Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase

Article abstract of DOI:10.1016/j.bmcl.2008.01.121

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vi

Full text of DOI:10.1016/j.bmcl.2008.01.121

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1945–1951
Identification of pyrrolo[2,1-f][1,2,4]triazine-based
inhibitors of Met kinase
Gretchen M. Schroeder,* Xiao-Tao Chen, David K. Williams, David S. Nirschl,
Zhen-Wei Cai, Donna Wei, John S. Tokarski, Yongmi An, John Sack, Zhong Chen,
Tram Huynh, Wayne Vaccaro, Michael Poss, Barri Wautlet, Johnni Gullo-Brown,
Kristen Kellar, Veeraswamy Manne, John T. Hunt, Tai W. Wong, Louis J. Lombardo,
Joseph Fargnoli and Robert M. Borzilleri
Bristol-Myers Squibb Research and Development, PO Box 4000, Princeton, NJ 08543-4000, USA
Received 2 January 2008; revised 30 January 2008; accepted 30 January 2008
Available online 7 February 2008
Abstract—An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of
Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in
in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved
potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demon-
strated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to
the Met kinase domain in an ATP-competitive manner.
Ó 2008 Elsevier Ltd. All rights reserved.
Over the last decade, a paradigm shift has occurred in
cancer therapy. While cytotoxic agents have tradition-
ally been employed to combat aberrant cellular growth,
the advent of targeted therapies such as sunitinib1
(VEGFR, PDGFR), erlotinib2 (EGFR), and dasatinib3
(Bcr-Abl, Src) has provided new treatment options.
These new therapies function through targeted inhibi-
tion of protein kinases whose normal activity has be-
come deregulated. For example, protein kinase activity
can be altered due to ligand-dependent activation (auto-
crine or paracrine), gene amplification, gene mutation,
or cross-talk with other receptors (heterodimerization).4
factor (SF), is a disulfide-linked heterodimer which can
activate Met via autocrine or paracrine mechanisms.
HGF–Met signaling has been shown to trigger a variety
of cellular responses including cell growth, migration
and invasion, tumor metastasis, angiogenesis, wound
healing, and tissue regeneration. Deregulated Met ki-
nase signaling has been implicated in a variety of cancers
including prostate7 (ligand-dependent activation), gas-
tric8 (gene amplification/overexpression), and hereditary
papillary renal cell carcinoma9 (activating mutation).
The successful identification of pyrrolo[2,1-f][1,2,4]tri-
azine-based drug candidates in our VEGFR-210 and
EGFR/pan-Her11 programs prompted us to utilize this
versatile scaffold in search of Met kinase inhibitors
(Fig. 1). Early pyrrolotriazine leads were obtained from
focused amide libraries. Computer assisted molecular
modeling of these leads in the Met kinase domain sug-
gests they bind in the ATP-binding site. The pyrrolotri-
azine moiety mimics the adenine ring of ATP in forming
critical hydrogen bonds to the hinge region of the Met
protein. Substitution of the pyrrolotriazine core at C-5
was believed to provide access to the ribose pocket while
substitution at C-6 was expected to lead to surface-ex-
posed protein, consistent with previously reported
The Met receptor, also known as hepatocyte growth fac-
tor receptor, is a glycosylated, heterodimeric receptor
tyrosine kinase, predominately expressed in the epithe-
lium and endothelium.5 Met kinase was first identified
and derived its name as an activated oncogene in
N-methyl-N0-nitro-N-nitrosoguanidine-treated human
osteosarcoma cells.6 The ligand for the Met receptor,
hepatocyte growth factor (HGF) also known as scatter
Keywords: Met kinase; Hepatocyte growth factor receptor; HGFR;
Pyrrolotriazine; Gastric carcinoma.
*
Corresponding author. E-mail: gretchen.schroeder@bms.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.121

1946
G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
Reduction of the nitro group proceeded smoothly with
Zn/NH4Cl to afford aniline 4 in 51–95% yield. Finally,
amide formation was achieved using PyBOP to give
the desired compounds 5. Alternatively, intermediate 4
could be treated with 3-(4-fluorophenyl-amino)-3-oxo-
propanoic acid or 2-(4-fluoro-phenyl)acetyl isocyanate13
to afford malonamides 6 and acylureas 7, respectively.
