Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4- ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the met kinase superfamily

Article abstract of DOI:10.1021/jm801586s

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)- 1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met- dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Full text of DOI:10.1021/jm801586s

J. Med. Chem. 2009, 52, 1251–1254
1251
disclosed. Replacement of the pendent acylurea moiety by a
conformationally constrained 2-pyridone to give Met kinase
inhibitors with potent antiproliferative activity in a Met-
dependent human gastric carcinoma GTL-16 cell line was
subsequently reported.6 Herein, we describe application of the
2-pyridone fragment to the aminopyridine chemotype, which
ultimately led to identification of a compound for clinical
development. The synthesis, SAR, structural biology, and
preclinical assessment of aminopyridines are described.
Discovery of N-(4-(2-Amino-3-chloropyridin-
4-yloxy)-3-fluorophenyl)-4-ethoxy-1-
(4-fluorophenyl)-2-oxo-1,2-
dihydropyridine-3-carboxamide
(BMS-777607), a Selective and Orally
Efficacious Inhibitor of the Met Kinase
Superfamily
Gretchen M. Schroeder,* Yongmi An, Zhen-Wei Cai,
Xiao-Tao Chen, Cheryl Clark, Lyndon A. M. Cornelius,
Jun Dai, Johnni Gullo-Brown, Ashok Gupta,
Benjamin Henley, John T. Hunt, Robert Jeyaseelan,
Amrita Kamath, Kyoung Kim, Jonathan Lippy,
Louis J. Lombardo, Veeraswamy Manne,
Simone Oppenheimer, John S. Sack, Robert J. Schmidt,
Guoxiang Shen, Kevin Stefanski, John S. Tokarski,
George L. Trainor, Barri S. Wautlet, Donna Wei,
David K. Williams, Yingru Zhang, Yueping Zhang,
Joseph Fargnoli, and Robert M. Borzilleri
Pyridone 16 is a potent inhibitor of Met kinase (IC50 ) 1.8
nM) with excellent activity in the Met-driven GTL-16 cell
proliferation assay (IC50 ) 60 nM). Application of the confor-
mationally constrained 2-pyridone ring to the aminopyridine
Bristol-Myers Squibb Research and DeVelopment, P.O. Box 4000,
Princeton, New Jersey, 08543-4000
series gave parent pyridone 2 (IC50 ) 4.5 nM, GTL-16 IC50
)
170 nM), which served as a starting point for SAR analysis.
On the basis of X-ray crystal structures of related analogues,4-6
we hypothesized that these inhibitors would occupy the Met
ATP-binding site and that substitution of the pyridine C-3
position would provide access to the pocket occupied by the
ribose group of ATP. As described in an earlier series, additional
interactions with the Met protein could be gained by projecting
solubilizing groups such as alcohols and amines into the ribose
pocket.4 We initially sought to introduce such groups in the
aminopyridine series via a linker at the pyridine C-3 position
to simultaneously improve potency, solubility, and the CYP450
inhibition profile.
ReceiVed December 16, 2008
Abstract: Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-
1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were iden-
tified as potent and selective Met kinase inhibitors. Substitution of the
pyridine 3-position gave improved enzyme potency, while substitution
of the pyridone 4-position led to improved aqueous solubility and kinase
selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-
dependent GTL-16 human gastric carcinoma xenograft model following
oral administration. Because of its excellent in vivo efficacy and
favorable pharmacokinetic and preclinical safety profiles, 10 has been
advanced into phase I clinical trials.
Alcohols and amines were appended to the aminopyridine
core via a carbon linker, and representative analogues are
depicted in Table 1. N-Methylpiperazine 3 gave excellent
potency in the biochemical assay (IC50 ) 5.9 nM), demonstrat-
ing that substitution at the pyridine C-3 position was tolerated.
However, disappointing results were obtained in the GTL-16
cell-based assay (GTL-16 IC50 ) 2800 nM). In our previous
work, rigid amines with limited degrees of freedom gave
enhanced Met potency. Application of this concept as in
propargylamine 4 gave similar biochemical potency but im-
proved cellular activity (IC50 ) 3.8 nM, GTL-16 IC50 ) 210
nM). The corresponding propargyl alcohol 5 gave an exception-
ally potent Met inhibitor in the biochemical and cell-based
assays (IC50 ) 2.7 nM, GTL-16 IC50 ) 23 nM). Methanol
adduct 6 gave similar biochemical inhibition (IC50 ) 3.4 nM)
but was 10-fold less potent in cells relative to the propargyl
alcohol (GTL-16 IC50 ) 280 nM).
