Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.042

Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

Full text of DOI:10.1016/j.bmcl.2010.01.042

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2998–3002
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and structure–activity relationships of novel
biarylamine-based Met kinase inhibitors
*
David K. Williams , Xiao-Tao Chen, Christine Tarby, Robert Kaltenbach, Zhen-Wei Cai,
John S. Tokarski, Yongmi An, John S. Sack, Barri Wautlet, Johnni Gullo-Brown, Benjamin J. Henley,
Robert Jeyaseelan, Kristen Kellar, Veeraswamy Manne, George L. Trainor, Louis J. Lombardo,
Joseph Fargnoli, Robert M. Borzilleri
Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomo-
lar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human
gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive con-
formation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase
inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor
xenograft model.
Received 23 October 2009
Revised 8 January 2010
Accepted 11 January 2010
Available online 20 January 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
c-Met kinase inhibitor
Kinase inhibitor
HGFR inhibitor
Met kinase
Met (c-Met, HGFR-hepatocyte growth factor receptor) is a gly-
cosylated, heterodimeric receptor tyrosine kinase, which is primar-
ily expressed on epithelial and endothelial cells.1 The activation of
Met is ligand dependent with the natural ligand being HGF/SF
(hepatocyte growth factor/scatter factor).2 The high-affinity bind-
ing of HGF/SF to Met results in receptor autophosphorylation and
upregulation of Met kinase activity, which in turn activates a com-
plex cell-signaling pathway that stimulates mitogenesis, motogen-
esis, and morphogenesis. Met signaling is implicated in a wide
variety of human malignancies, including: colon, lung, prostate,
gastric, and breast cancers.3 This signaling contributes to oncogen-
esis and tumor progression in several human cancers and pro-
motes aggressive cellular invasiveness that is strongly linked to
tumor metastasis.4 The activation of Met-mediated oncogenic sig-
naling can occur through aberrant paracrine or autocrine HGF
stimulation and/or receptor overexpression. Other modes of acti-
vation for Met include: activating mutations, gene rearrangement
and/or amplification, as well as the interaction of Met with other
receptor families.4
(PP) templates have been shown, by X-ray crystal structure analy-
sis, to serve as a replacement for the adenine group of ATP in estab-
lishing the critical hydrogen-bonding array to the hinge region of
the Met protein. Kinetic analysis has further demonstrated that
these compounds bind in the Met kinase domain in a manner that
is ATP-competitive. The malonamide and acylurea groups of 1 and
2 approach planarity in the bioactive conformation, due to a hydro-
gen-bonding arrangement between the phenoxyaniline NH and the
distal carbonyl group of either the malonamide or acylurea. Com-
pound 3 appears to engage in a similar arrangement between the
aniline NH and the carbonyl oxygen of the pyridone ring. Con-
straining this region of the molecule, as an aromatic ring, might
serve to lock this portion of the molecule into a bioactive confor-
mation. Molecular modeling studies suggested that a biarylamine
motif could potentially be used to preorganize the molecule. To
adopt the bioactive conformation, however, the aromatic rings
would need to approach the coplanarity observed between the
We have previously disclosed potent Met kinase inhibitors,
which have focused on the utilization of malonamide 1, acylurea
2, or pyridone 3 groups tethered to suitable kinase inhibitor tem-
plates (Fig. 1).5–7 These aminopyridine (AP) and pyrrolopyridine
H
H
N
F
F
X
Y
N
N
O
O
O
O
O
F
O
F
1: X = CH2, Y = NH
2: X = NH, Y = CH2
3
N
N
H2N
H
N
* Corresponding author.
E-mail address: david.williams@bms.com (D.K. Williams).
