OD) δ 8.75 (d, J ) 2.2 Hz, 1H), 8.12 (s, 2H),
7.97 (d, J ) 12.1 Hz, 1H), 7.64-7.61 (m, 2H), 7.51 (dd, J ) 2.7,
11.0 Hz, 1H), 7.42-7.38 (m, 3H), 7.32 (t, J ) 8.8 Hz, 1H), 7.23
(t, J ) 8.8 Hz, 1H), 7.10 (t, J ) 8.8 Hz, 2H), 6.66 (d, J ) 6.0 Hz,
1H), 6.51 (d, J ) 3.3 Hz, 1H), 3.88 (s, 3H); MS (ESI) 583 (M +
H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-(4-
chloro-3-fluorophenyl)-5-(4-fluorophenyl)-4-oxo-1,4-dihydropy-
ridine-3-carboxamide (43). HPLC purity 98%; 1H NMR (CD3OD)
δ 8.82 (d, J ) 2.2 Hz, 1H), 8.19 (s, 1H), 8.14 (d, J ) 6.6 Hz, 1H),
7.98 (d, J ) 12.6 Hz, 1H), 7.71 (dd, J ) 2.2, 9.3 Hz, 1H), 7.67 (t,
J ) 8.3 Hz, 1H), 7.64-7.61 (m, 2H), 7.48 (d, J ) 8.3 Hz, 1H),
7.42-7.38 (m, 2H), 7.33 (t, J ) 8.2 Hz, 1H), 7.11 (t, J ) 8.8 Hz,
2H), 6.67 (d, J ) 6.6 Hz, 1H), 6.51 (d, J ) 3.8 Hz, 1H); MS (ESI)
587 (M + H)+.
6-Phenylpicolinic Acid (45). A mixture of 6-bromopicolinic acid
44 (2.02 g, 10 mmol), phenylboronic acid (1.22 g, 10 mmol), cesium
carbonate (5.0 g, 15 mmol), and PdCl2 [(t-Bu)2P(OH)]2 (180 mg,
0.36 mmol, CombiPhos Catalyst, Inc.) in DMF (20 mL) and water
(3 mL) was purged with Ar gas and heated at 110 °C for 24 h. The
reaction did not proceed any further despite remaining starting
materials. The mixture was acidified to pH 6 using 1 N aqueous
HCl, and the mixture was extracted with EtOAc (150 mL). The
EtOAc layer was dried over Na2SO4 and concentrated in vacuo,
and the residue was purified by preparative HPLC to obtain 45 as
a white solid (350 mg, 18%). HPLC purity 96%; 1H NMR (CDCl3)
δ 10.46 (br s, 1H), 8.10 (d, J ) 7.7 Hz, 1H), 8.03 (m, 4H), 7.53
(m, 3H); MS (ESI) 200 (M + H)+.
2-Carboxy-6-phenylpyridine 1-Oxide (46). To a solution of
phenypicolinic acid 45 (150 mg, 0.75 mmol) in dichloroethane (8
mL) at room temperature was added a solid of m-CPBA (300 mg,
∼77% purity, Aldrich), and the mixture was heated at 60 °C
overnight. Solid K2HPO4 (600 mg) was added to the reaction
mixture, and the suspension was heated at 60 °C for 90 min. With
addition of more m-CPBA (600 mg), the mixture continued to be
heated at 60 °C for 30 min. The mixture was directly purified by
preparative HPLC to obtain 46 as a white solid (110 mg, 68%).
HPLC purity 99%; 1H NMR (DMF-d7) δ 8.58 (s, 1H), 8.42 (dd, J
) 7.7, 2.2 Hz, 1H), 8.10-7.88 (m, 4H), 7.60-7.58 (m, 3H); MS
(ESI) 216 (M + H)+.
2.2 Hz, 1H), 7.90 (d, J ) 2.2 Hz, 1H), 7.62-7.58 (m, 2H), 7.12 (t,
J ) 8.8 Hz, 2H), 4.12 (t, J ) 5.0 Hz, 2H), 3.86 (t, J ) 5.0 Hz,
2H), 3.83 (s, 3H); MS (ESI) 292 (M + H)+.