NH
F
HO
H2N
HN
O
O
N
N
N
HO
O
N
N
N
N
BMS-540215
BMS-690514
VEGFR-2 Inhibitor
pan-HER Inhibitor
Analogs with solubilizing basic amines at C-5 were pre-
pared via one of two routes (Scheme 2). In the first ap-
proach, tetraalkylammonium salt 811 was reacted with
commercially available 4-amino-2-fluorophenol fol-
lowed by a secondary amine to rapidly yield intermedi-
ate 9. Acylurea formation as described above gave the
desired compounds 10.
Figure 1. Pyrrolo[2,1-f][1,2,4]triazine-based kinase inhibitors.
observations.10,11 This paper describes our efforts to ac-
cess the ribose pocket and surface-exposed protein of
the Met receptor using the pyrrolotriazine core as an
ATP mimetic.
Alternatively, treatment of 5-methyl pyrrolotriazine 1112
with sodium thiomethoxide gave the corresponding thi-
oether. Reaction of the intermediate thioether with one
equivalent of NBS gave a benzylic bromide which could
be displaced with various secondary amines. The result-
ing amine-substituted pyrrolotriazine 12 was then trea-
ted with 1 equiv of mCPBA to generate the
corresponding sulfoxide. TFA was added to the oxida-
tion to coordinate the basic amine, leaving the mCPBA
free to perform its desired function. The resulting sulfox-
ide was then displaced with the sodium anion of 4-ami-
no-2-fluorophenol to give aniline 9, which was readily
converted to compound 10.
SAR at the pyrrolotriazine C-6 position was explored
using the chemistry sequence shown in Scheme 1 where-
in the protected hydroxyl group in 112 served as a handle
for introduction of polar substituents. Treatment of
compound 1 with phosphorous oxytrichloride and Hu-
nig’s base gave rise to chloroimidate 2 in 78% yield. Dis-
placement of the chlorine atom at C-4 of the
pyrrolotriazine with 2-fluoro-4-nitrophenol took place
using K2CO3 to yield the coupled product. Subsequent
hydrolysis of the pivaloyl-protecting group at the C-6
position was achieved in 85% yield to give 3. The result-
ing alcohol was derivatized using Mitsunobu conditions.
H
R2
F
NO2
F
NH2
F
N
O
O
N
Cl
N
O
O
O
5
NH
N
N
O
N
6
N
b,c
d,e
f
a
O
O
O
O
HO
O
O
N
N
N
N
N
R1
N
R1
N
N
F
1
2
3
4
5
h
g
H
N
H
N
H
N
H
N
F
F
O
O
O
O
F
O
N
N
O
O
7
N
N
6
R1
R1
N
N
Scheme 1. Reagents and conditions: (a) POCl3, DIPEA, toluene, 110 °C, 4 h, 78%; (b) 2-fluoro-4-nitrophenol, K2CO3, DMF, rt, 24 h, 44%; (c)
NaOH, MeOH/THF, rt, 5 min, 85%; (d) R1CH2OH, DIAD, PPh3, 0 °C to rt, 12 h, 19–82%; (e) Zn, NH4Cl, MeOH/THF, rt, 7 h, 51–95%; (f)
R2CO2H, PyBOP, DIPEA, DMF, 70 °C, 5 h; (g) 3-(4-fluorophenylamino)-3-oxo-propanoic acid, PyBOP, DIPEA, THF, rt, 24 h, 13–80%; (h) 2-(4-
fluorophenyl)acetyl isocyanate, DCM, rt, 2 h, 15–46%.
Et3N+
Br-
Cl
N
H
N
O
a
N
F
NH2
F
NH
O
N
F
R2N
O
8
R2N
O
N
b
N
N
N
Cl
R2N
SCH3
N
N
9
10
N
N
N
c,d
N
N
N
e
12
11
Scheme 2. Reagents and conditions: (a) NaHMDS, 4-amino-2-fluorophenol, DMF, 2 h, then HNR2, DIPEA, 130 °C, 4 h, 20–30%; (b) 2-(4-
fluorophenyl)acetyl isocyanate, DCM, 1 h, 60%; (c) NaSMe, THF, 0 °C, 18 h, 90%; (d) NBS, AIBN, CCl4, 80 °C, 1 h, then HNR2, DIPEA, 18 h, 50–
70%; (e) mCPBA (1.1 equiv), 1.5 equiv TFA, DCM, 0 °C, 1 h, then NaHMDS, 4-amino-2-fluorophenol, THF, 0 °C, 1 h, 30–50%.