Figure 1. Previously disclosed Met kinase inhibitors.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.042

D. K. Williams et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2998–3002
2999
followed by treatment with bis(trifluoroacetoxy)iodobenzene and
pyridine afforded compound 8.10
H
N
O
F
NH O
F
Pyrrolo[2,3-b]pyridine analog 9 was prepared in a similar man-
ner. Thus, microwave-assisted reaction between 4-(1H-pyrrol-
o[2,3-b]pyridine-4-yloxy)-3-fluorobenzenamine7 and the desired
2-chloro- or 2-fluoro-nicotinamide, in the presence of p-toluene-
sulfonic acid monohydrate, afforded compound 9, albeit in low
(18–27%) yield. A subsequent optimization study on the synthesis
of the pyrrolopyridine analogs determined preferred conditions,
utilizing 4 N HCl in dioxane and the desired 2-fluoronicotinamide,
under thermal conditions, which afforded compound 9b in 72%
yield.11a
Initial structure–activity relationship (SAR) studies centered on
determining the effect of substitution on the nicotinamide nitro-
gen, with both the aminopyridine (AP) and pyrrolopyridine (PP)
based scaffolds (Table 1). Two assays were employed to evaluate
the activity of the compounds. A Met kinase substrate based assay
was used to assess the ability of these compounds to inhibit the
enzymatic activity of the Met receptor.6 Compounds exhibiting
sufficient activity were evaluated for their ability to inhibit the
proliferation of the GTL-16 human gastric carcinoma cell line,
which expresses high levels of constitutively-activated Met ki-
nase.6 Studies have shown this cell line to be very sensitive to
small molecule inhibitors of Met.12 As such, it was utilized as the
primary in vitro cell line for the screening of Met inhibitors.
In the Met kinase assay, utilization of the aminopyridine core
with an initial p-chlorophenyl amide (8a) resulted in a compound
demonstrating potent activity. Incorporating a 2,4-dihalogenated
amine, in the form of the 2,4-difluorophenylamide (8b), afforded
O
H2N
N
Ar
NH O
F
Figure 2. X-ray structure of acylurea 2 in the Met binding domain.
fluorophenyl ring and attached acylurea group seen in compound 2
(Fig. 2). Further analysis suggested that utilization of a diphenyl-
amine in this region, would force the rings out-of-plane by approx-
imately 30°. Incorporation of an aromatic nitrogen ortho to the
diarylamino nitrogen, was proposed to relieve this potential con-
formational energy strain.
Herein, we describe the synthesis, structure–activity relation-
ships, and in vivo activity of novel biarylamine-based Met kinase
inhibitors.
The synthesis of compounds in these series centered upon the
acid-catalyzed addition of an appropriate 2-fluoro-4-aryloxyaniline
to a desired 2-halonicotinamide (Scheme 1). Thus, 2-chloro- or
2-fluoronicotinamide 5 could be readily generated from the precur-
sor nicotinic acid via generation of the acid chloride and coupling
that acid chloride with a suitable amine. Addition of the resultant
halonicotinamide to 4-(4-amino-2-fluorophenoxy)picolinamide
6,8 in the presence of p-toluenesulfonic acid monohydrate,9
CO2H
N
X
X = Cl, F
4
a, b
NRR'
O
N
X
5
F
NH2
F
NH2
O
O
c, d
e or f
N
N
7
H2NOC
N
6
H
N
NRR'
N
NRR'
F
NH
O
F
NH
O
O
O
H2N
N
N
N
9
8
H
Scheme 1. Reagents and conditions: (a) (ClCO)2, THF, DMF, reflux; (b) RR0NH, DMF, pyr, 55–100% (c) p-TsOHꢀH2O, NMP, MW (300 W), 160 °C, 31–66%; (d) PhI(OCOCF3)2, H2O,
pyr, DMF 25–93%; (e) p-TsOHꢀH2O, NMP, MW (300 W), 160 °C, 18–27%; (f) 4 N HCl/dioxane (4 equiv), NMP, 110 °C, 60–72%.

3000
D. K. Williams et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2998–3002
Table 1
Table 2
SAR of 2-aminopyridine (AP) and 1H-pyrrolo[2,3-b]pyridine (PP) nicotinamidesa
SAR of heterocyclic portion of the diarylaminea
Core
NR1R2
Het
F
Het
F
N
H
H
N
N
NH
O
F
O
O
NH
F
NH
F
F
F
N
H
N
N
H2N
O
O
O
AP
PP
Core
Compound–core
–NR1R2
IC50
,
l
M
H2N
N
N
PP
AP
IC50
N
H
Met
GTL-16
Compound–core
Het
,
l
M
HN
F
Cl
F
8a–AP
8b–AP
0.011
1.16
Met
GTL-16
H3C
Br
15–PP
0.018
0.013
2.40
0.008
0.007
0.006
0.024
0.90
0.21
0.42
1.20
N
HN
F
16–AP
0.59
0.64
9b–PP
9c–PP
9d–PP
HN
H3C
HN
Cl
F
N
Br
17–PP
0.009
N
N
HN
18–AP
19–PP
3.50
0.27
—
H3C
N
9e–PP
9f–PP
9g–PP
0.19
5.58
0.27
0.46
F
F
N
N
>10.00
0.034
0.021
HN
N
N
20–AP
21–PP
0.069
0.055
2.73
7.15
N
N
NH
OH
9h–PP
0.009
0.40
HN
N
N
a
Assay conditions are detailed in Ref. 6.