Intrigued by the excellent cellular potency of propargylic
alcohol 5 and with the desire to keep the pyridine C-3 substituent
small, we prepared the unsubstituted propargylic compound 7.
This analogue also demonstrated potent inhibition of Met kinase
in biochemical and cell-based assays (IC50 ) 1.0 nM, GTL-16
IC50 ) 31 nM) but suffered from potent CYP450 inhibition
(3A4, IC50 < 1 µM), a common problem among the propargyl
analogues. Replacement of the propargyl unit with a simple
Met kinase, also known as hepatocyte growth factor receptor,
is an oncogenic transmembrane receptor tyrosine kinase that
has been implicated in a variety cancers.1,2 Binding of its
endogenous ligand hepatocyte growth factor (HGF), also known
as scatter factor (SF), results in dimerization of the Met kinase
receptor and intermolecular transphosphorylation of multiple
tyrosine residues in the intracellular domain. The resulting
activated enzyme binds and phosphorylates adaptor proteins at
a multifunctional docking site, which in turn activates signal
transducers to mediate a variety of cellular responses including
cell growth, migration and invasion, tumor metastasis, angio-
genesis, wound healing, and tissue regeneration. While normal
Met/HGF signaling plays a critical role in placental development
and liver regeneration,3 aberrant Met signaling can lead to
oncogenic transformation. Met kinase can become dysregulated
via a variety of mechanisms including ligand-dependent activa-
tion, activating mutations, Met overexpression, and interactions
with other receptor families (heterodimerization/cross-talk).1
The structure-activity relationships (SARs) of several Met
kinase inhibitors derived from pyrrolo[2,1-f][1,2,4]triazine,4
pyrrolopyridine,5 and aminopyridine5 scaffolds were recently
* To whom correspondence should be addressed. Phone: 609-252-3965.
Fax: 609-252-7410. E-mail: gretchen.schroeder@bms.com.
10.1021/jm801586s CCC: $40.75
2009 American Chemical Society
Published on Web 02/13/2009

1252 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Letters
Scheme 1a
Table 1. Enzymatic and Cellular Activities of Select 2-Aminopyridine
Analoguesa
a Reagents and conditions: (a) LiTMP, ether, -78 °C, then CO2, 39%;
(b) SOCl2, 80 °C, then NH4OH, 0 °C, 76%; (c) 4-amino-2-fluorophenol,
KO-t-Bu, DMF, 50 °C, 79%; (d) TMSI, CH3CN, 90%; (e) 4-fluorophe-
nylboronic acid, Cu(OAc)2, 2,6-lutidine, myristic acid, toluene, 68%; (f)
NaOCl, 2-methyl-2-butene, NaH2PO4, THF/t-BuOH/H2O, 70%; (g) SOCl2,
toluene; (h) DIPEA, THF/DMF, 89% (two steps); (i) NaOEt, EtOH, THF,
95%; (j) PhI(OAc)2, EtOAc/CH3CN/H2O, 74%.
improved aqueous solubility, attractive in vitro profile, and
desirable metabolic stability/CYP450 profile (IC50 values > 20
µM), 10 was selected for further characterization in kinase
selectivity, PK, and in vivo efficacy studies.
Compound 10 was prepared according to the synthetic
sequence outlined in Scheme 1. Commercially available 3,4-
dichloropyridine 13 was converted to acid 14 by deprotonation
with LiTMP and quenching with dry ice.8 The resulting acid
was converted to amide 15 under standard conditions. Nucleo-
philic displacement of the C-4 chloride of 15 with 4-amino-2-
fluorophenol gave key intermediate 16. The pyridone subunit
19 was synthesized from 4-iodo-2-methoxynicotinaldehyde.9
Thus, the methyl ether of 17 was cleaved with TMSI and the
resulting amide was arylated with 4-fluorophenylboronic acid
using copper(II) acetate.10 Oxidation of aldehyde 18 to the
corresponding acid followed by acid chloride formation gave
intermediate 19. Amine 16 and acid chloride 19 were joined
under standard conditions, and the iodide was reacted with
sodium ethoxide to give ethyl ether 20. Finally, Hoffman
rearrangement delivered 10 in high yield.