G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
Table 1. Pyrrolo[2,1-f][1,2,4]triazine benzamidesa
1947
Finally, a conformationally constrained C-5 analog 17
was prepared as shown in Scheme 3. Alkenyl boronate
ester 14 was prepared from the known ketone 1314 via
ruthenium-catalyzed cross-metathesis.15 Palladium-cat-
alyzed Suzuki coupling with 5-chloro pyrrolotriazine
15,12 followed by acylurea formation, gave ketone 16.
Finally, reductive amination with ammonium acetate
furnished analog 17.16
O
R2
NH
F
O
N
R1
N
N
Compound R1
R2
Met IC50 (lM)
Initial efforts were directed at exploring SAR of the
benzamide moiety of a C-6 unsubstituted pyrrolotri-
azine via the synthesis of a small library.12 Unfortu-
nately, Met kinase inhibitory activity of these analogs
was disappointing with IC50’s in the low to mid micro-
molar range (for example, 5a, Table 1). Dramatic
improvement in potency could be achieved upon addi-
tion of a polar substituent to C-6 of the pyrrolotriazine
core. For example, N-methyl piperidine was appended
to C-6 of the pyrrolotriazine core to give compound
5b. This analog demonstrated an 8-fold improvement
in activity with an IC50 of 0.09 lM as compared to the
unsubstituted compound 5a. The appendage of a polar
group such as a basic amine also benefited from a signif-
icant increase in aqueous solubility, a common problem
encountered in ATP-competitive kinase inhibitor pro-
grams. The 2,5- and 2,6-difluorobenzamides 5c and 5d
gave improved potency relative to the 2-fluoro analog
with IC50’s of 0.04 and 0.03 lM, respectively. This trend
mirrors that observed in the C-6 unsubstituted series of
benzamides (data not shown). The 2-fluoro-5-methyl
benzamide 5e and 3-acetyl benzamide 5f showed good
activity against Met kinase each with an IC50 of
0.09 lM. While the 3-chloro-6-fluorobenzamide analog
5g was also potent with an IC50 of 0.07 lM, heteroaryla-
mide analogs 5h and 5i were relatively less potent. The
heteroaryl analogs compare favorably to the unsubsti-
tuted phenyl compound where R1 = H and R2 = Ph
(IC50 > 2 lM, data not shown).
F
5a
5b
5c
5d
5e
5f
H
0.76
0.09
0.04
0.03
0.09
0.09
0.07
0.16
0.19
0.51
0.03
0.07
F
O
N
N
N
N
N
N
N
N
N
F
F
O
O
O
O
O
O
O
N
N
N
N
N
N
N
F
F
F
F
O
5g
5h
5i
Cl
N
S
F
F
O
5j
O
N
The C-6 solubilizing group was varied and the morpho-
line analog 5j was found to be less potent against Met
kinase than the corresponding piperazine 5e (0.51 vs
0.09 lM). On the other hand, a 3-dimethylamino group
gave compounds with excellent potency. For example,
analog 5k possessing a 2,6-difluorophenyl amide gave
an IC50 of 0.03 lM.
N
N
O
O
5k
F
5l
N
a For assay conditions see Ref. 23.
Next, we sought to replace the benzamide moiety with
malonamides and acylureas (Table 2).17 Malonamide
6a was only marginally potent in the Met kinase assay
(IC50 = 0.78 lM). Fortunately, as seen with the benzam-
ides, incorporation of a polar substituent at the pyrrol-
H
H
N
O
N
O
F
NH
O
F
NH
O
F
F
O
H2N
O
O
O
O
d
a
b,c
N
N
B
O
O
N
N
N
N
13
14
16
17
Scheme 3. Reagents and conditions: (a) (Z)-4,4,5,5-tetramethyl-2-(prop-1-enyl)-1,3,2-dioxaborolane, Ru catalyst (Ref. 15), DCM, reflux, 6 h, 24%;
(b) 4-(5-chloropyrrolo[2,1-f][1,2,4]triazin-4-yloxy)-3-fluoroaniline 15, Pd(OAc)2, X-Phos, K3PO4, t-BuOH, 80 °C, 8 h, 18%; (c) 2-(4-fluoro-
phenyl)acetyl isocyanate, DCM, rt, 1 h, 38%; (d) NH4OAc, NaCNBH3, MeOH, THF, 0 °C, 1 h, 19%.