(B) A solution of methyl 5-(4-fluorophenyl)-1-(2-hydroxyethyl)-
4-oxo-1,4-dihydropyridine-3-carboxylate (160 mg, 0.55 mmol) in
MeOH (3 mL) and 6 N aqueous NaOH (0.5 mL) was stirred for
2 h at room temperature. The reaction mixture was acidified with
1 N aqueous HCl solution and the product was extracted with ethyl
acetate. The organic layer was washed with brine, dried over
MgSO4, and concentrated to give 36 as a light-yellow solid (135
mg, 89%), which was used directly in the next step without further
purification. MS (ESI) 278 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-5-(4-
fluorophenyl)-1-(2-hydroxyethyl)-4-oxo-1,4-dihydropyridine-3-
carboxamide (37). To a mixture of 36 (125 mg, 0.45 mmol) in
anhydrous DMF (1.5 mL) were added HATU (206 mg, 0.54 mmol)
and i-Pr2NEt (0.5 mL) followed by aniline 8 (115 mg, 0.0.47 mmol)
under nitrogen. The reaction mixture was stirred for 1.5 h at room
temperature and purified by preparative HPLC to give 37 as a white
solid (105 mg, 67%), mp180-182 °C. HPLC purity 96%; 1H NMR
(CD3OD) δ 8.76 (d, J ) 2.2 Hz, 1H), 8.31 (d, J ) 6.6 Hz, 1H),
8.08-8.06 (m, 2H), 7.67-7.65 (m, 2H), 7.57 (d, J ) 3.3 Hz, 1H),
7.51-7.43 (m, 2H), 7.19 (t, J ) 8.8 Hz, 2H), 6.91 (d, J ) 6.6 Hz,
1H), 6.69 (d, J ) 3.3 Hz, 1H), 4.25 (t, J ) 5.0 Hz, 2H), 3.93 (t, J
) 5.5 Hz, 2H); HRMS (ESI) calcd for C27H20F2N4O4 (M + H)+
503.1526, found 503.1524.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-5-(4-
fluorophenyl)-4-oxo-4H-pyran-3-carboxamide (40). (A) To a
solution of aldehyde 38 (218 mg, 1.0 mmol) in THF (5 mL) and
water (1 mL) was added oxone (1.84 g, 3 mmol). The reaction
mixture was stirred for 2 h at room temperature and concentrated
in vacuo. The residue was triturated with water/ether, and the solid
that formed was collected by filtration and dried to give 5-(4-
fluorophenyl)-4-oxo-4H-pyran-3-carboxylic acid as a white solid
(183 mg, 78%), mp 175-177 °C. HPLC purity 97%; 1H NMR
(DMSO-d6) δ 13.60 (br s, 1H), 9.04 (s, 1H), 8.67 (s, 1H), 7.68-7.60
(m, 2H), 7.30 (t, J ) 8.8 Hz, 2H); MS (ESI) 235 (M + H)+.
(B) To a solution of 5-(4-fluorophenyl)-4-oxo-4H-pyran-3-car-
boxylic acid (200 mg, 0.85 mmol) in CH2Cl2 (10 mL) at 0 °C was
added thionyl chloride (202 mg, 1.7 mmol). The reaction mixture
was stirred for 1 h at room temperature and concentrated in vacuo.
The residue was dissolved in a mixed solvent of DMF (2 mL) and
CH2Cl2 (4 mL) at 0 °C. A solution of 8 (207 mg, 0.85 mmol) in
CH2Cl2 (1 mL) was added dropwise to the acyl chloride solution
followed by the addition of 2,6-lutidine (0.20 mL, 1.70 mmol). The
reaction mixture was stirred for 1 h at 0 °C and concentrated in
vacuo. Upon addition of cold water (5 mL), solid was precipitated,
which was collected and dried to obtain 40 (202 mg, 52%) as a
white solid. HPLC purity 98%; 1H NMR (CD3OD) δ 8.83 (d, J )
2.2 Hz, 1H), 8.34 (d, J ) 2.2 Hz, 1H), 8.15 (d, J ) 6.0 Hz, 1H),
7.83 (dd, J ) 11.0, 2.8 Hz, 1H), 7.70-7.66 (m, 2H), 7.60 (d, J )
10.5 Hz, 1H), 7.55 (t, J ) 8.2 Hz, 1H), 7.39 (d, J ) 3.3 Hz, 1H),
7.17 (t, J ) 8.8 Hz, 2H), 6.70 (d, J ) 6.0 Hz, 1H), 6.51 (d, J ) 3.3
Hz, 1H); MS (ESI) 460 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-1-cy-
clopropyl-5-(4-fluorophenyl)-4-oxo-1,4-dihydropyridine-3-car-
boxamide (41). To a solution of 40 (46 mg, 0.1 mmol) in THF (2.0
mL) at room temperature was added cyclopropanamine (29 mg, 0.5
mmol). The reaction mixture was stirred for 14 h, and concentrated in
vacuo. The residue was purified by preparative HPLC to obtain 41 as
a white solid (30 mg, 61%), mp 212-214 °C (dec). HPLC purity 98%;
1H NMR (CD3OD) δ 8.65 (d, J ) 2.2 Hz, 1H), 8.16 (d, J ) 6.6 Hz,
1H), 7.98 (d, J ) 2.8 Hz, 1H), 7.95 (dd, J ) 11.0, 2.8 Hz, 1H),
7.57-7.53 (m, 2H), 7.42 (d, J ) 3.3 Hz, 1H), 7.36 (d, J ) 8.8 Hz,
1H), 7.31 (t, J ) 8.3 Hz, 1H), 7.09 (t, J ) 8.8 Hz, 2H), 6.70 (d, J )
6.6 Hz, 1H), 6.52 (d, J ) 3.8 Hz, 1H), 3.75-3.70 (m, 1H), 1.12-1.08
(m, 4H); MS (ESI) 499 (M + H)+.