An X-ray crystal structure of 10 complexed to the Met kinase
domain confirmed that 10 resides in the ATP-binding site with
the activation loop in an inactive, DFG-out conformation (Figure
1).11 The 2-aminopyridine core anchors inhibitor 10 to the hinge
region via two key hydrogen bonding interactions with Met1160;
the backbone NH of Met1160 donates a hydrogen bond to the
pyridine nitrogen, while the backbone carbonyl of Met1160
accepts a hydrogen bond from the 2-amino group. As postulated,
the 3-chloro group is directed toward the ribose pocket. The
central phenyl ring π-stacks with Phe1223 (DFG motif) and is
flanked on the opposite face by gatekeeper residue Leu1157.
The pyridone carbonyl forms an intramolecular hydrogen bond
with the amide NH and a second hydrogen bond with the
backbone NH of Asp1222. Finally, the terminal phenyl ring
occupies a deep hydrophobic pocket defined by Phe1134,
Leu1195, and Phe1200.
a See ref 6 for a description of assay conditions. b IC50 values are reported
as the mean of at least three independent determinations. c Met-dependent
human gastric carcinoma cell line.
branched alkyl group such as isopropyl provided no benefit (8,
IC50 ) 3.4 nM, GTL-16 IC50 ) 120 nM). Finally, 3-chloro
analogue 9 was found to potently inhibit Met kinase with an
IC50 of 1.0 nM while maintaining excellent cellular potency
(GTL-16 IC50 ) 57 nM). Chloro-substituted aminopyridine 9
benefited from excellent stability in mouse liver microsomes
(MLM ) 0.012 (nmol/min)/mg protein) and an encouraging
CYP450 profile (3A4 IC50 ) 4 µM; 2C9 IC50 ) 7 µM; 2C19,
2D6, 1A2 IC50 values of >20 µM); therefore, it was selected
as a scaffold for pyridone SAR exploration to improve kinase
selectivity and the CYP450 profile.
A series of alkoxy groups was appended to the pyridone ring
at R2 (Table 1). Ethoxy analogue 10 inhibited Met kinase with
an IC50 of 3.9 nM (GTL-16 IC50 ) 100 nM). In addition,
compound 10 demonstrated >40-fold improvement in aqueous
solubility at pH 1.0 (aqueous solubility ) 400 µg/mL, crystal-
line).7 The dramatic improvement in aqueous solubility can be
attributed to a deviation from planarity induced by the R2
substituent which results in disruption of crystal packing forces
(vide infra). Replacement of the R1 chlorine atom of 10 with
other halogens (e.g., F, Br) or a methyl substituent provided
analogues with inferior cellular potency. Additional alkoxy
groups were examined at R2 with isopropoxy analogue 11 giving
an in vitro potency (IC50 ) 3.2 nM, GTL-16 IC50 ) 140 nM)
similar to that of 10. Likewise, introduction of an alcohol
provided no benefit (12, IC50 ) 2.6 nM, GTL-16 IC50 ) 250
nM). On the basis of its relatively low molecular weight,
Compound 10 was evaluated in an in-house kinase selectivity
panel (Table 2). The Ki was determined for Met kinase (Ki )
4.6 ( 0.3 nM), and while 10 was found to be a potent inhibitor
of Met family member Ron and three phylogenetically related

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1253
Figure 3. Phospho Met protein levels in GTL-16 cell lysates.
Table 3. Pharmacokinetic Parameters for 10 in Mouse, Rat, and Doga
parameter
mouseb
ratc
dog
po dose (mg/kg)
iv dose (mg/kg)
Cmax (µM), po
Tmax (h), po
10
5
17.6
0.5
90
4.5
4.4
1.2
100
10
5
5c
1d
8.7 ( 4.2
1.7 ( 0.6
37.7 ( 19.2
4.8 ( 1.5
12.1 ( 1.4
1.0 ( 0.1
100
29.0 ( 6.9
2.3 ( 1.5
198 ( 68
4.9
0.82
0.22
Figure 1. X-ray crystal structure of 10 bound to the Met kinase domain
(1067-1378, triple mutant Y1212F, Y1252F, Y1253D).11
AUC0-24h (µM·h), po
t1/2 (h), iv
Table 2. Kinase Selectivity Profile of 10
Cl (mL min-1 kg-1), iv
Vss (L kg-1), iv
Fpo (%)
kinase
Met
Axl
Ron
Tyro3
Mer
Flt-3
AurB
Lck
VEGFR-2
TrkB
TrkA
PKA
enzyme IC50, nM
kinase
BTK
CDK2E
EGFR
GSK-3ꢀ
IGFR-1R
IKK1/2
InsR
Jak2
MK2
P38R
Pim-1
Plk-1/3
enzyme IC50, nM
100
3.9
1.1
1.8
4.3
14
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
a Vehicle: 70% PEG400-30% water. b Data reported as an average of
six animals. c Data reported as an average of three animals. d Data reported
as an average of two animals.