1948
G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
Table 2. SAR of malonamides/acylureasa
contains an amplified gene locus for the Met receptor,
resulting in expression of high levels of constitutively-
activated Met kinase. Disappointingly, compound 5b
had only marginal activity in the GTL-16 assay
(IC50 = 0.84 lM) and compounds 5c and 5d had
IC50’s greater than 1.0 lM. Compounds 5g and 5k
showed good cellular potency in the GTL-16 assay
with IC50’s of 0.27 and 0.17 lM, respectively. Malona-
mide 6e also demonstrated good potency with an IC50
of 0.27 lM. Finally, acylurea 7c gave the best cellular
activity with an IC50 of 0.068 lM. Unfortunately,
these compounds were deemed unsuitable for further
development as they possessed high rates of metabo-
lism in mouse- and human-liver microsomes (data
not shown).
O
X
Y
F
NH
O
F
O
N
R
N
N
Compound
X
Y
R
Met IC50 (lM)
6a
CH2 NH
CH2 NH
CH2 NH
CH2 NH
CH2 NH
H
0.78
O
6b
6c
6d
6e
0.04
0.13
0.14
0.03
N
N
O
Since the acylurea moiety gave the best cellular potency, it
was held constant as substitution at the C-5 position was
explored to gain access to the ribose pocket. As shown in
Table 3, 5-chloro substituted pyrrolotriazine 10a was
found to have poor activity against Met kinase
(IC50 > 2.0 lM). Replacement of the 5-chloro substituent
with a phenyl ring gave a compound with a greater than
10-fold increase in potency (10b, IC50 = 0.26 lM). While
these initial results were encouraging, the aryl and hetero-
aryl (data not shown) substituted pyrrolotriazines suf-
fered from poor aqueous solubility (typically <1 lg/mL
at pH 6.5). To address this issue, we sought to prepare
analogs that would impart improved aqueous solubility
in addition to enzyme potency through the incorporation
of basic amines. To that end, the conformationally flexible
diamine 10c gave disappointing results (IC50 = 1.4 lM),
however, the ring constrained analog 10d gave encourag-
ing activity (IC50 = 0.41 lM). Importantly, piperazine
10d demonstrated dramatically improved aqueous solu-
bility (290 lg/mL, pH 6.5) compared to the previous
aryl/heteroaryl analogs. Removal of the piperazine N–H
as in N-methyl analog 10e (IC50 = 0.86 lM) and morpho-
line 10f (IC50 = 0.99 lM) resulted in 2-fold loss of potency
relative to 10d suggesting a pivotal role for the amine.
Contraction of the piperazine to give a 3-amino pyrroli-
dine ring was well tolerated (10g and 10h). Absolute con-
figuration of the pyrrolidine amine had little effect on
enzyme potency with the (S)-enantiomer 10h giving
slightly improved Met kinase inhibitory activity
(IC50 = 0.19 lM). Further ring contraction to give azeti-
dine 10i was also tolerated and gave potent Met activity
(IC50 = 0.18 lM). Encouraged by the activity of primary
amine analogs 10h and 10i, we explored extension to ami-
no piperidines. Gratifyingly, 4-aminopiperidine 10j gave
O
N
N
O
N
N
O
O
7a
7b
7c
NH
NH
NH
CH2
0.05
0.15
0.04
N
O
CH2
CH2
N
N
O
a For assay conditions see Ref. 23.
otriazine C-6 position gave dramatically improved re-
sults. For example, malonamide 6b with a piperazine
group attached at C-6 of the pyrrolotriazine core dem-
onstrated almost 20-fold improvement in potency in
the Met kinase assay (IC50 = 0.04 lM). As observed in
the amide series, morpholine substitution also improved
the potency of Met kinase inhibition, but less efficiently
than with a piperazine (compound 6c, IC50 = 0.13 lM).
The acyclic 2-dimethylaminoethoxy analog 6d had sim-
ilar
potency
to
morpholine
compound
6c
(IC50 = 0.14 lM). Homologation of the basic amine
gave improved potency with the 3-dimethylaminoprop-
oxy analog 6e affording an IC50 of 0.03 lM. Replace-
ment of the malonamide with an acylurea substituent
was examined and the SAR was found to parallel that
of the malonamide series. Selected compounds from this
effort are shown in Table 2. As observed in the malona-
mide series, the 2-piperazine derivative 7a and the 3-
dimethylamino-propoxy derivative 7c were almost equi-
potent, while the 2-dimethylaminoethoxy analog 7b was
inferior in inhibiting Met kinase (IC50 = 0.15 lM).