N-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl)-6-
phenylpicolinamide (47). To a solution of acid 45 (48 mg, 0.24
mmol) in DMF (5 mL) at room temperature were added EDCI ·HCl
(75 mg, 0.39 mmol) and HOBt ·hydrate (25 mg, 0.16 mmol)
followed by pyrrolopyridine 8 (65 mg, 0.27 mmol). The reaction
mixture was stirred at room temperature over the weekend, and
the mixture was purified directly by preparative HPLC to obtain
1
47 as a light-brown solid (25 mg, 19%). HPLC purity 97%; H
NMR (DMF-d7) δ 11.93 (br s, 1H), 11.01 (s, 1H), 8.37-8.17 (m,
7H), 7.51-7.57 (m, 6H), 6.52 (d, J ) 5.5 Hz, 1H), 6.41(d, J ) 3.3
Hz, 1H); MS (ESI) 441 (M + H)+.
2-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenylcar-
bamoyl)-6-phenylpyridine 1-Oxide (4). To a solution of N-oxide
46 (25 mg, 0.12 mmol) in DMF (3.5 mL) at room temperature
were added EDCI ·HCl (42 mg, 0.22 mmol) and HOBt ·H2O (18
mg, 0.12 mmol) followed by pyrrolopyridine 8 (40 mg, 0.16 mmol).
The reaction mixture was stirred at room temperature overnight,
and the mixture was purified directly by preparative HPLC to obtain
4 as a light-brown solid (36 mg, 54%). HPLC purity 96%; 1H NMR
(DMF-d7): δ 14.12 (br s, 1H), 12.10 (br s, 1H), 8.48 (dd, J ) 7.7,
2.2 Hz, 1H), 8.24 (d, J ) 5.5 Hz, 1H), 8.18 (dd, J ) 12.5, 2.2 Hz,
1H), 7.94-7.51 (m, 10H), 6.61 (d, J ) 5.5 Hz, 1H), 6.44 (d, J )
3.3 Hz, 1H); MS (ESI) 425 (M + H)+.
2-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenylcar-
bamoyl)-6-(4-fluorophenyl)pyridine 1-Oxide (49). (A) 6-(4-
Fluorophenyl)picolinic Acid. A solution of 6-bromopicolinic acid
(2.02 g, 10 mmol) in DME containing 4 mL of 10% aqueous
Na2CO3 was purged with Ar gas. To this mixture was added

5340 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Kim et al.
Pd(PPh3)4 followed by 2-(4-fluorophenyl)-5,5-dimethyl-1,3,2-di-
oxaborinane (2.40 g, 11.5 mmol) and EtOH (20 mL). This mixture
was purged with Ar gas. The reaction mixture was heated at 100
°C for 2.5 h in a sealed tube. Additional 2-bromopicolinic acid
(900 mg) and Pd(OAc)2 were added to the mixture, and it was
heated at 100 °C for 4.5 h. Trifluoroactic acid (20 mL) was added
to the reaction mixture and concentrated in vacuo, and MeOH (150
mL) was added to the residue. The insoluble material was filtered,
the filtrate solution was concentrated, and the residue was purified
by flash column chromatography using silica gel and eluting with
9:1 EtOAc/MeOH to 1:2.1:0.3 EtOAc/MeOH/HOAc to provide the
desired product (1.0 g, 40% based on borinane starting material)
Each compound was tested at eight different concentrations in
triplicate, as was a control sample. Growth inhibition of 50% (IC50)
is calculated by analysis of the data in Excel using a four-parameter
logistic equation with data fitted using the Levenburg-Marquardt
algorithm.