22–AP
12–AP
0.022
0.009
1.93
0.44
H3CS
N
similar activity in both assays. With a change to the pyrrolopyri-
dine core (9b), potency in the Met kinase assay was maintained,
while a greater than fourfold improvement in activity in the GTL-
16 cellular proliferation assay was observed. Monosubstitution in
the 2-position of the aniline (9c and 9d) afforded compounds that,
while still potent in the Met kinase assay, exhibited somewhat de-
creased cellular activity, compared to 9b. Methylation of the nico-
tinamide nitrogen (9e) resulted in compounds with significantly
reduced activity in both assays. As expected, smaller substitutions
on the nicotinamide nitrogen, such as the primary carboxamide or
the N-methyl nicotinamide, also resulted in compounds with
reduced activity in the Met kinase assay (data not shown). Hetero-
aromatic replacement of the phenyl ring, in the form of N-pyridin-
2-yl-nicotinamide 9f, afforded potent cellular activity. Extension of
the terminal phenyl via N-benzyl-nicotinamide (9g) and incorpora-
tion of polar substituents, such as hydroxyethyl (9h) also resulted
in compounds exhibiting potency against Met kinase as well as cel-
lular activity.
N
CH3
N
N
H3C
14–AP
0.12
>10.00
N
a
Assay conditions are detailed in Ref. 6.
various biarylamine targets, wherein the heterocycle was defined
as a pyridine ring, were generally carried out similarly to the route
previously described in Scheme 1.8 In the case of the pyrimidine
analogs a slightly different route was employed (Scheme 2).8
Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate was
treated with 4-(4-amino-2-fluorophenoxy)-picolinamide, under
acidic conditions, to give ethyl 4-(4-(2-carbamoylpyridin-4-yloxy)-
3-fluoro-phenylamino)-2-(methylthio)-pyrimidine-5-carboxylate.
Saponification with NaOH, afforded the carboxylate salt which was
thencoupledwith 2,4-difluoroanilineto give intermediate11. Treat-
For the purpose of exploring the SAR about the central hetero-
cycle, the terminal 2,4-diflourophenyl group was held constant
while varying the central heterocycle (Table 2). The synthesis of

D. K. Williams et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2998–3002
3001
MeS
N
MeS
N
F
F
H
H
N
N
N
N
SMe
N
F
NH
O
F
NH
O
N
O
F
F
a−d
f
Cl
OEt
O
O
11
12
NH2
N
N
NH2
10
O
e
O
O
N
N
S
N
F
F
H
N
H
N
N
N
F
NH
O
F
NH
O
F
F
g, h
O
O
13
14
NH2
N
NH2
N
O
Scheme 2. Reagents and conditions: (a) 6, 4 N HCl/dioxane, NMP, 93%; (b) 4 N NaOH, MeOH/H2O, 83%; (c) SOCl2, reflux; (d) 2,4-difluoroaniline, 45%; (e) m-CPBA, DMF, 81%;
(f) PhI(OCOCF3)2, H2O, pyr, DMF, 53%; (g) dimethylamine, THF, 78%; (h) PhI(OCOCF3)2, H2O, pyr, DMF, 36%.
ment with bis(trifluoroacetoxy)iodobenzene afforded the 2-
(methylthio)pyrimidine product 12.11b Alternatively, intermediate
11 was oxidized with m-CPBA to afford sulfone 13, which was then
displaced by a variety of nucleophiles, including dimethylamine.
Subsequent treatment with bis(trifluorocetoxy)iodobenzene affor-
ded compound 14.
The SAR about the heterocyclic portion of the biarylamines is
shown in Table 2. Compounds which incorporated a nitrogen in
the heterocycle, ortho to the amine juncture, showed preferred
activity in the Met kinase assay. Consistent with the activity ob-
served for the 2-aminonicotinamides 8b and 9b, compounds
substituted in the 5- or 6-position (15–17) also exhibited potency
against Met kinase regardless of the core being utilized. The
4-aminonicotinamides, 18 and 19, were less potent in the kinase
assay, presumably due to the necessity of that pyridine ring to lie
out-of-plane with the fluorophenylamino ring. Replacement of
the pyridine ring of the nicotinamide with pyrazine (20 and 21),
led to an eightfold drop in kinase activity, relative to 8b and 9b.