dramatically
improved
enzyme
potency
(IC50 = 0.045 lM). Conversion of the amino to an alcohol
(10k) or acetamide (10l) gave significantly reduced po-
tency (IC50 = 0.90 and IC50 = 0.44 lM, respectively)
again revealing the critical nature of this amine. The ori-
entation of the piperidine substituent was reversed to give
analog 10m and this compound demonstrated a 3-fold
reduction in activity relative to 10j. Extension of the
hydrogen bond donor by insertion of a methylene was
not well tolerated (10n, IC50 = 1.0 lM). As observed in
our pan-Her program,11 addition of a second hydrogen
bond donor as in amino alcohol 10o resulted in slightly
improved potency (IC50 = 0.037 lM) relative to 10j.19
With compounds of increased potency, we examined
their ability to inhibit proliferation of the GTL-16
human gastric carcinoma cell line.18 This cell line

G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
1949
Table 3. SAR of 5-substituted pyrrolotriazinesa
enzyme with the activation loop of the protein in an
inactive, DFG-out conformation (Fig. 2).20 The pyrrol-
otriazine core engages in key hydrogen-bonding interac-
tions with the hinge region of the Met protein. For
example, backbone NH of Met1160 donates a hydrogen
bond to the pyrrolotriazine N1 nitrogen and backbone
carbonyls of Pro1158 and Met1160 accept hydrogen
bonds in the form of aromatic C–H interactions from
the pyrrolotriazine C-2 and C-7 hydrogens, respectively.
The piperidine ring occupies the ribose pocket and
forms a key ionic interaction with Asp1164. The central
fluorinated phenyl ring is flanked on one side by gate-
keeper residue Leu1157 and on the opposite face by
Phe1223 (DFG motif) in an edge-face p-stacking inter-
action. The acylurea portion of 10j forms an intramolec-
ular hydrogen bond. In addition, the terminal acylurea
carbonyl hydrogen bonds with the backbone NH of
Asp1222 and the terminal acylurea NH hydrogen bonds
with the conserved Glu1127 of helix aC. Finally, the ter-
minal 4-fluorophenyl ring resides in a mostly hydropho-
bic pocket and interacts with several aromatic residues
including Phe1134 and Phe1200.
H
N
O
F
NH
O
F
O
R
N
N
N
Compound
R
Met IC50 (lM)
10a
Cl
>2.0
10b
10c
10d
10e
10f
10g
10h
10i
Ph
0.26
1.4
H
N
H
N
2
HN
N
0.41
0.86
0.99
0.44
0.19
0.18
0.045
0.90
0.44
0.15
1.0
N
N
Based on the X-ray crystal structure of 10j, we postu-
lated that a more conformationally rigid analog could
optimize interactions in the ribose pocket. Indeed, con-
strained analog 17 gave dramatically improved results
with an IC50 against Met kinase of 1.5 nM (Table 3).
Compound 17 was tested in the GTL-16 gastric carci-
noma cell proliferation assay and was determined to
have an IC50 of 140 nM.21
O
H
N
N
2
N
N
H N
2
In pharmacokinetic studies, compound 10j demon-
strated high stability in mouse-liver microsome
(MLM = 0 nmol/min/mg protein) and mouse hepatocyte
(2 pmol/min/106 cells) assays. The compound demon-
strated low clearance after iv dosing (25 mL/min/kg)
and 81% bioavailability from ip administration both at
doses of 20 mg/kg, respectively. Low exposures were ob-
H N
2
N
H N
2
10j
N
served following
a 50 mg/kg oral dose (AUC0–
HO
8h = 1.1 lM h, Cmax = 0.78 lM) and the poor PK was
attributed to limited absorption. Consistent with this
10k
10l
N
H
N
O
N
HN
10m
10n
10o
17
N
H
H N
2
N
OH
H N
2
0.037
0.0015
N
H N
2
a For assay conditions see Ref. 23.
An X-ray crystal structure of compound 10j complexed
with the Met kinase domain demonstrated that pyrrolo-
triazine inhibitors bind in the ATP-binding site of the
Figure 2. X-ray co-crystal structure of compound 10j bound to the
Met kinase domain (triple mutant). The hinge region of the Met
protein is shaded blue.