16
78
concentration. A result of <1% control correlates with a Kd <
1-3 µM. As expected, Met-related kinases (Ron, Axl, Tyro-3,
Mer) and several other kinases from Table 2 (AurB, Flt-3, Lck,
TrkA/B) registered binding of <1% control. A handful of other
kinases registered <1% control and were followed up in a two-
point assay where IC50 values were estimated to be between 50
and 500 nM (LOK, Tie-2, Mnk2, Musk) or >500 nM (Blk,
Epha3, Kit, Src). A cellular proliferation assay was used to
determine the IC50 for PDGFRꢀ (IC50 ) 1 µM). To conclude,
our in-house kinase selectivity panel and the Ambit binding
assay confirmed 10 to be a potent and selective inhibitor of the
Met kinase superfamily.
120
180
190
290
550
kinases (Axl, Tyro-3, and Mer), it was more than 40-fold
selective versus Lck, VEGFR-2, and TrkA/B and more than
500-fold selective versus all other receptor and nonreceptor
kinases in the panel. To further examine its selectivity, 10 was
profiled against more than 200 kinases in the Ambit phage-
based competition binding assay (Figure 2).12 In this assay,
results are reported as % control at a 10 µM screening
The antiproliferative activity of 10 was explored in additional
cellular settings, and 10 demonstrated selective inhibition of
proliferation in Met-driven tumor cell lines. In addition to potent
inhibition of proliferation of the GTL-16 cell line, which
contains an amplified Met gene locus, 10 also inhibited
proliferation in Met-driven H1993 (non-small-cell lung cancer,
Met amplification, IC50 ) 150 nM) and U87 (glioblastoma,
autocrine loop, IC50 ) 160 nM) cell lines. Importantly, 10 lacked
activity against the Met-independent N87 cell line (gastric, IC50
> 10 000 nM).
Phosphorylation levels of the Met protein in GTL-16 cell
lysates was determined following a 30 min incubation period
(Figure 3). Phosphorylation of the Met receptor was inhibited
in a concentration-dependent manner, consistent with the
observed in vitro activity and also consistent with the activity
in GTL-16 cells being a result of Met kinase inhibition.
The pharmacokinetic parameters obtained for 10 in mouse,
rat, and dog are summarized in Table 3. After iv administration,
10 demonstrated a moderate steady-state volume of distribution
(Vss) in mouse and rat and low systemic clearance (Cl) compared
to hepatic blood flow for all species. The compound was well
absorbed after oral administration of drug from solution
formulation with favorable half-lives (t1/2) and mean residence
times (MRT). Furthermore, the measured oral bioavailabilities
(Fpo) were excellent in all three species (100%).13
Figure 2. Radial plot of Ambit competition binding assay. The logit
transformation was applied [logit ) x/(100 - x)]. Abbreviations: TK,
tyrosine kinase; TKL, tyrosine kinase-like; AGC, protein kinase A, G,
and C families; CAMK, calcium-calmodulin dependent kinases; CMGC,
cyclin dependent kinases, mitogen-activated protein kinases, glycogen
synthase kinase-3, cyclin dependent kinase-like; STE, homologues of
yeast sterile 7, 11, 20 kinases.

1254 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Letters
References
(1) Recent reviews on Met kinase: (a) Liu, X.; Yao, W.; Newton, R. C.;
Scherle, P. A. Targeting the c-Met signaling pathway for cancer. Expert.
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Targeting the c-Met signaling pathway in cancer. Clin. Cancer Res.
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(2) Jarvis, L. M. Targeting the c-Met signaling pathway for cancer therapy.