A decrease in the cellular activity was also observed. Kinase and
cellular activities observed with pyrimidine substitution (22, 12,
and 14) were found to be sensitive to the type of group located
in the 2-position of the pyrimidine. Attempts to improve the solu-
bility of these compounds, by incorporating a 2-position substitu-
ent having a basic nitrogen, resulted in a decrease in activity.
The X-ray crystal structure of 8b13 revealed the compound re-
sides in the Met kinase domain (Fig. 3) with a conformation consis-
tent with that of pyridone 3.7 Of particular note is how the
biarylamine rings adopt a conformation in which they approach
planarity mimicking the hydrogen-bond between the amide NH
and the 2-pyridone carbonyl oxygen in 3.
Figure 3. Comparison of X-ray co-crystal structures of compounds 3 (green) and 8b
(orange) bound to the Met Kinase Domain [Met(1067-1378)/Y1212F, Y1252F,
Y1253D].
daily over 15 days. Figure 4 illustrates the dose-dependent tumor
growth inhibition of compound 9b in the GTL-16 gastric carcinoma
model. The compound demonstrated activity at multiple dose lev-
els, resulting in complete tumor stasis at the lowest dose tested
(25 mg/kg).
In summary, biarylamine-based inhibitors of Met kinase were
identified. This template was found to serve as an effective surro-
gate for the hydrogen-bonded array previously observed in acylu-
reas, malonamides, and pyridones. When suitably tethered to an
aminopyridine or pyrrolopyridine, these compounds demonstrated
potent activity in the kinase and cellular proliferation assays. Based
on the X-ray crystal structure, compound 8b was shown to adopt a
conformation consistent with that previously seen for compound
3, in the Met kinase binding domain. Additionally, in vivo studies
involving compound 9b demonstrated that these compounds can
exhibit potent antitumor activity.
Based on its potency against Met kinase,14 as well as its activity
in the GTL-16 cellular proliferation assay, 9b was evaluated in a
preliminary pharmacokinetic study in mice. The compound dem-
onstrated reasonable exposures in an 8 h mouse oral exposure
study at 50 mpk (Cmax = 9.3 lM, AUC0–8 h = 42 lM h) and was sub-
sequently taken into the GTL-16 human gastric carcinoma model.5
In the study, anthymic mice are implanted with GTL-16 xenografts.
The compound was administered orally at various dose levels, once

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D. K. Williams et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2998–3002
3. A tabular summary of the role of HGF/SF and Met in various cancers, with links
to appropriate references, is available at: http://www.vai.org/met/.
4. Danilkovitch-Miagkova, A.; Zbar, B. J. Clin. Invest. 2002, 109, 863.
5. Cai, Z.-W.; Wei, D.; Schroeder, G. M.; Cornelius, L. A. M.; Kim, K.; Chen, X.-T.;
Schmidt, R. J.; Williams, D. K.; Tokarski, J. S.; An, Y.; Sack, J. S.; Manne, V.;
Kamath, A.; Zhang, Y.; Marathe, P.; Hunt, J. T.; Lombardo, L. J.; Fargnoli, J.;
Borzilleri, R. M. Bioorg. Med. Chem. Lett. 2008, 18, 3224.
6. Schroeder, G. M.; Chen, X.-T.; Williams, D. K.; Nirschl, D. S.; Cai, Z.-W.; Wei, D.;
Tokarski, J. S.; An, Y.; Sack, J.; Chen, Z.; Huynh, T.; Vaccaro, W.; Poss, M.;
Wautlet, B.; Gullo-Brown, J.; Kellar, K.; Manne, V.; Hunt, J. T.; Wong, T. W.;
Lombardo, L. J.; Fargnoli, J.; Borzilleri, R. M. Bioorg. Med. Chem. Lett. 2008, 18,
1945.