Metabolic Stability. The compound was incubated with human
liver microsomes purchased from In Vitro Technologies (Baltimore,
MD) and pooled from 10 individual donors. The rates of oxidative
metabolism were measured under the following conditions: com-
pound (substrate) at 3 µM final concentration; final microsomal
protein concentration of approximately 1 mg/mL; 1 mM NADPH;
56 mM of pH 7.4 potassium phosphate buffer. Incubations were
performed at 37 °C and were initiated by the addition of the
substrate. Incubations were quenched by the addition of one volume
of acetonitrile after 10 min. Samples were then analyzed for the
parent compound by LC/MS. The rate of metabolism was calculated
by determining the nanomoles of parent compound oxidized and
dividing it by the time of incubation and the milligrams of protein.
Pharmacokinetic Parameters Obtained in Mice. To evaluate
the oral bioavailability of compound 2 in male Balb/C mice, a single
dose was administered as a solution in PEG 400/water (70:30) by
either tail vein injection (iv, 5 mg/kg) or by oral gavage (po, 10
mg/kg). The mice were fasted overnight prior to dosing and fed
4 h postdose. A total of 18 mice were used in the study (n ) 9
each for iv and po group). Three serum samples were collected
from each mouse, the first two samples by retro-orbital bleed (∼100
µL/20-25 g mouse) and the third sample by cardiac puncture.
Blood samples were collected at 0.17, 0.5, 1, 3, 6, 8, 10, 12, and
24 h time points following iv dosing and at 0.25, 0.5, 1, 3, 6, 8,
10, 12, and 24 h following oral dosing. Blood samples were allowed
to clot on ice and were centrifuged, and serum was harvested. Serum
samples were stored at -20 °C until analysis. Concentrations of
parent compound were later determined by LC/MS/MS. Composite
serum concentration-time profiles were constructed for pharma-
cokinetic analysis.
Serum Protein Binding of Compound 2. The extent of protein
binding of compound 2 was determined in mouse and human sera
using the equilibrium dialysis method. All experiments were carried
out using pooled serum (n ) 10 for mice, n ) 3 for human) obtained
from Bioreclamation Inc. (Hicksville, NY). Compound 2 (1 mM)
in acetonitrile was added to serum at a ratio of 1:100 to give a
final concentration of 10 µM. Serum samples were dialyzed against
134 mM phosphate buffer (pH 7.4). The Micro-Equilibrium
Dialyzer (500 µL chamber volume, Amika Corp., Holliston, MA)
containing spiked serum was incubated in a shaking water bath
maintained at 37 °C for 4 h. A 10 000 molecular weight cutoff
dialysis membrane (Amika Corp., Holliston, MA) was used. All
experiments were carried out in triplicate. Aliquots of buffer and
serum were taken at 4 h and analyzed by LC/MS/MS. Compound
2 was stable under these conditions over the 4 h incubation period
in mouse and human serum. From each dialysis cell, the free and
bound drug percentages were calculated as follows: % free ) 100
× (concentration in buffer)/(concentration in serum); % bound )
100 - (% free).
1
as a white solid. H NMR (CD3OD) δ 8.01 (d, J ) 7.7 Hz, 1H),
7.94-7.87 (m, 3H), 7.73 (d, J ) 7.7 Hz, 1H), 7.13 (t, J ) 8.8 Hz,
2H); MS (ESI) 234 (M + H)+.
(B) 6-(4-Fluorophenyl)picolinic Acid N-Oxide. A mixture of
picolinic acid derivative (1.0 g, 4.6 mmol), Na2HPO4 (1.2 g), and
m-CPBA (1.1 g, ∼70%) in CH2ClCH2Cl (30 mL) was stirred at
room temperature for 2 h. Additional Na2HPO4 (0.8 g) and m-CPBA
(1.0 g, ∼70% from Aldrich) was added to the reaction mixture,
and it was stirred for 3 h at room temperature. More Na2HPO4
(0.5 g) and m-CPBA (0.5 g, ∼70%) were added to the reaction
mixture, and the mixture was stirred at room temperature overnight.