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(4) Schroeder, G. M.; Chen, X.-T.; Williams, D. K.; Nirschl, D. S.; Cai,
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Borzilleri, R. M. Identification of pyrrolo[2,1-f][1,2,4]triazine-based
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(5) Cai, Z.-W.; Wei, D.; Schroeder, G. M.; Cornelius, L. A. M.; Kim, K.;
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D. K.; Lombardo, L. J.; Trainor, G. L.; Xie, D.; Zhang, Y.; An, Y.;
Sack, J. S.; Tokarski, J. S.; D’Arienzo, C.; Kamath, A.; Marathe, P.;
Zhang, Y.; Lippy, J.; Jeyaseelan, R.; Wautlet, B.; Henley, B.; Gullo-
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Figure 4. In vivo efficacy of 10 versus GTL-16 human tumor
xenografts in athymic mice (1qd x 14).
On the basis of its encouraging pharmacokinetic profile, 10
was evaluated in in vivo efficacy studies (Figure 4). A GTL-16
xenograft model was examined in which tumor cells were
implanted subcutaneously and staged to approximately 125 mm3
prior to commencement of once daily oral dosing of compound
for 14 consecutive days. Compound 10 was active at all dose
levels tested as defined by >50% tumor growth inhibition (TGI)
over one tumor volume doubling time (TVDT).14 The minimum
efficacious dose of 6.25 mg/kg corresponded to a TGI of 66%,
and complete tumor stasis was observed throughout the dosing
period at 25 and 50 mg/kg doses (TGI ) 95% and 102%,
respectively). The antitumor activity of 10 was dose responsive,
and the measured drug exposures at each dose level were dose
proportional (6.25 mg/kg, Cmax ) 4.5 µM, AUC0-24h ) 37
µM·h; 50 mg/kg, Cmax ) 43.7 µM, AUC0-24h ) 567 µM·h).
No overt toxicity was observed at any of these dose levels as
defined by weight loss or morbidity.
(7) Aqueous solubility at pH 6.5 is 2 µg/mL (crystalline).
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6143.
In conclusion, novel substituted N-(4-(2-aminopyridin-4-
yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydro-
pyridine-3-carboxamides were identified as potent inhibitors of
Met kinase activity. Optimization of the series SAR led to
identification of 10, a highly selective and orally bioavailable
Met kinase inhibitor. Complete tumor stasis was achieved in
the GTL-16 human gastric carcinoma xenograft model following
oral administration with no observed toxicity. On the basis of
its favorable in vivo efficacy, pharmacokinetic profile, and safety
profile, 10 has been advanced into clinical trials.
(10) Antilla, J. C.; Buchwald, S. L. Copper-catalyzed coupling of arylbo-
ronic acids and amines. Org. Lett. 2001, 3, 2077–2079.
(11) The structure was deposited in the PDB as 3F82.
(12) Fabian, M. A.; Biggs, W. H., III.; Treiber, D. K.; Atteridge, C. R.;
Azimioara, M. D.; Benedetti, M. G.; Carter, T. A.; Ciceri, P.; Edeen,
P. T.; Floyd, M.; Ford, J. M.; Galvin, M.; Gerlach, J. L.; Grotzfeld,
R. M.; Herrgard, S.; Insko, D. E.; Insko, M. A.; Lai, A. G.; Lelias,
J.-M.; Mehta, S. A.; Milanov, Z. V.; Velasco, A. M.; Wodicka, L. M.;
Patel, H. K.; Zarrinkar, P. P.; Lockhart, D. J. A small molecule-
kinase interaction map for clinical kinase inhibitors. Nat. Biotechnol.
2005, 23, 329–336.
Acknowledgment. We thank Gerry Everlof for determina-
tion of aqueous solubility, Brian Claus and Stephen Johnson
for generation of the radial plot, and Discovery Analytical
Sciences for compound characterization efforts.
(13) Protein binding was >99% across species.
(14) TGI ) {(Ct- Tt)/(Ct- C0)} × 100, where Ct ) the median tumor
volume (mm3) of vehicle-treated control mice at time t, Tt ) median
tumor volume of treated mice at time t, and C0 is the median tumor
volume of control mice at time 0.
Supporting Information Available: Characterization data for
2-12, experimental procedures for 10, and detailed description of
pharmacokinetic assays. This material is available free of charge
via the Internet at http://pubs.acs.org.
JM801586S