7. Kim, K. S.; Zhang, L.; Schmidt, R.; Cai, Z.-W.; Wei, D.; Williams, D. K.; Lombardo,
L. J.; Trainor, G. L.; Xie, D.; Zhang, Y.; An, Y.; Sack, J. S.; Tokarski, J. S.; Darienzo,
C.; Kamath, A.; Marathe, P.; Zhang, Y.; Lippy, J.; Jeyaseelan, R.; Wautlet, B.;
Henley, B.; Gullo-Brown, J.; Manne, V.; Hunt, J. T.; Fargnoli, J.; Borzilleri, R. M. J.
Med. Chem. 2008, 51, 5330.
8. Borzilleri, R. M.; Chen, X.-T.; Williams, D. K.; Tokarski, J. S.; Kaltenbach, R. F. U.S.
Patent Application 2007/0078140, 2007.
9. (a) Galeeva, R. N.; Gavrilov, M. Y.; Nazmetdinov, F. Y.; Drovosekova, L. P.; Kolla,
V. E.; Kon’shin, M. E. Khim. Farm. Zh. 1998, 32, 31; (b) Kermack, W. O.;
Weatherhead, A. P. J. Chem. Soc. 1942, 726.
10. Yu, C.; Jiang, Y.; Liu, B.; Hu, L. Tetrahedron Lett. 2001, 42, 1449.
11. (a) Representative analytical data for: 9b (HCl salt): 1H NMR (400 MHz, DMSO-
d6) d 12.83 (s, 1H), 10.75 (s, 1H), 10.52 (s, 1H), 8.28–8.41 (m, 3H), 8.15 (dd, 1H,
J = 13.6, 2.2 Hz), 7.32–7.56 (m, 5H), 7.09–7.13 (m, 1H), 6.98–7.01 (m, 1H), 6.72
(d, 1H, J = 6.5 Hz), 6.48–6.49 (m, 1H); HRMS for C25H17F3N5O2 (M+H)+: calcd:
476.1334, found: 476.1350.; (b) 12 (HCl salt):1 H NMR (400 MHz, DMSO-d6) d
11.06 (s, 1H), 10.51 (s, 1H), 8.94 (s, 1H), 8.12 (dd, 1H, J = 13.0, 2.3 Hz), 7.98 (d,
1H, J = 7.2 Hz), 7.90 (br s, 2H), 7.61–7.40 (m, 4H), 7.18 (m, 1H), 6.74 (dd, 1H,
J = 7.3, 2.5 Hz), 6.18 (d, 1H, J = 2.4 Hz), 2.57 (s, 3H); MS(ESI+) m/z 499 (M+H)+.
12. Christensen, J. G.; Schreck, R.; Burrows, J.; Kuruganti, P.; Chan, E.; Le, P.; Chen,
J.; Wang, X.; Ruslim, L.; Blake, R.; Lipson, K. E.; Ramphal, J.; Do, S.; Cui, J. J.;
Cherrington, J. M.; Mendel, D. B. Cancer Res. 2003, 63, 7345.
Figure 4. In vivo activity of compound 9b versus GTL-16 xenografts implanted in
anthymic mice (arrows denote days of dosing).
References and notes
1. For recent reviews on Met kinase see: (a) Migliore, C.; Giordano, S. Eur. J. Cancer
2008, 44, 641; (b) Peruzzi, B.; Bottaro, D. P. Clin. Cancer Res. 2006, 12, 3657; (c)
Christensen, J. G.; Burrows, J.; Salgia, R. Cancer Lett. 2005, 225, 1; (d) Ma, P. C.;
Maulik, G.; Christensen, J.; Salgia, R. Cancer Metastasis Rev. 2003, 22, 309.
2. (a) Maulik, G.; Shrikhande, A.; Kijima, T.; Ma, P. C.; Morrison, P. T.; Salgia, R.
Cytokine Growth Factor Rev. 2002, 13, 41; (b) Bottaro, D. P.; Rubin, J. S.; Faletto,
D. L.; Chan, A. M.; Kmiecik, T. E.; VandeWoude, G. F.; Aaronson, S. A. Science
1991, 251, 802.
13. The atomic coordinates have been deposited in the RCSB Protein Data Bank
(PDB code: 3L8V).
14. Compound 9b was further evaluated in an in-house kinase selectivity panel,
where it was found to be more than 17-fold selective for Met versus Flt-3
(IC50 = 0.12
(IC50 = 0.35
l
l
M) and greater than 50-fold selective against TrkA
M). Other kinases tested in the assay, including Lck, IFGR-1R,
InsR, MK2, CDK2E and PKC-
50 M.
a, were found to have IC50 values of greater than
l