The next morning, CHCl3 (160 mL) and 2 N aqueous HCl (50 mL)
were added to the reaction mixture. The organic layer was separated,
dried over MgSO4, and concentrated. The residue was purified by
flash column chromatography using silica gel and eluting with 7:2.4:
0.6 EtOAc/MeOH/HOAc to provide the desired product that was
contaminated by a small amount of m-CPBA. This impure material
was purified by preparative HPLC to obtain the desired N-oxide
product (175 mg, 16%) as a white solid. 1H NMR (DMF-d7) δ
8.45 (dd, J ) 8.3, 2.2 Hz, 1H), 8.15 (d, J ) 2.2 Hz, 1H), 8.13-8.00
(m, 4H), 7.45 (t, J ) 8.7 Hz, 2H).
(C) 2-(4-(1H-Pyrrolo[2,3-b]pyridin-4-yloxy)-3-fluorophenyl-
carbamoyl)-6-(4-fluorophenyl)pyridine 1-Oxide (49). To a solu-
tion of the N-oxide obtained above (23 mg, 0.1 mmol) and HOBt
(10 mg) in DMF (2 mL) at room temperature was added EDCI ·HCl
(30 mg, 0.16 mmol) followed by 4-(4-amino-2-fluorophenoxy)py-
ridine-2-amine (22 mg, 0.1 mmol). The reaction mixture was stirred
at room temperature overnight. Direct purification of the reaction
mixture by preparative HPLC afforded 49 as a white solid (25 mg,
1
46%). HPLC purity 98%; H NMR (DMF-d7) δ 14.00 (br s, 1H),
12.14 (br s, 1H), 8.43 (dd, J ) 8.0, 2.2 Hz, 1H), 8.15 (dd, J )
12.8, 2.4 Hz, 1H), 8.08 (d, J ) 7.1 Hz, 1H), 7.99-7.37 (m, 9H),
6.72 (dd, J ) 7.0, 2.4 Hz, 1H), 6.32 (d, J ) 2.3 Hz, 1H), 3.7 (br
s, 1H); MS (ESI) 417 (M + H)+.
Met Kinase Assay. The kinase reaction consisted of baculovirus
expressed GST-Met, 3 µg of poly(Glu/Tyr) (Sigma), 0.12 µCi 33
P
γ-ATP, 1 µM ATP in 30 µL of kinase buffer (20 mM Tris-Cl, 5
mM MnCl2, 0.1 mg/mL BSA, 0.5 mM DTT). Reactions were
incubated for 1 h at 30 °C and stopped by the addition of cold
trichloroacetic acid (TCA) to a final concentration of 8%. TCA
precipitates were collected onto GF/C unifilter plates (Packard
Instrument Co., Meriden, CT) using a Filtermate universal harvester
(Packard Instrument Co., Meriden, CT), and the filters were
quantitated using a TopCount 96-well liquid scintillation counter
(Packard Instrument Co., Meriden, CT). Dose response curves were
generated to determine the concentration required to inhibit 50%
of substrate phosphorylation (IC50). Compounds were dissolved at
10 mM in dimethylsulfoxide (DMSO) and evaluated at 10
concentrations, in duplicate.
In Vivo Antitumor Activity in the Subcutaneously Implanted
GTL-16 Xenograft Model in Nude Mice. Female Balb/C athymic
(nu+/nu+)mice,6-8weeksold,wereobtainedfromSprague-Dawley
Co. (Indianapolis, IN). Animals were provided with food and water
ad libitum and housed five per cage. Mice were maintained in
accordance with Bristol-Myers Squibb’s Institutional Animal Care
and Use Committee in accordance with the American Association
for Accreditation of Laboratory Animal Care (AAALAC) guidelines
for the humane treatment and care of laboratory mice.
GTL-16 tumor fragments maintained by serial passage in vivo
were implanted subcutaneously in the hind flank using an 18 g
trocar. Approximately 2 weeks after implant, when tumor sizes
reached 100-150 mm3, oral dosing was initiated using gavage
needles with either compound at the indicated concentrations or
vehicle (7:3 PEG 400/H2O) in the control group. Tumor growth
was assessed twice weekly by vernier caliper measurement. Group
sizes were n ) 8 or 9.
Cellular Proliferation Assay. Inhibition of cell proliferation was
assessed by a MTS assay using a CellTiter 96 aqueous nonradioac-
tive proliferation assay kit (Promega). Cells were inoculated into
96-well microtiter plates containing 0.5% fetal calf serum and
incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity
for 24 h prior to addition of drug. At the time of drug addition,
one plate of the cell line was processed using the above kit to
represent a measurement of the cell population at the time of drug
addition. Following drug addition, the plates were incubated for
an additional 72 h before processing to measure the cell population.