1950
G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
A.; Vyas, V.; Marathe, P.; D’Arienzo, C.; Derbin, G.;
Fargnoli, J. J. Med. Chem. 2006, 49, 2143, and references
therein.
hypothesis, 10j demonstrated poor cell permeability in a
Caco-2 assay (Pc < 15 nm/s).22
11. (a) Gavai, A. V.; Chen, P.; Norris, D.; Fink, B. E.;
Mastalerz, H.; Zhao, Y.; Han, W.-C.; Zhang, G.; John-
son, W.; Ruediger, E.; Dextraze, P.; Daris, J.-P.; Kim,
S.-H.; Leavitt, K.; Kim, K.; Lu, S.; Zheng, P.; Mathur, A.;
Vyas, D.; Tokarski, J. S.; Yu, C.; Oppenheimer, S.; Zhang,
H.; Lee, F.; Wong, T.W.; Vite, G. D. Abstracts of Papers,
233rd National Meeting of the American Chemical
Society, Chicago, IL, March 25–29, 2007; American
Chemical Society: Washington, DC, 2007; MEDI-017;
(b) Gavai, A. V.; Han, W.-C.; Chen, P.; Ruediger, E. H.;
Mastalerz, H.; Fink, B. E.; Norris, D.J. U.S. Patent US
7,064,203, 2006.
In summary, potent C-5 and C-6 substituted pyrrolotri-
azine inhibitors of Met kinase were identified. Initial
studies explored benzamides with solubilizing groups
at the pyrrolotriazine C-6 position. Acylureas and malo-
namides were found to be suitable benzamide replace-
ments and provided compounds with excellent cellular
potency. The series suffered from poor PK properties
making the compounds unsuitable for in vivo efficacy
studies. Further SAR studies to address liabilities of
the present series of compounds will be discussed in
due course.
12. Borzilleri, R. M.; Chen, Z.; Hunt, J. T.; Huynh, T.; Poss,
M. A.; Schroeder, G. M.; Vaccaro, W.; Wong, T. W.;
Chen, X.-T.; Kim, K. S. U.S. Patent US 7,173,031, 2007.
Examples from the amide library can be found in Table 2.
13. The known acyl isocyanate was prepared by the method
described in Shaw-Ponter, S.; Mills, G.; Robertson, M.;
Bostwick, R. D.; Hardy, G. W.; Young, R. J. Tetrahedron
Lett. 1996, 37, 1867.
References and notes
1. Sun, L.; Liang, C.; Shirazian, S.; Zhou, Y.; Miller, T.; Cui,
J.; Fukuda, J. Y.; Chu, J.-Y.; Nematalla, A.; Wang, X.;
Chen, H.; Sistla, A.; Luu, T. C.; Tang, F.; Wei, J.; Tang,
C. J. Med. Chem. 2003, 46, 1116.
2. Moyer, J. D.; Barbacci, E. G.; Iwata, K. K.; Arnold, L.;
Boman, B.; Cunningham, A.; Diorio, C.; Doty, J.; Morin,
M. J.; Moyer, M. P.; Neveu, M.; Pollack, V. A.; Pustilnik,
L. R.; Reynolds, M. M.; Sloan, D.; Theleman, A.; Miller,
P. Cancer Res. 1997, 57, 4838.
3. Lombardo, L. J.; Lee, F. J.; Chen, P.; Norris, D.; Barrish,
J. C.; Behnia, K.; Castaneda, S.; Cornelius, L. A. M.; Das,
J.; Doweyko, A. M.; Fairchild, C.; Hunt, J. T.; Inigo, I.;
Johnston, K.; Kamath, A.; Kan, D.; Klei, H.; Marathe, P.;
Pang, S.; Peterson, R.; Pitt, S.; Schieven, G. L.; Schmidt,
R. J.; Tokarski, J.; Wen, M.-L.; Wityak, J.; Borzilleri, R.
M. J. Med. Chem. 2004, 47, 6658.
4. For recent reviews of small molecule inhibitors of tyrosine
kinases see (a) Madhusudan, S.; Ganesan, T. S. Clin.
Biochem. 2004, 37, 618; (b) Sawyer, T. K. Expert Opin.
Investig. Drugs 2004, 13, 1; (c) Laird, A. D.; Cherrington,
J. M. Expert Opin. Investig. Drugs 2003, 12, 51; (d)
Grosios, K.; Traxler, P. Drugs Future 2003, 28, 679.
5. For recent reviews on Met kinase see (a) Peruzzi, B.;
Bottaro, D. P. Clin. Cancer Res. 2006, 12, 3657; (b)
Christensen, J. G.; Burrows, J.; Salgia, R. Cancer Lett.