Pyrrolopyridine-Pyridone Based Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5341
cancer. Clin. Cancer Res. 2006, 12, 3657–3650.
(6) http://www.vai.org/vari/metandcancer/index.as.
Antitumor activity was determined by calculating the maximum
percent tumor growth inhibition (TGI) of treated animals using the
formula % TGI ) {(Ct - Tt)/(Ct - C0)} × 100, where Ct is the
median tumor volume (mm3) of vehicle treated control (C) mice
at time t. Tt is the median tumor volume of treated mice (T) at
time t. C0 is the median tumor volume of control mice at time 0.
Activity is defined as a continuous % TGI > 50% for at least one
tumor volume doubling time after the start of drug treatment.
Measurement of Phospho Met Protein Levels in GTL-16
Tumor Extracts. GTL-16 human gastric carcinoma cell line
xenografts were implanted in nude mice and allowed to grow to
200-300 mm3 as previously described. Mice were given a single
oral dose of compound 2. At the indicated time points, mice were
sacrificed and tumors immediately excised and snap-frozen in liquid
nitrogen. Tumors were homogenized and lysed in ice cold buffer
containing 20 mM Tris, 0.12 M NaCl, 0.2 mM EDTA, 1% Triton,
5% glycerol, 1 mM sodium vanadate, and 40 µM ammonium
molybdate, with protease inhibitors (Complete Tabs, Boehringer
Mannheim). The protein concentration of tumor lysates was
determined using a BCA protein assay (Pierce). Approximately 500
µg of total protein was immunoprecipitated using anti-hMet clone
C-12 antibody (Santa Cruz Biotechnology) linked to immunopure
immobilized protein A/G resin (Pierce) for 3 h in lysis buffer,
shaking at 4 °C. Lysates were washed 3 times and resuspended in
Lane Marker Reducing Sample Buffer (Pierce). Protein was
denatured and separated on a 7.5% polyacrylamide gel (BioRad)
and transferred onto pretreated nitrocellulose membrane (BioRad).
Membranes were blocked using LiCor Odyssey blocking buffer.
Antibodies were diluted in blocking solution containing 0.1%
Tween-20. Two color Western blots were performed using rabbit
anti-hMet C-12 antibody (Santa Cruz Biotechnology) and mouse
anti-phosphotyrosine PY-20 antibodies (BD Transduction Labora-
tories). For detection, goat antimouse Alexa Fluor 680 (Molecular
Probes) and goat antirabbit IRDye 800 (Rockland Immunochemi-
cals) antibodies diluted 1:10000 were utilized. Blots were incubated
with primary antibodies simultaneously for 2 h at room temperature,
washed with Tris-buffered saline, and incubated with both IR-
labeled secondary antibodies for 1 h at room temperature. Blots
were imaged and quantified using the Li-Cor Odyssey imaging
system.
(7) Cui, J. J. Inhibitors targeting hepatocyte growth factor receptor and
their potential therapeutic applications. Expert Opin. Ther. Pat. 2007,
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Wang, L. M.; Vande Woude, G. F. Neutralizing monoclonal antibodies
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Crystal Structure of Met Kinase Complexed with Compound
2. Crystals of the Met kinase domain (1067-1378) complexed with
compound 2 were grown at room temperature by the hanging drop
vapor diffusion method. The hanging drop consisted of 10λ of the
concentrated Met kinase complex mixed with an equal volume of
reservoir solution containing 12% MEPEG 5k, 0.1 M Hepes (pH
7.1), and 11% 2-propanol. Crystals were flash-frozen in cryopro-
tectant containing 15% glycerol, and data were collected at the
IMCA-CAT beamline ID17 at the Advanced Photon Source,
Argonne National Laboratory, Argonne, IL (Table 6).
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Vaula, G.; Comoglio, P. M. c-Met is amplified but not mutated in a
cell line with an activated met tyrosine kinase. Oncogene 1991, 6,
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Nallasura, V.; Fox, E. A.; Hansen, M.; Schaefer, E.; Naoki, K.; Lader,
A.; Richards, W.; Sugarbaker, D.; Husain, A. N.; Christensen, J. G.;
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Note Added after ASAP Publication. This manuscript was
released ASAP on August 9, 2008 with an error in Scheme 1. The
correct version was released on August 19, 2008.
Supporting Information Available: HPLC and elemental
analysis data for key compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
(22) 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. Amplification
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