2005, 225, 1; (c) Ma, P. C.; Maulik, G.; Christensen, J.;
Salgia, R. Cancer Metastasis Rev. 2003, 22, 309.
6. Cooper, C. S.; Park, M.; Blair, D. G.; Tainsky, M. A.;
Huebner, K.; Croce, C. M.; Vande Woude, G. F. Nature
1984, 311, 29.
7. (a) Knudsen, B. S.; Magnus, E. Adv. Cancer Res. 2004, 91,
31; (b) Humphrey, P. A.; Zhu, X.; Zarnegar, R.; Swanson,
P. E.; Ratliff, T. L.; Vollmer, R. T.; Day, M. L. Am. J.
Pathol. 1995, 147, 386.
8. Heideman, D. A. M.; Snijders, P. J. F.; Bloemena, E.;
Meijer, C. J. L. M.; Offerhaus, G. J. A.; Meuwissen, S. G.
M.; Gerritsen, W. R.; Craanen, M. E. J. Pathol. 2001, 194,
428.
9. Schmidt, L.; Junker, K.; Nakaigawa, N.; Kinjerski, T.;
Weirich, G.; Miller, M.; Lubensky, I.; Neumann, H. P.;
Brauch, H.; Decker, J.; Vocke, C.; Brown, J. A.; Jenkins,
R.; Richard, S.; Bergerheim, U.; Gerrard, B.; Dean, M.;
Linehan, W. M.; Zbar, B. Oncogene 1999, 18, 2343, and
references therein.
10. Bhide, R. S.; Cai, Z.-W.; Zhang, Y.-Z.; Qian, L.; Wei, D.;
Barbosa, S.; Lombardo, L. J.; Borzilleri, R. M.; Zheng,
X.; Wu, L. I.; Barrish, J. C.; Kim, S.-H.; Leavitt, K.;
Mathur, A.; Leith, L.; Chao, S.; Wautlet, B.; Mortillo, S.;
Jeyaseelan, R.; Kukral, D.; Hunt, J. T.; Kamath, A.; Fura,
14. Frontier, A. J.; Danishefsky, S. J.; Koppel, G. A.; Meng,
D. Tetrahedron 1998, 54, 12721.
15. Morrill, C.; Grubbs, R. H. J. Org. Chem. 2003, 68, 6031.
16. Representative characterization data: (a) 7c: aq sol. (pH
6.5) = 23 lg/mL (crystalline material); 1H NMR (CD3OD)
d 10.77 (s, 1H), 7.80 (s, 1H), 7.69 (s, 1H), 7.40–7.23 (m,
5H), 7.11–7.02 (m, 3H), 4.15–4.12 (m, 2H), 3.71 (s, 2H),
3.41–3.36 (m, 2H), 2.97 (s, 6H), 2.44 (s, 3H), 2.31–2.22 (m,
2H); HRMS for C27H28F2N6O4 (M+H)+ Calcd 539.2218.
Found: 539.2218; (b) 10j, aq sol. (pH 6.5) = 1500 lg/mL
(crystalline material); 1H NMR (DMSO-d6) d 11.01 (s,
1H), 10.57 (s, 1H), 8.21–8.18 (m, 2H), 7.73 (dd, 1H,
J = 12.8, 2.4 Hz), 7.48–7.33 (m, 4H), 7.18–7.11 (m, 3H),
4.57 (m, 1H), 3.69 (s, 2H), 3.44 (s, 2H), 3.59–3.48 (m, 2H),
3.13–3.06 (m, 2H), 2.05–1.98 (m, 2H), 1.71–1.55 (m, 2H);
MS(ESI+) m/z 535.29 (M+H)+.
17. (a) Fujiwara, Y.; Senga, T.; Nishitoba, T.; Osawa, T.;
Miwa, A.; Nakamura, K. PCT Int. Patent Appl. WO
2003/000660, 2003; (b) Fujiwara, Y.; Senga, T.; Nishitoba,
T.; Osawa, T.; Nakamura, K. U.S. Patent 2004/0242603,
2004; (c) Bannen, L. C.; Chan, D. S.-M.; Chen, J.;
Dalrymple, L. E.; Forsyth, T. P.; Huynh, T. P.; Jamma-
lamadaka, V.; Khoury, R. G.; Leahy, J. W.; Mac, M. B.;
Mann, G.; Mann, L. W.; Nuss, J. M.; Parks, J. J.;
Takeuchi, C. S.; Wang, Y.; Xu, W. PCT Int. Patent Appl.
WO 2005/030140, 2005; (d) Kung, P.-P. PCT Int. Patent
Appl. WO 2005/121125, 2005; (e) Borzilleri, R. M.;
Cornelius, L. A. M.; Schmidt, R. J.; Schroeder, G. M.;
Kim, K. S. U.S. Patent 2005/0245530, 2005; (f) Saavedra,
O. M.; Claridge, S. W.; Zhan, L.; Raeppel, F.; Vaisburg,
A.; Raeppel, S.; Deziel, R.; Mannion, M.; Zhou, N. Z.;
Gaudette, F.; Isakovic, L.; Wahhab, A.; Granger, M.-C.;
Bernstein, N. U.S. Patent 2007/0004675, 2007.
18. Inhibition of GTL-16 cell growth was assessed by a
tetrazolium-based colorimetric assay (490 nm) using a
CellTiter 96 Aqueous Non-Radioactive Proliferation
Assay kit from Promega. GTL-16 cells were inoculated
into 96-well microtiter plates in 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 treatment, 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
treatment, the plates were incubated for an additional 72 h
before processing, and measuring cell populations. Each
compound was tested at 8 different concentrations in

G. M. Schroeder et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1945–1951
1951
triplicate, as was the untreated control sample. Growth
inhibition of 50% (IC50) is calculated by analysis of the
data in Excel that uses a 4 parameter logistic equation with
data fitted using the Levenburg Marquardt algorithm.
19. Compound 10j was tested against a variety of protein
kinases. The IC50 was determined to be greater than 5 lM
for IGF1R, InsR, LCK, VEGFR2, CDK2, PKC, PKA,
HER2, and MK2. Moderate activity was observed for
TrkA (IC50 = 200 nM).
20. The structure was deposited in the PDB as 3C1X.
21. The discrepancy between enzymatic and cell activity is
likely attributed to poor cell permeability (Caco-2
Pc < 15 nm/s).
22. For experimental details see Borzilleri, R. M.; Zheng, X.;
Qian, L.; Ellis, C.; Cai, Z.-W.; Wautlet, B. S.; Mortillo, S.;
Jeyaseelan, R.; Kukral, D. W.; Fura, A.; Kamath, A.;
Vyas, V.; Tokarski, J. S.; Barrish, J. C.; Hunt, J. T.;
Lombardo, L. J.; Fargnoli, J.; Bhide, R. S. J. Med. Chem.
2005, 48, 3991.
23. Met kinase assay: The entire cytoplasmic domain incor-
porating the enzymatic function of the human c-Met
protein form was expressed as an n-terminal Glutathione-
S-Transferase (GST) recombinant fusion protein in High
Five insect cells using the Baculovirus Expression Vector
System. Glutathione-S-Sepharose (Pharmacia Biotech)
was used for rapid affinity purification to high homoge-
neity. Glutathione (Sigma Chemicals) eluted protein was
further purified with buffer exchange using CentriconTM
size exclusion filter centrifugal units (Amicon Biosepara-
tions) and then stored at ꢀ80 °C. Kinase assays were
performed in 96-well microtiter plates using the synthetic
polymer poly(Glu4/Tyr) (Sigma Chemicals) as a phosp-
hoacceptor substrate. The c-Met kinase reaction mixture
contained 10 ng GST-Met enzyme, 100 lg/mL poly(Glu4/
Tyr), 1 lM ATP and 0.2 lCi [c-33P]ATP in 50 ll total
reaction volume (kinase buffer: 20 mM Tris–HCl, pH 7.0,
1 mM MnCl2, 0.5 mM dithiothreitol and 100 lg/mL
BSA). Reaction mixtures were incubated for 60 min at
27 °C and stopped by the addition of cold trichloroacetic
acid (TCA) to a 4% final concentration. TCA precipitates
were collected onto GF/C unifilter plates (Packard Instru-
ment Co., Meriden, CT) using a FiltermateTM universal
harvester (Packard Instrument Co., Meriden, CT). Filters
were quantitated using a TopCount 96/384-well liquid
scintillation counter (Packard Instrument Co., Meriden,
CT). Dose–response curves were generated to determine
the concentration required to inhibit 50% of the kinase
activity (IC50). Compounds were dissolved to a stock
concentration of 10 mM in dimethyl sulfoxide (DMSO)
and evaluated at six concentrations each in quadruplicate.
The final concentration of DMSO in the assay was 1%.
IC50 values were derived by non-linear regression analysis.